WO2022229378A1 - Production biotechnologique de terpènes - Google Patents

Production biotechnologique de terpènes Download PDF

Info

Publication number
WO2022229378A1
WO2022229378A1 PCT/EP2022/061458 EP2022061458W WO2022229378A1 WO 2022229378 A1 WO2022229378 A1 WO 2022229378A1 EP 2022061458 W EP2022061458 W EP 2022061458W WO 2022229378 A1 WO2022229378 A1 WO 2022229378A1
Authority
WO
WIPO (PCT)
Prior art keywords
synthase
microorganism
nucleic acid
cytochrome
seq
Prior art date
Application number
PCT/EP2022/061458
Other languages
English (en)
Inventor
Christoph Schwarz
Christian PREUSS
Amira ANTELMANN
Original Assignee
S2B Gmbh & Co. Kg
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by S2B Gmbh & Co. Kg filed Critical S2B Gmbh & Co. Kg
Publication of WO2022229378A1 publication Critical patent/WO2022229378A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P15/00—Preparation of compounds containing at least three condensed carbocyclic rings

Definitions

  • the present invention relates to microorganisms, which are genetically modified to produce pant derived triterpenes.
  • the present invention provides a microorganism of the class Labyrinthulomycota comprising at least one transgene encoding a heterologous 2,3-oxidosqualene cyclase.
  • 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.
  • 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).
  • MEV mevalonate
  • MEP methyl-D-erythrol-4-phosphate
  • 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 centuries 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).
  • 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).
  • 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.
  • CDW maximum cellular dry weight
  • CSQ squalene content
  • YSQ squalene titer
  • PSQ productivities
  • 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 facednet 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.
  • Thraustochytrids 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).
  • PUFAs polyunsaturated fatty acids
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • AA arachidonic acid
  • 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.
  • 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 - NeossanceTM Squalane.
  • 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.
  • this multistep process is costly and mainly used to provide intermediates for the synthesis of derivatives.
  • squalene is extensively exploited in dermatology and cosmetic industry, since it demonstrates exceptional performance as emulsifier, emollient and hydratation.
  • squalene 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.
  • 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).
  • CAGR 4,01 % a global market volume of approx. 5 tonnes / a
  • 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.
  • oleanolic formulations are marketed to fight hyperlipidemia, liver and lymphatic diseases (Schmandke 2009).
  • oleanolic acid and derivatives e.g., Bardoxolon [2-cyano-3,12-dioxooleana-1 ,9-dien-28-oic acid; CCDO], Bardoxolon-Methyl
  • Bardoxolon-Methyl Bardoxolon [2-cyano-3,12-dioxooleana-1 ,9-dien-28-oic acid; CCDO], Bardoxolon-Methyl
  • Both oleanolic as well as ursolic acid exhibit antioxidative, anti-inflammatory, anti-microbial, angiogenic and immunomodulatory activity.
  • 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.
  • 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).
  • 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).
  • 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).
  • betulinic acid may amiliorate artheroscerosis at an early stage (Song et al. 2020, Yoon et al. 2017).
  • 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.
  • 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.
  • HSV1 Herpes simplex virus 1
  • 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. Lup
  • 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.
  • 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.
  • 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).
  • 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.
  • the present invention relates to a microorganism of the class Labyrinthulomycota comprising at least one transgene encoding a heterologous 2,3- oxidosqualene cyclase.
  • 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.
  • the microorganism described above is selected from the group consisting of Thraustochytrids, in particular Schizochytrium, Aurantiochytrium, Thraustochytrium and Hondea.
  • 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.
  • ER endoplasmatic reticulum
  • 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.
  • 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.
  • 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- mevalon
  • 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.
  • at least one endogenous gene selected from the group consisting of lanosterol synthase, b-carotene synthase, ku80 and anthranilate synthase is inactivated.
  • At least one transgene 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.
  • 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
  • a squalene synthase and/or a squalene epoxidase preferably a hybrid squalene synthase/farnesyl pyrophosphate synthase and/or a hybrid squalene synthase/squalene epoxidase;
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • certain regulatory factors may lead to limited expression of the protein at all times or under certain conditions.
  • 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).
  • DMAPP dimethylallyl pyrophosphate
  • FPP farnesyl pyrophosphate
  • 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).
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • 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).
  • AATC acetyl-CoA-acetyltransferase
  • HMGS 3-hydroxy-3-methylglutaryl-CoA synthase
  • HMGR 3-hydroxy-3-methylglutaryl-CoA reductase
  • MVK mevalonate kinase
  • PMK phosphomevalonate kinase
  • PMD dihydrophosphomevalonate decarboxylase
  • Idi isopen
  • “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.
  • 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.
  • 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).
  • NHEJ non-homologous end joining
  • 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.
  • 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.
  • nucleic acid or amino acid sequences 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.
  • 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.
  • AACT acetyl-CoA-acetyltransferase
  • HMG-CoA 3-hydroxy-3-methylglutaryl- CoA
  • HMGR HMG-CoA-reductase
  • GPP geranyl pyrophosphate
  • FPP farnesyl pyrophosphate
  • FPPS farnesyl pyrophosphate synthase
  • GGPP geranylgeranyl pyrophosphate
  • SQS squalene synthase
  • aSQE alternative squalene epoxidase
  • LanS lanosterol synthase.
  • 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.
  • oriV ColdE1
  • 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.
  • PrPK pyruvate kinase gene promoter from A.limacinum MYA-1381
  • 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
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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. (2016) or into the mitochondria.
  • the N-terminal targeting signal of the oxidase CrAO was exchanged for the ER-targeting sequence of calreticulin from Aurantiochytrium limacinum MYA-1381.
  • 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).
  • FIG. 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.
  • IPP isopentenyl pyrophosphat
  • DMAPP dimethylallyl pyrophosphate
  • 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 di
  • HMGS HMG-CoA-synthase
  • MVA mevalonate
  • MVP mevalonate-5phosphate
  • MVPP mevalonate-5-diphosphate
  • 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.
  • 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 ColdE1: origin of replication for maintenance in in E.coli
  • bla ampicillin/carbenicillin resistance marker
  • 2- Micron replicon for maintenance in S.
  • 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
  • bla ampicillin/carbenicillin resistance marker
  • 2-Micron replicon for maintenance in S.
  • 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.
  • 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 ColdE1: origin of replication for maintenance in in E.coli
  • bla ampicillin/carbenicillin resistance marker
  • 2-Micron replicon for maintenance in S.
  • FIG. 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.
  • 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: 5 amino acid sequence of cytochrome P450 monooxygenase from Lagerstroemia speciosa (LsCYP716) (CYP5)
  • 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-
  • 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-
  • 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-
  • SEQ ID NO: 43 nucleic acid sequence of plasmid pAKU20888-S3 with vector backbone pAKU20888 and insert PrTub-NeoR-2A-XylE-2A-LupS-
  • SEQ ID NO: 44 nucleic acid sequence of plasmid pAKU20888-S4 with vector backbone pAKU20888 and insert PrTub-NeoR-2A-XylE-2A-MdAS-
  • SEQ ID NO: 45 nucleic acid sequence of plasmid pAKU20888-S5 with vector backbone pAKU20888 and insert PrTub-NeoR-2A-XylE-2A-LupS-
  • SEQ ID NO: 46 nucleic acid sequence of plasmid pAKU20888-S6 with vector backbone pAKU20888 and insert PrTub-NeoR-2A-XylE-2A-MdAS-
  • 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: 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: 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: 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: 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
  • 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.
  • triterpenes such as lupeol, oleanolic acid, ursolic acid, betulinic acid, corosolic acid and maslinic acid
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ATCC American Type Culture Collection
  • 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).
  • transgenes 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.
  • 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.
  • 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.
  • ER endoplasmatic reticulum
  • cytochrome P450 oxidase or hybrid cytochrome P450 oxidase/reductase 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • squalene represents a precursor for the production of the desired triterpenes (figure 1).
  • FPPS farnesyl pyrophosphate synthase
  • GFP farnesyl pyrophosphate synthase
  • FPP farnesyl pyrophosphate
  • SQL squalene synthase
  • 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.
  • 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
  • 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 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 .
  • 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.
  • 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 decarbox
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • At least one transgene are integrated into the genome of the microorganism.
  • 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.
  • 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.
  • 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.
  • the present invention relates to a nucleic acid construct or set of nucleic acid constructs encoding
  • 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
  • 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;
  • 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.
  • 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.
  • the construct or set of constructs is codon optimized to be expressed in Labyrinthulomycota, in particular in Thraustochytrids, preferably Schizochytrium, Aurantiochytrium, Thraustochytrium and Hondea.
  • the enzymes encoded in (i) to (iv) are under the control of a strong constitutive promoter.
  • the at least one 2,3-oxidosqualene cyclase and the at least one cytochrome P450 oxidase 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.
  • 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.
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the microorganism provided in step a) is selected from the group consisting of Thraustochytrids, in particular Schizochytrium, Aurantiochytrium, Thraustochytrium and Hondea.
  • 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.
  • the nucleic acid construct or the set of nucleic acid constructs is inserted into the genome of the microorganism by homologous recombination.
  • 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.
  • 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.
  • 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.
  • the enzymes of the methyl erythrol phosphate pathway (MEP) found in chloroplasts in the host microorganism, preferably in the mitochondria (see figure 6).
  • 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).
  • Thraustochytrids in particular Schizochytrium, Aurantiochytrium, Thraustochytrium and Hondea
  • Schizochytrium sp. S31 ATCC 20888
  • Aurantiochytrium sp. T66 ATCC-PRA-276
  • Schizochytrium sp. S31 ATCC 20888
  • Aurantiochytrium sp. T66 ATCC-PRA-276
  • Thraustochytrium sp. S-3 ATCC-26185
  • Aurantiochytrium limacinum SR 21 ATCC- MYA-1381
  • 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 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 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.
  • 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.
  • 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.
  • 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.
  • 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
  • Plasmid extraction was essentially accomplished using the QIAprep spin plasmid miniprep kit (Qiagen) with minor modifications.
  • 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.
  • 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.
  • 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 1 ⁇ 4 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.
  • protease XV 10 mg/mL
  • Snailase 10 mg/mL
  • Viscozym L hen egg lysozyme
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 ProtranTM 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.
  • 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.
  • Example 10A Agrobacterium-based transformation
  • 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.
  • 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.
  • IM induction medium
  • 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.
  • 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).
  • CRISPRdirect software Naito et al. 2014.
  • 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).
  • RNAs were purified using the MEGAclearTM 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
  • flanking homology regions to the genomic cutting site generated by the CRSIPR/Cas9 ribonucleoprotein complex were cotransformed into thraustochytrids via electroporation.
  • 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.
  • 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® UltraTM 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, Kunststoff) 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.
  • 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, Düsseldorf, Germany) and 1 ml of methanol/chloroform (2:1 , v:v) in a 14 ml screw cap glass tubes.
  • internal standard phenyloctadecane Sigma-Aldrich, St. Louis, MO, USA
  • the lipid fraction 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, Düsseldorf, 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, Düsseldorf, Germany) for subsequent HPLC analysis.
  • 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.
  • the lipid fraction 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, Düsseldorf, 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, Düsseldorf, Germany) for subsequent HPLC analysis.
  • 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.
  • Thraustochytrids as production organisms for docosahexaenoic acid (DHA), squalene, and carotenoids. Appl Microbiol Biotechnol 100, 4309-4321.
  • Brendolise C., Yauk, Y.-K., Eberhard, E.D., Wang, M., Chagne, D., Andre, C., Greenwood, D.R., and Beuning, L.L. (2011).
  • Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 27, 753-759.
  • AMPK regulating energy balance at the cellular and whole body levels.
  • Betulinic acid inhibits high-fat diet-induced obesity and improves energy balance by activating AMPK. Nutr Metab Cardiovasc Dis 29, 409- 420.
  • Betulinic acid derivatives that target gp120 and inhibit multiple genetic subtypes of human immunodeficiency virus type 1. Antimicrob Agents Chemother 52, 128-136.
  • virus 2A and 2A-like sequences provide a solution. Future Virology 8, 983-996.
  • 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.
  • Botryococcus braunii a rich source for hydrocarbons and related ether lipids. Appl Microbiol Biotechnol 66, 486-496.
  • 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., Hyundai, 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.
  • 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.
  • 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.
  • 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 (Srochu kasu) by Cellulase-Producing Thraustochytrids. J Oleo Sci 66, 31-40.
  • 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.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des micro-organismes génétiquement modifiés pour produire des triterpènes dérivés de plantes. En particulier, la présente invention fournit un micro-organisme de la classe Labyrinthulomycota comprenant au moins un transgène codant pour une 2,3-oxydosqualène cyclase hétérologue. L'invention concerne en outre une construction d'acide nucléique codant pour au moins une 2,3-oxydosqualène cyclase et au moins une autre enzyme. La présente invention concerne également un procédé de production d'un micro-organisme selon l'invention et fournit en outre un procédé de production d'un ou plusieurs triterpènes dérivés de plantes et/ou de leurs dérivés comprenant une étape de culture d'un micro-organisme selon la présente invention dans des conditions favorisant la production de triterpènes dérivés de plantes.
PCT/EP2022/061458 2021-04-29 2022-04-29 Production biotechnologique de terpènes WO2022229378A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EPPCT/EP2021/061342 2021-04-29
PCT/EP2021/061342 WO2022228687A1 (fr) 2021-04-29 2021-04-29 Production biotechnologique de terpènes

