TITLE

YEAST WITH ENHANCED ASTAXANTHIN

TECHNICAL FIELD

This invention relates to production of astaxanthin (AX). More particularly, the invention relates to biological production of AX such as using modified strains of Xanthophyllomyces dendrorhous.

BACKGROUND

Astaxanthin (AX) is a carotenoid of the xanthophyll group known for its red-pinkish pigmentation. Due to its strong antioxidant properties, AX has significant application in health supplement and pharmaceutical industries. AX is also associated with desirable flesh colour in certain marine animals consumed as seafood such as shrimp, krill, crayfish, salmon, and trout. Generally, such animals cannot synthesise AX de-novo, and AX supplementation is used during production by aquaculture.

Currently, AX is synthesised primarily using petrochemical feedstock by a double Wittig reaction. AX can also be biologically synthesised, for example using the microalgae Haematococcus pluvialis, the gram-negative bacteria Paracoccus carotinifaciens, and the yeast Xanthophyllomyces dendrorhous (alternatively known as Phaffia rhodozyma). At present, the market size for pure AX is about 670 metric tonnes per annum, valued at about US$1.1 billion, with this market expected to exceed sales of US$2.25 billion by 2025. Synthetically produced AX is priced from about US$1,000 per kilogram, while biologically produced AX is priced from about US$7,000 per kilogram. Synthetic AX dominates the current global market due to its significantly lower cost, although health and safety concerns, environmental issues associated with the synthetic production process, and observed higher antioxidant activity of biologically produced AX as compared to synthetic AX, along with the trends towards use of natural products, sees significant current interest in the biological production of AX.

X. dendrorhous is a basidiomycetous yeast that produces AX as its main fermentation product using the mevalonate pathway. This yeast is typically preferred to other microbial AX producers for industrial purposes in view of superior growth rate, productivity, and robustness, and an ability to assimilate a wide diversity of carbon sources from feedstock or waste products including sucrose, glucose, fructose, xylose, glycerol, molasses, and bagasse hydrolysate (among others). Nevertheless, wild-type strains of X. dendrorhous produce relatively low yields of AX (200-400 pg/gDCW) which limits industrial efficacy. A detailed evaluation has suggested that AX yield, biomass density, and fermenter volume parameters of above 4,000 pg/gDCW, 60 g/L, and 1,500 L, respectively, could result in biological production of AX from A. dendrorhous that would be industrially competitive with existing synthetic approaches. With the preceding in mind, new approaches for biological production of AX would be desirable. It would be particularly desirable, in at least some instances, to develop new approaches suitable for production of AX from yeast, particularly X. dendrorhous.

Reference to prior art in the background is not, and should not be taken to be, a suggestion that the prior art forms part of the common general knowledge in any jurisdiction.

SUMMARY

A first aspect of the invention provides an isolated nucleic acid comprising a nucleotide sequence set forth in SEQ ID NOs: 1240-12684 or a nucleotide sequence at least 80% identical thereto, or a fragment of the isolated nucleic acid.

Suitably, the nucleotide sequence of the isolated nucleic acid is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 12685-19331.

In embodiments, the nucleotide sequence of the isolated nucleic acid is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

In embodiments, the nucleotide sequence of the isolated nucleic acid is or comprises a variant of a nucleotide sequence set forth in SEQ ID NOs:12685-19331. In embodiments, the nucleotide sequence is or comprises a variant of a nucleotide sequence set forth in SEQ ID NOs: 1- 1239.

The nucleotide sequence of the isolated nucleic acid may be a variant of a genic, regulatory, or intergenic region sequence as set out in Table 12. In embodiments, the variant nucleotide sequence is associated with a change in an amino acid sequence encoded by and/or expression of one or more CDS sequences set out in Table 12.

The nucleotide sequence of the isolated nucleic acid may be a variant of a genic, regulatory, or intergenic region sequence as set out in Table 13. In embodiments, the variant nucleotide sequence is associated with a change in amino acid sequence encoded by and/or expression of one or more CDS sequences set out in Table 13.

The nucleotide sequence of the isolated nucleic acid may be a variant of a genic, regulatory, or intergenic sequence as set out in Table 14. In embodiments, the variant is associated with a change in amino acid sequence encoded by and/or expression of one or more CDS sequences set out in Table 14.

The nucleotide sequence of the isolated nucleic acid may be a variant of a genic, regulatory, or intergenic sequence as set out in Table 15. In embodiments, the variant is associated with a change in amino acid sequence encoded by and/or expression of one or more CDS sequences set out in Table 15.

In embodiments, the nucleotide sequence of the isolated nucleic acid is a variant of a sequence encoding a transcript set out in Table 16. In embodiments, the nucleotide sequence of the isolated nucleic acid is a variant of a regulatory sequence for a transcript as set out in Table 16.

In embodiments, the nucleotide sequence of the isolated nucleic acid is a variant of a sequence encoding a transcript set out in Table 17.

In embodiments, the nucleotide sequence of the isolated nucleic acid is a variant of a regulatory sequence for a transcript as set out in Table 17.

In embodiments, the nucleotide sequence of the isolated nucleic acid is a variant of a CDS sequence set forth in Table 18.

In embodiments, the nucleotide sequence of the isolated nucleic acid is a variant of a regulatory sequence for a CDS sequence set forth in Table 18.

In embodiments, the nucleotide sequence of the isolated nucleic acid is a variant of a CDS sequence set forth in Table 19.

In embodiments, the nucleotide sequence of the isolated nucleic acid is a variant of a regulatory sequence for a CDS sequence set forth in Table 19.

In embodiments, the nucleotide sequence of the isolated nucleic acid comprises a nucleotide change at a position of a variation X223-X8395, as set out in Table 12. In embodiments, the nucleotide change is at a different position than any of the variations X1-X222, as set out in Table 12.

In embodiments, the nucleotide sequence of the isolated nucleic acid comprises a nucleotide change of a variation X223-X8395, as set out in Table 12. In embodiments, the nucleotide change is different than any of the variations X1-X222, as set out in Table 12.

In embodiments, the nucleotide sequence of the isolated nucleic acid comprises a nucleotide change at a position of a variation Y1-Y368, as set out in Table 13. In embodiments, the nucleotide sequence comprises a nucleotide change of a variation Y1-Y368, as set out in Table 13.

In embodiments, the nucleotide sequence of the isolated nucleic acid comprises a nucleotide change at a position of a variation Z1-Z25, as set out in Table 14. In embodiments, the nucleotide sequence comprises a nucleotide change of a variation Z1-Z25, as set out in Table 14.

In embodiments, the nucleotide sequence of the isolated nucleic acid comprises a nucleotide change at a position of a V1-V26, as set out in Table 15. In embodiments, the nucleotide sequence comprises a nucleotide change of a variation V1-V26, as set out in Table 15.

In embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs: 1240-2795 or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs:12685-19331. In embodiments, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239. In embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs: 2796-4360 or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs:12685-19331. In embodiments, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

In embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID N0s:4361-5500or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs:12685-19331. In embodiments, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

In embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs:5501-6921 or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs:12685-19331. In embodiments, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

In embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs: 6922-8057 or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs:12685-19331. In embodiments, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

In embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs: 8058-9311 or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs:12685-19331. In embodiments, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

In embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs:9312-11033 or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs:12685-19331. In embodiments, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

In embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs: 11034-12684 or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 12685-19331. In embodiments, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239. A second aspect of the invention provides an isolated protein encoded by an isolated nucleic acid comprising a nucleotide sequence set forth in SEQ ID NOs: 1240-12684 or a nucleotide sequence at least 80% identical thereto, or a fragment or derivative of the isolated protein. Suitably, the isolated protein of the second aspect is encoded by the isolated nucleic acid of the first aspect.

In embodiments, the isolated protein of the first aspect is of a protein classification selected from: short-chain dehydrogenase; deoxyribodipyrimidine photolyase/cryptochrome; nuclear transport factor 2; 60s ribosomal protein 132; armadillo/beta-catenin-like repeat-containing protein; 60s ribosomal protein 110a; pyridoxamine 5'-phosphate oxidase-like, FMN-binding domain; glutaredoxin-related protein; glycosyl transferase, family 8-glycogenin; mitochondrial carrier; nucleosome assembly protein; sterile alpha motif, type 2; snare protein ykt6; UDP-glucose dehydrogenase; predicted translation factor, contains W2 domain; G-protein beta subunit-like protein; heat shock protein HSS1 ; 40s ribosomal protein s7; ATP synthase fl beta subunit; catalase 1; stress responsive alpha-beta barrel; cytokinin riboside 5 '-monophosphate phosphoribohydrolase LOG; EF-hand domain pair; 20s proteasome subunit; ferrochelatase; glycine hydroxymethyltransferase; carboxypeptidase s; NADH-ubiquinone oxidoreductase 304 kDa subunit precursor; phytoene dehydrogenase; ribosomal protein L49/IMG2; nopl Op-domain- containing protein; thioredoxin/protein disulfide isomerase; predicted dehydrogenase; 6- phosphogluconate dehydrogenase; NADH-dehydrogenase (ubiquinone); COPII vesicle protein; ornithine aminotransferase; ER-associated protein catabolism-related protein; isocitrate dehydrogenase; AAA atpase; probable NADP-dependent dehydrogenase acting on 3 -hydroxy acids; CNDP dipeptidase; actin-related protein Arp2/3 complex, subunit ARPC2; branched-chain amino acid aminotransferase ii; carbon-nitrogen hydrolase; aspartate aminotransferase; NADPH oxidase; 26s proteasome subunit p45; pre-mRNA-splicing factor rsel; porphobilinogen deaminase; prolyl oligopeptidase; ABC transporter; 40s ribosomal protein s9; polyadenylate- binding protein; ATP-dependent RNA helicase dhx8; fatty acid synthase complex subunit alpha; glycosyltransferase family 35 protein; WD repeat protein; heat shock protein 60; succinate dehydrogenase; translocase of outer mitochondrial membrane complex, subunit TOM70/TOM72; nucleic acid-binding protein; nucleotide excision repair factor NEF2, RAD23 component; t- complex protein alpha subunit (tcp-1 -alpha); fk506-binding protein 2; aromatic amino acid aminotransferase; adenylate kinase; alpha-aminoadipate reductase lyslp; coatomer protein subunit alpha; 40s ribosomal protein s21; carbamoyl-phosphate synth; histone acetyltransferase SAGA, TRRAP/TRA1 component, PI-3 kinase superfamily; SAM-dependent RNA methyltransferase; related to 2 -hydroxy-3 -oxopropionate reductase; transcriptional coactivator pl 00; 60s ribosomal protein 113a; ornithine carbamoyltransferase; eukaryotic translation initiation factor 5b; aconitate hydratase; RNA 2-o-methyltransferase fibrillarin; t-complex protein beta subunit (tcp-1 -beta); voltage-dependent ion-selective channel; coatomer beta subunit; succinate-ligase (adp-forming); carbamoyl-phosphate synthase; related to ste23 -metalloprotease involved in a- factor processing; microtubule binding protein; pyridoxalphosphate-dependent enzyme/predicted threonine synthase; fact complex subunit SPT16; SLY1 vesicle trafficking seel -like protein; cytoplasm protein; NADH dehydrogenase; phosphoglycerate kinase; arm repeat-containing protein; ribonuclease III domain; GTP binding protein 4; peptidyl-prolyl cis-trans isomerase b; Translation initiation factor 4F, ribosome/mRNA- bridging subunit (eIF-4G); eukaryotic polypeptide chain release factor 3; asparagine synthase (glutamine-hydrolyzing); splicing factor U2AF, large subunit (RRM superfamily); NADH-cytochrome b5 reductase; histidine biosynthesis trifunctional-protein; Enoyl-CoA hydratase; alcohol; imidazoleglycerol phosphate synthase; thioredoxin-like fold; ef- hand; electron-transferring-flavoprotein dehydrogenase; MDF1 -domain-containing protein; transcription factor IIS, N-terminal; heat shock protein 70; pyruvate carboxylase; homoaconitate hydratase; uncharacterized conserved coiled-coil protein; alternative splicing factor SRp55/B52/SRp75 (RRM superfamily); eukaryotic translation initiation factor 3 subunit 7; threonyl -tma synthetase; RmlC-like jelly roll fold; 60s ribosomal protein 120; mRNA splicing factor; pre-mma-processing protein 45; atp-dependent rma helicase rrp3; dihydrolipoyllysine- residue acetyltransferase; Acyl-CoA synthetase; ribosomal protein S5; phenylalanyl-tRNA synthetase subunit beta; wd40 repeat-like protein; vacuolar ATP synthase subunit d; phosphatidylserine decarboxylase; vigilin; RNA recognition motif domain; plasma membrane h( )-atpase 1; RRM motif-containing protein; predicted GTPase-activating protein; Fl -ATP synthase assembly protein; acetyl-hydrolase; peptidyl-prolyl cis-trans isomerase; antiviral helicase; acetyl CoA carboxylase; age pka protein kinase; ATP-dependent RNA helicase pitchoune; Microtubule- associated protein; cell-cycle nuclear protein, contains WD-40 repeats; phosphoserine aminotransferase; vacuolar protein sorting-associated protein; GMP synthase; translational regulator gcn20-like abc transporter; GDP-mannose pyrophosphorylase; acetyl CoA acyltransferase 2; phosphoketolase; delta 12 fatty acid desaturase; vacuolar protein 8; predicted haloacid-halidohydrolase and related hydrolases; class iii adh enzyme; t-complex protein 1; isocitrate lyase; atpase; 6-phosphogluconolactonase; mitochondrial inner membrane protein; t- complex protein 1 subunit delta; adaptor protein complex ap-1 gamma subunit; rRNA processing protein Rrp5; succinate:fumarate antiporter; predicted proline-serine-threonine phosphatase- interacting protein (PSTPIP); phospho-2-dehydro-3 -deoxyheptonate aldolase; RNA-binding domain-containing protein; epsilon DNA polymerase; cullins; asparaginyl-tRNA synthetase; dihydroxy-acid dehydratase; SNARE protein SED5/Syntaxin 5; centromere microtubule binding protein cbf5; histidyl-tma synthetase; endoplasmic reticulum protein EP58, contains fdamin rod domain and KDEL motif; 3 -isopropylmalate dehydrogenase; Glycosyl transferase, family 1; eukaryotic translation initiation factor 3 subunit 6; phosphoglycerate mutase family; chromatin remodelling complex ATPase chain; predicted hydrolases or acyltransferases (alpha/beta hydrolase superfamily); NADH dehydrogenase subunits 2, 5, and related proteins; synaptobrevin-like protein; 40s ribosomal protein s6; ubiquitin C-terminal hydrolase UCHL1 ; polyC-binding proteins alphaCP- 1 and related KH domain proteins; nucleolar RNA-associated protein (NRAP); WD40 repeat-containing protein; pyruvate decarboxylase; RhoGEF GTPase; Ca2-dependent lipid- binding protein CLBl/vesicle protein vpl l5/Granuphilin A, contains C2 domain; molecular cochaperone STI1; vacuolar H- ATPase VI sector, subunit E; p-loop containing nucleoside triphosphate hydrolase protein; spliceosome subunit; microtubule-binding protein involved in cell cycle control; karyopherin (importin) beta 3; DNA-dependent RNA polymerase ii second largest subunit; coatomer subunit gamma; dehydrogenase kinase; mitochondrial pyruvate dehydrogenase el component beta subunit; glycoside hydrolase family 13 protein; NAD-specific glutamate dehydrogenase; mitochondrial 50s ribosomal protein 13; Ran GTPase-activating protein; FKBP- type peptidyl-prolyl cis-trans isomerase; 60s ribosomal protein 119; small nuclear ribonucleoprotein splicing factor; mannosyltransferase; dUTP pyrophosphatase; GST, gst; glutamate-tma ligase; mov34-domain-containing protein; mitochondrial nuclease; 1,4- benzoquinone reductase-like; thiamine biosynthetic bifunctional enzyme; protein of unknown function DUF3602; upf0041 -domain-containing protein; 60s ribosomal protein 111; serine/threonine protein phosphatase 2A, regulatory subunit; argininosuccinate lyase; elongation factor 1 beta delta chain; bar-domain-containing protein; uridylate kinase; phosphatidyl ethanolamine n-methyltransferase; stomatin family protein; ubiquitin-conjugating enzyme; glycosyltransferase family 2 protein; signal recognition particle protein; B-cell receptor- associated protein and related proteins; RNA-binding S4 domain; Drebrins and related actin binding proteins; small gtpase-binding protein; gtp cyclohydrolase i; psi 6 protein; predicted hydrolase related to dienelactone hydrolase; nuclear localization sequence binding protein; SWI SNF complex protein; GTP-binding protein yptl; ATPase, F0 complex, subunit H; metal resistance protein ycfl; outer membrane protein, MIM1/T0M13, mitochondrial; ubiquitin-protein ligase molybdopterin-converting factor; GTP-binding protein; predicted mitochondrial carrier protein; 28 kda golgi snare protein; dead-domain-containing protein; trehalose-phosphate synthase (UDP-forming); ran protein binding protein; pkinase-domain-containing protein; ribosome recycling factor domain; phosphatase; nucleic acid-binding, OB-fold; ATP-dependent RNA helicase dbp5; mRNA export protein (contains WD40 repeats); protein phosphatase 2A regulatory subunit A and related proteins; glutaminyl-tRNA synthetase; prolactin regulatory element-binding protein/protein transport protein SEC12p; ribosome assembly protein; C4-type Zn-fmger protein; exosomal 3'-5' exoribonuclease complex subunit Rrp40; transcription regulator HTH, APSES-type DNA-binding domain; RIB7, arfC; 60s ribosomal protein 112; guanylate kinase; predicted membrane protein; glycerol-3 -phosphate o-acyltransferase; cactin; translation initiation factor eif3 subunit; biotin holocarboxylase synthetase/biotin-protein ligase; 60s ribosomal protein 123; Inositol monophosphatase; RAS-domain-containing protein;maltase glucoamylase and related hydrolases, glycosyl hydrolase family 31; ribosomal protein S24/S35, mitochondrial, conserved domain; peptide methionine sulfoxide reductase; NAD-dependent formate dehydrogenase; molecular chaperone (DnaJ superfamily); immunoglobulin-like fold; translational repressor pumilio/PUF3 and related RNA-binding proteins (PUF superfamily); urease accessory protein; modular protein with glycoside hydrolase family 13 and glycosyltransferase family 5 domains; orotidine-5-phosphate decarboxylase; phosphoprotein/predicted coiled-coil protein; nucleosome remodeling subunit cafl nurf55 msil; zinc finger, RING/FYVE/PHD-type; prefoldin subunit 6, KE2 family; thioredoxin h; ADF-like domain-containing protein; alcohol dehydrogenase, class V; 60s ribosomal protein 113; glycoside hydrolase family 3 protein; delta 9 fatty acid desaturase; predicted regulator of rRNA gene transcription (MYB-binding protein); regulator of ribosome synthesis; hexose transport-related protein; protein-histidine kinase; DNA-directed RNA polymerase II subunit I; inositol-3 -phosphate synthase; protein transport protein sec22; taurine catabolism dioxygenase TauD/TfdA; ATPase inhibitor, IATP, mitochondria; and glycoside hydrolase family 32 protein.

In embodiments, the isolated protein of the second aspect is of a protein classification selected from: ferredoxin/adrenodoxin reductase; cytochrome; ATP synthase; NADH dehydrogenase; fatty acid desaturase; Acyl-CoA-oxidase; pantothenate kinase; polyphosphate multikinase; G protein-coupled receptor; and succinate dehydrogenase.

In embodiments, the isolated protein of the second aspect is selected from ferredoxin/adrenodoxin reductase; mitochondrial cytochrome b2; cytochrome b; cytochrome c oxidase subunit 1 ; ATP synthase subunit 6; NADH dehydrogenase subunit 4; cytochrome c oxidase subunit 2; cytochrome c oxidase subunit 3; NADH dehydrogenase subunit 2; NADH dehydrogenase subunit 5; NADH dehydrogenase subunit 6; cytochrome c oxidase subunit 3; delta 9 fatty acid desaturase; Acyl-CoA-oxidase; pantothenate kinase PanK; geranylgeranyl pyrophosphate synthase; fumarate reductase; sucrose transporter; inositol polyphosphate multikinase, ARGR transcription regulatory complex component; G protein-coupled receptor, rhodopsin-like; succinate dehydrogenase; and ATP synthase subunit mitochondrial.

A third aspect of the invention provides a method of modifying a nucleic acid or protein, including a step of changing one or more nucleotides or amino acids of the nucleic acid or protein, to produce: an isolated nucleic acid comprising a nucleotide sequence set forth in SEQ ID NOs:1240- 12684 or a nucleotide sequence at least 80% identical thereto, or a fragment of the isolated nucleic acid, or an isolated protein encoded by an isolated nucleic acid comprising a nucleotide sequence set forth in SEQ ID NOs: 1240-12684 or a nucleotide sequence at least 80% identical thereto, or a fragment or derivative of the isolated protein.

Suitably, the method of the third aspect is a method of producing the isolated nucleic acid of the first aspect or the isolated protein of the second aspect.

In embodiments, the method of modifying the nucleic acid or protein according to the third aspect is a method of mutagenising the nucleic acid or protein.

A fourth aspect of the invention provides a nucleic acid vector or construct comprising the isolated nucleic acid of the first aspect.

In embodiments, the vector or construct of the fourth aspect is an expression vector or construct. In embodiments, the vector or construct is adapted for protein expression in yeast.

In embodiments, the vector or construct of the fourth aspect is a silencing vector or construct. In embodiments, the vector or construct is adapted for gene silencing in yeast.

In embodiments, the vector or construct of the fourth aspect is an editing construct. In embodiments, the editing construct is adapted for gene editing in yeast.

A fifth aspect of the invention provides a cell comprising the nucleic acid of the first aspect, the protein of the second aspect, or the vector or construct of the fourth aspect.

In embodiments, the cell of the fifth aspect is a prokaryotic cell. The prokaryotic cell may be a bacterial cell. In embodiments, the bacterial cell is a Paracoccus cell. The Paracoccus cell may be Paracoccus carotinifaciens .

In embodiments, the cell is a eukaryotic cell. The eukaryotic cell may be selected from a plant cell, an animal cell, an algal cell, and a fungal cell.

In embodiments, the algal cell is a microalgae cell. The microalgae cell may be a Haematococcus cell. In embodiments, the Haematococcus cell is Haematococcus pluvialis.

In embodiments the fungal cell is a yeast cell. The yeast cell may be a Xanthophyllomyces cell. In embodiments, the Xanthophyllomyces cell is Xanthophyllomyces dendrorhous.

A sixth aspect of the invention provides an isolated organism comprising the cell of the fifth aspect. Suitably, the isolated organism is selected from abacterial strain, algal strain, fungal strain, yeast strain, plant, or animal

In embodiments, the organism of the sixth aspect is a yeast strain. In embodiments, the yeast strain is a Xanthophyllomyces dendrorhous strain. A seventh aspect of the invention provides a method of producing astaxanthin including a step of expressing the isolated nucleic acid of the first aspect or the isolated protein of the second aspect, to thereby produce the astaxanthin.

In embodiments, the expression of the nucleic acid or protein according to the method of the seventh aspect is in vitro expression.

In embodiments, the expression of the nucleic acid or protein according to the method of the seventh aspect is in vivo expression.

An eighth aspect of the invention provides a method of producing astaxanthin including a step of performing metabolism with the cell of the fifth or the organism of the sixth aspect, to thereby produce the astaxanthin.

In embodiments, the step of performing metabolism according to the eighth aspect is a step of performing fermentation with the cell of the fifth aspect or the organism of the sixth aspect.

In embodiments, the cell according to the method of the eighth aspect isXanthophyllomyces dendrorhous cell.

In embodiments, the method of the eighth aspect includes a step of combining the cell of the fifth aspect or the organism of the sixth aspect with one or more metabolites.

In embodiments, the one or more metabolites combined with the cell or organism comprise a nitrogen source metabolite. The nitrogen source metabolite may be selected from urea, ammonium sulphate, yeast extract, malt extract, bactopeptone, and dried com steep liquor. In embodiments, the nitrogen source metabolite is or comprises malt extract.

In embodiments, the one or more metabolites combined with the cell or organism comprise a carbon source metabolite. The carbon source metabolite may be selected from molasses, glucose, glycerol, and sucrose. In embodiments, the carbon source metabolite is or comprises molasses.

In a ninth aspect, the invention provides astaxanthin produced according to the method of the seventh or eighth aspect.

In a tenth aspect, the invention provides a non-astaxanthin by-product of the method of the seventh or eighth aspect. In embodiments, the by-product is an invertase enzyme.

In an eleventh aspect, the invention provides a formulation comprising the cell of the fifth aspect or a part thereof, the organism of the sixth aspect or a part thereof, the astaxanthin of the ninth aspect, and/or the by-product of the tenth aspect.

