Method for genetically modifying microorganisms with high CG content

Abstract

The present invention relates to a method for genetically modifying a GC-rich microorganism. The present invention further relates to a genetically modified GC-rich microorganism. Furthermore, the present invention relates to a composition comprising an RNA-guided endonuclease, at least one guide RNA (gRNA), and optionally a donor DNA. The present invention also relates to a method for preparing a target compound, such as an acetyl-CoA-based hydrophobic compound and / or an oil with a specific fatty acid profile, such as a high oleic oil, using the genetically modified GC-rich microorganism.

Description

[Technical field]

[0001] The present invention relates to a method for genetically modifying a GC-rich microorganism. The present invention further relates to a genetically modified GC-rich microorganism. Furthermore, the present invention relates to a composition comprising an RNA-guided endonuclease, at least one guide RNA (gRNA), and optionally a donor DNA. The present invention also relates to a method for preparing a target compound, such as an acetyl-CoA-based hydrophobic compound and / or an oil with a specific fatty acid profile, such as a high oleic oil, using the genetically modified GC-rich microorganism. [Background technology]

[0002] The increasing global demand for animal and plant-based lipids in the biofuel, pharmaceutical, and oleochemical industries has negatively impacted biodiversity and resulted in land use changes. As a result, microbial oils have attracted attention as an alternative to vegetable oils. Oleaginous microorganisms such as the oleaginous yeast Cutaneotrichosporon oleaginosus ATCC20509 can accumulate lipids through a "de novo" lipid biosynthetic pathway. Oleaginous microorganisms such as C. oleaginosus can be used to generate microbial lipids because they are cost-effective and grow on complex, non-detoxified waste biomass streams.

[0003] Genetic engineering is a route to improve and diversify high-value compounds produced by microorganisms, such as triglycerides, terpenoids, and other metabolically-derived high-value compounds. Improvement and optimization of high-value products produced by oleaginous yeasts, such as tailor-made lipids, have been reported [1]. However, the lack of efficient genetic tools limits their productivity and product diversity.

[0004] To date, Agrobacterium-mediated transformation (AMT) has been established for stable random integration of expression cassettes into the genome of GC-rich microorganisms, especially oleaginous microorganisms such as C. oleaginosus [1]. Such random integration of genes into the genome results in different and unpredictable gene expression levels, which mainly depend on the variable insertion locus and the number of insertion events. In GC-rich microorganisms, especially oleaginous microorganisms such as C. oleaginosus, targeted and extensive genetic manipulation has been hindered due to the predominance of non-homologous end joining (NHEJ) over homologous recombination (HDR) and the lack of suitable plasmids for GC-rich microorganisms, e.g., artificial plasmids for oleaginous yeast [2]. In response to this, there is a need to establish efficient genome editing tools for targeted genetic modification to enable more precise and advanced manipulation of GC-rich microorganisms, especially oleaginous microorganisms with high GC content, e.g., C. oleaginosus.

[0005] The dominance of non-homologous end joining (NHEJ) over homologous recombination (HDR) has been an obstacle to the genetic modification of GC-rich microorganisms such as C. oleaginosus. In particular, genetic engineering of GC-rich microorganisms, such as oleaginous microorganisms, having a high GC content, e.g., 50% or more or 60% or more GC content, and having many repetitive sequences, is very difficult due to the presence of such repetitive sequences and the high GC content that leads to off-target effects and the formation of nucleic acid secondary structures.

[0006] The reported protocols for genetic modification of GC-rich microorganisms, such as oleaginous microorganisms, have only described random gene insertion into the genome and random mutagenesis to date. Thus, there is a need for a method for targeted genetic modification of GC-rich microorganisms, such as oleaginous microorganisms. Furthermore, there is a need for a method for genetically modifying GC-rich microorganisms, particularly oleaginous microorganisms with high GC content, such as C. oleaginosus. There is also a need for a means for targeted genetic modification of such GC-rich microorganisms to increase the production of target compounds, such as, for example, acetyl-CoA-based hydrophobic compounds and / or specific fatty acids. Summary of the Invention

[0007] The elements of the present invention are described below. Although the elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to produce further embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The specification should be understood to support and encompass embodiments combining two or more of the explicitly described embodiments or combining one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application unless the context indicates otherwise.

[0008] In a first aspect, the present invention relates to a method for genetically modifying an optionally GC-rich microorganism in order to increase the production of acetyl-CoA-based hydrophobic compounds in said microorganism, the method comprising the steps of: i) providing a GC-rich microorganism, preferably an oleaginous microorganism, more preferably an oleaginous yeast; ii) optionally pretreating the GC-rich microorganism; preferably, the pretreatment comprises spheroplasting the GC-rich microorganism; iii) transforming the GC-rich microorganism with an RNA-guided endonuclease, at least one guide RNA, and optionally donor DNA; iv) optionally selecting transformed cells of said GC-rich microorganism; v) Obtaining a genetically modified GC-rich microorganism.

[0009] In one embodiment, the GC-rich microorganism has a guanine-cytosine (GC) content of at least 50%, preferably at least 55%, more preferably at least 58%, even more preferably at least 60%.

[0010] In one embodiment, the GC-rich microorganism is selected from yeast, fungi, bacteria, and microalgae; preferably, the GC-rich microorganism is an oleaginous microorganism; more preferably, the GC-rich microorganism is an oleaginous yeast; even more preferably, the GC-rich microorganism is selected from Rhodosporidium spp., Yarrowia spp., Rhodotorula spp., Candida spp., Lipomyces spp., Cutaneotrichosporon spp., Trichosporon spp., preferably Cutaneotrichosporon spp., more preferably Cutaneotrichosporon oleaginosus (ATCC20509); and / or The GC-rich microorganism has a guanine-cytosine (GC) content of at least 50%, preferably at least 55%, more preferably at least 58%, even more preferably at least 60%.

[0011] In one embodiment, the RNA-guided endonuclease is a CRISPR-associated (Cas) endonuclease, preferably selected from Cas9, Cas1, Cas2, Cas4, Cas3, Cas10, Cas12, Cas13, Csm, scf1, or variants thereof, such as Cas12a, dCas9, D10A CAS9 nickase, or H840A CAS9 nickase.

[0012] In one embodiment, the transformation in step iii) comprises applying the RNA-guided endonuclease to the GC-rich microorganism in the form of an RNA-guided endonuclease protein, in the form of a DNA encoding the RNA-guided endonuclease, or in the form of an mRNA encoding the RNA-guided endonuclease, preferably in the form of an mRNA encoding the RNA-guided endonuclease.

[0013] In one embodiment, the at least one guide RNA comprises a CRISPR RNA (crRNA), a trans-activating CRISPR RNA (tracrRNA), and / or a single guide RNA (sgRNA); preferably, the at least one guide RNA comprises a first guide RNA and a second guide RNA, wherein the sequence of the first guide RNA is different from the sequence of the second guide RNA.

[0014] In one embodiment, the donor DNA includes a DNA repair template, DNA encoding a selectable marker, and / or a gene or sequence of interest, e.g., a gene involved in the production of a target compound, such as a fatty acid.

[0015] In one embodiment, said transformation in step iii) comprises applying said RNA-guided endonuclease and said at least one guide RNA, preferably together, in the form of a ribonucleoprotein complex.

[0016] In one embodiment, the transformation in step iii) comprises applying an RNA-guided endonuclease in the form of an mRNA encoding said RNA-guided endonuclease, wherein said at least one guide RNA comprises an sgRNA.

[0017] In one embodiment, the pretreatment in step ii) comprises an enzymatic pretreatment, a chemical pretreatment, and / or another spheroplasting procedure; Preferably, the pretreatment in step ii) comprises an enzymatic pretreatment carried out with at least one enzyme selected from glycosyl hydrolase, cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, glucosidase, arabinase, amylase, fructanase, laminase, hydrolase, protease, or any combination thereof, preferably a combination of cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, and glucosidase.

[0018] In one embodiment, the pretreatment in step ii) comprises treatment of the microorganism with a hydrolase alone or a combination of a hydrolase and a protease / hydrolase followed by a protease; Optionally, the hydrolase is selected from hydrolases produced by fungi, preferably filamentous fungi, more preferably fungi selected from Trichoderma spp., Aspergillus spp., Penicillium spp., Aureobasillium spp., and Fusarium spp., even more preferably Trichoderma reesei; and / or Optionally, the protease is selected from proteases produced by Aspergillus species, Streptomyces species, or Bacillus species.

[0019] In one embodiment, the transformation in step iii) is carried out using electroporation; PEG-based or other nanoscale carrier-based transformation; biological ballistic flight; glass bead transformation; vesicle-mediated delivery; viral transfection systems, such as lentivirus (LV), adenovirus (AdV), or adeno-associated virus (AAV); liposomal delivery, such as lipofection; chemical transfection techniques; or combinations thereof; Preferably, this is done using electroporation and / or PEG-based transformation; more preferably, using electroporation.

[0020] In one embodiment, the method comprises said selecting in step iv), wherein said selecting in step iv) comprises exposing said microorganism to a selection agent selective for a selection marker, optionally a selection marker encoded by said donor DNA; Preferably, the selection comprises exposing the microorganism to a concentration of the selection agent in the range of 10% to 90%, preferably 10% to 70%, more preferably 10% to 60% of the minimum selectable concentration; and / or exposing the microorganism to a concentration of the selection agent in the range of 90% to 100%, preferably 99% to 100% of the minimum selectable concentration; More preferably, the selection comprises exposing the microorganism to the selective agent in a first medium, such as a liquid or solid medium, preferably an agar underlayer, at a concentration in the range of 10% to 90%, preferably 10% to 70%, more preferably 10% to 60% of the minimum selectable concentration, and optionally thereafter exposing the microorganism to the selective agent in a second medium, such as a liquid or solid medium, preferably an agar underlayer, at a concentration in the range of 90% to 100%, preferably 99% to 100%.

[0021] In a further aspect, the present invention relates to a genetically modified GC-rich microorganism, preferably an oleaginous microorganism, more preferably an oleaginous yeast, even more preferably Cutaneotrichosporon oleaginosus, comprising an RNA-guided endonuclease, at least one guide RNA, and optionally donor DNA; optionally, said RNA-guided endonuclease and said at least one guide RNA are present in said cell in the form of a ribonucleoprotein complex.

[0022] In one embodiment, the genetically modified GC-rich microorganism is obtained using the methods defined herein.

[0023] In this aspect, the GC-rich microorganism, the oleaginous microorganism, the oleaginous yeast, the RNA-guided endonuclease, the at least one guide RNA, the donor DNA, and the ribonucleoprotein complex are as defined herein.

[0024] In a further aspect, the present invention provides a method for producing a composition comprising the steps of: - an RNA-guided endonuclease; optionally an RNA-guided endonuclease protein, a DNA encoding said RNA-guided endonuclease, or an mRNA encoding an RNA-guided endonuclease, preferably an mRNA encoding an RNA-guided endonuclease; an RNA-guided endonuclease, preferably a CRISPR-associated (Cas) endonuclease, more preferably selected from Cas9, Cas1, Cas2, Cas4, Cas3, Cas10, Cas12, Cas13, Csm, scf1, or variants thereof, such as Cas12a, dCas9, D10A CAS9 nickase, or H840A CAS9 nickase, and even more preferably a Cas9 endonuclease, such as a Cas9 endonuclease having the sequence of any of SEQ ID NOs: 1 to 4; - at least one guide RNA (gRNA); preferably a crRNA, a tracrRNA and / or an sgRNA; Optionally, a gRNA comprising the tracrRNA sequence of SEQ ID NO:5 and / or comprising any one of the crRNA sequences of SEQ ID NOs:14 to 26; - optionally, a donor DNA; preferably a donor DNA comprising a DNA repair template, DNA encoding a selectable marker, and / or a gene or sequence of interest, e.g. a gene involved in the production of a target compound, such as a fatty acid; Optionally, the selectable marker is Ura5; Optionally, the gene or sequence of interest encodes a delta-9-desaturase, a delta-12-desaturase, an elongase, an oleate hydratase, an aldo-ketoreductase promoter, an aldo-ketoreductase terminator, a transcription elongation factor 2 promoter, a TEF promoter, and / or a TEF terminator; Optionally, the donor DNA is a donor DNA comprising any of the sequences of SEQ ID NOs: 6 to 13 and 27 to 36, and optionally any of SEQ ID NOs: 6 to 13 and 27. The present invention relates to a composition comprising:

[0025] In one embodiment, the Cas9 endonuclease having a sequence of any of SEQ ID NOs: 1 to 4 has an amino acid sequence of any of SEQ ID NOs: 1 to 2 and / or a nucleic acid sequence of any of SEQ ID NOs: 3 to 4.

[0026] In this embodiment, the RNA-guided endonuclease, the at least one guide RNA, and the donor DNA are as defined herein.

[0027] In a further aspect, the present invention provides a method for producing a composition comprising the steps of: - an mRNA encoding an RNA-guided endonuclease; an RNA-guided endonuclease that is preferably a Cas9 endonuclease, and more preferably a Cas9 endonuclease having any of the sequences of SEQ ID NOs: 1 to 4; - at least one guide RNA (gRNA); preferably a crRNA, a tracrRNA and / or an sgRNA; Optionally, a gRNA comprising the tracrRNA sequence of SEQ ID NO:5 and / or comprising any one of the crRNA sequences of SEQ ID NOs:14 to 26; - optionally a donor DNA, such as a DNA repair template; Optionally, a donor DNA having any of SEQ ID NOs: 6 to 13 and 27 to 36, and optionally any of SEQ ID NOs: 6 to 13 and 27. The present invention relates to a plasmid or a collection of plasmids comprising the plasmid.

[0028] In one embodiment, the mRNA encoding the RNA-guided endonuclease comprises or consists of an mRNA encoding a Cas9 endonuclease, the Cas9 endonuclease having the amino acid sequence of any of SEQ ID NOs: 1-2 and / or an mRNA encoding a Cas9 endonuclease, the mRNA having the nucleic acid sequence of any of SEQ ID NOs: 3-4.

[0029] In one embodiment, the mRNA encoding the RNA-guided endonuclease comprises or consists of any of the mRNAs encoding a Cas9 endonuclease, the Cas9 endonuclease having the amino acid sequence of any of SEQ ID NOs: 1-2 and the mRNA encoding the Cas9 endonuclease, the mRNA having the nucleic acid sequence of any of SEQ ID NOs: 3-4.

[0030] In one embodiment, the plasmid or plasmid collection comprises: - an mRNA encoding an RNA-guided endonuclease; Preferably, the RNA-guided endonuclease is Cas9 endonuclease; more preferably, the mRNA is any one of mRNAs encoding Cas9 endonuclease, the Cas9 endonuclease having an amino acid sequence of any one of SEQ ID NOs: 1 to 2 and an mRNA encoding Cas9 endonuclease, the mRNA having a nucleic acid sequence of any one of SEQ ID NOs: 3 to 4; - at least one guide RNA (gRNA); preferably a crRNA, a tracrRNA and / or an sgRNA; Optionally, a gRNA comprising the tracrRNA sequence of SEQ ID NO:5 and / or comprising any one of the crRNA sequences of SEQ ID NOs:14 to 26; - optionally a donor DNA, such as a DNA repair template; Optionally, a donor DNA having any of SEQ ID NOs: 6 to 13 and 27 to 36, and optionally any of SEQ ID NOs: 6 to 13 and 27. Includes.

[0031] In this embodiment, the RNA-guided endonuclease, the at least one guide RNA, and the donor DNA are as defined herein.

