Engineered polyol lipid-producing microorganism

Abstract

Provided are non-naturally occurring polyol lipid producing microorganisms having altered expression of endogenous genes or expressing heterologous genes. The microorganisms can be further engineered to obtain a functional property of interest, or can be used to enhance production of a product of interest, such as polyol fatty acid esters (PEFA).

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 594,874, filed October 31, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to genetically modified microbial cells for the production of polyol lipids, particularly microorganisms containing altered expression of endogenous genes and / or introduced and expressing heterologous genes. Background Technology

[0004] Polyol lipid compounds have a variety of uses, including as surfactants. Surfactants (including petroleum-based and bio-based) are examples of compounds that can be used as emulsifiers, detergents, dispersants, foam control agents, and wetting agents in a wide range of consumer applications, including agricultural, nutritional, cosmetic, veterinary, therapeutic, paint, ink, and industrial applications. However, such surfactants perform poorly in terms of sustainability, bioaccumulation, ecotoxicity, and / or biodegradability. Therefore, there is a need to develop alternative surfactants and compositions for use in consumer and industrial products in these applications.

[0005] Several yeast species are attractive chassis strains for producing polyol lipids, such as fatty acid polyol esters (PEFAs), which have applications as surfactants. (See phylogeny in Garay et al. (2018) Biotechnology Advances 36:397–414, doi:10.1016 / j.biotechadv.2018.01.003). Many yeast species that naturally produce and secrete polyol lipids have been described. However, to improve the fermentation yield and production of such lipids, methods for introducing heterologous DNA into the organism's genome are needed. Selection markers, transformation methods, heterologous promoters, and codon optimization methods suitable for producing recombinant strains of these species are required.

[0006] PEFAs can be produced using glucose as the sole carbon source. Their biosynthesis likely begins with the production of (1) fatty acids and (2) polyols, such as mannitol and arabinitol, from glucose. Therefore, PEFA yields can be increased by enhancing the expression of dedicated PEFA biosynthetic pathways, increasing intracellular pools of fatty acids, and / or increasing intracellular pools of polyols. Furthermore, to enhance PEFA yields, competitive biosynthetic pathways can be blocked, as can PEFA degradation pathways.

[0007] Enhancing the PEFA production pathway can improve the yield of PEFA produced by the strain. The PEFA production pathway is expected to be (1) formation of mannitol and arabinitol, (2) formation of acyl-CoA, (3) hydroxylation of acyl-CoA, (4) ester bond formation, (5) acetylation of 3-OH fatty acids and polyols, and (6) secretion. (Garay et al., 2018) The core PEFA biosynthetic pathway involves the steps of hydroxylation of acyl-CoA, ester bond formation, acetylation, and secretion. Previous researchers have identified a putative acetyltransferase in the genome of *Rhodotorula truncatula* MD1149 based on its homology with the acetyltransferase of *Starmarella bombicola*. (PCT Publication No. WO2018 / 148465 A1). Similarly, candidate genes for ester bond formation between polyols and 3-OH fatty acids can be located by analogy to liamocin biosynthesis, which includes the gene EST1 (A0A385XI13) from *Aureobasidium pullulans* (Xue et al., 2020). β-hydroxylation of long-chain and short-chain fatty acids is known to originate from flocculin biosynthesis. Although the enzymes responsible for hydroxylation are unknown, α-hydroxylation of long-chain fatty acids is catalyzed by the gene UmAhd1 (A0A0D1DT68) from *Ustilago maydis*, while β-hydroxylation of short-chain fatty acids is catalyzed by UmUhd1 (A0A0D1BUI1) (Teichman et al., 2011).

[0008] Increasing the overall flux of de novo fatty acid synthesis can improve the yield of PEFA production. Several reviews have been published describing methods to improve the yield of fatty acid-derived molecules. (See Wang et al., 2020.) Several studies in *Rhodotorula toruloides* have also shown improved intracellular fatty acid accumulation. (Zhang et al., 2016.) Notably, increasing fatty acid biosynthetic flux often involves overexpression of acetyl-CoA carboxylase, which enhances the availability of malonyl-CoA (a substrate for fatty acid biosynthesis). Another effective strategy may involve blocking lipid degradation in peroxisomes and / or mitochondria, for example, by deleting PEX10 (peroxisome biogenesis factor), removing fatty acyl-CoA oxidase, or deleting acyl-CoA dehydrogenase. (Coradetti et al., 2018.) Metabolic modeling suggests that transketolases play an important role in the production of acetyl-CoA for lipid biosynthesis in *Rhodotorula*. Therefore, overexpression of transketolases can achieve higher yields. (Rekena et al., 2023.)