Publications (1)

Publication Number Publication Date
WO2022229378A1 true WO2022229378A1 (fr) 2022-11-03

Family

ID=75728859

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/EP2021/061342 WO2022228687A1 (fr) 2021-04-29 2021-04-29 Production biotechnologique de terpènes
PCT/EP2022/061458 WO2022229378A1 (fr) 2021-04-29 2022-04-29 Production biotechnologique de terpènes

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/EP2021/061342 WO2022228687A1 (fr) 2021-04-29 2021-04-29 Production biotechnologique de terpènes

Country Status (1)

Country Link
WO (2) WO2022228687A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116891871A (zh) * 2023-09-08 2023-10-17 云南农业大学 一种采用葫芦二烯醇合酶合成葫芦二烯醇的方法及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6607900B2 (en) 2000-01-28 2003-08-19 Martek Biosciences Boulder Corporation Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
WO2008130372A2 (fr) * 2006-09-28 2008-10-30 Microbia, Inc. Production de stérols dans des levures et des champignons oléagineux
US20120107893A1 (en) * 2009-11-10 2012-05-03 Massachusetts Institute Of Technology Microbial engineering for the production of chemical and pharmaceutical products from the isoprenoid pathway
JP2017131202A (ja) * 2016-01-29 2017-08-03 国立大学法人大阪大学 ２２α位水酸化五環系トリテルペンの生産およびその利用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6607900B2 (en) 2000-01-28 2003-08-19 Martek Biosciences Boulder Corporation Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors
WO2008130372A2 (fr) * 2006-09-28 2008-10-30 Microbia, Inc. Production de stérols dans des levures et des champignons oléagineux
US20120107893A1 (en) * 2009-11-10 2012-05-03 Massachusetts Institute Of Technology Microbial engineering for the production of chemical and pharmaceutical products from the isoprenoid pathway
JP2017131202A (ja) * 2016-01-29 2017-08-03 国立大学法人大阪大学 ２２α位水酸化五環系トリテルペンの生産およびその利用

Non-Patent Citations (159)

