YUZBASHEVA EVGENIYA (GB)
Claims 1. A recombinant microorganism producing at least one carotenoid, comprising and expressing: one or more copies of a nucleotide sequence encoding a fusion protein of: geranylgeranyl pyrophosphate synthase; or mutated farnesyl pyrophosphate synthase having geranylgeranyl pyrophosphate synthase activity; with phytoene synthase. 2. A composition for producing at least one carotenoid, comprising: a fusion protein of: geranylgeranyl pyrophosphate synthase; or mutated farnesyl pyrophosphate synthase having geranylgeranyl pyrophosphate synthase activity; with phytoene synthase. 3. The recombinant microorganism or composition of claim 1 or 2, wherein the geranylgeranyl pyrophosphate synthase comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, wherein the geranylgeranyl pyrophosphate synthase has retained geranylgeranyl pyrophosphate synthase activity. 4. The recombinant microorganism or composition of any one of claims 1-3, wherein the mutated farnesyl pyrophosphate synthase comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, wherein the mutated farnesyl pyrophosphate synthase has retained geranylgeranyl pyrophosphate synthase activity. 5. The recombinant microorganism or composition of any one of claims 1-4, wherein the phytoene synthase comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3, wherein the phytoene synthase has retained phytoene synthase activity. 6. The recombinant microorganism or composition of any one of claims 1-5, wherein the fusion protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 14-17. 7. The recombinant microorganism or composition of any one claims 1-5, further comprising a lycopene cyclase. 8. The recombinant microorganism or composition of claim 7, wherein the lycopene cyclase and phytoene synthase are a bifunctional lycopene cyclase-phytoene synthase. 9. The recombinant microorganism or composition of claim 8, wherein the bifunctional lycopene cyclase-phytoene synthase comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 4-5 or 33, wherein the bifunctional lycopene cyclase-phytoene synthase has retained bifunctional lycopene cyclase-phytoene synthase activity. 10. The recombinant microorganism or composition of claim 8 or 9, wherein the fusion protein is geranylgeranyl pyrophosphate synthase with bifunctional lycopene cyclase-phytoene synthase. 11. The recombinant microorganism or composition of claim 8 or 9, wherein the fusion protein is mutated farnesyl pyrophosphate synthase with bifunctional lycopene cyclase- phytoene synthase. 12. The recombinant microorganism or composition of claim 8 or 9, wherein the fusion protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 6-13 or 34-37. 13. The recombinant microorganism or composition of claims 1-12, further comprising: one or more copies of genes that encode at least one enzyme generating NADPH; or at least one enzyme generating NADPH; selected from the list of enzymes consisting of: NADP+-dependent malic enzyme; NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase; glucose-6-phosphate dehydrogenase; a NADP+-dependent formate dehydrogenase; and a NAD+/NADH kinase. 14. The recombinant microorganism or composition of claim 13, wherein the one or more copies of genes that encode at least one enzyme generating NADPH, or at least one enzyme generating NADPH, comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least one selected from the list of enzymes consisting of: NADP+-dependent malic enzyme of SEQ ID NO:18; NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase of SEQ ID NO:19; glucose-6- phosphate dehydrogenase of SEQ ID NOs:20-21; a NADP+-dependent formate dehydrogenase of SEQ ID NO:22; and a NAD+/NADH kinase of SEQ ID NO:23. 15. The recombinant microorganism or composition of any one of claims 1-14, wherein the at least one carotenoid is selected from the group consisting of: β-carotene, lycopene, lutein, phytoene, β-cryptoxanthin, zeaxanthin, violaxanthin, neoxanthin, fucoxanthin, canthaxanthin, astaxanthin and modifications thereof. 16. The recombinant microorganism or composition of any one of claims 1-15, further comprising at least one enzyme or functionally equivalent variant thereof selected from: a) HMGR; b) ERG12; c) CarRP; d) CarB; e) GGS1; f) HpBKT; g) HpCrtZ; h) GapC; i) Mce2; j) ERG20; k) any combination of (a)-(j). 17. The recombinant microorganism or composition of claim 16, wherein the at least one enzyme or functionally equivalent variant thereof comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: a) HMGR of SEQ ID NO:24 or 30; b) ERG12 of SEQ ID NO:25; c) CarRP of SEQ ID NO:4; d) CarB of SEQ ID NO:26; e) GGS1 of SEQ ID NO:27; f) HpBKT of SEQ ID NO:28; g) HpCrtZ of SEQ ID NO:29; h) GapC of SEQ ID NO:19; i) Mce2 of SEQ ID NO:18; j) ERG20 of SEQ ID NO: 38; k) any combination of (a)-(j). 18. The recombinant microorganism or composition of any one of claims 1-17, wherein the recombinant microorganism is selected from the group consisting of: bacteria, fungi, yeasts, algae, and archaea. 19. The recombinant microorganism or composition of any one of claims 1-18, wherein the recombinant microorganism is a yeast. 20. The recombinant microorganism or composition of claim 19, wherein the yeast is oleaginous. 21. The recombinant microorganism or composition of claim 20, wherein the yeast is selected from the genera Rhodosporidium, Rhodotorula, Yarrowia, Cryptococcus, Candida, Lipomyces and Trichosporon. 22. The recombinant microorganism or composition of claim 21, wherein said yeast is a Yarrowia lipolytica, a Lipomyces starkey, a Rhodosporidium toruloides, a Rhodotorula glutinis, a Trichosporon fermentans or a Cryptococcus curvatus. 23. A cell free method of producing at least one carotenoid, comprising subjecting the composition of any one of claims 1-22 to conditions to produce the at least one carotenoid. 24. A method for producing at least one carotenoid, comprising culturing the microorganism of any one of claims 1-22 under conditions to produce the at least one carotenoid. 25. A composition for producing at least one carotenoid, comprising the recombinant microorganism of any one of claims 1-22. 26. A fusion protein of geranylgeranyl pyrophosphate synthase or mutated farnesyl pyrophosphate synthase with bifunctional lycopene cyclase-phytoene synthase; or geranylgeranyl pyrophosphate synthase or mutated farnesyl pyrophosphate synthase with phytoene synthase. 27. The fusion protein of claim 26, wherein the fusion protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 6-13, 14-17 or 34-37. 28. A nucleic acid construct, comprising one or more copies of a nucleotide sequence encoding the fusion protein of claim 27. 29. At least one carotenoid produced using the method of claim 23 or 24. |