[0009] PEFA is produced by yeast, which also produces large amounts of intracellular triglycerides (TAGs), common storage molecules in eukaryotes (Garay et al., 2017). Since both TAGs and PEFAs originate from de novo fatty acid biosynthesis, a direct approach to increasing PEFA yield is to block TAG biosynthesis or increase the recycling of TAGs to acetyl-CoA molecules, which can then be incorporated into PEFAs (US Patent No. 10,443,047 B2). In fungi, TAG biosynthesis ends with the transfer of the acyl moiety on the acyl-CoA carrier to the diacylglycerol molecule via diacylglycerol acyltransferase (DAGT). In *Rhodotorula buergerianum*, the dominant enzyme responsible for this is DGA1 (Schultz et al., 2022), while the role of LRO1 is unclear; LRO1 is involved in TAG formation in another lipogenic yeast, *Yarrowia lipolytica* (Athenstaedt, 2011). Conversely, diacylglycerol lipases break down TAG molecules, allowing their acyl groups to be metabolized. Metabolic modeling showed that knocking out these lipases improved TAG molecule accumulation (Dinh et al., 2019). Overexpression of these enzymes improved PEFA production in strains that also accumulated TAG.

[0010] Mannitol is derived from fructose in fungi via one of two pathways (Solomon et al., 2007), while arabinose is produced via the pentose phosphate pathway. In liamomicin biosynthesis (a structure-associated glycolipid), the absence of enzymes encoding mannitol-1-phosphate dehydrogenase, mannitol dehydrogenase, and arabinose dehydrogenase eliminates liamomicin production (Xue et al., 2020). Overexpression of these enzymes may lead to higher PEFA yields.

[0011] NADPH is a key cofactor in fatty acid production. In Rhodotorula species, the oxidative branch of the pentose phosphate pathway plays a crucial role in NADPH generation (Pinheiro et al., 2020). By systematically altering the expression of the pentose phosphate pathway, an appropriate balance can be achieved between the availability of substrates for arabinitol and other biosynthetic processes.

[0012] PEFA is reportedly consumed post-production. (Lyman et al., 2018) Complex carbon molecules (such as TAG) are typically degraded extracellularly to allow their components to be absorbed more efficiently. Secretory lipases are known to enable oleogenetic yeasts (such as Yersinia lipophila) to digest extracellular TAG. (Thevenieau et al., 2010) Similar molecules may play a role in the degradation of PEFA. Removing these molecules can increase the yield of PEFA. Summary of the Invention

[0013] A non-naturally occurring microorganism for producing polyol lipids is provided, along with a method for preparing the microorganism and a polynucleotide construct, and a method of using the microorganism for producing polyol lipids in a microbial culture.

[0014] In one aspect, a non-naturally occurring polyol lipid-producing microorganism is provided. The non-naturally occurring microorganism comprises: (i) at least one knocked-out endogenous gene; (ii) altered expression of at least one endogenous gene compared to a naturally occurring microorganism from which the non-naturally occurring microorganism is derived or compared to a parent microorganism from which the non-naturally occurring microorganism is derived; and / or (iii) at least one expressed heterologous polynucleotide.

[0015] In some embodiments, the non-naturally occurring microorganism produces one or more polyol lipids selected from the group consisting of: esterification of a 3-hydroxy fatty acid moiety of 6 to 24 carbon atoms to a sugar alcohol moiety; esterification of an acetylated 3-hydroxy fatty acyl moiety to a sugar alcohol moiety; esterification of a sugar alcohol moiety containing a non-esterified hydroxyl group to the carboxyl terminus of the 3-hydroxy fatty acyl moiety; esterification of a sugar alcohol moiety containing a mannitol or arabinitol backbone to the carboxyl terminus of the 3-hydroxy fatty acyl moiety; and fatty acid polyol esters (PEFAs). In one embodiment, the polyol lipid is PEFA.

[0016] In some embodiments, the yield of at least one polyol lipid produced by the non-naturally existing microorganism is increased (e.g., when grown in a liquid culture medium) compared to the naturally existing microorganism from which the non-naturally existing microorganism is derived or compared to the parent microorganism from which the non-naturally existing microorganism is derived.