* Cited by examiner, † Cited by third party
Title
AALBERS, F.S.FRAAIJE, M.W.: "Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes", CHEMBIOCHEM, vol. 20, 2019, pages 20 - 28
AASEN, I.M.ERTESVAG, H.HEGGESET, T.M.B.LIU, B.BRAUTASET, T.VADSTEIN, O.ELLINGSEN, T.E.: "Thraustochytrids as production organisms for docosahexaenoic acid (DHA), squalene, and carotenoids", APPL MICROBIOL BIOTECHNOL, vol. 100, 2016, pages 4309 - 4321, XP035870418, DOI: 10.1007/s00253-016-7498-4
AGRA, L.C.FERRO, J.N.S.BARBOSA, F.T.BARRETO, E.: "Triterpenes with healing activity: A systematic review", J DERMATOLOG TREAT, vol. 26, 2015, pages 465 - 470
AJAIYEOBA, E.O.ONOCHA, P.A.NWOZO, S.O.SAMA, W.: "Antimicrobial and cytotoxicity evaluation of Buchholzia coriacea stem bark", FITOTERAPIA, vol. 74, 2003, pages 706 - 709, XP008048104, DOI: 10.1016/S0367-326X(03)00142-4
AJIKUMAR, P.K., TYO, K., CARLSEN, S., MUCHA, O., PHON, T.H.,STEPHANOPOULOS, G.: "Terpenoids: Opportunities for Biosynthesis of Natural Product Drugs Using Engineered Microorganisms", MOL. PHARMACEUTICS, vol. 5, 2008, pages 167 - 190, XP002617175, DOI: 10.1021/MP700151B
ANDRE, C.M.LEGAY, S.DELERUELLE, A.NIEUWENHUIZEN, N.PUNTER, M.BRENDOLISE, C.COONEY, J.M.LATEUR, M.HAUSMAN, J.-F.LARONDELLE, Y. ET A: "Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids", NEW PHYTOL, vol. 211, 2016, pages 1279 - 1294
ARAFILES, K.H.V.IWASAKA, H.ERAMOTO, Y.OKAMURA, Y.TAJIMA, T.MATSUMURA, Y.NAKASHIMADA, Y.AKI, T.: "Value-added lipid production from brown seaweed biomass by two-stage fermentation using acetic acid bacterium and thraustochytrid", APPL MICROBIOL BIOTECHNOL, vol. 98, 2014, pages 9207 - 9216, XP055473579, DOI: 10.1007/s00253-014-5980-4
ARENDT, P.MIETTINEN, K.POLLIER, J.DE RYCKE, R.CALLEWAERT, N.GOOSSENS, A.: "An endoplasmic reticulum-engineered yeast platform for overproduction of triterpenoids", METABOLIC ENGINEERING, vol. 40, 2017, pages 165 - 175, XP029945566, DOI: 10.1016/j.ymben.2017.02.007
ATTA-UR-RAHMANZAREEN, S.CHOUDHARY, M.I.AKHTAR, M.N.KHAN, S.N.: "α-Glucosidase Inhibitory Activity of Triterpenoids from Cichorium intybus", J. NAT. PROD., vol. 71, 2008, pages 910 - 913
AYELESO, T.B.MATUMBA, M.G.MUKWEVHO, E.: "Molecules", 2017, article "Oleanolic Acid and Its Derivatives: Biological Activities and Therapeutic Potential in Chronic Diseases", pages: 22
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.: "SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing", J COMPUT BIOL, vol. 19, 2012, pages 455 - 477, XP002764171, DOI: 10.1089/cmb.2012.0021
BAYNE, A.-C.V., BOLTZ, D., OWEN, C., BETZ, Y., MAIA, G., AZADI, P., ARCHER-HARTMANN, S.,ZIRKLE, R., LIPPMEIER, J.C.: "Vaccination against Influenza with Recombinant Hemagglutinin Expressed by Schizochytrium sp. Confers Protective Immunity", PLOS ONE, vol. 8, 2013, pages e61790, XP055062273, DOI: 10.1371/journal.pone.0061790
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 AZEVED: "Lupeol, a Dietary Triterpene, Enhances Wound Healing in Streptozotocin-Induced Hyperglycemic Rats with Modulatory Effects on Inflammation, Oxidative Stress, and Angiogenesis", OXID MED CELL LONGEV, vol. 2019, 2019, pages 3182627
BOLGER, A.M.LOHSE, M.USADEL, B.: "Trimmomatic: a flexible trimmer for Illumina sequence data", BIOINFORMATICS, vol. 30, 2014, pages 2114 - 2120, XP055862121, DOI: 10.1093/bioinformatics/btu170
BOTH, D.M.GOODTZOVA, K.YAROSH, D.B.BROWN, D.A.: "Liposome-encapsulated ursolic acid increases ceramides and collagen in human skin cells", ARCH DERMATOL RES, vol. 293, 2002, pages 569 - 575, XP002265521
BRENDOLISE, C.YAUK, Y.-K.EBERHARD, E.D.WANG, M.CHAGNE, D.ANDRE, C.GREENWOOD, D.R.BEUNING, L.L.: "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, vol. 278, 2011, pages 2485 - 2499
BROKER, J.N.MULLER, B.VAN DEENEN, N.PRUFER, D.SCHULZE GRONOVER, C.: "Upregulating the mevalonate pathway and repressing sterol synthesis in Saccharomyces cerevisiae enhances the production of triterpenes", APPL MICROBIOL BIOTECHNOL, vol. 102, 2018, pages 6923 - 6934
BURJA, A.M.RADIANINGTYAS, H.WINDUST, A.BARROW, C.J.: "Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species: screening of strains and optimization of omega-3 production", APPL MICROBIOL BIOTECHNOL, vol. 72, 2006, pages 1161 - 1169
CHANG, M.-H.KIM, H.-J.JAHNG, K.-Y.HONG, S.-C.: "The isolation and characterization of Pseudozyma sp. JCC 207, a novel producer of squalene", APPL MICROBIOL BIOTECHNOL, vol. 78, 2008, pages 963 - 972, XP019586370
CHEN, A.H.SILVER, P.A.: "Designing biological compartmentalization", TRENDS IN CELL BIOLOGY, vol. 22, 2012, pages 662 - 670
CHEN, J.J.GENEREUX, J.C.WISEMAN, R.L.: "Endoplasmic reticulum quality control and systemic amyloid disease: Impacting protein stability from the inside out", IUBMB LIFE, vol. 67, 2015, pages 404 - 413
CHENG, R.MA, R.LI, K.RONG, H.LIN, X.WANG, Z.YANG, S.MA, Y.: "Agrobacterium tumefaciens mediated transformation of marine microalgae Schizochytrium", MICROBIOLOGICAL RESEARCH, vol. 167, no. 3, 2012, pages 179 - 186, XP055617890, Retrieved from the Internet <URL:https://doi.Org/10.1016/j.micres.2011.05.003> DOI: 10.1016/j.micres.2011.05.003
CHI, Z.HU, B.LIU, Y.FREAR, C.WEN, Z.CHEN, S.: "Production of omega-3 polyunsaturated fatty acids from cull potato using an algae culture process", APPL BIOCHEM BIOTECHNOL, vol. 137-140, 2007, pages 805 - 815, XP055457326, DOI: 10.1007/s12010-007-9099-2
CHU, L.L.MONTECILLO, J.A.V.BAE, H.: "Recent Advances in the Metabolic Engineering of Yeasts for Ginsenoside Biosynthesis", FRONT. BIOENG. BIOTECHNOL., vol. 8, 2020, pages 139
D'ADAMO, S.SCHIANO DI VISCONTE, G.LOWE, G.SZAUB-NEWTON, J.BEACHAM, T.LANDELS, A.ALLEN, M.J.SPICER, A.MATTHIJS, M.: "Engineering the unicellular alga Phaeodactylum tricornutum for high-value plant triterpenoid production", PLANT BIOTECHNOL J, vol. 17, 2019, pages 75 - 87, XP055822420, DOI: 10.1111/pbi.12948
DAHLIN, P.SRIVASTAVA, V.BULONE, V.MCKEE, L.S.: "The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product", FRONT. MICROBIOL., vol. 7, 2016
DAI, L.LIU, C.ZHU, Y.ZHANG, J.MEN, Y.ZENG, YSUN, Y.: "Functional Characterization of Cucurbitadienol Synthase and Triterpene Glycosyltransferase Involved in Biosynthesis of Mogrosides from Siraitia grosvenorii", PLANT CELL PHYSIOL, vol. 56, 2015, pages 1172 - 1182
DAI, Z., WANG, B., LIU, Y., SHI, M., WANG, D., ZHANG, X., LIU, T., HUANG, L., ZHANG, X.: "Producing aglycons of ginsenosides in bakers' yeast", SCI REP, vol. 4, 2015, pages 3698, XP055471751, DOI: 10.1038/srep03698
DAI, Z.LIU, Y.ZHANG, X.SHI, M.WANG, B.WANG, D.HUANG, L.ZHANG, X.: "Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides", METABOLIC ENGINEERING, vol. 20, 2013, pages 146 - 156, XP055471754, DOI: 10.1016/j.ymben.2013.10.004
DING, Y.WANG, K.-F.WANG, W.-J.MA, Y.-R.SHI, T.-Q.HUANG, H.JI, X.-J.: "Increasing the homologous recombination efficiency of eukaryotic microorganisms for enhanced genome engineering", APPL MICROBIOL BIOTECHNOL, vol. 103, 2019, pages 4313 - 4324, XP036776481, DOI: 10.1007/s00253-019-09802-2