[0060] In certain embodiments, the mutated farnesyl pyrophosphate synthase comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2, wherein the mutated farnesyl pyrophosphate synthase has retained geranylgeranyl pyrophosphate synthase activity. In certain embodiments, the mutated farnesyl pyrophosphate synthase comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:2, wherein the mutated farnesyl pyrophosphate synthase has retained geranylgeranyl pyrophosphate synthase activity. The sequence for Yarrowia lipolytica mutated farnesyl pyrophosphate synthase YlERG20 F88C is shown herein, however the skilled person would understand that homologs may also be suitable. Examples of YlERG20 F88C homologs are shown in Table 2. For the SEQ IDS described herein, mutations are shown with a solid underline. The F88C mutation provides farnesyl pyrophosphate with the ability to produce geranylgeranyl pyrophosphate. T ABLE 2: Y L ERG20 F88C H OMOLOGS [0061] In certain embodiments, the phytoene synthase comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:3, wherein the phytoene synthase has retained phytoene synthase activity. In certain embodiments, the phytoene synthase comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:3, wherein the phytoene synthase has retained phytoene synthase activity. The sequence for Synechococcus sp. phytoene synthase сrtB is shown herein, however the skilled person would understand that homologs may also be suitable. Examples of сrtB homologs are shown in Table 3. TABLE 3: СRTB HOMOLOGS [0062] In certain embodiments, the recombinant microorganism or composition further comprises a lycopene cyclase. In certain embodiments, the phytoene synthase and the lycopene cyclase are separate. In alternative embodiments, the lycopene cyclase and phytoene synthase are a bifunctional lycopene cyclase-phytoene synthase. In certain embodiments, the bifunctional lycopene cyclase-phytoene synthase may be produced together with either or both of a separate phytoene synthase or lycopene cyclase. Lycopene cyclase (aka lycopene beta- cyclase) is useful for the conversion of acyclic lycopene to bicyclic beta-carotene. As the skilled person would understand, there are four known families of lycopene cyclases: CrtY, CrtL (beta-ionone end group producing), CrtL (eta-ionone end group producing) and CrtL (capsanthin/capsorubin synthase). The appropriate family may be chosen depending on the desired carotenoid. [0063] In certain embodiments, the bifunctional lycopene cyclase-phytoene synthase comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOS:4 & 5, wherein the bifunctional lycopene cyclase-phytoene synthase has retained bifunctional lycopene cyclase-phytoene synthase activity. In certain embodiments, the bifunctional lycopene cyclase-phytoene synthase comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 4, 5 & 33, wherein the bifunctional lycopene cyclase- phytoene synthase has retained bifunctional lycopene cyclase-phytoene synthase activity. The sequences for Mucor circinelloides bifunctional lycopene cyclase-phytoene synthase CarRP (SEQ ID NO: 4), Xanthophyllomyces dendrorhous bifunctional lycopene cyclase-phytoene synthase crtYB (SEQ ID NO: 5), and Blakeslea trispora CarRA (SEQ ID NO: 33) are shown herein, however the skilled person would understand that homologs may also be suitable. Examples of CarRP homologs are shown in Table 4 and examples of CarRA homologs are shown in Table 5.
TABLE 4: CARRP HOMOLOGS T ABLE 5: CAR RA H OMOLOGS [0064] In certain embodiments, the fusion protein is geranylgeranyl pyrophosphate synthase with bifunctional lycopene cyclase-phytoene synthase. In certain embodiments, the fusion protein is mutated farnesyl pyrophosphate synthase with bifunctional lycopene cyclase- phytoene synthase. In certain embodiments, the fusion protein is geranylgeranyl pyrophosphate synthase with phytoene synthase. In certain embodiments, the fusion protein is mutated farnesyl pyrophosphate synthase with phytoene synthase. In certain embodiments, the order of the proteins in the fusion protein may be as shown above, or reversed. For example, mutated farnesyl diphosphate synthase fused by its N-terminus to the C-terminus of bifunctional lycopene cyclase-phytoene synthase, or mutated farnesyl diphosphate synthase fused by its C- terminus to the N-terminus of bifunctional lycopene cyclase-phytoene synthase. [0065] In certain embodiments, the fusion protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 6-17 or 34-37. In certain embodiments, the fusion comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a subset of any one of SEQ ID NOS: 6-17 or 34-37, such as SEQ ID NOS: 14-17, SEQ ID NOS: 6-9, SEQ ID NOS: 10-13, SEQ ID NOS: 34-37, or any combination thereof. [0066] The amino acid sequence for the fusion of CarRP-GS-YlERG20 F88C is shown in SEQ ID NO:6. The amino acid sequence for the fusion of YlERG20 F88C -GS-CarRP is shown in SEQ ID NO:7. The amino acid sequence for the fusion of GGPPs7-GS-CarRP is shown in SEQ ID NO:8. The amino acid sequence for the fusion of CarRP-GS-GGPPs7 is shown in SEQ ID NO:9. The amino acid sequence for the fusion of YlERG20 F88C -GS-crtYB is shown in SEQ ID NO:10. The amino acid sequence for the fusion of crtYB-GS-YlERG20 F88C is shown in SEQ ID NO:11. The amino acid sequence for the fusion of GGPPs7-GS-crtYB is shown in SEQ ID NO:12. The amino acid sequence for the fusion of crtYB-GS-GGPPs7 is shown in SEQ ID NO:13. The amino acid sequence for the fusion of сrtB-GS-YlERG20 F88C is shown in SEQ ID NO:14. The amino acid sequence for the fusion of YlERG20 F88C -GS-crtB is shown in SEQ ID NO:15. The amino acid sequence for the fusion of GGPPs7-GS-crtB is shown in SEQ ID NO:16. The amino acid sequence for the fusion of crtB-GS-GGPPs7 is shown in SEQ ID NO:17. The amino acid sequence for the fusion of CarRA-GS-YlERG20F88C is shown in SEQ ID NO:34. The amino acid sequence for the fusion of YlERG20F88C-GS-CarRA is shown in SEQ ID NO:35. The amino acid sequence for the fusion of GGPPs7-GS-CarRA is shown in SEQ ID NO:36. The amino acid sequence for the fusion of CarRA-GS-GGPPs7 is shown in SEQ ID NO:37. [0067] The enzymes in the fusion protein may be joined by any suitable linker. The role of the linker is to join the two functional domains while allowing each to function. Linkers may be flexible, rigid, or in vivo cleavable. Flexible and rigid linkers are particularly suitable for joining the enzymes of the fusion proteins disclosed herein. Preferable amino acids for use in the linker include, eg, polar uncharged or charged residues. Examples may include arginine (Arg), threonine (Thr), serine (Ser), phenylalanine (Phe), proline (Pro), glutamic acid (Glu), glycine (Gly), aspartic acid (Asp), lysine (Lys), glutamine (Gln), asparagine (Asn), and alanine (Ala). In certain embodiments, the linker comprises GGGGS, (GGGGS) n (n=1, 2, 3 4), eg, (GGGGS) 3 , GSAGSAAGSGEF, (Gly) 8 , (Gly) 6 , (EAAAK) 3 , (EAAAK) n (n=1-3), A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro)n (10 – 34 aa), LE, KESGSVSSEQLAQFRSLD or EGKSSGSGSESKST. In particular embodiments, the linker is a GlySer linker, comprising (GGGGS) n (n=1, 2, 3 4), such as GGGGS, (GGGGS) 2 or (GGGGS) 3 .