[0017] In some embodiments, the non-naturally occurring microorganism is yeast, for example, from the subkingdom of Dikaryotic Bacteria. The microorganism may belong to the phylum Ascomycota or Basidiomycota. The microorganism may belong to the order Basidiomyales. In some embodiments, the microorganism belongs to genera selected from the group consisting of: *Rhodotorula*, *Rhodotorula*, and *Rhodotorula*. The microorganism may be, but is not limited to, species selected from the group consisting of: *Rhodotorula beggivora*, *Rhodotorula diodovoidea*, *Rhodotorula kratochevilova*, *Rhodotorula granatum*, *Rhodotorula simulans*, *Rhodotorula squarrosa*, *Rhodotorula colostrum*, *Rhodotorula daliani*, *Rhodotorula rouxii*, *Rhodotorula taiwanensis*, *Rhodotorula mucilaginosa*, and *Rhodotorula natans*. The microorganisms may be, but are not limited to, strains selected from the following groups: *Rhodotorula biciliae* strain NRRL Y-67018, *Rhodotorula biciliae* strain NRRL Y-67017, *Rhodotorula biciliae* strain UCDFST 68-916.1, *Rhodotorula biciliae* strain UCDFST67-458, *Rhodotorula biciliae* strain UCDFST 05-736, *Rhodotorula biciliae* strain UCDFST 04-830, *Rhodotorula diopside* strain NRRL Y-67015, *Rhodotorula kratochevilova* strain NRRL Y-67016, *Rhodotorula sphaeroides* strain NRRLY-67012, *Rhodotorula sphaeroides* strain UCDFST 82-646.2, *Rhodotorula sphaeroides* strain UCDFST 81-492, *Rhodotorula sphaeroides* strain NRRL Y-67009, *Rhodotorula sphaeroides* strain NRRL Y-67010, and *Rhodotorula dalianense* strain NRRL Y-67011, NRRL Y-67014 (similar to Rhodotorula colostrum), NRRL Y-67013 (similar to Rhodotorula natans), NRRL Y-17302 (similar to Rhodotorula roximans), MD1149 (similar to Rhodotorula taiwanensis), and 50-3-19 / 208 (similar to Rhodotorula mucilaginosa).

[0018] In some implementations, the expression level of the endogenous gene is altered compared to the expression level of the endogenous gene in a naturally occurring microorganism from which the non-naturally occurring microorganism is derived, or compared to the expression level of the endogenous gene in a parental microorganism from which the non-naturally occurring microorganism is derived.

[0019] For example, the promoter of an endogenous gene can be replaced with different promoters, and / or a 3' untranslated region (UTR) and / or a 5' UTR can be added, thereby increasing, decreasing, or altering the regulation of endogenous gene expression and / or the regulation of mRNA stability transcribed from the endogenous gene. In one embodiment, the promoter of the endogenous gene is replaced by homologous recombination, CRISPR, zinc finger nucleases, or TALEN. In another embodiment, the endogenous gene is fused with a peptide, such as, but not limited to, the auxin-inducible degrader IAA7, the N-terminal fusion of ubiquitin, or the tobacco etched viral degrader tag TDegF, to increase, decrease, or alter the regulation of endogenous gene expression.

[0020] In another example, endogenous genes may be knocked out, silenced, or have their expression levels reduced. In one implementation, endogenous genes are knocked out or edited via homologous recombination, CRISPR, zinc finger nucleases, or TALEN. In another implementation, endogenous genes are silenced via RNA interference, RNA silencing, antisense oligonucleotides, ribozymes, or CRISPR-based methods (e.g., but not limited to dCas9 and Cas13).

[0021] In some embodiments, the non-naturally occurring microorganism contains an endogenous knockout gene, a silenced gene, or a reduced expression level of an endogenous gene encoding an enzyme in the biosynthetic pathway for triacylglycerol (TAG) production. The microorganism produces at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% less TAG compared to a naturally occurring microorganism from which the non-naturally occurring microorganism was derived, or compared to a parent microorganism from which the non-naturally occurring microorganism was derived. For example, the enzyme in the biosynthetic pathway for TAG production can be an acyltransferase, such as, but not limited to, diacylglycerol acyltransferase.

[0022] In other embodiments, the non-naturally occurring microorganism contains endogenous knockout genes, silenced genes, or endogenous genes encoding enzymes in the biosynthetic pathway for carotenoid production with reduced expression levels. For example, the enzyme in the biosynthetic pathway for carotenoid production could be a bifunctional lycopene cyclase / hydrolycopene synthase.

