Process for producing jasmonate

Abstract

The present invention relates to a method for producing jasmonate using a fungal cell, wherein the expression of the polyketide synthase (PKS) gene in the fungal cell has been disrupted. In one embodiment, the fungal cell is a yeast cell Malassezia species.

Description

Technical Field

[0001] The present invention generally relates to the field of microbial technology. In particular, the present invention relates to a method for producing jasmonate in fungal cells. Background Art

[0002] Biological control agents (BCAs) are living organisms and / or derivatives that protect plants against diseases and pests by direct antipathogen effects or indirectly by inducing host resistance. Jasmonic acid (JA) and methyl-Jasmonate (MeJA) are plant hormones belonging to the large phytooxylipin family, collectively referred to as jasmonates. Jasmonates are derived from the enzymatic oxygenation of polyunsaturated fatty acids (PUFAs), exhibit direct biocidal activity, and participate in a variety of signal transduction processes, including regulating the defense mechanisms of plants to insect and pathogen attacks and reducing stress. In addition, the application of JA or MeJA to crops induces a variety of beneficial effects, including anti-infection, increased production of antioxidant activity, improved growth, seed germination, enhanced stress tolerance and drought tolerance, and photosynthesis and transpiration rates. Jasmonates have a strong effect, and their picomolar effective range is sufficient to induce downstream effects. Jasmonates are also important flavor and fragrance compounds for a variety of food and consumer care products.

[0003] There is a growing awareness of environmental safety and sustainability around the world, including concerns about the overuse of pesticides and fertilizers in agricultural practices. To reduce the impact of overuse of pesticides and fertilizers on our environment, the alternative use of BCAs such as JA to induce resistance to pathogens and insects in crops has attracted significant investment from many companies. Unfortunately, jasmonates can be prohibitively expensive to produce, resulting in jasmonates costing thousands of dollars per gram, which has limited the widespread adoption of jasmonates as an alternative agrochemical. Furthermore, in order to produce 1 kg of essential oil containing approximately 1 g of MeJA, 500 kg of jasmine petals, equivalent to 10,000 flowers, are required. This time-consuming and laborious process not only results in a high price, but the production method is also extremely unsustainable.

[0004] It is therefore generally desirable to overcome or ameliorate one or more of the above-mentioned difficulties. Summary of the invention

[0005] Disclosed herein is a method for producing jasmonate using fungal cells, the method comprising a) culturing the fungal cells under conditions suitable for producing the jasmonate, wherein the expression of a polyketide synthase (PKS) gene in the fungal cells has been disrupted; and b) isolating the jasmonate produced by the fungal cells.

[0006] Disclosed herein are methods for enhancing jasmonate production in fungal cells, the methods comprising disrupting expression of a polyketide synthase (PKS) gene in the fungal cells.

[0007] Disclosed herein are engineered fungal cells for producing jasmonate, wherein a polyketide synthase (PKS) gene has been disrupted in the fungal cell.

[0008] Disclosed herein are microbiome compositions comprising fungal cells, wherein a polyketide synthase (PKS) gene has been disrupted in the fungal cells.

[0009] Disclosed herein is a microbiome composition as defined herein for use as a cosmetic or a pharmaceutical.

[0010] Disclosed herein is the use of a microbiome composition as defined herein for producing jasmonates. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Embodiments of the present invention are described below, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0012] Figure 1 Shown is the LC-MS / MS chromatogram of jasmonic acid (JA) isolated from Malassezia.

[0013] Figure 2 Malassezia JA levels showing inter- and intra-species variability are shown.

[0014] Figure 3 Figure 1 is a heat map cluster analysis of lipid mediator (oxylipin) profiles of wild-type (WT) M. furfur CBS14141 and PKS-deficient (KO) M. furfur strains. The left three columns represent gene expression of the WT strain, while the right three columns represent gene expression of the KO strain. The boxed portion represents an increase in the level of oxylipin species, while the unboxed portion represents a decrease in the level of oxylipin species. The increase in JA levels in the absence of PKS in the KO strain is highlighted by the light-colored box.

[0015] Figure 4 Quantification of JA levels in WT and PKS-KO strains is shown. All data are mean ± SE of n = 3. DETAILED DESCRIPTION

[0016] This specification teaches a method for producing jasmonate using fungal cells, the method comprising a) culturing fungal cells under conditions suitable for producing the jasmonate, wherein the expression of a polyketide synthase (PKS) gene in the fungal cells has been destroyed; and b) isolating the jasmonate produced by the fungal cells. Jasmonate can be secreted by the fungal cells and can be separated from the cell culture medium or from the gas phase of the cell culture system during or after culturing the fungal cells. Suitable fungal cells contain PKS genes and can produce jasmonate.

[0017] "Oxylipins" of the present disclosure are biologically active oxygen-containing derivatives of polyunsaturated fatty acids, formed by the oxidative metabolism of the fatty acids. "Jasmonates" as used herein are members of the oxylipin family derived from jasmonic acid (JA) or related thereto. Jasmonates include, but are not limited to, jasmonic acid (JA), methyl jasmonate, 7-isojasmonic acid, 9,10-dihydrojasmonic acid, 2,3-didehydrojasmonic acid, 3,4-didehydrojasmonic acid, 3,7-didehydrojasmonic acid, 4,5-didehydrojasmonic acid, 4,5-didehydro-7-isojasmonic acid, cucurbic acid, 6-epi-cucurbic acid, 6-epi-cucurbic acid-lactone, 12-hydroxy-jasmonic acid, 12-hydroxy-jasmonic acid-lactone, 11-hydroxy-jasmonic acid, 8-hydroxy-jasmonic acid, homojasmonic acid, dihomo-jasmonic acid, 11-hydroxy-diohomojasmonic acid, 8-hydroxy-diohomojasmonic acid, tuberonic acid, acid), tuberic acid-O-β-pyranoglucoside, cucurbitic acid-O-β-pyranoglucoside, 5,6-didehydrojasmonic acid, 6,7-didehydrojasmonic acid, 7,8-didehydrojasmonic acid, methyl dihydroisojasmonate, amino acid conjugates of jasmonic acid, and lower alkyl esters, salts and stereoisomers thereof.