[0072] SEQ ID NO:11 crtYB-GS-YlERG20 F88C
[0082] In certain embodiments, the recombinant microorganism or composition further comprises at least one enzyme or one or more copies of genes that encode at least one enzyme generating NADPH selected from the list of enzymes consisting of: NADP+-dependent malic enzyme; NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase; glucose-6- phosphate dehydrogenase; a NADP+-dependent formate dehydrogenase; and a NAD+/NADH kinase. Increased generation of NADPH increases the pool of NADPH available for use as a cofactor by the enzymes involved in carotenoid precursors biosynthesis, as would be understood by skilled person, such as mevalonate synthesized from HMG-CoA and NADPH. Additionally, β-carotene hydroxylase converting β-carotene to zeaxanthin or canthaxanthin to astaxanthin via introduction of an oxygen atom from molecular oxygen in their substrates requires an electron donor to reduce the second oxygen atom to water. The final donor of electrons is NADPH. In most cases electrons are transferred from NADPH to ferredoxin by a ferredoxin oxidoreductase. β-carotene hydroxylase, in turn, uses a reduced ferredoxin cluster. Finally, β-carotene ketolase converting from β-carotene to canthaxanthin or zeaxanthin to astaxanthin, also uses reduced electron donors for oxidation reaction, however, the nature of electron donors remains unclear. [0083] Examples of sequences for each of these enzymes are shown below; however the skilled person would understand that homologs or functionally equivalent variants thereof may also be suitable. In certain embodiments, the one or more copies of genes that encode at least one enzyme generating NADPH, or at least one enzyme generating NADPH, comprises a polypeptide or a polynucleotide encoding a polypeptide, having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least one selected from the list of enzymes consisting of: NADP+-dependent malic enzyme Mce2 of SEQ ID NO:18; NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase GapC of SEQ ID NO:19; glucose-6-phosphate dehydrogenase from Yarrowia lipolytica and Saccharomyces cerevisiae of SEQ ID NO:20 and SEQ ID NO: 21, respectively; a Burkholderia stabilis NADP+-dependent formate dehydrogenase of SEQ ID NO:22, and a Saccharomyces cerevisiae NAD+/NADH kinase of SEQ ID NO:23. [0084] Examples of Mucor circinelloides NADP+-dependent malic enzyme Mce2 homologs are shown in Table 6. Examples of Clostridium acetobutylicum NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase GapC homologs are shown in Table 7. Examples of Yarrowia lipolytica glucose-6-phosphate dehydrogenase homologs are shown in Table 8. Examples of Saccharomyces cerevisiae glucose-6-phosphate dehydrogenase homologs are shown in Table 9. Examples of Burkholderia stabilis NADP+-dependent formate dehydrogenase homologs are shown in Table 10. Examples of Saccharomyces cerevisiae NAD+/NADH kinase homologs are shown in Table 11. As mentioned elsewhere herein, homologs or variants can be identified by the person skilled in the art by conducting, eg, BLAST searches using the disclosed sequences, or literature searches using gene, enzyme, substrate or product names, and those variants can be tested using the methods of the Examples disclosed herein. T ABLE 6: M CE 2 H OMOLOGS
TABLE 7: GAPC HOMOLOGS TABLE 8: YARROWIA LIPOLYTICA GLUCOSE-6-PHOSPHATE DEHYDROGENASE HOMOLOGS TABLE 9: SACCHAROMYCES CEREVISIAE GLUCOSE-6-PHOSPHATE DEHYDROGENASE HOMOLOGS
T ABLE 10: B URKHOLDERIA STABILIS NADP+ DEPENDENT FORMATE DEHYDROGENASE H OMOLOGS TABLE 11: Saccharomyces cerevisiae NAD+/NADH kinase Homologs [0091] In certain embodiments, the recombinant microorganism or composition further comprises at least one enzyme or functionally equivalent variant thereof, or at least one polynucleotide encoding the at least one enzyme or functionally equivalent variant thereof, selected from: a) 3-hydroxy-3-methylglutaryl coenzyme A reductase; b) mevalonate kinase; c) bifunctional lycopene cyclase-phytoene synthase; d) phytoene dehydrogenase; e) geranylgeranyl pyrophosphate synthase; f) β-carotene ketolase; g) β-carotene hydroxylase; h) NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase; i) NADP+-dependent malic enzyme; j) farnesyl pyrophosphate synthase; or any combination of (a)-(j). [0092] Examples of polypeptide sequences for each of these enzymes are shown below; however the skilled person would understand that homologs or functionally equivalent variants thereof may also be suitable. Those exemplified herein include: a) 3-hydroxy-3-methylglutaryl coenzyme A reductase HMGR of SEQ ID NO:24 or 30; b) mevalonate kinase ERG12 of SEQ ID NO:25; c) bifunctional lycopene cyclase-phytoene synthase CarRP of SEQ ID NO:4; d) phytoene dehydrogenase CarB of SEQ ID NO:26; e) geranylgeranyl pyrophosphate synthase GGS1 of SEQ ID NO:27; f) β-carotene ketolase HpBKT of SEQ ID NO:28; g) β-carotene hydroxylase HpCrtZ of SEQ ID NO:29; h) NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase GapC of SEQ ID NO:19; i) NADP+-dependent malic enzyme is Mce2 of SEQ ID NO:18; and j) farnesyl pyrophosphate synthase ERG20 of SEQ ID NO: 38. [0093] Examples of HMGR homologs are shown in Table 12. Examples of ERG12 homologs are shown in Table 13. Examples of CarRP homologs are shown in Table 4. Examples of CarB homologs are shown in Table 14. Examples of GGS1 homologs are shown in Table 15. Examples of HpBKT homologs are shown in Table 16. Examples of HpCrtZ homologs are shown in Table 17. Examples of GapC homologs are shown in Table 7. Examples of Mce2 homologs are shown in Table 6. Examples of ERG20 homologs are shown in Table 18. [0094] High levels of FPP production are dependent on adequate mevalonate production. Hydroxymethylglutaryl-CoA reductase (HMGR) catalyses the production of mevalonate from HMG-CoA and NADPH. HMGR is a rate limiting step in the FPP pathway in yeast. Accordingly, overexpressing HMGR may increase flux through the pathway and increase the production of FPP. HMGR is a FPP pathway gene. Other FPP pathway genes include those genes that are involved in the FPP pathway, the products of which either directly produce FPP or produce intermediates in the FPP pathway, for example, ERG10, ERG13, ERG12, ERG8, ERG19, IDI1 or ERG20, The HMGR1 sequence from Y. lipolytica consists of 999 amino acids (aa) (SEQ ID NO:24), of which the first 500 aa harbor multiple transmembrane domains and a response element for signal regulation. The remaining 499 C-terminal residues contain a catalytic domain and an NADPH-binding region. Truncated HMGR1(tHmgR) has been generated by deleting the N-terminal 500 aa (Gao et al.2017). tHMGR is able to avoid self- degradation mediated by its N-terminal domain and is thus stabilized in the cytoplasm, which increases flux through the FPP pathway. The N-terminal 500 aa are shown with a dashed underline in SEQ ID NO:24. The N-terminal 500 aa are deleted in SEQ ID NO:30. [0095] High levels of carotenoid production are dependent on adequate production of intermediates at particular steps in the biosynthetic pathway. Accordingly, expression or overexpression of, eg, bifunctional lycopene cyclase-phytoene synthase (eg CarRP) may increase flux through the pathway and increase production of phytoene from GGPP and increase production of β-carotene from lycopene. Expression or overexpression of phytoene dehydrogenase (eg CarB) increases flux