[0023] In a further embodiment, the expression levels of endogenous genes from non-naturally occurring microorganisms, including knockout genes, silenced genes, or genes encoding orotidine 5-phosphate decarboxylase, cytosine deaminase, Ku70 homolog, Lig4 homolog, 3-hydroxyacyl-CoA dehydrogenase / enoyl-CoA hydratase, acyl-CoA oxidase, peroxisome transporter, peroxisome fatty acyl-CoA synthase, secretory lipase, enoyl-CoA hydratase, phosphatidic acid hydrolase, glycerol-3-phosphate dehydrogenase, phospholipid:diacylglycerol acyltransferase, lipid droplet-coated protein-like proteins, or seipin transmembrane proteins, are reduced. Those skilled in the art can identify further targets for reducing TAG production based on functional genomics studies (e.g., the study reported by Coradetti et al., 2018). Any gene reported by Coradetti et al. that reduces lipid accumulation in *Rhodotorula buergerianum* is a good target for reducing TAG accumulation in PEFA-producing yeasts.

[0024] In a further embodiment, the non-naturally occurring microorganism contains endogenous genes with altered expression levels encoding mannitol dehydrogenase, mannitol-1-phosphate phosphatase, mannitol-1-phosphate dehydrogenase, d-xylulose kinase, arabinose dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphate gluconate lactonease, 6-phosphate gluconate dehydrogenase, phosphoglucose isomerase, transaldolase, transketoolase, triglyceride lipase, PEFA synthase, acetyltransferase, long-chain fatty acid α-hydroxylase, PEFA transporter, fatty acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, or malate dehydrogenase. Those skilled in the art can identify further targets for increasing lipid production based on functional genomics studies (e.g., studies reported by Coradetti et al.). Any gene reported by Coradetti et al. that increases lipid accumulation in *Rhodotorula* is a good target for increasing lipid accumulation in PEFA-producing yeasts.

[0025] In some embodiments, the non-naturally occurring microorganism expresses a heteropolynucleotide encoding a protein that provides a selectable marker. For example, the selectable marker may be an enzyme that provides antibiotic resistance, a fluorescent protein, or produces a desired metabolite.

[0026] In some implementations, non-naturally occurring microorganisms express heteropolynucleotides encoding triglyceride lipase, PEFA synthase, acetyltransferase, long-chain fatty acid α-hydroxylase, PEFA transporter, fatty acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, mannitol dehydrogenase, mannitol-1-phosphate phosphatase, d-xylulose kinase, arabinose dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphate gluconate lactonease, 6-phosphate gluconate dehydrogenase, phosphoglucose isomerase, transaldolase, transketoolase, or malate dehydrogenase.

[0027] In another aspect, a microbial culture is provided, comprising: one or a combination of two or more non-naturally occurring microorganisms described herein; a culture medium; and at least about 1 g / L of polyol lipids produced by said microorganisms. In some embodiments, the microorganisms secrete said polyol lipids into the culture medium.

[0028] On the other hand, polynucleotide constructs are provided for altering the expression of endogenous genes or introducing heterologous genes in a host microorganism producing polyol lipids. In one example, the polynucleotide construct includes: a promoter; a nucleotide sequence homologous to at least a portion of a target gene sequence in the genome of the microorganism; a nucleotide coding sequence encoding a protein providing a selection marker; and a terminator, wherein the promoter and terminator sequences are capable of driving gene expression of the coding sequence in the microorganism. In another example, the polynucleotide construct includes: a promoter; a nucleotide coding sequence of a heterologous gene; and a nucleotide coding sequence encoding a protein providing a selection marker, wherein the promoter is capable of driving gene expression of the coding sequence in the microorganism.

[0029] In some embodiments, the nucleotide coding sequence is codon-optimized for expression in the host microorganism, for example, using codons as described in Table 1 or Table 2. In some embodiments, at least about 50% of the codons in the nucleotide coding sequence are the most common or second most common codons in the host microorganism, for example, the most common or second most common codons described in Table 1 or Table 2.

[0030] In some implementations, the promoter is inducible in non-naturally occurring microorganisms.

[0031] In some implementations, the selection marker is an enzyme that provides antibiotic resistance, a fluorescent protein, or produces the desired metabolite.