[0018] Fungi, including the skin-resident yeast Malassezia, can produce JA, but while the plant pathways leading to JA production have been characterized to some extent, details about JA synthesis in fungi are scarce. For example, in plants, JA biosynthesis via peroxidation of α-linolenic acid by 13-lipoxygenase occurs in plastids, an organelle not present in fungi. Interestingly, fungal plant pathogens increase their virulence by downregulating plant host defense mechanisms at the metabolic level through the secretion of JA.

[0019] Malassezia is a genus of fungi that is a major eukaryotic member of the skin microbiome. The inventors have found that various Malassezia species are able to produce JA ( Figure 2In addition to JA, Malassezia species produce a number of other oxylipins when grown in vitro, many of which can be detected on human skin. Jasmonates may affect human skin immunology and, therefore, human health and disease, similar to their immunomodulatory functions in plants.

[0020] The present inventors have found that the production of jasmonate in Malassezia can be enhanced by altering the activity of certain genes involved in fatty acid metabolism. In particular, the present inventors have found that disrupting the expression of polyketide synthase (PKS) genes can increase JA production in Malassezia by several folds ( Figure 4 ).

[0021] Polyketide synthase is a multidomain enzyme complex involved in the biosynthesis of polyketides, a large class of secondary metabolites in bacteria, fungi, plants, and some animal lineages. The biosynthesis of polyketides has similarities to fatty acid biosynthesis, the pathway responsible for the production of oxylipins and jasmonates. The present inventors have found that disruption of PKS expression in Malassezia results in increased biosynthesis of certain oxylipins, including JA, but decreased synthesis of other oxylipins, including polyunsaturated fatty acids (PUFAs), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Figure 3 Without wishing to be bound by theory, this may indicate that in Malassezia, the PKS complex is not directly involved in the synthesis of jasmonate family oxylipins, but that jasmonate production is limited by competition with other PUFA pathways. Loss of PKS-mediated PUFA and oxylipin biosynthesis by disruption of PKS genes may eliminate substrate competition and / or stimulate biosynthetic pathways involved in the production of jasmonate and other oxylipin subgroups in Malassezia.

[0022] Therefore, in some embodiments of the present invention, the fungal cell for producing jasmonate is a Malassezia species, wherein the expression of polyketide synthase (PKS) gene in the Malassezia cell has been destroyed. The Malassezia species mentioned in the present disclosure include all strains, isolates and serotypes classified under the species, and also include any new species that can be classified under the Malassezia, and any species that can be reclassified under the Malassezia. Specific Malassezia species is not limited, and can be appropriately selected according to its ability to produce jasmonate. In a preferred embodiment, the Malassezia cell endogenously produces one or more jasmonates, such as by natural metabolic pathways. The Malassezia cell can also be engineered to produce one or more jasmonates, such as by introducing genes encoding proteins involved in the metabolic pathways producing jasmonate.

[0023] The non-limiting examples of suitable Malassezia species include M.arunalokei, M.dermatis, M.furfur, M.globosa, M.japonica, M.obtusa, M.ochoterenai, M.pachydermatis, M.restricta, M.sympodialis, M.tropica, M.yamatoensis, etc. Two or more strains can be co-cultured for the production of jasmonates. Malassezia yeast can be isolated and identified from a sample (e.g., a skin sample from a human or animal), or can be obtained from a cell collection (e.g., American Type Culture Collection (ATCC)) or a provider of biological genetic resources.

[0024] In some embodiments, the fungal cell is Malassezia furfur, Malassezia sympodialis, Malassezia pachydermatis, Malassezia ajaponica, Malassezia yamtoensis and / or Malassezia dermatis. In one embodiment, the fungal cell is a strain or isolate of Malassezia furfur, such as Malassezia furfur CBS14141.

[0025] Polyketide synthases (PKS) disclosed herein include any one of a family of enzymes that catalyze the formation of polyketide compounds and are encoded by PKS genes. The PKS enzyme can be a known or naturally occurring PKS, or a polypeptide homologous thereto or derived therefrom and exhibiting one or more enzymatic activities characteristic of a polyketide synthase. The PKS herein can be a type I, type II or type III PKS. The PKS is encoded by a single gene or a gene cluster.

[0026] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0027] The terms "encode" and "encoding" include reference to nucleotides and / or amino acids that correspond to other nucleotides or amino acids in a transcriptional and / or translational sense.

[0028] The terms "destruction" and "destroyed" are used interchangeably herein and refer to any genetic or cellular modification that reduces or eliminates the expression and / or functional activity of a nucleic acid or its expression product of a polyketide synthase (PKS) gene. For example, the destruction of a PKS gene includes within its scope any genetic modification, whether naturally occurring or engineered, that reduces or eliminates the expression of a PKS gene and / or the functional activity of a corresponding gene product (e.g., mRNA and / or protein). Genetic modification includes complete or partial inactivation, suppression, deletion, interruption, blocking or downregulation of a nucleic acid (e.g., gene). Genetic modification can be accomplished by forced evolution, random mutagenesis or more targeted genetic engineering methods, followed by appropriate selection or screening to identify desired mutants. Exemplary genetic modifications include, but are not limited to, gene knockout, inactivation or mutation (e.g., insertion mutations, deletion mutations, point mutations or frameshift mutations that destroy the expression or activity of a gene product). Gene disruption also includes the use of inhibitory nucleic acids (e.g., inhibitory RNA, such as sense or antisense RNA, molecules that mediate RNA interference, such as siRNA, shRNA, miRNA, etc.), inhibitory polypeptides (e.g., antibodies, polypeptide binding partners, dominant negative polypeptides, enzymes, etc.), or any other molecule that inhibits gene activity or the level or functional activity of a gene expression product.