through the pathway and increases production of lycopene from phytoene. Expression or overexpression of geranylgeranyl pyrophosphate synthase (eg GGS1) increases flux through the pathway and increases production of GGPP from FPP. Expression or overexpression of β-carotene ketolase (eg HpBKT) increases flux through the pathway and increases production of canthaxanthin from β-carotene, or astaxanthin from zeaxanthin. Expression or overexpression of β-carotene hydroxylase (eg HpCrtZ) increases flux through the pathway and increases production of astaxanthin from canthaxanthin or zeaxanthin from β-carotene. As mentioned elsewhere herein, expression or overexpression of NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (eg GapC) or NADP+- dependent malic enzyme (eg Mce2) increases flux through the pathway and increases production of NADPH, which increases the pool of NADPH available for use as a cofactor by the enzymes involved in carotenoid biosynthesis. [0096] The skilled person could readily choose the particular combinations of these enzymes or polynucleotides encoding the enzymes, depending on the desired carotenoid. [0097] In certain embodiments, the at least one enzyme or functionally equivalent variant thereof comprises a polynucleotide encoding a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: HMGR of SEQ ID NO: 24 or 30; ERG12 of SEQ ID NO:25; CarRP of SEQ ID NO:4; CarB of SEQ ID NO:26; GGS1 of SEQ ID NO:27; HpBKT of SEQ ID NO:28; HpCrtZ of SEQ ID NO:29; GapC of SEQ ID NO:19; Mce2 of SEQ ID NO:18; ERG20 of SEQ ID NO:38; any combination thereof. [0098] In certain embodiments, the at least one enzyme or functionally equivalent variant thereof comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to: HMGR of SEQ ID NO: 24 or 30; ERG12 of SEQ ID NO:25; CarRP of SEQ ID NO:4; CarB of SEQ ID NO:26; GGS1 of SEQ ID NO:27; HpBKT of SEQ ID NO:28; HpCrtZ of SEQ ID NO:29; GapC of SEQ ID NO:19; Mce2 of SEQ ID NO:18; ERG20 of SEQ ID NO: 38; any combination of thereof. [0099] Genes producing at least one enzyme or functionally equivalent variant may be expressed using, for example, a constitutive TEF intron promoter, EXP1 promoter, GAPDH promoter or other native or hybrid promoters (Wong et al.2017, Blazeck et al.2011) and LIP2 terminator, TEF terminator, XPR2 terminator or other native or synthesized short terminators (Curran et al.2015, Larroude et al.2019). [00100] In certain embodiments, specific combinations of the disclosed enzymes are used. For example, in certain embodiments, the combination comprises CarB together with either CarRP-GSsh-YlERG20( F88C ) or/and CarRP-GSsh-GGPPs7. In certain embodiments, the combination comprises 2 copies of CarB together with either CarRP-GSsh-YlERG20( F88C ) or/and CarRP-GSsh-GGPPs7. In certain embodiments, the combination comprises one or more copies of CarB, one or more copies of HpBKT, one or more copies of HpCrtZ together with either CarRP-GSsh-YlERG20( F88C ) or/and CarRP-GSsh-GGPPs7. In certain embodiments, the combination comprises HMGR, ERG12, ERG20, CarRP, one or more copies of CarB, GGS1 together with either CarRP-GSsh-YlERG20( F88C ) or/and CarRP-GSsh-GGPPs7. In certain embodiments, the combination comprises HMGR, ERG12, ERG20, CarRP, CarB, GGS1, one or more copies of HpBKT, one or more copies of HpCrtZ together with either CarRP-GSsh- YlERG20( F88C ) or/and CarRP-GSsh-GGPPs7. In certain embodiments, the combination comprises one or two copies of CarB, CarRP-GSsh-YlERG20( F88C ) or/and CarRP-GSsh- GGPPs7, together with either GapC or Mce2. In certain embodiments, the combination comprises HMGR, ERG12, ERG20, CarRP, one or more copies of CarB, GGS1, and fusion CarRP-GSsh-GGPPs7 together with either GapC or Mce2. In certain embodiments, the combination comprises one or more copies of CarB, one or more copies of HpBKT, one or more copies of HpCrtZ, CarRP-GSsh-YlERG20( F88C ) or/and CarRP-GSsh-GGPPs7 together with either GapC or Mce2. In certain embodiments, the combination comprises HMGR, ERG12, ERG20, CarRP, one or more copies of CarB, GGS1, and fusion CarRP-GSsh- GGPPs7, one or more copies of HpBKT, one or more copies of HpCrtZ, and fusion CarRP- GSsh-GGPPs7 together with either GapC or Mce2. [00101] In certain embodiments, additional enzymes may be added to those described above to, for example, produce other carotenoids which may be derived from those produced by the enzymes described above. TABLE 12: HMGR HOMOLOGS TABLE 13: ERG12 HOMOLOGS TABLE 14: CARB HOMOLOGS T ABLE 15: Y L GGS1 H OMOLOGS
TABLE 16: HPBKT HOMOLOGS TABLE 17: HPCRTZ HOMOLOGS T ABLE 18: ERG20 H OMOLOGS [00104] The production of fatty acids and fats in yeast may be increased by expressing rate limiting genes in the lipid biosynthesis pathway. Y. lipolytica naturally produces Acetyl- CoA. The overexpression of ACC1 increases the amount of Malonyl-CoA, which is the first step in fatty acid production. In certain embodiments, the recombinant microorganism comprises one or more genetic modifications that may result in increased production of fatty acids or fats. In certain embodiments, genetic modifications comprise one or more copies of a nucleotide sequence encoding Acetyl-CoA carboxylase (ACC1) or Diacylglyceride acyl- transferase (DGA1). The sequences for the native Y. lipolytica genes are shown herein, however the skilled person would understand that homologs may also be suitable. Examples of ACC1 homologs as shown in Table 18. Examples of DGA1 homologs as shown in Table 19. In certain embodiments, ACC1 comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:31. In certain embodiments, ACC1 comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:31. In certain embodiments, DGA1 comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:32. In certain embodiments, DGA1 comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:32. [00105] ACC1 and DGA1 may be overexpressed in yeast by adding extra copies of the genes driven by native or stronger promoters. Alternatively, native promoters may be substituted by stronger promoters such as TEFin, hp4d, hp8d and others, as would be appreciated by the person skilled in the art. The overexpression of ACC1 and DGA1 may be determined by quantitative PCR, Microarrays, or next generation sequencing technologies, such as RNA-seq. Alternatively, the product of increased enzyme levels will be increased production of fatty acids. Fatty acid production may be determined using chemical titration, thermometric titration, measurement of metal-fatty acid complexes using spectrophotometry, enzymatic methods or using a fatty acid binding protein. [00106] Variants of the fatty acid and fat producing proteins, such as ACC1 retain the ability to produce malonyl-CoA from acetyl-CoA plus bicarbonate. For example, a variant of a fatty acid and fat producing protein, such as ACC1, must retain the ability to produce malonyl-CoA from acetyl-CoA plus bicarbonate with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% efficacy compared to the original sequence. In preferred embodiments, a variant of a fatty acid and fat producing protein, such as ACC1, has improved activity over the sequence from which it is derived in that the improved variant common carotenoid protein has more than 110%, 120%, 130%, 140%, or and 150% improved activity in producing malonyl-CoA from acetyl-CoA plus bicarbonate, as compared to the sequence from which the improved variant is derived.
