Technical Field

The present invention relates to microorganisms, which are genetically modified to produce pant derived triterpenes. In particular, the present invention provides a microorganism of the class Labyrinthulomycota comprising at least one transgene encoding a heterologous 2,3-oxidosqualene cyclase. Further provided is a nucleic acid construct encoding at least one 2,3-oxidosqualene cyclase and at least one further enzyme. The present invention also relates to a method for the production of a microorganism according to the invention and further provides a method for the production of one or more plant derived triterpenes and/or derivatives thereof comprising the step of cultivating a microorganism of the present invention under conditions, which facilitate the production of plant derived triterpenes.

Background

With more than 40 000 structurally distinct compounds, terpenes are the largest and most diverse class of secondary metabolites. Chemically terpenes are based on multiple units of the core C5-building block isoprene which is biosynthesized from the universal precursor molecules isopentenyl pyrophosphate and its isomer dimethylallyl pyrophosphate via the mevalonate (MEV) - and/or methyl-D-erythrol-4-phosphate (MEP) pathways (Maschek & Baker 2008). Terpenes are assembled to linear or cyclic hydrocarbon skeletons and formally classified into hemi (C5)-, mono (C10)-, sesqui (C15)-, di (C20)-, tri (C30), tetra (C40)- and Polyterpenes, while the terpenoids additionally carry diverse functional groups and modifications. Terpenes have been used by mankind since ancient times as fragrance, flavors, as bioactive component of medication and cosmetics and are going to reach progressive impact in future platform chemicals- and biofuels markets. Prominent examples are besides the antimalaria drug Artemisinin, the antitumor therapeutic taxol, the anti-inflamatory, anti-apoptotic Tanshinone IIA, the antibiotic compounds fusidic acid, casbene and pentalenolacton, the polyterpene rubber as well as many flavours and fragrances like menthol, b-ionone, nootkatone, limonene, camphor, myrcene or pinene (Peralta-Yahya et al. 2011). However, the complexity of their chemical synthesis and the large requirement of raw material from natural resources challenged intense efforts to establish efficient biotechnological processes for their renewable and environmental friendly production (Ajikumar et al. 2008). While highly efficient bioprocesses for biotechnological manufacturing of particular terpenes have been established, e.g., for Artemisinin in Saccharomyces cerevisiae (Westfall et al. 2012, Paddon et al. 2013) or taxol in plant cell culture (Phyton Biotech GmbH), the majority of engineering investigations in microorganisms, in the past decades, did not generate exceptionally high or commercially viable terpene productivities. The few naturally occurring microbes known for their efficient terpene metabolism such as the green algae Dunaliella salina, Botryococcus braunii or Haematococcus pluvialis, which are capable of accumulating tetraterpenes (mainly carotinoids) in quantities of up to 14, 20 and 8% per dry weight, respectively (Borowitzka & Borowitzka 1988, Metzger & Largeau 2005) depict slow growth rates and are poorly accessible to genetic engineering. A direct comparison of technologies for squalene production (see table 1) clearly demonstrates that even recombinant microorganisms improved by iterative metabolic engineering did not exceed the exceptional levels found in wildtype strains of Thraustochytrids. Especially, Schizochytrium and Aurantiochytrium, proved to be capable of accumulating up to 30 % squalene (dry weight) with remarkable productivities in the g/L*day range (Nakazawa et al. 2014, Ren et al. 2014).

Table 1 : Overview of bioprocess technology for the production of squalene in natural and genetically modified (3 bottom lines) microorganims. Maximum cellular dry weight (CDW), squalene content (CSQ), squalene titer (YSQ) and productivities (PSQ) achieved in batch processes.

Thraustochytrids are unicellular eukaryotic marine microorganisms with saprophytic or parasitic life style, which form biflagellate zoospores and characteristic ectoplasmic net structures - thus received the trivial name „net slime molds". Taxonomically, the family Thraustochytridae belongs to the class Labyrinthulomycota, superphylum Stramelophila (Heterokonta). Historically, they were often denoted as heterotrophic microalgae, based on the evolutionary proximity to their photosynthetic relatives. At low incubation temperature and high C/N ratio (N-depreviation) Thraustochytrids accumulate lipid droplets with up to 50 % of lipids per dry weight and very high contents of polyunsaturated fatty acids (PUFAs), mainly docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (AA). For this reason, representatives of biotech industry extensively exploit the Thraustochytrids Schizochytrium and LZ/ken/aforthe heterotrophic production of DHA since decades. Nevertheless, Thraustochytrids also depict a rich source for carotinoids, e.g. b- carotene, astaxanthin, canthaxanthin, zeaxanthin, echinenone and phoenicioxanthin (Aasen et al. 2016), the triterpene squalene orthe compatible solutes quercitol and glycine betaine (Jakobsen et al. 2015). The industrial high cell density bioprocess achieves final biomass densities of up to 200 g/L with a DHA-content of > 20 % (Bailey et al. 2003; US Patent US6607900B2). In continuous culture average biomass- and DHA-productivities of 3.88 g/L*day and 0.52 g/L*day, respectively, were reached (Ethier et al. 2011). Thraustochytrids synthesize a variety of extracellular catabolic enzymes, e.g., cellulase, chitinase, amylase, which enable the utilization of complex polysaccharides (Nagano et al. 2014). In the past decade, several organic side-streams have successfully been adopted as alternative substrates in thraustochytridial fermentation, e.g., hemp hydrolysate (Gupta et al. 2015), whey and wastewater from dairy industry (Humhal et al. 2017), crude glycerol, a byproduct of biofuel refinery (Pyle et al. 2008), molasse (Hong et al. 2011), corn syrup and syrup from food processing industry (Yu et al 2015, Iwasaka et al. 2013), brewer grains (Taoka et al. 2017), potato cull (Chi et al. 2007) or brown algal biomass (Arafiles et al. 2014, Scullin et al. 2015).

The global squalene market has reached a volume of 2.500 tonnes in 2013, equivalent to an annual turnover of 160 M USD (CAGR > 10 %). Around 40 % of the squalene on the market is derived from fish oil, in particular shark cod liver oil. Further 42 % is produced from amaranth or palm oil, but primarily from deodorizer distillates of olive oil refining. Currently, the biotechnological production of squalene is only carried out by the Californian company Amyris. Amyris employs an indirect procedure using recombinant strains of the yeast Saccharomyces cerevisiae for the biosynthesis of farnesene which is chemically transformed into squalene - Neossance™ Squalane. Other bioprocesses, e.g., the production in recombinant tobacco (SynShark LLC) or the direct biosynthesis in Saccharomyces cerevisiae or Yarrowia lipolytica, developed by Synshark, Organobalance GmbH (now Novozymes) and Mycogen Corp. (now Dow Chemicals), respectively, have been patented but did not reach commercially viable efficiencies. The Japanese company Kuraray utilizes a total chemical synthesis. However, this multistep process is costly and mainly used to provide intermediates for the synthesis of derivatives. As a natural constituent of human skin, squalene is extensively exploited in dermatology and cosmetic industry, since it demonstrates exceptional performance as emulsifier, emollient and hydratation. Based on its anti-oxidative and lipid peroxidation inhibiting activity, squalene has also entered the nutritional supplements market. In the animal model, squalene feeding could significantly lower glucose, cholesterol-, and leptin levels in blood, while in contrast leading to an increase in testosterone accompanied by an increase in fertility of roosters. Furthermore, squalene-based emulsions are applied as adjuvants in vaccines or as carrier for the administration of pharmacological agents (Spanova & Daum 2011 , Huang et al. 2009). The triterpenes oleanolic and ursolic acid, as well as derivatives thereof, are widely used as bioactive additives in cosmetics formulations, in nutritional supplements and pharmaceutical preparations. According to a forecast for 2022, ursolic acid is going to have a global market volume of approx. 5 tonnes / a (CAGR 4,01 %) corresponding to a turnover of 9 Mio USD and an average value of 350-1800 USD / kg, depending on product quality (purity). Currently, their commercial production exclusively relies on the extraction from plant sources, mainly rosemary, apple and olive or argan oil. Oleanolic and ursolic acid are primarily used as emulsifier or pharmaceutical agent in nutritional supplements, cosmetics and pharmaceutical formulations. Based on its positive impact on structure and elasticity of collagen fibres in the skin, ursolic acid decreases the occurence of wrinkles and age spots and facilitates natural repair after UV light damage. Ursolic acid inhibits the enzymes elastase, cyclooxygenase and lipooxygenase and is therefore recognized as anti-aging and anti-inflammatory substance. In-vitro it also increases the ceramide content in epidermal keratinocytes and of collagen in fibroblasts of skin (Both et al. 2001 , Yarosh et al. 2002). Usually ursolic acid is applied in concentrations of 0,2-3% in cremes, lotions, lip balm, shampoo and gels. The application of triterpenes as pharmaceutical agent is subject to more than 25 clinical studies and approx. 10 products are already available on the market. In asia, oleanolic formulations are marketed to fight hyperlipidemia, liver and lymphatic diseases (Schmandke 2009). Furthermore, oleanolic acid and derivatives (e.g., Bardoxolon [2-cyano-3,12-dioxooleana-1 ,9-dien-28-oic acid; CCDO], Bardoxolon-Methyl) are currently investigated for their potential in tumor therapy and to treat chronic renal diseases (Ayeleso et al. 2017). Both oleanolic as well as ursolic acid exhibit antioxidative, anti-inflammatory, anti-microbial, angiogenic and immunomodulatory activity. Studies in the mouse model revealed a reduction of glucose, cholesterol and triacyl glycerol in blood, an increase in formation of skeletal muscles, along with a reduced occurrence of adipohepatic diseases (Kunkel et al. 2012) upon ursolic acid co-feeding. Both oleanolic and ursolic acid demonstrate significant potential in the treatment of snakebites (Preciado et al. 2018). For a detailed picture of the broad action spectrum of oleanolic and ursolic acid, see review articles by Wozniak et al. (2015) and Lopez-Hortaz et al. (2018).

Betulinic acid and its pharmacological application have been subject to intense investigations in the past decades (Mertens-Talcott et al. 2013, Pal et al. 2015). Betulinic acid induces apoptosis in human blastoma and inhibits human melanoma, malignant brain tumors, ovarian carcinoma and human leukemia cells (Tan et al. 2003, Zuco et al. 2002, Selzer et al. 2002, Ehrhardt et al. 2004). Derivatives of betulinic acid, eg., the orally administered Berivimat, also known as PA-457 inhibit the replication of HIV virus by blocking p25 maturation. The novel synthetic derivatives IC9564 or A43-D proved to be capable of significantly inhibiting HIV-1 subtypes A, B, C at concentration of 0,2-1 ,5 pM (Lai et al. 2008, Huang et al. 2018). Moreover, recent findings (Kim et al. 2019) discovered that betulinic acid may inhibit high fat diet induced obesity by activation of the AMP-activated protein kinase, which regulates cellular and whole-body energy balance (Hardie 2014). Since betulinic acid inhibits several enzymes of carbohydrate and lipid absorption metabolism such as a-amylase (Kumar et al., 2013), a-glucosidase (Zareen et al., 2008), glycogen phosphorylase, diacylglycerol acetyl-transferase, increases insulin and leptin secretion, it results in reduced body weight, abdominal fat accumulation, blood glucose, plasma triglyceride and cholesterol levels in rats (Melo et al., 2009, Xie at al. 2017). It is therefore suggested as potential antidiabetic agent in the treatment of diabetes type 2 and associated metabolic syndrome (Rios & Manez 2018). Experiments in mouse demonstrated that betulinic acid may amiliorate artheroscerosis at an early stage (Song et al. 2020, Yoon et al. 2017). In contrast to common antidiabetic drugs like sulfonylurea, a- glucosidase inhibitors, biguanides or thiazolidinediones, betulinic acid did not show adverse side effects. Lupeol, which is naturally found in Shea butter, elm plant, aloe leaves, the mango fruit or Tamarindus indica and Hemidesmus indicus, is of particular interest in wound healing (Harrish et al. 2008, Beserra et al. 2019) and especially well-suited for dermatological applications (Agra et al. 2015). The mechanism of action is based on the induction of differentiation (Hata etal. 2000), the activation of the p38 mitogen-activated protein kinases (MAPK) (Hata et al. 2003) and actin cytoskeleton remodeling (Hata et al. 2005) among others. Lupeol further exerts a broad antibiotic action spectrum. The antimicrobial action of lupeol against bacterial and fungal pathogens proved to be superiorto traditional antibiotics such as ampicillin or tioconazole (Ajaiyeoba et al. 2003, Hernandez-Perez et al. 1994). Similar to betulinic acid, lupeol exerts antiviral activity eg., against Herpes simplex virus 1 (HSV1) and Influenza A virus (Hernandez-Perez et al. 1994). It is further known to inhibit the proliferation of Plasmodium falciparum, the causative agent of malaria disease, by blocking the invasion of the merozoite stages into erythrocytes. Lupeol as well as synthetic derivatives thereof were also able to inhibit the amastogote stages of Leishmania amazonensis and Trypanosoma cruzi (Fournet et al. 1992, Machado et al. 2018). Triterpenes naturally occur in a broad variety of plants. However, the content of these substances in plant material rarely exeeds 0,1% per dry weight (Jager et al. 2009). Low concentrations in the plant material dramatically increase the cost for extraction and has so far hampered commercial production. An exception to this rule is the bark of white birch (Betula pubescens) which may accumulate up to > 30% of betulin (Laszczyk 2009). In plants, the yield of certain biosynthetic products which rely on complex, multi-step biosynthetic pathways is limited by several factors. 1) The desired product competes with branching metabolic routes 2) several biosynthetic enzymes are promiscuous and accept different substrates 3) It may be limited by the availability of precursors and cofactors. The natural cell is programmed to avoid wasting energy and nutrient resources by 4) regulation of transcription and translation, posttranslational control and 5) feedback inhibition of enzymatic activity. 6) Metabolic engineering approaches frequently result in metabolic imbalance and several pathway intermediates have been observed to be toxic to the cell. To overcome these limitations, recombinant strain development generally combines a set of engineering strategies e.g., the deletion ordownregulation of competing pathway genes, the overepression of heterologous genes, the deregulation of feedback inhibited enzymes, the deregulation of transcriptional and translational control by promoter exchange or the alteration of regulators, the engineering of bi- or multifunctional fusion proteins or the attachment to scaffolds which enable intramolecular channeling of intermediate or redox energy, thereby improving substrate to product conversion efficiency at metabolic branching points (Zhao et al. 2016, Dueber et al. 2009) or the redirection of metabolic enzymes to different compartments of the cell (Arendt et al. 2017, Hammer & Avalos 2017, Huttanus & Feng 2017, Chen & Silver 2012). During the past decade systems metabolic engineering investigations were able to achieve significant improvements in heterologous biosynthesis of terpenes in microbial host systems, in particular of sesquiterpenes. By reiterative strain development, a-farnesene titers of > 25 g/L in Yarrowia lipolytica (Liu et al. 2019) and 130 g/L in Saccharomyces cerevisiae (Meadows et al. 2016) could be reached. Similarly, Amorphadiene, a precursor in the biosynthesis of the antimalarial drug Artemisinin yielded product titers of 27,4 g/L in E.coli (Tsuruta et al 2009) and 40 g/L in Saccharomyces cerevisiae (Westfall et al. 2012). In contrast, triterpene engineering approaches have so far only generated comparatively low productivities (see table 2), likely because of the cytotoxic nature of the molecules.

Table 2: Top scoring recombinant triterpene engineering investigations in microbes ln order to be able to produce heterologous triterpenes on an economically relevant scale in a host organism, it is necessary to identify a suitable host organism, which can be engineered to produce the heterologous triterpenes as well as to overcome any production limiting factors such as feedback inhibition, competing metabolic pathways and toxicity of the product. In a next step, a suitable strategy needs to be established for the host organism to address all relevant issues that might hamper the productivity. The development of production strains for a specific compound or class of compounds comprises a number of challenges, which cannot always be foreseen and for which individual solutions need to be developed.

It was an objective of the present invention to provide means and methods to produce relevant quantities of plant derived triterpenes without having to rely on the isolation from plant material. In particular, it was an objective of the present invention to identify and engineer host organisms, which are able to produce plant derived triterpenes in high yields and which are suitable for industrial production.

Summary of the Invention

In one aspect, the present invention relates to a microorganism of the class Labyrinthulomycota comprising at least one transgene encoding a heterologous 2,3- oxidosqualene cyclase.

In one embodiment, in the microorganism described above, the heterologous 2,3- oxidosqualene cyclase is selected from the group consisting of an alpha-amyrin synthase, a beta-amyrin synthase, a lupeol synthase, a dammarenediol synthase and a cucurbitadienol synthase.

In another embodiment, the microorganism described above is selected from the group consisting of Thraustochytrids, in particular Schizochytrium, Aurantiochytrium, Thraustochytrium and Hondea.

In one embodiment of the microorganism according to any of the embodiments described above, the microorganism further comprises at least one transgene encoding a heterologous cytochrome P450 oxidase and, optionally, a heterologous cytochrome P450 reductase or a ferredoxin, preferably a hybrid cytochrome P450 oxidase/reductase or a hybrid cytochrome P450 oxidase/ferredoxin, and further preferably the at least one transgene encoding a cytochrome P450 oxidase and/or a cytochrome P450 reductase or a hybrid cytochrome P450 oxidase/reductase is fused to an endoplasmatic reticulum (ER) targeting sequence and/or an ER retention sequence or the ferredoxin or the hybrid cytochrome P450 oxidase/ferredoxin is fused to a mitochondrial targeting sequence.

In a further embodiment of the microorganism according to any of the embodiments described above, the microorganism further comprises a transgene for the overexpression of a squalene synthase and/or a squalene epoxidase under the control of a strong constitutive promoter, preferably a transgene for the overexpression of a hybrid squalene synthase/farnesyl pyrophosphate synthase and/or a hybrid squalene synthase/squalene epoxidase.

In yet another embodiment of the microorganism according to any of the embodiments described above, the microorganism carries at least one further transgene for the overexpression of enzymes of the mevalonate pathway under the control of a strong constitutive promoter, preferably selected from the group consisting of acetyl-CoA- acetyltransferase, 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl- CoA reductase, mevalonate kinase, phosphomevalonate kinase, dihydrophospho- mevalonate decarboxylase and isopentenyl pyrophosphate isomerase. In one embodiment of the microorganism according to any of the embodiments described above, the microorganism carries a transgene encoding a truncated 3-hydroxy-3- methylglutaryl-CoA reductase, which is missing the regulatory sensor domain and/or a transgene encoding a heterologous mevalonate kinase derived from Methanosarzina mazeii. In another embodiment of the microorganism according to any of the embodiments described above, at least one endogenous gene selected from the group consisting of lanosterol synthase, b-carotene synthase, ku80 and anthranilate synthase is inactivated.

In a further embodiment of the microorganism according to any of the embodiments described above, at least one transgene, preferably all transgenes, are integrated into the genome of the microorganism, particularly preferably the transgene(s) is/are inserted into one or more of the lanosterol synthase, the b-carotene synthase, the ku80, the anthranilate synthase and the 18 S rDNA locus in the genome of the microorganism.

In another aspect, the present invention relates to a nucleic acid construct or set of nucleic acid constructs encoding (i) at least one 2,3-oxidosqualene cyclase heterologous to

Labyrinthulomycota, preferably selected from the group consisting of an alpha-amyrin synthase, a beta-amyrin synthase, a lupeol synthase, a dammarenediol synthase and a cucurbitadienol synthase; and at least one of (ii) a cytochrome P450 oxidase heterologous to Labyrinthulomycota and, optionally, a cytochrome P450 heterologous to Labyrinthulomycota reductase or a ferredoxin, preferably a hybrid cytochrome P450 oxidase/reductase or a hybrid cytochrome P450 oxidase/ferredoxin, and further preferably wherein the at least one transgene encoding a cytochrome P450 oxidase and/or a cytochrome P450 reductase or a hybrid cytochrome P450 oxidase/reductase is fused to an endoplasmatic reticulum (ER) targeting sequence and/or an ER retention sequence or wherein the ferredoxin or the hybrid cytochrome P450 oxidase/ferredoxin is fused to a mitochondrial targeting sequence; and

(iii) a squalene synthase and/or a squalene epoxidase, preferably a hybrid squalene synthase/farnesyl pyrophosphate synthase and/or a hybrid squalene synthase/squalene epoxidase; and

(iv) at least one enzyme selected from the group consisting of acetyl-CoA- acetyltransferase, 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3- methylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, dihydrophosphomevalonate decarboxylase and isopentenyl pyrophosphate isomerase, preferably a truncated 3-hydroxy-3- methylglutaryl-CoA reductase, which is missing the regulatory sensor domain and/or a heterologous mevalonate kinase derived from Methanosarzina mazeii ; wherein the sequence of the nucleic acid construct or set of constructs is codon optimized to be expressed in Labyrinthulomycota, and preferably wherein two or more of the genes recited in (i) to (iv) are fused to encode multienzymatic translational fusion proteins separated by self-cleavable 2A peptides.