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.: "Co-expression of squalene epoxidases with triterpene cyclases boosts production of triterpenoids in plants and yeast", METABOLIC ENGINEERING, vol. 49, 2018, pages 1 - 12, XP085507108, DOI: 10.1016/j.ymben.2018.07.002
DUEBER, J.E.WU, G.C.MALMIRCHEGINI, G.R.MOON, T.S.PETZOLD, C.J.ULLAL, A.V.PRATHER, K.L.J.KEASLING, J.D.: "Synthetic protein scaffolds provide modular control over metabolic flux", NAT BIOTECHNOL, vol. 27, 2009, pages 753 - 759, XP008144815
EHRHARDT, H.FULDA, S.FUHRER, M.DEBATIN, K.M.JEREMIAS, I.: "Betulinic acid-induced apoptosis in leukemia cells", LEUKEMIA, vol. 18, 2004, pages 1406 - 1412, XP037782004, DOI: 10.1038/sj.leu.2403406
ETHIER, S.WOISARD, K.VAUGHAN, D.WEN, Z.: "Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid", BIORESOURCE TECHNOLOGY, vol. 102, 2011, pages 88 - 93, XP055315438, DOI: 10.1016/j.biortech.2010.05.021
FOURNET, A.ANGELO, A.MUNOZ, V.ROBLOT, F.HOCQUEMILLER, R.CAVE, A.: "Biological and chemical studies of Pera benensis, a Bolivian plant used in folk medicine as a treatment of cutaneous leishmaniasis", J ETHNOPHARMACOL, vol. 37, 1992, pages 159 - 164, XP023857034, DOI: 10.1016/0378-8741(92)90074-2
GAO, S.TONG, Y.ZHU, L.GE, M.JIANG, Y.CHEN, D.YANG, S.: "Production of β-carotene by expressing a heterologous multifunctional carotene synthase in Yarrowia lipolytica", BIOTECHNOL LETT, vol. 39, 2017, pages 921 - 927, XP036239645, DOI: 10.1007/s10529-017-2318-1
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.ALGI: "Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome", SCIENCE, vol. 319, 2008, pages 1215 - 1220, XP009101716, DOI: 10.1126/science.1151721
GOLDMAN, A.HARPER, S.SPEICHER, D.W.: "Detection of Proteins on Blot Membranes", CURR PROTOC PROTEIN SCI, vol. 86, 2016
GRAHAME HARDIE, D.: "AMP-activated protein kinase: a key regulator of energy balance with many roles in human disease", J INTERN MED, vol. 276, 2014, pages 543 - 559
GUHLING, O.HOBL, B.YEATS, T.JETTER, R.: "Cloning and characterization of a lupeol synthase involved in the synthesis of epicuticularwax crystals on stem and hypocotyl surfaces of Ricinus communis", ARCH BIOCHEM BIOPHYS, vol. 448, 2006, pages 60 - 72, XP055560806, DOI: 10.1016/j.abb.2005.12.013
GUPTA, A.ABRAHAM, R.E.BARROW, C.J.PURI, M.: "Omega-3 fatty acid production from enzyme saccharified hemp hydrolysate using a novel marine thraustochytrid strain", BIORESOURCE TECHNOLOGY, vol. 184, 2015, pages 373 - 378, XP029205302, DOI: 10.1016/j.biortech.2014.11.031
GUREVICH, A.SAVELIEV, V.VYAHHI, N.TESLER, G.: "QUAST: quality assessment tool for genome assemblies", BIOINFORMATICS, vol. 29, 2013, pages 1072 - 1075
HAMMER, S.K.AVALOS, J.L.: "Harnessing yeast organelles for metabolic engineering", NAT CHEM BIOL, vol. 13, 2017, pages 823 - 832, XP055712903, DOI: 10.1038/nchembio.2429
HARDIE, D.G.ASHFORD, M.L.J.: "AMPK: regulating energy balance at the cellular and whole body levels", PHYSIOLOGY (BETHESDA, vol. 29, 2014, pages 99 - 107
HARISH, B.G.KRISHNA, V.SANTOSH KUMAR, H.S.KHADEER AHAMED, B.M.SHARATH, R.KUMARA SWAMY, H.M.: "Wound healing activity and docking of glycogen-synthase-kinase-3-beta-protein with isolated triterpenoid lupeol in rats", PHYTOMEDICINE, vol. 15, 2008, pages 763 - 767
HATA, K.HORI, K.MURATA, J.TAKAHASHI, S.: "Remodeling of actin cytoskeleton in lupeol-induced B16 2F2 cell differentiation", J BIOCHEM, vol. 138, 2005, pages 467 - 472
HATA, K.HORI, K.TAKAHASHI, S.: "Role of p38 MAPK in lupeol-induced B16 2F2 mouse melanoma cell differentiation", J BIOCHEM, vol. 134, 2003, pages 441 - 445
HATA, K.ISHIKAWA, K.HORI, K.KONISHI, T.: "Differentiation-inducing activity of lupeol, a lupane-type triterpene from Chinese dandelion root (Hokouei-kon), on a mouse melanoma cell line", BIOL PHARM BULL, vol. 23, 2000, pages 962 - 967, XP002980461
HERNANDEZ-PEREZ, M.LOPEZ-GARCIA, R.E.RABANAL, R.M.DARIAS, V.ARIAS, A.: "Antimicrobial activity of Visnea mocanera leaf extracts", J ETHNOPHARMACOL, vol. 41, 1994, pages 115 - 119, XP025544319, DOI: 10.1016/0378-8741(94)90065-5
HOANG, M.H., HA, N.C., THORN, L.T., TAM, L.T., ANH, H.T.L., THU, N.T.H., HONG, D.D.: "Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 118, 2014, pages 632 - 639, XP055903175, DOI: 10.1016/j.jbiosc.2014.05.015
HONG, W.-K.RAIRAKHWADA, D.SEO, P.-S.PARK, S.-Y.HUR, B.-K.KIM, C.H.SEO, J.-W.: "Production of lipids containing high levels of docosahexaenoic acid by a newly isolated microalga, Aurantiochytrium sp. KRS101", APPL BIOCHEM BIOTECHNOL, vol. 164, 2011, pages 1468 - 1480, XP019922295, DOI: 10.1007/s12010-011-9227-x
HU, W.LIU, N.TIAN, Y.ZHANG, L.: "Molecular cloning, expression, purification, and functional characterization of dammarenediol synthase from Panax ginseng", BIOMED RES INT, vol. 2013, 2013, pages 285740
HUANG, J.ZHA, W.AN, T.DONG, H.HUANG, Y.WANG, D.YU, R.DUAN, L.ZHANG, X.PETERS, R.J. ET AL.: "Identification of RoCYP01 (CYP716A155) enables construction of engineered yeast for high-yield production of betulinic acid", APPL MICROBIOL BIOTECHNOL, vol. 103, 2019, pages 7029 - 7039, XP036859179, DOI: 10.1007/s00253-019-10004-z
HUANG, L.LI, J.YE, H.LI, C.WANG, H.LIU, B.ZHANG, Y.: "Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus", PLANTA, vol. 236, 2012, pages 1571 - 1581
HUANG, Q.-X.CHEN, H.-F.LUO, X.-R.ZHANG, Y.-X.YAO, X.ZHENG, X.: "Structure and Anti-HIV Activity of Betulinic Acid Analogues", CURR MED SCI, vol. 38, 2018, pages 387 - 397, XP036617316, DOI: 10.1007/s11596-018-1891-4
HUANG, Z.-R.LIN, Y.-K.FANG, J.-Y.: "Biological and Pharmacological Activities of Squalene and Related Compounds: Potential Uses in Cosmetic Dermatology", MOLECULES, vol. 14, 2009, pages 540 - 554, XP055147095, DOI: 10.3390/molecules14010540
HUMHAL, T.KASTANEK, P.JEZKOVA, Z.CADKOVA, A.KOHOUTKOVA, J.BRANYIK, T.: "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, vol. 40, 2017, pages 395 - 402
HUTTANUS, H.M.FENG, X.: "Compartmentalized metabolic engineering for biochemical and biofuel production", BIOTECHNOL J, vol. 12, 2017
IWASAKA, H.AKI, T.ADACHI, H.WATANABE, K.KAWAMOTO, S.ONO, K.: "Utilization of waste syrup for production of polyunsaturated fatty acids and xanthophylls by Aurantiochytrium", J OLEO SCI, vol. 62, 2013, pages 729 - 736, XP055247270, DOI: 10.5650/jos.62.729
IWASAKA, H.KOYANAGI, R.SATOH, R.NAGANO, A.WATANABE, K.HISATA, K.SATOH, N.AKI, T.: "A Possible Trifunctional β-Carotene Synthase Gene Identified in the Draft Genome of Aurantiochytrium sp. Strain KH105", GENES, vol. 9, 2018, pages 200
J. CHEM. TECHNOL. BIOTECHNOL., vol. 47, pages 181 - 182
JAGER, S.TROJAN, H.KOPP, T.LASZCZYK, M.N.SCHEFFLER, A.: "Pentacyclic triterpene distribution in various plants - rich sources for a new group of multi-potent plant extracts", MOLECULES, vol. 14, 2009, pages 2016 - 2031