TABLE 18: ACC1 HOMOLOGS TABLE 19: DGA1 HOMOLOGS RECOMBINANT MICROORGANISMS [00107] As described above, the microorganism of the present disclosure, the microorganism contained in the composition of the present disclosure or employed in a method of the present disclosure is a recombinant microorganism. Thus, in certain embodiments, the recombinant microorganism has been genetically modified to have an increased activity of the described enzymes or increased expression of the described enzymes for the production of at least one carotenoid. This can be achieved e.g. by transforming the microorganism with a nucleic acid encoding a corresponding enzyme. In certain embodiments, the nucleic acid molecule introduced into the microorganism is a nucleic acid molecule which is heterologous with respect to the microorganism, i.e. it does not naturally occur in said microorganism. In certain embodiments, a native gene is modified to produce the enzyme, to increase activity of the enzyme and/or increase expression of the enzyme. As would be understood by the skilled person, this can be performed using technology described elsewhere herein, such as standard molecular biology techniques or CRISPR-Cas9 genome editing technology. [00108] The term “microorganism” in the context of the present disclosure refers to bacteria, as well as to fungi, such as yeasts, and also to algae and archaea. Accordingly, in certain embodiments, the recombinant microorganism is selected from: bacteria, fungi, yeasts, algae, and archaea. In particular embodiments, the recombinant microorganism is a yeast. In certain embodiments, wherein the yeast is oleaginous. [00109] In certain embodiments, the yeast is selected from the genera Rhodosporidium, Rhodotorula, Yarrowia, Cryptococcus, Candida, Lipomyces and Trichosporon. In certain embodiments, the yeast is a Yarrowia lipolytica, a Lipomyces starkey, a Rhodosporidium toruloides, a Rhodotorula glutinis, a Trichosporon fermentans or a Cryptococcus curvatus. [00110] It is also conceivable to use a combination of microorganisms in any of the aspects of the present disclosure, wherein different microorganisms express different enzymes as described above. [00111] In the context of the present disclosure, an “increased activity” means that the expression and/or the activity of an enzyme in the recombinant microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferred at least 90% or 100% higher than in the corresponding non-modified microorganism. In even more preferred embodiments, the increase in expression and/or activity may be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments the expression is at least 10-fold, more preferably at least 100-fold and even more preferred at least 1000-fold higher than in the corresponding non-modified microorganism. [00112] The term “increased” expression/activity also covers the situation in which the corresponding non-modified microorganism does not express a corresponding enzyme so that the corresponding expression/activity in the non-modified microorganism is zero. Preferably, the concentration of the overexpressed enzyme is at least 5%, 10%, 20%, 30%, or 40% of the total host cell protein. Additionally, as would be appreciated by the person skilled in the art, increased expression of a gene may provide increased the activity of the gene product. In certain embodiments, overexpression of a gene can increase the activity of the gene product by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 95%, or about 200%. [00113] Methods for measuring the level of expression of a given protein in a cell are well known to the person skilled in the art. In one embodiment, the measurement of the level of expression is done by measuring the amount of the corresponding protein. Corresponding methods are well known to the person skilled in the art and include Western Blot, ELISA etc. In another embodiment the measurement of the level of expression is done by measuring the amount of the corresponding RNA. Corresponding methods are well known to the person skilled in the art and include, e.g., Northern Blot. [00114] In addition, it is possible to insert different mutations into the polynucleotides by methods usual in molecular biology (see for instance Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA), leading to the synthesis of polypeptides possibly having modified biological properties. The introduction of point mutations is conceivable at positions at which a modification of the amino acid sequence for instance influences the biological activity or the regulation of the polypeptide. Similarly, CRISPR-Cas9 genome editing technology can be used to modify the disclosed sequences to produce enzyme variants. [00115] The transformation of the host cell with a polynucleotide or vector as described above can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. The host cell is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc. [00116] The nucleotide sequences encoding the disclosed enzymes may be under the control of any suitable promoter. Many native promoters are available, for example, for Y. lipolytica, native promoters are available from the genes for translational elongation factor EF−1 alpha, acyl-CoA: diacylglycerol acyltransferase, acetyl-CoA-carboxylase 1, ATP citrate lyase 2, fatty acid synthase subunit beta, fatty acid synthase subunit alpha, isocitrate lyase 1, POX4 fatty-acyl coenzyme A oxidase, ZWF1 glucose-6-phosphate dehydrogenase, cytosolic NADP-specific isocitrate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, the TEF intron promoter or native promoter (Wong et al.2017), or the alcohol dehydrogenase II promoter of Y. lipolytica. Any suitable terminator may be used. Short synthetic terminators are particularly suitable and are readily available, see for example, MacPherson et al. CS Synth. Biol.6(1)130–138, 2017. Terminators could also include yeast translational elongation factor EF−1 alpha (TEF), iso-2-cytochrome c (CYC) gene, or others. [00117] Methods of detecting increased production of carotenoids may be determined using high-performance liquid chromatography (HPLC) or Liquid chromatography–mass spectrometry (LC/MS). [00118] The disclosed recombinant microorganism may produce various carotenoids from a simple nutrient source, for example, where the main carbon source available is a sugar (glucose, galactose, fructose, sucrose, honey, molasses, raw sugar, etc.) or glycerol and may optionally include amino acid sources, such as peptone and/or yeast extract. The benefit of this method is that once the yeast is engineered, the production of the carotenoid is low cost and reliable, only a specific carotenoid is produced or a subset is produced, depending on the organism and the genetic manipulation. The purification of the carotenoids is straightforward as they are readily extracted by solvent-based techniques or ultrasound under pressure. The process is a sustainable process which is more environmentally friendly than synthetic production. [00119] In the past, there have been multiple attempts to produce carotenoids in yeasts. At present, no one has been able reach a reasonable price for production due to extremely low yield. The present inventors have identified how the yield can be increased. [00120] In certain embodiments, the biosynthetic pathway shown in Figure 1 is produced in yeast, such as for example, Y. lipolytica. [00121] Additionally and as described below, it is also proposed (1) add additional genes from the carotenoid production pathway in combination with genes from pathways that produce carotenoid intermediates, such as for example genes from the mevalonate production pathway; (2) increase production of GGPP by, for example, genetically