[0032] In some embodiments, the nucleotide coding sequence of the heterologous gene further encodes an amino acid sequence that facilitates the detection or recovery of the heterologous gene expression product. For example, the amino acid sequence that facilitates detection or recovery may include a His-tag, such as a sequence of at least six (e.g., six to nine) consecutive histidine residues, a FLAG-tag sequence having the amino acid sequence DYKDDDDK (SEQ ID NO: 1), or a Myc-tag sequence having the amino acid sequence EQKLISEEDL (SEQ ID NO: 2).

[0033] On the other hand, the polynucleotide construct replaces the promoter of an endogenous gene and / or adds an untranslated region and / or expression tag, for example, to facilitate detection or recovery. In one example, the polynucleotide construct comprises: a nucleotide sequence homologous to at least a portion of a target gene sequence in a microbial genome; and a promoter sequence, a 3' UTR, and / or a polynucleotide sequence encoding an N-terminal tag. In another example, the polynucleotide construct comprises: a nucleotide sequence homologous to at least a portion of a target gene sequence in a microbial genome; and a promoter sequence, a terminator sequence, a 5' UTR, and / or a polynucleotide sequence encoding a C-terminal tag.

[0034] On the other hand, a recombinant polyol lipid production microorganism is provided, which has been transformed with any of the polynucleotide constructs described herein.

[0035] On the other hand, a method is provided for producing a non-naturally occurring polyol lipid-producing microorganism, which, compared to a naturally occurring microorganism from which the non-naturally occurring microorganism is derived or to a parent microorganism from which the non-naturally occurring microorganism is derived, contains at least one knocked-out gene. The method includes transforming a polyol lipid-producing host microorganism with a polynucleotide construct described herein, the polynucleotide construct comprising: a promoter; a nucleotide sequence homologous to at least a portion of a target gene sequence in the microbial genome; a nucleotide-coding sequence encoding a protein providing a selection marker; and a terminator, wherein the promoter and terminator sequences are capable of driving gene expression of the coding sequence in the microorganism, wherein the nucleotide sequence homologous to at least a portion of the target gene sequence in the microbial genome disrupts the gene sequence through homologous recombination, wherein the disrupted gene is inactivated or deleted in the microorganism.

[0036] On the other hand, a method is provided for producing a non-naturally occurring polyol lipid-producing microorganism, the microorganism comprising at least one expressed heterologous polynucleotide compared to a naturally occurring microorganism from which the non-naturally occurring microorganism is derived or compared to a parent microorganism from which the non-naturally occurring microorganism is derived. The method includes converting a polyol lipid-producing host microorganism with a polynucleotide construct comprising: a promoter; a nucleotide-coding sequence of a heterologous gene; and a nucleotide-coding sequence encoding a protein providing a selection marker, wherein the promoter is capable of driving gene expression of the coding sequence in the microorganism, and wherein the nucleotide-coding sequence of the heterologous gene is expressed in the microorganism.

[0037] On the other hand, a method for producing polyol lipids is provided. The method involves culturing one or more non-naturally occurring microorganisms, as described herein, in an environment containing a culture medium (e.g., a bioreactor) under conditions and nutrients suitable for microbial growth and the production of biosynthetic products, wherein the polyol lipid product is produced by the microorganisms and secreted into the culture medium. In some embodiments, the method is carried out as a batch, fed-batch, or continuous process.

[0038] In some embodiments, the method further includes recovering the polyol lipid product from the culture medium. In some embodiments, the polyol lipid is recovered from the culture medium without lysing microorganisms or extracting with organic solvents. For example, polyol lipids may be denser than water, and the recovery of polyol lipids may involve separation from the culture medium by mechanical methods such as centrifugation, continuous decantation, or passive sedimentation.

[0039] In some embodiments, at least about 1 g / L of polyol lipids is secreted into the culture medium. In some embodiments, the density of the polyol lipids is about 1.00 g / mL to about 1.10 g / mL. In some embodiments, the polyol lipid product includes one or more PEFAs.

[0040] On the other hand, a polyol lipid is provided for production by the method described herein in a non-naturally occurring microorganism. In some embodiments, the polyol lipid is selected from the group consisting of: esterification of a 3-hydroxy fatty acid moiety of 6 to 24 carbon atoms to a sugar alcohol moiety; esterification of an acetylated 3-hydroxy fatty acyl moiety to a sugar alcohol moiety; esterification of a sugar alcohol moiety containing a non-esterified hydroxyl group to the carboxyl terminus of the 3-hydroxy fatty acyl moiety; esterification of a sugar alcohol moiety containing a mannitol or arabinitol backbone to the carboxyl terminus of the 3-hydroxy fatty acyl moiety; and fatty acid polyol esters (PEFAs). In some embodiments, the polyol lipid is PEFA. Attached Figure Description

[0041] Figure 1 The image shows Rhodotorula babjevae transformed with a norsulin selection cassette and plated on different concentrations of norsulin.