[0032] In a further aspect, the present invention relates to the use of the composition, the plasmid and / or the plasmid collection defined herein for genetically modifying a GC-rich microorganism, in particular a microorganism selected from the species of the genus Cutaneotrichosporon.

[0033] In this aspect, the composition, the plasmid, the plasmid collection, the GC-rich microorganism, and the microorganism selected from a species of the genus Cutaneotrichosporon are as defined herein.

[0034] In a further aspect, the present invention provides a method for preparing a target compound, such as acetyl-CoA or acetyl-CoA based hydrophobic compounds and / or an oil having a specific fatty acid profile, such as a high oleic oil, using a genetically modified GC-rich microorganism, preferably an oleaginous microorganism, more preferably an oleaginous yeast, comprising: a) providing a genetically modified GC-rich microorganism, preferably an oleaginous microorganism, more preferably an oleaginous yeast, using a method as defined above; said method comprising transforming said microorganism with a donor DNA, said donor DNA comprising a gene or sequence of interest, e.g. a gene involved in the production of said target compound and / or a specific fatty acid; b) growing the genetically modified GC-rich microorganism; c) obtaining an oil having said target compounds and / or a particular fatty acid profile; Preferably, the target compound is selected from saturated short-chain fatty acids, saturated medium-chain fatty acids, saturated long-chain fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, functionalized fatty acids, ergosterol, ergosterol derivatives, terpenes, alkaloids, acetyl-CoA based synthetic compounds, tocochromanols, such as α-tocopherol and α-tocotrienol, monoterpenoids, sesquiterpenoids, diterpenoids, squalene, carotenoids, triterpenes, pheophytins, vitamins, citric acid, volatile fatty acids, oxalic acid, lactic acid, malic acid, and exopolysaccharides. The present invention relates to a method comprising the steps of:

[0035] In this aspect, the GC-rich microorganism, oleaginous microorganism, oleaginous yeast, and donor DNA are as defined herein. [Brief description of the drawings]

[0036] The invention will now be further described by reference to the following drawings. All methods described in the figure legends below were carried out as detailed in the Examples. [Figure 1] Figure 1 shows that transfection with mRNA results in more colonies. Positive colonies are marked with a circle. All numbered colonies on the plate appeared during the effective period of the selection agent. The small unnumbered colonies are non-specific colonies after the selection agent has lost its effect. It has been demonstrated that spheroplasting allows efficient transfection of GC-rich microorganisms, such as oleaginous yeasts, such as C. oleaginosus. Furthermore, it has been shown that transfection with RNA-guided endonuclease in the form of mRNA is even more efficient than transfection with RNA-guided endonuclease in the form of protein. It has also been shown that combining pretreatment of GC-rich microorganisms, such as oleaginous microorganisms, with spheroplasting with transfection of the organisms with RNA-guided endonuclease in the form of mRNA synergistically increases the efficiency of genetic modification of GC-rich microorganisms. [Diagram 2] Figure 2 shows that no transformants are obtained when spheroplasting with commercially available lyticase enzyme. Thus, spheroplasting GC-rich microorganisms such as oleaginous yeast with an enzyme mix adapted to the respective microorganism is surprisingly superior to spheroplasting with commercially available enzymes. Unexpectedly, the spheroplasting procedure used in the method of the present invention allows for highly efficient transfection of GC-rich microorganisms, such as oleaginous yeast, using RNA-guided endonucleases. [Diagram 3]FIG. 3 shows an exemplary targeted site modification at the Ura5 locus in genomic DNA. A) Genetic modification with Cas wild type. In this example, CAS9 is used to knock out the ura5 gene by creating a targeted frameshift. This method allows for targeted gene knockout at a selected locus. B) Genetic modification with Cas nickase. In this example, CAS nickase is used to knock out the ura5 gene by creating a targeted frameshift. This method allows for targeted gene knockout at a selected locus. [Figure 4] Figure 4 shows DNA gel electrophoresis of wild type and mutant (engineered with nickase) Ura5 genes after restriction digestion. Agarose gel electrophoresis: wild type ura5 gene and edited ura5 locus, both treated with restriction digestion enzymes (non-cutter enzymes for WT ura5 gene). Donor DNA for the nickase ura5 knockout in this example contained a non-cutter restriction digestion site. Mutant and wild type ura5 genes were amplified and digested with the respective enzymes. [Diagram 5] 5 shows an exemplary spheroplasting procedure used in the methods of the invention. The spheroplasting procedure allows the pretreatment of microorganisms so that they can be efficiently transfected. [Figure 6] 6 shows a schematic diagram of targeted genetic modification of GC-rich microorganisms using CRISPR-Cas. The method of the present invention, including spheroplast formation, allows efficient transfection of GC-rich microorganisms, such as oleaginous microorganisms with high GC content. [Figure 7] Figure 7 shows the fatty acid profiles of the strains engineered via CRISPR and wild type in minimal nitrogen medium. Replacing the delta-9 desaturase promoter with the TEF promoter resulted in less C18:1 and more C18:0. Thus, the method of the present invention makes it possible to effectively modify the fatty acid profile of GC-rich microorganisms and prepare oils with specific fatty acid profiles. [Figure 8]8 shows that knocking out the delta-12 desaturase gene in GC-rich microorganisms, particularly oleaginous microorganisms, results in the absence of C18:2 and C18:3 in the final fatty acid profile of the mutant. Thus, the method of the present invention makes it possible to effectively modify the fatty acid profile of GC-rich microorganisms and prepare oils with specific fatty acid profiles. [Figure 9] FIG. 9 shows the fatty acid profile of the engineered strain in which the D9 promoter was replaced with the AKR promoter compared to wild-type fatty acids after 96 hours of growth under controlled conditions in a bioreactor using minimal nitrogen medium supplemented with glucose. [Figure 10] FIG. 10 shows the fatty acid profile of the engineered strain in which the D9 promoter was replaced with the AKR promoter compared to wild-type fatty acids after 96 hours of growth under controlled conditions in a bioreactor using nitrogen-rich medium supplemented with glucose and acetate. [Figure 11] Figure 11 shows the fatty acid profile of the engineered strain in which the D9 promoter was replaced with the TEF promoter compared to the wild-type fatty acids after 96 hours of growth in a bioreactor under controlled conditions using minimal nitrogen medium supplemented with glucose. Replacing the delta-9 desaturase promoter with the TEF promoter resulted in less C18:1 and more C18:0. [Figure 12] FIG. 12 shows the fatty acid profile of the engineered strain in which the D9 promoter was replaced with the TEF promoter compared to wild-type fatty acids after 96 hours of growth under controlled conditions in a bioreactor using nitrogen-rich medium supplemented with glucose and acetate. [Figure 13] FIG. 13 shows the fatty acid profiles of D9 overexpression engineered strains and wild type after 96 hours of cultivation under controlled conditions in a bioreactor using minimal nitrogen medium. [Figure 14] FIG. 14 shows the fatty acid profiles of D9 overexpression engineered strains and wild type after 96 hours of cultivation under controlled conditions in bioreactors using nitrogen-rich medium supplemented with glucose and acetate. [Figure 15] Figure 15 shows the fatty acid profile of the engineered strain overexpressing D12 desaturase compared to wild-type fatty acids after 96 hours of culture in shake flasks in minimal nitrogen medium supplemented with glucose. A slight increase in C18:2 content can be observed in the engineered strain. [Figure 16] Figure 16 shows the fatty acid profile of an engineered strain with a full knockout of the D12 gene compared to wild type fatty acids after 96 hours of growth in a bioreactor under controlled conditions in minimal nitrogen medium supplemented with glucose. Knocking out the delta-12 desaturase gene resulted in the absence of C18:2 and C18:3 in the final fatty acid profile of this mutant. [Figure 17] FIG. 17 shows the fatty acid profile of the engineered D12 full knockout strain compared to wild type fatty acids after 96 hours of growth under controlled conditions in a bioreactor using nitrogen-rich medium supplemented with glucose and acetate. [Figure 18] FIG. 18 shows the lipid content and lipid yield of engineered and wild-type strains grown in minimal nitrogen medium supplemented with glucose for 96 hours in bioreactors. [Figure 19] FIG. 19 shows lipid content and lipid yield of engineered and wild type strains grown in nitrogen-rich medium supplemented with glucose and acetate for 96 hours in bioreactors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] In GC-rich microorganisms, e.g., oleaginous microorganisms such as C. oleaginosus, non-homologous end joining (NHEJ) predominates over homologous recombination (HDR), preventing widespread targeted genetic engineering of oleaginous microorganisms. Furthermore, genetic engineering of oleaginous microorganisms has also been hindered by the lack of artificial plasmids for GC-rich microorganisms such as oleaginous microorganisms. Furthermore, delivery of genes and / or endonucleases into the cell wall containing GC-rich microorganisms, e.g., oleaginous microorganisms, has also been hindered by the presence of the cell wall.

[0038] The object of the present invention is to provide an enhanced transformation system, e.g., a method for genetically modifying GC-rich microorganisms in a targeted manner. In particular, the object of the present invention is to provide an enhanced means for genetically modifying GC-rich microorganisms. Furthermore, the object of the present invention is to provide a genetically modified microorganism capable of efficiently producing a target compound, e.g., an acetyl-CoA-based hydrophobic compound, and / or to provide a genetically modified microorganism having an altered acetyl-CoA pool. Also, the object of the present invention is to efficiently produce a target compound, e.g., an acetyl-CoA-based hydrophobic compound, using a GC-rich microorganism, e.g., an oleaginous microorganism. A further object of the present invention is to efficiently transform a GC-rich microorganism, e.g., to genetically modify a GC-rich microorganism to produce a target compound, e.g., an acetyl-CoA-based hydrophobic compound. Also, the object of the present invention is to provide a method for genetically modifying a GC-rich microorganism at a defined position in the genome. Furthermore, the object of the present invention is to increase the transformation efficiency of a GC-rich microorganism.

[0039] The method of genetically modifying GC-rich microorganisms according to the present invention is highly advantageous in that the cell wall is degraded before transformation. For example, enzymatic pretreatment is used to degrade the cell wall of GC-rich microorganisms, preferably oleaginous microorganisms. Degrading the cell wall of GC-rich microorganisms facilitates gene transfer, RNA transfer, protein transfer, and / or transfer of ribonucleoprotein complexes. Advantageously, the enzymatic pretreatment and / or spheroplasting procedure can be specifically adapted to each microorganism to be treated, for example, the enzymes used can be adapted to the components of the cell wall of each microorganism. Thus, advantageously, the method of the present invention makes it possible to provide a tailor-made enzyme system for pretreatment of GC-rich microorganisms.

[0040] Furthermore, the genetic modification method of the present invention advantageously allows even ribonucleoprotein complexes to be transferred into GC-rich microorganisms. The present inventors have successfully developed an efficient genome editing technique mediated by Cas through the adoption of CAS mRNA and ribonucleoprotein (RNP). The advantage of using RNA-guided endonuclease in the form of mRNA or in the form of protein, particularly ribonucleoprotein, is that it is not necessary to consider the promoter efficiency and expression level of the RNA-guided endonuclease gene, such as the CAS gene. Therefore, advantageously, the genetic modification method of the present invention can be carried out in the form of a DNA-free genome editing technique. The advantage of the RNA-guided endonuclease in the form of mRNA or RNP is that the RNP is degraded over time and the Cas mRNA transiently expresses the CAS protein, which results in a significant reduction in cloning effort and mitigation of off-target effects. The genetic modification method of the present invention is highly advantageous in that it can genetically modify even microorganisms with high GC content. Advantageously, when the transformation in step iii) comprises applying the RNA-guided endonuclease in the form of an mRNA encoding the RNA-guided endonuclease, the Cas protein is transiently expressed. The inventors have found that applying the RNA-guided endonuclease in the form of an mRNA reduces off-target effects compared to the DNA or protein forms. The inventors have found that when GC-rich microorganisms such as species of the genus Cutaneotrichosporon are pretreated, preferably spheroplasted, and when an RNA-guided endonuclease is used, especially in the form of an mRNA, the microorganisms can be efficiently genetically modified with very low off-target effects.

[0041] To further increase the specificity of genetic modification, multiple sgRNA molecules can be used to transform GC-rich microorganisms. For example, sgRNA molecules targeting the same or different target sequences can be used to increase specificity. In one embodiment, the method for genetically modifying GC-rich microorganisms comprises transforming the microorganism with one or more sgRNA molecules, preferably multiple sgRNA molecules targeting different target sequences. In one embodiment, the method for genetically modifying GC-rich microorganisms comprises transforming the microorganism with one or more tracrRNA and one or more crRNA molecules, preferably multiple crRNA molecules targeting different target sequences.

[0042] The genomes of GC-rich microorganisms, e.g., oleaginous microorganisms, such as C. oleaginosus, are rich in GC content (e.g., about 61%), which complicates the design of guide RNAs, e.g., sgRNA design. Although a 20-bp sgRNA is believed to specify an RNA-guided endonuclease, e.g., CAS9, to the targeted sequence, it has been shown that it can tolerate 3–5 mismatches in the PAM-distal portion. In addition to the PAM, the seed sequence of the sgRNA (10–12 bp directly adjacent to the PAM) generally plays a role in on-target binding of the CAS9:sgRNA complex. Thus, the presence of similar repeat sequences in the genomes of GC-rich microorganisms, e.g., oleaginous microorganisms with high GC content, e.g., C. oleaginosus, contributes to the off-target effects of CAS9:sgRNA. Derivatives of CAS9 nickase (D10A, H840A) were created by mutating one of the two Cas9 nuclease domains, resulting in single-stranded breaks in DNA. These CAS9 derivatives therefore feature two adjacent gRNAs that target opposite DNA strands to generate the required double-stranded breaks, thus reducing off-target effects and facilitating gRNA design.

[0043] Previously established methods cannot be used to directly deliver CAS proteins / mRNA, synthetic sgRNA, and donor DNA into oleaginous microorganisms with high GC content, such as C. oleaginosus. Thus, a more flexible transformation approach for GC-rich microorganisms, such as oleaginous yeast, specifically C. oleaginosus, was established, which allows efficient RNA-guided endonuclease delivery, preferably Cas delivery. Electroporation of spheroplasts, such as C. oleaginosus spheroplasts, offers a less laborious and more rapid procedure for delivering any type of genetic element, such as ssDNA / dsDNA fragments, DNA, mRNA, small RNA, and proteins such as nucleases. Optimized and efficient spheroplast isolation was achieved by treating GC-rich microorganisms, such as oleaginous microorganisms, preferably oleaginous yeast cells, with an enzyme mixture isolated from an enzyme-producing organism, preferably a filamentous fungus, even more preferably Trichoderma reesei. For example, hydrolase enzyme systems used for spheroplast preparation were generated by fermenting T. reesei on C. oleaginosus biomass cultured under non-limiting conditions. Additionally, hydrolase enzyme systems for spheroplasting were generated by culturing T. reesei on Co biomass generated under oil-forming culture conditions. Additionally, in exemplary hydrolase production for downstream spheroplasting, such as C. oleaginosus spheroplasting, the oil-containing oleaginous microbial biomass was subjected to organic phase (i.e., methanol) and water washes, either sequentially or in combination, prior to fermentation with T. reesei.