mutating ERG20 and/or by using equivalent genes from alternative pathways; (3) add additional genes encoding enzymes generating NADPH. CELL-FREE PRODUCTION [00122] In a third aspect, there is provided a cell-free (eg in vitro) method for producing at least one carotenoid, comprising subjecting the composition described herein to conditions to produce the at least one carotenoid. [00123] An in vitro reaction is understood to be a reaction in which no cells are employed, i.e. an acellular reaction. Thus, in vitro preferably means in a cell-free system. The term “in vitro” in certain embodiments means in the presence of isolated enzymes (or enzyme systems optionally comprising possibly required cofactors). In particular embodiments, the enzymes employed in the method are used in purified form. [00124] The “conditions” to produce the at least one carotenoid means carrying out the method such that the enzymes are incubated under conditions (buffer, temperature, cosubstrates, cofactors etc.) allowing the enzymes to be active and the enzymatic conversion to occur. The reaction is allowed to proceed for a time sufficient to produce the respective product. The production of the respective products can be measured by methods known in the art, such as gas chromatography possibly linked to mass spectrometry detection. [00125] The enzymes described herein may be in any suitable form allowing the enzymatic reaction to take place. They may be purified or partially purified or in the form of crude cellular extracts or partially purified extracts. It is also possible that the enzymes are immobilized on a suitable carrier. METHODS OF PRODUCTION [00126] In a fourth aspect, there is provided a method for producing at least one carotenoid, comprising culturing the microorganism described herein under conditions to produce the at least one carotenoid. In a fifth aspect, there is provided a composition for producing at least one carotenoid, comprising the recombinant microorganism described herein. As would be appreciated by the skilled person, the composition could be used to produce one or more carotenoids by the culturing the composition (and hence the microorganism as described herein) under conditions to produce the at least one carotenoid. In certain embodiments of these aspects, the conditions comprise contacting the microorganism with a carbohydrate source under conditions and for a time sufficient to produce the at least one carotenoid. [00127] Specifically, examples of the conditions for producing the at least one carotenoid include a batch process and a fed batch or repeated fed batch process in a continuous manner, but are not limited thereto. Carbon sources that may be used for producing the at least one carotenoid may include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, xylose and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, chicken fat and beef tallow; fatty acids such as palmitic acid, stearic acid, oleic acid and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as gluconic acid, acetic acid, malic acid and pyruvic acid, but these are not limited thereto. These substances may be used alone or in a mixture. Nitrogen sources that may be used in the present disclosure may include peptone, yeast extract, meat extract, malt extract, corn steep liquor, defatted soybean cake, and urea or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate, but these are not limited thereto. These nitrogen sources may also be used alone or in a mixture. Phosphorus sources that may be used in the present disclosure may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or corresponding sodium- containing salts, but these are not limited thereto. In addition, the culture medium may contain a metal salt such as calcium chloride, zinc chloride, magnesium sulfate, iron sulfate, manganese sulfate, copper sulfate, or sodium molybdate, which may be required for the growth. Lastly, in addition to the above-described substances, essential growth factors such as amino acids and vitamins may be used. Such a variety of culture methods is disclosed, for example, in the literature ("Biochemical Engineering" by James M. Lee, Prentice-Hall International Editions, pp 138-176). [00128] Basic compounds such as sodium hydroxide, potassium hydroxide, or ammonia, or acidic compounds such as phosphoric acid or sulfuric acid may be added to the culture medium in a suitable manner to adjust the pH of the culture medium. In addition, an anti- foaming agent such as fatty acid polyglycol ester may be used to suppress the formation of bubbles. In certain embodiments, the culture medium is maintained in an aerobic state, accordingly, oxygen or oxygen-containing gas (e.g., air) may be injected into the culture medium. The temperature of the culture medium may be usually 20°C to 35°C, preferably 25°C to 32°C, but may be changed depending on conditions. The culture may be continued until the maximum amount of a desired the carotenoid is produced, and it may generally be achieved within 5 hours to 160 hours. The at least one carotenoid may be released into the culture medium or contained in the recombinant microorganisms. [00129] Methods of the present disclosure for producing the at least one carotenoid may include a step of recovering the at least one carotenoid from the microorganism or the medium. Methods known in the art, such as centrifugation, filtration, anion-exchange chromatography, crystallization, HPLC, etc., may be used for the method for recovering the at least one carotenoid from the microorganism or the culture, but the method is not limited thereto. The step of recovering may include a purification process. Specifically, following an overnight culture, 1L cultures are pelleted by centrifugation, resuspended, washed in PBS and pelleted. The cells are lysed by either chemical or mechanical methods or a combination of methods. Mechanical methods can include a French Press or glass bead milling or other standard methods. Chemical methods can include enzymatic cell lysis, solvent cell lysis, or detergent based cell lysis. A liquid-liquid extraction of the at least one carotenoid is performed using the appropriate chemical solvent in which the at least one carotenoid is highly soluble and the solvent is not miscible in water. Examples include hexane, ethyl acetate, and cyclohexane, preferably solvents with straight or branched alkane chains (C5-C8) or mixtures thereof. [00130] In certain embodiments, the at least one carotenoid is selected from the group consisting of: β-carotene, lycopene, lutein, phytoene, β-cryptoxanthin, zeaxanthin, violaxanthin, neoxanthin, fucoxanthin, canthaxanthin, astaxanthin and modifications thereof. As such, the at least one carotenoid could be a single carotenoid, such as β-carotene only, or could be two, three or more different carotenoids. Modifications may include, eg, modifications to increase polarity, such as the addition of a hydroxyl group, an acidic group or oxidation of a methyl group to the corresponding carboxylic acidic function, or modifications of other functional groups, such as ketones, or substituent additions, such as the addition of sugars (eg glycosylation), or fatty acids (esterification). [00131] The production of at least one carotenoid may be determined using a variety of methods as described herein. An example protocol for analysing a carotenoid is as follows: 1. Add 0.3 mL of 0.5–0.75 mm acid-washed glass beads to 10 mg of lyophilized cells followed by the addition of 0.5 mL of acetate supplemented with 0.01% 3,5-di-tert-4- butylhydroxytoluene (BHT). 