[0042] Figure 2A-2C This diagram illustrates a design for replacing genomic fragments with selection markers. 2A: A plasmid containing DNA sequences homologous to genomic regions flanking the selection markers. 2B: Homologous recombination between a linear DNA fragment generated by the plasmid and the organism's genome. 2C: The genomic sequence replaced by the selection cassette.

[0043] Figures 3A-3BModular assembly of plasmids used for gene knockout is shown. 3A: Schematic diagram showing the input plasmid specified as a component type with a conserved 4 bp BsaI to generate overhangs. 3B: Assembly of component plasmids to generate the target plasmid in the Golden Gate reaction; schematic diagram showing the assembly of the left plasmid for CAR2 knockout.

[0044] Figures 4A-4B Modular assembly of expression constructs for heterologous gene expression is shown. 4A: Schematic diagram showing the assembly of the left plasmid designed for integration into the CAR2 locus, containing an *E. coli* GFP expression cassette flanked by an outward-flanked SapI site (SapI detachment). During cloning of the expression construct, the replacement of the detached sequence causes the *E. coli* colonies to appear white instead of green. 4B: Schematic diagram of the modular assembly of the expression construct using the SapI gate. The promoter, coding sequence, and terminator are assigned 3 bp overhangs generated by the flanking SapI sites. Combining these component plasmids with an appropriate backbone assembles the left plasmid designed for integration into the CAR2 locus, containing an expression construct for expressing the coding sequence (CDS) in *Rhodotorula*.

[0045] Figures 5A-5D The images show a *Rhodotorula biceps* strain with CAR2 knocked out. 5A: A representative view of *Rhodotorula biceps* 04-877 transformed with the cleavage marker norsinidin cassette targeting the CAR2 gene. 5B: An agar plate streaked with three white colonies (top left, bottom left, and bottom right) and one red colony (top right) to illustrate the colony color phenotype. 5C: A diagram of PCR primer binding sites used to examine CAR2 locus disruption. 5D: A gel PCR using these primers demonstrating removal of CAR2 DNA via homologous recombination. Note that these lanes are from... Figure 7B The gels in the solution; they are included here for clarity.

[0046] Figure 6 Representative fields of view of transmitted light and fluorescence microscopy images of Red yeast rice 04-877, which is either carrying a selection marker or a GFP expression construct with the RPS3A promoter of Red yeast rice integrated at the CAR2 locus.

[0047] Figures 7A-7B The DGA1 knockout in *Rhodotorula basilicum* 04-877 is shown. 7A shows a diagram of the knockout construct and the primer locations used to screen for gene disruption. 7B shows the results of PCR using internal primers, demonstrating the removal of the wild-type DGA1 sequence.

[0048] Figures 8A-8BThe images show a decrease in TAG and an increase in PEFA production / g glucose in *Rhodotorula basheerii* strain 04-877 with the DGA1 gene (dga1) disrupted. Image 8A shows a representative field of view of photomicrographs of 04-877 cells with wild-type or disrupted DGA1 genes after fermentation in nitrogen-limited medium. Image 8B shows PEFA production / g glucose during shake-flask fermentation.

[0049] Figures 9A-9B The diagram shows a design for replacing an endogenous gene promoter with a new promoter, with or without an N-terminal peptide tag or degradation terminator.

[0050] Figures 10A-10B The diagram shows a design to replace the endogenous gene terminator with a new terminator, with or without a C-terminal peptide or degradation tag. Detailed Implementation

[0051] This article presents non-naturally occurring polyol lipid-producing microorganisms engineered to express heterologous selection markers to alter the expression of endogenous genes, including knockout or silencing, and / or to improve the yield and / or production of polyol lipids in microbial cultures. Transformation methods and polynucleotide constructs for producing such microbial strains are described. Methods for culturing the engineered microorganisms described herein and for producing polyol lipids in microbial cultures are also described.