In yet another aspect, the present invention provides a vector or a set of vectors encoding a nucleic acid construct or set of nucleic acid constructs as described above. In one aspect, the present invention relates to a method for the production of a microorganism, preferably a microorganism according to any of any of the embodiments described above, comprising a) providing a microorganism of the class Labyrinthulomycota; and b) transforming the microorganism of step a) with a nucleic acid construct or set of nucleic acid constructs or a vector or set of vectors as described in any of the embodiments above.

In one embodiment of the method described above, the nucleic acid construct orthe set of nucleic acid constructs is inserted into the genome of the microorganism by homologous recombination, preferably the nucleic acid construct or the nucleic acid constructs of the set of nucleic acid constructs is/are inserted at one or more of the lanosterol synthase, the b-carotene synthase, the ku80, the anthranilate synthase and the 18 S rDNA locus in the genome of the microorganism.

In yet another aspect, the present invention provides a method for the production of one or more plant derived triterpene(s) and/or derivatives thereof comprising the steps: i) providing a microorganism according to any of the embodiments described above; and ii) cultivating the microorganism of step i) under conditions, which facilitate the production of plant derived triterpenes.

In one embodiment of the method for the production of one or more plant derived triterpene(s) and/or derivatives thereof, the plant derived triterpene(s) is/are selected from the group consisting of lupeol, oleanolic acid, ursolic acid, betulinic acid, corosolic acid, maslinic acid and dammarenediol.

Definitions

A “transgene” in the context of the present invention is a gene, which has been introduced into an organism by genetic engineering such as transformation with a genetic construct and optionally integration into the genome of the organism, e.g. by homologous recombination. The transgene may be “heterologous” to the organism, i.e. it does not naturally occur in the organism, or it may be “homologous”, i.e. a copy of the gene may naturally exist in the microorganism. If the transgene is homologous to the organism, the organism may comprise two copies of the gene, one in its natural genetic context and a transgene at a different locus and optionally under the control of a different promoter and/or terminator. The transgene can be introduced together with a promoter and/or a terminator controlling the expression of the transgene as well as with other transgenes, e.g. as part of an expression cassette. A “2,3-oxidosqualene cyclase” or “OSC” is an enzyme, which catalyzes the cyclisation of 2,3-oxidosqualene to form triterpenes. In the context of the present invention, an 2,3- oxidosqualene cyclase is preferably a plant derived 2,3-oxidosqualene cyclase, which does not naturally occur in the microorganisms of the present invention. In particular, a heterologous 2,3-oxidosqualene cyclase is not a lanosterol synthase, which naturally occurs in the microorganisms of the present invention. “Cytochrome P450 oxidases” or “CYPs” are a diverse class of HEME-dependent enzymes which catalyze specific monoxygenation reactions of non-reactive carbon-hydrogen bonds in intermediates of the biosynthesis of terpenes, steroids and fatty acids or in the detoxification of xenobiotics. “Cytochrome P450 reductases” or“CPRs”, also referred to as “NADPH-cytrochrom P450 oxidoreductases”, are FMN/FAD-containing redox proteins which mediate the transfer of electrons from the universal reducing equivalent NADPH to cytochrom P450, thereby constantly providing cytochrome P450 oxidases with reducing power.

A “hybrid cytochrome P450 oxidase/reductase” is an enzyme, which provides oxidase and reductase activity and thus can directly transfer electrons intramolecularly from the reductase domain to the cytochrome P450 oxidase domain.

A “ferredoxin” is an iron-sulfur cluster protein, which mediates electron transfer in metabolic reactions such as the respiratory or photosynthetic electron transport chain and functions as a reductase in the context of the present invention. In mitochondria, ferredoxin further serves as an electron donor for the membrane bound cytochrome P450 oxidases involved in steroid biosynthesis (Midzak & Papadoupulos 2016).

An “endoplasmatic reticulum (ER) targeting sequence” in the context of the present invention refers to a peptide sequence or a nucleic acid sequence encoding a peptide sequence, which is a signal for the attached polypeptide to be transported to the ER. An “endoplasmatic reticulum (ER) retention sequence” refers to a peptide sequence or a nucleic acid sequence encoding a peptide sequence, which provides for the attached polypeptide to be retained in the ER.

A “mitochondrial targeting sequence” in the context of the present invention refers to a peptide sequence or a nucleic acid sequence encoding a peptide sequence, which is a signal for the attached polypeptide to be transported to the mitochondria of the microorganism.

Overexpression” in the context of the present invention relates to the increased expression of a protein, in particular an enzyme, with respect to it expression in a natural context. In the natural context, certain regulatory factors may lead to limited expression of the protein at all times or under certain conditions. When the protein is overexpressed, it is taken out of this natural context and as a result the expression is increased. For example, the gene encoding for a protein may be provided under the control of a strong promoter, which is active at all times and under any conditions. A “transgene for overexpression” is therefore provided together with a strong constitutive promoter. This setup is able to circumvent naturally occurring regulatory mechanisms. A “strong constitutive promoter” is a promoter, which provides high expression levels and is always active. A “farnesyl pyrophosphate synthase” or “FPPS” catalyzes sequential condensation reactions of dimethylallyl pyrophosphate (DMAPP) with 2 units of 3-isopentenyl pyrophosphate to form farnesyl pyrophosphate (FPP). Two molecules FPP are converted into squalene by a "squalene synthase”. “Squalene epoxidase” or “squalene monooxygenase” oxidizes squalene to 2,3-oxidosqualene or squalene epoxide. Hybrids, i.e. enzymes with two activities, can be provided namely a “hybrid squalene synthase/farnesyl pyrophosphate synthase” and a “hybrid squalene synthase/squalene epoxidase”, making the process more efficient.

The “mevalonate pathway” is an essential metabolic pathway present in many organisms, which produces isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) from acetyl co-enzyme A (acetyl CoA). Enzymes involved in the mevalonate pathway are acetyl-CoA-acetyltransferase (AATC), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), mevalonate kinase (MVK), phosphomevalonate kinase (PMK), dihydrophosphomevalonate decarboxylase (PMD) and isopentenyl pyrophosphate isomerase (Idi). An “endogenous” gene is a gene, which naturally occurs in an organisms and is present in its natural genetic context, i.e. in its natural gene locus and under the control of its natural regulatory factors.

“Lanosterol synthase” is an enzyme, which converts 2,3-oxidosqualene to lanosterol, displaying the first committed step in the biosynthesis of sterols, e.g., Zymosterol, Fecosterol, Episterol and Ergosterol (Jiang et al. 2020). The “b-carotene synthase” plays a role in the synthesis of tetraterpenes, in particular b-Carotene and its secondary carotenoid astaxanthin. Similar to Aurantiochytrium KH105 (Iwasaka et al. 2018) the b-carotene synthase in Schizochytrium ATCC 20888 is a trifunctional enzyme catalyzing the activities of geranylgeranyl phytoene synthase, phytoene desaturase and lycopene cyclase, which are encoded by three separate genes (crtl, crtB, crtY) in most organisms (Gao et al. 2017). A gene is “inactivated” in the context of the present invention, when its expression is no longer possible due to a deletion of the gene or relevant parts thereof or due to the insertion of another gene disrupting the locus of the gene to be inactivated.

In the context of the present invention, genes can be fused, i.e. combined into one expression construct to encode “multienzymatic translational fusion proteins”. The encoded proteins are separated by “self-cleavable 2A peptides” and the construct may require only one promoter and one terminator to express all proteins of the fusion. The self-cleavable 2A peptides are used to cleave the polypeptide being translated from the construct into the separate functional gene products of the fused genes. ..Homologous recombination" refers to a recombination mechanism, by which a sequence can be inserted by double cross-over integration e.g. into a genome. This is achieved by providing the sequence to be inserted, preferentially as linear DNA fragment, with flanking sequences, which are homologous to corresponding sequences in the genome.

The heterodimer composed of “Ku80” and Ku70 proteins is considered as initiating key player in DNA double strand repair by a mechanisms known as non-homologous end joining (NHEJ). In most organisms NHEJ competes with homologous recombination, thereby lowering the efficiency of genomic integration for recombinant DNA. A deletion of ku80 or ku70 genes has been reported to stimulate homologous recombination frequency by approximately 10 fold (Ding et al. 2019). The genomic “18S rDNA locus”, which encodes 18S ribosomal RNA, represents a popular site for integration of recombinant DNA for two reasons. The extremely high sequence conservation of 18S rDNA clusters among related species enables the generation of integration vectors with broad host range (Klabunde et al. 1993). Some genomes further feature two or more copies of the 18S rDNA locus which may lead to a multicopy integration of the transgenic DNA (Marx et al. 2009). In this context, the present 18SrDNA vectors derived from the 18S rDNA sequence of Schizochytrium sp. ATCC 20888 may be applied to transform several Schizochytrium and Aurantiochytrium strains as well.

Plant derived triterpenes are triterpenes, which naturally only occur in plants. Derivatives of such triterpenes may carry certain functional or non-functional modifications. Examples of plant derived triterpenes include lupeol, oleanolic acid, ursolic acid, betulinic acid, corosolic acid, maslinic acid, cucurbitadienol and dammarenediol. Whenever the present disclosure relates to the percentage of identity of nucleic acid or amino acid sequences to each otherthese values define those values as obtained by using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) programme (www.ebi.ac.uk/Tools/psa/ emboss_water/nucleotide.html) nucleic acids or the EMBOSS Water Pairwise Sequence Alignments (protein) programme (www.ebi.ac.uk/Tools/psa/emboss_water/) for amino acid sequences. Alignments or sequence comparisons as used herein refer to an alignment over the whole length of two sequences compared to each other. Those tools provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) for local sequence alignments use a modified Smith-Waterman algorithm (see www.ebi.ac.uk/Tools/psa/ and Smith, T.F. & Waterman, M.S. "Identification of common molecular subsequences" Journal of Molecular Biology, 1981 147 (1):195-197). When conducting an alignment, the default parameters defined by the EMBL-EBI are used. Those parameters are (i) for amino acid sequences: Matrix = BLOSUM62, gap open penalty = 10 and gap extend penalty = 0.5 or (ii) for nucleic acid sequences: Matrix = DNAfull, gap open penalty = 10 and gap extend penalty = 0.5.

The skilled person is well aware of the fact that, for example, a sequence encoding a protein can be "codon-optimized" if the respective sequence is to be expressed in another organism than the sequence originates from. Brief description of the Figures

Figure 1 shows the natural terpene biosynthetic pathway in Schizochytrium sp. ATCC20888 wildtyp and recombinant, heterologous plant type triterpene biosynthesis branch (grey shaded box) which was heterologously expressed in Thraustochytrids. MEV- pathway: (AACT: acetyl-CoA-acetyltransferase), HMG-CoA: 3-hydroxy-3-methylglutaryl- CoA (HMGS: HMG-CoA-synthase), MVA: mevalonate (HMGR: HMG-CoA-reductase), MVP: mevalonate-5-phosphate (MVK: mevalonate kinase), MVPP: mevalonate-5- diphosphate (PMK: phosphomevalonate kinase), IPP: Isopentenyl pyrophosphate und DMAPP: dimethylallyl pyrophosphate (PMD: phosphomevalonate decarboxylase), (IDI: isopentenyl diphosphate-isomerase. Key enzymes with known feedback regulation are marked with *. GPP: geranyl pyrophosphate (GPPS: geranyl pyrophosphate synthase), FPP: farnesyl pyrophosphate (FPPS: farnesyl pyrophosphate synthase), GGPP: geranylgeranyl pyrophosphate (GGPPS: geranylgeranyl pyrophosphate synthase), SQS: squalene synthase, aSQE: alternative squalene epoxidase, LanS: lanosterol synthase. Heterologously installed plant triterpene biosynthetic pathway: MdAS: amyrin synthase (oxidosqualene cyclase OSC1) from Malus domestica, CYP716A52: C28 cytochrome P450 oxidase; CYP716C55: C2 cytochrome P450 oxidase from Lagerstroemia speciosa. CarS: trifunctional beta-carotene synthase. Non verified branching pathways are depicted in grey color. Not present in Schizochytrium 20888 are the MST: monoterpene synthase, STS: sesquiterpene synthase DTS: diterpene synthase frequently found in other organisms.

Figure 2 shows a schematic drawing of the integration vector series for transformation of Thraustochytrids. The flanking regions A and B are homologous to a specific locus within the acceptor genome and enable double cross-over integration of expression cassettes by homologous recombination. All constructions of expression cassettes (see Fig. 3, 4 and 5) were conducted in at least two integration vectors to obtain a variation of expression levels and compensate for detrimental positional effects. The following integration sites were used: 1) lanosterol synthase gene lanS in A.limacinum MYA-1381 (1800 bp each, integration causes a deletion of the entire lanS gene) , 2) 18S rDNA locus in Schizochytrium ATCC 20888 (~850 bp each; flanking regions are applicable for a broad set of strains within the Schizochytrium and A.limacinum based on sequence conservation, 3) beta-carotene synthase deletion in Schizochytrium ATCC20888 (2000 bp each, partial gene deletion caused by the vector integration enables visual selection by loss of color), 4) anthranilate synthase gene in Schizochytrium ATCC20888 (2000 bp each, integration causes a deletion of the entire gene and results in auxotrophy on minimal media) 5) KU80 in Schizochytrium ATCC20888 and A.limacinum (1800 bp each, integration causes a deletion of the entire

KU80 gene, thereby altering non homologous end joining). oriV (ColE1): origin of replication for maintenance in in E.coli, bla: ampicillin/ca rbenicillin resistance marker, 2-Micron: replicon for maintenance in S. cerevisiae, Ura3: orotidine decarboxylase selection marker for application in uracil auxotrophic background strains of S. cerevisiae. Promoters A) PrPK: pyruvate kinase gene promoter from A.limacinum MYA-1381 , B) PraTub gene promoter from Schizochytrium ATCC 20888, PrPolyU: polyubiquitin gene promoter from Schizochytrium ATCC 20888, PrG3P: glycerol-3-phosphate dehydrogenase gene promoter from A. limacinum MYA-1381 , Prhsp70/Prelf1cr hybrid of heat shock gene hsp70 promoter and elongation factor 1 gene promoter from A.limacinum MYA-1381). Figure 3a and 3b show a schematic drawing of DNA cassettes for heterologous expression of triterpene biosynthetic features. The sequence numbers on the left indicate the respective plasmids in table 5. MdAS: 2,3oxidosqualene cyclase / amyrin synthase from Malus domestica, CrAO: C28 cytochrome P450 oxidase from Cantharanthus roseus, AtCPR: cytochrom P450 reductase from Arabidopsis thaliana. The enzyme encoding genes were codonoptimized for expression in the acceptor strains and expressed as monocistronic or polycistronic transcripts encoding translational fusion proteins, which split into the respective single enzymes cotranslationally based on viral 2A linker peptides. PrPK: pyruvate kinase gene promoter from A.limacinum MYA-1381 , PrG3P: glycerol-3- phosphate dehydrogenase gene promoter from A.limacinum MYA-1381 , PrS35: Cauliflower mosaic virus (CaMV) S35 promoter, PrPolyU: polyubiquitin gene promoter from Schizochytrium ATCC 20888, Prhsp70/Prelf1 : hybrid dual promoter consisting of hsp70 gene promoter and elongation factor 1 a gene promoter from A.limacinum MYA-1381 , NeoR and KanMX: aminoglycoside phosphotransferase genes conferring resistance to Geneticin (G418), erg1 : squalene epoxidase from S.cerevisiae, SQSMYA: squalene synthase from A. limacinum MYA-1381 , ERsig-CrAO-AtCPR-ERret: synthetic fusion of C28 cytochrome P450 oxidase from Cantharanthus roseus with cytochrom P450 reductase from Arabidopsis thaliana equipped with N-terminal ER targeting and C-terminal ER retention signal sequence from calreticulin (MYA-1381). Mitosig-CrAO-fdx: synthetic fusion of C28 cytochrome P450 oxidase from Cantharanthus roseus with mitochondrial ferredoxin from A. limacinum MYA-1381 equipped with a mitochondrial targeting signal sequence from ferredoxin (MYA-1381). ERsig-LsCYP-BbCPR: synthetic fusion of C2 cytochrome P450 oxidase from Lagerstoemia speciosa with cytochrom P450 reductase from Botryococcus braunii equipped with N-terminal ER targeting signal sequence from hsp70 (MYA-1381). MdAS: amyrin synthase from Malus domestica, LupS: lupeol synthase from Rhizinus communis, rpl44*: ribosomal protein 44 mutant P56Q from Aurantiochytrium, ShBle: Bleomycin resistance gene from Streptomyces hindustanicus, gfp: green fluorescence protein Mgfp5, NeoR: aminoglycoside phosphotransferase = G418 resistance determinant, XylE: catechol-2, 3-dioxigenase from Pseudomonas putida. Spotted bars indicate viral 2A- linker peptides, which enable self-cleavage of the translational fusion proteins. CYC-T: cytochrome C terminator from Saccharomyces cerevisiae, NosT: nopalin gene terminator from Agrobacterium tumefaciens Ti plasmid, EF1-T: elongation factor 1 terminator from Schizochytrium ATCC 20888. Mottled grey sections between arrows represent the 2A linkers.

Figure 4a and 4b show a schematic drawing of DNA cassettes for overexpression of structural features of terpene precursor biosynthesis. The sequence numbers on the left indicate the respective plasmids in table 5. NosT: Nos terminator, PrPK: Pyruvate kinase gene promoter and PrG3P: glycerol-3-, phosphate gene promoter from Aurantiochytrium limacinum MYA-1381 , PrTEF: yeast translation elongation factor 1 gene promoter, tHMGR: truncated HMG-CoA reductase (Sequ 3 and 9: tHMG-1 from S.cerevisiae, Sequ 38: tHMGR from Schizochytrium ATCC20888), erg10: acetyl-CoA acetyltransferase from S.cerevisiae, erg 13: HMG-CoA synthase from S. cerevisiae, erg20: farnesyl pyrophosphate synthase from S.cerevisiae, erg1 : squalene epoxidase from S.cerevisiae, SQSMYA: squalene synthase from A. limacinum MYA-1381 , IdiMYA: isopentenyl pyrophosphate isomerase from A. limacinum MYA-1381 , TeSQS: squalene synthase from Thermosynechococcus elongatus, LupsS: lupeol synthase from Rhizinus communis, MmMevK: mevalonate kinase from Methanosarcina mazeii, erg8: phosphomevalonate kinase from S.cerevisiae, erg19: mevalonate diphosphate decarboxylase from S.cerevisiae, erg10: acetyl-CoA- acetyltransferase from S.cerevisiae, erg13: hydroxymethylglutaryl-CoA synthase from S.cerevisiae, Idi1 : isopentenylpyrophosphate isomerase from S.cerevisiae, PrPK: pyruvate kinase gene promoter from A.limacinum MYA-1381 , PrG3P: glycerol-3-phosphate dehydrogenase gene promoter from A.limacinum MYA-1381 , PrS35: Cauliflower mosaic virus (CaMV) S35 promoter, PrPolyU: polyubiquitin gene promoter from Schizochytrium ATCC 20888, Prhsp70/Prelf1 : hybrid dual promoter consisting of hsp70 gene promoter and elongation factor 1a gene promoter from A.limacinum MYA-1381 . rpl44*: ribosomal protein 44 mutant P56Q from A. limacinum MYA-1381 , ShBle: Bleomycin resistance gene from Streptomyces hindustanicus, gfp: green fluorescence protein mgfp5. CYC-T: cytochrome C terminator from S. cerevisiae, NosT: nopalin gene terminator from Agrobacterium tumefaciens Ti plasmid, EF1-T: elongation factor 1 terminator from Schizochytrium ATCC 20888. Spotted bars indicate viral 2A-linker peptides, which enable self-cleavage of the translational fusion proteins. Mottled grey sections between arrows represent the 2A linkers.