[0044] In one embodiment, an enzyme-producing microorganism, such as a fungus, preferably a filamentous fungus, produces at least one enzyme used in the pretreatment in step ii). In one embodiment, the at least one enzyme used in the pretreatment in step ii) is obtained from an enzyme-producing microorganism, such as a fungus, preferably a filamentous fungus, for example, any enzyme selected from glycosyl hydrolase, cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, glucosidase, arabinase, amylase, fructanase, laminaase, hydrolase, protease, or any combination thereof, is obtained from a fungus, preferably a filamentous fungus. In one embodiment, the enzyme is produced by the enzyme-producing microorganism, such as a fungus, preferably a filamentous fungus, and then obtained from the fermentation broth of the fungus by salting out the enzyme, by chromatography, and / or by filtration, such as nanofiltration and / or ultrafiltration, and the obtaining optionally further comprises a drying procedure, such as spray drying. In one embodiment, the pretreatment, preferably spheroplasting, in step ii) is carried out with a tailor-made enzyme system, the tailor-made enzyme system being the at least one enzyme produced by an enzyme-producing filamentous fungus cultivated with an induction system, the induction system comprising or consisting of the GC-rich microorganism or a component thereof. The inventors have found that the method for genetically modifying a GC-rich microorganism, in particular a microorganism comprising a cell wall, such as a species of the genus Cutaneotrichosporon, is highly efficient, and allows the microorganism to be genetically modified specifically and efficiently, when the microorganism is pretreated, in particular spheroplasted. Advantageously, by spheroplasting the microorganism, in particular a microorganism comprising a cell wall, prior to the transformation, the microorganism becomes susceptible to efficient transformation with an RNA-guided endonuclease, for example an RNA-guided endonuclease in the form of an mRNA. Advantageously, by pretreating the microorganism and transforming the microorganism with an RNA-guided endonuclease, the donor DNA can be efficiently integrated into the genome of the microorganism in a targeted and specific manner.The inventors have found that GC-rich microorganisms, particularly selected from the species of the genus Cutaneotrichosporon, can be genetically modified with surprisingly high efficiency when pretreated with a tailor-made enzyme system. Moreover, the synergistic effect of pretreatment with the tailor-made enzyme system and the RNA-guided endonuclease, particularly when applied in the form of mRNA, resulted in very high efficiency and yield. The inventors have shown that GC-rich microorganisms, for example selected from the species of the genus Cutaneotrichosporon, can be modified to produce microbial oils, such as microbial oils with specific fatty acid compositions. Thus, advantageously, the composition of the microbial oil produced by the GC-rich microorganisms can be changed, for example by genetically modifying the microorganisms, so that it contains less unhealthy trans-unsaturated fatty acids and more healthy fatty acids.

[0045] The present inventors have developed a gene editing system using an RNA-guided endonuclease for GC-rich microorganisms, particularly the CRISPR / CAS system, for targeted engineering of oleaginous microorganisms with high GC content, such as C. oleaginosus. The method of the present invention is advantageous because it reduces off-target effects. Off-target effects were reduced by sequence alignment between the crRNA sequence and the genomic DNA of GC-rich microorganisms, such as C. oleaginosus. Specifically, off-targets were identified by sequence alignment between the crRNA sequence and the genomic DNA, and then crRNA sequences without non-specific targets were selected. After constructing a library of crRNA sequences, the library was screened by sequence alignment with genomic DNA to select those without non-specific targeting. An RNA-guided endonuclease, such as the CAS:sgRNA RNP complex, plus its variants, such as D10A, together with two sgRNAs, was successfully delivered to GC-rich microorganisms, such as C. oleaginosus. Furthermore, targeted gene editing was successfully achieved by transforming spheroplasts, such as yeast spheroplasts, with an RNA-guided endonuclease in the form of mRNA (e.g., Cas mRNA) and sgRNA. Furthermore, new sequences were introduced through simultaneous transfer of single-stranded or double-stranded donor DNA, resulting in ura5 knockout mutants. Mutants were selected on YNB-5foa plates containing uracil. Further analysis was performed by gene sequencing. In one embodiment, the guide RNA, e.g., crRNA, is selected such that no sites other than the target site are detected on the genomic DNA. In one embodiment, the off-target activity of the endonuclease is reduced by selecting the guide RNA, e.g., crRNA, such that no sites other than the target site are detected on the genomic DNA. In one embodiment, the off-target effect is further reduced by using a nickase. In one embodiment, delivery of an endonuclease protein or an mRNA encoding an endonuclease reduces off-target effects compared to delivery of a gene encoding a nuclease that is integrated into the genome and remains permanently active.In a preferred embodiment, the two sgRNAs are delivered to a GC-rich microorganism in combination with an endonuclease variant, such as D10A CAS9 nickase or H840A CAS9 nickase, which further increases the efficiency of the method.

[0046] The methods of the invention using RNA-guided endonucleases, such as CRISPR / CAS or its variants such as D10A and H840A, provide a precise, flexible, and convenient approach for targeted engineering of GC-rich microorganisms, particularly oleaginous yeasts such as C. oleaginosus. The methods of the invention allow these important microorganisms, such as C. oleaginosus, to be fully utilized as industrial cell factories, optimizing metabolically derived products such as acetyl-CoA or acetyl-CoA-based hydrophobic compounds for a broader range of applications. For example, the methods of the invention can be used to prepare microbial oils with customized fatty acid profiles, medically active compounds such as ergosterol, ergosterol derivatives, tocochromanols (such as α-tocopherol and α-tocotrienol), mono-, sesqui-, and diterpenoids, squalene, carotenoids, triterpenes, pheophytins, and vitamins. Furthermore, the methods of the present invention allow for the preparation of genetically modified GC-rich microorganisms that have high resistance to toxic compounds and / or the ability to efficiently utilize a wide range of complex feedstocks and waste streams.

[0047] In one embodiment, the term "genetically modifying" as used herein relates to modifying the genetic information of an organism, preferably a GC-rich microorganism, such as an oleaginous microorganism. For example, such genetic modification may include gene knock-in, gene knock-out, and / or point mutation. By genetically modifying a GC-rich microorganism, such as an oleaginous microorganism, a characteristic of interest can be modified, for example, the production of acetyl-CoA-based hydrophobic compounds can be increased and / or a specific fatty acid profile of the microbial lipids produced by the GC-rich microorganism can be achieved. In one embodiment, the method of genetically modifying the present invention is advantageous because it allows a GC-rich microorganism, preferably an oleaginous microorganism with a high GC content, to be modified to have a characteristic of interest, such as increased production of acetyl-CoA-based hydrophobic compounds and / or the production of a specific fatty acid profile. In one embodiment, the method of genetically modifying a GC-rich microorganism of the present invention is a method of increasing the production of acetyl-CoA-based hydrophobic compounds in the microorganism and / or modifying or regulating the acetyl-CoA pool of the microorganism. For example, one or more genes involved in acetyl-CoA synthesis can be modified to alter acetyl-CoA levels, which is the precursor of many acetyl-CoA-derived products, such as acetyl-CoA-based hydrophobic compounds. Additionally, the production of acetyl-CoA based compounds can be regulated and altered by modifying one or more genes involved in the metabolism of these compounds.

[0048] In one embodiment, the method for genetically modifying a GC-rich microorganism allows for gene modifications, such as increased or decreased expression of a modified gene, gene deletion, gene insertion, frameshift, point mutation, and / or gene replacement.

[0049] The term "GC-rich microorganism" as used herein relates to a microorganism having a guanine-cytosine (GC) content of at least 50%, preferably at least 55%, more preferably at least 58%, even more preferably at least 60%, for example about 61%. GC content (or guanine-cytosine content) is the percentage of nitrogenous bases in a molecule, e.g. a genome, of DNA or RNA, that are either guanine (G) or cytosine (C). In one embodiment, GC content refers to the proportion of G and C bases out of the total nucleic acid bases, which also include adenine and thymine for DNA and adenine and uracil for RNA. In one embodiment, the term "GC content" as used herein relates to the GC content of the genome of a GC-rich microorganism and / or the GC content of all nucleic acids contained in said GC-rich microorganism. In one embodiment, the GC content is

[0050]

number

[0051] In one embodiment, the GC content is measured by sequencing the genome of the microorganism and / or by the total nucleic acid content of the microorganism. In one embodiment, the GC-rich microorganism comprises a cell wall. In a preferred embodiment, the GC-rich microorganism is an oleaginous microorganism, such as an oleaginous yeast. In one embodiment, the GC-rich microorganism is a microorganism having a genome with a GC content of at least 50%, preferably at least 55%, more preferably at least 58%, even more preferably at least 60%, such as about 61%. In one embodiment, the GC-rich microorganism is an oleaginous microorganism having a genome with a GC content of at least 50%, preferably at least 55%, more preferably at least 58%, even more preferably at least 60%, such as about 61%.

[0052] In a preferred embodiment, the GC-rich microorganism is an oleaginous microorganism, i.e., a GC-rich oleaginous microorganism, such as an oleaginous yeast, such as Cutaneotrichosporon oleaginosus. Oleaginous microorganisms are known to those skilled in the art. Oleaginous microorganisms, such as, for example, oleaginous yeast, oleaginous fungi, oleaginous bacteria, and oleaginous microalgae, are typically microorganisms capable of producing microbial lipids, for example, microorganisms that accumulate more than 20% w / w lipids on a cell dry weight basis. In one embodiment, the GC-rich microorganism is selected from an oleaginous microorganism, preferably selected from an oleaginous yeast, oleaginous fungi, oleaginous bacteria, and oleaginous microalgae. In one embodiment, the oleaginous yeast is selected from Rhodosporidium spp., Yarrowia spp., Rhodotorula spp., Candida spp., Lipomyces spp., Cutaneotrichosporon spp., Trichosporon spp., preferably Cutaneotrichosporon spp., more preferably Cutaneotrichosporon oleaginosus. When the oleaginous microorganism is a yeast, it is an oleaginous yeast, preferably the oleaginous yeast is selected from Rhodosporidium spp., Yarrowia spp., Rhodotorula spp., Candida spp., Lipomyces spp., Cutaneotrichosporon spp., Trichosporon spp., preferably Cutaneotrichosporon spp., more preferably Cutaneotrichosporon oleaginosus. When the oleaginous microorganism is a fungus, it is an oleaginous fungus, preferably the oleaginous fungus is selected from the genera Cunninghamella, Aspergillus, Mortierella, and Humicola. When the oleaginous microorganism is a bacterium, it is an oleaginous bacterium, preferably the oleaginous bacterium is selected from the genera Rhodococcus, Acinetobacter, and Bacillus. Preferred oleaginous bacteria species are Rhodococcus opacus, Acinetobacter calcoaceticus, and Bacillus alcalophilus.When the oleaginous microorganism is a microalgae, it is an oleaginous microalgae, preferably the microalgae is selected from the genera Chlorella, Pseudochlorococcum, Nannochloris, Nannochloropsis, Isochrysis, Tribonema such as Tribonema minus, Dunaliella, Ankistrodesmus, Botryococcus such as Botryococcus braunii, Pavlova, Scenedesmus, Skeletonema, and Nitzschia.

[0053] In one embodiment, the GC-rich microorganism is an oleaginous yeast selected from Rhodosporidium spp., Yarrowia spp., Rhodotorula spp., Candida spp., Lipomyces spp., Cutaneotrichosporon spp., Trichosporon spp., preferably Cutaneotrichosporon spp., more preferably Cutaneotrichosporon oleaginosus. A particularly preferred oleaginous yeast species is Cutaneotrichosporon oleaginosus. In one embodiment, the Cutaneotrichosporon oleaginosus is Cutaneotrichosporon oleaginosus ATCC20509. In one embodiment, the oleaginous microorganism has a guanine-cytosine (GC) content of at least 50%, preferably at least 55%, more preferably at least 58%, even more preferably at least 60%, for example about 61%.

[0054] The term "acetyl-CoA-based hydrophobic compound" as used herein relates to a hydrophobic compound that contains acetyl-CoA and / or is produced with acetyl-CoA and / or with acetyl-CoA metabolism. In one embodiment, the acetyl-CoA-based compound, preferably the acetyl-CoA-based hydrophobic compound, is any compound selected from saturated short-chain fatty acids, saturated medium-chain fatty acids, saturated long-chain fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, functionalized fatty acids, ergosterol, ergosterol derivatives, terpenes, alkaloids, acetyl-CoA-based synthetic compounds, tocochromanols, such as α-tocopherol and α-tocotrienol, monoterpenoids, sesquiterpenoids, diterpenoids, squalene, carotenoids, triterpenes, pheophytins, vitamins, citric acid, volatile fatty acids, oxalic acid, lactic acid, malic acid, and exopolysaccharides. In one embodiment, the term "short-chain fatty acid" relates to a fatty acid having 5 or less carbon atoms. In one embodiment, the term "medium chain fatty acid" refers to a fatty acid having 6-12 carbon atoms. In one embodiment, the term "long chain fatty acid" refers to a fatty acid having 12 or more carbon atoms. In one embodiment, the target compound is an acetyl-CoA based hydrophobic compound and / or a fatty acid.

[0055] In one embodiment, the term "spheroplasting" as used herein relates to the partial or complete removal of the cell wall of a microbial cell, preferably a GC-rich microorganism, such as an oleaginous microorganism. For example, a spheroplast is a microbial cell that is partially or completely devoid of a cell wall. In one embodiment, spheroplasting a GC-rich microorganism removes sugars, lipids, and proteins from the cell wall of the GC-rich microorganism. In one embodiment, removing and / or dissolving the cell wall of a GC-rich microorganism, such as an oleaginous microorganism, renders the microorganism accessible to transfection. Typically, when the cell wall of a microorganism is digested to obtain a spheroplast, the tension of the membrane causes the cell to assume a spherical shape. Advantageously, spheroplasting the GC-rich microorganism, such as an oleaginous microorganism, renders the GC-rich microorganism accessible to transfection. The inventors have surprisingly found that after pretreating GC-rich microorganisms, such as oleaginous microorganisms, to provide spheroplasts of the GC-rich microorganisms, the GC-rich microorganisms can be efficiently transfected, for example by transfecting with an RNA-guided endonuclease in the form of mRNA. In one embodiment, the GC-rich microorganism is a microorganism that comprises a cell wall. In one embodiment, the GC-rich microorganism has a cell wall. In one embodiment, when the GC-rich microorganism comprises a cell wall, the method for genetically modifying the GC-rich microorganism comprises pretreating the microorganism, preferably comprising spheroplasting the microorganism.

[0056] In one embodiment, the term "pretreatment" as used herein relates to treating a GC-rich microorganism, preferably an oleaginous microorganism, prior to transfection of said GC-rich microorganism, preferably to increase the susceptibility of said GC-rich microorganism to transfection. In one embodiment, such pretreatment in step ii) increases the transfection efficiency of the transfection of such a pretreated GC-rich microorganism, e.g., such a pretreated oleaginous microorganism, compared to the transfection of a GC-rich microorganism without such pretreatment. In one embodiment, said pretreatment in step ii) comprises an enzymatic pretreatment, a chemical pretreatment, and / or another spheroplasting procedure. In one embodiment, said pretreatment is an enzymatic pretreatment comprising contacting said GC-rich microorganism, preferably an oleaginous microorganism, with said at least one enzyme. Preferably, such at least one enzyme, e.g., an enzyme mix, is obtained from another microorganism, preferably a filamentous fungus, selected from filamentous fungi and bacteria. In one embodiment, the fungus is selected from the genera Trichoderma, Aspergillus, Penicillium, Aureobasillium, and Fusarium. In a more preferred embodiment, the filamentous fungus is Trichoderma reesei, since it has been shown to produce a particularly efficient enzyme mix that allows the pretreatment of the cell walls of GC-rich microorganisms, such as oleaginous microorganisms. In one embodiment, the at least one enzyme is obtained from a fungus, preferably a filamentous fungus, cultured in the presence of an induction system, preferably the induction system being a component of the GC-rich microorganism, preferably one or several cell wall components of the GC-rich microorganism, such that an enzyme preparation is obtained that allows the pretreatment of the cell walls of the GC-rich microorganism to improve the permeability of the cell walls for transfection. Preferably, such an enzyme preparation is produced by culturing the filamentous fungus in the presence of cell wall fragments of the GC-rich microorganism.Without wishing to be bound by any theory, the inventors believe that exposing a filamentous fungus, such as T. reesei, to the presence of such cell wall components of the GC-rich microorganism allows such filamentous fungus to produce the enzyme(s) suitable for completing the lysis of the cell wall of the GC-rich microorganism. In a particularly preferred embodiment, a filamentous fungus of the genus Trichoderma, such as T. reesei, is used, and in a particularly preferred embodiment, a mutant of T. reesei, such as the mutant with ATCC deposit number(s) 56765 and 13631, is used. Once the filamentous fungus is cultivated, the resulting culture may be subjected to further processing such as concentration, the biomass of the filamentous fungus itself may be removed, and the resulting supernatant may be used in such form, or may be lyophilized and kept for storage, and then reconstituted with a suitable aqueous solution.