[0029] In some embodiments, the disruption of a PKS gene in a fungal cell is heritable, that is, the disrupted PKS gene in a parent fungal cell can be passed on to all progeny cells derived from the daughter cells of the fungal cell.

[0030] In one embodiment, a fungal cell is provided, which has been modified so that the expression and / or function of a PKS has been reduced or eliminated. For example, the fungal cell can be modified to obtain a PKS knockout or knockdown. The term "knock-out" can refer to the elimination of a gene or gene expression. For example, a gene can be knocked out by missing or adding a nucleotide sequence that causes a reading frame to be destroyed. As another example, a gene can be knocked out by replacing part of a gene with an unrelated sequence. On the other hand, the term "knock-down" can refer to the reduction of gene expression or its gene product. The result of a gene knockdown is that protein activity or function may be weakened, or protein levels may be reduced or eliminated.

[0031] In some embodiments, the PKS gene has been disrupted by a deletion or insertion in the gene sequence.

[0032] In some embodiments, the PKS gene of the fungal cell has been destroyed by nuclease-mediated gene editing. It is known in the art that it is possible to use site-specific nucleases to produce DNA breaks in the genome of living cells, and this DNA break can be repaired by non-homologous end joining (NHEJ) of mutagenesis or by homologous recombination with exogenous DNA sequences to cause permanent modification of the genome. NHEJ can produce mutagenesis at the cleavage site, resulting in allele inactivation. Mutagenesis associated with NHEJ can inactivate alleles by generating early stop codons, generating abnormal non-functional protein frameshift mutations, or can trigger mechanisms such as nonsense-mediated mRNA decay. Using nucleases to induce mutagenesis by NHEJ can be used to target specific mutations or sequences present in wild-type alleles. It is known that the use of nucleases to induce double-strand breaks in the target locus can stimulate homology-directed repair (HDR), particularly homology-directed repair of transgenic DNA sequences flanked by sequences homologous to the genomic target. In this way, an exogenous nucleic acid sequence can be inserted into the target locus. This exogenous nucleic acid can encode any sequence of interest.

[0033] In different embodiments, a variety of different types of nucleases can be used to destroy PKS genes. In one embodiment, a recombinant meganuclease is used to destroy the gene. In another embodiment, a CRISPR nuclease is used to destroy the gene. Methods for making CRISPR that recognizes a predetermined DNA site are known in the art. In another embodiment, zinc finger nucleases (ZFNs) are used to destroy the gene. In another embodiment, a transcription activator-like effector nuclease (TALEN) or Compact TALEN is used to destroy the gene. In another embodiment, megaTAL is used to destroy the gene. In yet another embodiment, ARCUS nucleases are used to destroy the gene.

[0034] In various embodiments, the homing endonuclease or meganuclease is engineered to bind to the PKS gene and introduce a single-strand nick or double-strand break (DSB) therein. "Homing endonuclease" and "meganuclease" are used interchangeably and refer to naturally occurring nucleases or engineered meganucleases that recognize cleavage sites of 12-45 base pairs and are generally divided into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK. Non-limiting examples of meganucleases include I-Scel (from Saccharomyces cerevisiae), I-Crel (from Chlamydomonas reinhardtii), and I-Dmol (from Desulfurococcus mobilis). Homing endonuclease variants can be designed and / or modified from naturally occurring homing endonucleases or from another homing endonuclease variant. Homing endonuclease variants can also include one or more additional functional domains, for example, an end processing enzyme domain of an end processing enzyme that exhibits 5-3' exonuclease, 5-3' alkaline exonuclease, 3-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, or template-independent DNA polymerase activity.

[0035] Illustrative examples of LAGLIDADG homing endonucleases include, but are not limited to, I-Crel, I-Scel, I-AabMI, I-AaeMI, I-Anil, I-ApaMI, I-CapIII, I-CapIV, IKaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, IEjeMI, I-GpeMI, I-Gpil, I-GzeMI, I-GzeMII, I -GzeMIII, I-HjeMI, 5I-Ltrll, I-Ltrl, ILtrWI, I-MpeMI, I-MveMI, I-Ncrll, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-Oso MI, IOsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, ISmaMI, I-SscMIh and I-Vdil41I.

[0036] Some embodiments herein contemplate megaTAL nucleases that bind and cleave a target region of one or more target sites. "megaTAL" refers to an engineered nuclease comprising an engineered TALE DNA binding domain and an engineered meganuclease, and optionally one or more linkers.

[0037] A "TALE DNA binding domain" is the DNA binding portion of a transcription activator-like effector (TALE or TAL effector). The TALE DNA binding domain contemplated in specific embodiments is engineered de novo or from a naturally occurring TALE, e.g., AvrBs3 from Xanthomonas campestris pv. vesicatoria, Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforan, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria, and Xanthomonas oryzae, and brgl 1 and hpxl7 from Ralstonia solanacearum.

[0038] In specific embodiments, the megaTAL includes a TALE DNA binding domain, which includes one or more repeat sequence units that participate in the binding of the TALE DNA binding domain to its corresponding target DNA sequence. The length of a single "repeat sequence unit" (also called a "repeat sequence") is typically 33-35 amino acids. Each TALE DNA binding domain repeat sequence unit includes 1 or 2 DNA binding residues, which constitute the repeat variable di-residue (RepeatVariable Di-Residue, RVD), which is usually located at position 12 and / or 13 of the repeat sequence. The natural (classical) coding for DNA recognition of these TALE DNA binding domains has been determined, so that the HD sequences at positions 12 and 13 result in binding to cytosine (C), NG binds to T, NI binds to A, NN binds to G or A, and NG binds to T. In certain embodiments, non-classical (atypical) RVDs are considered.