JAKOBSEN, A.N.AASEN, I.M.STROM, A.R.: "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, vol. 73, 2007, pages 5848 - 5856, XP008153839, DOI: 10.1128/AEM.00610-07
JIANG, Y.ZHU, Q.LIAO, Y.WANG, Q.LI, Y.DONG, X.PENG, J.YUAN, J.WANG, J.: "The Delta 5, 7-STEROLS and Astaxanthin in the Marine Microheterotroph Schizochytrium sp. S31", J AM OIL CHEM SOC, vol. 97, 2020, pages 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.: "Direct transfer of whole genomes from bacteria to yeast", NAT METHODS, vol. 10, 2013, pages 410 - 412, XP055602448, DOI: 10.1038/nmeth.2433
KATABAMI, A.LI, L.IWASAKI, M.FURUBAYASHI, M.SAITO, K.UMENO, D.: "Production of squalene by squalene synthases and their truncated mutants in Escherichia coli", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 119, 2015, pages 165 - 171
KAYA, K.NAKAZAWA, A.MATSUURA, H.HONDA, D.INOUYE, I.WATANABE, M.M.: "Thraustochytrid Aurantiochytrium sp. 18W-13a Accummulates High Amounts of Squalene", BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, vol. 75, 2011, pages 2246 - 2248, XP055531938, DOI: 10.1271/bbb.110430
KHAN, N.E.NYBO, S.E.CHAPPELL, J.CURTIS, W.R.: "Triterpene hydrocarbon production engineered into a metabolically versatile host--Rhodobacter capsulatus", BIOTECHNOL BIOENG, vol. 112, 2015, pages 1523 - 1532, XP071101916, DOI: 10.1002/bit.25573
KIM, J.-E.JANG, I.-S.SON, S.-H.KO, Y.-J.CHO, B.-K.KIM, S.C.LEE, J.Y.: "Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway", METAB ENG, vol. 56, 2019, pages 50 - 59, XP085876189, DOI: 10.1016/j.ymben.2019.08.013
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.: "Betulinic acid inhibits high-fat diet-induced obesity and improves energy balance by activating AMPK", NUTR METAB CARDIOVASC DIS, vol. 29, 2019, pages 409 - 420
KLABUNDE, J.KUNZE, G.GELLISSEN, G.HOLLENBERG, C.P.: "Integration of heterologous genes in several yeast species using vectors containing a Hansenula polymorpha-derived rDNA-targeting element", FEMS YEAST RES, vol. 4, 2003, pages 185 - 193, XP008136433, DOI: 10.1016/S1567-1356(03)00148-X
KOUPRINA, N.LARIONOV, V.: "Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology", CHROMOSOMA, vol. 125, 2016, pages 621 - 632, XP036055097, DOI: 10.1007/s00412-016-0588-3
KUMAR, S.KUMAR, V.PRAKASH, O.: "Enzymes inhibition and antidiabetic effect of isolated constituents from Dillenia indica", BIOMED RES INT, vol. 2013, 2013, pages 382063
KUNKEL, S.D.ELMORE, C.J.BONGERS, K.S.EBERT, S.M.FOX, D.K.DYLE, M.C.BULLARD, S.A.ADAMS, C.M.: "Ursolic acid increases skeletal muscle and brown fat and decreases diet-induced obesity, glucose intolerance and fatty liver disease", PLOS ONE, vol. 7, 2012, pages e39332
LAI, W.HUANG, L.HO, P.LI, Z.MONTEFIORI, D.CHEN, C.-H.: "Betulinic acid derivatives that target gp120 and inhibit multiple genetic subtypes of human immunodeficiency virus type 1", ANTIMICROB AGENTS CHEMOTHER, vol. 52, 2008, pages 128 - 136
LASZCZYK, M.: "Pentacyclic Triterpenes of the Lupane, Oleanane and Ursane Group as Tools in Cancer Therapy", PLANTA MED, vol. 75, 2009, pages 1549 - 1560
LEE, S.POULTER, C.D.: "Cloning, solubilization, and characterization of squalene synthase from Thermosynechococcus elongatus BP-1", J BACTERIOL, vol. 190, 2008, pages 3808 - 3816
LEIPOLD, D.WUNSCH, G.SCHMIDT, M.BART, H.-J.BLEY, T.EKKEHARD NEUHAUS, H.BERGMANN, H.RICHLING, E.MUFFLER, K.ULBER, R.: "Biosynthesis of ursolic acid derivatives by microbial metabolism of ursolic acid with Nocardia sp. strains—Proposal of new biosynthetic pathways", PROCESS BIOCHEMISTRY, vol. 45, 2010, pages 1043 - 1051
LIU, X.-B.LIU, M.TAO, X.-Y.ZHANG, Z.-X.WANG, F.-Q.WEI, D.-Z.: "Metabolic engineering of Pichia pastoris for the production of dammarenediol-II", JOURNAL OF BIOTECHNOLOGY, vol. 216, 2015, pages 47 - 55
LIU, Y.JIANG, X.CUI, Z.WANG, Z.QI, Q.HOU, J.: "Engineering the oleaginous yeast Yarrowia lipolytica for production of a-farnesene", BIOTECHNOL BIOFUELS, vol. 12, 2019, pages 296
LOESCHCKE, A.DIENST, D.WEWER, V.HAGE-HULSMANN, J.DIETSCH, M.KRANZ-FINGER, S.HUREN, V.METZGER, S.URLACHER, V.B.GIGOLASHVILI, T. ET : "The photosynthetic bacteria Rhodobacter capsulatus and Synechocystis sp. PCC 6803 as new hosts for cyclic plant triterpene biosynthesis", PLOS ONE, vol. 12, 2017, pages e0189816
LOPEZ-HORTAS, L.PEREZ-LARRAN, P.GONZALEZ-MUNOZ, M.J.FALQUE, E.DOMINGUEZ, H.: "Recent developments on the extraction and application of ursolic acid. A review", FOOD RES INT, vol. 103, 2018, pages 130 - 149
LU, C.ZHANG, C.ZHAO, F.LI, D.LU, W.: "Biosynthesis of ursolic acid and oleanolic acid in Saccharomyces cerevisiae", ALCHE J, vol. 64, 2018, pages 3794 - 3802, XP071013728, DOI: 10.1002/aic.16370
LUKE, G.A.RYAN, M.D.: "The protein coexpression problem in biotechnology and biomedicine: virus 2A and 2A-like sequences provide a solution", FUTURE VIROLOGY, vol. 8, 2013, pages 983 - 996
MA, H.KUNES, S.SCHATZ, P.J.BOTSTEIN, D.: "Plasmid construction by homologous recombination in yeast", GENE, vol. 58, 1987, pages 201 - 216, XP025705444, DOI: 10.1016/0378-1119(87)90376-3
MACHADO, V.R.SANDJO, L.P.PINHEIRO, G.L.MORAES, M.H.STEINDEL, M.PIZZOLATTI, M.G.BIAVATTI, M.W.: "Synthesis of lupeol derivatives and their antileishmanial and antitrypanosomal activities", NAT PROD RES, vol. 32, 2018, pages 275 - 281, XP018530852
MANTZOURIDOU, F.TSIMIDOU, M.Z.: "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, vol. 10, 2010, pages 699 - 707
MARX, H.MECKLENBRAUKER, A.GASSER, B.SAUER, M.MATTANOVICH, D.: "Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus", FEMS YEAST RES, vol. 9, 2009, pages 1260 - 1270, XP055867263, DOI: 10.1111/j.1567-1364.2009.00561.x
MASCHEK, J.A.BAKER, B.J.: "Algal Chemical Ecology", 2008, SPRINGER BERLIN HEIDELBERG, article "The Chemistry of Algal Secondary Metabolism", pages: 1 - 24
MATSUURA, H.WATANABE, M. M.KAYA, K.: "Squalene Quantification Using Octadecylbenzene as the Internal Standard", PROCEDIA ENVIRONMENTAL SCIENCES, vol. 15, 2012, pages 43 - 46, XP028498345, DOI: 10.1016/j.proenv.2012.05.008
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.: "Rewriting yeast central carbon metabolism for industrial isoprenoid production", NATURE, vol. 537, 2016, pages 694 - 697, XP055577914, DOI: 10.1038/nature19769
MERTENS-TALCOTT, S.U.NORATTO, G.D.LI, X.ANGEL-MORALES, G.BERTOLDI, M.C.SAFE, S.: "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, vol. 52, 2013, pages 591 - 602
METZGER, P.LARGEAU, C.: "Botryococcus braunii: a rich source for hydrocarbons and related ether lipids", APPL MICROBIOL BIOTECHNOL, vol. 66, 2005, pages 486 - 496, XP019331745, DOI: 10.1007/s00253-004-1779-z
MIDZAK APAPADOPOULOS V.: "Adrenal Mitochondria and Steroidogenesis: From Individual Proteins to Functional Protein Assemblies", FRONT ENDOCRINOL (LAUSANNE, vol. 7, 2016, pages 106
MONTAGUE, T. G.CRUZ, J. M.GAGNON, J. A.CHURCH, G. M.VALEN, E.: "CHOPCHOP: A CRISPR/Cas9 and TALEN web tool for genome editing", NUCLEIC ACIDS RESEARCH, vol. 42, no. W1, 2014, pages W401 - W407, XP055201392, Retrieved from the Internet <URL:https://doi.org/10.1093/nar/gku410> DOI: 10.1093/nar/gku410