[0057] In one embodiment, the pretreatment in step ii) comprises an enzymatic pretreatment, preferably with at least one enzyme selected from glycosyl hydrolase, cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, glucosidase, arabinase, amylase, fructanase, laminase, hydrolase, protease, or any combination thereof. In one embodiment, the pretreatment in step ii) comprises contacting the GC-rich microorganism with at least one enzyme, preferably with an enzyme mix, such as an enzyme mix produced by an enzyme-producing microorganism selected from filamentous fungi and bacteria. In one embodiment, the pretreatment in step ii) comprises treating the microorganism with a hydrolase alone or a combination of a hydrolase and a protease / hydrolase followed by a protease. Optionally, the hydrolase is selected from a hydrolase produced by a fungus, preferably a filamentous fungus, more preferably a fungus selected from Trichoderma spp., Aspergillus spp., Penicillium spp., Aureobacillium spp., and Fusarium spp., even more preferably Trichoderma reesei. Optionally, the protease is selected from a protease produced by Aspergillus spp., Streptomyces spp., or Bacillus spp.

[0058] In one embodiment, the at least one enzyme, e.g. enzyme mix and / or hydrolase, used to pretreat the microorganism is obtained from a fungus cultured in the presence of an induction system, preferably the induction system being a component of the GC-rich microorganism, more preferably one or several cell wall components of the GC-rich microorganism, so as to obtain an enzyme mix and / or hydrolase preparation that allows lysis of the cell wall of the GC-rich microorganism. In one embodiment, the at least one enzyme, e.g. enzyme mix and / or hydrolase, preferably at least one enzyme obtained from the fungus, is prepared separately (from the implementation of step ii) of the present invention) and used in step ii) either as a liquid preparation obtained directly from culturing the fungus or as a lyophilized preparation that is subsequently reconstituted in a solution and used in step ii). In one embodiment, the at least one enzyme, preferably an enzyme mix, has one or several enzyme activities, for example, but not limited to, selected from glycosyl hydrolase, cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, glucosidase, arabinase, amylase, fructanase, laminase, hydrolase, protease, or any combination thereof.

[0059] The term "at least one enzyme" as used herein refers to one or more enzymes, preferably an enzyme mix. In one embodiment, the at least one enzyme is capable of spheroplasting the GC-rich microorganism. In one embodiment, the at least one enzyme, preferably the enzyme mix, comprises one or more enzymes selected from glycosyl hydrolases, cellulases, hemicellulases, mannanases, xyloglucanases, xylanases, glucanases, glucosidases, arabinases, amylases, fructanases, laminases, hydrolases, proteases, and any combination thereof. In one embodiment, the at least one enzyme is an enzyme mix. In one embodiment, the enzyme mix comprises hydrolases and proteases, and optionally further comprises additional enzymes. In one embodiment, the enzyme mix comprises at least 2 or 3 enzymes, preferably at least 5, more preferably at least 6, and even more preferably at least 7 enzymes. In one embodiment, the at least one enzyme is adapted to the GC-rich microorganism, i.e., adapted by contacting at least one component of the GC-rich microorganism with the filamentous fungus as an induction system, and the filamentous fungus produces an enzyme that acts specifically on the GC-rich microorganism.

[0060] In one embodiment, the term "enzymatic pretreatment" as used herein relates to treating a GC-rich microorganism, e.g., an oleaginous microorganism, with at least one enzyme, preferably an enzyme mix. In one embodiment, the enzymatic pretreatment comprises contacting the GC-rich microorganism with said at least one enzyme, preferably an enzyme mix. In one embodiment, such enzymatic pretreatment results in the GC-rich microorganism being present in the form of a spheroplast. In one embodiment, such enzymatic pretreatment allows for efficient transfection of said GC-rich microorganism, e.g., efficient transfection of an oleaginous microorganism. In one embodiment, the enzymatic pretreatment comprises contacting the GC-rich microorganism with at least one enzyme selected from glycosyl hydrolase, cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, glucosidase, arabinase, amylase, fructanase, laminase, hydrolase, protease, or any combination thereof, e.g., hydrolase alone or a combination of hydrolase and protease / hydrolase followed by protease. Optionally, the hydrolase is selected from a hydrolase produced by a fungus, preferably a filamentous fungus, more preferably a fungus selected from Trichoderma spp., Aspergillus spp., Penicillium spp., Aureobacillium spp., and Fusarium spp., even more preferably Trichoderma reesei, and / or the protease is selected from a protease produced by Aspergillus spp., Streptomyces spp., or Bacillus spp.

[0061] In one embodiment, the pretreatment in step ii) is carried out at a temperature in the range of 16°C to 60°C. In one embodiment, the pretreatment in step ii) is carried out at a pH in the range of pH3 to pH11. In one embodiment, the pretreatment in step ii) is carried out under stirring, preferably under stirring at a rotation speed in the range of 0.5 to 120 rpm. In one embodiment, the pretreatment in step ii) comprises treating the GC-rich microorganism with at least one enzyme, preferably an enzyme mix, for 10 minutes to 72 hours at a temperature in the range of 16°C to 60°C, in a medium comprising a buffer system and / or at a pH in the range of pH3 to pH11. In one embodiment, the buffer system comprises any of water, tripotassium phosphate, citrate buffer, MES buffer, HEPES buffer, and any combination thereof.

[0062] In one embodiment, the at least one enzyme is produced by a microorganism selected from filamentous fungi and bacteria, preferably filamentous fungi, In one embodiment, the microorganism selected from filamentous fungi and bacteria is capable of producing at least one enzyme, preferably an enzyme mix, adapted to GC-rich microorganisms, e.g. adapted to digest the cell walls of GC-rich microorganisms. In one embodiment, the microorganism producing at least one enzyme is selected from filamentous fungi and bacteria, and the filamentous fungi are selected from Ceratocystis species, such as Ceratocystis fimbriata, Ceratocystis moniliformis, and Ceratocystis paradoxa, preferably Ceratocystis paradoxa; Trichoderma species, such as Trichoderma reesei and Trichoderma harzianum, preferably Trichoderma reesei; Aspergillus species, such as Aspergillus oryzae, Aspergillus tubingensis, Aspergillus spp. tubingensis, and Aspergillus niger; Neurospora species, for example Neurospora intermedia; Monascus species, for example Monascus purpureus; Rhizopus species, for example Rhizopus oryzae; Fusarium species, for example Fusarium venenatum; Thermomyces species; Penicillium species; Aureobasillium species; Ischnoderma species, for example Ischnoderma benzoinum; Polyporus species, for example Polyporus duras. durus);Pycnoporus species, e.g., Pycnoporus cinnabarinus; Phanerochaete species, e.g., Phanerochaete chrysosporium; chrysosporium); and Xylaria species; preferably selected from Ceratocystis species, Trichoderma species, Aspergillus species, and Fusarium species; more preferably selected from Trichoderma species, such as Trichoderma reesei; the bacterium is selected from Clostridium species, Halobacillus species, Halomonas species, Rhodothermus species, Streptomyces species, and Bacillus species. In a preferred embodiment, the microorganism producing the at least one enzyme is selected from Trichoderma species, preferably Trichoderma reesei;

[0063] In one embodiment, to obtain the at least one enzyme for pretreating the GC-rich microorganism, the enzyme-producing microorganism selected from filamentous fungi and bacteria, for example Trichoderma reesei, is cultured with the GC-rich microorganism or a component thereof as an induction system. In one embodiment, the cultivation of the enzyme-producing microorganism with the induction system is carried out at a temperature in the range of 16°C to 60°C, preferably in the range of 20°C to 50°C, more preferably in the range of 25°C to 40°C, for example at about 30°C. In one embodiment, the cultivation of the enzyme-producing microorganism is carried out at a pH in the range of pH3 to pH11. In one embodiment, the cultivation of the enzyme-producing microorganism is carried out for 1 minute to 12 days, preferably for 10 minutes to 72 hours. In one embodiment, the cultivation of the enzyme-producing microorganism is carried out in a medium comprising water, tripotassium phosphate, cited buffer, and / or MES buffer. In one embodiment, the cultivation of the enzyme-producing microorganism is carried out at pO2>20%. In one embodiment, culturing the enzyme-producing microorganism with the GC-rich microorganism comprises culturing the enzyme-producing microorganism selected from a filamentous fungus and a bacterium with the GC-rich microorganism, thereby causing the enzyme-producing microorganism to produce at least one enzyme.

[0064] In one embodiment, when an enzyme-producing microorganism selected from filamentous fungi and bacteria, such as Trichoderma reesei, is cultured with the GC-rich microorganism or a component thereof to obtain the at least one enzyme, the GC-rich microorganism, such as an oleaginous microorganism, is nutrient-limited or not nutrient-limited. For example, the GC-rich microorganism, such as an oleaginous microorganism, is cultured under non-limiting or oil-forming conditions. In one embodiment, before culturing the enzyme-producing microorganism with the GC-rich microorganism, the GC-rich microorganism is subjected to culture under non-limiting or oil-forming conditions. By culturing the enzyme-producing microorganism with a GC-rich microorganism, such as an oleaginous microorganism, exposed to non-limiting or oil-forming conditions, at least one enzyme adapted to the cell wall of the GC-rich microorganism under non-limiting conditions or at least one enzyme adapted to the cell wall of the GC-rich microorganism under oil-forming conditions, respectively, is produced.

[0065] In one embodiment, the at least one enzyme produced by the enzyme-producing microorganism selected from filamentous fungi and bacteria is suitable for hydrolyzing GC-rich microorganisms as carbohydrate sources.In one embodiment, the at least one enzyme produced comprises glycosyl hydrolase, cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, glucosidase, arabinase, amylase, fructanase, laminase, hydrolase, protease, or any combination thereof, preferably comprising a combination of cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, and glucosidase.In one embodiment, the pretreatment in step ii) comprises pretreating the GC-rich microorganism under limiting or non-limiting conditions.In one embodiment, when the at least one enzyme is produced using nutrient-limited GC-rich microorganism, the pretreatment in step ii) comprises pretreating the GC-rich microorganism under limiting conditions. In one embodiment, when at least one enzyme is produced using a GC-rich microorganism that is not nutrient-limited, the pretreatment in step ii) comprises pretreating the GC-rich microorganism under non-limiting conditions. In one embodiment, under non-limiting conditions, the microorganism is provided with all necessary nutrients. In one embodiment, under oil-forming conditions, the culture medium is nitrogen, sulfur, and / or phosphorus limited.

[0066] In one embodiment, the method comprises obtaining the at least one enzyme and pretreating the GC-rich microorganism with the at least one enzyme to obtain spheroplasts of the GC-rich microorganism. In one embodiment, the pretreatment comprises contacting the GC-rich microorganism with the at least one enzyme, for example in the form of a liquid enzyme preparation, preferably directly obtained from culturing the enzyme-producing microorganism with the induction system, or in the form of a lyophilized enzyme preparation, optionally reconstituted in a solution. In one embodiment, obtaining the at least one enzyme comprises obtaining the at least one enzyme in liquid form or in the form of a lyophilized enzyme preparation. In one embodiment, such a lyophilized enzyme preparation of the at least one enzyme is dissolved before pretreating the second substrate with the at least one enzyme.

[0067] In one embodiment, the enzymatic pretreatment is carried out using at least one enzyme selected from glycosyl hydrolase, cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, glucosidase, arabinase, amylase, fructanase, laminase, hydrolase, protease, or any combination thereof, such as a combination of cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, and glucosidase. In one embodiment, the enzymatic pretreatment comprises contacting the GC-rich microorganism with the at least one enzyme at a temperature in the range of 16°C to 60°C, preferably in the range of 20°C to 50°C, more preferably in the range of 25°C to 40°C, for example, for 10 minutes to 72 hours. In one embodiment, the enzymatic pretreatment comprises contacting the GC-rich microorganism with the at least one enzyme in a medium containing a buffer having a pH in the range of preferably pH 3 to pH 11. In one embodiment, the buffer comprises or consists of potassium phosphate buffer, citrate buffer and / or MES buffer.In one embodiment, the enzymatic pretreatment comprises contacting the GC-rich microorganism with at least one enzyme selected from, for example, glycosyl hydrolase, cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, glucosidase, arabinase, amylase, fructanase, laminase, hydrolase, protease, or any combination thereof, preferably a combination of cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, and glucosidase, for example, at a temperature in the range of 16°C to 60°C, preferably in the range of 20°C to 50°C, more preferably in the range of 25°C to 40°C, for 10 minutes to 72 hours.

[0068] In one embodiment, the term "chemical pretreatment" as used herein relates to treating a GC-rich microorganism, e.g., an oleaginous microorganism, with at least one chemical. In one embodiment, such chemical pretreatment renders the GC-rich microorganism susceptible to transfection, e.g., by partially or completely removing the cell wall of the GC-rich microorganism or by permeabilizing the cell wall to facilitate the passage of nucleic acids and proteins. For example, complete or partial removal of the cell wall of the GC-rich microorganism using a chemical such as lithium acetate increases the permeability of the GC-rich microorganism, facilitating the transfer of nucleic acids, e.g., DNA or RNA, and proteins into the GC-rich microorganism. In one embodiment, the chemical pretreatment is a pretreatment with lithium acetate, CaCl2, 2-mercaptoethanol, EDTA, dithiothreitol (DTT), DMSO, ethanol, or any combination thereof. In one embodiment, such chemical pretreatment, e.g., using lithium acetate, is carried out at a temperature ranging from 16°C to 60°C, preferably from 25°C to 50°C. In one embodiment, such chemical pretreatment, e.g., using lithium acetate, has a duration of 30 seconds to 24 hours.

[0069] The term "another spheroplasting procedure" as used herein refers to a method for spheroplasting a GC-rich microorganism, such as an oleaginous microorganism, other than the enzymatic or chemical pretreatment described above, such as a mechanical pretreatment.