[0039] Illustrative examples of non-classical RVDs suitable for use in specific megaTALs contemplated in specific embodiments include, but are not limited to: HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN for recognizing guanine (G); NI, KI, RI, HI, SI for recognizing adenine (A); NG, HG, KG, RG for recognizing thymine (T); RD, SD, HD, ND, KD, YG for recognizing cytosine (C); NV, HN for recognizing A or G; and H*, HA, KA, N*NA, NC, NS, RA, S* for recognizing A or T or G or C, where (*) means that the amino acid at position 13 is absent. In specific embodiments, the megaTALs contemplated herein comprise a TALE DNA binding domain comprising 3 to 30 repeat units.

[0040] In a specific embodiment, the engineered nuclease is a TALEN. "TALEN" refers to an engineered nuclease comprising an engineered TALE DNA binding domain and an endonuclease domain (or endonuclease half-domain thereof), and optionally one or more linkers. In specific embodiments, the TALEN contemplated herein comprises an N-terminal domain, a TALE DNA binding domain, a C-terminal domain, and an endonuclease domain or half-domain, wherein the TALE DNA binding domain comprises about 3.5 to 30.5 repeat units, such as about 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, 15.5, 16.5, 17.5, 18.5, 19.5, 20.5, 21.5, 22.5, 23.5, 24.5, 25.5, 26.5, 27.5, 28.5, 29.5, or 30.5 repeat units. In one embodiment, the TALEN contemplated herein comprises an endonuclease domain of a Type IIS restriction endonuclease. In one embodiment, the Type IIS restriction endonuclease is Fok I.

[0041] In a specific embodiment, the engineered nuclease is a zinc finger nuclease (ZFN). "ZFN" refers to an engineered nuclease comprising one or more zinc finger DNA binding domains and an endonuclease domain (or its endonuclease half domain), and optionally comprises one or more linkers. In a specific embodiment, the ZFN comprises a zinc finger DNA binding domain having one, two, three, four, five, six, seven or eight or more zinc finger motifs and an endonuclease domain (or endonuclease half domain). Typically, a single zinc finger motif is about 30 amino acids in length. Zinc finger motifs include classical C2H2 zinc fingers and non-classical zinc fingers, such as C3H zinc fingers and C4 zinc fingers. The zinc finger binding domain can be engineered to bind to any DNA sequence.

[0042] A single zinc finger motif binds three or four nucleotide sequences. Candidate zinc finger DNA binding domains for a given 3 bp DNA target sequence have been identified, and a modular assembly strategy has been designed to connect multiple domains into multi-finger peptides targeting corresponding composite DNA target sequences. Other suitable methods known in the art can also be used to design and construct nucleic acids encoding zinc finger DNA binding domains, such as phage display, random mutagenesis, combinatorial libraries, computer / rational design, affinity selection, PCR, cloning from cDNA or genomic libraries, synthetic construction, etc.

[0043] In specific embodiments, the ZNFs contemplated herein comprise a zinc finger DNA binding domain comprising two, three, four, five, six, seven, or eight or more zinc finger motifs, and an endonuclease domain or half-domain from at least one Type IIS restriction enzyme. In one embodiment, the endonuclease domain or half-domain is from a Fok I Type IIS restriction endonuclease.

[0044] In various embodiments, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated) nuclease system is engineered to bind to one or more target sites and introduce a single-strand nick or double-strand break (DSB) in one or more target sites. The CRISPR / Cas nuclease system is a recent engineered bacterial system-based nuclease system that can be used for mammalian genome engineering. Engineered endonucleases based on the CRISPR / Cas9 system are also known in the art. The CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically a microbial Cas9; and (2) a short "guide RNA" comprising a nucleotide targeting sequence that guides the nuclease to the desired location in the genome.

[0045] The term "CRISPR" refers to a caspase-based endonuclease comprising a caspase, such as Cas9, and a guide RNA that directs DNA cleavage by the caspase by hybridizing to a recognition site in genomic DNA.

[0046] In some embodiments, zinc finger proteins, nucleases containing transcription activator-like effector (TALEN) nucleases, or CRISPR / Cas systems are used to disrupt the PKR gene.

[0047] In some embodiments, the CRISPR / Cas system is CRISPR / Cas9. In some embodiments, the CRISPR / Cas system comprises at least one nucleic acid encoding a CRISPR nuclease and at least one nucleic acid encoding a guide RNA. The two nucleic acids can be introduced separately, such as into different vectors or mRNAs, or together, such as into a polycistronic construct separated by a viral self-cleaving peptide or an IRES element.

[0048] In some embodiments, the CRISPR / Cas system comprises an exogenous sequence for integration into the yeast genome by homologous recombination after nuclease cleavage. The design of exogenous sequences for replacing native sequences in CRISPR / Cas gene editing is well known in the art.

[0049] Selection of successful knockout or knockdown strains can be performed by introducing a noursothricin (NAT) selection cassette into fungal cells during gene editing. Noursothricin (NTC) is a metabolite produced by Streptomyces noursei, which belongs to the streptomycin class of aminoglycoside antibiotics that inhibit protein synthesis. NTC N-acetyltransferase (NAT) from Streptomyces noursei inactivates NTC by acetylation of the β-amino group of β-lysine residues on NTC, so cells that have been successfully gene-edited will contain a NAT resistance cassette and will be able to grow in the presence of NTC.

[0050] In some embodiments, expression of a PKS gene is disrupted using an inhibitory nucleic acid or small molecule that inhibits expression of the PKS gene.