[0052] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Singleton et al. Dictionary of Microbiology and Molecular Biology Second edition, John Wiley and Sons, New York (1994), and Hale and Markham, The Harper Collins Dictionary of Biology Harper Perennial, New York (1991) provides a general dictionary of many terms used in this invention for those skilled in the art. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention.

[0053] Unless otherwise stated, the practice of this invention will employ conventional techniques from molecular biology (including recombinant technologies), microbiology, cell biology, and biochemistry, which are within the scope of this art. Such techniques are well explained in the literature, for example, Molecular Cloning: A Laboratory Manual Second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, editor, 1984;) Current Protocols in Molecular Biology(FM Ausubel et al., eds., 1994); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Gene Transfer and Expression: A Laboratory Manual (Kriegler, 1990).

[0054] The range of numbers provided in this article includes the numbers that define that range.

[0055] Unless otherwise stated, nucleic acids are written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction.

[0056] definition

[0057] “A,” “an,” and “the” include plural references unless the context clearly specifies otherwise.

[0058] In this article, the term “about” means plus or minus ten percent (10%) of a value. For example, “about 100” refers to any number between 90 and 110.

[0059] "Acetylated 3-hydroxy fatty acid" refers to a fatty acid compound in which the hydroxyl group on the third carbon of the acid terminus of the fatty acid is acetylated.

[0060] As used herein, the term "similar sequence" refers to a polypeptide sequence within a protein that provides similar function, tertiary structure, and / or conserved residues relative to a reference protein. For example, in epitope regions containing α-helical or β-sheet structures, substituted amino acids in a similar sequence retain the same structural elements. In some embodiments, a similar sequence is provided that results in the variant enzyme exhibiting similar or improved function relative to its derived parent protein.

[0061] The phrase “and / or” as used herein in the specification and claims should be understood to mean “any one or both” of the elements so connected, i.e., elements that coexist in some cases and exist separately in others. Unless expressly stated otherwise, other elements may optionally be present in addition to those expressly indicated by the “and / or” clause, whether related to or unrelated to the expressly indicated elements. Thus, as a non-limiting example, when used in conjunction with open-ended language such as “comprising,” reference to “A and / or B” may in one embodiment refer to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on.

[0062] The terms "bioreactor" or "fermenter" refer to a closed or semi-closed container used for cell growth and maintenance. Cells may, but are not necessarily, held in a liquid suspension. In some embodiments, cells may instead be grown and / or maintained in a liquid suspension in, or in, or in other non-liquid matrices (including, but not limited to, solid growth support materials).

[0063] "Code sequence" or "CDS" refers to a polynucleotide sequence that encodes the amino acid sequence of a polypeptide.

[0064] The term "culture" refers to the cultivation of a group of cells, such as microbial cells, in a liquid or solid culture medium under suitable growth conditions.

[0065] The term “derived from” includes the terms “originating from,” “derived from,” “available from,” “separated from,” and “created from,” and generally indicates that a particular material is derived from another particular material or has features that can be described by reference to another particular material.

[0066] As used in this article, "expression" refers to the process by which a gene's nucleic acid sequence produces a polypeptide. This process includes transcription and translation.

[0067] As used herein, an "expression vector" refers to a DNA construct containing a DNA coding sequence (e.g., a gene sequence) operatively linked to one or more suitable control sequences capable of enabling the expression of the coding sequence in a host. Such control sequences include promoters influencing transcription, optional operon sequences controlling such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. The vector can be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, can integrate into the genome itself. Plasmids are the most commonly used form of expression vector. However, this invention aims to include other forms of expression vectors that have equivalent functionality and are known or to be known in the art.

[0068] A "gene" is a DNA segment that is involved in the production of polypeptides, including regions before and after the coding region and intron sequences between individual coding segments (exons).

[0069] "Gene knockout" or "knockout" refers to the loss of function of endogenous genes through genome editing. For example, genetic engineering can be used to inactivate or remove one or more specific gene sequences in a host microorganism from which a non-naturally occurring microorganism is derived.

[0070] "Gene silencing" or "gene knockdown" refers to regulating gene expression in cells, such as regulating the expression of endogenous genes to prevent gene expression at the transcriptional or translational level.

[0071] The term "heterologous" or "exogenous," in relation to polynucleotides or proteins, refers to polynucleotides or proteins that are not naturally present in a particular cell (e.g., a host cell). This term is intended to encompass proteins encoded by naturally occurring genes, mutant genes, and / or synthetic genes. Conversely, the term "homologous" or "endogenous," in relation to polynucleotides or proteins, refers to polynucleotides or proteins that are naturally present in a cell.