[0070] The terms "transform" and "transformation" as used herein refer to the genetic change of a cell that occurs by the direct uptake and incorporation of foreign genetic material from the surroundings through the cell membrane(s), e.g., the introduction of nucleic acids, such as DNA and RNA, into the cell. Such introduction of foreign nucleic acids using various chemical, biological, or physical methods can be used to change the properties of the cell, e.g., altering the gene function and protein expression of the cell. In one embodiment, the term "transformation" refers to any artificial introduction of foreign nucleic acid into a cell, e.g., any form of gene transfer, such as transfection or transduction. In a preferred embodiment, transformation of the GC-rich microorganism, e.g., oleaginous microorganism, comprises or consists of transfecting the GC-rich microorganism. In one embodiment, the terms "transform" and "transfection" are used interchangeably. In one embodiment, the terms "transform" and "transfect" are used interchangeably. Transfection is the process of deliberately introducing a nucleic acid into a cell, such as a cell of a GC-rich microorganism. The two main objectives of transfection are to produce recombinant proteins or to specifically enhance or inhibit gene expression in transfected cells, e.g., to specifically enhance the production of acetyl-CoA-based hydrophobic compounds or specific fatty acids in GC-rich microorganisms such as oleaginous microorganisms.

[0071] In one embodiment, the transformation in step iii) comprises transferring an RNA-guided endonuclease, at least one guide RNA, and optionally a donor DNA into the GC-rich microorganism. In a preferred embodiment, the transformation in step iii) comprises transferring an RNA-guided endonuclease, more than one, preferably at least two or three, guide RNAs, and optionally a donor DNA into the GC-rich microorganism. When referring to "more than one" guide RNA, such as "at least two or three" guide RNAs, it means more than one type of guide RNA and at least two or three types of guide RNAs. The "type" of guide RNA refers to a guide RNA having a specific sequence. That is, when there are "at least two" guide RNAs, such "at least two" guide RNAs include a first guide RNA and a second guide RNA, and the sequence of the first guide RNA is different from the sequence of the second guide RNA. In one embodiment, the first and second guide RNAs target opposite DNA strands on the genome of the GC-rich microorganism. The inventors have surprisingly found that the use of more than one guide RNA, for example at least two guide RNAs, greatly increases the specificity and efficiency of transformation. The inventors have found that the use of more than one guide RNA is particularly useful in the transformation of microorganisms with a high guanine-cytosine (GC) content of at least 50%, preferably at least 55%, more preferably at least 58%, even more preferably at least 60%, for example about 61%, since the use of more than one guide RNA reduces off-target effects. Transformation of GC-rich microorganisms, for example oleaginous microorganisms with a GC content of at least 50% or 60%, is problematic, since primers with high melting temperatures are usually required to perform PCR. GC base pairs have three hydrogen bonds, while AT base pairs have two. Thus, double-stranded DNA with a high number of GC base pairs binds more tightly to each other, is more stable, and has a higher melting temperature.Furthermore, primers used for GC-rich microorganisms, such as oleaginous microorganisms with at least 50% or 60% GC content, typically form secondary structures, which may result in low or no product yield. Furthermore, repetitive sequences in GC-rich microorganisms, such as oleaginous microorganisms with at least 50% or 60% GC content, result in off-target effects. The inventors have surprisingly found that such obstacles can be overcome by using more than one guide RNA when transforming a GC-rich microorganism, such as an oleaginous microorganism, for example in step iii) of the method of the present invention. In a preferred embodiment, the method of the present invention comprises transforming the GC-rich microorganism with two or more guide RNAs, such as two or more sgRNAs with different sequences. The use of two gRNAs further increases the fidelity of specifically targeting the region of interest.

[0072] In one embodiment, the transformation in step iii) comprises applying the RNA-guided endonuclease to the GC-rich microorganism in the form of an RNA-guided endonuclease protein, in the form of a DNA encoding the RNA-guided endonuclease, or in the form of an mRNA encoding the RNA-guided endonuclease, preferably in the form of an mRNA encoding the RNA-guided endonuclease. In one embodiment, the transformation in step iii) comprises applying the RNA-guided endonuclease and the at least one guide RNA, preferably together, in the form of a ribonucleoprotein complex. Advantageously, by applying the RNA-guided endonuclease in the form of a ribonucleoprotein complex, off-target activity can be reduced. In one embodiment, the transformation in step iii) comprises applying the RNA-guided endonuclease in the form of an mRNA encoding the RNA-guided endonuclease, and the at least one guide RNA comprises an sgRNA. In one embodiment, when it is said that at least one guide RNA "comprises" an sgRNA, the at least one guide RNA comprises or consists of an sgRNA. In one embodiment, the sgRNA comprises or consists of the crRNA and the tracrRNA. In a preferred embodiment, the transformation in step iii) comprises transforming the GC-rich microorganism with an RNA-guided endonuclease in the form of an mRNA encoding the RNA-guided endonuclease. In a preferred embodiment, the transformation in step iii) comprises transforming the GC-rich microorganism with more than one guide RNA, preferably at least two or three guide RNAs. In one embodiment, the at least one guide RNA comprises a first guide RNA and a second guide RNA, the sequence of the first guide RNA being different from the sequence of the second guide RNA. In one embodiment, the at least two or three guide RNAs comprise a first guide RNA and a second guide RNA, and in the case of the at least three guide RNAs, a third guide RNA, the sequence of the first guide RNA being different from the sequence of the second guide RNA and, if present, also different from the sequence of the third guide RNA.

[0073] In one embodiment, the transformation in step iii) is carried out using electroporation; PEG-based or other nanoscale carrier-based transformation; biological ballistic flight; glass bead transformation; vesicle-mediated delivery; viral transfection systems, such as lentivirus (LV), adenovirus (AdV), or adeno-associated virus (AAV); liposomal delivery, such as lipofection; chemical transfection techniques; or combinations thereof. In a preferred embodiment, the transformation in step iii) is carried out using electroporation and / or PEG-based transformation, preferably using electroporation. In one embodiment, the transformation in step iii) comprises targeted genetic modification of the chromosome or episome of the GC-rich microorganism, preferably GC-rich oleaginous microorganism. In one embodiment, the PEG-based transformation comprises PEG, which promotes the association of nucleic acid (the nucleic acid to be transferred to the cell) with the surface of the cell. Optionally, the PEG-based transformation further comprises lithium ion and heat shock, which promote the passage of DNA into the cell. In one embodiment, the nanoscale carrier-based transformation is polymer-based transformation and / or nanoparticle-based transformation, including carriers such as carrier DNA, polyDMAEMA carrier, and BG2 carrier. In one embodiment, when the transformation in step iii) comprises applying the RNA-guided endonuclease and the at least one guide RNA in the form of a ribonucleoprotein complex, the ribonucleoprotein complex is applied using vesicle-mediated delivery. In one embodiment, when "applying" in the context of transformation, such term is meant to refer to the introduction and / or administration of compounds such as the RNA-guided endonuclease and at least one guide RNA.

[0074] The term "RNA-guided endonuclease" as used herein relates to any RNA-guided endonuclease known to those skilled in the art. In one embodiment, the RNA-guided endonuclease is a CRISPR-associated (Cas) endonuclease, preferably selected from Cas9, Cas1, Cas2, Cas4, Cas3, Cas10, Cas12, Cas13, Csm, scf1, or variants thereof, such as Cas12a, dCas9, D10A CAS9 nickase, or H840A CAS9 nickase. A "variant" of an RNA-guided endonuclease relates to an RNA-guided endonuclease that is artificially modified by mutations, such as point mutations, to include desired features, such as a modified cleavage site that only cuts single strands and not double strands, or that naturally includes modifications due to its origin from a different organism, such as an analog, homolog, and ortholog of an endonuclease. For example, the variant of Cas9 is dCas9, D10A CAS9 nickase, or H840A CAS9 nickase, and the variant of Cas12 is Cas12a. In one embodiment, reference to an endonuclease, such as Cas9, refers to the endonuclease obtained from any microorganism, such as Cas9 isolated from Streptococcus pyogenes (SpCas9), Staphylococcus aureus (SaCas9), Streptococcus thermophilus (StCas9), Streptococcus canis (ScCas9), Neisseria meningitidis (NmCas9), Francisella novicida (FnCas9), or Campylobacter jejuni (CjCas9).In one embodiment, the RNA-guided endonuclease is used in the method of the invention, such as in step iii), in the form of an RNA-guided endonuclease protein, in the form of a DNA encoding said RNA-guided endonuclease, or in the form of an mRNA encoding said RNA-guided endonuclease, preferably in the form of an mRNA encoding said RNA-guided endonuclease. In one embodiment, said transformation in step iii) comprises applying said RNA-guided endonuclease in the form of an mRNA and said at least one guide RNA in the form of an sgRNA or in the form of a crRNA and a tracrRNA, preferably more than one guide RNA. The advantage of the method of the invention is that the RNA-guided endonuclease can be applied in the form of an mRNA and at least one guide RNA can be applied in the form of an sgRNA or in the form of a crRNA and a tracrRNA, providing it with a highly efficient method with a high transformation yield. Furthermore, by using more than one type of guide RNA, i.e., at least two guide RNAs with different sequences, for example, at least two sgRNAs with different sequences, the target specificity for microorganisms with repeat and GC-rich sequences in the genome is increased, so that a reduced frequency of off-target activity is observed. Thus, the advantage of the method for genetically modifying GC-rich microorganisms is that GC-rich microorganisms, such as oleaginous microorganisms with high GC content, can be efficiently transformed despite the abundance of repeat sequences. In one embodiment, the RNA-guided endonuclease is Cas9, for example, Cas9 comprising any of the sequences of SEQ ID NOs: 1 to 4.

[0075] The term "guide RNA" or "gRNA" as used herein relates to an RNA that functions as a guide for an RNA or DNA targeting enzyme, for example by forming a complex with an RNA-guided endonuclease, e.g., an sgRNA. In one embodiment, the at least one guide RNA comprises any of CRISPR RNA (crRNA), trans-activating CRISPR RNA (tracrRNA), and single guide RNA (sgRNA), preferably comprising a) sgRNA and / or b) crRNA and tracrRNA. In one embodiment, the at least one guide RNA comprises or consists of crRNA and tracrRNA, and / or comprises or consists of sgRNA. The term "at least one guide RNA" as used herein relates to one or more guide RNAs, preferably one or more types of guide RNA, e.g. guide RNAs with different sequences. In one embodiment, when the at least one guide RNA comprises two or more guide RNAs, the sequence of the second or further guide RNA is different from the sequence of the first guide RNA. Furthermore, the sequence of the further guide RNA is different from the sequences of the first and second guide RNAs. In one embodiment, the transformation in step iii) comprises transferring one or more, preferably several, molecules of each of the at least one guide RNA, e.g., each of the one or more guide RNAs, into the GC-rich microorganism. Advantageously, the use of at least two guide RNAs can further increase the efficiency of transformation of the method of the invention. In one embodiment, the at least one guide RNA comprises or consists of a CRISPR RNA (crRNA), a trans-activating CRISPR RNA (tracrRNA), and / or a single guide RNA (sgRNA). In a preferred embodiment, the at least one guide RNA comprises or consists of a sgRNA. In one embodiment, the sgRNA comprises a crRNA and a tracrRNA. In a preferred embodiment, the at least one guide RNA comprises a first guide RNA and a second guide RNA, and the sequence of the first guide RNA is different from the sequence of the second guide RNA.In one embodiment, the tracrRNA comprises or consists of a sequence having SEQ ID NO:5.

[0076] In one embodiment, the term "donor DNA" as used herein refers to any DNA of interest that is transferred to the GC-rich microorganism, preferably integrated into the genome of the GC-rich microorganism. In one embodiment, the donor DNA comprises a DNA repair template, a DNA encoding a selection marker, and / or a gene or sequence of interest, such as a gene involved in the production of a target compound, such as acetyl-CoA or an acetyl-CoA-based hydrophobic compound or a fatty acid. For example, when using a DNA repair template, specific mutations, such as point mutations and / or knockouts, can be integrated into the genome of the GC-rich microorganism. In one embodiment, the gene or sequence of interest encodes delta-9-desaturase, delta-12-desaturase, elongase, oleate hydratase, aldo-ketoreductase promoter, aldo-ketoreductase terminator, transcription elongation factor 2 promoter, TEF promoter, and / or TEF terminator. In one embodiment, the donor DNA is single-stranded or double-stranded. In one embodiment, the donor DNA comprises or consists of any of SEQ ID NOs: 6 to 13 and 27, and / or a sequence having at least 80%, preferably at least 90%, or at least 99% sequence identity to any of SEQ ID NOs: 6 to 13 and 27. In one embodiment, the donor DNA comprises DNA encoding genes involved in any of: i) cellular processes and signaling, e.g., involved in cell wall / membrane / envelope biosynthesis, cell motility, post-translational modification, protein turnover, chaperones, signal transduction mechanisms, intracellular transport, secretion, vesicle trafficking, defense mechanisms, extracellular structures, nuclear structure, and / or the cytoskeleton; ii) information storage and processing, e.g., involved in RNA processing and modification, chromatin structure and dynamics, translation, ribosome structure and biogenesis, transcription, and / or replication, recombination and repair; iii) metabolism, e.g., involved in energy production and conversion, cell cycle control, cell division, chromosome segregation, amino acid transport and metabolism, nucleotide transport and metabolism, carbohydrate transport and metabolism, coenzyme transport and metabolism, lipid transport and metabolism, inorganic ion transport and metabolism, and / or secondary metabolite biosynthesis, transport, and catabolism.

[0077] In one embodiment, the term "selecting transformed cells" as used herein refers to selecting GC-rich microorganisms that have been successfully transformed. If the GC-rich microorganism comprises the RNA-guided endonuclease, the at least one guide RNA, and optionally the donor DNA; preferably, if the GC-rich microorganism is genetically modified, for example, by DNA cleavage cleaved by the RNA-guided endonuclease and / or by integration of the donor DNA into the genome of the GC-rich microorganism, if present, the GC-rich microorganism is successfully transformed. In one embodiment, the selection of the transformed cells in step iv) comprises selecting the transformed GC-rich microorganism, for example a transformed oleaginous microorganism, using a selection marker and / or a selection agent, if present. In one embodiment, the method comprises the selection in step iv), and the selection in step iv) comprises exposing the microorganism to a selection agent that is selective for the selection marker, optionally the selection marker encoded by the donor DNA. In one embodiment, the selection comprises exposing the microorganism to a concentration of the selection agent in the range of 10% to 90%, preferably 10% to 70%, more preferably 10% to 60% of the minimum selectable concentration; and / or exposing the microorganism to a concentration of the selection agent in the range of 90% to 100%, preferably 99% to 100% of the minimum selectable concentration.

[0078] In one embodiment, the selection in step iv) if present is a two-step selection, in the first step, the GC-rich microorganism is exposed to a medium having a lower selection strength than the medium in the second step, and in the second step, the GC-rich microorganism is exposed to a medium having a higher selection strength than the medium in the first step. In one embodiment, the selection in step iv) if present is a two-step selection, in the first step, the GC-rich microorganism is exposed to an upper layer of agar having a lower selection strength than the lower layer of agar, and in the second step, the GC-rich microorganism is exposed to a lower layer of agar having a higher selection strength than the upper layer of agar. In a preferred embodiment, the upper layer of agar and the lower layer of agar are placed on top of each other on a plate. In one embodiment, the selection in step iv) comprises exposing the GC-rich microorganism, if present, to an upper layer of agar having a lower selection strength than the lower layer of agar, and if the GC-rich microorganism is successfully transformed, the GC-rich microorganism grows on the upper layer. In one embodiment, if the GC-rich microorganism growing on the upper layer grows further towards the lower layer, preferably towards and on the lower layer, the lower layer has a higher selection strength than the upper layer, and the GC-rich microorganism is a successfully transformed GC-rich microorganism.