[0051] In one embodiment, the fungal cell comprises a PKS inhibitor or has been contacted with a PKS inhibitor. The inhibitor can be, for example, a small molecule or a polypeptide. Non-limiting examples of small molecule PKS inhibitors include kraussianone 6, kraussianone 1, neodiospyrin, clionamine D, bromotopsentin, isodiospyrin, spongotine A, kraussianone 3, 14-β-hydroxybufa-3,5,20,22-tetraenolide, and kraussianone 7.

[0052] In another embodiment, the fungal cell comprises or has been contacted with an inhibitory nucleic acid, such as an inhibitory RNA, such as sense or antisense RNA, or a nucleic acid that mediates RNA interference, such as siRNA, shRNA, miRNA. RNA-mediated gene disruption methods are well known in the art.

[0053] Fungal cells can be cultured in any nutrient medium that supports cell growth and cell production of jasmonate. Cultivation and fermentation incubation of fungal strains are usually completed in aqueous medium in the presence of essential nutrients (carbon source, nitrogen source, inorganic salts and growth factors). Examples of inorganic salts that can be included in the nutrient medium include, but are not limited to, phosphates and / or sulfates of sodium, calcium, magnesium and potassium. Other nutrients can also be added, such as one or more B vitamins, one or more trace minerals such as iron, manganese, cobalt, copper, zinc, etc., as those skilled in the art are familiar with. Fungal growth hormones, such as 10-oxo-trans-8-decenoic acid and histidine betaine (hercynine), can also be included in the nutrient medium. Culture media commonly used for fungal culture include, for example, brain-heart infusion medium, Czapek-Dox medium, potato dextrose medium, Sabouraud's heart infusion medium, Sabouraud's dextrose medium, dermatophyte medium and bird seed medium. The present inventors have found that Dixon's medium and modified Dixon's medium, both of which contain malt extract, glycerol, desiccated ox-bile, peptone, oleic acid and Tween 40, are particularly suitable for culturing Malassezia. The composition of Dixon's medium is provided in the Examples.

[0054] In some embodiments, the method herein is included in step a) and a fatty acid source is provided to the fungal cell. The source of fatty acid can include, for example, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, butyric acid, valeric acid, caproic acid, enanthic acid, sad, nonanoic acid, capric acid, undecanoic acid, undecylenic acid, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, margaric acid, stearic acid, oleic acid, linolenic acid, linolenic acid, arachidonic acid and erucic acid. The fatty acid source can improve the jasmonate production of fungal growth and / or fungi.

[0055] In a typical process, fungal cells are first grown in an inoculum to produce a mature culture in a nutrient medium. The culture is inoculated into a fermenter nutrient medium and allowed to grow on its own. Fermentation is continued until a steady concentration of jasmonate product is present.

[0056] Cultivation can be carried out in batches or in a continuous manner. In batch fermentation, the nutrient medium is combined with the culture and fermented to a constant amount of the jasmonic acid product in the culture. In a continuous process, the nutrient medium can be continuously recirculated through the fermentation reactor, and the product can be removed from the recycled culture medium.

[0057] The Malassezia fermentation can be carried out under stirring at about 150 rpm. The culture temperature can be about 32° C. The culture and incubation can be carried out under aerobic conditions at a pH of about 6. The jasmonate product can be isolated after at least 2 days of culture, or when no additional jasmonate is produced.

[0058] The jasmonate product can be separated from the nutrient medium and / or the fungal cell fraction using any method known in the art, for example, by liquid-liquid extraction with an extraction solvent such as methanol to form a jasmonate extract. The extraction solvent can be removed to provide a concentrated jasmonate extract. Fractionation can be performed, for example, using silica gel, to further purify the jasmonate or to separate different isomers. The jasmonate product can also be separated from the gaseous space of the culture or fermentation chamber, for example, by passing the gas over an adsorbent (e.g., filter trap) and eluting the jasmonate retained on the adsorbent with a suitable solvent (e.g., methanol, dichloromethane or ether).

[0059] In some embodiments of the methods herein, the jasmonate produced by the yeast is jasmonic acid, methyl jasmonate, or jasmonic acid conjugated to an amino acid. Jasmonic acid can be naturally conjugated to any amino acid, such as isoleucine, leucine, valine, or methionine.

[0060] In some embodiments, the jasmonate is secreted by a fungal cell, and step b) comprises separating the secreted jasmonate. The jasmonate may be naturally secreted by the fungal cell, or the fungal cell may be engineered to secrete jasmonate or improve the secretion of jasmonate. The secreted jasmonate may be separated from the culture medium or the airspace above the culture medium.

[0061] Also disclosed herein is a method for enhancing the production of jasmonate by fungal cells, the method comprising destroying the expression of a polyketide synthase (PKS) gene in the fungal cell. Suitable fungal cells contain PKS genes and can produce jasmonate. The fungal cell can be a yeast cell, such as a Malassezia species.

[0062] In some embodiments, the method further comprises culturing the fungal cell under conditions suitable for producing jasmonate, and isolating the jasmonate produced by the fungal cell.

[0063] Provided herein are engineered fungal cells for producing jasmonate, wherein a polyketide synthase (PKS) gene has been disrupted in the fungal cell. Suitable fungal cells contain PKS genes and can produce jasmonate. The engineered fungal cell can be a yeast cell, such as a Malassezia species.

[0064] Disclosed herein is a microbiome composition comprising a fungal cell, wherein a polyketide synthase (PKS) gene has been disrupted in the fungal cell. The fungal cell is preferably a cell that produces jasmonate.

[0065] In some embodiments, the microbiome composition is a skin microbiome composition. The microbiome composition may include fungal cells and one or more skin commensal microorganisms. "Skin commensal microorganisms" refer to prokaryotes and eukaryotes that live and reproduce on the skin (preferably human skin) or temporarily inhabit the skin (preferably human skin) in the body. Non-limiting examples of skin commensal microorganisms include Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Propionibacteria, Corynebacteria, Clostridiales, Lactobacillales, Staphylococcus, Bacillus, Micrococcus, Streptococcus, Bacteroidales, Flavobacteriales, Enterococcus, Pseudomonas, Malassezia, Maydida, Rhodotorula, Epicoccum, and Cryptococcus. The one or more skin commensal microorganisms may help support or enhance the growth of fungal cells and / or increase jasmonate production in fungal cells.