In a twelfth aspect, the invention provides a method of supplementing an animal with the astaxanthin of the ninth aspect or the formulation of the eleventh aspect.

In embodiments, the animal supplemented according to the method of the twelfth aspect is a farmed animal. The farmed animal may be an aquaculture animal. In embodiments, the aquaculture animal according to the method of the ninth aspect is a crustacean or a fish. The crustacean may be selected from shrimp, krill, crab, and crayfish. The fish may be selected from salmon and trout.

In embodiments, the animal supplemented according to the method of the twelfth aspect is a domestic animal or a companion animal. The domestic animal or companion animal may be selected from a canine animal (e.g. a dog), a feline animal (e.g. a cat), and an equine animal (e.g. a horse).

In embodiments, the animal supplement according to the method of the twelfth aspect is a human.

A thirteenth aspect of the invention provides a method of treating or preventing a disease or disorder in a subject, including a step of administering the astaxanthin of the ninth aspect or the formulation of the eleventh aspect to the subject.

Suitably, the subject according to the thirteenth aspect is an animal subject. In embodiments, the subject according to the method of the thirteenth aspect is a human subject.

A fourteenth aspect of the invention provides a method of co-cultivating the cell of the fifth aspect or the organism of the sixth aspect with a further cell or organism. In embodiments of the method of the fourteenth aspect, the cell of the fifth aspect or the organism of the sixth aspect is a yeast cell or organism, and the further cell organism is an algal cell or organism. In embodiments, the cell of the fifth aspect is a Xanthophyllomyces dendrorhous cell or the organism of the sixth aspect is Xanthophyllomyces dendrorhous.

A fifteenth aspect of the invention provide a co-cultivated cell or organism produced according to the method of the fourteenth aspect. In embodiments, the co-cultivated cell or organism is a yeast cell or organism. In embodiments, the co-cultivated cell is a Xanthophyllomyces dendrorhous cell or the co-cultivated organism is Xanthophyllomyces dendrorhous.

BRIEF DESCRIPTION OF THE FIGURES

In order that the invention may be readily understood and put into practical effect, typical embodiments will now be described by way of example with reference to the accompanying figures, wherein:

Figure 1 sets forth a schematic of methodology developed to obtain and analyse mutant strains of X. dendrorhous producing enhanced AX.

Figure 2 sets forth relative carotenoid production (as a multiple of wild-type production) in mutant strains of A. dendrorhous identified using screening with antimycin.

Figure 3 sets forth relative carotenoid production (as a multiple of wild-type production) in mutant strains of A. dendrorhous identified using screening with 0-ionone.

Figure 4 sets forth relative carotenoid production (as a multiple of wild-type production) in mutant strains of A dendrorhous identified using screening with diphenyamine. Figure 5 sets forth relative carotenoid production (as a multiple of wild-type production) in mutant strains of X. dendrorhous identified using screening with flow cytometry and YM plates. Strains MYMO to MYM13 and MYM18 to MYM40 screened with NTG-FACS; Strains MYM16 to MYM17 UV-FACS and MYM42-MYM55 screened with UV-FACS.

Figure 6 sets forth relative carotenoid production (as a multiple of wild-type production) in mutant strains of X. dendrorhous identified using screening with flow cytometry and YM plates. Strains MYM56-MYM65 screened with UV-FACS; strains MYM66 to MYM89 screened with MS-FACS; strains MYM91 to MYM96 screened with NTG-FACS.

Figure 7 sets forth relative carotenoid production in shake flask culture (as a multiple of wild-type production) in fifteen selected mutant strains of X. dendrorhous. Strains with the index MYM were isolated using FACS-YM screening; strains with the index MAMY were isolated using YM supplemented with antimycin; strains with the index MB were isolated using YM plates supplemented with P-ionone; strains with the index MDHA were isolated using YM plates supplemented with diphenylamine.

Figures 8 and 9 set forth kinetic model validation for fermentation using mutant X. dendrorhous strain MYMO in batch culture. Circle = experimental biomass; square = experimental carotenoids; rhombus = experimental sugars. Smooth black line = simulated biomass; smooth light grey line = simulated carotenoids; smooth grey line = simulated sugars.

Figures 10 and 11 set forth kinetic model validation for fermentation using mutant X. dendrorhous strain MYMO in fed-batch culture. Circle = experimental biomass; square = experimental carotenoids; rhombus = experimental sugars. In Figure 10, smooth black line = simulated biomass; smooth light grey line = simulated carotenoids; smooth grey line = simulated sugars. In Figure 11 , smooth black line = simulated flow rate; smooth grey line = simulated volume.

Figure 12 sets forth a schematic of variants detected across the genomes of the sequenced mutant strains and the re-sequenced A. dendrorhous CBS 6938 strain. Conserved mutations across the selected mutant strains, but not in the re-sequenced A. dendrorhous CBS 6938 strain, are shown as ‘Shared’.

Figure 13 sets forth transmembrane helices (as predicted using TMHMM Server 2.0) of a mutant GPCR protein with a premature stop codon identified in all sequenced mutant strains of A. dendrorhous CBS 6938. A. Protein in wild-type A dendrorhous CBS 6938. B. Protein in sequenced mutant strains of A dendrorhous CBS 6938.

Figures 14 to 17 set forth fermentation data for the mutant A dendrorhous strain BPAX- A1 (MYMO) and wild-type A dendrorhous strain CBS6938. The data represent the average of three biological replicates. Figure 14 = fermentation profile for A dendrorhous BPAX-A1; Figure 15 = specific rates for A dendrorhous BPAX-A1; Figure 16 = fermentation profile for A. dendrorhous CBS6938; Figure 17 = specific rates for A dendrorhous CBS6938. In Figure 14 and Figure 16: orange line and circle = glucose; blue line and circle = biomass; red line and circle = total carotenoids; red line and triangle = P-carotene; red line and asterisk = cantaxanthin; red line and square = astaxanthin. In Figure 15 and Figure 17: orange circle = glucose; blue circle = biomass; red circle = total carotenoids; red triangle = P-carotene; red asterisk = cantaxanthin; red square = astaxanthin.

Figure 18 sets forth principal component analyses of metabolites at four growth phases of wild-type X. dendrorhous strain CBS6938 and mutant X. dendrorhous strain BPAX-A1 (MYMO).

Figure 19 sets forth metabolite profile heat maps for A. dendrorhous BPAX-A1 (MYMO). For each metabolite, the response ratio of mutant strain to wild-type strain (X. dendrorhous CBS6938) was normalized to Log2. Each column represents one of the growth phases studied: Phase 1, Phase 2, Phase 3, and Phase 4 - these phases representing points across the kinetic of AX production. Blue colour indicates decreased expression and red colour indicates increased expression across a heat map representation as shown in Figure 19D. Amino acid (Figure 19A), fatty acid (Figure 19C) and central carbon (Figure 19B) metabolites were assessed.

Figure 20 sets forth metabolic pathway representation of the central carbon metabolism, carotenoid biosynthesis, and electron transport chain. Bar charts represent metabolite abundance normalized to 100 across Phase 1, Phase 2, Phase 3, and Phase 4 or RNA-seq data in FPKM for Phase 3. Red bar = metabolites in the mutant strain A dendrorhous BPAX-A1 (MYMO); Light blue bar = metabolites in the wild-type strain A dendrorhous strain CBS6938; Dark red bar = transcripts in the mutant strain A dendrorhous BPAX-A1 (MYMO); Green bar = transcripts in the wild-type strain A. dendrorhous strain CBS6938. An asterisk below a bar indicates statistical significance (p < 0.05) for metabolites or (q < 0.05) for transcripts.

Figure 21 sets forth fatty acid profile for exemplary AX-containing formulations as described in Example 3 herein.

Figure 22 sets forth a volcano plot of a comparison of protein expression between A dendrorhous strain BPAX-A1 (MYMO) and wild-type A. dendrorhous strain CBS6938 during the Phase 3 growth phase. Dashed line is adjusted p-value cutoff (0.05). Blue circles are down- regulated proteins. Red circles are up-regulated proteins. A subset of CDS IDs corresponding to the differentially regulated proteins are given - the full list of CDS IDs for differentially regulated proteins is provided in Table 18.

Figure 23 sets forth a volcano plot of a comparison of protein expression between X. dendrorhous strain BPAX-A1 (MYMO) and wild-type X. dendrorhous strain CBS6938 during the Phase 4 growth phase. Dashed line is adjusted p-value cutoff (0.05). Blue circles are down- regulated proteins. Red circles are up-regulated proteins. A subset of CDS IDs corresponding to the differentially regulated proteins are given - the full list of CDS IDs for differentially regulated proteins is provided in Table 19.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs: 1-1239: Genomic sequence contigs from re-sequencing of a sample of A. dendrorhous CBS 6938.

SEQ ID NOs: 1240-2795 : Genomic sequence contigs of mutant X. dendrorhous strain

MAMY3.

SEQ ID NOs:2796-4360: Genomic sequence contigs of mutant X. dendrorhous strain

MAMY6.

SEQ ID N0s:4361-5500: Genomic sequence contigs of mutant X. dendrorhous strain

MB 18.

SEQ ID NOs:5501-6921 : Genomic sequence contigs of mutant X. dendrorhous strain

MB24.

SEQ ID NOs:6922-8057: Genomic sequence contigs of mutant X. dendrorhous strain

MYMO.

SEQ ID NOs:8058- 9311: Genomic sequence contigs of mutant X. dendrorhous strain

MYM6.

SEQ ID NOs: 9312- 11033 : Genomic sequence contigs of mutant X. dendrorhous strain

MYM44.

SEQ ID NOs: 11034-12684: Genomic sequence contigs of mutant X. dendrorhous strain MYM92.

SEQ ID NOs: 12685-12950: Genomic sequence scaffolds of X. dendrorhous CBS 6938 (ATCC 96594) as published by Sharma et al. BMC genomics 16.1 (2015): 1-13 and publicly available via htt s : //fungi . ensembl . org/in fo/data/ftp/ dex. html .

SEQ ID NOs:12951-19331: CDS sequences of X. dendrorhous CBS 6938 (ATCC 96594) as published by Sharma et al. BMC genomics 16.1 (2015): 1-13 and publicly available via

BIOLOGICAL DEPOSITS

The following biological deposits are incorporated herein under the provisions of the Budapest Treaty. To avoid doubt, this incorporation is not, and is not to be interpreted as, a suggestion that the incorporated deposits are required to work the invention.

CBS 145279: Phaffia rhodozyma BPAX-A1 (alternatively referred to herein as Xanthophyllomyces dendrorhous MYMO) deposited with Westerdijk Fungalbio Diversity Institute, an International Depository Authority at Uppsalalaan 8, 3584 CT Utrecht, Netherlands, on 6 December 2018. CBS 145280: Phaffia rhodozyma BPAX-A2 (alternatively referred to herein as Xanthophyllomyces dendrorhous MYM92) deposited with Westerdijk Fungalbio Diversity Institute, an International Depository Authority at Uppsalalaan 8, 3584 CT Utrecht, Netherlands, on 6 December 2018.

DETAILED DESCRIPTION

This invention is at least partly predicated on the identification of mutations in yeast associated with increased accumulation of astaxanthin.

One aspect of the invention is directed to isolated nucleic acids. In typical embodiments, the isolated nucleic acids are associated with increased accumulation of astaxanthin, as herein described.

For the purposes of this invention, by “isolated’ is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

The term “nucleic acid’ as used herein designates single or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and/or thiouridine, although without limitation thereto.

A “polynucleotide” is a nucleic acid having eighty (80) or more contiguous nucleotides, while an “oligonucleotide” has less than eighty (80) contiguous nucleotides.

A “probe” may be a single or double-stranded oligonucleotide or polynucleotide, suitably labelled for the purpose of detecting complementary sequences in northern or Southern blotting, for example.

A "primer" is usually a single-stranded oligonucleotide, typically having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid ‘template’ and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™.

A typical embodiment of this aspect provides an isolated nucleic acid comprising a nucleotide sequence set forth in SEQ ID NOs: 1240-12684 or a nucleotide sequence at least 80% identical thereto, or a fragment of the isolated nucleic acid. Reference is made herein to sequence identity, in the context of comparisons of nucleotide and/or amino acid sequences. Terms used generally herein to describe sequence relationships between respective sequences of nucleic acids and proteins may include "comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”, and the like.

Because respective nucleic acids/proteins or sequences thereof may each comprise (1) only one or more portions that are shared, and (2) one or more portions which are divergent between the nucleic acids/proteins, sequence comparisons are typically performed by comparing sequences over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences.

Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection, and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al, 1997, Nucl. Acids Res. 25 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999). Other assessment of sequence alignment approaches can be found, for example, in Thompson et al, 2011, PLOS ONE. 6 (3): el 8093, and Num et al, 2006, BMC Bioinformatics. 7: 471.

The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program, Version 2.5 for Windows, as was made available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA. Typically, the isolated nucleic acid according to this aspect comprises a nucleotide sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence set forth in SEQ ID NOs: 1240-12684.

As set out hereinabove, this aspect is also directed to fragments of the isolated nucleic acid. More generally, reference is made herein to fragments of nucleic acids and proteins. It will be understood that, as used herein, a nucleic acid or protein ''fragment" includes a nucleotide sequence or amino acid sequence, respectively, of less than 100% of that of the full nucleic acid or protein, respectively.

The nucleic acid or protein fragment may comprise about, or at least about, 10, 20, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 5000, or 10000 contiguous nucleotides or amino acids of the nucleic acid or protein, respectively. In embodiments, the nucleic acid or protein fragment comprises about, or at least about, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90-99% of the full length of the nucleic acid or protein, respectively.

Suitably, the nucleotide sequence of the isolated nucleic acid of this aspect is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 12685-12950.

The skilled person will appreciate that SEQ ID NOs: 12685-12950 correspond to the published genome sequence of the sequenced wild-type yeast strain Xanthophyllomyces dendrorhous CBS 6938 as reported in Sharma et al. BMC genomics 16.1 (2015): 1-13.

Suitably, the nucleotide sequence of the isolated nucleic acid of this aspect is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 12951-19331.

The skilled person will appreciate that SEQ ID NOs:12951-19331 correspond to the published CDS annotations of the sequenced wild-type yeast strain Xanthophyllomyces dendrorhous CBS 6938 as reported in Sharma et al. BMC genomics 16.1 (2015): 1-13.

Typically, the nucleotide sequence of the isolated nucleic acid according to this aspect is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

As set out in Example 1, SEQ ID NOs: 1-1239 correspond to the re-sequenced genome of wild-type Xanthophyllomyces dendrorhous CBS 6938.

The skilled person will appreciate that various annotations for Xanthophyllomyces dendrorhous CBS 6938 are publicly available as at the filing date, including those annotations hosted at To avoid doubt, all such publicly available annotation data is incorporated herein in full, by reference.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid of this aspect is a variant of a nucleotide sequence set forth in SEQ ID NOs: 12685-19331. As used herein, a nucleotide or amino acid sequence “variant" will be understood to have one or more nucleotides or amino acids changes, respectively, inclusive of substitutions and deletions. Said changes may be referred to as “variations” of the nucleotide or amino acid sequence.

Typically, variants of a nucleotide sequence or amino acid sequence share at least about 70% or 75%, more typically at least about 80% or 85%, and even more typically at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence or amino acid sequence.

In some embodiments, a nucleic acid comprising a variant of a nucleotide sequence will hybridize to isolated nucleic acids comprising the nucleotide sequence, under at least low stringency conditions, typically under at least medium stringency conditions, more typically under high stringency conditions.

“Hybridize” and “hybridization” is used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA-DNA, RNA-RNA or DNA-RNA hybrid. Hybrid sequences comprising complementary nucleotide sequences occur through base-pairing between complementary purines and pyrimidines as is well known in the art. In this regard, it will be appreciated that modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (thiouridine and methylcytosine) may also engage in base pairing.

“Stringency” as used herein, refers to temperature and ionic strength conditions, and presence or absence of certain organic solvents and/or detergents during hybridisation. The higher the stringency, the higher will be the required level of complementarity between hybridizing nucleotide sequences.

In general terms, “high stringency conditions” designate those conditions under which only nucleic acid having a high frequency of complementary bases will hybridize.

Reference herein to high stringency conditions includes and encompasses:

(i) from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridisation at 42°C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42°C;

(n) 1% BSA, 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65°C, and (a) 0.1 x SSC, 0.1% SDS; or (b) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO ₄ (pH 7.2), 1% SDS for washing at a temperature in excess of 65°C for about one hour; and

(iii) 0.2 x SSC, 0.1% SDS for washing at or above 68°C for about 20 minutes.

In general, washing is carried out at T _m = 69.3 + 0.41 (G+ C) % -12°C. In general, the T _m of a duplex DNA decreases by about 1°C with every increase of 1% in the number of mismatched bases. Notwithstanding the above, stringent conditions are well known in the art, such as described in Chapters 2.9 and 2.10 of. Ausubel et al, supra. The skilled person will also recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization.

It will be appreciated by the skilled person that isolated nucleic acids comprising a variant nucleotide sequence may be producing using a nucleic acid amplification technique. Suitable nucleic acid amplification techniques are well known to the skilled person and include polymerase chain reaction (PCR), strand displacement amplification (SDA), rolling circle replication (RCR), nucleic acid sequence-based amplification (NASBA), Q-P replicase amplification, and helicasedependent amplification, although without limitation thereto.

As used herein, an "amplification product” refers to a nucleic acid product generated by nucleic acid amplification.

Particularly for analytical purposes, nucleic acid amplification techniques may include quantitative and semi-quantitative techniques such as qPCR, real-time PCR and competitive PCR, as are well known in the art.

Suitably, isolated nucleic acids comprising a variant of a nucleotide sequence may be produced using nucleic acid amplification techniques using one or more degenerate primers based on, or derived from, an isolated nucleic acid comprising the nucleotide sequence.

It is well understood in the art that some nucleotide changes do not change an encoded amino acid sequence (f‘ synonymous” changes), while some nucleotide sequences change an encoded amino acid sequence (‘‘non-synonymous” changes). In some typical embodiments, the nucleotide sequence of the isolated nucleic acid is a variant of a nucleotide sequence set forth in SEQ ID NOs: 12685-19331 comprising a non-synonymous nucleotide change.

It is well understood in the art that some amino acid changes do not substantially change affect activity (“conservative" changes), while some amino acid changes substantially affect protein activity (“non-conservative” changes). In some typical embodiments, the nucleotide sequence of the isolated nucleic acid is a variant of a nucleotide sequence set forth in SEQ ID NOs: 12685-19331 encoding a non-conservative amino acid change.

It will be further understood that nucleotide and/or amino acid variations may be more specifically characterised in a range of ways as will be known to the skilled person. For the purposes of this invention, nucleotide changes may (by way of non-limiting example) be characterised with reference to a gene or CDS region as: ‘synonymous' (resulting in no change to an encoded amino acid sequence); ''missense'' (resulting in a change to an encoded amino acid sequence); ‘upstream' (occurring upstream of the gene or CDS); ‘upstream less than or equal to 100 base pairs' (occurring within 100 base pairs of the start of the gene or CDS), ‘downstream' (occurring downstream of the gene or CDS), ‘ downstream less than or equal to 100 bp’ (occurring within 100 base pairs of the end of the gene or CDS); ‘intergenic’ (occurring in a region between respective genes or CDSs); ‘intron varianf (occurring in an intron of the gene or unspliced sequence containing the CDS); ‘splice varianf (causing a variation in splicing of the gene or unspliced sequence containing the CDS); ‘stop gained’’ (introducing a stop codon into the gene or CDS); ‘start lost’ (removing a start codon from the gene or CDS).

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid is a variant of a nucleotide sequence set forth in SEQ ID NOs: 12685-19331 characterised as ‘synonymous’, ‘missense’, ‘upstream’, ‘upstream less than or equal to 100 base pairs’, ‘downstream’, ‘downstream less than or equal to 100 bp’, ‘intergenic’, ‘intron variant’, ‘splice variant’, ‘stop gained’, or ‘start lost’, with reference to one or more suitable genes or CDSs.

Tables 12, 13, 14, and 15 set out hereinbelow show nucleotide sequence variations identified relative to genomic scaffolds published by Sharma et al. BMC genomics 16.1 (2015): 1- 13, which scaffolds are incorporated herein as SEQ ID NOs: 12685-12950, as hereinabove described. CDS sequences as published by Sharma et al. BMC genomics 16.1 (2015): 1-13 in or near to which the nucleotide sequence variations are located (as applicable) are also shown in Tables 12-15, which CDS sequences are incorporated herein as SEQ ID NOs:12951-19331, as hereinabove described.

With reference to Sharma et al. BMC genomics 16.1 (2015): 1-13, SEQ ID NOs:12685- 12950, and SEQ ID NOs:12951-19331, the skilled person will readily appreciate that each respective nucleotide sequence variation as set out in Tables 12-15 can be characterised as a variation of: a genic region, i.e. a region from the transcription start site to the end of the 3’ UTR for a given gene; a regulatory region, i.e. a region associated with regulation of expression of a given gene; or an intergenic region, i.e. a region that is not a genic or regulatory region.

By way of example, as per Table 12: variation X223 is a variation of a nucleotide sequence in a genic region of CDS CED80056; variation X246 is a variation of a nucleotide sequence in a regulatory region for CDS CED80058; variation X260 is a variation of a nucleotide sequence in an intergenic region located towards the start of genomic scaffold 7 as per Sharma et al, supra.

By way of another example, as per Table 13: variation Y24 is a variation of a nucleotide sequence in a genic region of CDS CED80060; variation Y322 is a variation of a nucleotide sequence in a regulatory region for CDS CED85243.

By way of another example, as per Table 14: variation Z9 is a variation of a nucleotide sequence in a genic region of CDS CDZ96153; variation Z24 is a variation of a nucleotide sequence in a regulatory region for CDS CED83975.

By way of another example, as per Table 15: variation V3 is a variation of a nucleotide sequence in a genic region of CDS CDZ98193; variation V23 is a variation of a nucleotide sequence in a regulatory region for CDZ96151.

It will be further appreciated that variations as set forth in Tables 12-15 may be more specifically characterised, such as in the manner hereinabove described including characterisations such as synonymous, missense, upstream, downstream, intergenic, intron variant, splice variant, stop gained, or start lost. Characterisation in said (or similar) manner is provided in Tables 12-15.

With reference to Example 1 and Table 12, it will be appreciated that variations X1-X222 are variations identified in the re-sequenced wild-type Xanthophyllomyces dendrorhous strain relative to the published genome sequence in Sharma et al., supra.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid of this aspect is a variant of a genic, regulatory, or intergenic sequence as set out in Table 12. The nucleotide sequence variant may be associated with a change in an encoded amino acid sequence and/or expression of a CDS sequence set out in Table 12.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid of this aspect is a variant of a genic, regulatory, or intergenic region as set out in Table 13. The nucleotide sequence variant may be associated with a change in an encoded amino acid sequence and/or expression of a CDS sequence set out in Table 13.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid of this aspect is a variant of a genic, regulatory, or intergenic region as set out in Table 14. The nucleotide sequence variant may be associated with a change in an encoded amino acid sequence and/or expression of a CDS sequence set out in Table 14.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid of this aspect is a variant of a genic, regulatory, or intergenic region as set out in Table 15. The nucleotide sequence variant may be associated with a change in an encoded amino acid sequence and/or expression of a CDS sequence set out in Table 15.

Tables 16 and 17 set out hereinbelow show transcript expression in a mutant Xanthophyllomyces dendrorhous strain (MYMO) relative to the wild-type CBS 6938 strain. In some typical embodiments, the nucleotide sequence of the isolated nucleic acid of this aspect is a variant of a nucleotide sequence encoding a transcript set out in Table 16. In some typical embodiments, the nucleotide sequence of the isolated nucleic acid of this aspect is a variant of a nucleotide sequence encoding a transcript set forth in Table 17.

With reference to Sharma et al. BMC genomics 16.1 (2015): 1-13, SEQ ID NOs: 12685- 12950, and SEQ ID NOs: 12951-19331, at least certain regulatory sequences associated with control of expression of the transcripts set out in Table 16 and Table 17 will be apparent to or readily determinable by the skilled person. The skilled person is further directed to all annotations available at https://fijn gr.ensembj .org/info/data/ftp/i for the wild-type CBS 6938 strain, incorporated herein by reference.

Accordingly, in some typical embodiments, the nucleotide sequence of the isolated nucleic acid of this aspect is a regulatory sequence for a transcript set out in Table 16. In some typical embodiments, the nucleotide sequence of the isolated nucleic acid of this aspect is a regulatory sequence for a transcript set out in Table 17.