[0079] In one embodiment, the selection intensity is related to the presence of a particular concentration of the selection agent, for example, a selection intensity of 60% is related to the presence of a selection agent at a concentration of 60% of the minimum selectable concentration of the selection agent or at a concentration of 60% of the minimum lethal concentration of the selection agent. In one embodiment, the selection comprises exposing the microorganism to a selection agent at a concentration ranging from 10% to 90%, preferably 10% to 70%, more preferably 10% to 60% of the minimum selectable concentration in a first medium, such as a liquid or solid medium, preferably an agar underlayer, and then exposing the microorganism to a selection agent at a concentration ranging from 90% to 100%, preferably 99% to 100%, in a second medium, such as a liquid or solid medium, preferably an agar underlayer. In one embodiment, the selection in step iv) comprises a two-step selection, i.e., the selection comprises exposing the microorganism to a first agar and a second agar. Such a two-stage selection involves selecting a first agar containing a lower concentration of the selective agent than a second agar, e.g., a low but selectable range (e.g., IC4 of a compound with toxic effects, e.g., 5-FOA). 50 The advantage of the present invention is that it allows the regeneration of microorganisms, preferably spheroplasts, in an environment with a temperature lower than that of the conventional method. Such regeneration of microorganisms is particularly advantageous for microorganisms that undergo transformation in a subsequent step, e.g., to obtain a gene editing / selectable marker gene of interest in a subsequent step. In one embodiment, the term "minimum selectable concentration" as used herein refers to a concentration of an agent, e.g., a selective agent, that allows visible or measurable selection. In one embodiment, the minimum selectable concentration refers to a minimum inhibitory concentration. In one embodiment, the minimum selectable concentration is determined by preparing plates with serial dilutions of the selective agent and observing the growth of the microorganism.

[0080] In one embodiment, the term "genetically modified GC-rich microorganism" as used herein relates to a transformed and / or transfected GC-rich microorganism, such as an oleaginous microorganism, preferably a GC-rich microorganism genetically modified using the method of the present invention. For example, the genetic material of the genetically modified GC-rich microorganism has been modified using genetic engineering. In one embodiment, the genetically modified GC-rich microorganism has a GC content of at least 50%, preferably at least 55%, more preferably at least 58%, even more preferably at least 60%, such as about 61%. In one embodiment, the genetically modified GC-rich microorganism is a genetically modified oleaginous microorganism, such as an oleaginous yeast, preferably selected from the species of the genus Cutaneotrichosporon, more preferably Cutaneotrichosporon oleaginosus. In one embodiment, the genetically modified GC-rich microorganism comprises an RNA-guided endonuclease, at least one guide RNA, and optionally a donor DNA. In one embodiment, the RNA-guided endonuclease and the at least one guide RNA are present in the cell in the form of a ribonucleoprotein complex. In one embodiment, the method further comprises the step of sequencing the genome of the genetically modified GC-rich microorganism obtained in step v).

[0081] In one embodiment, the term "genetically modified oleaginous microorganism" as used herein relates to a transformed and / or transfected oleaginous microorganism, preferably a genetically modified oleaginous microorganism using the methods of the present invention. For example, the genetic material of the genetically modified oleaginous microorganism has been altered using genetic engineering. In one embodiment, the genetically modified oleaginous microorganism is an oleaginous microorganism having a GC content of at least 50%, preferably at least 55%, more preferably at least 58%, even more preferably at least 60%. In one embodiment, the genetically modified oleaginous microorganism is a genetically modified oleaginous yeast, preferably selected from the species of the genus Cutaneotrichosporon, more preferably Cutaneotrichosporon oleaginosus. In one embodiment, the genetically modified oleaginous microorganism comprises an RNA-guided endonuclease, at least one guide RNA, and optionally donor DNA. In one embodiment, the RNA-guided endonuclease and the at least one guide RNA are present in the cell in the form of a ribonucleoprotein complex. In a preferred embodiment, the genetically modified GC-rich microorganism of the present invention has a GC content of at least 50%, preferably at least 55%, more preferably at least 58%, even more preferably at least 60%, for example about 61%.

[0082] The term "selection agent" as used herein relates to an agent that affects the ability of an organism to survive in a given environment, such as an antibiotic. For example, after successful transfection, the GC-rich microorganism may contain a selection marker, so that successfully transfected cells can be selected using a selection agent specific for said selection marker. The selection agent, such as an antibiotic, allows for specific selection of transfected cells that contain the respective antibiotic resistance gene. In one embodiment, the selection agent used in the method of the present invention is selected from 5-fluoroorotic acid, 5-fluoroanthranilic acid, hygromycin B, geneticin, phleomycin, and any combination thereof.

[0083] In one embodiment, the term "selection marker" as used herein relates to a nucleic acid sequence, e.g., a gene or a compound, contained in a cell that confers a trait suitable for selection. In one embodiment, the selection marker is a gene introduced into a cell that confers a trait suitable for artificial selection. Typically, the selection marker indicates the success of transformation, transfection, or any procedure intended to introduce foreign DNA into a cell. The selection marker may be an antibiotic resistance gene. For example, the selection marker may be a reporter gene and / or an antibiotic resistance gene to indicate the success of a transfection or other procedure intended to introduce foreign DNA into a cell. In a preferred embodiment, the selection marker is an antibiotic resistance gene. The GC-rich microorganism transformed in step iii) can grow on a medium containing a corresponding selection agent, e.g., the antibiotic, and the colonies that can grow have successfully taken up and expressed the introduced genetic material, e.g., the donor DNA. In one embodiment, the selection marker is a positive or negative selection marker. In one embodiment, the selection marker is encoded by the donor DNA. In one embodiment, the selection marker is selected from marker genes encoding essential enzymes for de novo nucleic acid and amino acid synthesis, marker genes involved in nutrient utilization such as acetamidase, and antibacterial resistance genes. In one embodiment, the selection marker is selected from URA5, URA3, HIS, LEU, TRP, MET, ADE, LYS, URA, ARG, ALA, sulfonylurea, sulfometuron, acetamide, formamide, hygromycin B resistance gene, KanMx resistance gene, zeocin resistance gene, nourseothricin, phleomycin, puromycin, aureobasidin, cycloheximide, and erythromycin. Furthermore, endogenous genes can also be used as selection markers, for example, a compound or drug may be toxic to untransformed GC-rich microorganisms due to the expression of a particular endogenous gene, but transformed cells survive due to their inability to form toxic compounds. Mutations that have occurred on these target genes can be selected by plating the transformants on a medium supplemented with the appropriate inhibitor.In one embodiment, the selectable marker is introduced into the cell, for example, via transformation as part of donor DNA, or is endogenous to the cell.

[0084] In one embodiment, the term "DNA repair template" as used herein refers to a DNA sequence of interest, e.g., DNA comprising a fragment of a region of genomic DNA centered on the location of a desired modification. For example, such a DNA repair template can be used to incorporate a genetic modification, such as a point mutation, into the genome of an organism. In one embodiment, the DNA repair template comprises a sequence of a genome of a GC-rich microorganism, such sequence comprising a desired mutation. In one embodiment, the term "gene or sequence of interest" as used herein refers to a nucleic acid of interest, e.g., a gene involved in the production of a target compound, such as an acetyl-CoA pool or an acetyl-CoA-based hydrophobic compound or fatty acid, a promoter or terminator involved in the production of the target compound, and / or a selection marker. In one embodiment, the gene or sequence of interest encodes a product of interest, such as a protein or enzyme. In one embodiment, the gene or sequence of interest encodes any gene involved in i) cellular processes and signal transduction, e.g., involved in cell wall / membrane / envelope biosynthesis, cell motility, post-translational modification, protein turnover, chaperones, signal transduction mechanisms, intracellular transport, secretion, vesicle trafficking, defense mechanisms, extracellular structures, nuclear structure, and / or cytoskeleton; ii) information storage and processing, e.g., involved in RNA processing and modification, chromatin structure and dynamics, translation, ribosome structure and biogenesis, transcription, and / or replication, recombination, and repair; iii) metabolism, e.g., involved in energy production and conversion, cell cycle control, cell division, chromosome segregation, amino acid transport and metabolism, nucleotide transport and metabolism, carbohydrate transport and metabolism, coenzyme transport and metabolism, lipid transport and metabolism, inorganic ion transport and metabolism, and / or secondary metabolite biosynthesis, transport, and catabolism. In one embodiment, the reference to "a gene involved in the production of the target compound and / or a specific fatty acid" refers to a gene or sequence of interest. In one embodiment, the gene or sequence of interest is selected from the group consisting of delta-9-desaturase; delta-12-desaturase; elongase; oleate hydratase; aldo-ketoreductase promoter; aldo-ketoreductase terminator; transcription elongation factor 2 promoter; TEF promoter; TEF terminator;1,2-α-mannosidase;1,4-α-glucan branching enzyme / starch branching enzyme II;15-hydroxyprostaglandin dehydrogenase and related dehydrogenases;17β-hydroxysteroid dehydrogenase;1-acyl-sn-glycerol-3-phosphate acyltransferase;2-enoyl-CoA hydratase / 3-hydroxyacyl-CoA dehydrogenase / peroxisomal 3-ketoacyl-CoA-thiolase;sterol-binding domain and related enzymes;2-oxoglutarate dehydrogenase;3-hydroxy-3- Methylglutaryl-CoA (HMG-CoA) reductase; 3-hydroxyacyl-CoA dehydrogenase; 3-ketosterol reductase; 3-methylcrotonyl-CoA carboxylase; propionyl-CoA carboxylase; α-chain / acetyl-CoA carboxylase; 3-methylcrotonyl-CoA carboxylase; acetyl-CoA carboxylase carboxyltransferase; 3-oxoacyl-CoA thiolase; 3-oxoacyl-(acyl-carrier-protein) synthase (I and II); 3-phosphoglyceryl 5'-AMP-activated protein kinase;5'-phosphoribosylglycine amidoformyltransferase;6-phosphogluconate dehydrogenase;6-phosphogluconolactonase;Acetyl-CoA acetyltransferase;Acetyl-CoA carboxylase;Acetylglucosaminyltransferase EXT1 / exostosin 1;Acid sphingomyelinase and PHM5 phosphate metabolism protein;Aconitase / homoaconitase (aconitase superfamily);Acyl carrier protein / NADH-ubiquitin nonoxidoreductase;acyl-CoA oxidase;acyl-CoA synthase;acyl-CoA thioesterase;acyl-CoA:diacylglycerol acyltransferase (DGAT);acyl-CoA binding protein;acylphosphatase;alcohol dehydrogenase;aldehyde dehydrogenase;alkaline ceramidase;α-amylase;α-1;4-N-acetylglucosaminyltransferase;α-amylase;α-mannosidase;amidase;aquaporin;ATP-binding protein;ATP-citrate lyase;β-1,6-N-acetylglucosaminyltransferase;β-fructofuranosidase (invertase);β-glucosidase;lactase phlorizin hydrolase;and related proteins;β-glucuronidase GUSB (glycosyl hydrolase superfamily 2);betaine aldehyde dehydrogenase;β-N-acetylhexosaminidase;bifunctional leukotriene A4 hydrolase / aminopeptidase LTA4H;branched-chain α-keto acid dehydrogenase complex;C-3 sterol dehydrogenase / 3-β-hydroxysteroid dehydrogenase and related dehydrogenases aldolase;C-4 sterol methyloxidase;carnitine O-acyltransferase;CDP-alcohol phosphatidyltransferase / phosphatidylglycerol-phosphate synthase;CDP-diacylglycerol synthase;ceramide glucosyltransferase;chitinase;cholesterol transport protein;choline transporter-like protein;cis-prenyltransferase;citrate synthase;class II aldolase / adducin N-terminal domain protein;cytochrome b5;cytochrome c;cytochrome c oxidase;cytochrome c1;cytochrome P450 CYP4 / CYP19 / CYP26 subfamily; delta 6-fatty acid desaturase / delta 8 sphingolipid desaturase; dihydrolipoamide acetyltransferase; dihydrolipoamide dehydrogenase; dihydrolipoamide succinyltransferase (2-oxoglutarate dehydrogenase; E2 subunit); dihydrolipoamide transacylase (α-ketoacid dehydrogenase E2 subunit); dihydroxyacetone kinase / glycerone kinase; dimeric dihydrodiol dehydrogenase dolochinase;dolichol kinase;dolichyl pyrophosphate phosphatase and related acid phosphatases;d-ribulose-5-phosphate 3-epimerase;dTDP-glucose 4-6-dehydratase / UDP-glucuronic acid decarboxylase;electron transfer flavoprotein ubiquinone oxidoreductase;electron transfer flavoprotein;enolase;enoyl-CoA hydratase;enoyl-CoA hydratase / isomerase;enoyl-CoA isomerase;exopolyphosphatase and related proteins;F0F1-type ATP synthase;F1-ATP synthase assembly protein;Fatty acid desaturase;Fatty acyl-CoA elongase / polyunsaturated fatty acid-specific elongase;Ferredoxin;Flavohem protein b5+b5R;Fructose-1,6-bisphosphate aldolase;Fructose-1,6-bisphosphatase;Fructose-6-phosphate 2-kinase / fructose-2,6-biphosphatase;Fumarate reductase;Fumarylacetoacetase;Galactose-1-phosphate uridylyltransferase;Galactosyl Transferase;γ-butyrobetaine;2-oxoglutarate dioxygenase;GDP-mannose pyrophosphorylase;Globins and related hemoproteins;Gluconate kinase;Gluconate transport inducer protein;Glucosamine-6-phosphate isomerase;Glucose-6-phosphate 1-dehydrogenase;Glucose-6-phosphate isomerase;Glucose-6-phosphate / phosphate and phosphoenolpyruvate / phosphate antiporters;Glucosidase I;Glucosidase II catalytic (α) subunit and related enzymes;Glycosyl hydrolase family Lee 31;Glyceraldehyde 3-phosphate dehydrogenase;Glycerol-3-phosphate dehydrogenase;Glycerol-3-phosphate dehydrogenase / dihydroxyacetone 3-phosphate reductase;Glycerophosphoryl diester phosphodiesterase;Glycogen phosphorylase;Glycogen synthase;Glycogen synthase kinase;Glycolate oxidase;Glycolipid 2-α-mannosyltransferase (α-1,2-mannosyltransferase);Glycolipid transfer protein;Glycosyl hydrolase;Glycosyltransferase hydroxylase;glyoxalase;glyoxylate / hydroxypyruvate reductase (D-isomer-specific 2-hydroxyacid dehydrogenase superfamily);hexokinase;holocytochrome c synthase / heme-lyase;hormone-sensitive lipase HSL;hydroxyacyl-CoA dehydrogenase / enoyl-CoA hydratase;hydroxymethylglutaryl-CoA lyase;hydroxymethylglutaryl-CoA synthase;inorganic pyrophosphatase;inositol monophosphatase;inositol phospholipid synthesis protein;Scs3p;Inositol polyphosphate multikinase;isoamylacetate hydrolyzing esterase;isocitrate lyase;isovaleryl-CoA dehydrogenase;kynurenine 3-monooxygenase;lecithin:cholesterol acyltransferase (LCAT) / acylceramide synthase;lipid exporter ABCA1 and related proteins;ABC superfamily;lipid phosphate phosphatase and related enzymes of the PAP2 family;lipoyltransferase;long-chain acyl-CoA synthetase (AMP formation);low-density lipoprotein B-like protein;lyso Phosphatidic acid acyltransferase endophilin / SH3GL;Lysophosphatidic acid acyltransferase LPAAT and related acyltransferases;Lysophospholipase;Malate synthase;Maltase glucoamylase and related hydrolases;Glycosyl hydrolase family 31;MAM33;Mitochondrial matrix glycoprotein;Mannose-6-phosphate isomerase;Mannosyltransferase;Medium-chain acyl-CoA dehydrogenase;Methylmalonate semialdehyde dehydrogenase;Mevalonate Pyrophosphate decarboxylase;Mitochondrial ADP / ATP carrier protein;Mitochondrial aspartate / glutamate carrier protein Aralar / Citrin;Mitochondrial carnitine-acylcarnitine carrier protein;Mitochondrial carrier protein;Mitochondrial F1F0-ATP synthase;Mitochondrial FAD carrier protein;Mitochondrial Fe-S cluster biogenesis protein ISA2;Mitochondrial oxaloacetate carrier protein;Mitochondrial oxodicarboxylate carrier protein;Mitochondrial oxoglutarate / malate carrier protein;Mitochondrial phosphate carrier protein;Mitochondrial protein Surfeit1 / SURF1 / SHY1;Mitochondrial solute carrier protein;Mitochondrial tricarboxylate / dicarboxylate carrier protein;Mitochondrial / plastidial β-ketoacyl-ACP reductase monocarboxylate transporter;Monooxygenase involved in coenzyme Q (ubiquinone) biosynthesis;Myo-inositol-1-phosphate synthase;N-acetylglucosamine kinase;N-acetylglucosamine-6-phosphate deacetylase;N-acetylglucosaminyltransferase complex;NAD-dependent malate dehydrogenase;NADH:flavin oxidoreductase / 12-oxophytodienoate reductase;NADH:ubiquinone oxidoreductase;NADH-cytochrome b-5 reductase;NADH-dehy; drogenase;NADP / FAD-dependent oxidoreductase;NADP+-dependent malic enzyme;NADP-dependent flavoprotein reductase;NADP-dependent isocitrate dehydrogenase;NADPH oxidase;neutral trehalase;N-myristoyltransferase;nucleotide-sugar transporter;oligoketide cyclase / lipid transfer protein;O-linked N-acetylglucosamine transferase (OGT);oxidosqualene-lanosterol cyclase;oxysterol-binding protein;palmitoyl protein thioesterase ase;peroxisomal 3-ketoacyl-CoA-thiolase P-44 / SCP2;peroxisomal long-chain acyl-CoA transporter;ABC superfamily;peroxisomal multifunctional beta-oxidation protein;peroxisomal phytanoyl-CoA hydroxylase;phosphate acyltransferase;phosphatidic acid-preferring phospholipase A1;phosphatidylglycerol phosphate synthase;phosphatidylinositol synthase;phosphatidylinositol transfer protein PDR16 and related proteins;phosphatidylinositol transfer protein S EC14 and related proteins;phosphatidylserine decarboxylase;phosphoglucomutase;phosphoglucomutase / phosphomannomutase;phosphoglycerate mutase;phosphoinositide phosphatase SAC1;phospholipase;phospholipase A2;phospholipase A2-activating protein;phospholipase D1;phospholipid methyltransferase;phosphomannomutase;phosphomevalonate kinase;phosphorylcholinetransferase / cholinephosphate cytidylyltransferase;phytoene / squalene synthase;pfkB family leucine carbohydrate kinase;2-oxoglutarate dehydrogenase;β-mannosidase;dehydrogenase;γ-butyrobetaine, 2-oxoglutarate dioxygenase;L-carnitine dehydratase / α-methylacyl-CoA racemase;lipase / calmodulin-binding heat shock protein;mitochondrial carrier protein;mitochondrial cholesterol transporter;mutarotase;oxidoreductase;phosphoglucosamine acetyltransferase;phosphoglycerate mutase;phospholipase;quinone oxidoreductase;Sugar kinases; sugar transporters; UDP-galactose transporters; pristanoyl-CoA / acyl-CoA oxidases; prohibitins and stomatins of the PID superfamily; proteins containing FAD-binding domains; purple acid phosphatase; cytochrome C oxidase assembly proteins; lipases, e.g., lipases essential for the degradation of autophagic bodies in the vacuole; NAD+-dependent epimerases; phosphoinositide phosphatases; trehalases; pyrophosphate-dependent phosphofructo-1-kinases; pyruvate carboxylases; pyruvate dehydrogenases drogenase E1;pyruvate kinase;ribokinase;ribose 5-phosphate isomerase;ribulose kinase and related carbohydrate kinases;SAM-dependent methyltransferase;serine / threonine kinase receptor;serine / threonine protein kinase;serine / threonine-specific protein phosphatase;short-chain acyl-CoA dehydrogenase;sn-1,2-diacylglycerolethanolamine and choline phosphotransferase;soluble epoxide hydrolase;sphingoid base-phosphate phosphatase;sphingolipids Fatty acid hydroxylase;Sphingolipid hydroxylase;Sphingosine kinase;Squalene monooxygenase;Squalene synthase;Steroid reductase;Sterol C5 desaturase;Sterol O-acyltransferase / diacylglycerol O-acyltransferase;Sterol reductase / lamin B receptor;Succinate dehydrogenase;Succinyl-CoA synthetase;Succinyl-CoA:α-ketoacid-CoA transferase;Sucrose transporter and related proteins;Sugars (pentulose and hexasaccharides) (ose) kinase;sugar transporter / spinster transmembrane protein;sulfatase;sulfide:quinone oxidoreductase / flavo-binding protein;transaldolase;transketolase;transthyretin and related proteins;trehalose-6-phosphate synthase;triglyceride lipase-cholesterol esterase;triosephosphate isomerase;ubiquinol cytochrome c reductase assembly protein;ubiquinol cytochrome c reductase;UDP-galactose transporter;UDP-glucose pyrophosphorylase;The proteins encode any of UDP-glucose / GDP-mannose dehydrogenase; UDP-glucose:glycoprotein glucosyltransferase; UDP-glucuronosyl and UDP-glucosyltransferase; UDP-N-acetylglucosamine transporter; aldose 1-epimerase; vacuolar H+-ATPase; very long chain acyl-CoA dehydrogenase; vigilin; voltage-gated Shaker-like K+ channel; zinc-binding oxidoreductase; and any combination thereof;