MORRONE, D.LOWRY, L.DETERMAN, M.K.HERSHEY, D.M.XU, M.PETERS, R.J.: "Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering", APPL MICROBIOL BIOTECHNOL, vol. 85, 2010, pages 1893 - 1906, XP019778553
MOSER, S.STROHMEIER, G.A.LEITNER, E.PLOCEK, T.J.VANHESSCHE, K.PICHLER, H.: "Whole-cell (p)-ambrein production in the yeast Pichia pastoris", METABOLIC ENGINEERING COMMUNICATIONS, vol. 7, 2018, pages e00077
NAGANO, N.MATSUI, S.KURAMURA, T.TAOKA, Y.HONDA, D.HAYASHI, M.: "The distribution of extracellular cellulase activity in marine Eukaryotes, thraustochytrids", MAR BIOTECHNOL (NY, vol. 13, 2011, pages 133 - 136, XP019896327, DOI: 10.1007/s10126-010-9297-8
NAITO, Y.HINO, K.BONO, H.UI-TEI, K.: "CRISPRdirect: Software for designing CRISPR/Cas guide RNA with reduced off-target sites", BIOINFORMATICS, vol. 31, no. 7, 2015, pages 1120 - 1123, XP055186272, Retrieved from the Internet <URL:https://doi.org/10.1093/bioinformatics/btu743> DOI: 10.1093/bioinformatics/btu743
NAKAZAWA, A.KOKUBUN, Y.MATSUURA, H.YONEZAWA, N.KOSE, R.YOSHIDA, M.TANABE, Y.KUSUDA, E.VAN THANG, D.UEDA, M. ET AL.: "TLC screening of thraustochytrid strains for squalene production", J APPL PHYCOL, vol. 26, 2014, pages 29 - 41, XP055374842, DOI: 10.1007/s10811-013-0080-x
NAKAZAWA, A.MATSUURA, H.KOSE, R.KATO, S.HONDA, D.INOUYE, I.KAYA, K.WATANABE, M.M.: "Optimization of culture conditions of the thraustochytrid Aurantiochytrium sp. strain 18W-13a for squalene production", BIORESOURCE TECHNOLOGY, vol. 109, 2012, pages 287 - 291, XP002678627, DOI: 10.1016/j.biortech.2011.09.127
OKINO, N.WAKISAKA, H.ISHIBASHI, Y.ITO, M.: "Visualization of Endoplasmic Reticulum and Mitochondria in Aurantiochytrium limacinum by the Expression of EGFP with Cell Organelle-Specific Targeting/Retaining Signals", MAR BIOTECHNOL, vol. 20, 2018, pages 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.: "High-level semi-synthetic production of the potent antimalarial artemisinin", NATURE, vol. 496, 2013, pages 528 - 532, XP055112673, DOI: 10.1038/nature12051
PAL, A.GANGULY, A.CHOWDHURI, S.YOUSUF, M.GHOSH, A.BARUI, A.K.KOTCHERLAKOTA, R.ADHIKARI, S.BANERJEE, R.: "Bis-arylidene oxindole-betulinic Acid conjugate: a fluorescent cancer cell detectorwith potent anticancer activity", ACS MED CHEM LETT, vol. 6, 2015, pages 612 - 616
PERALTA-YAHYA, P.P.OUELLET, M.CHAN, R.MUKHOPADHYAY, A.KEASLING, J.D.LEE, T.S.: "Identification and microbial production of a terpene-based advanced biofuel", NAT COMMUN, vol. 2, 2011, pages 483
POLAKOWSKI, T.STAHL, U.LANG, C.: "Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 49, 1998, pages 66 - 71, XP001167014, DOI: 10.1007/s002530051138
POLLIER, J.VANCAESTER, E.KUZHIUMPARAMBIL, U.VICKERS, C.E.VANDEPOELE, K.GOOSSENS, A.FABRIS, M.: "A widespread alternative squalene epoxidase participates in eukaryote steroid biosynthesis", NAT MICROBIOL, vol. 4, 2019, pages 226 - 233, XP036679931, DOI: 10.1038/s41564-018-0305-5
PRECIADO, L.M.REY-SUAREZ, P.HENAO, I.C.PEREANEZ, J.A.: "Betulinic, oleanolic and ursolic acids inhibit the enzymatic and biological effects induced by a P-I snake venom metalloproteinase", CHEM BIOL INTERACT, vol. 279, 2018, pages 219 - 226, XP085312399, DOI: 10.1016/j.cbi.2017.12.001
PRIMAK, Y.A.DU, M.MILLER, M.C.WELLS, D.H.NIELSEN, A.T.WEYLER, W.BECK, Z.Q.: "Characterization of a feedback-resistant mevalonate kinase from the archaeon Methanosarcina mazei", APPL ENVIRON MICROBIOL, vol. 77, 2011, pages 7772 - 7778, XP055109562, DOI: 10.1128/AEM.05761-11
PROSCHEL, M.DETSCH, R.BOCCACCINI, A.R.SONNEWALD, U: "Engineering of Metabolic Pathways by Artificial Enzyme Channels", FRONT BIOENG BIOTECHNOL, vol. 3, 2015, pages 168
PYLE, D.J.GARCIA, R.A.WEN, Z.: "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., vol. 56, 2008, pages 3933 - 3939, XP002634289, DOI: 10.1021/JF800602S
QIAO, W.ZHOU, Z.LIANG, Q.MOSONGO, I.LI, C.ZHANG, Y.: "Improving lupeol production in yeast by recruiting pathway genes from different organisms", SCI REP, vol. 9, 2019, pages 2992
RAHMAT ENDANG ET AL: "Yeast metabolic engineering for the production of pharmaceutically important secondary metabolites", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER BERLIN HEIDELBERG, BERLIN/HEIDELBERG, vol. 104, no. 11, 8 April 2020 (2020-04-08), pages 4659 - 4674, XP037126188, ISSN: 0175-7598, [retrieved on 20200408], DOI: 10.1007/S00253-020-10587-Y *
RAYMOND, C.K.POWNDER, T.A.SEXSON, S.L.: "General method for plasmid construction using homologous recombination", BIOTECHNIQUES, vol. 26, 1999, pages 134 - 138,140-141
RAYMOND, C.K.SIMS, E.H.OLSON, M.V.: "Linker-mediated recombinational subcloning of large DNA fragments using yeast", GENOME RES, vol. 12, 2002, pages 190 - 197, XP055532111, DOI: 10.1101/gr.205201
REMANS, T.SCHENK, P.M.MANNERS, J.M.GROF, C.P.L.ELLIOTT, A.R.: "A Protocol for the Fluorometric Quantification of mGFP5-ER and sGFP(S65T) in Transgenic Plants", PLANT MOLECULAR BIOLOGY REPORTER, vol. 17, 1999, pages 385 - 395
REN, L.-J.SUN, G.JI, X.-J.HU, X.HUANG, H.: "Compositional shift in lipid fractions during lipid accumulation and turnover in Schizochytrium sp", BIORESOURCE TECHNOLOGY, vol. 157, 2014, pages 107 - 113, XP055937114, DOI: 10.1016/j.biortech.2014.01.078
RIOS, J.L.MANEZ, S.: "New Pharmacological Opportunities for Betulinic Acid", PLANTA MED, vol. 84, 2018, pages 8 - 19, XP018531033
SALA-TREPAT, J.M.EVANS, W.C.: "The meta Cleavage of Catechol by Azotobacter Species. 4-Oxalocrotonate Pathway", EUR J BIOCHEM, vol. 20, 1971, pages 400 - 413
SANDEEP, NULLMISRA, R.C.CHANOTIYA, C.S.MUKHOPADHYAY, P.GHOSH, S.: "Oxidosqualene cyclase and CYP716 enzymes contribute to triterpene structural diversity in the medicinal tree banaba", NEW PHYTOL, vol. 222, 2019, pages 408 - 424
SCHMANDKE, H: "Triterpenoide in Oliven", ERNAHRUNGS UMSCHAU, vol. 56, 2009, pages 92 - 95
SCULLIN, C.STAVILA, V.SKARSTAD, A.KEASLING, J.D.SIMMONS, B.A.SINGH, S.: "Optimization of renewable pinene production from the conversion of macroalgae Saccharina latissima", BIORESOUR TECHNOL, vol. 184, 2015, pages 415 - 420
SEEMANN, T, SHOVILL: FASTER SPADES ASSEMBLY OF ILLUMINA READS, Retrieved from the Internet <URL:Https://Github.Com/Tseemann/Shovill>
SELZER, E.PIMENTEL, E.WACHECK, V.SCHLEGEL, W.PEHAMBERGER, H.JANSEN, B.KODYM, R.: "Effects of betulinic acid alone and in combination with irradiation in human melanoma cells", J INVEST DERMATOL, vol. 114, 2000, pages 935 - 940, XP008072456, DOI: 10.1046/j.1523-1747.2000.00972.x
SHANKS, R.M.Q.KADOURI, D.E.MACEACHRAN, D.P.O'TOOLE, G.A.: "New yeast recombineering tools for bacteria", PLASMID, vol. 62, 2009, pages 88 - 97, XP026351365
SIMAO, F.A., WATERHOUSE, R.M., LOANNIDIS, P., KRIVENTSEVA, E.V., ZDOBNOV, E.M.: "BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs", BIOINFORMATICS, vol. 31, 2015, pages 3210 - 3212