2. Disrupt cells using a Precellys R 24 homogenizer (Bertin Corp., Montigny-le- Bretonneux, France) in four cycles of 5500 rpm for 30 s. 3. Place the tubes on ice between each lysis cycle for 1 min. 4. Precipitate the cells by centrifugation and collect acetate fraction. 5. Repeat extraction two times 6. HPLC Method a. Instrument Waters, MA, USA equipped with a NOVA-PAK C18150 mm×3.9 mm column. b. The column oven temperature 30° C с. Theflow rate 1.0 mL/min d. Detection at 450nm for β-carotene and echinenone, at 470nm for astaxanthin and canthaxanthin [00132] In a sixth aspect, there is provided a fusion protein of geranylgeranyl pyrophosphate synthase or mutated farnesyl pyrophosphate synthase with bifunctional lycopene cyclase-phytoene synthase; or geranylgeranyl pyrophosphate synthase or mutated farnesyl pyrophosphate synthase with phytoene synthase. [00133] In certain embodiments, the fusion protein comprises a polynucleotide encoding a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 6-13, 14-17 or 34-37. In certain embodiments, the fusion protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 6-13, 14-17 or 34-37. [00134] In a seventh aspect, there is provided a nucleic acid construct, comprising one or more copies of a polynucleotide sequence encoding a fusion protein of the sixth aspect, such as a fusion protein comprising a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 6-13, or 34-37. [00135] In an eighth aspect, there is provided at least one carotenoid produced using the methods described herein. In certain embodiments, the at least one carotenoid is selected from the group consisting of: β-carotene, lycopene, lutein, phytoene, β-cryptoxanthin, zeaxanthin, violaxanthin, neoxanthin, fucoxanthin, canthaxanthin, astaxanthin and modifications thereof. EXAMPLES Example 1: Engineering platform strains Yarrowia lipolytica synthesizing carotenoids [00136] Y. lipolytica expression plasmids comprise one or two transcriptional units (promoter-gene-terminator), homologous flanks for integration into precise locus in genome, and bacteria replicative backbone. Fragments for construction of these plasmids were synthesized by Twist Bioscience or amplified from Y. lipolytica genomic DNA. Plasmids were constructed by Gibson Assembly, Golden gate assembly, ligation or sequence-and ligation- independent cloning (SLIC). Genomic DNA isolation from bacteria (E. coli) and yeast (Y. lipolytica) were performed using Wizard Genomic DNA purification kit according to manufacturer’s protocol (Promega, USA). Synthetic genes were codon-optimized using GeneGenie or Genscript (USA) and assembled from gene fragments purchased from TwistBioscience. All the engineered Y. lipolytica strains were constructed by integration of the corresponding expression cassettes released from the plasmids by treatment with endonuclease restriction. All gene expression cassettes were constructed using a TEF intron promoter or XPR2 promoter or GAPDH promoter and synthesized short terminator or TEF promoter or XPR2 promoter or CYC1 promoter. [00137] E. coli minipreps were performed using the Zyppy Plasmid Miniprep Kit (Zymo Research Corporation). Transformation of E. coli strains was performed using Mix & Go Competent Cells (Zymo research, USA). Transformation of Y. lipolytica was performed using LiOAc method. Briefly, Y. lipolytica strains were grown overnight at 30°C on YPD agar medium containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 20 g/L of agar. For each transformation a full loop of cells were resuspended in 100 uL of sterile water in a sterile microcentrifuge tube, spun down and resuspended in 100 uL of transformation mixture (90μL 50% PEG, 5μL 2M LiAc pH 6, 5μL 2M DTT) . 2-2.5μL of DNA carrier (5 mg/mL salmon sperm DNA) were boiled for 7 min at 99°C, cooled and then mixed with 0.5-3μg of expression DNA. All DNA mixture was transferred to the cells transformation mixture. Cells were incubated with shaking at 28°C for 30 min, heat-shocked for 30 min in a 39°C water bath, spun down and resuspended in 100 ml sterile water. Cells (100 µl) were plated on appropriate selection plates. [00138] E. coli strain DH10B was used for cloning and plasmid propagation. DH10B was grown at 37°C with constant shaking in Luria–Bertani Broth supplemented with 100 mg/L of ampicillin for plasmid propagation. Y. lipolytica was cultivated at 30°C with constant agitation. Cultures of Y. lipolytica used in large-scale screens were grown in a shaking incubator at speed 250 rpm for 5 days in 50 ml tubes with working volume 10 mL, and larger culture volumes were shaken in flasks or fermented in a bioreactor. [00139] For colony screening and cell propagation, Y. lipolytica grew on YPD liquid or agar media. Medium was often supplemented with 150 to 300 mg/L hygromycin B or 250 to 500 mg/L nourseothricin for selection, as appropriate. [00140] Constructed Yarrowia lipolytica base strains contain overexpression and heterologous expression of the genes encoding enzymes of the mevalonate and astaxanthin biosynthesis pathways, namely HMGR (Y. lipolytica 3-hydroxy-3-methylglutaryl coenzyme A reductase), Erg12 (Y. lipolytica mevalonate kinase), Ggs1 (Y. lipolytica geranylgeranyl pyrophosphate synthase), CarRP (Mucor circinelloides bifunctional lycopene cyclase- phytoene synthase), CarB (M. circinelloides phytoene dehydrogenase), HpBKT (Haematococcus pluvialis β-carotene ketolase), HpCrtZ (H. pluvialis β-carotene hydroxylase) (Figure 1, marked by underlining). Example 2: Increased carotenoid production by introducing nucleotide sequence encoding the fusion protein of geranylgeranyl synthase and phytoene synthase [00141] Two genes encoding geranylgeranyl pyrophosphate synthase were tested in fusion variants, namely Synechococcus sp. GGPPs7 and mutated variant of YlERG20 F88C . In the current example, the phytoene synthase that is provided is the bifunctional lycopene cyclase-phytoene synthase (CarRP), the enzyme from Mucor circinelloides encoded by the CarRP gene. Coexpression of the fusion variant of geranylgeranyl synthase and bifunctional lycopene cyclase-phytoene synthase together with genes encoding phytoene dehydrogenase (CarB), β-carotene ketolase (HpBKT), and β-carotene hydroxylase (HpCrtZ) leads to enhanced β-carotene, canthaxanthin, and astaxanthin production (Figure 3, 4). [00142] Coexpression of the fusion variant of geranylgeranyl synthase and bifunctional lycopene cyclase-phytoene synthase with phytoene dehydrogenase gene leads to enhanced β- carotene production. The bifunctional lycopene cyclase-phytoene synthase can be from other microbial sources, e.g. crtYB Xanthophyllomyces dendrorhous or CarRA Blakeslea trispora. Heterologous expression of the fusion variant of geranylgeranyl synthase and phytoene synthase (for instance, crtB gene from Pantoea ananatis) in Y. lipolytica leads to enhanced phytoene production. Coexpression of the fusion variant of geranylgeranyl synthase and phytoene synthase (for instance, crtB gene from Pantoea ananatis) with phytoene dehydrogenase gene leads to enhanced lycopene production. [00143] The parental strain BMCY506 harbors overexpression and heterologous expression of the following genes: HMGR, ERG12, ERG20, CarRP, CarB, GGS1, 3 copies of HpBKT, 2 copies of HpCrtZ. TEFin promoter and XPR2 terminator were used for expression of HMGR, ERG20 genes. TEFin promoter and TEF terminator were used for ERG12, CarRP, HpBKT, and HpCrtZ genes’ expressions. EXP1 promoter and CYC1 terminator were used for CarB gene expression. GAPDH promoter and XPR2 terminator were used for GGS1 gene expression. All cultivation was performed in 50-ml test tubes with 10 ml of working volume in rich peptone and yeast extract containing medium for 5 days. [00144] Carotenoid production was analysed using HPLC. The sample preparation was as follows: 1. 