[0085] The term "oil with a particular fatty acid profile, e.g. high oleic oil" as used herein relates to an oil with a desired fatty acid profile, e.g. an oil containing a particular type of fatty acid, such as unsaturated fatty acids. In one embodiment, a high oleic oil is an oil containing at least 50%, preferably at least 60%, e.g. at least 70% oleic acid.

[0086] In one embodiment, the method of genetically modifying a GC-rich microorganism, such as an oleaginous microorganism, and / or the pretreatment in step ii) comprises: a) The GC-rich microorganism, e.g., oleaginous microorganism, is cultured to an OD of 0.2 to 5.0, e.g., 0.2×10 7 ~2×10 8 to cells / mL; b) harvesting the cells expanded in step a); c) washing the cells collected in step b), for example with sterile water followed by sorbitol (1M); d) treating the cells washed in step c) with said at least one enzyme, preferably in a suitable buffer; e) harvesting the cells treated in step d); f) resuspending the cells collected in step e) in a suitable buffer, such as a Tris-HCl buffer containing sorbitol or mannitol and CaCl2. Includes.

[0087] In one embodiment, the GC-rich microorganism is grown, washed and treated with the at least one enzyme prior to the transformation in step iii). In one embodiment, the pretreatment in step ii) preferably comprises washing the microorganism with an organic phase, such as methanol, ethanol, butanol and / or amyl alcohol; a buffer; and / or water, prior to contacting the microorganism with at least one enzyme selected from, for example, glycosyl hydrolases, cellulases, hemicellulases, mannanases, xyloglucanases, xylanases, glucanases, glucosidases, arabinases, amylases, fructanases, laminases, hydrolases, proteases, or any combination thereof. In one embodiment, the pretreatment in step ii) comprises an enzymatic pretreatment and / or another spheroplasting procedure, comprising contacting the microorganism with at least one enzyme produced by an enzyme-producing filamentous fungus; optionally, the enzymatic pretreatment and / or the another spheroplasting procedure comprises washing the microorganism with an organic phase, a buffer, and / or water prior to the contacting. In one embodiment, the method of the invention comprises, for example, a step of washing the GC-rich microorganism with an organic phase, a buffer, and / or water prior to the pretreatment in step ii). In one embodiment, the method of the invention preferably comprises a step of washing the GC-rich microorganism with an organic phase, a buffer, and / or water prior to the contacting of the GC-rich microorganism with at least one enzyme.

[0088] In one embodiment, the organic phase comprises or consists of methanol, amyl alcohol, ethanol, butanol, or any combination thereof. In one embodiment, the enzyme-producing filamentous fungus is selected from the group consisting of Ceratocystis species, such as Ceratocystis fimbriata, Ceratocystis moniliformis, and Ceratocystis paradoxa, preferably Ceratocystis paradoxa; Trichoderma species, such as Trichoderma reesei and Trichoderma harzianum, preferably Trichoderma reesei; Aspergillus species, such as Aspergillus oryzae, Aspergillus tubingensis, and Aspergillus niger; Neurospora species, such as Neurospora intermedia; Monascus species, For example, the protease is selected from Monascus purpureus; Rhizopus species, such as Rhizopus oryzae; Fusarium species, such as Fusarium venenatum; Thermomyces species; Penicillium species; Aureobasillium species; Ischnoderma species, such as Ischnoderma benzoinum; Polyporus species, such as Polyporus duras; Pycnoporus species, such as Pycnoporus cinnabarinus; Phanerochaete species, such as Phanerochaete chrysosporium; and Xylaria species; preferably, it is produced by Trichoderma reesei.

[0089] In one embodiment, at least one enzyme produced by a fungus, e.g., a hydrolase or protease produced by a fungus, is produced by growing the fungus; harvesting the cells of the fungus; and optionally washing the cells with water and / or sorbitol. In one embodiment, the pretreatment in step ii) comprises using an enzyme, e.g., in an enzymatic pretreatment and / or another spheroplasting procedure. In one embodiment, the pretreatment in step ii) comprises using an enzyme produced by and / or obtained from a filamentous fungus, e.g., Trichoderma species, Aspergillus species, e.g., A. niger and A. awamori; Penecillium species; Aureobacillium species; Phanerochaete species; Fusarium species; Streptomyces species; or Bacillus species, e.g., in an enzymatic pretreatment and / or another spheroplasting procedure.

[0090] In one embodiment, the composition of the invention is a composition for genetically modifying GC-rich microorganisms, in particular for genetically modifying Cutaneotrichosporon species. In one embodiment, the at least one guide RNA, in particular the crRNA and / or the sgRNA, comprises a nucleic acid sequence of a microorganism selected from GC-rich microorganisms, in particular Cutaneotrichosporon species. In one embodiment, the at least one guide RNA, in particular the crRNA and / or the sgRNA, comprises a nucleic acid sequence of a part of the genome of a GC-rich microorganism, in particular a microorganism selected from Cutaneotrichosporon species. In one embodiment, the at least one guide RNA, in particular the crRNA and / or the sgRNA, is configured to target a nucleic acid sequence of the genome of a GC-rich microorganism, in particular a microorganism selected from Cutaneotrichosporon species. In one embodiment, the composition is used for genetically modifying GC-rich microorganisms, in particular a microorganism selected from Cutaneotrichosporon species. The present invention also relates to the use of a composition as defined herein for genetically modifying GC-rich microorganisms, in particular a microorganism selected from Cutaneotrichosporon species.

[0091] In one embodiment, the plasmid or plasmid collection is a plasmid or plasmid collection for genetically modifying GC-rich microorganisms, in particular for genetically modifying Cutaneotrichosporon species. In one embodiment, the at least one guide RNA, in particular the crRNA and / or the sgRNA, comprises a nucleic acid sequence of a microorganism selected from GC-rich microorganisms, in particular Cutaneotrichosporon species. In one embodiment, the at least one guide RNA, in particular the crRNA and / or the sgRNA, comprises a nucleic acid sequence of a part of the genome of a microorganism selected from GC-rich microorganisms, in particular Cutaneotrichosporon species. In one embodiment, the at least one guide RNA, in particular the crRNA and / or the sgRNA, is configured to target a nucleic acid sequence of the genome of a microorganism selected from GC-rich microorganisms, in particular Cutaneotrichosporon species. In one embodiment, the plasmid or plasmid collection is used for genetically modifying a microorganism selected from GC-rich microorganisms, in particular Cutaneotrichosporon species. The present invention also relates to the use of a plasmid or a collection of plasmids as defined herein for genetically modifying a GC-rich microorganism, in particular a microorganism selected from the species of the genus Cutaneotrichosporon.

[0092] As used herein, the terms "of the invention," "in accordance with the invention," "inventive," and the like are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0093] As used herein, the term "comprising" is to be interpreted as encompassing both "including" and "consisting of," both of which are specifically contemplated according to the present invention and therefore are separately disclosed embodiments. As used herein, "and / or" should be interpreted as specifically disclosing each of the two specified features or components, with or without the other feature or component. For example, "A and / or B" should be interpreted as specifically disclosing each of (i) A, (iii) B, and (iii) A and B, as if each were individually described herein. In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature of interest. This term typically indicates a deviation of ±20%, ±15%, ±10%, e.g., ±5% from the specified numerical value. As a person skilled in the art would understand, such a specific deviation of the numerical value for a given technical effect depends on the nature of that technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than an artificial or engineered technical effect. Where an indefinite or definite article is used when referring to a singular noun e.g. "a", "an", "the", etc., the plural of that noun is also included unless specifically stated otherwise. EXAMPLES

[0094] Example 1: Transfection of oleaginous microorganisms with RNA-guided endonucleases. Transfections were performed as shown in FIG.

[0095] Spheroplasts were mixed with the endonuclease mRNA or protein along with donor DNA and sgRNA, electroporated, and plated on selection plates in an agar overlay containing the selection agent. Plates were incubated at 28-30°C until colonies were obtained.

[0096] The present inventors have demonstrated that transfection with Cas mRNA is more efficient than transfection with Cas protein. The present inventors have further demonstrated that transfection with two sgRNAs is more efficient than transfection with only one sgRNA.

[0097] Example 2: Spheroplasting enhances transfection efficiency. Spheroplast formation was carried out as shown in FIG.

[0098] Spheroplasts were prepared using commercial enzymes and tailor-made enzyme mixes. Both spheroplasts were mixed with the mRNA or protein of the endonuclease along with donor DNA and sgRNA, electroporated, and plated on selection plates in an agar overlay containing the selection agent. Plates were incubated at 28-30 degrees until colonies were obtained.

[0099] The inventors have demonstrated that pre-treatment of GC-rich microorganisms, especially oleaginous microorganisms, for example by spheroplasting, increases the transfection efficiency. No transformants were obtained from microorganisms treated with commercial enzymes. In particular, spheroplasting with a tailor-made enzyme mix, such as an enzyme mix produced by a fungus, allows for a much higher transfection efficiency than transfection without prior spheroplasting or spheroplasting with a commercial lyticase enzyme.

[0100] Example 3: The method of the present invention successfully integrates donor DNA into the genomic DNA of oleaginous microorganisms. Spheroplasts were mixed with the endonuclease mRNA or protein along with donor DNA and sgRNA, electroporated, and plated on selection plates in an agar overlay containing the selection agent. Plates were incubated at 28-30°C until colonies were obtained.

[0101] The exemplary target site was genetically modified as shown in Figure 3. Sequencing confirmed successful modification of the exemplary target site at the Ura5 locus of C. oleaginosus genomic DNA.