Tables 18 and 19 set out hereinbelow show CDS sequences encoding proteins differentially expressed (based on proteomic analysis) mXanthophyllomyces dendrorhous strain MYMO relative to the wild-type CBS 6938 strain. In some typical embodiments, the nucleotide sequence of the isolated nucleic acid of this aspect is a variant of a CDS sequence set out in Table 18 and/or Table 19. The nucleotide sequence variant may be associated with a change in an encoded amino acid sequence and/or expression of a CDS sequence set out in Table 18 and/or Table 19.

With reference to Sharma et al. BMC genomics 16.1 (2015): 1-13, SEQ ID NOs: 12685- 12950, and SEQ ID NOs: 12951-19331, at least certain regulatory sequences associated with control of expression of CDS sequences and encoded proteins set out in Table 18 and Table 19 will be apparent to or readily determinable by the skilled person. The skilled person is further directed to all annotations available at htps: //fungi . ensembl org/info/data/ftp/index.html for the wild-type CBS 6938 strain, incorporated herein by reference.

Accordingly, in some typical embodiments, the nucleotide sequence of the isolated nucleic acid of this aspect is a regulatory sequence for a CDS sequence and/or encoded protein set out in Table 18 and/or Table 19.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid of this aspect comprises a nucleotide change at a position of one or more variations X223-X8395, as set forth in Table 12, relative to SEQ ID NOs: 12685-12950. Typically, the nucleotide change comprises a change at a different position than any of the variations X1-X222, as set forth in Table 12, relative to SEQ ID NOs: 12685-12950.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid comprises a nucleotide change of one or more variations X223-X8395, as set forth in Table 12, relative to SEQ ID NOs: 12685-12950. Typically, the nucleotide change comprises a change that is different than any of the variations X1-X222, as set forth in Table 12, relative to SEQ ID NOs: 12685-12950.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid comprises a nucleotide change at a position of one or more variations Y 1-Y368, as set forth in Table

13, relative to SEQ ID NOs: 12685-12950.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid comprises a nucleotide change of one or more variations Y1-Y368, as set forth in Table 13, relative to SEQ ID NOs: 12685-12950.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid comprises a nucleotide change at a position of one or more variations Z1-Z25, as set forth in Table

14, relative to SEQ ID NOs: 12685-12950.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid comprises a nucleotide change of one or more variations Z1-Y25, as set forth in Table 14, relative to SEQ ID NOs: 12685-12950.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid comprises a nucleotide change at a position of one or more variations V1-Z26, as set forth in Table

15, relative to SEQ ID NOs:12685-12950.

In some typical embodiments, the nucleotide sequence of the isolated nucleic acid comprises a nucleotide change of one or more variations V1-V26, as set forth in Table 15, relative to SEQ ID NOs: 12685-12950.

SEQ ID NOs: 1240-2795 are genomic sequence contigs of mutant X. dendrorhous strain MAMY3. In some typical embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs: 1240-2795 or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 12685- 12950 or SEQ ID NOs: 12951-19331. Typically, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

SEQ ID NOs:2796-4360 are genomic sequence contigs of mutant X. dendrorhous strain MAMY6. In some typical embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs:2796-4360 or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 12685- 12950 or SEQ ID NOs: 12951-19331. Typically, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

SEQ ID N0s:4361-5500 are genomic sequence contigs of mutant X. dendrorhous strain MB 18. In some typical embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID N0s:4361-5500 or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 12685- 12950 or SEQ ID NOs: 12951-19331. Typically, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

SEQ ID NOs:5501-6921 are genomic sequence contigs of mutant A. dendrorhous strain MB24. In some typical embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs:5501-6921 or a variant thereof, wherein the nucleotide sequence is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 12685-12950 or SEQ ID NOs: 12951-19331. Typically, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

SEQ ID NOs: 6922-8057 are genomic sequence contigs of mutant A. dendrorhous strain MYMO. In some typical embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs:6922-8057 or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 12685- 12950 or SEQ ID NOs: 12951-19331. Typically, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

SEQ ID NOs: 8058-9311 are genomic sequence contigs of mutant A dendrorhous strain MYM6. In some typical embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs: 8058-9311 or a variant thereof, wherein the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 12685- 12950 or SEQ ID NOs: 12951-19331. Typically, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

SEQ ID NOs:9312-11033 are genomic sequence contigs of mutant A dendrorhous strain MYM44. In some typical embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs:9312-11033 or a variant thereof, wherein the nucleotide sequence is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 12685-12950 or SEQ ID NOs: 12951-19331. Typically, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

SEQ ID NOs:l 1034-12684 are genomic sequence contigs of mutant A dendrorhous strain MYM92. In some typical embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in SEQ ID NOs: 11034-12684 or a variant thereof, wherein the nucleotide sequence is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 12685-12950 or SEQ ID NOs: 12951-19331. Typically, the nucleotide sequence or variant thereof is not identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-1239.

Another aspect of the invention provides an isolated protein encoded by the isolated nucleic acid of the preceding aspect, or a fragment, variant, or derivative of the isolated protein. By “protein" is meant an amino acid polymer. The amino acids may be natural or nonnatural amino acids, D- or L-amino acids as are well understood in the art.

A “ peptide” is a protein having no more than fifty (50) amino acids.

A “polypeptide” is a protein having more than fifty (50) amino acids.

As used herein, “derivative” proteins have been altered, for example by conjugation or complexing with other chemical moieties, by post-translational modification (e.g. phosphorylation, acetylation etc.), modification of glycosylation (e.g. adding, removing or altering glycosylation) and/or inclusion of additional amino acid sequences as is understood in the art.

Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may assist in detection and/or purification of the isolated fusion protein. Non-limiting examples include metalbinding (e.g. polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g. GFP), epitope tags such as Myc, FLAG and haemagglutinin tags.

For the particular purpose of fusion polypeptide purification by affinity chromatography, relevant matrices for affinity chromatography include glutathione-, amylose-, and nickel- or cobalt conjugated resins respectively. Many such matrices have been made available in kit form, such as the QIAexpress™ system (Qiagen) useful with (HIS6) fusion partners and the Pharmacia GST purification system.

Preferably, the fusion partners also have protease cleavage sites, such as for Factor X or Thrombin, which allow the relevant protease to partially digest the fusion polypeptide of the invention and thereby liberate the recombinant polypeptide of the invention therefrom. The liberated polypeptide can then be isolated from the fusion partner by subsequent chromatographic separation.

Other protein derivatives include, but are not limited to, modification to amino acid side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the isolated protein, fragments and variants disclosed herein.

In embodiments, the isolated protein of this aspect is of a protein classification selected from: cytochrome c oxidase subunit 1; ATP synthase subunit 6; NADH dehydrogenase; NADH dehydrogenase subunit 4; cytochrome c oxidase subunit 2; NADH dehydrogenase subunits 2, 5; cytochrome b; NADH dehydrogenase subunit 6; cytochrome c oxidase subunit 3; V-SNARE; DNA polymerase zeta; mRNA splicing factor; ubiquitin-conjugating enzyme E2 -binding protein; transcription coactivator protein; phosphoenolpyruvate carboxykinase; mercaptopyruvate sulfurtransferase/thiosulfate sulfurtransferase; HLH transcription factor EBF/Olf-1; peptidase S9; capl-related protein; C4-type Zn-finger protein; endonuclease MUS81; p-loop containing nucleoside triphosphate hydrolase protein; inorganic phosphate transporter; nuclear AAA ATPase (VCP subfamily); leucine-tma ligase; xanthine uracil permease; basic-leucine zipper domain protein; DNA-binding centromere protein B (CENP-B); dimeric dihydrodiol dehydrogenase; pre- mRNA splicing factor prpl; ATP-dependent clp protease proteolytic subunit; myosin 5; MYND Zn-finger protein; splicing factor 3b, subunit 2; 0CH1; phosphoinositide phosphatase; ATP synthase subunit mitochondrial kinase-like protein; small molecule transporter protein; B-block binding subunit of TFIIIC; pre-mRNA-splicing factor clfl; transcriptional regulator protein; DnaJ superfamily molecular chaperone; atypical pikk frap protein kinase; 1 ,4-alpha-glucan branching enzyme/starch branching enzyme II; cyclin-dependent kinase regulatory subunit; heat shock protein 60; FOG protein; density -regulated protein related to translation initiation factor 1 (elF- 1/SUI1); CDC12-septin; MRG-domain-containing protein; mitochondrial cytochrome b2; eukaryotic translation initiation factor 2 subunit alpha; Zn-finger protein; DNA mismatch repair protein; ATPase; RNA polymerase II, subunit POLR2C/RPB3; ATP-dependent metallopeptidase HFL; sexual differentiation process protein ISP4; extracellular protein SEL-1; nucleic acidbinding, OB-fold protein; dead-domain-containing protein; 60s ribosomal protein 121; protein phosphatase; regulatory subunit PPP1R3C/D; ATP-dependent RNA helicase dhx8; RmlC-like jelly roll fold protein; white collar 1 protein; UV radiation resistance associated protein; SAP family cell cycle dependent phosphatase-associated protein; exosomal 3'-5' exoribonuclease complex, subunit Rrp44/Dis3; ABC transporter; peptidase M28; delta 9 fatty acid desaturase; microtubule binding protein; DNA replication factor large subunit; beta and beta-prime subunits of DNA dependent RNA-polymerase; aspartate-semialdehyde dehydrogenase; metalloexopeptidase; acyl-oxidase; E3 ubiquitin ligase; TPR repeat-containing protein; peroxisomal half ABC transporter; nucleosome assembly protein; mRNA cleavage and polyadenylation factor II complex, subunit CFT2 (CPSF subunit); inositol polyphosphate multikinase, component of the ARGR transcription regulatory complex; ATP-dependent RNA helicase DBP5; SMAD/FHA domain; RNA polymerase II transcription initiation/nucleotide excision repair factor TFIIH, subunit TFB2; immunoglobulin- like; pre-mRNA splicing factor; g-protein alpha-subunit; F-box protein containing LRR; DRIM (down-regulated in metastasis)-like proteins; acid phosphatase; extracellular matrix glycoprotein Laminin subunits alpha and gamma; GTP-binding protein yptl; DNA repair protein; mRNA (guanine-n7-)-methyltransferase; ataxia telangiectasia protein; ornithine decarboxylase antizyme; aldo/keto reductase family proteins; spermidine/spermine synthases family; membrane protein; high-affinity cell membrane calcium channel; WD40/YVTN repeat-like-containing domain; Golgi apparatus membrane protein TVP23; conserved hypothetical protein CHP02453; AAA-type ATPase; HSP20-like chaperone; pantothenate kinase PanK; mediator complex, subunit Med4; Ml 3 family peptidase; Ca-transporting ATPase; histone tail methylase containing SET domain; ribonuclease iii; zinc finger, RING-type; 40s ribosomal protein si 5; nuclear export receptor crml; voltage-gated chloride channel; phosphatidylinositol 4-kinase; geranylgeranyl pyrophosphate synthase; membrane coat complex retromer, subunit VPS5/SNX1 ; iron permease FTR1 ; t-complex protein alpha subunit (tcp-1 -alpha); short chain-type dehydrogenase; polyadenylation factor I complex, subunit, Ythl (CPSF subunit); lipase; RNA polymerase III, subunit C34; cytoplasm protein; plant ascorbate peroxidase; lysine-tRNA ligase; retrotransposon tyl-copia subclass; GTP- binding protein; ring finger protein; cytochrome b5; tetraspanin/peripherin; snf2 family aminoterminal protein; arginyl -tRNA synthetase; dihydropteroate synthase; START-like domain protein; WD40 repeat-containing protein; alanine-tRNA ligase; protein kinase essential for the initiation of DNA replication; palp-domain-containing protein; arginase deacetylase; ATP-NAD kinase; fumarate reductase; peptidase clb bleomycin hydrolase; cytochrome c oxidase, subunit IV/COX5b; 1 -aminocyclopropane- 1 -carboxylate synthase, and related proteins; endosomal p24a protein; sirtuin 4 and related class II sirtuins (SIR2 family); protein-tyrosine/dual specificity phosphatase; calcineurin responsive transcription factor przl; acyl-n-acyltransferase; maintenance of telomere capping protein 1, Mtcl; beta-l,6-N-acetylglucosaminyltransferase; Zn(2)-C6 fungal -type DNA- binding domain; nuclear pore complex, Nupl55 component; E3 ubiquitin protein ligase; transmembrane protein; major facilitator superfamily domain, general substrate transporter; protein kinase; glucosyltransferase-Alg8p; Golgi-associated protein/Nedd4 WW domain-binding protein; origin recognition complex, subunit 1; cysteine proteinase; related to C2H2 zinc finger protein FLBC; C-14 reductase; hydroxymethylglutaryl-CoA reductase; RNA polymerase II transcription initiation/nucleotide excision repair factor TFIIH, subunit TFB4; microfibrillar-associated protein MF API; tetratricopeptide-like helical; SNF2-family ATP dependent chromatin remodeling factor SNF21; arsenical pump-driving atpase; guanylate kinase; fatty acid-2 hydroxylase; sucrose transporter; RNA polymerase I termination factor, MYB superfamily; ferredoxin/adrenodoxin reductase; WD40 repeat-like protein; HSP90 co-chaperone CNS1 (contains TPR repeats); RhoGEF GTPase; vacuole import and degradation protein; mitochondrial Fe2 transporter MMT1 and related transporters (cation diffusion facilitator superfamily); glycoside hydrolase, superfamily; CLASP N-terminal domain; pinin/SDK/MemA protein; G protein-coupled receptor, rhodopsin-like; histone deacetylase clr6; methylase protein; and RAB protein geranylgeranyltransferase component A.

It will be appreciated that the preceding protein classifications correspond to proteins encoded by one or more nucleotide sequences as herein described with reference to Examples 1 and 2 and Tables 12 and 13. In some embodiments, the isolated protein of this aspect is of a protein classification selected from: short-chain dehydrogenase; deoxyribodipyrimidine photolyase/cryptochrome; nuclear transport factor 2; 60s ribosomal protein 132; armadillo/beta-catenin-like repeat-containing protein; 60s ribosomal protein 110a; pyridoxamine 5'-phosphate oxidase-like, FMN-binding domain; glutaredoxin-related protein; glycosyl transferase, family 8-glycogenin; mitochondrial carrier; nucleosome assembly protein; sterile alpha motif, type 2; snare protein ykt6; UDP-glucose dehydrogenase; predicted translation factor, contains W2 domain; G-protein beta subunit-like protein; heat shock protein HSS1 ; 40s ribosomal protein s7; ATP synthase fl beta subunit; catalase 1; stress responsive alpha-beta barrel; cytokinin riboside 5 '-monophosphate phosphoribohydrolase LOG; EF-hand domain pair; 20s proteasome subunit; ferrochelatase; glycine hydroxymethyltransferase; carboxypeptidase s; NADH-ubiquinone oxidoreductase 304 kDa subunit precursor; phytoene dehydrogenase; ribosomal protein L49/IMG2; nopl Op-domain- containing protein; thioredoxin/protein disulfide isomerase; predicted dehydrogenase; 6- phosphogluconate dehydrogenase; NADH-dehydrogenase (ubiquinone); COPII vesicle protein; ornithine aminotransferase; ER-associated protein catabolism-related protein; isocitrate dehydrogenase; AAA atpase; probable NADP-dependent dehydrogenase acting on 3 -hydroxy acids; CNDP dipeptidase; actin-related protein Arp2/3 complex, subunit ARPC2; branched-chain amino acid aminotransferase ii; carbon-nitrogen hydrolase; aspartate aminotransferase; NADPH oxidase; 26s proteasome subunit p45; pre-mRNA-splicing factor rsel; porphobilinogen deaminase; prolyl oligopeptidase; ABC transporter; 40s ribosomal protein s9; polyadenylate- binding protein; ATP-dependent RNA helicase dhx8; fatty acid synthase complex subunit alpha; glycosyltransferase family 35 protein; WD repeat protein; heat shock protein 60; succinate dehydrogenase; translocase of outer mitochondrial membrane complex, subunit TOM70/TOM72; nucleic acid-binding protein; nucleotide excision repair factor NEF2, RAD23 component; t- complex protein alpha subunit (tcp-1 -alpha); fk506-binding protein 2; aromatic amino acid aminotransferase; adenylate kinase; alpha-aminoadipate reductase lyslp; coatomer protein subunit alpha; 40s ribosomal protein s21; carbamoyl-phosphate synth; histone acetyltransferase SAGA, TRRAP/TRA1 component, PI-3 kinase superfamily; SAM-dependent RNA methyltransferase; related to 2 -hydroxy-3 -oxopropionate reductase; transcriptional coactivator pl 00; 60s ribosomal protein 113a; ornithine carbamoyltransferase; eukaryotic translation initiation factor 5b; aconitate hydratase; RNA 2-o-methyltransferase fibrillarin; t-complex protein beta subunit (tcp-1 -beta); voltage-dependent ion-selective channel; coatomer beta subunit; succinate-ligase (adp-forming); carbamoyl-phosphate synthase; related to ste23 -metalloprotease involved in a- factor processing; microtubule binding protein; pyridoxalphosphate-dependent enzyme/predicted threonine synthase; fact complex subunit SPT16; SLY1 vesicle trafficking seel -like protein; cytoplasm protein; NADH dehydrogenase; phosphoglycerate kinase; arm repeat-containing protein; ribonuclease III domain; GTP binding protein 4; peptidyl-prolyl cis-trans isomerase b; Translation initiation factor 4F, ribosome/mRNA- bridging subunit (eIF-4G); eukaryotic polypeptide chain release factor 3; asparagine synthase (glutamine-hydrolyzing); splicing factor U2AF, large subunit (RRM superfamily); NADH-cytochrome b5 reductase; histidine biosynthesis trifunctional-protein; Enoyl-CoA hydratase; alcohol; imidazoleglycerol phosphate synthase; thioredoxin-like fold; ef- hand; electron-transferring-flavoprotein dehydrogenase; MDF1 -domain-containing protein; transcription factor IIS, N-terminal; heat shock protein 70; pyruvate carboxylase; homoaconitate hydratase; uncharacterized conserved coiled-coil protein; alternative splicing factor SRp55/B52/SRp75 (RRM superfamily); eukaryotic translation initiation factor 3 subunit 7; threonyl -tma synthetase; RmlC-like jelly roll fold; 60s ribosomal protein 120; mRNA splicing factor; pre-mma-processing protein 45; atp-dependent rma helicase rrp3; dihydrolipoyllysine- residue acetyltransferase; Acyl-CoA synthetase; ribosomal protein S5; phenylalanyl-tRNA synthetase subunit beta; wd40 repeat-like protein; vacuolar ATP synthase subunit d; phosphatidylserine decarboxylase; vigilin; RNA recognition motif domain; plasma membrane h( )-atpase 1; RRM motif-containing protein; predicted GTPase-activating protein; Fl -ATP synthase assembly protein; acetyl-hydrolase; peptidyl-prolyl cis-trans isomerase; antiviral helicase; acetyl CoA carboxylase; age pka protein kinase; ATP-dependent RNA helicase pitchoune; Microtubule- associated protein; cell-cycle nuclear protein, contains WD-40 repeats; phosphoserine aminotransferase; vacuolar protein sorting-associated protein; GMP synthase; translational regulator gcn20-like abc transporter; GDP-mannose pyrophosphorylase; acetyl CoA acyltransferase 2; phosphoketolase; delta 12 fatty acid desaturase; vacuolar protein 8; predicted haloacid-halidohydrolase and related hydrolases; class iii adh enzyme; t-complex protein 1; isocitrate lyase; atpase; 6-phosphogluconolactonase; mitochondrial inner membrane protein; t- complex protein 1 subunit delta; adaptor protein complex ap-1 gamma subunit; rRNA processing protein Rrp5; succinate:fumarate antiporter; predicted proline-serine-threonine phosphatase- interacting protein (PSTPIP); phospho-2-dehydro-3 -deoxyheptonate aldolase; RNA-binding domain-containing protein; epsilon DNA polymerase; cullins; asparaginyl-tRNA synthetase; dihydroxy-acid dehydratase; SNARE protein SED5/Syntaxin 5; centromere microtubule binding protein cbf5; histidyl-tma synthetase; endoplasmic reticulum protein EP58, contains fdamin rod domain and KDEL motif; 3 -isopropylmalate dehydrogenase; Glycosyl transferase, family 1; eukaryotic translation initiation factor 3 subunit 6; phosphoglycerate mutase family; chromatin remodelling complex ATPase chain; predicted hydrolases or acyltransferases (alpha/beta hydrolase superfamily); NADH dehydrogenase subunits 2, 5, and related proteins; synaptobrevin-like protein; 40s ribosomal protein s6; ubiquitin C-terminal hydrolase UCHL1 ; polyC-binding proteins alphaCP- 1 and related KH domain proteins; nucleolar RNA-associated protein (NRAP); WD40 repeat-containing protein; pyruvate decarboxylase; RhoGEF GTPase; Ca2-dependent lipid- binding protein CLBl/vesicle protein vpl l5/Granuphilin A, contains C2 domain; molecular cochaperone STI1; vacuolar H-ATPase VI sector, subunit E; p-loop containing nucleoside triphosphate hydrolase protein; spliceosome subunit; microtubule-binding protein involved in cell cycle control; karyopherin (importin) beta 3; DNA-dependent RNA polymerase ii second largest subunit; coatomer subunit gamma; dehydrogenase kinase; mitochondrial pyruvate dehydrogenase el component beta subunit; glycoside hydrolase family 13 protein; NAD-specific glutamate dehydrogenase; mitochondrial 50s ribosomal protein 13; Ran GTPase-activating protein; FKBP- type peptidyl-prolyl cis-trans isomerase; 60s ribosomal protein 119; small nuclear ribonucleoprotein splicing factor; mannosyltransferase; dUTP pyrophosphatase; GST, gst; glutamate-tma ligase; mov34-domain-containing protein; mitochondrial nuclease; 1,4- benzoquinone reductase-like; thiamine biosynthetic bifunctional enzyme; protein of unknown function DUF3602; upf0041 -domain-containing protein; 60s ribosomal protein 111; serine/threonine protein phosphatase 2A, regulatory subunit; argininosuccinate lyase; elongation factor 1 beta delta chain; bar-domain-containing protein; uridylate kinase; phosphatidyl ethanolamine n-methyltransferase; stomatin family protein; ubiquitin-conjugating enzyme; glycosyltransferase family 2 protein; signal recognition particle protein; B-cell receptor- associated protein and related proteins; RNA-binding S4 domain; Drebrins and related actin binding proteins; small gtpase-binding protein; gtp cyclohydrolase i; psi 6 protein; predicted hydrolase related to dienelactone hydrolase; nuclear localization sequence binding protein; SWI SNF complex protein; GTP-binding protein yptl; ATPase, F0 complex, subunit H; metal resistance protein ycfl; outer membrane protein, MIM1/T0M13, mitochondrial; ubiquitin-protein ligase molybdopterin-converting factor; GTP-binding protein; predicted mitochondrial carrier protein; 28 kda golgi snare protein; dead-domain-containing protein; trehalose-phosphate synthase (UDP-forming); ran protein binding protein; pkinase-domain-containing protein; ribosome recycling factor domain; phosphatase; nucleic acid-binding, OB-fold; ATP-dependent RNA helicase dbp5; mRNA export protein (contains WD40 repeats); protein phosphatase 2A regulatory subunit A and related proteins; glutaminyl-tRNA synthetase; prolactin regulatory element-binding protein/protein transport protein SEC12p; ribosome assembly protein; C4-type Zn-fmger protein; exosomal 3'-5' exoribonuclease complex subunit Rrp40; transcription regulator HTH, APSES-type DNA-binding domain; RIB7, arfC; 60s ribosomal protein 112; guanylate kinase; predicted membrane protein; glycerol-3 -phosphate o-acyltransferase; cactin; translation initiation factor eif3 subunit; biotin holocarboxylase synthetase/biotin-protein ligase; 60s ribosomal protein 123; Inositol monophosphatase; RAS-domain-containing protein;maltase glucoamylase and related hydrolases, glycosyl hydrolase family 31; ribosomal protein S24/S35, mitochondrial, conserved domain; peptide methionine sulfoxide reductase; NAD-dependent formate dehydrogenase; molecular chaperone (DnaJ superfamily); immunoglobulin-like fold; translational repressor pumilio/PUF3 and related RNA-binding proteins (PUF superfamily); urease accessory protein; modular protein with glycoside hydrolase family 13 and glycosyltransferase family 5 domains; orotidine-5-phosphate decarboxylase; phosphoprotein/predicted coiled-coil protein; nucleosome remodeling subunit cafl nurf55 msil; zinc finger, RING/FYVE/PHD-type; prefoldin subunit 6, KE2 family; thioredoxin h; ADF-like domain-containing protein; alcohol dehydrogenase, class V; 60s ribosomal protein 113; glycoside hydrolase family 3 protein; delta 9 fatty acid desaturase; predicted regulator of rRNA gene transcription (MYB-binding protein); regulator of ribosome synthesis; hexose transport-related protein; protein-histidine kinase; DNA-directed RNA polymerase II subunit I; inositol-3 -phosphate synthase; protein transport protein sec22; taurine catabolism dioxygenase TauD/TfdA; ATPase inhibitor, IATP, mitochondria; glycoside hydrolase family 32 protein.