Figure 5 shows the intracellular targeting of heterologously expressed enzymes to the endoplasmic reticulum in accordance to previous work published by Zhao et al. (2016) and Okino et al. (2018) or into the mitochondria. A) Fusion of cytochrome P450 oxidase CrAO from Cantharantus roseus with a truncated version of cytochrome P450 reductase A45ATR1 from Arabidopsis thaliana. The N-terminal targeting signal of the oxidase CrAO was exchanged for the ER-targeting sequence of calreticulin from Aurantiochytrium limacinum MYA-1381. To retain the protein in the ER an ER retention signal was engineered at the C-terminus of the fusion protein. The same synthetic enzyme fusion strategy was applied to the C2 cytochrome P450 oxidase from Lagerstoemia speciosa which was linked to the cytochrom P450 reductase from Botryococcus braunii and equipped with an N-terminal ER targeting signal sequence from hsp70 (MYA-1381). B) Fusion of cytochrome P450 oxidase CrAO from Cantharantus roseus with ferredoxin from Aurantiochytrium limacinum MYA-1381. Similarly, the N-terminal targeting sequence of ferredoxin was used to guide the redox couple to the mitochondria of the acceptor strain. Figure 6 shows that the terpene biosynthesis in plants and certain algae has evolved in two independent pathways for supply of C5 isoprene precursor molecules isopentenyl pyrophosphat (IPP) and dimethylallyl pyrophosphate (DMAPP): 1) the cytoplasmic mevalonate (MEV) pathway and 2) the methylerythrol phosphate pathway (MEP) While MEV enzymes are localized in the cytosol, the MEP pathway is found in the chloroplast. MEP pathway: DXP: 1desoxy-D-xylulose-5-phosphate (DXS: DX-synthase), MEP: 2-C- methyl-D-erythrol-4-phosphate (DXR: DXP-reductoisomerase), CDP-ME: 4- diphosphocytidyl-2C-methyl-D-erythrol (MCT: methylerythrol-cytidyl-transferase), CDP- MEP: 4-diphosphocytidyl-2C-methyl-D-erythritol-2-P (CMK: Cytidylmethyl-Kinase), MME- cPP: 2C-Methyl-D-erythritol-2,4-cyclodiphosphat (MDS: Methylerythritol-cyclo- diphosphate-synthase), HMBPP: (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HDS: hydroxy-methyl-butenyldiphosphat-synthase), (HDR: HMBPP-reductase). MEV-pathway: (AACT: acetyl-CoA-acetyltransferase), HMG-CoA: 3-hydroxy-3methylglutaryl-CoA

(HMGS: HMG-CoA-synthase), MVA: mevalonate (HMGR: HMG-CoA-reductase), MVP: mevalonate-5phosphate (MVK: mevalonate kinase), MVPP: mevalonate-5-diphosphate (PMK: phosphomevalonate kinase), IPP: Isopentenyl pyrophosphate und DMAPP: dimethylallyl pyrophosphate (PMD: phosphomevalonate decarboxylase), (IDI: isopentenyl diphosphate-isomerase.

Figure 7 shows the plasmid map for the integration vector pdtrpE-BASIC. The vector enables stable genomic integration in Schizochytrium ATCC 20888. pdtrpE-BASIC may be used as a control vector expressing a 2A-linker peptide fusion of neoR (aminglycosid phsophotransferase gene) with the reporter gene xylE (catechol-2, 3-dioxygenase), both codonoptimized for the acceptor strain. The unique restriction sites Kpnl and Xbal further enable the exchange of the reporter gene neoR-2A-xylE for novel genes or gene clusters of interest. The vector is designed for application of in vivo gap-repair cloning in S.cerevisiae. dtrpE(A) & dtrpE(B): 2000 bp of upstream and downstream sequences flanking the anthranilate synthase (trpE) gene in Schizochytrium ATCC20888. The latter enable genomic integration of expression cassettes via homologous recombination. Genomic integration of the vector via double cross-over recombination leads to a deletion of the trpE gene. NeoR: aminoglycoside phosphotransferase (G418 resistance determinant), XylE: catechol-2, 3-dioxigenase from Pseudomonas putida, Prom(Tub): a- tubulin gene promoter from Schizochytrium ATCC20888, PrPolyU: polyubiquitin gene promoter from Schizochytrium ATCC 20888, NOS-Terminator: nopalin gene terminator from Agrobacterium tumefaciens Ti plasmid, ShBle: Bleomycin resistance gene from Streptomyces hindustanicus, gfp: green fluorescence protein mgfp5, oriV (ColE1): origin of replication for maintenance in in E.coli, bla: ampicillin/carbenicillin resistance marker, 2- Micron: replicon for maintenance in S. cerevisiae, Ura3: orotidine decarboxylase selection marker for application in uracil auxotrophic backround strains of S. cerevisiae. 2A: viral 2A- linker peptides, which enable self-cleavage of the translational fusion proteins. Spel restriction sites flanking dtrpE(A) upstream and dtrpE(B) downstream sequences enable the release of the expression cassette for subsequent transformation as linear DNA fragment.

Figure 8 shows the Plasmid map for the integration vector pdKU80(20888). The vector enables stable genomic integration in Schizochytrium ATCC 20888. pdKU80(20888) may be used as a control vector expressing a 2A-linker peptide fusion of neoR (aminglycosid phsophotransferase gene) with the reporter gene xylE (catechol-2, 3-dioxygenase), both codonoptimized for the acceptor strain. The unique restriction sites Hind III, Kpnl and Xbal further enable the exchange of the reporter gene expression cassette for novel genes or gene clusters of interest or additional 2A-based fusion genes. The vector is designed for appication of in vivo gap-repair cloning in S. cerevisiae. dKU80(20888A) & dKU80(20888B): 1800 bp of upstream and downstream sequences flanking the ku80 gene in Schizochytrium ATCC20888. The latter enable genomic integration of expression cassettes via homologous recombination. Genomic integration of the vector via double cross-over recombination leads to a deletion of the ku80 gene. NeoR: aminoglycoside phosphotransferase (G418 resistance determinant), XylE: catechol-2, 3-dioxigenase from Pseudomonas putida, Prom(Tub): a-tubulin gene promoter from Schizochytrium ATCC20888, EF1-T: elongation factor 1 terminator from A. limacinum ATCC MYA-1381 , oriV (ColE1): origin of replication for maintenance in in E.coli, bla: ampicillin/carbenicillin resistance marker, 2-Micron: replicon for maintenance in S. cerevisiae, Ura3: orotidine decarboxylase selection marker for application in uracil auxotrophic backround strains of S. cerevisiae. 2A: viral 2A-linker peptides, which enable self-cleavage of the translational fusion proteins. Spel restriction sites flanking KU80(20888A) upstream and KU80(20888B) downstream sequences enable the release of the expression cassette for subsequent transformation as linear DNA fragment.

Figure 9 shows the plasmid map for the integration vector pdKU80mya. The vector enables stable genomic integration in Aurantiochytrium limacinum ATCC MYA-1381. pdKU80mya may be used as a control vector expressing a fusion of neoR (aminglycosid phsophotransferase gene) with the reporter gene xylE (catechol-2, 3-dioxygenase). The unique restriction sites Xho I and Mlul further enable the exchange of the reporter gene xylE for novel genes or gene clusters of interest or additional 2A-based fusion genes. The vector is designed for appication of in vivo gap-repair cloning in S. cerevisiae. dKU(A) & dKU(B): 1800 bp of upstream and downstream sequences flanking the KU80 gene in Aurantiochytrium limacinum ATCC MYA-1381. The latter enable genomic integration of expression cassettes via homologous recombination. Genomic integration of the vector via double cross-over recombination leads to a deletion of the ku80 gene. NeoR: aminoglycoside phosphotransferase (G418 resistance determinant), XylE: catechol-2, 3- dioxigenase from Pseudomonas putida, Prom(Tub): Prhsp70/Prelfl : hybrid dual promoter consisting of hsp70 gene promoter and elongation factor 1a gene promoter from Aurantiochytrium limacinum MYA-1381 , NOS-Terminator: nopalin gene terminator from Agrobacterium tumefaciens Ti plasmid, oriV (ColE1): origin of replication for maintenance in in E.coli, bla: ampicillin/carbenicillin resistance marker, 2-Micron: replicon for maintenance in S. cerevisiae, Ura3: orotidine decarboxylase selection marker for application in uracil auxotrophic backround strains of S. cerevisiae. Spel restriction sites flanking dKU(A) upstream and dKU(B) downstream sequences enable the release of the expression cassette for subsequent transformation as linear DNA fragment. Figure 10 shows the Plasmid map for the integration vector pdCarS-BASIC. The vector enables stable genomic integration in Schizochytrium ATCC 20888. pdCarS-BASIC may be used as a control vector expressing a 2A-linker peptide fusion of neoR (aminglycosid phsophotransferase gene) with the reporter gene xylE (catechol-2, 3-dioxygenase), both codonoptimized forthe acceptor strain. The unique restriction sites Hind III and Xbal further enable the exchange of the reporter gene (neoR-2A-xylE) expression cassette for novel genes or gene clusters of interest or additional 2A-based fusion genes. The vector is designed for application of in vivo gap-repair cloning in S. cerevisiae. dCarS(A) &dCarS(B): 2000 bp of upstream and downstream sequences flanking the b-carotene synthase (carS) gene in Schizochytrium ATCC20888. The latter enable genomic integration of expression cassettes via homologous recombination. Genomic integration of the vector via double cross-over recombination leads to partial deletion of the carS gene. NeoR: aminoglycoside phosphotransferase (G418 resistance determinant), XylE: catechol-2, 3-dioxigenase from Pseudomonas putida, Prom(Tub): a-tubulin gene promoter from Schizochytrium ATCC20888, CYC1 -Terminator: cytochrome C terminator from S. cerevisiae, oriV (ColE1): origin of replication for maintenance in E.coli, bla: ampicillin/carbenicillin resistance marker, 2-Micron: replicon for maintenance in S. cerevisiae, Ura3: orotidine decarboxylase selection marker for application in uracil auxotrophic backround strains of S. cerevisiae. 2A: viral 2A-linker peptides, which enable self-cleavage of the translational fusion proteins. Spel restriction sites flanking dCarS(A) upstream and dCarS(B) downstream sequences enable the release of the expression cassette for subsequent transformation as linear DNA fragment. List of Sequences

SEQ ID NO: 1 amino acid sequence of amyrin synthase from Malus domestica (MdAS)

SEQ ID NO: 2 amino acid sequence of lupeol synthase from Rhicinus communis (LupS)

SEQ ID NO: 3 amino acid sequence of cytochrome P450 oxidase from Cantharantus roseus (CrAO) (CYP716AL1)

SEQ ID NO: 4 amino acid sequence of NADPH dependent cytochrome P450 reductase from Arabidopsis thaliana (ATR1)

SEQ ID NO: 5 amino acid sequence of cytochrome P450 monooxygenase from Lagerstroemia speciosa (LsCYP716) (CYP5)

SEQ ID NO: 6 amino acid sequence of NADPH dependent cytochrome P450 reductase from Botryococcus braunii (BbCPR)

SEQ ID NO: 7 amino acid sequence of HMG CoA-reductase from Schizochytrium ATCC20888 (HMG1)

SEQ ID NO: 8 amino acid sequence of the synthetic fusion protein ERSig(hsp70)- LsCYP716-A43BbCPR

SEQ ID NO: 9 amino acid sequence of the synthetic fusion protein ErSig(Calreticulin)- CrAO-45ATR-ERretSig(Calreticulin)

SEQ ID NO: 10 amino acid sequence of the synthetic fusion protein MdAS-2A- CrC28CYP/A45AtCPR

SEQ ID NO: 11 amino acid sequence of the synthetic fusion protein Erg1-(2A)-MdAS- (2A)-CrAO-(2A)-AtCPR

SEQ ID NO: 12 amino acid sequence of the synthetic fusion protein Fdx(MYA)Sig- CrAO-fdx(MYA)

SEQ ID NO: 13 amino acid sequence of the synthetic fusion protein LupS-2A-rpl44*- ShBle-gfp

SEQ ID NO: 14 amino acid sequence of the synthetic fusion protein NeoR-2A-XylE SEQ ID NO: 15 amino acid sequence of the synthetic fusion protein HMG1 (ATCC20888)-2A-erg9/erg20

SEQ ID NO: 16 amino acid sequence of the synthetic fusion protein MsmzMevK-2A- erg8-2A-erg19-2A-ldi1 -2A-erg10-2A-erg13

SEQ ID NO: 17 nucleic acid sequence of plasmid p18S-P1 with vector backbone p18S and insert IPPmya, tHMGR (D3 nt-AS) LupS-rpl44-ShBle-gfp

SEQ ID NO: 18 nucleic acid sequence of plasmid p18S-P2 with vector backbone p18S and insert IPPmya, Sc-tHMGR, LupS-rpl44-ShBle-gfp SEQ ID NO: 19 nucleic acid sequence of plasmid p18S-P3 with vector backbone p18S and insert Sc-tHMGR, LupS-rpl44-ShBle-gfp

SEQ ID NO: 20 nucleic acid sequence of plasmid p18S-P4 with vector backbone p18S and insert Sc-tHMGR (K12->M)), erg10-(2A)-erg13 (R1042->H), LupS- rpl44-ShBle-gfp

SEQ ID NO: 21 nucleic acid sequence of plasmid p18S-P5 with vector backbone p18S and insert tHMGR (A2AS OK, erg10-(2A)-erg13 (A1AS), LupS-rpl44- ShBle-gfp

SEQ ID NO: 22 nucleic acid sequence of plasmid p18S-P6 with vector backbone p18S and insert SQSmya-Erg1 (D4 AS), MITOSig(fdx)CrAO-fdx, LupS-rpl44- ShBle-gfp

SEQ ID NO: 23 nucleic acid sequence of plasmid p18S-P7 with vector backbone p18S and insert SQSmya-Erg1 present (D4 AS), (ERCalRet)CrAO- ATR(CalRetERret) LupS-rpl44-ShBle-gfp

SEQ ID NO: 24 nucleic acid sequence of plasmid p18S-P8 with vector backbone p18S and insert SQSmya-erg1 (A4AS), (ERCalRet)CrAO-ATR(CalRetERret) (AZAS), LupS-rpl44-ShBle-gfp

SEQ ID NO: 25 nucleic acid sequence of plasmid p18S-P9 with vector backbone p18S and insert Sc-tHMGR, LupS-rpl44-ShBle-gfp

SEQ ID NO: 26 nucleic acid sequence of plasmid p18S-P13 with vector backbone p18S and insert PrPK-LupS-rpl44*-ShBle-gfp

SEQ ID NO: 27 nucleic acid sequence of plasmid p18S-P15 with vector backbone p18S and insert SQSmya-erg20-(2A)-tHMGR (CO)

SEQ ID NO: 28 nucleic acid sequence of plasmid p18S-P18 with vector backbone p18S and insert SQSmya-erg1 (W806®R), LupS-rpl44-ShBle-gfp

SEQ ID NO: 29 nucleic acid sequence of plasmid p18S-3g with vector backbone p18S and insert TeSQS-2A-SctHMGR, LupS-rpl44-ShBle-gfp

SEQ ID NO: 30 nucleic acid sequence of plasmid p18S-2A(ll) with vector backbone p18S and insert PrTub-Erg10-erg13-tHMGR

SEQ ID NO: 31 nucleic acid sequence of plasmid p18SS-3(4) with vector backbone p18S and insert PrTub-tHMGR-erg9/erg20

SEQ ID NO: 32 nucleic acid sequence of plasmid pALanS-PP18 with vector backbone pALanS and insert PrPK-gfp-KanMX fusion (N185®S)

SEQ ID NO: 33 nucleic acid sequence of plasmid pALanS-PP20 with vector backbone pALanS

SEQ ID NO: 34 nucleic acid sequence of plasmid pALanS-PP15 with vector backbone pALanS and insert PrPK-LupS-rpl44*-ShBle-gfp SEQ ID NO: 35 nucleic acid sequence of plasmid pALanS-PP17 with vector backbone pALanS and insert PrG3P-TriT-rpl44*-ShBle-gfp

SEQ ID NO: 36 nucleic acid sequence of plasmid pALanS-PP13 with vector backbone pALanS and insert PrPK-SQSmya-erg1 , LupS-rpl44*-ShBle-gfp

SEQ ID NO: 37 nucleic acid sequence of plasmid pAKU80mya-M1 with vector backbone pAKU80mya and insert Prhsp70/elfl-NeoR-MdAS-

Term(elf1)/PrS35-CrCYP-A45AtATR-NosTerm

SEQ ID NO: 38 nucleic acid sequence of plasmid pAKU80mya-M2 with vector backbone pAKU80mya and insert Prhsp70/elfl-NeoR-xylE -NosTerm

SEQ ID NO: 39 nucleic acid sequence of plasmid pAKU80mya-M3 with vector backbone pAKU80mya and insert Prhsp70/elfl-NeoR-2A-rad52-

NosTerm

SEQ ID NO: 40 nucleic acid sequence of plasmid pAKU80mya-M4 with vector backbone pAKU80mya and insert Prhsp70/elf1-NeoR-MdAS- Term(elf1)/PrS35-CrCYP-fdx(mya)-NosTerm

SEQ ID NO: 41 nucleic acid sequence of plasmid pAKU20888-S1 with vector backbone pAKU20888 and insert PrTub-NeoR-2A-XylE-Term(elf1)

SEQ ID NO: 42 nucleic acid sequence of plasmid pAKU20888-S2 with vector backbone pAKU20888 and insert PrTub-NeoR-2A-XylE-2A-MdAS-

Term(elf1)/PrS35-CrCYP-A45AtATR-NosTerm

SEQ ID NO: 43 nucleic acid sequence of plasmid pAKU20888-S3 with vector backbone pAKU20888 and insert PrTub-NeoR-2A-XylE-2A-LupS-

Term(elf1)/PrS35-CrCYP-A45AtATR-NosTerm

SEQ ID NO: 44 nucleic acid sequence of plasmid pAKU20888-S4 with vector backbone pAKU20888 and insert PrTub-NeoR-2A-XylE-2A-MdAS-

Term(elf1)/PrS35-CrCYP-A45AtATR-NosTerm-Rr-LsCYP-BbCPR

SEQ ID NO: 45 nucleic acid sequence of plasmid pAKU20888-S5 with vector backbone pAKU20888 and insert PrTub-NeoR-2A-XylE-2A-LupS-

Term(elf1)/PrS35-CrCYP-A45AtATR-NosTerm-Pr(PolyU)-LsCYP-

BbCPR

SEQ ID NO: 46 nucleic acid sequence of plasmid pAKU20888-S6 with vector backbone pAKU20888 and insert PrTub-NeoR-2A-XylE-2A-MdAS-

Term(elf1 )/PrS35-CrCYP-Fdx(mya)-NosT erm

SEQ ID NO: 47 nucleic acid sequence of plasmid pAtrp-BASIC with vector backbone pAtrp and insert PrTub-NeoR-XylE-Pr(PolyU)-ShBle-gfp SEQ ID NO: 48 nucleic acid sequence of plasmid pAtrp-2A(ll) with vector backbone pAtrp and insert PrTub-Erg10-erg13-tHMGR

SEQ ID NO: 49 nucleic acid sequence of plasmid pAtrp-3(4) with vector backbone pAtrp and insert PrTub-tHMGR-erg9/erg20

SEQ ID NO: 50 nucleic acid sequence of plasmid pAtrp-Mev6(2) with vector backbone pAtrp and insert PrTub-HMG1 (20888)-erg9/erg20, MsmzMevK-erg8- 2A-erg19-2A-ldi1 -2A-erg10-2A-erg13-Pr(PolyU)-ShBle-gfp

SEQ ID NO: 51 nucleic acid sequence of plasmid pACarS-BASIC with vector backbone pACarS and insert PrTub-NeoR-XylE

SEQ ID NO: 52 nucleic acid sequence of plasmid pACarS-15b(A3) with vector backbone pACarS and insert PrTub-Erg1-MdAS

SEQ ID NO: 53 nucleic acid sequence of plasmid pACarS-14(1) with vector backbone pACarS and insert PrTub-Erg1-MdAS-CrCYP-AtCPR

SEQ ID NO: 54 nucleic acid sequence of plasmid p18S-P38 SEQ ID NO: 55 nucleic acid sequence of plasmid p18-P39 SEQ ID NO: 56 amino acid sequence of viral 2A linker peptide F2A from Foot & Mouth Disease Virus

SEQ ID NO: 57 amino acid sequence of viral 2A linker peptide P2A from Porcine Teschovirus 1

SEQ ID NO: 58 amino acid sequence of viral 2A linker peptide T2A from Thosea Asigna Virus

SEQ ID NO: 59 amino acid sequence of viral 2A linker peptide E2A from Equine Rhinitis A Virus