[0102] Example 4: The method of the present invention successfully integrates donor DNA into the genomic DNA of oleaginous microorganisms, allowing for the efficient production of lipids. A species of the genus Cutaneotrichosporon was prepared by spheroplastization. The microorganism was transformed with exemplary donor DNA, for example, as follows: 1) AKRp-D9: replacement of the delta 9 desaturase promoter with the aldo-keto reductase promoter (SEQ ID NO: 28) A donor DNA containing the knock-in gene of interest (a novel promoter of the D9 desaturase gene), a selection marker (ura5 gene-orotate phosphoribosyltransferase), and homology arms. Homology arm upstream 800bp 7...806 Ura5 gene (promoter, coding sequence, and terminator) 807...2106 Aldo-keto reductase promoter 2107...2906 Homology arm downstream 800bp 2907...3706 2) TEFp-D9: replacement of the delta 9 desaturase promoter with the transcription elongation factor 2 promoter (SEQ ID NO:29). The knock-in gene of interest (a novel promoter for the D9 desaturase gene), a selection marker (the ura5 gene orotate phosphoribosyltransferase), and homology arms. Homology arm upstream 800bp 7...806 ura5 gene (promoter, coding sequence, and terminator) 807...2106 Transcription elongation factor 2 promoter 2107...3019 Homology arm downstream 800bp 3020...3819 3) AKRp-D12: replacement of the delta 12 desaturase promoter with the aldo-keto reductase promoter (SEQ ID NO: 30). A donor DNA containing the knock-in gene of interest (a novel promoter of the D12 desaturase gene), a selection marker (ura5 gene-orotate phosphoribosyltransferase), and homology arms. Homology arm upstream 800bp 1...798 ura5 gene (promoter, coding sequence, and terminator) 799...2098 Aldo-keto reductase promoter 2099...2898 Homology arm downstream 800bp 2899...3698 4) AKRp-D12: replacement of the delta 9 desaturase promoter with the TEF promoter (SEQ ID NO: 31). A donor DNA containing the knock-in gene of interest (a novel promoter of the D12 desaturase gene), a selection marker (ura5 gene-orotate phosphoribosyltransferase), and homology arms. Homology arm upstream 800bp 1...798 Ura5 gene (promoter, coding sequence, and terminator) 799...2098 Promoter TEF 2099...3011 Homology arm downstream 800bp 3012...3811 5) Contains the knock-in gene of interest (elongase coding sequence, aldo-keto reductase promoter and terminator), a selection marker (ura5 gene - orotate phosphoribosyltransferase), and homology arms (SEQ ID NO: 32). Donor DNA . Homology arm upstream 800bp 7...799 Ura5 gene (promoter, coding sequence, and terminator) 808...1896 Target gene (elongase) 1897...3795 Promoter 1897...2696 Elongase coding sequence 2697...3524 Terminator 3525...3795 Homology arm downstream 800bp 3796...4595 6) Contains the knock-in gene of interest (oleate hydratase coding sequence, promoter and terminator of transcription elongation factor 2), a selection marker (ura5 gene-orotate phosphoribosyltransferase), and homology arms (SEQ ID NO: 33). Donor DNA . Homology arm upstream 800bp 7...799 Ura5 gene (promoter, coding sequence, and terminator) 808...1896 Promoter TEF 1897...2809 Oleate hydratase 2810...4489 TEF Terminator 4490...5108 Homology arm downstream 800bp 5109...5908 7) D9OE: overexpression of delta 9 desaturase (SEQ ID NO: 34). Donor DNA containing the knock-in gene of interest (delta 9 desaturase coding sequence, aldo-keto reductase promoter and terminator), a selection marker (ura5 gene - orotate phosphoribosyltransferase), and homology arms. Homology arm upstream 800bp 1...793 Ura5 gene (promoter, coding sequence, and terminator) 802...1890 Promoter 1891...2690 Delta 9 desaturase coding sequence 2691...4359 Terminator 4360...4630 Homology arm downstream 800bp 4631...5430 8) D12OE: overexpression of delta 12 desaturase (SEQ ID NO: 35). Donor DNA containing the knock-in gene of interest (delta-12 desaturase coding sequence, aldo-keto reductase promoter and terminator), a selection marker (ura5 gene - orotate phosphoribosyltransferase), and homology arms. Homology arm upstream 800bp 1...793 Ura5 gene (promoter, coding sequence, and terminator) 802...1890 Promoter 1891...2690 D12 desaturase coding sequence 2691...4512 Terminator 4513...4783 Homologous arm downstream 800bp 4784...5583 9) ΔD12: knockout of delta 12 desaturase (SEQ ID NO:36). Donor DNA containing a knock-in gene for a selection marker (ura5 gene - orotate phosphoribosyltransferase) and homology arms for delta-12 desaturase knock-out. Homology arm upstream 800bp 1...699 Ura5 gene (promoter, coding sequence, and terminator) 700...1999 Homology arm downstream 800bp 2000...2699

[0103] As shown in Figures 9-19, the method of the present invention allows for efficient transformation of GC-rich microorganisms, such as Cutaneotrichosporon species, with donor DNA. Cutaneotrichosporon species, such as Cutaneotrichosporon oleaginosus, transformed with donor DNA efficiently produce microbial lipids with high yields. Furthermore, the method of preparing target compounds of the present invention allows for the production of microbial lipids with well-defined compositions.

[0104] References [1] Gorner, C. et al.Genetic engineering and production of modified fatty acids by the non-conventional oleaginous yeast Trichosporon oleaginosus ATCC20509.Green Chemistry 18,2037-2046,doi:10.1039 / C5GC01767J(2016).

[0105] [2] Bracharz, F., Beukhout, T., Mehlmer, N. & Bruck, T. Opportunities and challenges in the development of Cutaneotrichosporon oleaginosus ATCC20509 as a new cell factory for custom tailored microbial oils. Microbial cell factories 16,178-178,doi:10.1186 / s12934-017-0791-9(2017).

[0106] The features of the invention disclosed in this specification, in the claims and / or in the accompanying drawings may, both individually and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Claims

1. 1. A method for genetically modifying an optionally GC-rich microorganism to increase the production of acetyl-CoA-based hydrophobic compounds in said microorganism, comprising the steps of: i) providing a GC-rich microorganism, preferably an oleaginous microorganism, more preferably an oleaginous yeast; ii) optionally pretreating the GC-rich microorganisms; preferably, the pretreatment comprises spheroplasting the GC-rich microorganisms; iii) transforming the GC-rich microorganism with an RNA-guided endonuclease, at least one guide RNA, and optionally donor DNA; iv) optionally selecting transformed cells of said GC-rich microorganism; v) Obtaining genetically modified GC-rich microorganisms.

2. The GC-rich microorganism is selected from yeast, fungi, bacteria, and microalgae; preferably, the GC-rich microorganism is an oleaginous microorganism; more preferably, the GC-rich microorganism is an oleaginous yeast; even more preferably, the GC-rich microorganism is selected from Rhodosporidium species, Yarrowia species, Rhodotorula species, Candida species, Lipomyces species, Cutaneotrichosporon species, Trichosporon species, preferably Cutaneotrichosporon species, more preferably Cutaneotrichosporon oleaginosus oleaginosus (ATCC 20509); and / or 2. The method of claim 1, wherein the GC-rich microorganism has a guanine-cytosine (GC) content of at least 50%, preferably at least 55%, more preferably at least 58%, and even more preferably at least 60%.

3. 3. The method of claim 1 or 2, wherein the RNA-guided endonuclease is a CRISPR-associated (Cas) endonuclease, preferably selected from Cas9, Cas1, Cas2, Cas4, Cas3, Cas10, Cas12, Cas13, Csm, scf1, or a variant thereof, such as Cas12a, dCas9, D10A CAS9 nickase, or H840A CAS9 nickase.

4. 3. The method according to claim 1 or 2, wherein the transformation in step iii) comprises applying the RNA-guided endonuclease to the GC-rich microorganism in the form of an RNA-guided endonuclease protein, in the form of DNA encoding the RNA-guided endonuclease, or in the form of mRNA encoding the RNA-guided endonuclease, preferably in the form of mRNA encoding the RNA-guided endonuclease.

5. 3. The method of claim 1 or 2, wherein the at least one guide RNA comprises a CRISPR RNA (crRNA), a transactivating CRISPR RNA (tracrRNA), and / or a single guide RNA (sgRNA); preferably, the at least one guide RNA comprises a first guide RNA and a second guide RNA, and the sequence of the first guide RNA is different from the sequence of the second guide RNA.

6. The method of claim 1 or 2, wherein the donor DNA comprises a DNA repair template, DNA encoding a selectable marker, and / or a gene or sequence of interest, e.g., a gene involved in the production of a target compound such as a fatty acid.

7. 3. The method according to claim 1 or 2, wherein the transformation in step iii) comprises applying the RNA-guided endonuclease and the at least one guide RNA, preferably together, in the form of a ribonucleoprotein complex.

8. 3. The method of claim 1 or 2, wherein the transformation in step iii) comprises applying the RNA-guided endonuclease in the form of an mRNA encoding the RNA-guided endonuclease, and wherein the at least one guide RNA comprises an sgRNA.

9. wherein the pretreatment in step ii) comprises an enzymatic pretreatment, a chemical pretreatment, and / or another spheroplasting procedure; 3. The method of claim 1 or 2, wherein the pretreatment in step ii) comprises an enzymatic pretreatment using at least one enzyme selected from glycosyl hydrolase, cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, glucosidase, arabinase, amylase, fructanase, laminase, hydrolase, protease, or any combination thereof, preferably a combination of cellulase, hemicellulase, mannanase, xyloglucanase, xylanase, glucanase, and glucosidase.

10. The pretreatment in step ii) comprises treatment of the microorganism with a hydrolase alone or a combination of a hydrolase and a protease / hydrolase followed by a protease; Optionally, the hydrolase is selected from hydrolases produced by fungi, preferably filamentous fungi, more preferably fungi selected from Trichoderma species, Aspergillus species, Penicillium species, Aureobasillium species, and Fusarium species, even more preferably Trichoderma reesei; and / or 3. The method of claim 1 or 2, wherein optionally the protease is selected from a protease produced by an Aspergillus species, a Streptomyces species, or a Bacillus species.

11. wherein the transformation in step iii) is performed using electroporation; PEG-based or other nanoscale carrier-based transformation; biological ballistic flight; glass bead transformation; vesicle-mediated delivery; viral transfection systems, such as lentivirus (LV), adenovirus (AdV), or adeno-associated virus (AAV); liposome delivery, such as lipofection; chemical transfection techniques; or a combination thereof; 3. The method of claim 1 or 2, preferably carried out using electroporation and / or PEG-based transformation; more preferably, using electroporation.

12. said selecting in step iv) comprising exposing said microorganism to a selection agent selective for a selection marker, optionally a selection marker encoded by said donor DNA; Preferably, the selection comprises exposing the microorganism to a concentration of the selection agent in the range of 10% to 90%, preferably 10% to 70%, more preferably 10% to 60% of the minimum selectable concentration; and / or exposing the microorganism to a concentration of the selection agent in the range of 90% to 100%, preferably 99% to 100% of the minimum selectable concentration; 3. The method of claim 1 or 2, wherein the selection comprises exposing the microorganism to a concentration of the selective agent in a first medium, such as a liquid or solid medium, preferably an agar underlayer, in the range of 10% to 90%, preferably 10% to 70%, more preferably 10% to 60% of the minimum selectable concentration, and optionally subsequently exposing the microorganism to a concentration of the selective agent in a second medium, such as a liquid or solid medium, preferably an agar underlayer, in the range of 90% to 100%, preferably 99% to 100%.

13. 1. A genetically modified GC-rich microorganism, preferably an oleaginous microorganism, more preferably an oleaginous yeast, even more preferably Cutaneotrichosporon oleaginosus, comprising an RNA-guided endonuclease, at least one guide RNA, and optionally donor DNA; and optionally, said RNA-guided endonuclease and said at least one guide RNA are present in said cell in the form of a ribonucleoprotein complex.

14. an RNA-guided endonuclease; optionally an RNA-guided endonuclease protein, a DNA encoding said RNA-guided endonuclease, or an mRNA encoding an RNA-guided endonuclease, preferably an mRNA encoding an RNA-guided endonuclease; an RNA-guided endonuclease which is preferably a CRISPR-associated (Cas) endonuclease, more preferably selected from Cas9, Cas1, Cas2, Cas4, Cas3, Cas10, Cas12, Cas13, Csm, scf1, or a variant thereof, such as Cas12a, dCas9, D10A CAS9 nickase, or H840A CAS9 nickase, and even more preferably a Cas9 endonuclease such as a Cas9 endonuclease having the sequence of any of SEQ ID NOs: 1-4; at least one guide RNA (gRNA); preferably a crRNA, a tracrRNA, and / or an sgRNA; Optionally, a gRNA comprising the tracrRNA sequence of SEQ ID NO: 5 and / or comprising the crRNA sequence of any one of SEQ ID NOs: 14-26; optionally, donor DNA; preferably donor DNA comprising a DNA repair template, DNA encoding a selectable marker, and / or a gene or sequence of interest, e.g. a gene involved in the production of a target compound such as a fatty acid, Optionally, the selectable marker is Ura5; Optionally, the gene or sequence of interest encodes a delta-9-desaturase, a delta-12-desaturase, an elongase, an oleate hydratase, an aldo-ketoreductase promoter, an aldo-ketoreductase terminator, a transcription elongation factor 2 promoter, a TEF promoter, and / or a TEF terminator; Optionally, the donor DNA comprises any of SEQ ID NOs: 6-13 and 27-36, optionally any of SEQ ID NOs: 6-13 and 27. A composition comprising:

15. - an mRNA encoding an RNA-guided endonuclease; an RNA-guided endonuclease, preferably a Cas9 endonuclease, more preferably a Cas9 endonuclease having the sequence of any of SEQ ID NOs: 1 to 4; at least one guide RNA (gRNA); preferably a crRNA, a tracrRNA, and / or an sgRNA; Optionally, a gRNA comprising the tracrRNA sequence of SEQ ID NO: 5 and / or comprising the crRNA sequence of any one of SEQ ID NOs: 14-26; - optionally donor DNA, e.g. a DNA repair template; Optionally, donor DNA having a sequence of any of SEQ ID NOs: 6 to 13 and 27 to 36, and optionally any of SEQ ID NOs: 6 to 13 and 27. A plasmid or a collection of plasmids comprising:

16. 1. A method for preparing a target compound, e.g., acetyl-CoA or acetyl-CoA-based hydrophobic compounds and / or an oil having a specific fatty acid profile, e.g., a high oleic oil, using a genetically modified GC-rich microorganism, preferably an oleaginous microorganism, more preferably an oleaginous yeast, comprising: a) providing a genetically modified GC-rich microorganism, preferably an oleaginous microorganism, more preferably an oleaginous yeast, using a method according to claim 1 or 2; said method comprising transforming said microorganism with donor DNA, said donor DNA comprising a gene or sequence of interest, e.g., a gene involved in the production of said target compound and / or a specific fatty acid; b) growing the genetically modified GC-rich microorganism; c) obtaining oils having said target compounds and / or specific fatty acid profiles; Preferably, the target compound is selected from saturated short-chain fatty acids, saturated medium-chain fatty acids, saturated long-chain fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, functionalized fatty acids, ergosterol, ergosterol derivatives, terpenes, alkaloids, acetyl-CoA-based synthetic compounds, tocochromanols such as α-tocopherol and α-tocotrienol, monoterpenoids, sesquiterpenoids, diterpenoids, squalene, carotenoids, triterpenes, pheophytins, vitamins, citric acid, volatile fatty acids, oxalic acid, lactic acid, malic acid, and exopolysaccharides. A method comprising:

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