It will be appreciated that the preceding protein classifications correspond to proteins differentially expressed (based on proteomic analysis) in Xanthophyllomyces dendrorhous strain MYMO relative to the wild-type strain, with reference to Example 3 and Tables 18 and 19.

In some typical embodiments, the isolated protein of this aspect is of a protein classification selected from: ferredoxin/adrenodoxin reductase; cytochrome; ATP synthase; NADH dehydrogenase; fatty acid desaturase; Acyl-CoA-oxidase; pantothenate kinase; polyphosphate multikinase; G protein-coupled receptor; and succinate dehydrogenase. In some typical embodiments, the isolated protein of a classification selected from ferredoxin/adrenodoxin reductase; mitochondrial cytochrome b2; cytochrome b; cytochrome c oxidase subunit 1; ATP synthase subunit 6; NADH dehydrogenase subunit 4; cytochrome c oxidase subunit 2; cytochrome c oxidase subunit 3; NADH dehydrogenase subunit 2; NADH dehydrogenase subunit 5; NADH dehydrogenase subunit 6; cytochrome c oxidase subunit 3; delta 9 fatty acid desaturase; Acyl-CoA- oxidase; pantothenate kinase PanK; geranylgeranyl pyrophosphate synthase; fumarate reductase; sucrose transporter; inositol polyphosphate multikinase, ARGR transcription regulatory complex component; G protein-coupled receptor, rhodopsin-like; succinate dehydrogenase; and ATP synthase subunit mitochondrial.

It will be appreciated that the preceding protein classifications correspond to proteins encoded by one or more nucleotide sequences in which variations considered of particular interest have been identified, with reference to Examples 1 and 2 and Tables 14 and 15.

A related aspect of the invention provides an antibody or antibody fragment that binds, or has been raised against, an isolated protein of the preceding aspect. As used herein an “antibody” is or comprises an immunoglobulin. The term “immunoglobulin” includes any antigen-binding protein product of a mammalian immunoglobulin gene complex, including immunoglobulin isotypes IgA, IgD, IgM, IgG and IgE and antigenbinding fragments thereof. Included in the term “immunoglobulin” are immunoglobulins that are chimeric or humanised or otherwise comprise altered or variant amino acid residues, sequences and/or glycosylation, whether naturally occurring or produced by human intervention (e.g. by recombinant DNA technology).

Antibody fragments include Fab and Fab'2 fragments, diabodies, triabodies and single chain antibody fragments (e.g. scVs), although without limitation thereto. Typically, an antibody comprises respective light chain and heavy chain variable regions that each comprise CDR 1, 2, and 3 amino acid sequences. A typical antibody fragment comprises at least one light chain variable region CDR and/or at least one heavy chain variable region CDR.

Antibodies and antibody fragments as described herein may be polycolonal or more typically monoclonal. Monoclonal antibodies may be produced using the standard method as for example, described in an article by Kohler & Milstein, 1975, Nature 256, 495, or by more recent modifications thereof as for example described in Chapter 2 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with an isolated protein or a fragment thereof. It will also be appreciated that antibodies may be produced as recombinant synthetic antibodies or antibody fragments by expressing a nucleic acid encoding the antibody or antibody fragment in an appropriate host cell. Recombinant synthetic antibody or antibody fragment heavy and light chains may be co-expressed from different expression vectors in the same host cell or expressed as a single chain antibody in a host cell. Non-limiting examples of recombinant antibody expression and selection techniques are provided in Chapter 17 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY and Zuberbuhler et al, 2009, Protein Engineering, Design & Selection 22 169.

In some embodiments, the antibody or antibody fragment is labelled. The label may be selected from a group including a chromogen, a catalyst, biotin, digoxigenin, an enzyme, a fluorophore, a chemiluminescent molecule, a radioisotope, a drug or other chemotherapeutic agent, a magnetic bead and/or a direct visual label.

It will be appreciated that the antibody or antibody fragment of this aspect may be used for the detection and/or purification of an isolated protein disclosed herein.

Another aspect of the invention provides a method of modifying a nucleic acid or protein, including a step of changing one or more nucleotides or amino acids of the nucleic acid or protein, to produce the isolated nucleic acid or the isolated protein of the preceding aspects. In embodiments, the method of modifying the nucleic acid or protein according to this aspect is a method of mutagenizing the nucleic acid or protein, or a nucleic acid encoding the protein.

In some typical embodiments, the method according to this aspect includes a step of mutagenising a cell or an organism to induce mutations in the genetic material of the cell or organism, to thereby modify the nucleic acid or protein.

The cell may be prokaryotic cell, such as a bacterial cell. In some typical embodiments, the bacterial cell is a Paracoccus cell. The Paracoccus cell may be Paracoccus carotinifaciens.

The cell may be eukaryotic cell, such as a plant cell, an animal cell, an algal cell, and a fungal cell.

In some typical embodiments, the algal cell is a microalgae cell. The microalgae cell may be a Haematococcus cell. In embodiments, the Haematococcus cell is Haematococcus pluvialis.

In some typical embodiments of the fungal cell is a yeast cell. The yeast cell may be a Xanthophyllomyces cell. In some typical embodiments, the Xanthophyllomyces cell is Xanthophyllomyces dendrorhous.

The terms “mutant”, “mutation” and “mutated’" are used herein generally to encompass synonymous, non- synonymous, conservative, and nonconservative nucleic acid base pair substitutions, deletions and/or insertions introduced into genetic material. For example, mutations may be introduced into chromosomal DNA and genomic DNA, RNA such as unspliced and spliced mRNA, tRNA and other forms of genetic material as are known in the art.

Mutagenesis of the genetic material of an organism may result in introduction of mutations in one or a plurality of nucleic acid molecules. Genome-wide mutagenesis of organisms is well- known in the art. In alternative embodiments, mutations can be introduced or induced by targeting specific loci or regions. It will be appreciated that gain-of-function and loss-of-function mutations may be achieved as a result of mutagenesis, although without limitation thereto.

Mutations may be induced or introduced using either non-specific methods such as random mutagenesis or alternatively by using specific methods such as targeted mutagenesis. Induced mutations may include single- or multiple -nucleotide substitutions, deletions and/or insertions, either alone or in combination. Mutagenesis methods of the present invention are inclusive of in vitro, in vivo and in situ methodology.

Chemical mutagenesis is a useful method of genome-wide random mutagenesis methods using alkylating agents such as ethylmethanesulfonate (EMS) and dimethyl sulfate (DMS) or other chemical mutagens such as ethidium bromide, formic acid, hydrazine, sodium bisulphite, and diepoxybutane. Physical mutagenesis using physical mutagens as for example irradiation using ionising radiation (such as , y or X-ray radiation), UV irradiation and fast neutron irradiation of cells may also be used for genome- wide random mutagenesis. It will be appreciated by a person skilled in the art that the time and dosage of exposure of the cell or organism, to a mutagen is dependent on the cell, organism, and mutagen that is used and can be readily determined by a skilled person.

Mutations may be introduced into nucleic acids by random or site-directed mutagenesis as are well known in the art. Non-limiting examples of nucleic acid mutagenesis methods are provided in Chapter 8 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds Ausubel et al. , (John Wiley & Sons , Inc. 1995-2008).

Mutagenesis methods can also include incorporation of dNTP analogs into nucleic acids (Zaccolo et al., 1996, J. Mol. Biol. 255 589) and PCR-based random mutagenesis such as described in Stemmer, 1994, Proc. Natl. Acad. Sci . USA 91 10747 or Shafikhani et al., 1997, Biotechniques 23 304. It is further noted that PCR-based random mutagenesis kits have been made commercially available, such as the Diversify™ kit (Clontech).

Mutations produced by a nucleic acid sequence amplification-based technique may be introduced into the genetic material of a cell.

As used herein, a “nucleic acid sequence amplification technique” includes but is not limited to polymerase chain reaction (PCR) as for example described in Chapter 15 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons NY USA 1995-2001) strand displacement amplification (SDA); rolling circle replication (RCR) as for example described in International Application WO 92/01813 and International Application WO 97/19193; nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et al . 1994, Biotechniques 17 1077; ligase chain reaction (LCR) as for example described in International Application W089/09385 and Chapter 15 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY supra; Q-P replicase amplification as for example described by Tyagi et al., 1996, Proc. Natl. Acad. Sci. USA 93 5395 and helicase dependent amplification as for example described in International Publication WO 2004/02025.

Region-specific mutagenesis and directed mutagenesis using PCR may also be employed to construct nucleic acid mutants according to the invention. Oligonucleotidemediated (or site- directed) mutagenesis may also be used. A non-limiting example of oligonucleotide-mediated site- directed mutagenesis procedures to introduce small clusters of point mutations throughout a target region is provided in Ausubel et al., supra. Briefly, mutations are introduced into a sequence by annealing a synthetic oligo nucleotide containing one or more mismatches to the sequence of interest cloned into a single-stranded Ml 3 vector. This template is grown in an Escherichia coli dut- ung- strain, which allows the incorporation of uracil into the template strand. The oligonucleotide is annealed to the template and extended with T4 DNA polymerase to create a double-stranded heteroduplex. Finally, the heteroduplex is introduced into a wild-type E. coli strain, which will prevent replication of the template strand due to the presence of apurinic sites (generated where uracil is incorporated), thereby resulting in plaques containing only mutated DNA. It is also noted that site-directed mutagenesis kits have been made commercially available, such as the QuikChange™ kit (Stratagene).

Alternatively, linker-scanning mutagenesis of DNA may be used to introduce clusters of point mutations throughout a sequence of interest that has been cloned into a plasmid vector. For example, reference may be made to Ausubel et al., supra, (in particular, Chapter 8) which describes a first protocol that uses complementary oligonucleotides and requires a unique restriction site adjacent to the region that is to be mutagenised. A nested series of deletion mutations is first generated in the region. A pair of complementary oligonucleotides is synthesised to fill in the gap in the sequence of interest between the linker at the deletion endpoint and the nearby restriction site. The linker sequence provides the desired clusters of point mutations as it is moved or ‘scanned’ across the region by its position at the varied endpoints of the deletion mutation series.

Mutations may also be induced or introduced by insertion of one or a plurality of nucleotides or base pairs into the genetic material. Transposon and retrotransposon mutagenesis (for example as described in Walbot 2000, Curr Opin Plant Biol 3 103; U.S. Pat. No. 6,720,479; Voytas 1996, Genetics 142 569) are possible methods for insertional mutagenesis. Other methods of insertional mutagenesis include targeted methods such as homologous recombination and sitespecific recombination. A non-limiting example of homologous recombination is the T-DNA system (for example as described in Wang et al. 2001, Gene 272 249; and lida & Terada 2005, Plant Mol. Biol. 59 205). An example of site-specific recombination is the cre-lox recombination system of bacteriophage Pl. Chimeric RNA/DNA oligonucleotide-directed gene targeting is also a useful technique for the generation of site-specific point mutations such as deletions, insertions and/or base changes in higher organisms including plants (see for example as described in lida & Terada, 2005, Plant Mol. Biol. 59 205; and Rice et al., 2000, Plant Physiol, 123 427).

Mutations may also be introduced by deletional mutagenesis of one or a plurality of nucleotides, or a region of a genetic locus. For example, fast neutron deletion mutagenesis can be effective for genome-wide deletional mutagenesis method and utilises fast neutron bombardment to create randomly mutagenised populations, and more particularly knockout mutations such as described Li et al., 2002, Comp. Funct. Genomics 3 158. It will be appreciated that targeted deletional mutagenesis may be achieved by using a variety of other nucleic acid-based mutagenesis methods as herein described, such as, but not limited to oligonucleotide-based mutagenesis. Targeting Induced Local Lesions in Genomes (‘TILLING’) is particularly amenable for random mutagenesis to generate point mutations in many organisms. TILLING combines traditional chemical mutagenesis following by high-throughput screening for point mutations. Reference is made to McCallum et al., 2000, Nat. Biotechnol. 18 455; Till et al., 2003, Methods Mol. Biol. 236 205; Henikoff et al., 2004, Plant Physiol. 135 630; and Till et al., 2003, Genome Res. 13 524 for non-limiting examples of TILLING methods that may be applicable to the present invention.

In certain embodiments, mutations are introduced into the genetic material of a cell or organism via “genome editing” .

“Genome editing” is a method for mutagenesis in which DNA is inserted, substituted, modified, or deleted from the genetic material of an organism in a targeted manner, typically using engineered nucleases.

Methods for genome editing include 'zinc finger nuclease" methods, as described for example by Miller et al., 2007, Nat. Biotech. 25 778; ‘ CRISPR/Cas" methods, as described for example by Cong et al., Science 339 819; and ‘TALEN" methods, as described for example by Bedell et al., Nature 491, 114.

As will be understood by those skilled in the art, genome editing typically comprises the transformation of a cell or tissue with one or more genetic constructs facilitating the expression of:

(i) one or more DNA nucleases; and

(ii) one or more molecules that guide the cleavage of DNA at a targeted region within the genetic material of an organism by said nuclease(s).

Targeted DNA breaks are thereby induced in the genetic material of the organism. These targeted DNA breaks are generally double stranded DNA breaks, although without limitation thereto.

In embodiments of genome editing wherein a zinc finger nuclease method is used, the one or more molecules that guide the cleavage of DNA at a targeted region within the genetic material of an organism by the nuclease(s) are proteins comprising a zinc finger DNA-binding domain. Generally, a plurality of the proteins are fused to the nuclease (s), and the plurality of zinc finger DNA-binding domains of the proteins bind with at least partial specificity to the targeted region, and thereby induce cleavage of the targeted region by the nuclease (s).

In embodiments of genome editing wherein a TALEN method is used, the one or more molecules that guide the cleavage of DNA at a targeted region within the genetic material of an organism by the nuclease(s) are proteins comprising a transcription activator-like effector DNA- binding (‘TALE’) domain. Generally, a plurality of the proteins are fused to the nuclease (s), and the plurality of TALE DNA-binding domains of the proteins bind with at least partial specificity to the targeted region, and thereby induce cleavage of the targeted region by said nuclease(s).

In embodiments of genome editing wherein a CRISPR/Cas method is used, the nuclease is a CRISPR-associated (‘Cas’) nuclease, and the one or more molecules that guide the cleavage of DNA at a targeted region is a “guide” RNA molecule (or ‘gRNA’) with homology to the targeted region. Generally, the gRNA molecule forms a complex with the Cas nuclease and guides binding of the Cas nuclease to the targeted region with at least partial specificity, and thereby induces cleavage of the targeted region by the Cas nuclease.

It will be further understood that targeted DNA breaks induced during genome editing can facilitate non homologous end joining or homology-dependent repair.

“Non-homologous end joining” is a cellular mechanism for DNA break repair wherein cleaved DNA ends are ligated, which is typically ‘error prone’, i.e. introduces nucleotide sequence variation, e.g. insertions or deletions, at the site of the DNA break. DNA breakage followed by error-prone non-homologous end joining induced by genome editing can be used to inactivate targeted regions within the genetic material of organisms (as described for example by Gaj et al., 2013 Trends Microbiol. 31 397).

“Homology-dependent repair” is a cellular mechanism for DNA break repair wherein a nucleic acid possessing homology to the region surrounding a DNA break is used as a template for repair of the DNA break. Genome editing can be used to introduce nucleic acid variants into targeted regions within the genetic material of organisms (as described for example by Gaj et al., 2013 Trends Microbiol 31 397) by inducing DNA breakage followed by homology-dependent repair in the presence of a "donor molecule", wherein said donor molecule comprises homology to the region surrounding the DNA break.

As will be understood by those skilled in the art, genome editing comprising homologydependent repair can be used for "allele replacement" , wherein a nucleic acid sequence of the genetic material of an organism is ‘substituted’, ‘exchanged’, or ‘replaced’ with a variant or variation of the nucleic acid sequence.

In some typical embodiments, the step of mutagenising a cell or organism to induce mutations in the genetic material of the cell or organism to thereby modify the nucleic acid or protein according to this aspect is a step of randomly mutagenising the cell using a chemical and/or physical mutagen.

In some typical embodiments, the chemical mutagen is or includes N-methyl-N'-nitro-N- nitrosoguanidine and/or ethyl methanesulfonate.

In some typical embodiments, the physical mutagen is or includes ultra violet light. Another aspect of the invention provides a nucleic acid vector or construct comprising the isolated nucleic acid of the invention, and one or more additional nucleotide sequences. Typically, the genetic construct is in the form of, or comprises genetic components of, a plasmid, bacteriophage, cosmid, or yeast or bacterial artificial chromosome, as are well known in the art.

Genetic constructs may be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or yeast or other host cells, for manipulation by recombinant DNA technology and/or expression of the nucleic acid or an encoded protein.

For the purposes of host cell expression, the genetic construct will be an expression construct. Suitably, the expression construct comprises one or more isolated nucleic acids or fragments disclosed herein operably linked to one or more additional sequences in an expression vector.

It will be understood that an “expression vector” may be either a self-replicating extra- chromosomal vector such as a plasmid, or a vector that integrates into a host genome.

By “operably linked” is meant that said additional nucleotide sequence(s) is/are positioned relative to the isolated nucleic acid, typically to initiate, regulate or otherwise control transcription.

In some embodiments, the additional nucleotide sequences are regulatory sequences. Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

The one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences.

Constitutive or inducible promoters as known in the art may be used. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.

In some embodiments, the additional nucleotide sequence is a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

The expression construct may also include an additional nucleotide sequence encoding a fusion partner (typically provided by the expression vector) so a protein of the invention is expressed as a fusion protein, as hereinbefore described.

By way of example only, an isolated protein of the invention may be produced by a method including the steps of: (i) preparing an expression construct which comprises an isolated nucleic acid of the invention, operably linked to one or more regulatory nucleotide sequences;

(ii) transfecting or transforming a suitable host cell with the expression construct;

(iii) expressing a recombinant protein in said host cell; and

(iv) isolating the recombinant protein from said host cell.

Suitable host cells for expression may be prokaryotic or eukaryotic. For example, suitable host cells may be mammalian cells, plant cells, yeast cells, insect cells or bacterial cells. In some typical embodiments, the host cell for expression of an isolated protein according to the invention is a bacterial cell. In some typical embodiments, the host cell for expression of an isolated protein according to the invention is an algal cell. In some typical embodiments, the host cell for expression of an isolated protein according to the invention is a yeast cell.

Introduction of genetic constructs into host cells (whether prokaryotic or eukaryotic) is well known in the art, as for example described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 9 and 16.

Recombinant proteins may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995- 2009), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 1, 5 and 6.

It will further be understood that, in some typical embodiments, the vector or construct of this aspect is a gene silencing or gene regulating vector or construct.

It will be appreciated by the skilled person that gene silencing or gene regulating vectors or constructs of the invention are typically adapted to produce non-coding sequences with the capacity to silence or regulate gene expression. Typically, the non-coding sequences are “small RNA” sequences.

As used herein, “small RNA” will be understood to refer to small, non-coding RNA molecules that have the capacity to bind to and regulate the expression, translation and/or replication of other nucleic acid molecules. The skilled person is directed to Ipsaro, J. J., & Joshua- Tor, L., 2015, Nature Struc. & Mol. Biol. 2220 for summary of small, non-coding RNA molecules.

It will be understood that, as used herein, the term small RNA encompasses all such molecules, regardless of the particular name that may be used in a scientific or research context. By way of non-limiting example, the skilled person will readily appreciate that, as used herein, the term small RNA encompasses small non-coding RNA molecules referred to as “miRNA” and “ siRNA'".

It will be further understood that small RNA molecules generally have a high degree of nucleotide sequence identity with a nucleic acid molecule for which they have the capacity to bind to and regulate the expression, translation, and/or replication of. However, it will also be understood that a small RNA molecule need not necessarily have 100% identity to such a sequence.

Nucleic acid vectors and constructs for gene silencing or regulation using small RNA molecules are widely available in a range of forms, as is well-known in the art.

In some typical embodiments, the gene silencing or gene regulating vector or construct is adapted for transformation of yeast. For a recent review of small RNA-based gene regulation for strain engineering in yeast, the skilled person is directed to Chen et al. Front. Bioeng. Biotechnol., 02 July 2020 https://doi.org/10.3389/fbioe.2020.00731.

It will also be understood that, in some typical embodiments, the vector or construct of this aspect is a genome editing construct. Details of genome editing and constructs therefor is provided hereinabove.

In some typical embodiments, the editing construct is adapted for genome editing in yeast. For recent review of genome editing approaches in yeast, the skilled person is directed to Yang, Z., & Blenner, M. (2020). Genome editing systems across yeast species. Current Opinion in Biotechnology, 66, 255-266.

Another aspect of the invention provides a cell comprising the nucleic acid, protein, or vector or construct of the preceding aspects.

In some embodiments, the cell of this aspect is a prokaryotic cell. The prokaryotic cell may be a bacterial cell. The bacterial cell may be Gram-negative or Gram-positive. The bacterial cell may be aerobic, anaerobic, or facultatively anaerobic.

In embodiments, the bacterial cell is of the order Enterobacterales. In embodiments, the bacterial cell is of the family Enterobacteriaceae. In embodiments, the bacterial cell is of the genus Escherichia. In an embodiment, the bacterial cell is Escherichia coli.

In embodiments, the bacterial cell is of the order Caulobacterales. In embodiments, the bacterial cell is of the family Caulobacteraceae. In embodiments, the bacterial cell is of the genus Brevundimonas . In an embodiment, the bacterial cell is Brevundimonas vesicularis.

In embodiments, the bacterial cell is of the order Sphingomonadales. In embodiments, the bacterial cell is of the family Sphingomonadaceae. In embodiments, the bacterial cell is of the genus Sphingomonas . In an embodiment, the bacterial cell is Sphingomonas astaxanthinifaciens . In embodiments, the bacterial cell is of the order Rhodobacterales. In embodiments, the bacterial cell is of the family Rhodobacteraceae. In embodiments, the bacterial cell is of the genus Paracoccus. In a typical embodiment, the Paracoccus cell is Paracoccus carotinifaciens .

In some typical embodiments, the cell is a eukaryotic cell. The eukaryotic cell may be selected from an animal cell, a plant cell, an algal cell, and a fungal cell.

The animal cell may be a marine animal cell. The animal cell may be a crustacean cell. In embodiments, the animal cell is prawn cell or shrimp cell. In embodiments, the animal cell is a krill cell.

The plant cell may be an angiosperm cell. The plant cell be a monocot or a dicot cell. The plant cell may be an algal cell. The algal cell may be a microalgae cell.

In embodiments, the plant cell is of the order Ranunculales. In embodiments, the plant cell is of the family Ranunculaceae. In atypical embodiment, the plant cell is of the genus Adonis. The plant cell may be of a species selected from Adonis aestivalis, Adonis aleppica, Adonis amurensis, Adonis annua, Adonis bobroviana, Adonis chrysocyathus, Adonis coerulea, Adonis Cyllene, Adonis davidii, Adonis dentata, Adonis distorta, Adonis flammea, Adonis macrocarpa, Adonis nepalensis, Adonis palaestina, Adonis pyrenaica, Adonis ramose, Adonis sibirica, Adonis sutchuenensis , Adonis tianschanica, Adonis vemalis, and Adonis volgensis.

In some embodiments, the algal cell is a microalgae cell. In embodiments, the microalgae cell is of the order Chlamydomonadales. In embodiments, the microalgae cell is of the family Haematococcaceae. In embodiments, the microalgae cell is of the genus Haematococcus. In a typical embodiment, the microalgae cell is Haematococcus pluvialis.

Typically, the cell is a fungal cell. More typically, the cell is a yeast cell.

In embodiments, the yeast cell is of the order Saccharomycetales. In embodiments, the yeast cell is of the family Saccharomycetaceae. In embodiments, the yeast cell is of the genus Saccharomyces. In an embodiment, the yeast cell is Saccharomyces cerevisiae.

In embodiments, the yeast cell is of the family Dipodascaceae. In embodiments, the yeast cell is of the genus Yarrowia. In an embodiment, the yeast cell is Yarrowia lipolytica.

In embodiments, the yeast cell is of the order Cystofdobasidiales. In embodiments, the yeast cell is of the family Cystofilobasidiaceae. In an embodiment, the yeast cell is of the genus Xanthophyllomyces. In a typical embodiment, the cell is Xanthophyllomyces dendrorhous.

A related aspect provides an organism comprising the cell of the preceding aspect. The organism may be any suitable organism inclusive of bacteria, plant, algae, animal, fungi, and yeast organisms. In some typical embodiments, the organism is an algal strain. More typically, the organism is a yeast strain. Exemplary yeast strains according to this aspect include mutant strains MAMY3, MAMY6, MB 18, MB24, MYMO, MYM6, MYM44, and MYM92 as described in Example 1.