SEQ ID NO: 60 nucleic acid sequence of primer P1 CHrpl/gfp/ SEQ ID NO: 61 nucleic acid sequence of primer P2CHrpl/gfp / SEQ ID NO: 62 nucleic acid sequence of primer P1 CH18S / SEQ ID NO: 63 nucleic acid sequence of primer P2CH18S/ SEQ ID NO: 64 nucleic acid sequence of primer P1 CHKU80mya/ SEQ ID NO: 65 nucleic acid sequence of primer P2CHKU80mya/ SEQ ID NO: 66 nucleic acid sequence of primer P1CHKU80(20888)/ SEQ ID NO: 67 nucleic acid sequence of primer P2CHKU80(20888)/ SEQ ID NO: 68 nucleic acid sequence of primer P1 CHtrpE SEQ ID NO: 69 nucleic acid sequence of primer P2CHtrpE/ SEQ ID NO: 70 nucleic acid sequence of primer P1 p-CarS/

SEQ ID NO: 71 nucleic acid sequence of primer P2p-CarS/ SEQ ID NO: 72 nucleic acid sequence of primer P1 CHIanS/ SEQ ID NO: 73 nucleic acid sequence of primer P2CHIanS/ SEQ ID NO: 74 amino acid sequence of dammarenediol synthase from Panax ginseng SEQ ID NO: 75 amino acid sequence of cucurbitadienol synthase from Siraitia grosvenorii

SEQ ID NO: 76 amino acid sequence of squalene synthase from Aurantiochytrium limacinum ATCC MYA1381

SEQ ID NO: 77 amino acid sequence of squalene synthase from Thermosynechococcus elongatus BP-1

SEQ ID NO: 78 amino acid sequence of squalene synthase from Saccharomyces cerevisiae ATCC 204508

SEQ ID NO: 79 nucleic acid sequence of plasmid pAKU20888-S1 with vector backbone pAKU20888 and insert PrTub-NeoR-2A-XylE-Term(elf1)

SEQ ID NO: 80 nucleic acid sequence of plasmid pAKU80mya-M2 with vector backbone pAKU80mya and insert Prhsp70/elf1-NeoR-xylE -NosTerm

SEQ ID NO: 81 nucleic acid sequence of plasmid pAKU20888-S1 with vector backbone pAKU20888 and insert PrTub-NeoR-2A-XylE-Term(elf1) SEQ ID NO: 82 nucleic acid sequence of plasmid pAtrp-BASIC with vector backbone pAtrp and insert PrTub-NeoR-XylE-Pr(PolyU)-ShBle-gfp

SEQ ID NO: 83 nucleic acid sequence of plasmid pACarS-BASIC with vector backbone pACarS and insert PrTub-NeoR-XylE

SEQ ID NO: 84 nucleic acid sequence of plasmid pAKU80mya-AmS with vector backbone pAKU80mya and insert AmS

SEQ ID NO: 85 nucleic acid sequence of plasmid pAKU80mya-DamDS with vector backbone pAKU80mya and insert DamDS SEQ ID NO: 86 nucleic acid sequence of plasmid pAKU80mya-CucDS with vector backbone pAKU80mya and insert CucDS

SEQ ID NO: 87 nucleic acid sequence of plasmid pAKU80mya-AmS/CYPC2/ATR-V1 with vector backbone pAKU80mya and insert AmS/CYPC2/ATR-V1 SEQ ID NO: 88 nucleic acid sequence of plasmid pAKU80mya-AmS/CYPC2/ATR-V2 with vector backbone pAKU80mya and insert AmS/CYPC2/ATR-V2

SEQ ID NO: 89 nucleic acid sequence of plasmid pAKU80mya-AmS/CYPC2/fdx with vector backbone pAKU80mya and insert AmS/CYPC2/fdx

SEQ ID NO: 90 nucleic acid sequence of plasmid pAKU20888-AmS/CYPC2/ATR with vector backbone pAKU80(20888) and insert AmS/CYPC2/ATR

SEQ ID NO: 91 nucleic acid sequence of plasmid pAKU20888-AmS/CYPC2/fdx with vector backbone pAKU80(20888) and insert AmS/CYPC2/fdx

SEQ ID NO: 92 nucleic acid sequence of plasmid pAKU20888-LupS/CYPC2/ATR with vector backbone pAKU80(20888) and insert LupS/CYPC2/ATR SEQ ID NO: 93 nucleic acid sequence of plasmid pAKU20888-LupS/CYPC2/fdx with vector backbone pAKU80(20888) and insert LupS/CYPC2/fdx

SEQ ID NO: 94 nucleic acid sequence of plasmid pCAMBIA-Yae-trpE-BASIC with vector backbone pCAMBIA-Yae and insert lacZ/AmS

SEQ ID NO: 95 nucleic acid sequence of plasmid pCAMBIA-Yae-KU80mya-neoR-lacZ- AmS with vector backbone pCAMBIA-Yae and insert KU80mya-neoR- lacZ-AmS

SEQ ID NO: 96 protein sequence of neomycin resistance protein

SEQ ID NO: 97 protein sequence of XylE

SEQ ID NO: 98 nucleic acid sequence of crRNA sequence of first oligonucleotide 1 in table 6

SEQ ID NO: 99 nucleic acid sequence of crRNA sequence of second oligonucleotide 1 in table 6 SEQ ID NO: 100 nucleic acid sequence of crRNA sequence of third oligonucleotide 1 in table 6

SEQ ID NO: 101 nucleic acid sequence of crRNA sequence of fourth oligonucleotide 1 in table 6

SEQ ID NO: 102 nucleic acid sequence of first oligonucleotide 1 in table 6 SEQ ID NO: 103 nucleic acid sequence of second oligonucleotide 1 in table 6 SEQ ID NO: 104 nucleic acid sequence of third oligonucleotide 1 in table 6 SEQ ID NO: 105 nucleic acid sequence of fourth oligonucleotide 1 in table 6 SEQ ID NO: 106 nucleic acid sequence of oligonucleotide 2 in table 6 SEQ ID NO: 107 part of the vector backbone carrying the 2 micron replicon SEQ ID NO: 108 nucleic acid sequence of PrTeF1-ShBle-CYCTerm-CEN6ArsH4 artificial chromosome sequence

SEQ ID NO: 109 nucleic acid sequence of panARS-oriT artificial chromosome sequence

Detailed Description The present invention relates to genetically modified strains and corresponding bioprocess for the production of heterologous terpenes, exemplified by but not limited to triterpenes such as lupeol, oleanolic acid, ursolic acid, betulinic acid, corosolic acid and maslinic acid as well as to metabolic modifications to improve the supply of biosynthetic precursor molecules. The invention further provides a novel genetic platform technology for metabolic engineering in Labyrinthulomycota, in particular Thraustochytrids based on the application of biosynthetic biobricks for combinatorial biosynthesis of various terpene molecules as well as vectors compatible with one step, multifragment, in vivo gap-repair cloning in Saccharomyces cerevisiae.

Several strategies have been explored in the context of the present invention: 1) the nuclear expression and ER targeting of triterpene biosynthetic enzymes from plant origin based on codonoptimized genes and polycistronic expression using translationally self-cleavable 2A- linker peptides. 2) the overexpression of several heterologous enzymatic features of the mevalonate pathway in addition to the biosynthetic repertoire already present in the wild type acceptor strains. 3) the deregulation of feedback inhibited key enzymatic steps such as the HMG-CoA reductase and the mevalonate kinase by expression of heterologous enzymes without feedback control 4) the channelling of metabolism at branching points by expression of novel, multifunctional fusion proteins such as hybrid squalene- synthase/farnesyl pyrophosphate synthase, squalene synthase/squalene epoxidase and cytochrome P450 oxidase/reductase to improve availability of precursors and direct metabolic flux towards the desired product molecules 5) the blocking of competing metabolic routes by gene deletions and 6) the nuclear expression and mitochondrial targeting of seven enzymes of the methyl erythrol phosphate pathway from the thermophilic cyanobacterium Mastigocladus laminosus.

Even though it was known that certain Labyrinthulomycota are able to produce significant quantities of squalene, it has not been attempted to employ these organisms to synthesize plant derived triterpenes. While the microorganisms have the natural ability to produce squalene as a precursor for the desired triterpenes, genetic engineering is required to provide them with the ability to convert squalene into different triterpenes. To this end, suitable enzymes need to be identified, which can be heterologously expressed in the host. Moreover, in order to reach a reasonable productivity, modification of native metabolic pathways is necessary in order to address production limiting factors such as feedback regulation mechanisms and competing metabolic pathways. It was not known, whether Labyrinthulomycota would be capable of providing the desired activities and could be engineered to synthesize the triterpenes with high productivity avoiding detrimental toxicity effects of the products.

In a fist aspect, the present invention provides a microorganism of the class Labyrinthulomycota comprising at least one transgene encoding a heterologous 2,3- oxidosqualene cyclase.

Labyrinthulomycota or Labyrinthulomycetes are a class of mostly marine protists, which produce an ectoplasmatic network of filaments. The two main groups of Labyrinthulomycota are labyrinthulids and thraustochytrids. Microorganisms of the class Labyrinthulomycota were found in the context of the present invention to be able to express a heterologous 2,3-oxidosqualene cyclase and form triterpenes, which naturally only occur in plants. Preferably, the heterologous 2,3-oxidosqualene cyclase is selected from the group consisting of an alpha-amyrin synthase, a beta-amyrin synthase, a lupeol synthase, a dammarenediol synthase and a cucurbitadienol synthase.

Expression of an alpha-amyrin synthase and a beta-amyrin synthase facilitates the formation of alpha-amyrin and beta-amyrin, respectively, from 2,3-oxidosqualene, which is naturally formed in the organism from squalene. Lupeol is formed from 2,3-oxidosqualene by lupeol synthase. Lupeol, alpha-amyrin and beta-amyrin can be converted to oleanolic acid, ursolic acid, betulinic acid, corosolic acid and maslinic acid. Dammarenediol, which is formed by a dammarenediol synthase, can be converted to protopanaxadiol by a cytochrome P450 oxidase and then - by glycosylation with glycosyl transferases - ginsenosides are formed, which represent a group of compounds with various biological effects.

A suitable heterologous beta-amyrin synthase to be introduced as a transgene is the amyrin synthase from Malus domestica (MdOSCI Oder MdAS, ACM89977.1) with the sequence represented by SEQ ID NO: 1. A suitable heterologous lupeol synthase to be introduced as a transgene is the lupeol synthase from Rhizinus communis (LupS, NP001310684.1) with the sequence represented by SEQ ID NO: 2. A suitable heterologous dammarenediol synthase to be introduced as a transgene is the dammarenediol synthase from Panax ginseng (AE027862.1) with the sequence represented by SEQ ID NO: 74. A suitable heterologous cucurbitadienol synthase to be introduced as a transgene is the cucurbitadienol synthase from Siraitia grosvenorii (AEM42982.1) with the sequence represented by SEQ ID NO: 75.

The above mentioned amyrin synthase, cucurbitadienol synthase, dammarenediol synthase and lupeol synthase are described in Andre et al. 2016, Dai et al. 2015, Hu et al. 2013 and Guhling et al. 2006.

In one embodiment of the microorganism according to any embodiment described above, the 2,3-oxidosqualene cyclase has an amino acid sequence selected from the sequences of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 74 and SEQ ID NO: 75 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95% to the sequences of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 74 or SEQ ID NO: 75. In a preferred embodiment, the microorganism described above is selected from the group consisting of Thraustochytrids, in particular Schizochytrium, Aurantiochytrium, Thraustochytrium and Hondea.

The order Thraustochytridiales comprises the genera Schizochytrium, Aurantiochytrium, Thraustochytrium, Botryochytrium, Parietichytrium, Aplanochytrium, Labyrinthuloides, Oblongichytrium, Sicyoidochytrium, Japonochytrium, Ulkenia and the novel genus Hondea as suggested by Dellero et al. (2018). Hondea has been suggested as a new genus and it is possible that Schizochytrium sp. S31 (ATCC 20888) is renamed as a Hondea strain. Therefore, Hondea is covered by the genera. Thraustochytrids represent an order of Labyrinthulomycetes, which includes the genera Schizochytrium, Aurantiochytrium and Thraustochytrium. Suitable strains of Thraustochytrids are Schizochytrium sp. S31 (ATCC 20888), Aurantiochytrium sp. T66 (ATCC-PRA-276), Thraustochytrium sp. S-3 (ATCC-26185) and Aurantiochytrium limacinum SR 21 (ATCC-MYA-1381). These strains are publicly available from the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110 USA.

In one embodiment, the microorganism in any of the embodiments described is selected from Schizochytrium sp. S31 (ATCC 20888), Aurantiochytrium sp. T66 (ATCC-PRA-276), Thraustochytrium sp. S-3 (ATCC-26185) and Aurantiochytrium limacinum SR 21 (ATCC- MYA-1381).

In orderto obtain a wider range of triterpenes from the microorganism, further enzymes are provided as transgenes, in particular to efficiently convert alpha-amyrin, beta-amyrin und lupeol to oleanolic acid, ursolic acid, betulinic acid, corosolic acid and maslinic acid.

In one embodiment, the microorganism described above further comprises at least one transgene encoding a cytochrome P450 oxidase and, optionally, a cytochrome P450 reductase or a ferredoxin, preferably a hybrid cytochrome P450 oxidase/reductase or a hybrid cytochrome P450 oxidase/ferredoxin. Preferably, the at least one transgene encoding a cytochrome P450 oxidase and/or a cytochrome P450 reductase or a hybrid cytochrome P450 oxidase/reductase is fused to an endoplasmatic reticulum (ER) targeting sequence and/or an ER retention sequence or the ferredoxin or the hybrid cytochrome P450 oxidase/ferredoxin is fused to a mitochondrial targeting sequence. Targeting and retaining the cytochrome P450 oxidase or hybrid cytochrome P450 oxidase/reductase to the endoplasmatic reticulum (ER) as shown in figure 5 increases the efficiency of the biosynthetic reaction (kinetics) by the concept of separation of consecutive enzymatic reactions into two compartments. ER membrane attachment facilitates folding and further stabilizes proteins (Chen et al. 2016) (Arendt et al. 2017, Kim et al. 2019).

A suitable cytochrome P450 oxidase may be derived from Cantharantus roseus (CrAO, CYP716AL1) and is represented by the sequence of SEQ ID NO: 3 or from Lagerstroemia speciosa (LsCYP716, CYP5) represented by the sequence SEQ ID NO: 5. A suitable cytochrome P450 reductase may be derived from Arabidopsis thaliana (ATR1) and is represented by the sequence of SEQ ID NO: 4 or from Botryococcus braunii (BbCPR) represented by the sequence of SEQ ID NO: 6.

The cytochrome P450 oxidase from Cantharantus roseus is described in Huang et al. 2012. The cytochrome P450 reductase from Botryococcus braunii is described in Tsou et al. 2017. A cytochrome P450 oxidase from banba tree is described in Sandeep et al. 2017. The cytochrome P450 reductase from Arabidopsis thaliana is described in Urban et al. 1997.

In one embodiment of the microorganism described above, the cytochrome P450 oxidase has an amino acid sequence selected from the sequences of SEQ ID NO: 3 and SEQ ID NO: 5 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95% to the sequences of SEQ ID NO: 3 or SEQ ID NO: 5.

The cytochrome P450 oxidase can be provided as a hybrid or fusion protein with a cytochrome P450 reductase or a ferredoxin, which is able to regenerate the cofactor and thus enhance the redox efficiency of the hybrid enzyme.

In one embodiment of the microorganism described above, the cytochrome P450 reductase has an amino acid sequence selected from the sequences of SEQ ID NO: 4 and SEQ ID NO: 6 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95% to the sequences of SEQ ID NO: 4 or SEQ ID NO: 6.

The amino acid sequence of a fusion protein cytochrome P450 monooxygenase LsCYP716 from Lagerstroemia speciosa with cytochrome P450 reductase BbCPR from Botyrococcus braunii with an ER-targeting signal is represented by the sequence of SEQ ID NO: 8. The amino acid sequence of a fusion protein cytochrome P450 oxidase CrAO from Cantharanthus roseus with cytochrome P450 reductase from Arabidopsis thaliana (ATR) with an ER-targeting and an ER-retention sequence is represented by the sequence of SEQ ID NO: 9.

In one embodiment of the microorganism described above, the hybrid cytochrome P450 oxidase/reductase has an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95% to the sequences of SEQ ID NO: 8 or SEQ ID NO: 9.

The amino acid sequence of a fusion protein cytochrome P450 oxidase CrAO from Cantharanthus roseus with a ferredoxin is represented by the sequence of SEQ ID NO: 12.

In one embodiment of the microorganism described above, the hybrid cytochrome P450 oxidase/ferredoxin has an amino acid sequence of SEQ ID NO: 12 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95% to the sequences of SEQ ID NO: 12.

The transgene encoding the 2,3-oxidosqualene cyclase may also be provided to express a fusion protein combining the 2,3-oxidosqualene cyclase with the cytochrome P450 oxidase or hybrid cytochrome P450 oxidase/reductase. The amino acid sequence of a synthetic fusion protein comprising amyrin synthase from Malus domestica with a hybrid cytochrome P450 oxidase/reductase is represented by the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 11.

In one embodiment, the microorganism described above therefore encodes a fusion protein having an amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95% to the sequences of SEQ ID NO: 10 or SEQ ID NO: 11.

The microorganisms of the present invention naturally have the ability to produce squalene, which represents a precursor for the production of the desired triterpenes (figure 1). In order to increase the availability of the precursor to a commercially viable level, however, one strategy is to overexpress the enzymes required for squalene synthesis, namely farnesyl pyrophosphate synthase (FPPS), which catalyzes the conversion of geranyl pyrophosphate (GPP) to farnesyl pyrophosphate (FPP) and squalene synthase (SQS), which catalyzes the conversion of FPP to squalene. Furthermore, two enzymes catalyzing subsequent reactions, may be provided as a hybrid or fusion protein providing both activities which may potentially lead to intramolecular transfer of intermediates or redox equivalents and thus more efficiently channel the metabolic flow at a biosynthetic branching point towards the desired product, as reviewed by Aalbers & Fraajie 2019. In one embodiment of the microorganism according of any of the embodiments described above, the microorganism further comprises a transgene for the overexpression of a squalene synthase and/or a squalene epoxidase under the control of a strong constitutive promoter, preferably a transgene for the overexpression of a hybrid squalene syn- thase/farnesyl pyrophosphate synthase and/or a hybrid squalene synthase/squalene epoxidase.

The squalene synthase, squalene epoxidase and farnesyl pyrophosphate synthase provided as transgene may be the same as the respective endogenous enzyme of the host microorganism. Alternatively, the enzymes can be derived from a different organism. Overexpression is achieved by providing the transgene(s) together with and under the control of a strong, constitutive promoter. Suitable promoters are PrPK: pyruvate kinase gene promoter from A. limacinum MYA-1381 , a-tubulin gene promoter from Schizochytrium ATCC 20888, PrPolyUbi: polyubiquitin promoter from Schizochytrium ATCC 20888, PrG3P: glycerol-3-phosphate dehydrogenase gene promoter from A. limacinum MYA- 1381 , Prhsp70/Prelf1cr hybrid of heat shock gene hsp70 promoter and elongation factor 1 gene promoter from A. limacinum MYA-1381 or Schizochytrium ATCC 20888. PrS35: Cauliflower mosaic virus (CaMV) S35 promoter.

A suitable squalene synthase may be derived from Aurantiochytrium limacinum ATCC MYA1381 , from Thermosynechococcus elongatus or from Saccharomyces cerevisiae ATCC 204508 as represented by the sequences of SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78.

The above mentioned squalene synthases are described in Zhu et al. 2014, Lee & Poulter 2008 and Zhang et al. 1993.

In one embodiment of the microorganism described above, the transgene encoding a squalene synthase encodes an amino acid sequence selected from any of SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95% to the sequences of SEQ ID NO: 76, SEQ ID NO: 77 or SEQ ID NO: 78.

A further strategy to increase the availability of triterpene precursors is the overexpression of enzymes of the mevalonate pathway depicted on the left side of figure 1 .

In one embodiment of the microorganism according of any of the embodiments described above, the microorganism carries at least one further transgene for the overexpression of enzymes of the mevalonate pathway under the control of a strong constitutive promoter, preferably selected from the group consisting of acetyl-CoA-acetyltransferase, 3-hydroxy- 3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, dihydrophosphomevalonate decarboxylase and isopentenyl pyrophosphate isomerase. In a preferred embodiment the microorganism described above carries at least two, at least three, at least four, at least five at least six or all seven of the transgenes for the overexpression of enzymes of the mevalonate pathway recited above.

The transgenes provided may be the same as the respective endogenous enzymes of the host microorganism. Again, overexpression of the respective transgene(s) is achieved by providing the transgene(s) together with and under the control of a strong, constitutive promoter. The amino acid sequence of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG CoA reductase of Schizochytrium ATCC 20888 is represented by the sequence of SEQ ID NO: 7.