SMITH, T.F.WATERMAN, M.S.: "Identification of common molecular subsequences", JOURNAL OF MOLECULAR BIOLOGY, vol. 147, no. 1, 1981, pages 195 - 197, XP024015032, DOI: 10.1016/0022-2836(81)90087-5
SONG, T.-J.PARK, C.-H.IN, K.-R.KIM, J.-B.KIM, J.H.KIM, M.CHANG, H.J.: "Antidiabetic effects of betulinic acid mediated by the activation of the AMP-activated protein kinase pathway", PLOS ONE, vol. 16, 2021, pages e0249109
SONG, X.WANG, X.TAN, Y.FENG, Y.LI, W.CUI, Q.: "High Production of Squalene Using a Newly Isolated Yeast-like Strain Pseudozyma sp. SD301", J. AGRIC. FOOD CHEM., vol. 63, 2015, pages 8445 - 8451
SOUZA-MOREIRA, T.M.NAVARRETE, C.CHEN, X.ZANELLI, C.F.VALENTINI, S.R.FURLAN, M.NIELSEN, J.KRIVORUCHKO, A.: "Screening of 2A peptides for polycistronic gene expression in yeast", FEMS YEAST RES, 2018, pages 18
SPANOVA, M.DAUM, G.: "Squalene - biochemistry, molecular biology, process biotechnology, and applications", EUR. J. LIPID SCI. TECHNOL., vol. 113, 2011, pages 1299 - 1320, XP002722160, DOI: 10.1002/ejlt.201100203
SUMMER, H.GRAMER, R.DROGE, P.: "Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE", JOURNAL OF VISUALIZED EXPERIMENTS, vol. 32, 2009, pages 1485, Retrieved from the Internet <URL:https://doi.org/10.3791/1485>
TAN, Y.YU, R.PEZZUTO, J.M.: "Betulinic acid-induced programmed cell death in human melanoma cells involves mitogen-activated protein kinase activation", CLIN CANCER RES, vol. 9, 2003, pages 2866 - 2875
TAOKA, Y.NAGANO, N.KAI, H.HAYASHI, M.: "Degradation of Distillery Lees (Shochu kasu) by Cellulase-Producing Thraustochytrids", J OLEO SCI, vol. 66, 2017, pages 31 - 40
TSOU, C.-Y.MATSUNAGA, S.OKADA, S.: "Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race", J BIOSCI BIOENG, vol. 125, 2018, pages 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.NEWMAN, J.D.: "High-Level Production of Amorpha-4,11-Diene, a Precursor of the Antimalarial Agent Artemisinin, in Escherichia coli", PLOS ONE, vol. 4, 2009, pages e4489, XP002516249, DOI: 10.1371/journal.pone.0004489
URBAN, P.MIGNOTTE, C.KAZMAIER, M.DELORME, F.POMPON, D.: "Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5", J BIOL CHEM, vol. 272, 1997, pages 19176 - 19186
VAN LEEUWEN, J.ANDREWS, B.BOONE, C.TAN, G.: "Rapid and Efficient Plasmid Construction by Homologous Recombination in Yeast", COLD SPRING HARB PROTOC, vol. 2015, 2015
WANG, P.WEI, W.YE, W.LI, X.ZHAO, W.YANG, C.LI, C.YAN, X.ZHOU, Z.: "Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at high-efficiency", CELL DISCOV, vol. 5, 2019, pages 5, XP055700596, DOI: 10.1038/s41421-018-0075-5
WATANABE, K., PEREZ, C. M. T., KITAHORI, T., HATA, K., AOI, M., TAKAHASHI, H., SAKUMA, T.,OKAMURA, Y., NAKASHIMADA, Y., YAMAMOTO, : "Improvement of fatty acid productivity of thraustochytrid, Aurantiochytrium sp. By genome editing", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 131, no. 4, 2021, pages 373 - 380, XP086525908, Retrieved from the Internet <URL:https://doi.Org/10.1016/j.jbiosc.2020.11.013> DOI: 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.: "Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin", PROC NATL ACAD SCI U S A, vol. 109, 2012, pages E111 - 118, XP055038329, DOI: 10.1073/pnas.1110740109
WOZNIAK, L.SKAPSKA, S.MARSZAFEK, K.: "Ursolic Acid--A Pentacyclic Triterpenoid with a Wide Spectrum of Pharmacological Activities", MOLECULES, vol. 20, 2015, pages 20614 - 20641
WU, Y.XU, S.GAO, X.LI, M.LI, D.LU, W.: "Enhanced protopanaxadiol production from xylose by engineered Yarrowia lipolytica", MICROB CELL FACT, vol. 18, 2019, pages 83
XIE, R.ZHANG, H.WANG, X.YANG, X.WU, S.WANG, H.SHEN, P.MA, T.: "The protective effect of betulinic acid (BA) diabetic nephropathy on streptozotocin (STZ)-induced diabetic rats", FOOD FUNCT, vol. 8, 2017, pages 299 - 306
XU, W.CHAI, C.SHAO, L.YAO, J.WANG, Y.: "Metabolic engineering of Rhodopseudomonas palustri s for squalene production", JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY, vol. 43, 2016, pages 719 - 725
YAROSH, D.B.BOTH, D.BROWN, D.: "Liposomal Ursolic Acid (Merotaine) Increases Ceramides and Collagen in Human Skin", HORM RES PAEDIATR, vol. 54, 2000, pages 318 - 321, XP009023309, DOI: 10.1159/000053280
YOKOYAMA, R.HONDA, D.: "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, vol. 1988, 2007, pages 199 - 211, XP019521654, DOI: 10.1007/s10267-006-0362-0
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.LEE, H.S.: "Protective effect of betulinic acid on early atherosclerosis in diabetic apolipoprotein-E gene knockout mice", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 796, 2017, pages 224 - 232, XP029894763, DOI: 10.1016/j.ejphar.2016.11.044
YU, X.-J.YU, Z.-Q.LIU, Y.-L.SUN, J.ZHENG, J.-Y.WANG, Z.: "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, vol. 177, 2015, pages 1229 - 1240
ZERBINO, D.R.BIRNEY, E.: "Velvet: algorithms for de novo short read assembly using de Bruijn graphs", GENOME RES, vol. 18, 2008, pages 821 - 829, XP008096312, DOI: 10.1101/gr.074492.107
ZHANG, D.JENNINGS, S.M.ROBINSON, G.W.POULTER, C.D.: "Yeast squalene synthase: expression, purification, and characterization of soluble recombinant enzyme", ARCH BIOCHEM BIOPHYS, vol. 304, 1993, pages 133 - 143, XP024753200, DOI: 10.1006/abbi.1993.1331
ZHANG, J.-L.BAI, Q.-Y.PENG, Y.-Z.FAN, J.JIN, C.-C.CAO, Y.-X.YUAN, Y.-J.: "High production of triterpenoids in Yarrowia lipolytica through manipulation of lipid components", BIOTECHNOL BIOFUELS, vol. 13, 2020, pages 133
ZHANG, Y.-N.ZHANG, W.HONG, D.SHI, L.SHEN, Q.LI, J.-Y.LI, J.HU, L.-H.: "Oleanolic acid and its derivatives: new inhibitor of protein tyrosine phosphatase 1 B with cellular activities", BIOORG MED CHEM, vol. 16, 2008, pages 8697 - 8705, XP025427665, DOI: 10.1016/j.bmc.2008.07.080
ZHAO, F.BAI, P.LIU, T.LI, D.ZHANG, X.LU, W.YUAN, Y.: "Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae: Optimization of a Cytochrome P450 Oxidation System", BIOTECHNOL. BIOENG., vol. 113, 2016, pages 1787 - 1795, XP055505407, DOI: 10.1002/bit.25934
ZHAO, Y.FAN, J.WANG, C.FENG, X.LI, C.: "Enhancing oleanolic acid production in engineered Saccharomyces cerevisiae", BIORESOURCE TECHNOLOGY, vol. 257, 2018, pages 339 - 343, XP055679880, DOI: 10.1016/j.biortech.2018.02.096
ZHOU, C.LI, J.LI, C.ZHANG, Y.: "Improvement of betulinic acid biosynthesis in yeast employing multiple strategies", BMC BIOTECHNOL, vol. 16, 2016, pages 59
ZHU, L.ZHANG, X.CHANG, L.WANG, A.FENG, P.HAN, L.: "Molecular cloning, prokaryotic expression and promoter analysis of squalene synthase gene from Schizochytrium Limacinum", APPL BIOCHEM MICROBIOL, vol. 50, 2014, pages 411 - 419
ZHUANG, X.CHAPPELL, J.: "Building terpene production platforms in yeast: Building Terpene Production Platforms in Yeast", BIOTECHNOL. BIOENG., vol. 112, 2015, pages 1854 - 1864, XP071153291, DOI: 10.1002/bit.25588
ZUCO, V.SUPINO, R.RIGHETTI, S.C.CLERIS, L.MARCHESI, E.GAMBACORTI-PASSERINI, C.FORMELLI, F.: "Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells", CANCER LETT, vol. 175, 2002, pages 17 - 25