It will be appreciated that the cell or organism according to this aspect may be a cell or organism produced according to the mutagenesis approaches for modifying nucleic acid or proteins as hereinabove described.

Another aspect of the invention provides a method of co-cultivating a cell or organism according to the preceding aspect with a further cell or organism. The further cell or organism may be any suitable cell or organism, including prokaryotic or eukaryotic cells and organisms. In some typical embodiments, the further cell or organism is an algal cell or organism. Typically, the algal cell or organism is a microalgae cell or organism. In a typical embodiment, the microalgae cell or organism is Haematococcus pluvialis.

Another aspect of the invention provides a method of producing astaxanthin including a step of expressing the isolated nucleic acid or the isolated protein of the preceding aspects, to thereby produce the astaxanthin.

The expression of the isolated nucleic acid or the isolated protein according to the method of this aspect may be in vitro expression, in vivo expression, or in situ expression. In embodiments, the isolated nucleic acid or isolated protein is expressed in a cell or organism, to thereby produce the astaxanthin. The cell or organism may be a prokaryotic, eukaryotic, animal, plant, algal, microalgal, fungal, or yeast cell or organism as set out in the preceding aspects. Typically, the cell or organism according to the method of this aspect is selected from a bacterial cell or organism, an algal cell or organism, a fungal cell or organism, and a yeast cell or organism.

In some typical embodiments, the method of this aspect includes a step of expressing the isolated nucleic acid or the isolated protein in a bacterial cell, to thereby produce the astaxanthin. In embodiments, the bacterial cell is a Paracoccus cell. The Paracoccus cell may >Q Paracoccus carotinifaciens .

In some typical embodiments, the method of this aspect includes a step of expressing the isolated nucleic acid or the isolated protein in a microalgae cell, to thereby produce the astaxanthin. The microalgae cell may be a Haematococcus cell. In embodiments, the Haematococcus cell is Haematococcus pluvialis.

In some typical embodiments, the method of this aspect includes a step of expressing the isolated nucleic acid or the isolated protein in a fungal cell or, more typically, a yeast cell, to thereby produce the astaxanthin. The yeast cell may be a Xanthophyllomyces cell. Typically, the Xanthophyllomyces cell is Xanthophyllomyces dendrorhous. In some typical embodiments, the astaxanthin is produced according to the method of this aspect at an increased or enhanced level or rate by expression of the isolated nucleic acid or isolated protein, as compared to a level or rate by expression of a corresponding wild-type nucleic acid or protein. Suitably, the corresponding wild-type nucleic acid or protein is a nucleic or protein of wildtype Xanthophyllomyces dendrorhous strain CBS 6938.

In some typical embodiments, the level or rate of expression of astaxanthin is increased or enhanced at least about 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 3000%, 4000%, or 5000%, as compared to the level or rate by expression of the corresponding wild-type nucleic acid or protein.

Another aspect of the invention provides a method of producing astaxanthin including a step of performing metabolism with a cell or organisms expressing an isolated nucleic acid, isolated protein, or vector or construct as hereinabove described, to thereby produce the astaxanthin.

In embodiments, the cell or organism according to this aspect is a prokaryotic, eukaryotic, animal, plant, algal, microalgal, fungal, or yeast cell or organism as hereinabove described. More typically, the cell or organism according to the method of this aspect is selected from a bacterial cell or organism, an algal cell or organism, a fungal cell or organism, and a yeast cell or organism.

In some typical embodiments, the cell is a Paracoccus bacterial cell. More typically, the bacterial cell is a Paracoccus carotinifaciens cell.

In some typical embodiments, the cell is Haematococcus algal cell. More typically, the algal cell is a Haematococcus pluvialis cell.

In some typical embodiments, the yeast cell is a Xanthophyllomyces cell. More typically, the yeast cell is a Xanthophyllomyces dendrorhous.

Typically, the step of performing metabolism with the cell or organism according to this aspect to produce the astaxanthin is a step of performing fermentation with the cell or organism.

Typically, the method of this aspect includes a step of combining the cell or organism with one or more metabolites.

In some typical embodiments, the one or more metabolites comprise a nitrogen source metabolite. Typically, the nitrogen source metabolite is selected from urea, ammonium sulphate, yeast extract, malt extract, bactopeptone, and dried com steep liquor. More typically, the nitrogen source metabolite is or comprises malt extract.

In some typical embodiments, the one or more metabolites comprise a carbon source metabolite. Typically, the carbon source metabolite is selected from molasses, glucose, glycerol, and sucrose. More typically, the carbon source metabolite is or comprises molasses.

In some typical embodiments, the astaxanthin is produced according to the method of this aspect at an increased or enhanced level or rate by metabolism with the cell expressing the isolated nucleic acid, isolated protein, or vector or construct, as compared to a level or rate by metabolism of a corresponding wild-type cell or organism. In some typical embodiments, the corresponding wild-type cell organism is, or is of, Xanthophyllomyces dendrorhous strain CBS 6938.

In some typical embodiments, the level or rate of expression of astaxanthin is increased or enhanced at least about 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 3000%, 4000%, or 5000%, as compared to the level or rate by metabolism of the corresponding wild-type cell.

In relation to aspects of the invention that can result in increased or enhanced astaxanthin production as hereinabove described, it will be appreciated with reference to Example 1 and Example 2, that increases of astaxanthin production of approaching 50 times (i.e. 5000%) have been achieved using mutant Xanthophyllomyces dendrorhous strains of the invention.

Another aspect of the invention provides a non-astaxanthin by-product or secondary product of the method of the preceding aspect.

The skilled person will readily appreciate that cellular metabolism to produce astaxanthin will typically produce a variety of by-products. In embodiments, the by-product according to this aspect is a by-product of fermentation.

In a typical embodiment, the by-product according to this aspect is an invertase enzyme. The skilled person will appreciate that invertase converts sucrose to glucose and fructose, and is one of the most widely used enzymes in food industry. Invertase is industrially produced by yeast fermentation, such that it could be desirable to produce invertase in conjunction with astaxanthin using the method of the preceding aspect.

Another aspect of the invention provides a formulation comprising the cell (or part thereof), organism (or part thereof), astaxanthin, and/or by-product of the preceding aspects. The formulation will suitably include one or more other components inclusive of buffers, excipients, and diluents, and/or one or more additional active agents.

In some typical embodiments, the formulation of this aspect is a substantially dry formulation, such as a granular or powdered formulation. Typically, the substantially dry formulation comprises astaxanthin.

In some typical embodiments, the formulation of this aspect is a liquid or semi-liquid formulation, such as an aqueous or oil-based formulation. Typically, the liquid or semi-liquid formulation comprises astaxanthin.

In some typical embodiments, the formulation of this aspect is a solid or semi-solid formulation, such as an oleoresin or encapsulated oleoresin formulation. Typically, the solid or semi-solid formulation comprises astaxanthin. In some typical embodiments, the formulation of this aspect, such as the substantially dry formulation, liquid or semi-liquid formulation, or solid or semi-solid formulation, comprises cell wall derivatives of the cell (such as the plant cell or more typically the yeast cell) of the cell or organism according to the invention as herein described.

The formulation of this aspect may be adapted for administration to or consumption by an animal.

In some typical embodiments, the formulation is adapted for administration to or consumption by a farmed animal. The farmed animal may be an aquaculture animal, typically a seafood aquaculture animal.

In some typical embodiments, the formulation is adapted for administration to or consumption by a pet animal, a domestic animal, or a companion animal. The pet animal, domestic animal, or companion animal may be a feline animal (e.g. cat), a canine animal (e.g. dog), or an equine animal (e.g. horse). The formulation may be a pet food formulation or the like.

In some typical embodiments, the formulation is adapted for administration to or consumption by a human. The formulation adapted for consumption by a human may comprise protein. In some typical embodiments, the formulation is an astaxanthin-supplemented protein formulation such as a protein bar, protein drink, or protein powder, or the like.

In some typical embodiments, the formulation of this aspect comprises beta-glucans.

In some typical embodiments, the formulation of this aspect comprises polyphenols.

Another aspect of the invention provides a method of supplementing an animal with the astaxanthin or formulation according to the preceding aspects.

In some typical embodiments of this aspect, the animal supplemented according to the method of this aspect is a farmed animal. Typically, the farmed animal is an aquaculture animal. Typically, the aquaculture animal is a crustacean or a fish. In some typical embodiments, the crustacean is selected from shrimp, krill, crab, and crayfish. In some typical embodiments, the fish is selected from salmon and trout. A related aspect provides a farmed animal product produced by or from a farmed animal, such as a seafood animal, supplemented according to the method of this aspect.

In some typical embodiments, the animal supplemented according to the method of this aspect is a pet animal, a domestic animal, or a companion animal. Typically, the pet animal, domestic animal, or companion animal is selected from a feline animal (e.g. domestic cat), a canine animal (e.g. domestic dog), and an equine animal (e.g. domestic horse).

In some typical embodiments, the supplemented animal is a human.

Another aspect of the invention provides a method of treating or preventing a disease or disorder in a subject, including a step of administering the astaxanthin or formulation according to the preceding aspects to the subject. Suitably, the subject is an animal subject. Typically, the animal subject is a human.

In some typical embodiments, the disease or disorder treated according to the method of this aspect is a wound. The method according to this aspect may be a method of treating, alleviating symptoms or, or controlling infection in a wound. The administration may be topical administration to the wound and/or oral administration to the subject.

A related aspect provides use of the astaxanthin, by-product, or secondary product according to the preceding aspect in the manufacture of a formulation, composition, or medicament for the treatment of a disease or disorder in a subject.

EXAMPLES

EXAMPLE 1: Enhanced astaxanthin production in mutant Xanthophyllomyces dendrorhous strains

Astaxanthin (AX) is a potent antioxidant with increasing biotechnological and commercial potential as a feed supplement. AX is also known for giving salmonids and crustaceans their characteristic pink colour. The red yeast Xanthophyllomyces dendrorhous (previously known as Phaffia rhodozymd) naturally produces AX as its main fermentation product but wild-type strains and those previously generated through classical random mutagenesis produce relatively low yields of AX, such that existing strains do not meet desired commercial requirements.

This example describes X. dendrorhous CBS 6938 mutant strains generated through chemical and ultraviolet radiation mutagenesis in combination with screening, the strains exhibiting comparatively high AX production. Additionally, this example describes enhancement of mutant X. dendrorhous strain AX production using culture media optimization and fed-batch culture kinetic modelling. Under optimised conditions, an approximately 50-fold increase in AX production as compared to the wild-type strain has been achieved, with a total biomass of around 100 gDCW/L and a carotenoid production of 1 g/L.

This example further describes whole genome sequencing of eight X. dendrorhous mutant strains showing substantially increased AX production to identify genomic changes. Genomic variant analyses found 368 conserved mutations across the selected strains with notable mutations including those identified in regulators and catalysts of AX precursors in the mevalonate pathway, the electron transport chain, oxidative stress mechanisms, and carotenogenesis.

MATERIALS AND METHODS

Strains, media, and growth conditions

The wild-type strain of X. dendrorhous CBS 6938 was obtained from the CBS Fungal Biodiversity Centre culture collection. The strain was kept on agar plates at 4°C for short term storage of up to one month or glycerol stock (glycerol 20%) at -80 °C for long term storage. YM media containing (g/L): yeast extract (3), malt extract (3), bactopeptone (5), and sucrose (20) as a carbon source was used for routine liquid culture and inoculum preparation. Agar (15 g/L) was supplemented to prepare YM agar plates. When specified, YM media was buffered with potassium hydrogen phthalate (20 g/L). For flask fermentations, 50 mL of liquid YM media in a 250 mL shake-flask was cultured on a rotary shaker at 250 rpm and 20 °C. Flasks for fermentation were inoculated with a pre-culture growing at the mid-exponential phase by using 10% of inoculum at A600.

Analytic techniques

• Optical Density (A600)

The absorbance of cultures with X. dendrorhous was measured at 600 nm in a spectrophotometer. The sample was diluted within a range of 0.1 - 1 absorbance units.

• Dried Cell Weight

One to two millilitres of cells were vacuum filtered using a Millipore HAWP04700 filter (pore size of 0.45 pm and diameter of 47 mm) with the filter humidified with distilled water before filtrating the cells. The filter with filtered cells was then dried in an oven at 80 °C for 48 h and weighed. The difference between the weight of the filter with and without cells, and the volume taken as a sample, were used to calculate the dried cell weight, represented as gDCW/L.

• Total Carotenoids

The DMSO technique was used to determine the total carotenoids of samples (Sedmak et al., 1990). For this, 5-50 pL of sample was placed into a 2 mL Eppendorf tube and centrifuged at 14,000 rpm for 1 min, in which the supernatant was discarded and the pellet kept. The cells were then washed two times with distilled water. Next, 0.5 mL of heated DMSO at 55 °C was added to the pellet and vortexed for 30 s. To ensure total disruption, the cells were incubated for an additional five minutes at 55 °C and vortexed again for 30 sec. Next, 0.1 mL of 0.01 M sodium phosphate at pH 7 and 1 mL of 1:1 hexane : ethyl acetate was added, followed by vortexing for 30 s. The tube was then centrifuged at 14,000 rpm for 1 min to separate the solution into two phases. Finally, 0.7 mL of the organic phase (the phase on the top with the pigments) was transferred into a 700 pL Quartz Cuvette to measure absorbance at 480 nm. The total carotenoids were calculated with the following equation: Total

A480: Absorbance at 480 nm; VI: Volume of the organic phase, mL; E: Extinction coefficient of 2150; L: Length of the Quartz Cuvette (generally 1 cm); V2: Volume of the sample, mL.

• HPLC analyses for astaxanthin and other carotenoids The samples were obtained using the DMSO extraction method for total carotenoid analyses. Samples and standards were diluted in hexane : ethyl acetate 1 : 1 (v/v) before injection of 30 pL. The column was a Luna® 3 pm Silica (2) 100 A, LC Column 150 x 4.6 mm (Phenomenex Cat. No. OOF-4162-EO). The column was equipped with a security guard cartridge holder (Phenomenex Cat. No. KJO-4282) and a security guard cartridge Silica 4 x 3 mm ID (Phenomenex Cat. No. AJO- 4348) so as to extend lifespan. The mobile phase was a mixture of hexane : acetone (82: 18, v/v) which was injected at a flow rate of 1.2 mL/min. The column temperature was ambient and carotenoids were detected in a UV/VIS detector at 474 nm. Standards of astaxanthin, p- Carotene, canthaxanthin, zeaxanthin, 9-cis-AX, 13-cis-AX were used to identify carotenoids in the sample. When required, the column was cleaned with 10 volumes each of hexane, methylene chloride, isopropanol, methylene chloride, and hexane : acetone (82:18, v/v). For water removal (when needed), the column was flushed with 60 mL of 2.5% of 2,2-dimethoxy propane, and 2.5% of glacial acetic in hexane. The column was stored in hexane or isopropanol.

• Carbon Source

- Glucose

The DNS method was used to measure the concentration of reducing sugars (Miller, 1959). The DNS solution consisted of (g/100 mL): DNS (1), NaOH (1.6), KOH (2.24), Na K Tartrate (30) to 100 mL of distilled water. The technique was adapted so as to be undertaken in microplates of 96-wells where 10 pL of each sample was placed into a 200 pL well of a PCR plate. Then 70 pL of water was added to each well. The blank used 80 pL of water. Next, 120 pL of the DNS solution was added to each well. This solution was centrifuged for 5 min at 4000 g and heated in a thermocycler for 5 min at 100 °C. Finally, 170 pL was transferred into a 96-well plate to measure absorbance at 540 nm in a microplate reader. A linear growth curve of glucose (in a range of 0.1 to 10 g/L) was created and used to calculate the concentration of glucose in the sample in g/L.

- Sucrose

Sucrose was measured using the DNS technique, as for glucose. However, the sucrose was firstly inverted using an acid treatment where 10 pL of the sample was treated with 2 pL of HC1 37% at 90 °C for 5 min. To neutralise the solution, 5 pL of NaOH 10 M was added. After the inversion, the technique was performed as for glucose. The concentration of sucrose was calculated using a linear curve in the linear range of 0.1 to 10 g/L.

Mutagenic and screening methods

• Mutagenic technique using N-methyl-N'-nitro-N-nitrosoguanidine (NTG)

Cells growing at the mid-exponential phase were washed twice with citrate buffer 0.1 M at pH 5.5. The cells were then treated with NTG at 0.1 g/L (dissolved in citrate buffer) for 30 min. These treated cells were then washed twice with a phosphate buffer 0.1 M at pH 7. Before plating on YM agar plates, treated cells were incubated on YM media on a rotary incubator at 20 °C and 250 rpm for 3 h. The plated cells were incubated at 20 °C for seven days. Cells with an increased red-colour were selected and the NTG treatment repeated. This protocol was performed until any increasing concentration of the red colour was no longer observed.

• Mutagenic technique using UV-light

Cells growing at the mid-exponential phase were washed twice with a phosphate buffer 0.1 M at pH 7 and centrifuged at 4000 rpm for 5 min. Cells adjusted to an Aeoo of 0.3, and 5 mL were placed in a plate and treated with the UV-light source of a biosafety cabinet for 10 min.

• Mutagenic technique using ethyl methyl sulfonate (EMS)

Cells growing at the mid-exponential phase were washed twice with a phosphate buffer 0.1 M at pH 7. Cells were centrifuged at 4000 rpm for 5 min. Cells were adjusted to an Aeoo of 0.3, and 5 mL were resuspended in the same buffer containing 4% EMS and treated for 2 h. Finally, treated cells were washed twice with the phosphate buffer and subjected to further screening analyses.

• Screening methods with selective pressure

Cells treated with the different mutagenic agents were incubated for 3 h in YM media at 20 °C and plated YM agar plates supplemented with 1 mM p-ionone (Bon et al., 1997), 75 pM diphenylamine (Chumpolkulwong et al., 1997), or 50 pM of antimycin A (Sigma Cat. No. A8674) (An et al., 1989). To perform screening with fluorescence activated cell sorting (FACS), treated cells were grown in a buffered YM media, stressed with 20 mM H2O2 after 1 day of incubation, supplemented with 20 g/L of sucrose after 3 days of incubation and allowed to grow for an additional 3 days.

Screening and selection of putatively superior strains using FACS was performed as described in Ukibe et al., 2008. Initially, the BD FACS Aria II flow cytometer, equipped with an ion laser emitting at 488 nm, was used to select putatively superior strains. Before analyses, cells were washed twice with a 10 mM potassium phosphate buffer (pH 7.4) and filtered through a 40 mm nylon mesh and placed into a 5 mL polystyrene 12 x 75 mm tube (BD Cat. No. 352063). Florescence emissions were measured in two channels at wavelengths of 490-550 nm and 665-685 nm. In further screenings, the cells were screened in a BD FACS ARIA III Cell Sorter using a yellow-green laser excited to 561 nm and a blue laser excited to 488 nm. The forward scatter signal (FS), side scatter signal (SC), and fluorescence intensities were measured simultaneously. Fluorescence emission was measured at 670/14 nm and 530/30 and cells with the highest fluorescence were collected in YM sterile medium. Sorted cells were then plated in YM plates and incubated for 7 days at 20 °C. In all strategies, colonies with an increased red colour were selected for further analyses.

• High throughput screening Selected colonies were cultivated in 24 deep well plates (Axygen Cat. No. AX-P-DW- 10ML-24-C) with 3 mL of YM buffered culture media in each well. The plates were covered with breathable paper (Axygen Cat. No. AX-BF-400-S-1) to allow the exchange of gasses. The plates were incubated in a shaker incubator at 250 rpm and 20 °C for 6 days. The cells were stressed with 20 mM of H2O2 at day 1 of incubation and allowed to grow for an additional 2 days before being supplemented with 30 g/L of sucrose and allowed to grow for an additional 3 days.

Culture media optimization

Nutritional requirements were studied in three steps. First, single factor designs were used to test the effect of different nitrogen and carbon sources. A response surface was then used to optimise the significant media components in a screening factorial design. Finally, a feeding profile was designed in a fed-batch culture to maximise AX production.

• Single factor design experiments

The media composition to test nitrogen sources was made of (g/L): magnesium sulphate (1.5), monobasic potassium phosphate (1.5), and sucrose (20). Potassium hydrogen phthalate was used as a buffer (20 g/L). The pH was adjusted to 5.5 with NaOH 2 M. The nitrogen sources tested were (g/L): yeast extract (5), malt extract (5), bactopeptone (5), dried com steep liquor (5), urea (2.14) and ammonium sulphate (4.71). The media to test different carbon sources was made of (g/L): ammonium sulphate (5), magnesium sulphate (1.5), monobasic potassium phosphate (1.5), yeast extract (3), dried com steep liquor (5), and malt extract (3). Potassium hydrogen phthalate was used as a buffer (20 g/L). The carbon sources tested were sucrose, glucose, molasses, and glycerol.

• Statistical experimental design and surface response

An initial screening test was conducted with seven components of the culture media using a factorial design 2 ^k ' ^p (k = factors = 7; p =fractionation = 4). The basal media to perform the design was made of (g/L): magnesium sulphate (1.5), calcium chloride (0.8), iron sulphate heptahydrate (0.019), and potassium hydrogen phthalate as a buffer (20). The media was supplemented with trace salts (mg/L): citric acid (15), ZnSO ₄ «7H ₂ O (5); CuSO ₄ «5H ₂ O (0.75); MnSO ₄ (0.60); H3BO3 (0.60); Na2MoO ₄ -2H2O (0.60); KI (0.15), and vitamins (mg/L): vitamin B3 (Niacin) (3); vitamin B5 (Pantothenic acid) (4.5); vitamin Bl (Thiamine) (3); vitamin B6 (Pyridoxine) (0.3); vitamin B7 (Biotin) (0.18); p-aminobenzoic acid (1.8). The pH was adjusted to 5.5 with sodium hydroxide 2 M. The factors tested were bactopeptone (Low Level 1 g/L; High Level 3 g/L), malt extract (Low Level 1 g/L; High Level 3 g/L), yeast extract (Low Level 3 g/L; High Level 5 g/L), dried com steep liquor (Low Level 2 g/L; High Level 5 g/L), potassium phosphate monobasic (KH2PO ₄ ) (Low Level 1 g/L; High Level 2 g/L), Sucrose (Low Level 20 g/L; High Level 30 g/L), or a vitamin cocktail (Low Level IX; High Level 2X). Further optimization was performed using a Central Composite Design and Response Surface Methodology.

Model development for fed-batch design

The model was first developed as a batch culture and then extrapolated to a fed-batch system. The parameters of the batch model were obtained from fermentations on flask culture using 10, 15, and 25 g/L in an optimised culture media. The model was developed on the following assumptions:

1. Sucrose is the only limiting carbon source;

2. There is no nitrogen limitation;

3. The pH is known and controlled throughout the fermentation at pH=5.5.

• Batch model

The differential mass balance equations (1) to (6) describe the dynamics of AX production in batch fermentation as follows: qs = (u/ Yxs) + ms (Specific substrate rate) (5) qp = (a * u) + f> (Specific production rate) (6)

Eq. (1) represents the growth rate and Eq. (4) its specific rate (p). Eq. (2) represents the consumption rate of sucrose and Eq. (5) its specific rate. Eq. (3) represents the carotenoid expression and Eq. (6) its specific production rate that considers growth associated and nonassociated production.

• Fed-batch model

The batch model was extrapolated to a fed-batch culture to design the feeding profile.

The fed- batch model is represented as equations (7) to (11) whereby a differential equation to represent volume (V) used to calculate the factor dilution (D) were added to the equations (1) to

(3). dX

—— = uX — DX (biomass) (7) dt dS

— = —qsX + D(So — S) (sucrose) (8) dt dP

— = qpX - DP (carotenoids) (9) dt — = F (volume) (10) at

D = F /V factor dilution) (11)

In equation (8), So represents sugar concentration in the feeding solution. Equations (12) and (13) were used to design an exponential feeding profile:

Where Xo and Vo represent the initial biomass and volume, respectively.

• Reliability of the model

The coefficient of determination (R ² ) was used to determine the reliability of the model.

The R ² was calculated as follows:

Parameter estimation of the model

The model parameters were obtained from batch fermentations at different initial carbon sources. The package SBPDgui of the System Biology Toolbox 2 (SBTOOLBOX2) (Schmidt and Jirstrand, 2006) was used to determine the model parameters.