By using a truncated HMG CoA reductase as transgene, which does not comprise the sensor domain, feedback inhibition of the enzyme can be circumvented and thus the productivity of the enzyme can be increased. Alternatively, a heterologous enzyme may be used, which does not naturally possess a significant feedback control such as the mevalonate kinase derived from Methanosarzina mazeii (Primak et al. 2011).

In one embodiment of the microorganism according of any of the embodiments described above, the microorganism carries a transgene encoding a truncated 3-hydroxy-3- methylglutaryl-CoA reductase, which is missing the regulatory sensor domain and/or a transgene encoding a heterologous mevalonate kinase derived from Methanosarzina mazeii.

A truncated 3-hydroxy-3-methylglutaryl-CoA reductase, which is missing the regulatory sensor domain is described in (Polakowski et al. 1998).

There are endogenous metabolic pathways present in the microorganisms of the present invention, which branch off from the synthesis of squalene and thus potentially result in reduced availability of squalene as precursor for the desired triterpenes. In addition, instead of being converted to the desired triterpenes, 2,3-oxidosqualene may be converted to lanosterol by the endogenous lanosterol synthase. In order to increase the availability of the precursors, such competing pathways can be blocked by inactivating the key enzymes. In one embodiment of the microorganism according to any of the embodiments described above, at least one endogenous gene selected from the group consisting of lanosterol synthase, b-carotene synthase, ku80 and anthranilate synthase is inactivated.

Inactivation of a gene may be achieved by deletion of the gene or a relevant part of the gene or, more preferably, by insertion of at least one of the transgenes into the endogenous genomic locus of the gene to be inactivated. The insertion of (several) genes disrupts the locus in such a way, that the expression of the endogenous gene is no longer possible.

Preferably, the microorganism according to the invention is able to stably pass the ability to produce plant derived triterpenes to its progeny. Therefore, it is desirable to integrate the transgene(s) into the genome.

In one embodiment of the microorganism according to any of the embodiments described above, at least one transgene, preferably all transgenes, are integrated into the genome of the microorganism. Particularly preferably, the transgene(s) is/are inserted into one or more of the lanosterol synthase, the b-carotene synthase, the ku80, the anthranilate synthase and the 18 S rDNA locus in the genome of the microorganism.

Integration into the genome can e.g. be achieved by homologous recombination as depicted in figure 2. In this case, the expression construct(s) encoding the transgene(s) is/are provided with flanking sequences, which are homologous to corresponding sequences in the genome of the host microorganism. The transgene(s) is/are inserted by double cross-over integration. Preferably, the flanking sequences of the expression construct(s) are homologous to the flanking sequences of the endogenous lanosterol synthase, b-carotene synthase, ku80, anthranilate synthase and/or the 18 S rDNA genes resulting in inactivation of at least one of these genes.

In a further aspect, the present invention relates to a nucleic acid construct or set of nucleic acid constructs encoding

(i) at least one 2,3-oxidosqualene cyclase, preferably selected from the group consisting of an alpha-amyrin synthase, a beta-amyrin synthase, a lupeol synthase, a dammarenediol synthase and a cucurbitadienol synthase; and at least one of (ii) a cytochrome P450 oxidase and, optionally, a cytochrome P450 reductase or a ferredoxin, preferably a hybrid cytochrome P450 oxidase/reductase or a hybrid cytochrome P450 oxidase/ferredoxin, and further preferably the at least one transgene encoding a cytochrome P450 oxidase and/or a cytochrome P450 reductase or a hybrid cytochrome P450 oxidase/reductase is fused to an endoplasmatic reticulum (ER) targeting sequence and/or an ER retention sequence or the ferredoxin or the hybrid cytochrome P450 oxidase/ferredoxin is fused to a mitochondrial targeting sequence; and

(iii) a squalene synthase and/or a squalene epoxidase, preferably a hybrid squa- lene synthase/farnesyl pyrophosphate synthase and/or a hybrid squalene syn- thase/squalene epoxidase; and

(iv) at least one enzyme selected from the group consisting of acetyl-CoA- acetyltransferase, 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3- methylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, dihydrophosphomevalonate decarboxylase and isopentenyl pyrophos-phate isomerase, preferably a truncated 3-hydroxy-3-methylglutaryl-CoA reductase, which is missing the regulatory sensor domain and/or a heterologous mevalonate kinase derived from Methanosarzina mazeii.

Preferably, two or more of the genes recited in (i) to (iv) are fused to encode multienzymatic translational fusion proteins separated by self-cleavable 2A peptides. Further preferably, the construct or set of constructs is codon optimized to be expressed in Labyrinthulomycota, in particular in Thraustochytrids, preferably Schizochytrium, Aurantiochytrium, Thraustochytrium and Hondea.

Further preferably, in the nucleic acid construct or set of nucleic acid constructs described above, the enzymes encoded in (i) to (iv) are under the control of a strong constitutive promoter.

In a preferred embodiment of a set of nucleic acid constructs as described above, the at least one 2,3-oxidosqualene cyclase and the at least one cytochrome P450 oxidase, preferably the hybrid cytochrome P450 oxidase/reductase, are encoded by the same construct and one or more of the enzymes listed under (iii) and/or (iv) are encoded on a separate construct.

The amino acid sequence of a fusion protein comprising the amyrin synthase from Malus domestica and a hybrid cytochrome P450 oxidase/reductase is represented by the amino acid sequence of SEQ ID NO: 10 and SEQ ID NO: 11. In one embodiment, the nucleic acid construct described above encodes a fusion protein having an amino acid sequence of SEQ ID NO: 10 orSEQ ID NO: 10 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95% to the sequences of SEQ ID NO: 10 or SEQ ID NO: 11 . The genes encoded by one construct as described above can be fused, i.e. combined to encode a multienzymatic translational fusion protein, in which the genes are separated by self-cleavable 2A peptides. This setup may require only one promoter upstream and one terminator downstream of the encoding sequences. The self-cleavable 2A peptides cleave the polypeptide being translated from the construct into the separate functional gene products of the fused genes. Since the cleavage efficiency of 2A-peptide linkers is usually in the range of 50-90% (Luke & Ryan 2013, Souza-Moreira et al. 2018), its application in metabolic engineering leads to a mixture of cleaved single enzymes and polyenzymatic fusion proteins, which positively affect the kinetics of metabolic substrate to product conversion. Figure 3 shows a schematic drawing of DNA cassettes for heterologous expression of triterpene biosynthetic features. Figure 4 shows a schematic drawing of DNA cassettes for overexpression of structural features of terpene precursor biosynthesis.

In a further aspect, the present invention relates to a vector or a set of vectors encoding a nucleic acid construct or set of nucleic acid constructs as described in any of the embodiments above.

In a preferred embodiment, a vector according to the invention has a sequence represented by a sequence selected from the sequences of SEQ ID NOs: 22 to 24, 35, 37, 40, 42, 44 to 46, 52 and 53, or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95% to the sequences of SEQ ID NOs: 22 to 24, 35, 37, 40, 42, 44 to 46, 52 and 53.

In a further preferred embodiment, a vector according to the invention has a sequence represented by a sequence selected from the sequences of SEQ ID NOs: 17 to 21 , 25 to 29, 33, 34, 36, 54 and 55 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95% to the sequences of SEQ ID NOs: 17 to 21 , 25 to 29, 33, 34 , 36 , 54 and 55. In a further aspect, the present invention provides a method for the production of a microorganism, preferably a microorganism according to any of the embodiment described above, comprising a) providing a microorganism of the class Labyrinthulomycota; and b) transforming the microorganism of step a) with a nucleic acid construct or set of nucleic acid constructs according to any of the embodiments described above or a vector or set of vectors according to any of the embodiments described above.

Preferably, the microorganism provided in step a) is selected from the group consisting of Thraustochytrids, in particular Schizochytrium, Aurantiochytrium, Thraustochytrium and Hondea.

In one embodiment, the microorganism provided in step a) is selected from Schizochytrium sp. S31 (ATCC 20888), Aurantiochytrium sp. T66 (ATCC-PRA-276), Thraustochytrium sp. S-3 (ATCC-26185) and Aurantiochytrium limacinum SR 21 (ATCC-MYA-1381).

Transformation of the microorganism can be achieved by various methods known to the person skilled in the art such as electroporation or biolistic transformation.

In one embodiment of the method for the production of a microorganism described above, the nucleic acid construct or the set of nucleic acid constructs is inserted into the genome of the microorganism by homologous recombination. Preferably, the nucleic acid construct or the nucleic acid constructs of the set of nucleic acid constructs is/are inserted at one or more of the lanosterol synthase, the b-carotene synthase, the ku80, the anthranilate synthase and the 18 S rDNA locus in the genome of the microorganism.

Inactivation of ku80 has been reported to stimulate homologous recombination.

In another aspect, the present invention relates to a method for the production of one or more plant derived triterpene(s) and/or derivatives thereof comprising the steps: i) providing a microorganism according to any of the embodiments described above; and ii) cultivating the microorganism of step i) under conditions, which facilitate the production of plant derived triterpenes. According to a preferred embodiment of the method for the production of one or more plant derived triterpene(s) and/or derivatives thereof, the plant derived triterpene(s) is/are selected from the group consisting of lupeol, oleanolic acid, ursolic acid, betulinic acid, corosolic acid, maslinic acid and dammarenediol. In order to provide microorganisms with the ability to produce plant derived triterpenes, it is also possible to express the enzymes of the methyl erythrol phosphate pathway (MEP) found in chloroplasts in the host microorganism, preferably in the mitochondria (see figure 6). In particular, the methyl erythrol phosphate pathway from the thermophilic cyanobacterium Mastigocladus laminosus can be expressed in a host selected from the group consisting of Thraustochytrids, in particular Schizochytrium, Aurantiochytrium, Thraustochytrium and Hondea, in particular is selected from Schizochytrium sp. S31 (ATCC 20888), Aurantiochytrium sp. T66 (ATCC-PRA-276), Thraustochytrium sp. S-3 (ATCC-26185) and Aurantiochytrium limacinum SR 21 (ATCC-MYA-1381).

Examples Example 1: Cultivation of thraustochytrids

Schizochytrium sp. S31 (ATCC 20888), Aurantiochytrium sp. T66 (ATCC-PRA-276), Thraustochytrium sp. S-3 (ATCC-26185) and Aurantiochytrium limacinum SR 21 (ATCC- MYA-1381) were obtained from the American Type Culture Collection (ATCC). All thraustochytrid strains were cultured in M50-V medium at 28°C under agitation (180 rpm). The medium containing NaCI (12.5 g/L), MgS04 * 7 H20 (2.5 g/L), KCI (0,5 g/L), proteose peptone (1 g/L), yeast extract (1 g/L), glucose (10 g/L), K2HP04 (30,5 mg/L), CaCI2 (51 ,45 mg/L), biotin (0,5 pg/L), thiamine (100 pg/L), cobalamin (0,5 pg/L), Fe-(NH4)-Citrat (6 mg/L), L-glutamate (0,1 g/L), HEPES pH 8 (595,8 mg/L), trace element solution (1 ml/L). The trace element solution contained: H3B03 (2.86 g/L), MnCI2 * 4 H20 (1.81 g/L), ZnS04 * 7 H20 (0.222 g/L), Na2Mo04 * 2 H20 (0.39 g/L), CuS04 * 5 H20 (0.079 g/L), CoCI2 * 6

H20 (0,035 g/L). Solid media were supplemented with 2% bacto agar. To prevent bacterial contaminations, carbenicillin at a concentration of 100 pg/mL was used in all M50-V agar plates.

Example 2: Next generation genome sequencing and assembly Schizochytrium sp. S31 (ATCC 20888) was sequenced to gain bioinformatics insight into the isoprene/terpene metabolism, to understand codon usage and intron/exon structures, to define promoter regions and to identify integrations sites or gene deletions. Schizochytrium sp. S31 (ATCC 20888) was obtained from the American Type Culture Collection (ATCC). Cells were cultured in M50-20 medium (Byne et al. 2013) at 28°C under agitation (170 rpm). Identity and axenity of the strain was verified by amplicon sequencing of the 18S rDNA using degenerate primers (Burja et al. 2006). Genomic DNA (gDNA) was extracted from 2 ml_ of an early log phase liquid culture using a CTAB DNA isolation protocol according to Ausuebel et al. (1999). gDNA was sheared on Covaris M220 with Covaris MicroCaps 50 pi (50 W Peak Incident Power, Duty Factor 20%, 200 Cycles/Burst, Treatment Time 30 sec, 20°C) to approx. 550 bp. Library preparation was performed with 700 ng sheared gDNA using the sparQ DNA Library Prep Kit (QuantaBio) according to the manufacturer's instructions. Libraries were quality controlled with DNA High Sensitivity DNA Kit on Bioanalyzer (Agilent) and quantified on Qubit 2.0 Fluorometer (ThermoFisher Scientific with ds HS Assay Kit). Genome sequencing was performed in the Genomics Service Unit (LMU Biocenter, Munich) on lllumina MiSeq with v3 chemistry (2 x 250 bp and 2 x 300 bp paired-end sequencing). Subsequent sequence data manipulation (processing) was exclusively carried out on the web platform Gallaxy Europe (usegalaxy.org). Paired- end reads (R1 & R2) from separate sequencing runs were subjected to quality assessment via FastQC v0.72. lllumina adapter sequences and reads with a quality threshold below 15 were removed (clipped) using Trimmomatic vO.36.5 (Bolger et al. 2014). The following settings were chosen for adapter clipping: 1. SLIDINGWINDOW (number of bases to average across: 4; average quality required: 15) 2. MINLEN (Minimum length of reads to be kept: 50) 3. LEADING (Minimum quality required to keep a base: 3) 4. TRAILING (Minimum quality required to keep a base: 3). Successful application of Trimmomatic was subsequently verified again via FastQC. De novo sequence assembly was performed using various assembly pipelines, among these, Velvet Optimizer (Zerbino et al. 2008), SPAdes (Bankevich et al. 2012) and Shovill (Seemann 2017). The final genome assembly was created via SPAdes (v3.12.0) based on trimmed data from several sequencing runs. The following settings were selected: no single cell; run only assembly (without read error correlation); no careful correction; automatically choose k-mer values; auto coverage cutoff. Sequence quality metrics and genome completeness of the different bioinformatic assembly approaches were assessed using QUAST v5.0.2 (Gurevich et al. 2013) and BUSCO v3.0.2 (Simao et al. 2015), respectively. The genome of Aurantiochytrium limacinum ATCC MYA-1381 (https://mycocosm.jgi.doe.gov/Aurli1/Aurli1.home.html) was used as a reference when using QUAST. Example 3: Construction of expression vectors

Strain development in this work essentially employed heterologous biosynthetic enzymes which originate from the plants Cantharantus roseus, Lagerstroemia speciosa, Malus domestica, Rhicinus communis, Arabidopsis thaliana, the green alga Botryococcus braunii, the yeast Saccharomyces cerevisiae or the cyanobacteria Synechococcus PCC7942 and Mastigocladus laminosus. The heterologous genes for triterpene biosynthesis as well as isoprenoid precursor biosynthesis were codon optimized for expression in Schizochytrium ATCC 20888 using a codon usage table derived from the highly expressed genes for PUFA biosynthesis (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species =164673). Additionally, several genes from the thraustochytrids Schizochytrium ATCC 20888 and Aurantiochytrium limacinum ATCC MYA-1381 were used. To enable multigene expression, a translational fusion strategy based on viral 2A-linker peptides (table 3) was applied. This strategy enables the translation of several enzymes as one translational fusion polypetide which is cotranslationally cleaved at C-terminus of the 2A-linkers by a mechanism known as „ribosomal skipping".

Table 3: Viral 2A-peptide linkers, their origin and amino acid sequence

Example 4: DNA-assembly via in vivo gap repair cloning in Saccharomyces cerevisiae

DNA-cloning was exclusively accomplished via one step, multifragment assembly of up to 20 pieces using in vivo gap repair cloning in Saccharomyces cerevisiae. S. cerevisiae in vivo gap-repair has proven as an efficient tool for denovo assembly of plasmids (Ma et al. 1987, Raymond et al. 1999, Raymond et al. 2002, van Leuwen et al. 2015, Shanks et al. 2009), the cloning of very large gene clusters or chromosomes directly from genomic DNA (Kouprina & Larionov 2016) as well as for the implantation of entire bacterial genomes or the design and assembly of synthetic genomes (Karas et al. 2013, Gibson et al. 2008). In contrast to the established gap repair protocols which rely on Lithium acetate / PEG transformation, an electroporation mediated protocol was used, exclusively. In brief, a single colony of S. cerevisiae strain BY4741 from a fresh plate was used to inoculate 4 ml YPD medium. After overnight culture at 30°C and 200 rpm the cell suspension was diluted 1/50 into 200 ml_ fresh YPD in 500 ml_ Erlenmeyer flasks and further grown for approximately 4-5 h. When the yeast culture reached early exponential growth phase (OD600 ~ 1 ,5) cells were harvested at 4000 g (4°C) and washed in 20 ml_ of 1M sorbitol. To weaken the cell walls of S. cerevisiae the pellets were then resuspended in buffer PreT (1 M sorbitol, 100 mM Lithium acetate, 100 mM DTT) and incubated for 20 min at 30°C and 100 rpm. Subsequently the cells were washed twice in cold 1 M Sorbitol, resuspended in 3 mL of the same and kept on ice until electroporation. Approximately 0,5 pg of linearized vector, equimolar amounts of insert DNA fragments and 4 pi single stranded salmon sperm DNA (2 mg/mL) were combined with 100 mI competent cells, mixed by pipetting and transferred to precooled 0,2 cm electroporation cuvettes. Electroporation was carried out at 1500 V (7500 V/cm), 200 W and 25 pF using the Biorad Gene Pulser device. To enable cell recovery and transgene expression the transformation mix was subsequently diluted in 1 mL YPDS and incubated overnight at 30°C and 180 rpm. Prior to plating, complex substrates were removed from the cell suspension by harvesting the yeast cells via centrifugation at 4000 g for 5 min, followed by three consecutive wash steps with 1 mL H20. The cell pellet was resuspended in 400 pi H20 and plated in 200 pi aliquots onto SC w/o Uracil plates. Colonies appeared after 2 days of growth at 30°C but were routinously grown for 4-5 days to develop thick colonies.