[0066] The microbiome composition may be formulated for topical application. The microbiome composition may be capable of producing jasmonates when topically applied. The microbiome composition may include one or more dermatologically acceptable carriers that are compatible with fungal cells and other skin commensal microorganisms. A dermatologically acceptable carrier is a carrier that is suitable for application to the skin or keratinous tissue without excessive toxicity, incompatibility, instability, or allergic reaction. Dermatologically acceptable carriers can be in a variety of forms, such as simple solutions (water-based or oil-based), solid forms (gels or sticks), and emulsions (water-in-oil or oil-in-water). Examples of dermatologically acceptable carriers include, but are not limited to: distilled or deionized water; propylene glycol; glycerin; silicones such as volatile silicones, amino or non-amino silicone gums or oils, and mixtures thereof; mineral oils; vegetable oils such as olive oil, castor oil, rapeseed oil, coconut oil, wheat germ oil, avocado oil, macadamia oil, almond oil, safflower oil, linseed oil, tamanu oil, lemon oil, and mixtures thereof; waxes; and organic compounds such as C 2 -C 10 Alkanes, acetone, methyl ethyl ketone, volatile C1 -C 12 Alcohol, C 1 -C 20 Acid ester and C 1 -C 8 Alcohols such as methyl acetate, butyl acetate, ethyl acetate and isopropyl myristate, dimethoxyethane, diethoxyethane, C 10 -C 30 Fatty alcohols such as lauryl alcohol, cetyl alcohol, stearyl alcohol and behenyl alcohol; C 10 -C 30 Fatty acids such as lauric acid and stearic acid; C 10 -C 30 Fatty amides such as lauric acid diethanolamide; C 10 -C 30 Fatty alkyl esters such as C 10 -C 30 Fatty alkyl benzoates; natural and synthetic hydrophilic polymers such as hydroxyalkyl cellulose, carboxymethyl cellulose, polyethylene glycol, polypropylene glycol, polyvinyl pyrrolidone and polyvinyl alcohol; poly(acrylic acid) polymers or copolymers such as products; and mixtures thereof.

[0067] The microbiome composition may also include one or more optional components known or otherwise effective for use in cosmetics or skin care products, provided that the optional components are physically and chemically compatible with the microbial components and carrier components described herein, or otherwise do not excessively impair product stability, aesthetics, or performance. Some non-limiting examples of these optional components include plasticizers, surfactants (which may be anionic, cationic, amphoteric, or nonionic), neutralizers, emollients, lubricants, and penetrants such as various lanolin compounds, vitamins, proteins, preservatives, dyes, colorants, antioxidants, reducing agents, sunscreens, thickeners (e.g., polymeric thickeners such as xanthan gum), non-polymeric thickeners including clays and flavors.

[0068] Disclosed herein are microbiome compositions as defined herein for use as cosmetics or medicines. The jasmonates produced by the fungal cells in the microbiome compositions can produce one or more cosmetic and / or pharmacological effects. The cosmetic or pharmacological effects of the jasmonates can be enhanced by the skin commensal microorganisms in the compositions.

[0069] The microbiome composition can be a cosmetic composition, i.e., a topically applied composition intended to improve the condition and / or appearance of the skin or keratinous tissue or otherwise provide a skin care benefit. Non-limiting examples of skin care benefits include improving the appearance of the skin by providing a smoother, more even appearance; increasing the thickness of one or more layers of the skin; improving the elasticity of the skin or hair; reducing the oiliness, shine, and / or dull appearance of the skin or hair; improving the hydration state of the skin or hair; improving the appearance of fine lines and / or wrinkles; improving skin barrier properties; reducing the appearance of redness or skin spots; and / or improving the brightness, radiance, or clarity of the skin.

[0070] The cosmetic composition of the invention may be in the form of a composition for hair care, in particular a shampoo, lotion, cream, gel or mousse. It may also be in the form of a composition for cleansing, protecting, treating or caring for the face, hands, feet, the large anatomical folds or the body, such as an ointment, cream, sunscreen, lotion, lotion, gel, moisturizer, shower gel, deodorant or aftershave. Alternatively, the composition may be a make-up composition for the body or face, such as a foundation; a composition against insect bites; or an analgesic or antipruritic composition.

[0071] Alternatively, the microbiome composition can be a dermatological or pharmaceutical composition, used as a medicament for treating certain skin diseases, such as eczema, rosacea, psoriasis, dermatitis, actinic keratosis, or severe itching. The pharmaceutical composition can also be used as an antiviral, antibacterial, antifungal, anti-aging, anti-inflammatory or analgesic composition, or for treating autoimmune or neurological diseases or cancer.

[0072] Disclosed herein is the use of a microbiome composition as defined herein for producing jasmonates. The microbiome composition can be used to produce jasmonates in culture, for example, the composition can be cultured in a culture vessel, a fermenter, or a bioreactor for producing jasmonates. Alternatively, when applied as a topical formulation, the microbiome composition can produce jasmonates.

[0073] The jasmonates produced according to the method of the present invention can be used in various applications in agriculture, food, spices and medicine. For example, methyl jasmonate can be used as a flavor ingredient in food and products such as perfumes, personal care products, home care products, oral consumables, etc.

[0074] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination in the alternative (or) interpretation.

[0075] As used in this application, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "agent" includes a plurality of agents, including mixtures thereof.

[0076] Throughout the specification and the claims that follow, unless the context requires otherwise, the word "comprise", "comprises" and variations (comprising), will be understood to imply the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps.