Also Published As

Publication number Publication date
WO2022228687A1 (fr) 2022-11-03

Similar Documents

Publication Publication Date Title
Li et al. Production of triterpene ginsenoside compound K in the non-conventional yeast Yarrowia lipolytica
D'Adamo et al. Engineering the unicellular alga Phaeodactylum tricornutum for high‐value plant triterpenoid production
Li et al. Metabolic engineering of Saccharomyces cerevisiae to overproduce squalene
Kim et al. Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway
Yu et al. Engineering Saccharomyces cerevisiae for high yield production of α-amyrin via synergistic remodeling of α-amyrin synthase and expanding the storage pool
Ma et al. Advances in the metabolic engineering of Yarrowia lipolytica for the production of terpenoids
Zhu et al. Metabolic compartmentalization in yeast mitochondria: Burden and solution for squalene overproduction
Li et al. Advanced strategies for the synthesis of terpenoids in Yarrowia lipolytica
Ma et al. Engineering Yarrowia lipolytica for sustainable production of the chamomile sesquiterpene (−)-α-bisabolol
Tang et al. Metabolic engineering of Yarrowia lipolytica for improving squalene production
Zhang et al. Refactoring β‐amyrin synthesis in S accharomyces cerevisiae
Wang et al. Efficient production of glycyrrhetinic acid in metabolically engineered Saccharomyces cerevisiae via an integrated strategy
Guo et al. Harnessing Yarrowia lipolytica peroxisomes as a subcellular factory for α-humulene overproduction
Du et al. Effects of methanol on carotenoids as well as biomass and fatty acid biosynthesis in Schizochytrium limacinum B4D1
Shang et al. Engineering Yarrowia lipolytica for enhanced production of arbutin
Zuo et al. Establishing Komagataella phaffii as a cell factory for efficient production of sesquiterpenoid α-Santalene
Li et al. Increase of betulinic acid production in Saccharomyces cerevisiae by balancing fatty acids and betulinic acid forming pathways
Wang et al. Recent advances in the biosynthesis of isoprenoids in engineered Saccharomyces cerevisiae
Du et al. Engineering Saccharomyces cerevisiae for hyperproduction of β-amyrin by mitigating the inhibition effect of squalene on β-amyrin synthase
Ahmed et al. Design and construction of short synthetic terminators for β-amyrin production in Saccharomyces cerevisiae
Yin et al. Engineering fungal terpene biosynthesis
ul Hassan et al. Engineered Saccharomyces cerevisiae for the de novo synthesis of the aroma compound longifolene
Zhu et al. Mitochondrial engineering of Yarrowia lipolytica for sustainable production of α-Bisabolene from waste cooking oil
WO2022229378A1 (fr) Production biotechnologique de terpènes
Zhao et al. Tuning geraniol biosynthesis via a novel decane-responsive promoter in Candida glycerinogenes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22726443

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22726443

Country of ref document: EP

Kind code of ref document: A1