Example 5: Colony plasmid rescue, vector amplification and sequence verification

For subsequent amplification in E.coli the plasmids were extracted from the yeast cells using a modified colony plasmid rescue procedure. Therefore, transformant colonies were collectively washed off the plates with 2 x 1 ,5 mL H20 using a 1 mL pipette, transferred to

1.5 mL reaction tubes and harvested by centrifugation at 4000 g for 1 min. Plasmid extraction was essentially accomplished using the QIAprep spin plasmid miniprep kit (Qiagen) with minor modifications. The cell pellet was resuspended in buffer P1 and combined with an equal volume of sterile glass beads (0 = 0,5 mm). To desintegrate the cell walls of the yeast, the precooled reaction tubes were subjected to a mechanical breakup at 2800 rpm for 10 min using a Disruptor Genie Digital device (Scientific Industries Inc.). All successive steps of the plasmid purification follow the instructions provided by the manufacturer. For very large plasmids > 30 kbp the mechanical cell desintegration was substituted for a partial enzymatic digestion of the cell walls using Zymolase 20T. Initial screening of the resultant plasmid vectors was performed by analytical restriction digestion. To verify exact sequence identity, all vectors used in subsequent transformations of Thraustochytrids were further subjected to next generation sequencing using lllumina Miseq technology. Sequencing was carried out by the Sequencing Serive Unit at the Gene Center of the Ludwig Maximilians University Munich. Example 6: Transformation of Thraustochytrids

Depending on the specific strain and the size of the vector to be transformed three different transformation protocols (A-C) based on electroporation with or without prior protoplasting or biolistic transformation were applied. Protocol A) Electroporation without enzymatic pretreatment: Cells from 50 mL culture were harvested by centrifugation at 4000 g for 10 min (4°C), washed once in 20 mL buffer SEP (1 M Sorbitol, 1 mM HEPES, pH 6,5) and resuspended in 10 mL of the same buffer. To break multicellular aggregates and weaken the cell wall structure the cells were subjected to a pretreatment with 25 mM Dithiothreitol for 20 min under continues agitation at 100 rpm (room temperature) followed by 20 sec of milling with ¼ Vol glass beads (0,5 mm) using a vortex at maximum speed. After settling of the glass beads by gravitational force the supernatant containing the cells was transferred to a fresh reaction tube and washed twice with 10 mL of buffer SEP. The final pellet was resuspended in 2 mL buffer SEP, split into 10Opl aliquots and combined with 1- 5 pg of linearized DNA in precooled 0,2 cm electroporation cuvettes. Electroporation was carried out by applying two consecutive high voltage pulses at 500-1000 V (2500-7500 V/cm), 200 W and 25 pF using the Biorad Gene Pulser device. To enable cell recovery and transgene expression the transformation mix was subsequently diluted in 1 mL M50V-S and incubated overnight at 28°C and 180 rpm prior to plating on M50 selective agar plates. Protocol B) Electroporation with enzymatic pretreatment: Cells from 50 mL culture were harvested by centrifugation at 4000 g for 10 min (4°C), washed once in 20 mL buffer SEP (1 M Sorbitol, 1 mM HEPES, pH 6,5) and resuspended in 10 mL buffer EPT (1 M Sorbitol, 10 mM CaCI2, 1 mM HEPES, pH 6,5). To initiate cell wall digestion the cell suspension was supplemented with 1/5 Vol of an enzyme cocktail consisting of protease XV (10 mg/mL), Snailase (10 mg/mL), Viscozym L and hen egg lysozyme (5 mg/mL) in buffer SEP. Samples were incubated at 28°C for 6-8 h under gentle shaking at 100 rpm. Protoplast formation was verified by bright field microscopy or fluorescence detection of Calcofluor White stained samples. The protoplasts were harvested by centrifugation at 1000 rpm for 5 min and washed twice with buffer SEP. All subsequent steps were carried out as described in protocol A. Protocol C) Biolistic transformation: Biolistic transformation also referred to as gene gun (micro-) particle bombardment or particle gun transformation was used to introduce recombinant DNA into Thraustochytrids, which were only poorly accessible by the electroporation procedures above. For the transformation gold particles with a diameter of 0.6 - 1 .6 pm were coated with linearized vector DNA and shot at cell material at high velocity using mechanical force with a particle gun. A vacuum of is applied in the bombardment chamber and a helium gas pressure is generated inside the upstream gas acceleration tube. A rupture disk, built into a retaining cap that seals the tube from the chamber, bursts at a defined pressure. The subsequent helium shock wave accelerates the macrocarrier with the DNA-coated microcarriers forwards in the bombardment chamber towards the target cells. Once particles have penetrated the cell, the DNA elutes from the particles and integrates into the host genome. To prepare the target for subsequent biolistic transformations, 300 pi of log phase liquid culture of the thraustochytridial acceptor strains were spread on M50V agar plates (alternatively on nitrocellulose membranes on M50V agar plates) and grown over night at 30 °C. Biolistic transformation was conducted using the PDS-1000/He Particle Delivery System (Bio-Rad). In particular, 3 mg (60 mg/ml) of gold microcarriers were coated with 5 pg linearized DNA by combining with 50 mI 2,5 M CaCI2 and 20 mI 0,1 M spermidine under constant vortexing. Upon a 10 min incubation on ice the DNA-coated particles were washed twice in 70% ethanol, followed by one washing in 100% ethanol and resuspension in a final volume of 50 pi of 100% ethanol. For each single bombardment, 10 pi of the microcarrier were then loaded onto macrocarrier discs and dried under air. The bombardments were carried out at a microcarrier to target distance of 6 cm using a 1350 psi rupture disc and a chamber vacuum of 25" Hg. To allow cell recovery the plates were incubated at 30°C for approximately 24 h post bombardment. Then the cells were washed from the plates using 2 ml_ M50 medium, harvested by centrifugation at 4000 g for 2 min, resuspended in 300 mI M50 medium and plated onto selective M50 agar plates.

Example 7: Visual Screening of transformants

To facilitate selection of transgene positive colonies on agar plates reporter genes were implemented into most of the recombinant constructions. The coexpression of Aqueoria victoria (jellyfish) green fluorescence protein GFP or the catechol-2, 3-dioxygenase XylE from Pseudomonas putida enables selection of positive clones via fluorescence imaging or colorimetric staining of the reporters. Green fluorescence was measured at an excitation wavelength of 395/473 nm and an emission filter of 507 nm using a fluorescence imaging device. For XylE detection agar plates containing mutant colonies were sprayed with 0,5M pyrocatechol (Sigma-Aldrich). The catabolic product of the XylE reaction 2-hydroxymuconic semialdehyde leads to a yellow staining of the colonies. In vitro XylE activity assays were conducted spectrophotometrically at 375 nm according to Sala-Trepat & Evans (1971). Successful genomic integration of the expression cassettes was verified via polymerase chain reaction using oligonuleotide primers listed in table 4. Polymerase chain reaction was exclusively performed using the LongAmp polymerase system (New England Biolabs) according to the protocols provided by the manufacturer.

Table 4: Primer sequences Example 8: Verification of translation and 2A-peptide cleavage

While codon optimization is capable of improving expression efficiency of heterologous genes by up to 1000-fold, the procedure may potentially cause unexpected interferences with transcription, translation or transcript and protein stability, in certain cases. To verify functional transcription and translation of the synthetic fusion gene constructions the respective gene cassettes were tested in Saccharomyces cerevisiae using vectors based on the galactose inducible bidirectional gall -gall 0 promoter. Since the GOIs were expressed as translational 2A peptide fusions with the respective antibiotic selection markers, Zeocin and G418 resistance could be employed as an indirect measure of efficient translation. Therefore S. cerevisiae strains containing the respective vectors were grown on SC w/o Uracil + 2% galactose and selected on increasing concentrations of Zeocin (200- 400 pg/ml) and G418 (200-300 pg/ml), respectively. S. cerevisae strain BY4741 was used as a negative control. Proteins samples extracted from S. cerevisiae or thraustochytridial mutant strains were separated on 4-12 % SDS-polyacrylamide gradient gels at 100 V constant voltage. Gels were stained using 0,5% Ponceau S (in 1% acetic acid) according to Goldman et al. (2016). Proteins were then transferred to nitrocellulose membrane (Amersham Protran™ 0,2 pm) by semi dry blotting using a Biometra Fastblot apparatus at 50 Volts (30 min transfer time). Immunological detection on western blots was carried out using the BM Chemiluminescence Western Blotting Kit (Roche) according to the instructions provided by the manufacturer. Chemiluminescence detection was carried out using a LI-COR cDiGit blot scanning device. For increased sensitivity the SuperSignal® West Femto substrate was used instead of the luminol solution.

Example 9: Plasmid vectors Table 5 gives a complete list of the plasmid vectors used to investigate improvements in triterpene biosynthesis as well as isoprene precursor provision. Combinatorial overexpression of isoprene precursor biosynthetic genes, gene fusions as well as sequential overexpression and selective intracellular targeting of triterpene biosynthetic core genes was investigated. The consecutive sequence numbers 1 to 39 in the table correspond to the schematic drawings of the respective gene expression cassettes in figures 3a-4b. Figures 7 to 10 show maps for the integration vectors. Final triterpene production strains were generated by chromosomal integration of a triterpene biosynthesis constructs (Fig 3a and 3b), preferentially at the ku80 or the cars locus, followed by a second round of transformation with constructs which integrate at the 18S rDNA, the lansS or the trpE locus to improve isoprene precursor formation (Fig. 4a and 4b). Plasmids and corresponding expression cassettes shown in Fig 4a and 4b were used to evaluate the effectivity of the coexpression of different combinations of heterologous genes of the mevalonate pathway, subsequent prenylation steps and enzyme-enzyme fusions at metabolic branching points on the final production of triterpenes. Some of the corresponding mutant strains (Fig. 4a) were directly evaluated for maximum productivity of lupeol. Table 5: Plasmid vectors

Plasmids of SEQ ID NOs: 37 to 53, can also be adapted to encode NeoR having the amino acid sequence of SEQ ID NO: 96 as exemplified with SEQ ID NOs: 80 to 83, which express NeoR having the sequence of SEQ ID NO: 96 as part of a fusion protein having amino acid sequence of SEQ ID NO: 79.

In certain cases, when episomal expression was superiorto genomic integration, sequence of SEQ ID NO: 107, present in all plasmid vectors, was exchanged for one of two artificial chromosome sequences (SEQ ID NO: 108 or SEQ ID NO: 109). Example 10: Integration of expression cassettes in thraustochytrids

Example 10A: Agrobacterium-based transformation

For transformation of very large DNA fragments into thraustochytrids or when other methods failed a binary vector strategy based on the yeast gap-repair compatible t-DNA vector pCAMBIA1303-Yae and Agrobacterium tumefaciens strain LBA4404 (Thermo Scientific), which harbors a disarmed octopine-type Ti plasmid (pAL4404) without selftransport features and encoding antibiotic selection marker genes conferring resistance to rifampicin and streptomycin, was employed. Transformation of t-DNA vectors into commercial electrocompetent Agrobacterium LBA4404 cells was performed via electroporation at 2kV, 200 W, 25 pF according to the instructions provided by Thermo Scientific. Agrobacterium transformants were selected on YM-agar plates (1 % Mannitol, 0.04 % yeast extract, 0.01 % NaCI, 0.02 % MgS04*7H20, 0.05 % K2HP04*3 H20, 1.5 % bacto agar; pH 7.0) supplemented with 100 pg/ml Streptomycin and 50 pg/ml Kanamycin. Cell culture, pre-preparation of cells and two-species mating were carried out according to Cheng et al. (2011) with modifications. A) To induce the Ti-plasmid transfer functions (vir genes) an Agrobacterium culture was harvested after 24 h precultivation in YM supplemented with 100 pg/ml Streptomycin and 50 pg/ml Kanamycin by centrifugation for 15 min at 4.500 g (RT) and resuspended in induction medium IM (TM, supplemented with 250 pM acetosyringone and 50 pg/ml Kanamycin) to a final OD600 of 0.4. Cultures were further incubated at 20 to 22°C for 4 h to establish distinctive transfer competence. B) Since the thraustochytridial cell is equipped with stable cell walls, which hamper access of Agrobacterium and consequently Ti-plasmid based transfer of nucleic acids, the acceptor cells require pretreatment. To partially digest the cell wall of thraustochytrids, cells were harvested by centrifugation for 5 min at 4.000 g (RT) and resuspended in enzyme medium (1 M Mannitol, 10 mM CaCI2, adjusted to pH 5.5, 0.25 mg/ml_ Protease XIV, 0.1 mg/ml_ Snailase) to an OD600 of 2. The reactions were subsequently incubated at 28 °C for 4 h at 180 rpm. Final protoplasts were washed twice in transfer buffer TM (YM, supplemented with 0.7 M KCI, 1 % (m/v) glucose, 0.5 g/L NaCI, 0.1 g/L MgS04*7H20, adjusted to pH 5.3) and harvested by centrifugation at 2.000 g (RT) for 5 min. The resulting pellet was resuspended in twice the original volume of induction medium IM (already containing induced Agro bacterium) to reach a cell density of approx. OD600 of 1. C) To enable mating of Agrobacterium with the thraustochytrids the coculture was incubated at 20 to 22 °C for 24 h and finally plated on M50 medium plates supplemented either with 0,5-1 ,5 mg/ml hygromycin for selection of genomic integration of the entire plasmid, alternatively with 75- 100 pg/ml zeocin or 100-200 pg/ml paromomycin for selection of t-DNA-insertions or integration via double cross-over homologous recombination. To suppress the growth of Agrobacterium, M50 plates were additionally supplemented with 100 pg/ml carbenicillin.

Example 10B: CRISPR/Cas9-gene editing

Gene editing was primarily applied to support site-specific genomic integration of target expression cassettes into lanS, cars, ku80, trpE orsqe gene loci. Specific PAM sequences and corresponding single guide-RNAs (sgRNAs) for targets in Schizochytrium ATCC20888 were selected using CRISPRdirect software (Naito et al. 2014). To avoid off-target effects a selection of the crRNA sequences were checked against the Schizochytrium genome for potential multiple occurrence or presence of highly homologous sequences using Blastn in Gallaxy.eu (https://usegalaxy.eu/). sgRNAs against targets in Aurantiochytrium limacinum were completely designed via the gene editing software ChopChop v3 (Montague et al. 2014) which has integrated access to the ATCC MYA-1381 genome to check for off-target effects. EnGen Spy Cas9 (New England Biolabs), a derivative of Streptococcus pyogenes Cas9 with C- and N-terminal nucleartargeting signals was used throughout all gene editing experiments to facilitate nuclear import of the CRISPR/Cas9 ribonucleoprotein complex upon transformation. The DNA-templates for production of guide-RNA (gRNA) via in vitro transcription were generated by hybridization of two overlapping oligonucleotides, which were elongated by a polymerase chain reaction. Oligonucleotide 1 (cf. table 6) comprises/encodes a bacteriophage T7-promoter, the target specific 20 bp crRNA and an overlap region to oligocucleotide 2, whereas the latter primarily encodes the Tracr-RNA sequence required for binding to the Cas9 endonuclease. Template-DNA synthesis and in vitro transcription were carried out using the EnGen sgRNA synthesis Kit (New England Biolabs). In vitro transcribed RNAs were purified using the MEGAclear™ Kit (Invitrogen) or the Monarch RNA Cleanup Kit (New England Biolabs), and analysed 8M-urea- polyacrylamide gel electrophoresis as described by Summer et al. (2009). Quantification of RNA was performed by at a wavelength of 260 nm using a NanoVue spectrophotometer (GE Healthcare). sgRNA was stored at -80 until further investigation. sgRNA, Cas9 endonuclease and linearized expression cassettes (eg., Spel-fragments of Ku80 vectors) with flanking homology regions to the genomic cutting site generated by the CRSIPR/Cas9 ribonucleoprotein complex were cotransformed into thraustochytrids via electroporation. In brief, 100 pmol of sgRNA (~4,3 pg) and 100 pmol EnGen Spy Cas9 enzyme (5 pi of a 20 mM stock solution = 17 pg) were combined in 1 x NEBuffer™ r3.1 (100 mM NaCI, 50 mM Tris-HCI, 10 mM MgCI2, 100 pg/ml Recombinant Albumin, pH 7.9) in a total reaction volume of 15 pi and incubate for 10 min at room temperature to assembly the CRISPR/Cas9 ribonucleoprotein complex. Samples were kept at 4°C until until shortly before subjecting to electroporation. Finally 15 mI CRISPR/Cas9 complex and 1-5 pg of linear DNA (integration construct) were mixed with the competent thraustochytrids on ice and immediately subjected to electroporation. Electroporations were carried out using a XCell gene pulser electroporation system (BIO-RAD Laboratories) and 2 mm electroporation cuvettes. Cell pretreatment and electroporation settings were already described earlier.

Table 6: Oligonucletide primer design used to generate template-DNA for subsequent in vitro synthesis of sgRNA as exemplyfied by oligonucleotides applied in sgRNA-targeting of lanosterol synthase gene lanS in Aurantiochytrium limacinum. underlined: T7 promoter region, italic: crRNA

Example 10C: RNA-preparation and trancriptome analysis Preparation of total RNA was essentially carried out using the RNeasy Plant Mini Kit (Qiagen) according to the instructions provided by the manufacturer. The optional extraction buffer RLT supplemented with 40 mM DTT (final cone.) was used for initial resuspension of cells. Qualitative analysis of RNA integrity was accomplished by denaturing agarose gel electrophoresis in 1 xTBE buffer supplemented with 1 ml/100ml of a 1M GITC (Guanidiniumisothiocyanate) solution. Trancriptomes of 10 RNA samples derived from cells in different growth stages or subject to abiotic stress factors such as heat shock, heavy metal stress or supplemented by multiple carbon sources were analysed to generate RNA-based evidence supporting the genome annotation process. Libraries for sequencing were generated from 100-1 .000 ng total RNA with NEBNext® Ultra™ II RNA Library Prep Kit for lllumina together with NEBNext® Poly(A) mRNA Magnetic Isolation Module according to the manufacturer's instructions. Libraries were quality controlled with High Sensitivity DNA Kit on Bioanalyzer (Agilent) and quantified using a Qubit 2.0 Fluorometer (ThermoFisher Scientific) with ds HS Assay Kit. Sequencing was performed in the Genomics Service Unit (LMU Biocenter, Munich) on lllumina MiSeq with v3 chemistry (2x 300 bp paired-end sequencing).

Example 10D: Southern Blotting & Hybridization To verify successful genomic integration of the respective expression plasmids, genomic DNA was extracted from 1 mL of mid log liquid cultures using a CTAB protocol (Ausuebel 1999). Approximately 10 pg aliquots of the genomic DNA were subjected to restriction digestion using enzymes that generate a target fragment size of 500 - 3500 bp. Digested DNA samples from wildtyp and mutant cell lines were applied to denaturing agarose gel electrophoresis using 1 ,2% gels in 1 x TBE (120 V constant voltage). Ethidium bromide was used for DNA visualization under UV. Agarose gels were equilibrated in denaturation buffer (0,5 M NaOH, 1 ,5 M NaCL) for 10 min twice, with buffer exchange, and subsequently washed in neutralization buffer (0,5 M Tris-HCI, 3M NaCI, pH7,5) again twice for 10 min. DNA was transfered to Hybond-N nylon membrane (Amersham) using an upward cappillary blotting procedure. Upon successful transfer, usually after 12 h, the nylon membrane was removed from the blotting setup and cross-linked by UV light using a Stratalinker2400 device. Target specific probes were labelled by DIG-dUTP incorporation using the DIG High Prime DNA labelling and detection starter Kit II (Roche). Hybridization was carried out at 50°C overnight in an hybridization oven OV2 (Biometra). Chemiluminescence detection based on an alcaline phosphatase-conjugated Anti-DIG antibody reaction with the substrate CSPD [Disodium 3-(4-methoxyspiro {l,2-dioxetane- 3,2'-(5'-chloro)tricyclo[3.3.1 13,7]decan}-4-yl) phenyl phosphate] was carried out using the cDiGit Blot Scanner (LI-COR).

Example 10E: Squalene extraction Extraction of squalene was performed following the protocol of Matsuura et al. (2012) with some modifications. Briefly, approximately 50 mg of lyophilized cells were mixed with 500 pg of internal standard phenyloctadecane (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 250 pi methanol/chloroform (2:1 , v:v), 1 ml of glass beads (0,25 - 0,5 mm, Carl Roth, Karlsruhe, Germany) and 1 ml of methanol/chloroform (2:1 , v:v) in a 14 ml screw cap glass tubes. Subsequently, 3 consecutive cycles of vortexing at maximum speed (2 min each) were applied using a Disruptor Genie (Scientific Industries Inc.). After addition of 2 ml methanol/chloroform (2:1 , v:v), the suspension was shaken overnight on an orbital shaker at 160 rpm (room temperature). The next day, phases were separated via centrifugation at 3000 g for 10 min and the supernatant was transferred into a fresh glass tube. The remaining pellet was extracted a second time with 2 ml methanol/chloroform (2:1 , v:v) and the supernatants of both extractions were combined. Upon evaporation of the solvent in a speedvac (Savant, USA), the lipid fraction was subjected to saponification by resuspension of the residue in 3 ml 0.5 M KOH (in ethanol) and subsequent incubation at 90 °C for 1 hour. After cooling to room temperature, 2 ml n-hexane (Carl Roth, Karlsruhe, Germany) and 1 ml ddH20 were added and the samples were briefly vortexed. In order to achieve most distinct phase separation, centrifugation at 3000 g for 10 min at room temperature was applied. The upper phase (n-hexane) was collected in a fresh glass tube. The hydrophilic phase was extracted again with 2 ml n-hexane, centrifuged and the solvent phases of both extractions were combined. Prior to the measurements the samples were dried in a speedvac and resuspended in a suitable volume of acetonitrile (Carl Roth, Karlsruhe, Germany) for subsequent HPLC analysis.

Example 10F: HPLC analysis

All analyte samples were filtered through a 0.22 pm syringe filter (Carl Roth, Karlsruhe, Germany) prior to injection. Analysis was conducted on a Shimadzu SCL-10A HPLC system equipped with a 270 x 4,7 mm RP C-18 (10 pm) column (Bischoff Analysetechnik GmbH) and a SPD-10A UV/VIS detector. Chromatographic separation was performed using acetonitrile as mobile phase at a flow rate of 1.5 ml / min and a column temperature of 30 °C. Injection volume was 20 pi. Signal detection was carried out at 195 nm. Peak integration and quantification of squalene were achieved using LC Solution software (Shimadzu) and the following formula: HPLC-standards were prepared by dissolving 10 mg/ml of squalene in ethanol (> 98 %, Sigma Aldrich, St. Louis, MO, USA) together with internal standard phenyloctadecane (> 99,5 %, Sigma Aldrich, St. Louis, MO, USA). The stock solution was diluted (1 :10) with mobile phase to achieve a concentration range of 5 to 100 pg/ml for squalene and 25 to 125 pg/ml for phenyloctadecane, respectively.