[0077] Reference in this specification to any prior publication (or information derived therefrom) or any known content is not and should not be taken as an acknowledgement or recognition or any form of suggestion that the prior publication (or information derived therefrom) or known content forms part of the common general knowledge in the field to which this specification relates.

[0078] Those skilled in the art will appreciate that the invention described herein is susceptible to variation and modification, except for those specifically described. It should be understood that the present invention includes all such variations and modifications that fall within the spirit and scope of the present invention. The present invention also includes all steps, features, compositions and compounds mentioned or indicated in this specification, either individually or in combination, and any and all combinations of any two or more of the steps or features.

[0079] Certain embodiments of the invention will now be described with reference to the following examples, which are intended for illustrative purposes only and are not intended to limit the general scope of the above description.

[0080] Example

[0081] method

[0082] Generation of a polyketide synthase (PKS)-deficient Malassezia furfur strain

[0083] In order to genetically modify commercially available Malassezia furfur strains (CBS14141), the Agrobacterium-mediated mutagenesis method using CRISPR / Cas9 was applied. The guide RNA sequence (sgRNA) 5'-ATACTTTGAGCTGCTCAAGG-3' (SEQ ID NO: 3) was cloned into a plasmid that allows CRISPR / Cas9 components to be expressed in Malassezia, and allows inducing specific double-strand breaks (DSBs) in the coding sequence of polyketide synthase (PKS) in Malassezia furfur. In order to effectively knock out (KO) Malassezia furfur PKS genes after inducing DSB in the PKS coding sequence, a homologous recombination (HR) repair template was generated, which contained 1.5kb flanking arms homologous to the upstream and downstream sequences of the PKS coding sequence. The PKS gene is located at the CP046234.1 locus in CBS14141 chromosome 1. The nucleic acid sequence of the PKS gene with flanking regions is provided as SEQ ID NO: 1 (in the attached sequence listing).

[0084] By knocking in (KI) the nourseothricin (NAT) selection cassette in place of the PKS gene, it is helpful to identify successful PKS gene KO strains. The sequence and position of the NAT selection cassette in the putative PKS gene (CP046234.1 locus) in chromosome 1 of CBS14141 are provided as SEQ ID NO:2 (in the attached sequence table). In short, the sequence from nucleotide position 1,746,826 to 1,751,948 in chromosome 1 of CBS14141 is replaced by the knocked-in NAT selection cassette sequence.

[0085] Generate and implement HR repair plasmid, Agrobacterium-mediated transformation and selection as previously described.In brief, plasmid is electroporated in Agrobacterium tumefaciens (A.tumefaciens) EHA105, and uses PCR and restriction enzyme to confirm successful transformation.Respectively harvest the logarithmic phase culture of Malassezia furfur CBS14141 and the Agrobacterium tumefaciens transformed, and described Agrobacterium tumefaciens has grown in the induction medium containing 100 μM acetosyringone (Sigma).Equal proportions of Malassezia and Agrobacterium cells are fully mixed, and by 0.45 μm mixed cellulose membrane (Merck, Millipore) and transferred to the induction medium agar supplemented with 200 μM acetosyringone.Cells are incubated at room temperature for 5 days, then cells are washed in 20mL sterile PBS, and transferred to the mDixon containing 100 μg / mL NAT, 200 μg / mL cefotaxime and 10 μg / mL tetracycline.

[0086] Growth conditions for in vitro production of jasmonates by Malassezia

[0087] All Malassezia strains were cultured in modified Dixon medium (36 g / L malt extract, 2 mL / L glycerol, 20 g / L dried ox bile, 6 g / L peptone, 2 mL / L oleic acid, 10 mL / L Tween 40, pH 6) and incubated in a rotary shaker at 32°C and 150 rpm. 15 ml triplicate cultures at late exponential growth were harvested by centrifugation, washed three times in PBS, transferred to 2 ml Eppendorf tubes, and stored at -80°C prior to jasmonate extraction.

[0088] Determination of jasmonates in Malassezia cultures

[0089] JA levels in the culture medium were monitored by extraction with methanol:water:acetic acid (20:80:0.02, v / v), JA-d4 was added as a deuterated internal standard, and enriched using a Strata-X 33 mm polymeric solid reversed phase (SPE) extraction column. The extracted samples were then analyzed using reversed phase high performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry (MS) analysis.

[0090] Reverse phase separation was performed on a Phenomenex, Kinetex C8 (2.1×100 mm ID×150 mm L., 2.6 μm) column and maintained at 40°C. The mobile phase consisted of (A) water / formic acid (100 / 0.1, v / v) and (B) ACN. A 30-min step gradient condition was performed as follows: 0 min, 10% solvent B; 0-5 min, 10-25% solvent B; 5-10 min, 25-35% solvent B; 10-20 min, 35-75% solvent B; 20-20.1 min, 75-98% solvent B; 20.1-28 min, 98% solvent B; 28-28.1 min, 98-10% solvent B; and finally 28.1-30 min, 10% solvent B. The flow rate was 0.4 mL / min, the injection volume was 10 μL, and all samples were maintained at 4°C throughout the analysis.

[0091] A representative mixture of native and internal standards was injected and run with the column to optimize source parameters. Electrospray ionization was performed in positive ion mode. The drying gas temperature was set at 270°C and the gas flow was 10 L / min. The thin layer gas temperature was set at 250°C and the gas flow was 10 L / min. The nebulizer gas flow was 230 kPa. The dynamic MRM option was used for all compounds, and transitions and collision energies were optimized. The MRM transitions (precursor and product ions) and collision voltages were as follows: cis-OPDA (293→81.15; -30 eV), jasmonic acid (211.1→133.15; -13 eV). All peaks were manually determined and integrated using LabSolutions Insight software. Peaks were smoothed before integration, and the peak-to-peak signal-to-noise ratio was determined using the area under the peak.