0.3 mL of 0.5–0.75 mm acid-washed glass beads was added to 10 mg of lyophilized cells followed by 0.5 mL of acetate supplemented with 0.01% 3,5-di-tert-4- butylhydroxytoluene (BHT); 2. Cells were disrupted using a Precellys R 24 homogenizer (Bertin Corp., Montigny-le-Bretonneux, France) in four cycles of 5500 rpm for 30 s; 3. Tubes were placed on ice between each lysis cycle for 1 min; 4. Cells were precipitated by centrifugation and acetate fraction was collected; and 5. Extraction was repeated two times. The HPLC protocol was as follows: a. Instrument was a Waters, MA, USA equipped with a NOVA-PAK C18150 mm×3.9 mm column; b. The column oven temperature was 30° C; с. Theflow rate was 1.0 mL/min; d. Detection was at 450nm for β-carotene and echinenone, at 470nm for astaxanthin and canthaxanthin. [00145] All nucleotide sequences encoding fusion of geranylgeranyl pyrophosphate synthase or mutated farnesyl pyrophosphate synthase and bifunctional lycopene cyclase- phytoene synthase were constructed with TEFin promoter and TEF terminator transcription elements. The expression cassette harbouring the nucleotide sequence, encoding fusion protein CarRP-GSsh-YlERG20( F88C ), where CarRP is fused by its C-terminus with N-terminal part of mutated YlERG20, was transformed into the strain BMCY506. GSsh is a GlySer linker inserted between two proteins. Introduction of fusion protein CarRP-GSsh-YlERG20( F88C ) gene product into the strain BMCY506, resulting in the engineered strain, BMCY508, demonstrated 2.o times higher production of β-carotene and 35% increase in canthaxanthin accumulation compared to parental BMCY506, respectively (Fig.3). Introduction of fusion protein CarRP- GSsh-GGPPs7 gene product, where CarRP is fused by its C-terminus with N-terminal part of GGPPs7, into the strain BMCY506, resulting in the strain BMCY509, provided a 4.1-, 3.1- fold, and 10% increase in production of β-carotene, canthaxanthin, and astaxanthin compared to parental BMCY506, respectively (Fig. 3). The increase in total β-carotene, canthaxanthin, and astaxanthin produced by BMCY509 was 2.8-fold compared with parental BMCY506. Introduction of the expression cassette P TEFin CarRP-T TEF -P EXP1 CarB-T CYC1 encoding two separated enzymes CarRP and CarB (strain BMCY507) increased total β-carotene, canthaxanthin, and astaxanthin production by 60% compared with parental BMCY506. Results are shown in Table 20. TABLE 20: Β-CAROTENE, CANTHAXANTHIN, AND ASTAXANTHIN PRODUCTION IN BMCY506 (PARENTAL STRAIN), BMCY507, BMCY508 & BMCY509 [00146] The parental strain BMCY510 harbors overexpression and heterologous expression of the following genes: HMGR, ERG12, ERG20, CarRP, CarB, GGS1, 3 copies of HpBKT, and 3 copies of HpCrtZ. The promoter and terminator regions for these genes were the same as described above. All cultivation was performed in 50-ml test tubes with 10 ml of working volume in rich peptone and yeast extract containing medium for 5 days. Carotenoid production was analysed using HPLC as described in Example 2. Introduction of fusion protein CarRP-GSsh-ERG20( F88C ) gene product, where CarRP is fused by its C-terminus with N-terminal part of ERG20( F88C ), into the strain BMCY510, resulting in the engineered the strain BMCY512, provided a 1.8- and 2.8-fold increase in production of β-carotene and canthaxanthin compared to the parental BMCY510 strain, respectively (Fig.4). Introduction of fusion protein CarRP- GSsh-GGPPs7 gene product, where CarRP is fused by its C-terminus with N-terminal part of GGPPs7, into the strain BMCY510, resulting in strain BMCY514, enhanced β- carotene and canthaxanthin production 2.9 and 4 times, respectively, as well as astaxanthin by 26% (Fig. 4). The increase in total β-carotene, canthaxanthin, and astaxanthin produced by BMCY514 was 2.1-fold compared with parental BMCY510. [00147] Introduction of the expression cassette PTEFinCarRP-TTEF-PEXP1CarB- TCYC1 encoding separated enzymes CarRP and CarB (strain BMCY511) increased total β-carotene, canthaxanthin, and astaxanthin production only by 6% compared with parental BMCY510. The results are shown in Table 21. TABLE 21: Β-CAROTENE, CANTHAXANTHIN, AND ASTAXANTHIN PRODUCTION IN BMCY510 (PARENTAL STRAIN), BMCY511, BMCY512, BMCY513 & BMCY514 Example 3: Bioreactor cultivation [00148] Production of carotenoids during fed-batch fermentation by the strain BMCY514 is depicted in (Figs.5, 6 & 7). Fermentation was conducted in a 3-L bioreactor with the initial cultivation volume of 0.75 mL. The medium contained yeast extract 20 g/L, peptone 40 g/L, glucose 6 g/L, and antifoam 0.5mL/L. The 70% glucose stock solution used for feeding which was started after 6 h after inoculation. The glucose concentration was maintained below 1 g/L. The temperature was kept constant at 28.5°C, aeration was set to 2 VVM, the agitation was set to 500-900 rpm, the dissolved oxygen was set to a minimum 20%. pH was automatically maintained at 5.5 by addition of 25% NH 4 OH. Carotenoid production was analysed using HPLC as described in Example 2. As seen in Fig. 5 and 6, astaxanthin accumulation has already reached 4.6 mg/g dcw and 290.1 mg/L at 39 h of fermentation representing 73.4% from the total carotenoids. To 133 h of cultivation the strain BMCY514 accumulated in biomass 8.4 mg/g dcw (594.9 mg/L) of astaxanthin and 34.2 mg/g dcw (2415.7 mg/L) of the mixture of β-carotene, canthaxanthin, and astaxanthin (Figs.5, 6). Cell lysis was also observed at 133 h that led to 132.0 mg/L of astaxanthin and 437.9 mg/L of the mixture of β-carotene, canthaxanthin, and astaxanthin detected in the supernatant (Fig. 7). Total astaxanthin produced by BMCY514 was 726.9 mg/L (Fig.7). Example 4: Increased carotenoid production by introducing genes that encode NADPH generating enzymes [00149] Schematic illustration of the engineered pathway for carotenoids biosynthesis in Y. lipolytica including pathways generating NADPH is presented in Figure 1. [00150] The parental strain BMCY509 harbors overexpression and heterologous expression of the following genes: HMGR, ERG12, ERG20, CarRP, CarB, GGS1, 3 copies of HpBKT, 2 copies of HpCrtZ, and fusion CarRP-GSsh-GGPPs7. Expression cassettes harbouring the TEF intron promoter + GapC or Mce2 gene + XPR2 or TEF terminator were integrated into the genome of BMCY509. All cultivation was performed in 50-ml test tubes with 10 ml of working volume in rich peptone and yeast extract containing medium for 5 days. Carotenoid production was analysed using HPLC as described in Example 2. Integration of Clostridium acetobutylicum GapC gene encoding NADP+-dependent glyceraldehyde-3- phosphate dehydrogenase into the strain BMCY509, resulting in the strain BMCY515, provided a 50% and 24% increase in production of β-carotene and astaxanthin, respectively, compared to parental BMCY509 (Fig. 8). Integration of Mucor circinelloides Mce2 gene encoding NADP+-dependent malic enzyme into the strain BMCY509, resulting in the strain BMCY516, enhanced β-carotene production by 54% compared to parental BMCY509 (Fig.8). The increase in total β-carotene, canthaxanthin, and astaxanthin produced by BMCY515 and BMCY516 compared to parental strain was 30% and 28%, respectively. The results are shown in Table 22. TABLE 22: Β-CAROTENE, CANTHAXANTHIN, AND ASTAXANTHIN PRODUCTION INBMCY509 (PARENTAL STRAIN), BMCY515 & BMCY516 [00151] Whilst not wishing to be bound by theory, the close proximity of the enzymes in the fusion protein is thought to enhance carotenoid production, as the substrates and products do not need to move far to be acted upon by each enzyme. [00152] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge. [00153] It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the disclosure as set forth and defined by the following claims.