- 56 - were applied using a Disruptor Genie (Scientific Industries Inc.). After addition of 2 ml methanol/chloroform (2:1 , v:v), the suspension was shaken overnight on an orbital shaker at 160 rpm (room temperature). The next day, phases were separated via centrifugation at 3000 g for 10 min and the supernatant was transferred into a fresh glass tube. The remaining pellet was extracted a second time with 2 ml methanol/chloroform (2:1 , v:v) and the supernatants of both extractions were combined. Upon evaporation of the solvent in a speedvac (Savant, USA), the lipid fraction was subjected to saponification by resuspension of the residue in 3 ml 0.5 M KOH (in ethanol) and subsequent incubation at 90 °C for 1 hour. After cooling to room temperature, 2 ml n-hexane (Carl Roth, Karlsruhe, Germany) and 1 ml ddH20 were added and the samples were briefly vortexed. In order to achieve most distinct phase separation, centrifugation at 3000 g for 10 min at room temperature was applied. The upper phase (n-hexane) was collected in a fresh glass tube. The hydrophilic phase was extracted again with 2 ml n-hexane, centrifuged and the solvent phases of both extractions were combined. Prior to the measurements the samples were dried in a speedvac and resuspended in a suitable volume of acetonitrile (Carl Roth, Karlsruhe, Germany) for subsequent HPLC analysis.

Example 10F: HPLC analysis

All analyte samples were filtered through a 0.22 pm syringe filter (Carl Roth, Karlsruhe, Germany) prior to injection. Analysis was conducted on a Shimadzu SCL-10A HPLC system equipped with a 270 x 4,7 mm RP C-18 (10 pm) column (Bischoff Analysetechnik GmbH) and a SPD-10A UV/VIS detector. Chromatographic separation was performed using acetonitrile as mobile phase at a flow rate of 1.5 ml / min and a column temperature of 30 °C. Injection volume was 20 pi. Signal detection was carried out at 195 nm. Peak integration and quantification of squalene were achieved using LC Solution software (Shimadzu) and the following formula:

HPLC-standards were prepared by dissolving 10 mg/ml of squalene in ethanol (> 98 %, Sigma Aldrich, St. Louis, MO, USA) together with internal standard phenyloctadecane (> 99,5 %, Sigma Aldrich, St. Louis, MO, USA). The stock solution was diluted (1 :10) with mobile phase to achieve a concentration range of 5 to 100 pg/ml for squalene and 25 to 125 pg/ml for phenyloctadecane, respectively. - 57 -

References:

Aalbers, F.S., and Fraaije, M.W. (2019). Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes. Chembiochem 20, 20-28.

Aasen, I.M., Ertesvag, H., Heggeset, T.M.B., Liu, B., Brautaset, T., Vadstein, O., and Ellingsen, T.E. (2016). Thraustochytrids as production organisms for docosahexaenoic acid (DHA), squalene, and carotenoids. Appl Microbiol Biotechnol 100, 4309-4321.

Agra, L.C., Ferro, J.N.S., Barbosa, F.T., and Barreto, E. (2015). Triterpenes with healing activity: A systematic review. J Dermatolog Treat 26, 465-470.

Ajaiyeoba, E.O., Onocha, P.A., Nwozo, S.O., and Sama, W. (2003). Antimicrobial and cytotoxicity evaluation of Buchholzia coriacea stem bark. Fitoterapia 74, 706-709.

Ajikumar, P.K., Tyo, K., Carlsen, S., Mucha, O., Phon, T.H., and Stephanopoulos, G. (2008b). Terpenoids: Opportunities for Biosynthesis of Natural Product Drugs Using Engineered Microorganisms. Mol. Pharmaceutics 5, 167-190.

Andre, C.M., Legay, S., Deleruelle, A., Nieuwenhuizen, N., Punter, M., Brendolise, C., Cooney, J.M., Lateur, M., Hausman, J.-F., Larondelle, Y., et al. (2016). Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids. New Phytol 211, 1279-1294.

Arafiles, K.H.V., Iwasaka, H., Eramoto, Y., Okamura, Y., Tajima, T., Matsumura, Y., Nakashimada, Y., and Aki, T. (2014). Value-added lipid production from brown seaweed biomass by two-stage fermentation using acetic acid bacterium and thraustochytrid. Appl Microbiol Biotechnol 98, 9207-9216.

Arendt, P., Miettinen, K., Pollier, J., De Rycke, R., Callewaert, N., and Goossens, A. (2017). An endoplasmic reticulum-engineered yeast platform for overproduction of triterpenoids. Metabolic Engineering 40, 165-175.

Atta-ur-Rahman, Zareen, S., Choudhary, M.I., Akhtar, M.N., and Khan, S.N. (2008). a- Glucosidase Inhibitory Activity of Triterpenoids from Cichorium intybus. J. Nat. Prod. 71, 910-913.

Ausubel, Frederick M., ed., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed (New York: Wiley, 1999)

Ayeleso, T.B., Matumba, M.G., and Mukwevho, E. (2017). Oleanolic Acid and Its Derivatives: Biological Activities and Therapeutic Potential in Chronic Diseases. Molecules 22.

Bailey R.B., D. DiMasi, Hansen J.M,, Mirrasoul P.J., Ruecker C.M., Veder G.T. Ill, Kaneko Tasuo, Barclay W.R. (2003) Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors. (US patent US6607900B2)

Bankevich, A., Nurk, S., Antipov, D., Gurevich, A.A., Dvorkin, M., Kulikov, A.S., Lesin, V.M., Nikolenko, S.I., Pham, S., Prjibelski, A.D., et al. (2012). SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19, 455-477.

Bayne, A.-C.V., Boltz, D., Owen, C., Betz, Y., Maia, G., Azadi, P., Archer-Hartmann, S., Zirkle, R., and Lippmeier, J.C. (2013). Vaccination against Influenza with Recombinant - 58 -

Hemagglutinin Expressed by Schizochytrium sp. Confers Protective Immunity. PLoS ONE 8, e61790.

Beserra, F.P., Vieira, A.J., Gushiken, L.F.S., de Souza, E.O., Hussni, M.F., Hussni, C.A., Nobrega, R.H., Martinez, E.R.M., Jackson, C.J., de Azevedo Maia, G.L., et al. (2019). Lupeol, a Dietary Triterpene, Enhances Wound Healing in Streptozotocin-lnduced Hyperglycemic Rats with Modulatory Effects on Inflammation, Oxidative Stress, and Angiogenesis. Oxid Med Cell Long ev 2019, 3182627.

Bolger, A.M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for lllumina sequence data. Bioinformatics 30, 2114-2120. Both, D.M., Goodtzova, K., Yarosh, D.B., and Brown, D.A. (2002). Liposome-encapsulated ursolic acid increases ceramides and collagen in human skin cells. Arch Dermatol Res 293, 569-575.

Brendolise, C., Yauk, Y.-K., Eberhard, E.D., Wang, M., Chagne, D., Andre, C., Greenwood, D.R., and Beuning, L.L. (2011). An unusual plant triterpene synthase with predominant a- amyrin-producing activity identified by characterizing oxidosqualene cyclases from Malus x domestica: Oxidosqualene cyclases from apple. FEBS Journal 278, 2485-2499.

Broker, J.N., Miiller, B., van Deenen, N., Priifer, D., and Schulze Gronover, C. (2018). Upregulating the mevalonate pathway and repressing sterol synthesis in Saccharomyces cerevisiae enhances the production of triterpenes. Appl Microbiol Biotechnol 102, 6923- 6934.

Burja, A.M., Radianingtyas, H., Windust, A., and Barrow, C.J. (2006). Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species: screening of strains and optimization of omega-3 production. Appl Microbiol Biotechnol 72, 1161-1169. Chang, M.-H., Kim, H.-J., Jahng, K.-Y., and Hong, S.-C. (2008). The isolation and characterization of Pseudozyma sp. JCC 207, a novel producer of squalene. Appl Microbiol Biotechnol 78, 963-972.

Chen, A.H., and Silver, P.A. (2012). Designing biological compartmentalization. Trends in Cell Biology 22, 662-670. Chen, J.J., Genereux, J.C., and Wiseman, R.L. (2015). Endoplasmic reticulum quality control and systemic amyloid disease: Impacting protein stability from the inside out. IUBMB Life 67, 404-413.

Cheng, R., Ma, R„ Li, K„ Rong, H., Lin, X., Wang, Z., Yang, S„ & Ma, Y. (2012). Agrobacterium tumefaciens mediated transformation of marine microalgae Schizochytrium. Microbiological Research, 167(3), 179-186. https://doi.Org/10.1016/j.micres.2011.05.003

Chi, Z., Hu, B., Liu, Y., Frear, C., Wen, Z., and Chen, S. (2007). Production of omega-3 polyunsaturated fatty acids from cull potato using an algae culture process. Appl Biochem Biotechnol 137-140, 805-815. Chu, L.L., Montecillo, J.A.V., and Bae, H. (2020). Recent Advances in the Metabolic Engineering of Yeasts for Ginsenoside Biosynthesis. Front. Bioeng. Biotechnol. 8, 139. - 59 -

D’Adamo, S., Schiano di Visconte, G., Lowe, G., Szaub-Newton, J., Beacham, T., Landels, A., Allen, M.J., Spicer, A., and Matthijs, M. (2019). Engineering the unicellular alga Phaeodactylum tricornutum for high-value plant triterpenoid production. Plant Biotechnol J 17, 75-87.

Dahlin, P., Srivastava, V., Bulone, V., and McKee, L.S. (2016). The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product. Front. Microbiol. 7.

Dai, L., Liu, C., Zhu, Y., Zhang, J., Men, Y., Zeng, Y., and Sun, Y. (2015a). Functional Characterization of Cucurbitadienol Synthase and Triterpene Glycosyltransferase Involved in Biosynthesis of Mogrosides from Siraitia grosvenorii. Plant Cell Physiol 56, 1172-1182.

Dai, Z., Liu, Y., Zhang, X., Shi, M., Wang, B„ Wang, D„ Huang, L, and Zhang, X. (2013). Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. Metabolic Engineering 20, 146-156.

Dai, Z., Wang, B., Liu, Y., Shi, M., Wang, D., Zhang, X., Liu, T., Huang, L., and Zhang, X. (2015b). Producing aglycons of ginsenosides in bakers’ yeast. Sci Rep 4, 3698.

Ding, Y., Wang, K.-F., Wang, W.-J., Ma, Y.-R., Shi, T.-Q., Huang, H., and Ji, X.-J. (2019). Increasing the homologous recombination efficiency of eukaryotic microorganisms for enhanced genome engineering. Appl Microbiol Biotechnol 103, 4313-4324.

Dong, L., Pollier, J., Bassard, J.-E., Ntallas, G., Almeida, A., Lazaridi, E., Khakimov, B., Arendt, P., de Oliveira, L.S., Lota, F., et al. (2018). Co-expression of squalene epoxidases with triterpene cyclases boosts production of triterpenoids in plants and yeast. Metabolic Engineering 49, 1-12.

Dueber, J.E., Wu, G.C., Malmirchegini, G.R., Moon, T.S., Petzold, C.J., Ullal, A.V., Prather, K.L.J., and Keasling, J.D. (2009). Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 27, 753-759.

Ehrhardt, H., Fulda, S., Fiihrer, M., Debatin, K.M., and Jeremias, I. (2004). Betulinic acid- induced apoptosis in leukemia cells. Leukemia 18, 1406-1412.

Ethier, S., Woisard, K., Vaughan, D., and Wen, Z. (2011). Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid. Bioresource Technology 102, 88-93.

Fournet, A., Angelo, A., Munoz, V., Roblot, F., Hocquemiller, R., and Cave, A. (1992). Biological and chemical studies of Pera benensis, a Bolivian plant used in folk medicine as a treatment of cutaneous leishmaniasis. J Ethnopharmacol 37, 159-164.

Gao, S., Tong, Y., Zhu, L., Ge, M., Jiang, Y., Chen, D., and Yang, S. (2017). Production of b-carotene by expressing a heterologous multifunctional carotene synthase in Yarrowia lipolytica. Biotechnol Lett 39, 921-927.

Gibson, D.G., Benders, G.A., Andrews-Pfannkoch, C., Denisova, E.A., Baden-Tillson, H., Zaveri, J., Stockwell, T.B., Brownley, A., Thomas, D.W., Algire, M.A., et al. (2008). Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome. Science 319, 1215-1220.

Goldman, A., Harper, S., and Speicher, D.W. (2016). Detection of Proteins on Blot Membranes. Curr Protoc Protein Sci 86, 10.8.1-10.8.11. - 60 -

Grahame Hardie, D. (2014). AMP-activated protein kinase: a key regulator of energy balance with many roles in human disease. J Intern Med 276, 543-559.

Guhling, O., Hobl, B., Yeats, T., and Jetter, R. (2006). Cloning and characterization of a lupeol synthase involved in the synthesis of epicuticular wax crystals on stem and hypocotyl surfaces of Ricinus communis. Arch Biochem Biophys 448, 60-72.

Gupta, A., Abraham, R.E., Barrow, C.J., and Puri, M. (2015). Omega-3 fatty acid production from enzyme saccharified hemp hydrolysate using a novel marine thraustochytrid strain. Bioresource Technology 184, 373-378.

Gurevich, A., Saveliev, V., Vyahhi, N., and Tesler, G. (2013). QUAST: quality assessment tool for genome assemblies. Bioinformatics 29, 1072-1075.

Hammer, S.K., and Avalos, J.L. (2017). Harnessing yeast organelles for metabolic engineering. Nat Chem Biol 13, 823-832.

Hardie, D.G., and Ashford, M.L.J. (2014). AMPK: regulating energy balance at the cellular and whole body levels. Physiology (Bethesda) 29, 99-107. Harish, B.G., Krishna, V., Santosh Kumar, H.S., Khadeer Ahamed, B.M., Sharath, R., and Kumara Swamy, H.M. (2008). Wound healing activity and docking of glycogen-synthase- kinase-3-beta-protein with isolated triterpenoid lupeol in rats. Phytomedicine 15, 763-767.

Hata, K., Ishikawa, K., Hori, K., and Konishi, T. (2000). Differentiation-inducing activity of lupeol, a lupane-type triterpene from Chinese dandelion root (Hokouei-kon), on a mouse melanoma cell line. Biol Pharm Bull 23, 962-967.

Hata, K., Hori, K., and Takahashi, S. (2003). Role of p38 MAPK in lupeol-induced B162F2 mouse melanoma cell differentiation. J Biochem 134, 441-445.

Hata, K., Hori, K., Murata, J., and Takahashi, S. (2005). Remodeling of actin cytoskeleton in lupeol-induced B162F2 cell differentiation. J Biochem 138, 467-472. Hernandez-Perez, M., Lopez-Garcia, R.E., Rabanal, R.M., Darias, V., and Arias, A. (1994). Antimicrobial activity of Visnea mocanera leaf extracts. J Ethnopharmacol 41, 115-119.

Hoang, M.H., Ha, N.C., Thom, L.T., Tam, L.T., Anh, H.T.L., Thu, N.T.H., and Hong, D.D. (2014). Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process. Journal of Bioscience and Bioengineering 118, 632-639.

Hong, W.-K., Rairakhwada, D., Seo, P.-S., Park, S.-Y., Hur, B.-K., Kim, C.H., and Seo, J.- W. (2011). Production of lipids containing high levels of docosahexaenoic acid by a newly isolated microalga, Aurantiochytrium sp. KRS101. Appl Biochem Biotechnol 164, 1468- 1480. Hu, W., Liu, N., Tian, Y., and Zhang, L. (2013). Molecular cloning, expression, purification, and functional characterization of dammarenediol synthase from Panax ginseng. Biomed Res Int 2013, 285740.

Huang, J., Zha, W., An, T., Dong, H., Huang, Y., Wang, D., Yu, R., Duan, L., Zhang, X., Peters, R.J., et al. (2019). Identification of RoCYPOI (CYP716A155) enables construction of engineered yeast for high-yield production of betulinic acid. Appl Microbiol Biotechnol 103, 7029-7039. - 61 -

Huang, L, Li, J„ Ye, H., Li, C., Wang, H„ Liu, B„ and Zhang, Y. (2012). Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus. Planta 236, 1571-1581.

Huang, Q.-X., Chen, H.-F., Luo, X.-R., Zhang, Y.-X., Yao, X., and Zheng, X. (2018). Structure and Anti-HIV Activity of Betulinic Acid Analogues. Curr Med Sci 38, 387-397.

Huang, Z.-R., Lin, Y.-K., and Fang, J.-Y. (2009). Biological and Pharmacological Activities of Squalene and Related Compounds: Potential Uses in Cosmetic Dermatology. Molecules 14, 540-554.

Humhal, T., Kastanek, P., Jezkova, Z., Cadkova, A., Kohoutkova, J., and Branyik, T. (2017). Use of saline waste water from demineralization of cheese whey for cultivation of

Schizochytrium limacinum PA-968 and Japonochytrium marinum AN-4. Bioprocess Biosyst Eng 40, 395-402.

Huttanus, H.M., and Feng, X. (2017). Compartmentalized metabolic engineering for biochemical and biofuel production. Biotechnol J 12. Iwasaka, H., Aki, T., Adachi, H., Watanabe, K., Kawamoto, S., and Ono, K. (2013). Utilization of waste syrup for production of polyunsaturated fatty acids and xanthophylls by Aurantiochytrium. J Oleo Sci 62, 729-736.

Iwasaka, H., Koyanagi, R., Satoh, R., Nagano, A., Watanabe, K., Hisata, K., Satoh, N., and Aki, T. (2018). A Possible Trifunctional b-Carotene Synthase Gene Identified in the Draft Genome of Aurantiochytrium sp. Strain KH105. Genes 9, 200.

Jager, S., Trojan, H., Kopp, T., Laszczyk, M.N., and Scheffler, A. (2009). Pentacyclic triterpene distribution in various plants - rich sources for a new group of multi-potent plant extracts. Molecules 14, 2016-2031.

Jakobsen, A.N., Aasen, I.M., and Stnzsm, A.R. (2007). Endogenously synthesized (-)-proto- quercitol and glycine betaine are principal compatible solutes of Schizochytrium sp. strain S8 (ATCC 20889) and three new isolates of phylogenetically related thraustochytrids. Appl Environ Microbiol 73, 5848-5856.

Jiang, Y., Zhu, Q., Liao, Y., Wang, Q., Li, Y., Dong, X., Peng, J., Yuan, J., and Wang, J. (2020). The Delta 5, 7-STEROLS and Astaxanthin in the Marine Microheterotroph Schizochytrium sp. S31. J Am Oil Chem Soc 97, 839-850.

Karas, B.J., Jablanovic, J., Sun, L., Ma, L., Goldgof, G.M., Stam, J., Ramon, A., Manary, M.J., Winzeler, E.A., Venter, J.C., et al. (2013). Direct transfer of whole genomes from bacteria to yeast. Nat Methods 10, 410-412.

Katabami, A., Li, L., Iwasaki, M., Furubayashi, M., Saito, K., and Umeno, D. (2015). Production of squalene by squalene synthases and their truncated mutants in Escherichia coli. Journal of Bioscience and Bioengineering 119, 165-171.

Kaya, K., Nakazawa, A., Matsuura, H., Honda, D., Inouye, I., and Watanabe, M.M. (2011). Thraustochytrid Aurantiochytrium sp. 18W-13a Accummulates High Amounts of Squalene. Bioscience, Biotechnology, and Biochemistry 75, 2246-2248. Khan, N.E., Nybo, S.E., Chappell, J., and Curtis, W.R. (2015). Triterpene hydrocarbon production engineered into a metabolically versatile host-Rhodobacter capsulatus. Biotechnol Bioeng 112, 1523-1532. - 62 -

Kim, J.-E., Jang, l.-S., Son, S.-H., Ko, Y.-J., Cho, B.-K., Kim, S.C., and Lee, J.Y. (2019a). Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway. Metab Eng 56, 50-59.

Kim, K.-D., Jung, H.-Y., Ryu, H.G., Kim, B., Jeon, J., Yoo, H.Y., Park, C.H., Choi, B.-H., Hyun, C.-K., Kim, K.-T., et al. (2019b). Betulinic acid inhibits high-fat diet-induced obesity and improves energy balance by activating AMPK. Nutr Metab Cardiovasc Dis 29, 409- 420.

Klabunde, J., Kunze, G., Gellissen, G., and Hollenberg, C.P. (2003). Integration of heterologous genes in several yeast species using vectors containing a Hansenula polymorpha-derived rDNA-targeting element. FEMS Yeast Res 4, 185-193.

Kouprina, N., and Larionov, V. (2016). Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology. Chromosoma 125, 621-632.