Instrumented fermenters

Laboratory optimization of AX production in X. dendrorhous was performed in a 2 L Biostat A fermenter. The fermenter was configured with 2 six-blade Rushton impellers with a diameter of 5 cm, three baffles, one ring sparger, and ports for acid, base, antifoam, feeding solution, and sampling. The fermenters were equipped with probes and controllers of pH, dCh, temperature, and antifoam to measure and control these parameters, respectively. Optimal fermenter conditions were as follows: the fermenter was inoculated using a 10% inoculum culture at an Aeoo from 5 to 10 (cells growing at the mid-exponential phase). The pH was maintained at 5.5 using 12.5% of Ammonium Hydroxide or 2 M of sulphuric acid. The foam was controlled by adding Antifoam C (Sigma Cat. No. A8011). The temperature was controlled at 20 °C using a heater jacket or chiller. The dissolved oxygen was controlled at 70% of air saturation by using cascade changes in agitation (400 - 1200 rpm), air flow rate (0.4 - 5 VVM), or pure oxygen flow rate (0- 0.5 WM).

Culture medium for fed-batch fermentation

The following media was used for high cell densities in fed-batch fermentations (BYM). The BYM media was made of (g/L): yeast extract (5), malt extract (5), monobasic potassium phosphate (5), magnesium sulphate (1.5), ammonium sulphate (4), FeSO ₄ »7H ₂ O (0.10), CaCl ₂ (0.4), sucrose (20). The media was supplemented with trace salts (mg/L): citric acid (225); ZnSO ₄ »7H ₂ O (75); CuSO ₄ «5H ₂ O (11.25); MnSO ₄ (9); H3BO3 (9); Na ₂ MoO ₄ *2H ₂ O (9); KI (2.25), and vitamins (mg/L): vitamin B3 (Niacin) (18.99); vitamin B5 (Pantothenic acid) (28.48); vitamin Bl (Thiamine) (18.99); vitamin B6 (Pyridoxine) (1.89); vitamin B7 (Biotin) (1.13); p- aminobenzoic acid (11.39).

Feeding solution and feeding strategy

The BYM 2X at 500 g/L of sucrose was used for the growth phase (0 - 3.5 days) of the fed- batch culture. The BYM 2X was made of (g/L): yeast extract (10), malt extract (10), monobasic potassium phosphate (10), FeSO ₄ »7H ₂ O (0.20), CaC12 (0.8), and sucrose (500). Carbon source and culture medium components were sterilised separately at 121 °C for 15 min. The media was supplemented with trace salts (mg/L): citric acid (345); ZnSO ₄ »7H ₂ O (5); CuSO ₄ »5H ₂ O (17.25); MnSO ₄ (13.8); H3BO3 (13.8); Na ₂ MoO ₄ *2H2O (13.8); KI (3.45), and vitamins (mg/L): vitamin B3 (Niacin) (30); vitamin B5 (Pantothenic acid) (45); vitamin Bl (Thiamine) (30); vitamin B6 (Pyridoxine) (3); vitamin B7 (Biotin) (1.8); p-aminobenzoic acid (18). The pH was adjusted to 5.5 with sodium hydroxide 2 M. From the 3.5 to 7 days of cultivation, the feeding solution consisted of sucrose at 800 g/L. The feeding strategy was designed using the kinetic model for a fed-batch culture. The feeding profile consisted of batch - fed-batch (exponential feed rate at 0.08 h- ¹ ) - fed- batch (constant feeding rate at 4.16 mL/h) - batch.

Genome sequencing and bioinformatics analyses

• DNA-sequencing

DNA was extracted using the YeaStar Genomic DNA Kit (Zymoresearch Cat. No. D2002) and quantified using the Nanodrop 1000 (Thermo Scientific) and Qubit dsDNA BR assay kit (Life Technologies Cat. No. Q32850). The DNA quality was determined by running a 1% agarose gel with a DNA gel stain SYBR safe (Life Technologies Cat. No. S33102). The gel was visualised using a ChemiDoc MP system (Bio-Rad). The Illumina platform was used to sequence the genomes of nine strains (wild-type A. dendrorhous CBS 6938 and eight mutant strains). Sequencing was performed using MiSeq V3 600 Cycle 300 PE (Illumina Cat. No. MS-102-3003). Libraries were prepared using the Illumina Nextera XT library preparation kit (Illumina Cat. No. FC-121-2003).

• Bioinformatics Sequenced genomes were analysed using the following bioinformatics tools. Firstly, FastQC was used to evaluate the quality of the Illumina reads (Andrews, 2010). Trimmomatic was then used to remove poor quality reads (Bolger et al., 2014). Assembly of reads was performed using a reference genome. For this step, Bowtie2 was used to align the reads against the publicly available genome A dendrorhous CBS 6938 (accession ids in the European Nucleotide Archive: LN483084- LN483350) (Langmead and Salzberg, 2012; Sharma et al., 2015). Then, the Velvet genome assembly algorithm was used to assemble the reads (Zerbino, 2010). To objectively determine genome similarity, the genome-to-genome distance calculator (GGDC 2.0) was used to calculate the genome distance between two genomes (Auch et al., 2010). The TMHMM server 2.0 was used to predict transmembrane helix proteins (Krogh et al., 2001). Variant analyses was performed with GATK by using the recommended best practices for a non-model organism (Auwera et al., 2013; Kryvokhyzha, 2016). Here, BWA-MEM was used to align the Illumina reads against the published genome X. dendrorhous CBS 6938 (Li and Durbin, 2010; Sharma et al., 2015). In further fdter analyses, the detected variants, namely SNPs and INDELs, were filtered out if the number of reads harbouring the mutation were below the total reads. BCFtools and Samtools were used to compare and manipulate the VCF files resulting from the variant analyses (Li et al., 2009). SnpEff was used to annotate the variants and SnpSift to filter them (Cingolani et al., 2012). IGV viewer was used for visualization (Thorvaldsdottir et al., 2013).

RESULTS

Mutant strains with enhanced carotenoid production

The wild-type X. dendrorhous CBS 6938 was submitted to recursive NTG mutagenic cycles and screening in YM plates until a visible plateau of pink-colour improvement was achieved. After four mutagenic cycles, a pool of the best strains was selected to perform further mutagenic cycles using either UV-light, EMS, or NTG, with different screening methods including fluorescence activated cell sorting, antimycin, (3-ionone, or diphenylamine (Fig. 1). From 187 selected strains, and after screening in deep-well liquid culture plates (see Figs. 2-6), 15 strains were selected fortesting in shake flask cultures: MYM0, MYM6, MAMY3, MAMY16, MAMY17, MDHA7, MDHA19, MYM19, MYM92, MB 10, MB 18, MB24, MYM42, MYM44, MYM66, and MYM8. Fig. 7 demonstrates that carotenoid production was increased 18 to 23-fold in these strains as compared to the wild-type strain.

Single factor design experiments

Different organic and inorganic nitrogen sources were evaluated for growth and production of AX by mutant A dendrorhous strain MYM0 (Table 2). Single factor statistical analyses gave significant results (p < 0.05) for the response variables of growth (Aeoo) and production (total carotenoids and intracellular carotenoid content (Ypx)). Here, except for malt extract, all nitrogen sources tested had similar growth performances (Aeoo) with growth from 18.60 to 21.76. Malt extract had the highest intracellular carotenoid content (Ypx) with 3.94, 1.85, 1.09, 1.19, and 1.40- fold improvements compared to urea, ammonium sulphate, yeast extract, bactopeptone, and dried com steep liquor, respectively. The best performing nitrogen sources in terms of carotenoid production were yeast extract and bactopeptone, followed by ammonium sulphate, dried com steep liquor, malt extract, and urea. Table 2 shows that urea led to the lowest specific carotenoid production, suggesting that it may be a sub-optimal nitrogen source for carotenoid production in this strain. The carbon sources molasses, glucose, glycerol, and sucrose were also tested for growth and carotenoid production. MYMO was able to grow and produce carotenoids in all carbon sources tested (Table 3). Here, the observed growth (Aeoo) was from 19.79 to 27.89, specific production from 3132 pg/gDCW to 4778 pg/gDCW, and carotenoid production from 36,047 to 53,395 pg/L. Except for molasses, all carbon sources had a similar growth performance. Glycerol and molasses led to similar levels of catotenoid production, with an improvement of 1.52-fold compared to glucose and sucrose. Of note is that the lag phase for growing on glycerol was extended for three days compared to six hours for the other carbon sources.

Factorial design and surface response

Initial screening was conducted with seven components of the culture media by using a factorial design of 2 ^k ' ^p (k = 7; p = 4). Tables 5, 6, and 7 show results for the analyses of variance for growth, carotenoid production, and specific yield, respectively. The significant factors were bactopeptone, malt extract, yeast extract, sucrose, and dried com steep liquor (p < 0.05). These factors were further used to perform a Central Composite Design (for the experiments) and Response Surface Methodology for identifying the optimal levels). The effects of malt extract, yeast extract, Sucrose, and dried com steep liquor were studied at five experimental levels (-1.6818, -1, 0, 1, 1.6818) for growth and production. Sucrose, malt extract, and yeast extract stimulated production. In contrast, dried com steep liquor was observed to be detrimental for carotenoid production. As a result, this media component was removed from further experiments. Experimental results were fitted to a predictive quadratic model and identify the conditions for maximal carotenoid production. Table 8 demonstrates the model was able to predict production with a Coefficient of Variance within 5%.

Fed-batch fermentation

After culture media optimization, production was scaled-up to instrumented fermenters using a non-structure model. This model was validated using kinetic data in a batch mode at 10 g/L and 25 g/L of initial sucrose concentration (Fig. 8 and Fig. 9) where the coefficients of determination were 0.95 and 0.93, respectively. The model was then extrapolated to a fed-batch culture and used to design feeding strategy. Growth and production was divided into four phases: Batch - Exponential Feeding Rate - Constant Feeding Rate - Batch. After seven days of fermentation, the MYMO strain was able to grow to 117 ± 4.24 gDCW/L and produce 967 ± 15 mg/L of carotenoids, wherein the carotenoid composition was 75% for AX, 5.5% for P-carotene, 4.5 % for cantaxanthin, and 15% for other carotenoids. There was no detectable zeaxanthin, 9- cis- AX, or 13 -cis- AX in the carotenoid fraction. The coefficient of determination was 0.94 suggesting a reliable model to predict growth and production in P. rhodozyma.

Bioinformatics analyses

From the strains screened for production (Fig. 2), X. dendrorhous MYM6 (coded for sequencing as MYM6 Y2), MYMO (coded as MYMO_Y11), MYM44 (coded as MYM44_Y14), MB 18 (coded as MB18_Y15), MAMY3 (coded as MAMY3 Y16), MB24 (coded as MB24_Y17), MYM92 (coded as MYM92_Y4), and MAMY6 (coded as MAMY6_Y12) were selected for whole genome sequencing using the Illumina platform. The wild-type A. dendrorhous CBS 6938 was resequenced as a control. Genome assembly was performed with Velvet, including adapter removal, trimming, and filtering of poor-quality reads. The contigs were then filtered to leave those with a length > 300 bp, and coverage above 10X. The genome size, number of contigs, N50, and GC content is presented in Table 4. The genome sizes were from 18.55 Mb to 19.11 Mb. The number of contigs were from 1239 to 1722. The N50 of the newly sequenced genomes were from 23,732 bp to 44,235 bp. The GC content for the newly sequenced genomes was 48.57% ± 0.17%.

• Variant analyses

The genome sequence and annotation of A. dendrorhous CBS 6938 was used as a reference to call and annotate the variants (Sharma et al., 2015). After filtering the variants using the criteria detailed hereinabove, there were 983, 1009, 1037, 994, 1015, 977, 1067, and 1091 SNPs and 52, 64, 47, 68, 76, 66, 68, and 61 INDELs detected for the strains MYM6_Y2, MYM0_Yl l, MYM44_Y14, MB18_Y15, MAMY3 Y16, MB24_Y17, MYM92_Y4, and MAMY6 Y12, respectively. Next, any mutations detected in the re-sequenced A. dendrorhous CBS6938 compared to the published sequence were removed from the newly sequenced mutant strains. Mutations present across all strains were extracted and used for variant analyses (Figure 12). After variant annotation, synonymous mutations were removed. These filtering criteria yielded 368 SNPs (see, Table 13). From these mutations, the following criteria were used for further analyses: non- synonymous missense variants in coding regions (144 mutations), upstream variants (126 mutations), upstream variants within 100 bp of a gene (5 mutations), stop gained (2 mutations), start lost (1 mutation), missense/splice variants (4 mutations), and splice donor/intron variants (6 mutations).

• Conserved variants Table 13 shows the types of mutations detected across the eight sequence mutant strains, and the genomic details location of the mutation in the chromosome, gene name, protein, gene ID, type of mutation, gene length, protein length, effect, and amino acid change. There were 144 non- synonymous missense mutations in coding regions found across all of the newly sequenced mutant strains. From these mutations, 27 in proteins annotated as hypothetical proteins while the remaining were in more specifically annotated proteins.

Of the mutations in specifically annotated proteins, several were found in subunits of complex proteins. Four SNPs were found in subunits of the gene encoding cytochrome c oxidase: subunit 1, subunit 2, subunit 3. In addition to this cytochrome-related protein, the cytochrome b2 gene encoding protein was also mutated. Five SNPs were found in subunits of the gene encoding to the NADH dehydrogenase protein. Among other mutations in subunits, the ATP synthase subunit 6 gene encoding protein was also mutated. In terms of mutations in genes encoding proteins related to transporters, a small molecule transporter, an ABC transporter, and a sucrose transporter were mutated. Five SNPs in Zn-finger proteins encoding genes were also mutated including C4- type Zn-finger, an uncharacterised MYND Zn-finger, Zn(2)-C6 fungal-type DNA-binding domain gene encoding protein, Zinc finger RING-type, and the related to C2H2 zinc finger protein FLBC. Fatty acid related gene encoding proteins were mutated including the delta 9 fatty acid desaturase, and the acyl-CoA oxidase. From mutations of the TCA cycle, the gene encoding to fumarate reductase was mutated. Mutations were also found in the pantothenate kinase (PanK), the calciumtransporting ATPase, the Snf2 family amino-terminal protein, the 1 -aminocyclopropane- 1- carboxylate synthase, the cysteine proteinase and the ferredoxin/adrenodoxin reductase encoding genes.

Notably, ‘high impact’ mutations included two SNPs leading to premature stop codons in a WD40 repeat-containing protein and a G protein-coupled receptor, rhodopsin- like, respectively. Additionally, a loss of start codon mutation was detected in one protein annotated as a hypothetical protein. Six mutations within 100 bp upstream of a gene were found including cytochrome b, inositol polyphosphate multikinase, components of the ARGR transcription regulatory complex, mRNA (guanine-n7-)- methyltransferase, and glucosyltransferase-Alg8p; two of these types of effect mutations were annotated as hypothetical proteins. Six mutations with the effect “Splice Donor and Intron Variant” were found. This type of mutation causes the loss of a splicing signal that defines the 3 '-end of an exon, the consequence being that the whole intron could be retained as the splicing machinery is unable to recognise the splice donor site (Jian et al., 2013). The mutations found with this effect included the predicted E3 ubiquitin ligase, acid phosphatase, predicted Zn- finger protein, palp- domain-containing protein, Zn(2)-C6 fungal-type DNA-binding domain, and uncharacterised conserved protein. Four mutations were found with the effect missense and splice region variant including mercaptopyruvate sulfurtransferase/thiosulfate sulfurtransferase, ATP- NAD kinase, snf2-family ATP dependent chromatin remodelling factor snf21 with G3713A, and arsenical pump-driving ATPase.

Overall, 25 mutations conserved across the sequenced strains were identified as of particular interest, as set out in Table 14.

DISCUSSION

Strain improvement

In this example, mutagenesis was applied using NTG combined with EMS and UV-light, and incorporated screening methods with selective pressure or cell sorting in the last mutagenic cycle, with the intention of producing mutagenized X. dendrorhous capable of enhanced AX production. This methodology resulted in strains capable of greater than 15-fold AX production as compared to the wild-type strain (e.g., Fig. 7). In addition to improving AX production, the combination of mutagenic agents and screening methods used reduced the number of mutagenic cycles 3-fold as compared to previous studies (see, e.g., Xie et al., 2014).

The use of antimycin, diphenylamine, or p-ionone in our screening methods appeared to assist with the selection of superior strains. Antimycin inhibits the cytochrome P450 enzyme responsible for providing electrons during the oxygenation of the AX molecule by the AX synthase (Bon et al., 1997; Ojima et al., 2006), diphenylamine inhibits the phytoene desaturase enzyme leading to an accumulation of the colourless carotenoid phytoene (Chumpolkulwong et al., 1997), and, p-ionone competes with the p -carotene molecule during its oxygenation by the AX synthase to produce AX (Lewis et al., 1990).

Cell sorting (FACS) was also used as a screening method to select superior strains. FACS used fluorescence properties of the AX molecule to select strains with an improved ability to produce AX. Additionally, as cells screened with FACS were stressed with H2O2, cells were selected for increased tolerance to oxidative stresses which is associated with an improved ability to produce AX (Schroeder and Johnson, 1995).

Culture media optimization and fed-batch fermentation

Classical random mutagenesis produces unique strains with specific phenotypic traits making it challenging to directly extrapolate culture media optimization results from other studies. Therefore, one of the selected strains, MYMO, was subjected to a culture media optimization process.

Different nitrogen sources were tested to grow X. dendrorhous cells including urea, ammonium sulphate, yeast extract (YE), malt extract (ME), bactopeptone (BP), and dried com steep liquor (DCSL) (see Table 2). While urea has been reported to be a low-cost and promising nitrogen source to grow and produce AX in A dendrorhous (An et al., 2001; Fontana et al., 1996), this example identified that urea inhibited carotenogenesis in our selected strain (Table 2). Ammonium sulphate is another low-cost nitrogen source used to grow X. dendrorhous cells (Flores-Cotera et al., 2001; Ni et al., 2007). The example provided here is consistent with these earlier studies, with ammonium sulphate resulting in the highest growth. YE, ME, and BP are part of the YM media used as a routine to grow A. dendrorhous cells. The evaluation of these media components as a sole nitrogen source confirmed its effectiveness to grow and produce AX. Of note is that ME produced the highest carotenoid content in our selected strain suggesting the importance of this media component. BP delivered improved growth and carotenoid production but its high cost that means this nitrogen source is sometimes replaced by other low-cost sources such as urea or ammonium sulphate (Ni et al., 2007).

Different carbon sources were also tested to grow and produce AX in the selected strain. Table 3 confirms that the selected strains grew in several carbon sources including glucose, sucrose, glycerol, and molasses and can be used to produce high cell densities. The best performing nitrogen and carbon sources were then optimised in a surface response experiment to determine optimal levels to maximise growth and AX production.

X. dendrorhous is a Crabtree positive yeast (high level of sugars produce fermentative products during aerobic fermentations) which limits the application of batch fermentations at a high concentration of carbon source (Reynders et al., 1997). To overcome this limitation, the optimised media and a nonstructure model were used to design the feeding profile for the fed-batch culture. Notably, after seven days of fermentation, the AX production of X. dendrorhous MYM0 increased 50-fold compared with the wild-type strain.

The final biomass achieved was above 100 gDCW/L and the carotenoid content was around 1 g/L (Fig. 10). This result was a 1.41-fold production improvement compared with previous fed- batch cultures that designed the feeding profile using either DO-stat, pH-stat, constant feeding rate, or feedback loops of online sugar measurements (Schewe et al., 2017; Schmidt et al., 2011).

Luna-Flores et al., 2010 used kinetic models to design the feeding profile of a fed-batch culture but the media used in that study was not optimised, rendering 3 -fold less growth than in the example presented here. The superiority of our approach highlights the significance of incorporating culture media optimisation in selected strains before using them in fed-batch fermentations.

While glycerol has been reported to be an effective carbon source to stimulate AX production in X. dendrorhous (Silva et al., 2012), and despite glycerol supported growth and production being similar to other carbon sources in a flask fermentation (Table 3), when tested in a fed-batch culture during the maturation phase (after day 3.5 of culture), glycerol in fact accumulated suggesting that it is a poor carbon source for the tested strain. This poor performance when using glycerol in fed-batch fermentations may well be associated with the long adaptation phase prior to assimilation of glycerol (Table 3).

Molasses has previously been tested in X. dendrorhous with two to three times more AX obtained than when using glucose or a synthetic blend of sugars that constitute molasses (Haard, 1988). Our results are consistent with this previous finding with molasses increasing AX production 1.52- fold compared with glucose or sucrose (Table 3) in the tested strain. The stimulation of AX production by molasses suggests it is a promising carbon source to maximise carotenoid production for strains obtained by this approach.

Genome and variant analyses

The genome assembly and filter method (remove contigs below 300 bp and depth below 10X) applied to the selected strains generated genome assembly sizes ranging from 18.55 Mb to 19.11 Mb (Table 4). The genome sizes without filtering the contigs using the mentioned criteria of size and depth resulted in assembly sizes of 19.14±0.15 for all the genomes.

The observed genome sizes were similar to the 19.50 Mb genome of the previously sequenced wild-type X dendrorhous CBS 6938 strain (Sharma et al., 2015). Similarly, the genome size of the re-sequenced A. dendrorhous CBS 6938 was 19.20 Mb. The GC contents in the new strains and re-sequenced wild-type strain were around 48% which were similar to the 47.3% GC content of the publicly available wild-type strain (Table 4) (Sharma et al., 2015). In previous studies with related strains, X. dendrorhous CBS 7918 yielded a 18.7 Mb genome and 47.2% GC content, andX dendrorhous CRUB 1149 an 18.9 Mb genome and 47.1% GC content (Libkind et al., 2011).

Genome to genome distance was calculated to objectively calculate the similarity between our selected strains and the wild-type strains (Auch et al., 2010). The genome distance calculated was 0.01 for the re-sequenced X. dendrorhous CBS 6938 and 0.02 for the selected strains (the closer to “0” the more similar the strains in comparison) suggesting that the wild-type strains are more similar between themselves than with our selected strains. This genome comparison confirmed the genome similarity among the X. dendrorhous strains used in this example and those with a genome sequence publicly available.

Genomic mutations associated with AX biosynthesis in X. dendrorhous

X. dendrorhous uses the precursor isopentenyl pyrophosphate (IPP) for AX biosynthesis, which is generated via the mevalonate (MV A) pathway, starting from acetyl-CoA. Here, a mutation was identified in the pantothenate kinase encoding gene, which is required in the first reaction of the conenzyme A (CoA) biosynthetic pathway (Table 14). Pantothenate kinase phosphorylates panthothenate to form phophopanthothenate at the expense of a molecule of ATP. This reaction is a limiting step in the biosynthesis of CoA, required by the MVA pathway in the form of acetyl- CoA. The MVA pathway uses five enzymes to produce IPP, in which 3- hydroxy-3 -methyl- glutaryl-CoA reductase (HMGR) is a critical regulator and the enzyme that catalyses the production of MVA (Goldstein and Brown, 1990). Here, a mutation was identified in the gene encoding to the HMGR enzyme (see Table 14). HMGR overexpression has been associated with an improvement of AX in X. dendrorhous indicating that the mutation found in the HMGR gene may influence the AX production increase in our selected strains. For example, the addition of ethanol to cultures of the mutant strains X. dendrorhous P-5-6 and Dp-41 overexpressed 3-fold the HMGR gene which was associated with the increase of AX production on those mutated strains (Gu et al., 1997). Similarly, the overexpression of three MVA synthetic pathway genes, namely acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMGR in X. dendrorhous increased AX production 2.1 fold (Hara et al., 2014).

Following the IPP synthesis through the MVA pathway, eight IPP molecules are then condensed through prenyltransferases in which IPP isomerase catalyses the isomerization of IPP to dimethylallyl-pyrophosphate (DMAPP), and then both molecules joined, generating geranyl pyrophosphate (GPP) (Kajiwara et al., 1997). The addition of a second molecule of IPP to GPP gives the precursor C-15 sesquiterpene, famesyl pyrophosphate (FPP), which is converted into geranylgeranyl-pyrophosphate (GGPP) by a further addition of IPP by the GGPP synthase (see Table 14) (Niklitschek et al., 2008). In this example, the new strains presented a mutation in the GGPP synthase encoding gene, which in abundance has been associated with a remarkable carotenoid production in ripening fruit (Sandmann, 1994). Because GGPPs are the building blocks of the carotenogenesis pathway, the mutation in the GGPP synthase gene might positively influence the synthesis of GGPPs and thus the carotenoid production in our selected strains.

Mutations associated with carbon source and nutrient assimilation

This example used sucrose as a carbon source in all screening and selection experiments and efficiently produced high cell densities in a fed-batch culture (Fig. 10). A mutation in a gene encoding a sucrose transporter, associated with sucrose assimilation, was identified. Due to its association with sucrose assimilation, the mutation in the sucrose transporter may have influenced the improved growth and carotenoid production in the selected strain.