[0092] Example 1: In vitro production of jasmonate (JA) by Malassezia species

[0093] It has been shown that Malassezia produces at least 46 oxylipins when cultured in vitro, many of which have been detected on human skin. In addition, several fungal species, including skin-resident Malassezia yeasts (see Figure 2 ) produces JA. Methods for in vitro cultivation and production of JA have been established (see Methods above). However, while the plant pathways leading to JA production have been characterized to some extent, there are few details about JA synthesis in fungi. Manipulation of the biosynthetic pathways involved in JA production in Malassezia can significantly increase production. In vitro cultured M. globosa species produce more JA than M. furfur species (see Figure 2 ), and thus may serve as a more promising basis for exploring increased JA production through genetic manipulation. Example 2: CRISPR / Cas9-mediated knockout (KO) of polyketide synthase (PKS) in Malassezia furfur significantly increases JA production

[0094] Malassezia yeasts are unable to produce polyunsaturated fatty acids (PUFAs) de novo due to the lack of the required Δ9 desaturase. However, Malassezia has been shown to accumulate several PUFAs, including α-linolenic acid (ALA), which is a substrate for JA synthesis, suggesting that alternative pathways for PUFA production in Malassezia may exist. Interestingly, under certain conditions, PKSs have been shown to produce PUFAs in both prokaryotes and eukaryotes. PKS activity in M. furfur was manipulated by genetic engineering to investigate how it might affect PUFAs and, in turn, JA levels. Interestingly, CRISPR / Cas9-mediated deletion of genes encoding polyketide synthases (PKSs), a multidomain enzyme complex involved in the production of related chemical structures, resulted in the production of certain oxylipins, including jasmonic acid ( Figure 3 and 4)) biosynthesis was significantly increased (3.6-fold), which was comparable to the decrease in similar oxylipins (including PUFAEPA, AA and DHA). Therefore, knocking out PKS activity in M. furfur using CRISPR / Cas9 technology generated a unique microbial strain with improved ability to enhance JA biosynthesis.

[0095] Surprisingly, the deletion of PKS genes in M. furfur did not reduce JA levels, but significantly enhanced JA production compared to the wild-type strain. This suggests that in Malassezia, the PKS complex is not directly involved in the jasmonate family of oxylipins, but their production is limited by competition with other PUFA pathways. The loss of PKS-mediated PUFA and oxylipin biosynthesis in M. furfur mutant strains may eliminate substrate competition and / or stimulate biosynthetic pathways involved in the production of jasmonic acid and other preferred oxylipin subgroups. The data also demonstrate that the deletion of PKS genes in other higher-yielding strains such as M. sphaerocephala will increase their jasmonate production.

[0096] It will be appreciated that numerous further modifications and permutations of the various aspects of the described embodiments may be made. Accordingly, the described aspects are intended to embrace all such alterations, modifications and variations that fall within the spirit and scope of the appended claims.

Claims

1. A method for producing jasmonate using a fungal cell, the method comprising a) culturing the fungal cell under conditions suitable for producing jasmonate, wherein the expression of a polyketide synthase (PKS) gene in the fungal cell has been disrupted; and b) isolating the jasmonate produced by the fungal cell.

2. The method according to claim 1, wherein the method comprises disrupting a PKS gene prior to step a).

3. The method according to claim 1 or 2, wherein the method comprises providing a fatty acid source to the fungal cells in step a).

4. The method according to any one of claims 1 to 3, wherein the fungal cell is a yeast cell.

5. The method according to claim 4, wherein the yeast cell is a Malassezia species.

6. A method according to any one of claims 1 to 5, wherein the PKS gene has been disrupted by deletion or insertion.

7. The method according to any one of claims 1 to 6, wherein the PKS gene of the fungal cell has been disrupted by nuclease-mediated gene editing.

8. The method according to claim 7, wherein the nuclease is a zinc finger protein, a nuclease containing a transcription activator-like effector (TALEN) nuclease, or a CRISPR / Cas system.

9. The method according to claim 8, wherein the CRISPR / Cas system is CRISPR / Cas9.

10. The method according to claim 8 or 9, wherein the CRISPR / Cas system comprises at least one nucleic acid encoding a CRISPR nuclease and at least one nucleic acid encoding a guide RNA.

11. The method according to any one of claims 8 to 10, wherein the CRISPR / Cas system comprises an exogenous sequence for integration into the yeast genome by homologous recombination after nuclease cleavage.

12. The method according to any one of claims 1 to 5, wherein the expression of the PKS gene is disrupted using an inhibitory nucleic acid or small molecule that inhibits the expression of the PKS gene.

13. The method according to any one of claims 1 to 12, wherein the jasmonate is jasmonic acid, methyl jasmonate or jasmonic acid conjugated to an amino acid.

14. The method according to any one of claims 1 to 13, wherein the jasmonate is secreted by fungal cells and step b) comprises isolating the secreted jasmonate.

15. A method for enhancing jasmonate production in a fungal cell, the method comprising disrupting the expression of a polyketide synthase (PKS) gene in the fungal cell.

16. The method according to claim 15, wherein the method further comprises culturing the fungal cells under conditions suitable for producing jasmonate, and isolating the jasmonate produced by the fungal cells.

17. An engineered fungal cell for producing jasmonate, wherein a polyketide synthase (PKS) gene has been disrupted in the fungal cell.

18. A microbiome composition comprising fungal cells, wherein a polyketide synthase (PKS) gene has been disrupted in the fungal cells.

19. A microbiome composition according to claim 18, wherein the microbiome composition is a skin microbiome composition.

20. The microbiome composition according to claim 18 or 19 for use as a cosmetic or as a medicament.

21. Use of a microbiome composition according to claim 18 or 19 for producing jasmonates.

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