Kumar, S., Kumar, V., and Prakash, O. (2013). Enzymes inhibition and antidiabetic effect of isolated constituents from Dillenia indica. Biomed Res Int 2013, 382063. Kunkel, S.D., Elmore, C.J., Bongers, K.S., Ebert, S.M., Fox, D.K., Dyle, M.C., Bullard, S.A., and Adams, C.M. (2012). Ursolic acid increases skeletal muscle and brown fat and decreases diet-induced obesity, glucose intolerance and fatty liver disease. PLoS One 7, e39332.

Lai, W., Huang, L., Ho, P., Li, Z., Montefiori, D., and Chen, C.-H. (2008). Betulinic acid derivatives that target gp120 and inhibit multiple genetic subtypes of human immunodeficiency virus type 1. Antimicrob Agents Chemother 52, 128-136.

Laszczyk, M. (2009). Pentacyclic Triterpenes of the Lupane, Oleanane and Ursane Group as Tools in Cancer Therapy. Planta Med 75, 1549-1560.

Lee, S., and Poulter, C.D. (2008). Cloning, solubilization, and characterization of squalene synthase from Thermosynechococcus elongatus BP-1. J Bacteriol 190, 3808-3816. van Leeuwen, J., Andrews, B., Boone, C., and Tan, G. (2015). Rapid and Efficient Plasmid Construction by Homologous Recombination in Yeast. Cold Spring Harb Protoc 2015, pdb.prot085100.

Leipold, D., Wiinsch, G., Schmidt, M., Bart, H.-J., Bley, T., Ekkehard Neuhaus, H., Bergmann, H., Richling, E., Muffler, K., and Ulber, R. (2010). Biosynthesis of ursolic acid derivatives by microbial metabolism of ursolic acid with Nocardia sp. strains — Proposal of new biosynthetic pathways. Process Biochemistry 45, 1043-1051 .

Liu, X.-B., Liu, M., Tao, X.-Y., Zhang, Z.-X., Wang, F.-Q., and Wei, D.-Z. (2015). Metabolic engineering of Pichia pastoris for the production of dammarenediol-ll. Journal of Biotechnology 216, 47-55.

Liu, Y., Jiang, X., Cui, Z., Wang, Z., Qi, Q., and Hou, J. (2019). Engineering the oleaginous yeast Yarrowia lipolytica for production of a-farnesene. Biotechnol Biofuels 12, 296.

Loeschcke, A., Dienst, D., Wewer, V., Hage-Hiilsmann, J., Dietsch, M., Kranz-Finger, S., Hiiren, V., Metzger, S., Urlacher, V.B., Gigolashvili, T., et al. (2017). The photosynthetic bacteria Rhodobacter capsulatus and Synechocystis sp. PCC 6803 as new hosts for cyclic plant triterpene biosynthesis. PLoS ONE 12, e0189816. - 63 -

Lopez-H ortas, L, Perez-Larran, P., Gonzalez-Munoz, M.J., Falque, E., and Dominguez, H. (2018). Recent developments on the extraction and application of ursolic acid. A review. Food Res Int 103, 130-149.

Lu, C., Zhang, C., Zhao, F., Li, D., and Lu, W. (2018). Biosynthesis of ursolic acid and oleanolic acid in Saccharomyces cerevisiae. AIChE J 64, 3794-3802.

Luke, G.A., and Ryan, M.D. (2013). The protein coexpression problem in biotechnology and biomedicine: virus 2A and 2A-like sequences provide a solution. Future Virology 8, 983-996.

Ma, H., Kunes, S., Schatz, P.J., and Botstein, D. (1987). Plasmid construction by homologous recombination in yeast. Gene 58, 201-216.

Machado, V.R., Sandjo, L.P., Pinheiro, G.L., Moraes, M.H., Steindel, M., Pizzolatti, M.G., and Biavatti, M.W. (2018). Synthesis of lupeol derivatives and their antileishmanial and antitrypanosomal activities. Nat Prod Res 32, 275-281 .

Mantzouridou, F., and Tsimidou, M.Z. (2010). Observations on squalene accumulation in Saccharomyces cerevisiae due to the manipulation of HMG2 and ERG6: Observations on squalene accumulation capacity in yeast. FEMS Yeast Research 10, 699-707.

Marx, H., Mecklenbrauker, A., Gasser, B., Sauer, M., and Mattanovich, D. (2009). Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus. FEMS Yeast Res 9, 1260-1270. Maschek, J.A., and Baker, B.J. (2008). The Chemistry of Algal Secondary Metabolism. In Algal Chemical Ecology, C.D. Amsler, ed. (Berlin, Heidelberg: Springer Berlin Heidelberg), pp. 1-24.

Matsuura, H., Watanabe, M. M., & Kaya, K. (2012) Squalene Quantification Using Octadecylbenzene as the Internal Standard. Procedia Environmental Sciences, 15, 43-46. Meadows, A.L., Hawkins, K.M., Tsegaye, Y., Antipov, E., Kim, Y., Raetz, L., Dahl, R.H., Tai, A., Mahatdejkul-Meadows, T., Xu, L., et al. (2016). Rewriting yeast central carbon metabolism for industrial isoprenoid production. Nature 537, 694-697.

Mertens-Talcott, S.U., Noratto, G.D., Li, X., Angel-Morales, G., Bertoldi, M.C., and Safe, S. (2013). Betulinic acid decreases ER-negative breast cancer cell growth in vitro and in vivo: role of Sp transcription factors and microRNA-27a:ZBTB10. Mol Carcinog 52, 591-602.

Metzger, P., and Largeau, C. (2005). Botryococcus braunii: a rich source for hydrocarbons and related ether lipids. Appl Microbiol Biotechnol 66, 486-496.

Midzak A, Papadopoulos V. (2016) Adrenal Mitochondria and Steroidogenesis: From Individual Proteins to Functional Protein Assemblies. Front Endocrinol (Lausanne) 7:106. Montague, T. G., Cruz, J. M., Gagnon, J. A., Church, G. M., & Valen, E. (2014). CHOPCHOP: A CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Research, 42(W1), W401-W407. https://doi.org/10.1093/nar/gku410

Morrone, D., Lowry, L., Determan, M.K., Hershey, D.M., Xu, M., and Peters, R.J. (2010). Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering. Appl Microbiol Biotechnol 85, 1893-1906. - 64 -

Moser, S., Strohmeier, G.A., Leitner, E., Plocek, T.J., Vanhessche, K., and Pichler, H. (2018). Whole-cell (p)-ambrein production in the yeast Pichia pastoris. Metabolic Engineering Communications 7, e00077.

Nagano, N., Matsui, S., Kuramura, T., Taoka, Y., Honda, D., and Hayashi, M. (2011). The distribution of extracellular cellulase activity in marine Eukaryotes, thraustochytrids. Mar Biotechnol (NY) 13, 133-136.

Naito, Y., Hino, K., Bono, H., & Ui-Tei, K. (2015). CRISPRdirect: Software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics, 31(7), 1120-1123. https://doi.org/10.1093/bioinformatics/btu743 Nakazawa, A., Matsuura, H., Kose, R., Kato, S., Honda, D., Inouye, I., Kaya, K., and Watanabe, M.M. (2012). Optimization of culture conditions of the thraustochytrid Aurantiochytrium sp. strain 18W-13a for squalene production. Bioresource Technology 109, 287-291.

Nakazawa, A., Kokubun, Y., Matsuura, H., Yonezawa, N., Kose, R., Yoshida, M., Tanabe, Y., Kusuda, E., Van Thang, D., Ueda, M., et al. (2014a). TLC screening of thraustochytrid strains for squalene production. J Appl Phycol 26, 29-41.

Okino, N., Wakisaka, H., Ishibashi, Y., and Ito, M. (2018). Visualization of Endoplasmic Reticulum and Mitochondria in Aurantiochytrium limacinum by the Expression of EGFP with Cell Organelle-Specific Targeting/Retaining Signals. Mar Biotechnol 20, 182-192. Paddon, C.J., Westfall, P.J., Pitera, D.J., Benjamin, K., Fisher, K., McPhee, D., Leavell, M.D., Tai, A., Main, A., Eng, D., et al. (2013). High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496, 528-532.

Pal, A., Ganguly, A., Chowdhuri, S., Yousuf, M., Ghosh, A., Barui, A.K., Kotcherlakota, R., Adhikari, S., and Banerjee, R. (2015). Bis-arylidene oxindole-betulinic Acid conjugate: a fluorescent cancer cell detector with potent anticancer activity. ACS Med Chem Lett 6, 612-

616.

Peralta-Yahya, P.P., Ouellet, M., Chan, R., Mukhopadhyay, A., Keasling, J.D., and Lee, T.S. (2011). Identification and microbial production of a terpene-based advanced biofuel. Nat Commun 2, 483. Polakowski, T., Stahl, U., and Lang, C. (1998). Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast. Applied Microbiology and Biotechnology 49, 66-71.

Pollier, J., Vancaester, E., Kuzhiumparambil, U., Vickers, C.E., Vandepoele, K., Goossens, A., and Fabris, M. (2019). A widespread alternative squalene epoxidase participates in eukaryote steroid biosynthesis. Nat Microbiol 4, 226-233.

Preciado, L.M., Rey-Suarez, P., Henao, I.C., and Pereahez, J.A. (2018). Betulinic, oleanolic and ursolic acids inhibit the enzymatic and biological effects induced by a P-l snake venom metalloproteinase. Chem Biol Interact 279, 219-226.

Primak, Y.A., Du, M., Miller, M.C., Wells, D.H., Nielsen, A.T., Weyler, W„ and Beck, Z.Q. (2011). Characterization of a feedback-resistant mevalonate kinase from the archaeon

Methanosarcina mazei. Appl Environ Microbiol 77, 7772-7778. - 65 -

Proschel, M., Detsch, R., Boccaccini, A.R., and Sonnewald, U. (2015). Engineering of Metabolic Pathways by Artificial Enzyme Channels. Front Bioeng Biotechnol 3, 168.

Pyle, D.J., Garcia, R.A., and Wen, Z. (2008). Producing Docosahexaenoic Acid (DHA)- Rich Algae from Biodiesel-Derived Crude Glycerol: Effects of Impurities on DHA Production and Algal Biomass Composition. J. Agric. Food Chem. 56, 3933-3939.

Qiao, W., Zhou, Z., Liang, Q., Mosongo, I., Li, C., and Zhang, Y. (2019). Improving lupeol production in yeast by recruiting pathway genes from different organisms. Sci Rep 9, 2992.

Raymond, C.K., Pownder, T.A., and Sexson, S.L. (1999). General method for plasmid construction using homologous recombination. Biotechniques 26, 134-138, 140-141. Raymond, C.K., Sims, E.H., and Olson, M.V. (2002). Linker-mediated recombinational subcloning of large DNA fragments using yeast. Genome Res 12, 190-197.

Remans, T., Schenk, P.M., Manners, J.M., Grof, C.P.L., and Elliott, A.R. (1999). A Protocol for the Fluorometric Quantification of mGFP5-ER and sGFP(S65T) in Transgenic Plants. Plant Molecular Biology Reporter 17, 385-395. Ren, L.-J., Sun, G., Ji, X.-J., Hu, X., and Huang, H. (2014). Compositional shift in lipid fractions during lipid accumulation and turnover in Schizochytrium sp. Bioresource Technology 157, 107-113.

Rios, J.L., and Manez, S. (2018). New Pharmacological Opportunities for Betulinic Acid. Planta Med 84, 8-19. Sala-Trepat, J.M., and Evans, W.C. (1971). The meta Cleavage of Catechol by Azotobacter Species. 4-Oxalocrotonate Pathway. Eur J Biochem 20, 400-413.

Sandeep, null, Misra, R.C., Chanotiya, C.S., Mukhopadhyay, P., and Ghosh, S. (2019). Oxidosqualene cyclase and CYP716 enzymes contribute to triterpene structural diversity in the medicinal tree banaba. New Phytol 222, 408-424. Schmandke, H. (2009). Triterpenoide in Oliven. Ernahrungs Umschau 56, 92-95.

Scullin, C., Stavila, V., Skarstad, A., Keasling, J.D., Simmons, B.A., and Singh, S. (2015). Optimization of renewable pinene production from the conversion of macroalgae Saccharina latissima. Bioresour Technol 184, 415-420.

Seemann, T. Shovill: Faster SPAdes assembly of lllumina reads. HttpsV/Github.Com/Tseemann/Shovill.

Selzer, E., Pimentel, E., Wacheck, V., Schlegel, W., Pehamberger, H., Jansen, B., and Kodym, R. (2000). Effects of betulinic acid alone and in combination with irradiation in human melanoma cells. J Invest Dermatol 114, 935-940.

Shanks, R.M.Q., Kadouri, D.E., MacEachran, D.P., and O’Toole, G.A. (2009). New yeast recombineering tools for bacteria. Plasmid 62, 88-97.

Simao, F.A., Waterhouse, R.M., loannidis, P., Kriventseva, E.V., and Zdobnov, E.M. (2015). BUSCO: assessing genome assembly and annotation completeness with singlecopy orthologs. Bioinformatics 31, 3210-3212. 66

Smith, T.F., and Waterman, M.S. (1981). Identification of common molecular subsequences. Journal of Molecular Biology 147, 195-197.

Song, T.-J., Park, C.-H., In, K.-R., Kim, J.-B., Kim, J.H., Kim, M., and Chang, H.J. (2021). Antidiabetic effects of betulinicacid mediated by the activation of the AMP-activated protein kinase pathway. PLoS ONE 16, e0249109.

Song, X., Wang, X., Tan, Y., Feng, Y., Li, W., and Cui, Q. (2015). High Production of Squalene Using a Newly Isolated Yeast-like Strain Pseudozyma sp. SD301 . J. Agric. Food Chem. 63, 8445-8451.

Souza-Moreira, T.M., Navarrete, C., Chen, X., Zanelli, C.F., Valentini, S.R., Furlan, M., Nielsen, J., and Krivoruchko, A. (2018). Screening of 2A peptides for polycistronic gene expression in yeast. FEMS Yeast Res 18.

Spanova, M., and Daum, G. (2011). Squalene - biochemistry, molecular biology, process biotechnology, and applications. Eur. J. Lipid Sci. Technol. 113, 1299-1320.

Summer, H., Gramer, R., & Droge, P. (2009). Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE). Journal of Visualized Experiments, 32, 1485. https://doi.Org/10.3791/1485

Tan, Y., Yu, R., and Pezzuto, J.M. (2003). Betulinic acid-induced programmed cell death in human melanoma cells involves mitogen-activated protein kinase activation. Clin Cancer Res 9, 2866-2875. Taoka, Y., Nagano, N., Kai, H., and Hayashi, M. (2017). Degradation of Distillery Lees (Shochu kasu) by Cellulase-Producing Thraustochytrids. J Oleo Sci 66, 31-40.

Trevan, M. (2007). Micro-algal biotechnology Edited by M. A. Borowitzka and L. J. Borowitzka, Cambridge University Press, Cambridge, 1988. pp. 477, price £45.00. ISBN 0- 521-323495. J. Chem. Technol. Biotechnol. 47, 181-182. Tsou, C.-Y., Matsunaga, S., and Okada, S. (2018). Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race. J Biosci Bioeng 125, 30-37.

Tsuruta, H., Paddon, C.J., Eng, D., Lenihan, J.R., Horning, T., Anthony, L.C., Regentin, R., Keasling, J.D., Renninger, N.S., and Newman, J.D. (2009). High-Level Production of Amorpha-4,11 -Diene, a Precursor of the Antimalarial Agent Artemisinin, in Escherichia coli. PLoS ONE 4, e4489.

Urban, P., Mignotte, C., Kazmaier, M., Delorme, F., and Pompon, D. (1997). Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5. J Biol Chem 272, 19176-19186.

Wang, P., Wei, W., Ye, W„ Li, X., Zhao, W., Yang, C., Li, C., Yan, X., and Zhou, Z. (2019). Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at high-efficiency. Cell Discov 5, 5.

Watanabe, K., Perez, C. M. T., Kitahori, T., Hata, K., Aoi, M., Takahashi, H., Sakuma, T., Okamura, Y., Nakashimada, Y., Yamamoto, T., Matsuyama, K., Mayuzumi, S., & Aki, T. (2021). Improvement of fatty acid productivity of thraustochytrid, Aurantiochytrium sp. By - 67 - genome editing. Journal of Bioscience and Bioengineering, 131(4), 373-380. https://doi.Org/10.1016/j.jbiosc.2020.11 .013

Westfall, P.J., Pitera, D.J., Lenihan, J.R., Eng, D., Woolard, F.X., Regentin, R., Horning, T., Tsuruta, H., Melis, D.J., Owens, A., et al. (2012). Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc Natl Acad Sci U S A 109, E111-118.

Wozniak, t., Skqpska, S., and Marszatek, K. (2015a). Ursolic Acid-A Pentacyclic Triterpenoid with a Wide Spectrum of Pharmacological Activities. Molecules 20, 20614- 20641. Wu, Y., Xu, S., Gao, X., Li, M., Li, D., and Lu, W. (2019). Enhanced protopanaxadiol production from xylose by engineered Yarrowia lipolytica. Microb Cell Fact 18, 83.

Xie, R., Zhang, H., Wang, X., Yang, X., Wu, S., Wang, H., Shen, P., and Ma, T. (2017). The protective effect of betulinic acid (BA) diabetic nephropathy on streptozotocin (STZ)- induced diabetic rats. Food Funct. 8, 299-306. Xu, W., Chai, C., Shao, L., Yao, J., and Wang, Y. (2016). Metabolic engineering of Rhodopseudomonas palustri s for squalene production. Journal of Industrial Microbiology and Biotechnology 43, 719-725.

Yarosh, D.B., Both, D., and Brown, D. (2000). Liposomal Ursolic Acid (Merotaine) Increases Ceramides and Collagen in Human Skin. Horm Res Paediatr 54, 318-321. Yokoyama, R., and Honda, D. (2007). Taxonomic rearrangement of the genus

Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov. Mycoscience 48, 199-211. Yoon, J.J., Lee, Y.J., Han, B.H., Choi, E.S., Kho, M.C., Park, J.H., Ahn, Y.M., Kim, H.Y., Kang, D.G., and Lee, H.S. (2017). Protective effect of betulinic acid on early atherosclerosis in diabetic apolipoprotein-E gene knockout mice. European Journal of Pharmacology 796, 224-232.

Yu, X.-J., Yu, Z.-Q., Liu, Y.-L, Sun, J., Zheng, J.-Y., and Wang, Z. (2015). Utilization of High-Fructose Corn Syrup for Biomass Production Containing High Levels of

Docosahexaenoic Acid by a Newly Isolated Aurantiochytrium sp. YLH70. Appl Biochem Biotechnol 177, 1229-1240.

Zerbino, D.R., and Birney, E. (2008). Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18, 821-829. Zhang, D., Jennings, S.M., Robinson, G.W., and Poulter, C.D. (1993). Yeast squalene synthase: expression, purification, and characterization of soluble recombinant enzyme. Arch Biochem Biophys 304, 133-143.

Zhang, J.-L, Bai, Q.-Y., Peng, Y.-Z., Fan, J., Jin, C.-C., Cao, Y.-X., and Yuan, Y.-J. (2020). High production of triterpenoids in Yarrowia lipolytica through manipulation of lipid components. Biotechnol Biofuels 13, 133. 68

Zhang, Y.-N., Zhang, W„ Hong, D., Shi, L, Shen, Q., Li, J.-Y., Li, J., and Hu, L.-H. (2008). Oleanolic acid and its derivatives: new inhibitor of protein tyrosine phosphatase 1B with cellular activities. Bioorg Med Chem 16, 8697-8705.

Zhao, F., Bai, P., Liu, T., Li, D., Zhang, X., Lu, W., and Yuan, Y. (2016). Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae: Optimization of a Cytochrome P450 Oxidation System. Biotechnol. Bioeng. 113, 1787-1795.

Zhao, Y., Fan, J., Wang, C., Feng, X., and Li, C. (2018). Enhancing oleanolic acid production in engineered Saccharomyces cerevisiae. Bioresource Technology 257, 339- 343.

Zhou, C., Li, J., Li, C., and Zhang, Y. (2016). Improvement of betulinic acid biosynthesis in yeast employing multiple strategies. BMC Biotechnol 16, 59.

Zhu, L., Zhang, X., Chang, L., Wang, A., Feng, P., and Han, L. (2014). Molecular cloning, prokaryotic expression and promoter analysis of squalene synthase gene from Schizochytrium Limacinum. Appl Biochem Microbiol 50, 411-419.

Zhuang, X., and Chappell, J. (2015). Building terpene production platforms in yeast: Building Terpene Production Platforms in Yeast. Biotechnol. Bioeng. 112, 1854-1864.

Zuco, V., Supino, R., Righetti, S.C., Cleris, L., Marchesi, E., Gambacorti-Passerini, C., and Formelli, F. (2002). Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer Lett 175, 17-25.