A mutation identified in a gene encoding a G protein-coupled receptor, rhodopsin-like protein (GPCR) had the effect “Stop Gained”. GPCR proteins comprise the largest class of membrane proteins in eukaryote genomes with a common denominator of seven-transmembrane domains. Yeast has three different GPCR proteins for pheromone and sugar sensing (Lengger and Jensen, 2020). Glycerol was a carbon source tested in the selected strains to grow and produce AX (Table 3). For glycerol, the lag phase lasted three days before the culture started to grow. In a fed- batch culture, glycerol failed as it accumulated during its feeding. Potentially, this GPCR mutation is negatively influencing the ability of the selected strain to grow under glycerol as a carbon source. Further analyses in the GPCR mutation using the TMHMM server 2.0 revealed that the transmembrane helices of the GPCR protein were not affected as these were found in the first 200 amino acid sequences - the stop gained was at the position 395/523 (see Table 14 and Fig. 13) (Krogh et al., 2001).

Five mutated genes encoding Zn-finger-type proteins (Table 14), comprising small a protein structural motif characterised by the coordination of one or more zinc ions in order to stabilise the fold, were also identified. These protein types interact with RNA, DNA, or proteins altering their binding specificity for a particular protein. Zn-fmger proteins have been associated with the regulation of nitrogen assimilation in Neurospora crassa or AspegiHius nidulans (Fu and Marzluf, 1990), so it is possible that mutations of Zn-fmger genes in the selected strain of P. rhodozyma resulted in superior use of ammonium sulphate as compared to urea as a nitrogen source (Table 2).

Mutations associated with oxidative phosphorylation and stress

Schroeder et. al., 1995 indicated that AX plays an important role against oxidative stress in X. dendrorhous (Schroeder and Johnson, 1995). This oxidative stress link with AX production is also supported by the evidence that AX production in X. dendrorhous is mainly associated with respiratory or aerobic fermentations rather than anaerobic ones (Luna-Flores et al., 2010). ROS tends to be generated during the respiration phase because of electron overflow in the respiratory chain caused by an imbalance of electron transfer during reduction of the ubiquinone pool and electron transfer occurring downstream in the respiratory chain. AX can quench ROS in a manner analogous to superoxide dismutase in which harmful oxygen molecules are break down and eliminated (Schroeder and Johnson, 1993).

ROS are produced via the respiratory electron chain and, in this example, mutations in subunits of the electron transport chain components affecting the complexes I, III, IV, and V (Table 14) were identified. Of these mutations, four were found in subunits of the cytochrome c oxidase (CoC) protein. This CoC protein is responsible for carrying the electrons from complex III to complex IV in the electron transport chain, the last step in the electron transport chain. In fact, this complex is inhibited by antimycin or potassium cyanide (KCN), a compound used in our studies as a screening method.

Evidence suggesting that A. dendrorhous shifted from KCN-sensitive to KCN- insensitive growth in later growth phases (carotenoid production phases) (Schroeder and Johnson, 1993) supports the importance of alternative pathways of oxygen utilization during AX biosynthesis. Another report suggests that mutant strains sensitive to antimycin, can create an imbalance in the flow of electrons during the electron transport chain, increasing the amount of ROS and so improving AX production (An et al., 1989). This overflow of electrons can also increase AX production by the action of AX synthase, an enzyme that belongs to the P450 family enzyme and requires an electron donor (P450 reductase or cytochrome b), to incorporate the oxygen functional groups to the molecule of p-carotene.

In this example, a mutation of a ferredoxin/adrenodoxin reductase encoding gene (Table 14) was also observed. Adrenodoxin is a cAMP-regulated ferredoxin that transports electrons from NADPH-dependent adrenodoxin reductase to P450 family enzymes (Grinberg et al., 2000). The electron donor capability of ferredoxin/adrenodoxin reductase that suggests a potential role as an electron donor required by AX synthase. Other mutations associated with the electron transport chain included a mutation in the cytochrome b, cytochrome b2 and ATP synthase encoding genes and five mutations in subunits of the NADH dehydrogenase protein (Table 14). These mutations are potentially associated with the improved AX production in the selected strains due to the abundance of oxidative phosphorylation enzymes such as NADH dehydrogenase and ATP synthase, found when X. dendrorhous cells were growing on succinate as a carbon source, and where production of AX was around 2-fold more than when grown on glucose (Martinez-Moya et al., 2015).

Another mutation potentially associated with increased AX production in the selected strains is in an acyl-CoA oxidase encoding gene (Table 14). This enzyme is associated with the fatty acid metabolism in a reaction involving the oxidation of acyl-CoA to tans- 2,3 -dehydroacyl - CoA and the reactive oxygen species H2O2, which can trigger an increase of AX production to protect the cells from an oxidative stress (Liu and Wu, 2006).

Another mutation associated with lipid metabolism was observed in a delta 9 fatty acid desaturase, which is reported to have preferences for substrates of Cl 8: 1 and Cl 6: 1 converting to C18:2 and C16:2 fatty acids (L. Zhang et al., 2020). Notably, the most abundant fatty acids in the X. dendrorhous pathway are linoleate (Cl 8:2), stearic acid (18:0), oleic acid (Cl 8: 1), palmitic acid (C16:l), and hexadecanoic acid (16:2) (Sharma et al., 2015). The proportion of fatty acid and its influence to produce AX (Miao et al., 2011) suggests that the mutation in the delta 9 fatty acid desaturase encoding gene may stimulated AX production in our selected strains of A. dendrorhous.

It is further notable that, in this example, antimycin, p-ionone, diphenylamine, or the stressor H2O2 were used in the screening methods to select superior strains of X. dendrorhous, which potentially increased selection for strains with the mutations in the electron transport chain to enhance AX production.

EXAMPLE 2: Metabolomic and transcriptomic analysis of a high astaxanthin producer strain of Xanthophyllomyces dendrorhous In this example, one of the dendrorhous mutant strains developed as per Example 1, and the wild-type as a control, were cultured in chemically defined media and instrumented fermenters, and differential kinetic, metabolomics, and transcriptomics data were obtained. The results obtained in this example suggest that carotenoid production primarily occurred during growth phase.

During the growth phase, the mutant strain showed positive regulation of central carbon metabolism metabolites associated with glycolysis, the pentose phosphate pathway, the TCA cycle, and amino acid and fatty acid biosynthesis. In the stationary phase, amino acids associated with the TCA cycle increased, but most of the fatty acids and central carbon metabolism metabolites decreased. TCA cycle metabolites such as succinate, fumarate, and a-ketoglutarate were abundant during both growth and stationary phases.

The overall observed metabolic changes in the central carbon metabolism and abundance of TCA cycle metabolites suggest an enhancement in the electron respiratory chain in the mutant, and in the provision of the electrons required for the AX synthesis by the AX synthase, may be primarily responsible for enhanced AX production. Transcriptomic data correlated with the metabolic data and found a positive regulation of genes associated with the electron respiratory chain.

MATERIALS AND METHODS

Strains, media, and growth conditions

The wild-type strain of A. dendrorhous CBS 6938 and the mutant strain A. dendrorhous MYMO, each as described in Example 1, were used in this example. In this example A dendrorhous MYMO is hereinafter referred to as X dendrorhous BPAX-A1. Storage of the strains was as described in Example 1. Chemically defined media for inoculum and fermenters contained (in g/L): glucose (20), (NH ₄ )2SO ₄ (6), KH ₂ PO ₄ (2), FeSO ₄ «7H ₂ O (0.019), MgSO ₄ «7H ₂ O (0.88), and CaCl- 2H ₂ O (0.2). Potassium hydrogen phthalate 20 was used as a buffer when growing in flasks. The pH was adjusted to 5.5 with NaOH 2M. The media was supplemented with trace salts (mg/L): ZnSO ₄ »7H ₂ O (5.01), CuSO ₄ «5H ₂ O (0.75), MnSO ₄ (0.48), H3BO3 (0.6), Na ₂ MoO ₄ *2H ₂ O (0.6), and KI (0.15) and vitamins (mg/L): myo-inositol (60), vitamin B3 (niacin) (3), vitamin B5 (pantothenic Acid) (3), vitamin Bl (thiamine) (3), vitamin B6 (pyridoxine) (3), vitamin B7 (biotin) (0.048), and p- aminobenzoic acid (1.8).

Analytical techniques

• Optical density (A600)

OD was measured as for Example 1.

• Dried cell weight

Dried cell weight was measured as for Example 1. • Total carotenoids

Total carotenoids were assessed as for Example 1.

• HPLC analyses for astaxanthin and other carotenoids

HPLC analyses for astaxanthin and other carotenoids were performed as for Example 1.

• HPLC analyses for sugars and organic acids

Supernatants for sugar and organic acid analyses was obtained by centrifugation of 1 mL of fermentation sample at 15,000 rpm for 5 min. Organic acids and carbohydrates were quantified by ion exchange chromatography using an Agilent 1200 HPLC system and an Agilent Hiplex H column (300 x 7.7 mm, PL1170-6830) with a guard column (PL Hi-Plex H 50 x 7.7 mm, PL1170- 1830). Sugars were monitored using a refractive index detector (Agilent RID, G1362A) set on positive polarity and optical unit temperature of 35 °C while organic acids were monitored at 210 nm (Agilent MWD, G1365B). 30 gL of each sample was injected onto the column using an autosampler (Agilent HiP-ALS, G1367B) and the column temperature was kept at 40°C using a thermostatted Column compartment (Agilent TCC, G1316A). Analytes were eluted isocratically with 5 mM H2SO4 at 0.4 mL/min for 40 min. Chromatograms were integrated using Chromeleon 7.2 software.

• Intracellular metabolite extraction and analyses

The following methods for quenching, extraction, and analyses were adapted from (Canelas et al., 2008, 2009; Martinez-Moya et al., 2015; Luna-Flores et al., 2018; Pan et al., 2020). Cells were quenched in cold methanol 60% and placed in a bath of ethanol/dry- ice. Cells were then centrifuged at 4,500 rpm for 5 min at -20 °C and pellet was washed with cold 60% methanol before to snap-freeze in liquid nitrogen. Cell pellets were kept at -80 °C until further use. The pellet was then dissolved in a mixture 1 : 1 of chloroform and methanol 50% and submitted to five cycles of 5 min of bead beating using acid washed glass beads (Sig. Cat. No. G1152-100G) and a tissue lyser (Qiagen TissueLyser II); the tubes were cooled down on ice for 5 min before start each cycle.

To separate polar and non-polar metabolites, the disrupted cells were centrifuged at 15,000 rpm for 15 min in which polar metabolites were in the top layer (methanol 50%) and non-polar metabolites were in the bottom layer (chloroform). The top layer was collected, freeze dried, and the pellet obtained was finally re-suspended in 2% acetonitrile. 5 uM of AZT, 3 ppm of 13-C valine, and 3 ppm of 13-C sorbitol were used as internal standards. LCMS was used to analyse central carbon metabolism using the method described in (Luna- Flores et al., 2018). Briefly, analyses were performed using a Dionex Ultimate 3000 HPLC system coupled to an ABSciex 4000 QTRAP mass spectrometer. Liquid chromatography was performed using a 50 min gradient with 0.3 mL/min flowrate, on a Phenomenex Gemini-NX C18 column (150 x 2 mm, 3 gm, 110 A), with a guard column (SecurityGuard Gemini-NX Cl 8, 4 x 2 mm), and column temperature of 55 °C. The mobile phases used were: 7.5 mM aqueous tributylamine (Sigma-Aldrich) with pH adjusted to 4.95 (±0.05) using acetic acid (Labscan) for Solvent A, and acetonitrile (Merck) for Solvent B. Samples were kept at 4 °C in the autosampler and 10 gL of various dilutions of samples were injected for analyses. The HPLC was controlled by Chromeleon 6.80 software (Dionex). Mass spectrometry was achieved using a scheduled multiple reaction monitoring (sMRM) method on the negative ionisation mode. Collected data were processed using MultiQuant 2.1 (AB Sciex). The amino acids profile was obtained using the protocol described in (Chen et al., 2020). In brief, Shimadzu LCMS 8050 was used for amino acid analyses. This instrument was equipped with three quadrupoles for mass analysers and collision. Liquid chromatography was performed by injecting 1 uL of sample to a F5 column (Sigma) and eluted in a 25 min gradient using as mobile phase acetonitrile with 0.1% of formic acid at 0.25 mL/min. Oven was set to 40 °C. Electrospray ionization was used to ionise the sample. Mass spectrometry was achieved using a scheduled multiple reaction monitoring (sMRM) method on the positive ionisation mode. Amino acid mix was used as standard for identification and quantification (Sig. Cat. No. A9906-1ML). Skyline daily was used to analyse amino acids detected in LC-MS (Adams et al., 2020). The fatty acid profile of yeast biomass was determined using the following procedure. A portion of dried yeast biomass (50 mg) was mixed with 2 mL solvents containing methanol/hydrochloric acid/chloroform (10:1:1, v/v/v). The mixture was heated at 90 °C for 1 h in a sealed glass tube to convert microbial oils to fatty acid methyl esters (FAMEs). Then the mixture was treated through mixing with 0.9% NaCl solution (1 mL). Afterwards, FAMEs were extracted through addition of 0.5 mL hexane, followed by centrifugation. Then, the supernatant hexane phase containing FAMEs were analyzed by an Agilent 6890 Series Gas Chromatography system equipped with a HP 5973 mass spectrometer detector and a HP-5MS capillary column (Agilent J&W 30 m x 0.25 mm x 0.25 [im). 1 [J.L of the sample was injected with a split ratio of 10:1. The injection port temperature was 230 °C. Initial column temperature was 90 °C and held for 1 min, followed by increasing the column temperature at a rate of 15 °C/min until 180 °C, 5 °C/min to 220 °C, 10 °C/min until 250 °C and held for 10 min. FAME Mix (Supelco® 37 Component, Sigma- Aldrich) was used as standard.

• RNA extraction, sequencing, and analyses

Cells sampled at Phase III were used for RNA extraction and analysis. For this, cells (50 ODs) were quenched in cold methanol 60% and placed in a bath of ethanol/dry-ice. The cells were then centrifuged at 4,500 rpm for 5 min at -20 °C and the pellet was snap- frozen in liquid nitrogen and finally storage at -80 °C until further use. ZymoBiomics DNA/RNA Miniprep Kit (Zymo Research Cat. No. R2002) was used for RNA extraction. For this, the pellets were first submitted to a cryogenic grinding step using a mortar, pestle and liquid nitrogen. To ensure extraction, using indications of the kit, the grinded pellet was submitted to three cycles of five minutes of bead beating using a Qiagen Tissue Lyser II. This grinded extract was then used for RNA extraction following the indications of the kit. Extracted RNA was quantified using a NanoDrop and Qubit 4.0. The library was prepared using the Illumina Stranded mRNA prep kit. The quality of the RNA and library were evaluated by a Fragment Analyser 5200. Finally, the samples were sequenced using the Illumina platform 100 bp Pair End using NovaSeq 6000 and a SP PEI 00 flow cell. Quality of the reads was evaluated using FASTQC (Andrews, 2010) andTrimmomatic (Bolger etal., 2014) was used to remove bad quality reads. Then, Tophat, Cufflinks, and CuffDiff were used to align the RNA-seq reads against the reference genome X. dendrorhous CBS6938 (Sharma et al., 2015), normalize and annotate the transcripts, and evaluate the differential expression, respectively (Trapnell et al., 2012). The cutoff for significant differentially expressed genes was q < 0.05.

• Statistical analyses of intracellular metabolomics

Metabolomics data were normalized and analysed for statistical significance (De Livera et al., 2012). Principal component analyses were used to profile all metabolites (Mendez etal., 2019).

• Instrumented fermenters

Instrumented fermenters were performed in a 1 L New Brunswick BioFlo/CelliGen 115. The fermenter was configured with one six-blade Rushton impeller with a diameter of 2.5 cm, three baffles, one ring sparger, and ports for acid, base, antifoam, and sampling. The fermenters were equipped with probes and controllers of pH, pCh, temperature, and antifoam to measure and control these parameters, respectively. Optimal fermenter conditions were the following. The fermenter was inoculated withl0% of cells at A600 of 5 growing at the mid-exponential phase. The pH was controlled at 5.5 using 2 M sodium hydroxide or 2 M of sulphuric acid. The foam was controlled by adding Antifoam C (Sigma Cat. No. A8011). The temperature was controlled at 20 °C using a heater jacket or chiller. The dissolved oxygen was controlled at 70% of air saturation by using cascade changes in agitation (400 - 1200 rpm), and a constant air flow rate of 1 VVM.

• Calculation of fermentation parameters

Specific growth rates (p) was calculated across all the growth phases using the logarithm method. Yield of sugar conversion to biomass (Yxs) was calculated using the total biomass produced over the consumed substrate. The specific consumption rate of glucose (qs) and the specific production rates of carotenoids (qp) and AX (qpax) were computed at all the growth phases by multiplying the specific growth rate by the linear correlations of sugar or carotenoids and AX with biomass. For the stationary phase in which no-growth was observed, the specific rates were calculated using the linear correlations of sugar or carotenoid production with an average of biomass multiplied by its time frame.

RESULTS

Instrumented fermenters and kinetic analyses The wild-type X. dendrorhous CBS 6938 and the mutant X. dendrorhous BPAX-A1 (referred to as MYMO in Example 1) strains were grown for three days in a chemically defined media and sampled for growth and production measurements. Comparatively, as can be seen in Table 9, the wild-type final biomass was 13.60 ± 0.29 gDCW/L, which represented 35.8% higher than that obtained in the mutant strain. The wild-type sugar to biomass yield (Yxs) was 0.62 g/gDCW being 44% higher than that obtained in the mutant strain. In terms of AX production, the mutant strain presented an improvement of around 11 -fold compared to the wild-type strain. The final proportion of AX was around 70% for both strains. In terms of specific rates, as can be seen in Table 10 and Figs. 14-17, the wild-type strain had a maximum specific growth of 0.12 h ^-1 which was 9% higher than that obtained in the mutant strain. The mutant and wild-type strain did not present a lag growth phase, but in the total carotenoid production (see Figs. 14-17). The maximum specific carotenoid production in the mutant strain was 12-fold higher than that obtained in the wild-type strain. In terms of strain specific changes for growth and production, as can be seen in Table 10 and Fig 14, the mutant strain had an increase of the specific carotenoid production of 2.44-fold from Phase 1 to Phase 2 and continued until the stationary phase. Interestingly, the specific AX production remained relatively constant across all the growth phases. At the stationary phase, the carotenoid production ceased but the accumulation of AX continued mainly due to an increase in the AX composition of the total carotenoids. For the wild-type strain, the specific carotenoid production was increased 7.92-fold from Phase 1 to Phase 2 and it was extended until the stationary phase. For the AX production specifically, it was increased 2.37-fold and 7.51 -fold from Phase 1 to Phase 2 and Phase 3, respectively.

Metabolite profiling of wild-type and mutant strains of X. dendrorhous

Figures 14 and 16 show the growth phases sampled for intracellular metabolite analyses. A total of 80 metabolites associated with primary metabolism were obtained. Before statistical analyses, the metabolite data was normalized based on biomass and internal standards. Principal component analyses (PCA) was then used for statistical grouping of the samples. As can be seen in Figure 18, the score and loading plots discerned between the different metabolites analysed grouping the four growth phases of the strains under analyses. Phase 3 suggested to be the most explanatory for differential comparison due to it was isolated inside each strain and between them. To compare the relative amounts of metabolites, the response ratio of each metabolite was obtained and converted to log2 (Fig. 19). The metabolomes of the wild-type and mutant strains were then compared at each growth phase. Here, central carbon metabolism metabolites presented the highest differences (more abundant in the mutant strain) for the first three growth phases analysed. In Phase 4, except pyruvate, fumarate, succinate, a-ketoglutarate, ribulose-5- phosphate, and glucose-6- phosphate all metabolites were similar or less abundant in the mutant strain. A similar pattern was observed with FAME metabolites, in which methyl laurete (C15:0) and methyl palmitoleate (16: 1) were more abundant in all the growth phases. In regards to amino acids, they were more abundant in the mutant strain during all the growth phases. To study the dynamic of metabolite profiles, the principal carbon, amino acid, FAME, electron transport chain, and carotenogenesis pathways were obtained using the KEGG database; to facilitate the comparison, the response ratios were normalized to 100 (Fig. 20).

Comparative transcriptomics analysis

Cells from Phase 3 were used for differential transcriptomic analysis. RNA-sequencing was used to compare the transcriptional profile of the strains. Our analyses identified 6363 transcribed genes, of which 690 were significantly different (q < 0.05). From these genes, 211 (132 < Log2 - 2) were downregulated and 479 (228 > Log2 2) were upregulated (Table 16). Figure 20 shows the gene expression on the pathways glycolysis, pentose phosphate pathway (PPP), TCA cycle, electron transport chain, and carotenoid biosynthesis (see also Tables 15 and 16; Fig. 20). Among the most relevant changes, the hexose carrier gene CED82529 was significantly downregulated in the mutant strain (Log2 -2.10, q < 0.05). There were not found significant differences on the expression of genes associated with the glycolytic pathway. However, this pathway seems to be more active in the wild-type strain than in the mutant strain. Similarly, the gluconate kinase and 6- phosphogluconate dehydrogenase PPP genes were significantly downregulated in the mutant strain (Fig. 20 and Table 17). For the TCA cycle, the gene isocitrate lyase was significantly downregulated (Log2 -1.24, q < 0.05) in the mutant strain and the genes ketoglutarate dehydrogenase and succinate dehydrogenase were significantly upregulated (Log2 1.55, q < 0.05) and (Log2 2.8, q < 0.05), respectively. Several genes associated with the electron transport chain were significantly upregulated in the mutant strain including the Complex I genes (CDZ96154, CED84925, CDZ96153, and CDZ96151), Complex II gene (CDZ98193), Complex III genes (CED80059 and CED80058), Complex IV genes (CED800856, CDZ96152, CED800061, CED84572, and

CED82283), and ATP-Synthase genes (CDZ96150 and CDZ96333) (see Table 17 and Fig. 20 for more details). Similarly, most of the genes associated with the AX biosynthesis were upregulated in the mutant strains including the AX synthase CDZ97194 (Log22.23, q < 0.05), the aldo/ketolase related proteins CDZ97194 (Log2 1.85, q < 0.05) and CDZ97021 (Log2 1.13, q < 0.05), and the P450 reductase CED84998 (Log2 4.05, q < 0.05) and CDZ98632 (Log2 1.44, q < 0.05); from these P450 reductase genes, the CED85015 belonging to the CYP2 family was significantly downregulated (Log2 -2.30, q < 0.05).

DISCUSSION In Example 1, comparative genomics was explored to link phenotype and genotype of the mutant strain X. dendrorhous BPAX-A1 (MYMO). However, metabolic changes associated with improved AX production remained unclear. In this example, to gain metabolic insight, the wildtype and mutant strains were grown in chemically defined media and sampled at four growth phases for kinetic and metabolic comparisons. No substantial lag-phase was observed in either strain, and carotenoid production appeared primarily associated with growth. Principal Component Analysis (PCA) of all metabolites grouped the growth phases between and among the strains under comparison. Growth Phase III was fully isolated in the PCA analysis (see Figure 18), and kinetically, presented the highest carotenoid production rate (see Table 10), indicating that this phase may be particularly relevant for transcriptomic comparisons. Accordingly, RNA-seq data was obtained and integrated with kinetic, metabolomics, and genomics data.

Comparative metabolomics

To study metabolic changes, a system-level characterization was performed, previously shown to be useful (Luna-Flores et al., 2016, 2018). Extracellular and intracellular metabolites were measured across the fermentation time course (Figs. 14-17). As can be seen in Tables 9 and 10, the mutant strain produced more carotenoids than the wild- type strain but had a lower specific growth rate and final biomass. These results are in agreement with other studies in which strains with enhanced AX production have displayed lower biomass (Cannizzaro et al., 2004; Gassel et al., 2013). These previous studies suggested that wild-type X dendrorhous used the PPP primarily for glucose degradation, leading to greater abundance of NADPH and pentoses used for lipid and nucleotide biosynthesis, respectively.

In this example, metabolites associated with the PPP were more abundant in the wild-type strain than in the mutant strain (Fig. 19 and Fig. 20). Of note is that NADPH was lower in the mutant than in the wild-type strain, providing a possible (at least partial) explanation for the observed decreased biomass. This decreased NADPH may well be associated with the improved AX in the mutant strain, with production of isoprenoid units of the AX molecule through the MVA pathway potentially competing with NADPH production. Also, the wild-type strain had a maximum glucose uptake rate 13% higher than the mutant strain. Notably, the hexose carrier gene CED82529 was significantly downregulated (Log2 -2.10, q < 0.05) in the mutant strain (Table 16 and Table 17), which may well be at least partly responsible for the lower growth rate in the mutant strain as compared to the wild-type strain.

During the growth phase, the mutant strain showed abundance of metabolites associated with glycolysis, TCA cycle, amino acids, and fatty acids metabolites, but at the stationary phase, except for amino acids, most of these metabolites were more abundant in the wild-type strain. Similarly, metabolites of the TCA cycle namely succinate, fumarate and a-ketoglutarate were more abundant in the mutant strain than the wild-type strain during all the growth phases studied (Fig. 19). These metabolic changes suggest a more active central carbon metabolism to grow and produce AX in the mutant strain in which the TCA cycle is playing an important role. The supplementation of TCA cycle metabolites, such as succinate or citrate, has been reported to increase AX production in A. dendrorhous (Flores-Coteraeta/., 2001; Martinez-Moya eta/., 2015). Similarly to oleaginous yeast, X. dendrorhous can use citrate as a carbon source for lipids and carotenoids biosynthesis (Flores-Cotera et al., 2001). It is reported that the use of succinate as a sole carbon source increased TCA cycle metabolites and proteins when compared to glucose as a carbon source suggesting that succinate can positively alter the whole synthesis of metabolites and translation of proteins (Martinez-Moya et al., 2015).

In regard to lipids, linoleic acid, palmitoleic acid, and stearic acid were the most abundant fatty acids in both strains. At Phase IV or stationary phase, the mutant strain presented 1.15-fold more palmitoleic acid, 1.17-fold more steric acid, and 1.41-fold less linoleic acid than the wildtype strain. Similarly to linoleic acid, all the other fatty acids were decreased in the mutant strain suggesting changes in the final fatty acid profile between the strains under study. Also, amino acids associated with the TCA cycle were more abundant in the mutant strain than in the wild-type strain. This Phase IV also matched with a switch in pH control - use of acid instead of base to maintain the pH at the desired level. The production of carotenoid ceased but AX continued increasing mainly due to a change in the composition of the total carotenoids. All collected data at Phase 4 suggest that the mutant strain was consuming lipids and peptides to obtain energy and metabolic precursors to continue producing AX.

Integrating transcriptomics Respiratory and energy metabolism

For Example 1, a genomic variant analysis was performed in mutant strains including X. dendrorhous BPAX-A1. This genomic analysis found mutations in the electron respiratory chain pathway and metabolomics analysis suggested changes in the TCA cycle associated with the electron respiratory chain. In Example 1, a subset of mutations that were considered of particular interest were identified. Notably, some of these mutated genes were overexpressed based on transcriptomic analysis (see Table 15, Table 16, and Table 17). For example, a mutation in the succinate dehydrogenase or complex II gene (CDZ981393) was identified; this gene was significantly upregulated in the mutant strain (Log2 2.8, q < 0.05). Also, the complex I or NADHmbiquinone/plastoquinone oxidoreductase, chain 3 (CDZ96154) gene was mutated with two intron variants and this gene was significantly upregulated in transcriptomics (Log2 4.73, q < 0.05). The complex I or the NADH dehydrogenase subunit 4 (CDZ96151) gene had two mutations and was significantly upregulated in transcriptomics (Log2 4.13, q < 0.05). Similarly, cytochrome b gene CED80058 was mutated in the upstream and in the downstream of the gene; these mutations might be responsible for the significantly upregulated gene CED80058 in transcriptomics (Log2 5.15, q < 0.05) (Fig 4). The complex IV or cytochrome c oxidase subunit 1 gene CED80056 had two mutations, the cytochrome c oxidase subunit 2 gene CDZ96152 had one mutation, and the cytochrome c oxidase subunit 3 gene CED80061 had two mutations. These genes were significantly upregulated in transcriptomics: CED80056 (Log2 5.57, q < 0.05), CDZ96152 (Log2 5.30, q < 0.05), and CED80061 (Log2 5.01, q < 0.05). Linked to these changes in the Complex IV gene, the cooper transporter gene CED82663 was significantly upregulated (Log2 2.38, q < 0.05) in the mutant strain probably increasing the copper availability inside the cell. As Complex

IV protein contains two copper catalytic centres (Cua and Cub), copper supplementation has been associated with an increase of AX production in X. dendrorhous (Srinivasan and Avadhani, 2012; Martinez-Cardenas et al., 2018). The ATP synthase subunit 6 gene CDZ96150 had two mutations in the upstream of the gene, and two other mutations in the downstream of the gene. Similarly, the ATP synthase subunit mitochondrial gene CDZ96333 had two mutations in the downstream of the gene. These CDZ96150 and CDZ96333 genes were significantly upregulated in transcriptomics (Log25.25, q < 0.05) and (Log24.65, q < 0.05), respectively. In A. dendrorhous, AX is synthesised by oxidation of P-carotene through the P450 enzyme AX synthase (Qjima et al., 2006). This reaction needs the adjunct activity of the cytochrome P450 reductase or the cytochrome b as an electron donor (Alcaino et al., 2008a). Although no mutation was found in the AX synthase gene (CED83940), this gene was significantly upregulated in transcriptomics (Log22.23, q < 0.05).

Similarly, the cytochrome P450 CYP3/CYP5/CYP6/CYP9 subfamilies gene (CED84998) and the cytochrome P450 CYP4/CYP19/CYP26 subfamilies gene (CDZ98632) were significantly upregulated (Log2 4.05, q < 0.05) and (Log2 1.45, q < 0.05), respectively. Interestingly, the cytochrome P450 CYP2 subfamily (CED85015) was significantly downregulated (Log2 -2.30, q < 0.05). Cytochrome P450 reductase (crtR) of A dendrorhous has been cloned and found to be an essential gene for AX biosynthesis (Alcaino et al., 2008b). That study suggested that only one crtR is required for AX biosynthesis. However, the disruption of that gene in X. dendrorhous was not lethal suggesting the existence of an alternative electron donor such as the cythochrome b5. Here, upregulation of two crtR genes (CED84998 and CDZ98632) and downregulation of another one (CED85015) was observed. No significant overexpression of cytochrome b5 was observed.

Significant upregulation of the Ferredoxin/adrenodoxin reductase gene (CDZ98521) (Log2 -2.30, q < 0.05) was observed. Ferredoxin/adrenodoxin reductase can transport electrons from a FADH2 coenzyme, produced during p-oxidation of lipids by the action of the acyl-CoA dehydrogenase, to a P450 systems of the mitochondria (Hanukoglu, 1992). Linked with this reaction step, here significant upregulation of an acyl-CoA dehydrogenase gene (CED84717) (Log2 1.36, q < 0.05) was observed, which is involved in the initial step of p-oxidation of lipids and production FADH2. This upregulated lipid degradation to provide FADH2 cofactors is potentially associated with the ongoing change in proportion of AX in the mutant strain BPAX-A1 after glucose was depleted and in its change of final lipids profile in which linoleic acid was 1.41- fold less than the wild-type.

Although not significant, all the other carotenoid-associated production genes (GGPP synthase, phytoene synthase, phytoene dehydrogenase, and lycopene cyclase) were nominally upregulated in transcriptomics in the mutant strain BPAX-A1 (Table 17). Linked to the TCA cycle, the a-ketoglutarate dehydrogenase gene (CED83799) was upregulated in transcriptomics in the mutant strain (Log2 1.55, q < 0.05) and downregulated the isocitrate lyase gene (CED85129)(Log2 1.24, q < 0.05). This suggests that the mutant strain is not using the glyoxylate shunt to deal with cell oxidative stress (Ahn et al., 2016), but the improved amounts of AX produced. Also, the asparagine synthase gene (CED83843) was upregulated in the mutant strain (Log22.11 , q < 0.05). Asparagine is a TCA cycle derived (from oxaloacetate) amino acid which was more abundant in the mutant strain than in the wild-type strain (Fig. 20). Contrary to TCA cycle intermediates, asparagine supplementation inhibited carotenogenesis in a hyper producing strain of X. dendrorhous impaired to assimilate nitrogen sources (An, 2001). This suggests that asparagine overproduction can be detrimental for AX production and potentially can be used as a metabolic strategy to improve AX production. Also, asparagine abundance is probably associated with a decrease in the Carbon/Nitrogen ratio which has been associated with a decrease in carotenoid production in A. dendrorhous (Fomtana et al., 1996). Overall, these metabolic differential analyses suggest a correlation between genome changes in the BPAX-A1 mutant strain and its transcriptome and metabolome profile, which potentially increased AX production (Figs. 14-17 and Table 9). The main driver for the improved AX production in the mutant BPAX-A1 strain seems likely to be a positive regulation of its respiratory electron transport chain (Fig. 20). This improved the availability of electrons required the incorporation of oxygen groups to the AX molecule and the increase the oxidative stress through ROS, which has been suggested to be a trigger of AX production in X. dendrorhous (Liu and Wu, 2006).

Oxidative stress response in X. dendrorhous

Carotenoids, including AX, present antioxidant properties that have been associated with survival mechanisms in X dendrorhous and other microorganisms (Schroeder and Johnson, 1995). Normally, ROS are generated by an overflow of electrons in the electron respiratory chain triggered by an imbalance of electrons transfer during the reduction and oxidation of the ubiquinone pool. AX can quench ROS analogously to the superoxide dismutase. In this example, it was observed that the mutant strain upregulated genes associated with the electron transport chain that in turn triggered mechanisms associated with protection against ROS. First, the AX and total carotenoids accumulation in the mutant strain was significantly higher (p < 05) than the ones found in the wildtype strain across all the growth and production phases (Figs. 14-17; Fig. 20; Table 9).

Second, in yeast, hydrogen peroxide is increased during p-oxidation of lipids in the peroxisome, which can be neutralized by catalases (Yin et al., 2009). It has been reported that X. dendrorhous shows low activity of this enzyme (Schroeder and Johnson, 1995). Here, at the transcriptome level, two catalases genes were significantly upregulated in the mutant strain: the CDZ96425 (Log2 2.89, q < 0.05) and CDZ98863 (Log2 1.91, q < 0.05) genes, respectively. This suggests that the mutant X. dendrorhous strain may use catalases as a mechanism to alleviate oxidative stresses. And third, a report suggests that A. dendrorhous uses an antioxidant core which is modulated by the carotenoid production including monooxygenases, a cytochrome P450 enzyme, phosphoglucomutase, and glyceraldehyde 3-phosphate (Flores-Cotera c/ a/., 2001; Martinez-Moya et al., 2015).

It is reported that X. dendrorhous showed low levels of superoxide dismutase, or glutathione peroxidase enzymes under inductions with single oxygen and peroxyl radicals (Schroeder and Johnson, 1995). However, in a separate study, at the proteome level, it was identified activity of the superoxide dismutase and some glutathione enzymes when grown on succinate as a carbon source, which increased AX by 2.33-fold (Martinez-Moya et al., 2015). In agreement with that study, here two glutathione-S-transferase genes were significantly upregulated in the mutant strain: CED84930 (Log26.09, q < 0.05) and CDZ97957 (Log2 5.72, q < 0.05) (Table 16 and Table 17). It is reported that the glutathione S- transferases counteract the mutagenic effect of aldehyde products of lipid peroxidation (Ames et al., 1993). Generally, glutathione, glutathione enzymes, and superoxide dismutase are used as oxidative stress biomarkers in trials to evaluated the antioxidant effect of certain chemicals or drugs (Ames et al., 1993). Using these markers, AX has shown to be effective to stimulate these oxidative stress mechanisms in rainbow trout (Elia et al., 2019). In addition, the highest overexpressed gene in the mutant strain was the conidiation- specific protein 6 gene (CED85080) (Log2 9.29, q < 0.05). This protein has been associated with survival mechanisms against environmental stresses (Zhang etal., 2016). Overall, the mutant strain showed upregulation of the electron transport chain which might increased ROS triggering some protective oxidative stress mechanisms including higher AX production, upregulation of AX synthase, P450 reductase, and glutathione-related genes.

Lipid biosynthesis

The fatty acid pathway in A. dendrorhous has been elucidated (Sharma et al., 2015). The synthesis starts with the acetyl CoA carboxylase and the acyl carrier protein (ACP) for the formation of acetyl-ACP and malonyl-ACP, which is followed by condensations, ketoacyl reduction, a dehydratase reaction, and enoyl reduction all the way to palmityl-CoA are catalyzed by two multi-enzyme complexes FAS1 and FAS2. The FAS1 has functional domains of malony transferase, enoly reductase, hydroxyacyl dehydratase, and malony/palmitoyl transferase andFAS2 has functional groups of ketoacyl reductase, ketoacyl synthase, and phosphopantetheinyl transferase. The additional genes involved in the elongation of Cl 6 to Cl 8 fatty acid by the fatty acid elongase, and the insertion of a delta-9 and a delta- 12 double bond by delta 9 fatty acid desaturase, and delta 12 fatty acid desaturase, respectively. From the mitochondrial fatty acid enzymes, palmitic acid can be synthesised by palmitoyl thioesterase from palmytil-ACP. Condensation, ketoacyl reduction, dehydratase, and enoyl-reduction can be also carried out by acetyl-CoA acyltransferase, 2 ketoacyl reductase, enoyl-CoA hydratase, or 3-hydroxyacyl CoA dehydrogenase, respectively.

In this example, another mutation was found in the delta 9 fatty acid desaturase. A report suggest that this enzyme has preferences for substrates of Cl 8:1 and Cl 6:1 converting to Cl 8:2 and C16:2 fatty acids (L. Zhang et al., 2020). The most abundant fatty acids in the P. rhodozyma pathway are linoleate (C18:2), stearic acid (18:0), oleic acid (C18:l), palmitic acid (C16:l), and hexadecanoic acid (16:2) (Sharma et al., 2015). Although not significant, the delta 9 fatty acid desaturase gene (CED83656) was nominally downregulated in the mutant strain (Log2 -0.53, q > 0.05). This suggests that delta 9 fatty acid desaturase might be influencing the fatty acid pathway in the novel strains of P. rhodozyma. A decrease in the fatty acid and ergosterol metabolism in P. rhodozyma increased AX production (Miao et al., 2011). Also, the FAS1 gene (CED83610) was significantly upregulated in the mutant strain (Log2 1.92, q < 0.05 ). Similarly, the delta- 6- desaturase gene (CED83550) was significantly upregulated (Log2 1.38, q < 0.05). This gene is the first step in the degradation of linoleic acid before to be utilised by the microorganism (Horrobin et al., 1993). Overall, these findings provide a possible explanation as to why linoleic acid was more abundant in the wild-type strain than in the mutant strain at the end of the fermentation.

EXAMPLE 3: Proteomic analysis of a high astaxanthin producer strain of Xanthophyllomyces dendrorhous

In this example, proteomic analysis was performed to complement the metabolomic and transcriptomic analysis of Example 2.

MATERIALS AND METHODS

Strains, media, and growth conditions

Strains, media, and growth conditions were as for Example 2, above.

Proteomic extraction, quantification, digestion, and SWATH analyses.

Samples equivalent to 20-50 A600 units were harvested from the instrumented fermenters at Growth Phase 3 and Growth Phase 4 as described in Example 2. Cells were pelleted at 14000 for 5 min at 4 °C. The supernatant was discarded, and the pellet was washed twice with ice-cold water. The washed pellet was then re-suspended in 2 mL of sodium dodecyl sulfate (SDS) lysis buffer and transferred to a tubes containing 0.5 g of acid washed glass beads (Sigma Cat. No. G1152-100G). The tubes were chilled on ice for 5 minutes and then disrupted using five beadbeating cycles of 5 min using a Qiagen Tissue Lyser II. After disruption, the tubes were centrifuged at 14000 *g for 10 min at 4 °C.

The extracted proteins were quantified using the Pierce BCA Protein Assay Kit (ThermoFisher Cat. No. 23225). The FASP and Cl 8 StageTip protocols were then applied to digest the extracted proteins and desalt the peptides, respectively (Jacek R Wisniewski, Alexandre Zougman, Nagarjuna Nagaraj, 2009). The peptides of digested samples were quantified using the Pierce Quantitative Colorimetric Peptide Kit (ThermoFisher Cat. No. 23275) and used to normalize their concentration before submitting the samples to mass spectrometry (MS) analysis. The sequential window acquisition of all theoretical mass spectra (SWATH) technique was then applied to the peptide samples using the Sciex 5600 QTOF (Yeo et al., 2016).

To prepare the library, a mixture of all peptide samples (PBQC) was MS analysed in Data Dependent Acquisition mode (DDA). For this, ProteinPilot 5.0.2 was first used to identify proteins using the Paragon method and the published and annotated P. rhodozyma CBS6938 genome using a quality threshold of 1.3 (95%) (Sharma et al., 2015). Skyline was then used to create the spectral library and SWATH mode analysed samples. Differential proteome expression was assessed between the mutant X. dendrorhous BPAX-A1 (referred to as MYM0 in Example 1) and wild-type X. dendrorhous CBS 6938 at Phase 3 and Phase 4.

RESULTS

Differential protein expression results are set out for Growth Phase 3 in Figure 22 and Table 18; and for Growth Phase 4 in Figure 23 and Table 19.

EXAMPLE 4: Products incorporating astaxanthin

This example provides details of certain typical formulation for supplementing domestic or companion animals, the formulation including astaxanthin produced as herein described. The formulation may be referred to as 'Na AX' , although without limitation thereto.

The formulations are also rich in dietary fibre, vitamins, beta-glucan, and amino acids. An analysis of fatty acid profile for the formulations is provided in Fig. 21. An analysis of amino acid profile is provided in Table 11.

Formulations for feline, canine, and equine use have been developed. All formulations include astaxanthin and beta-glucans.

In general, astaxanthin may provide benefits including:

• Antioxidant activity for supporting the immune system

• Mitochondrial protection against reactive oxygen species • Reduction in cellular damage and lipid peroxidation

• Improve response and processing of antigens

In general, beta-glucans may provide benefits including:

• Stimulation of the immune system

• Reduction of blood concentration of total cholesterol

• Antioxidant properties

• Reduction of inflammatory response

Equine variations comprise polyphenols, which may provide benefits including:

• Combat inflammation and the natural aging process (‘inflamm-aging’)

• Decrease the amount of non-steroidal anti-inflammatory drugs administered to older horses

• Antioxidant activity for reducing oxidative stress and improve immune function

• Support muscle recovery in endurance horses

Specifications for the feline, canine, and equine formulations are as follows:

Feline

Component percentages:

• Astaxanthin: 0.25% = 2.5 g/kg

• beta-Glucan: 15% = 150 g/kg

• Carriers: = 847.5% g/kg = 847.5 g/kg

Minimum and maximum recommended dose rates:

• Adult cats <5kg: 1 g/day

• Adult cats >5kg: 2 g/day

• Kittens and geriatric cats: 2 g/day

Canine

Component percentages:

• Astaxanthin: 0.25% = 2.5 g/kg

• beta-Glucan: 15% = 150 g/kg

• Carriers: = 847.5% g/kg = 847.5 g/kg

Minimum and maximum recommended dose rates:

• Extra small dogs (8 kg) = 1 g/day

• Small dogs (16 kg): 2 g/day

• Medium dogs (24 kg): 3 g/day

• Large dogs (32 kg): 4 g/day

• Extra large dogs (40 kg): 5 g/day (Puppies and geriatric dogs require double the recommended dose) Equine

Component percentages:

• Astaxanthin: 0.075% = 0.75 g/kg

• beta-Glucan: 4.22% = 42.2 g/kg

• Polyphenols: 2.5% = 25 g/kg

• Carriers: 93.21% g/kg = 932.05 g/kg

Minimum and maximum recommended dose rates:

• 100 g per day orally or mixed in feed

(Horses in poor condition or commencing training require double the recommended dose for the first 3 weeks)

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The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety.

Throughout the specification, the aim has been to describe typical aspects and embodiments without limiting the invention as disclosed herein to any one aspect, embodiment, or specific collection of features. It will be appreciated that various changes and modifications may be made relative to the exemplary disclosure provided herein without departing from the present invention.

In this specification, the use of the terms “suitable” and “suitably”, and similar terms, is not to be read as implying that a feature or step is essential, although such features or steps referred to as “suitable” may well be preferred.

In this specification, the indefinite articles “a” and “an” are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, “a” cell includes one cell, one or more cells, and a plurality of cells.

In this specification, the terms “comprises” , “comprising” , “includes” , “including” , and similar terms, are intended to denote the inclusion of a stated integer or integers, but not necessarily the exclusion of another integer or other integers, depending on context. That is, a product, composition, or method, etc., that comprises or includes stated integer(s) need not have those integer(s) solely, and may well have at least some other integers not stated, depending on context.

In this specification, the terms “consisting essentially of" and “consists essentially of" are intended to mean a non-exclusive inclusion only to the extent that, if additional elements are included beyond those elements recited, the additional elements do not materially alter basic and novel characteristics. That is, a composition, apparatus, system, or method that “consists essentially of one or more recited elements includes those elements only, or those elements and any additional elements that do not materially alter the basic and novel characteristics of the apparatus, system, or method.

TABLES

Table Descriptions

Table 1. Kinetic model parameters as per Example 1.

Table 2. Nitrogen sources assessed in relation to growth and carotenoid production as per Example 1.

Table 3. Carbon sources assessed in relation to growth and carotenoid production as per Example 1.

Table 4. Genomic characteristics of X. dendrorhous strains (referred to as P. rhodozyma in Table 4) sequenced as per Example 1.

Tables 5-7. Analyses of variance of the factorial design as per Example 1 to evaluate the effect on carotenoid production of the culture media components: Bactopeptone (BP), Malt Extract (ME), Yeast Extract (YE), Sucrose, Dried Com Steep Liquor (DCSL), Vitamins Cocktail, and Potassium Phosphate Monobasic (KH2PO4).

Table 8. Coefficients of the quadratic equation and model validation as per Example 1.

Table 9. Fermentation parameters of wild-type (CBS6938) and mutant (BPAX-A1) X. dendrorhous strains as per Example 2.

Table 10. Kinetic parameters of fermentation with wild-type (CBS6938) and mutant strains (BPAX-A1) of A. dendrorhous as per Example 2.

Table 11. Amino acid profile analysis for formulations as described in Example 3.

Table 12. Variants in re-sequenced X. dendrorhous CBS 6938 and eight mutant X. dendrorhous strains (MAMY3, MAMY6, MB 18, MB24, MYMO, MYM6, MYM44, andMYM92) as per Example 1, relative to SEQ ID NOs: 12685-12950. V = variation index; S = strain, wherein WT = re-sequenced CBS 6938, Ml = MAMY3, M2 = MAMY6, M3 = MB18, M4 = MB24, M5 = MYMO, M6 = MYM6, M7 = MYM44, M8 = MYM92); RS = reference scaffold in published genome assembly of Sharma et al. BMC genomics 16.1 (2015): 1-13, corresponding to SEQ ID NOs: 12685-12950, wherein the scaffold number is given, i.e. ‘62’ corresponds to ‘scaffold_62’ as per the published assembly - the correspondence of SEQ ID NOs: 12685-12950 to the reference scaffolds in the published genome assembly will be apparent or readily determinable by the skilled person; RB = reference scaffold base at the variation position; SB = strain base at the variation position; T = type of change of the variation, wherein 1 = synonymous, 2 = missense, 3 = upstream region of a CDS, 4 = downstream region of a CDS, 5 = stop gained, 6 = intergenic; 7 = intron variant, 8 = splice variant, 9 = start lost; CDS = coding sequence reference for affected CDS (if applicable) as per the published genome assembly of Sharma et al. BMC genomic 16.1 (2015): 1- 13, corresponding to SEQ ID NOs:12951-19331 - the correspondence of SEQ ID NOs:12951- 19331 to the CDS reference in the published genome assembly will be apparent or readily determinable by the skilled person.

Table 13. Variations (a subset of those in Table 12) shared across all eight sequenced mutant strains X. dendrorhous strains (MAMY3, MAMY6, MB18, MB24, MYMO, MYM6, MYM44, and MYM92) and not present in re-sequenced X. dendrorhous CBS 6938 as per Example 1, relative to SEQ ID NOs: 12685-12950. V, RS, RP, RB, SB, T, and CDS are as per Table 12. GC = gene characterisation; AA change = amino acid change of the variation, as applicable.

Table 14. Twenty-five selected variations (Zi to Z25; a subset of those in Table 13) considered of particular interest as per Example 1.

Table 15. Twenty-six selected variations (Vi to V26; a subset of those in Table 13) considered of particular interest as per Example 2.

Table 16. CDS transcripts differentially expressed between BPAX-A1 (MYMO) mutant X. dendrorhous strain and wild-type A. dendrorhous CBS 6938 as per Example 2.

Table 17. Differential expression analysis between BPAX-A1 (MYMO) mutant X. dendrorhous strain and wild-type A. dendrorhous CBS 6938 for selected CDS transcripts (Ti to T55) considered of particular interest.

Table 18. CDS sequences encoding proteins differentially expressed between BPAX-A1 (MYMO) mutant A dendrorhous strain and wild-type A dendrorhous CBS 6938 at Growth Phase

3, as per Example 3.

Table 19. CDS sequences encoding proteins differentially expressed between BPAX-A1 (MYMO) mutant A dendrorhous strain and wild-type A dendrorhous CBS 6938 at Growth Phase

4, as per Example 3.

TABLES 1-19 FOLLOW

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