[0072] As used herein, a “homology” or “homogeneous protein” refers to a protein that has a similar function and / or structure to a reference protein. Homologous proteins can originate from evolutionarily related or unrelated species. In some embodiments, homologous proteins have quaternary, tertiary, and / or primary structures similar to those of the reference protein, which may allow for the replacement of fragments or segments in the reference protein with similar fragments or segments derived from the homologous protein, with less disruption to the structure and / or function of the reference protein compared to replacing fragments or segments with sequences derived from non-homologous proteins.

[0073] Homologous recombination refers to the result of a DNA metabolic process characterized by excision, strand invasion, DNA synthesis to repair excised ends, and exchange. This process can lead to new polynucleotides containing genetic modifications, where the host organism's nucleotide sequence is altered by using a foreign polynucleotide sequence homologous to the target alteration region. The gene product encoded by the foreign polynucleotide sequence can be similar to or completely unrelated to the replaced endogenous nucleotide sequence. Homologous recombination can be used to remove, suppress, alter, or modify the expression level of endogenous polynucleotides, express gene products similar to or related to endogenous polynucleotides, or express gene products dissimilar to or unrelated to the gene product of the endogenous gene.

[0074] As used herein, the terms “host cell” and “parent cell” are used interchangeably and refer to a cell or cell line that can be transfected with a recombinant expression vector to produce a polypeptide. Host cells include the offspring of a single host cell, and due to natural, accidental, or intentional mutations, the offspring may not necessarily be identical to the original parent cell (in morphology or total genomic DNA composition). Host cells can be naturally occurring or non-natural. Host cells include cells transfected or transformed in vivo with an expression vector.

[0075] The term "hydrophilic carbon source" refers to an organic compound that is soluble in water at a concentration greater than 1 g / L.

[0076] The term "hydrophobic carbon source" refers to an organic compound that is insoluble in water or has a solubility in water of less than 1 g / L.

[0077] "3-hydroxy fatty acids" refer to fatty acids in which the third carbon atom at the acid terminus is hydroxylated.

[0078] The term “introduction,” in the context of inserting a nucleic acid sequence into a cell, including “transfection,” “conversion,” or “transduction,” refers to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell, wherein the nucleic acid sequence can be incorporated into the cell’s genome (e.g., chromosome, plasmid, plasmid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed.

[0079] "Naturally occurring" or "wild-type" microorganisms refer to unmodified microorganisms or microorganisms found in nature.

[0080] "Non-natural" microorganisms are those that contain one or more mutations or have been genetically modified compared to the naturally occurring microorganisms from which they are derived.

[0081] The term "operationally linked" refers to the juxtaposition or arrangement of specific elements so that they can work synergistically to produce an effect. For example, if a promoter controls the transcription of a coding sequence, then the promoter is operably linked to the coding sequence.

[0082] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length and any three-dimensional structure, and of single or multiple strands (e.g., single-stranded, double-stranded, triple-helical, etc.), comprising deoxyribonucleotides, ribonucleotides, and / or analogs or modifications of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or analogs thereof. Because the genetic code is degenerate, multiple codons can be used to encode specific amino acids, and this invention covers polynucleotides encoding specific amino acid sequences. Any type of modified nucleotide or nucleotide analog can be used, provided the polynucleotide retains the desired function under the conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2'-O-Me, phosphate thioester, etc.). Labels, such as radioactive or non-radioactive labels or anchors, such as biotin, can also be incorporated for detection or capture purposes. The term polynucleotide also includes peptide nucleic acids (PNAs). Polynucleotides can be naturally occurring or non-natural. The terms "polynucleotide," "nucleic acid," and "oligonucleotide" are used interchangeably herein. Polynucleotides may contain RNA, DNA, or both, and / or modified forms thereof and / or analogs. The nucleotide sequence may be interrupted by non-nucleotide components. One or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate group is replaced by P(O)S (“thio”), P(S)S (“dithio”), (O)NR2 (“amide”), P(O)R, P(O)OR', CO, or CH2 (“methylal”), wherein each R or R' is independently H or a substituted or unsubstituted alkyl group (1-20 C), optionally comprising an ether (--O--) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl group. All links in a polynucleotide are not necessarily identical. Polynucleotides may be linear or cyclic, or a combination of linear and cyclic portions.

[0083] The term "polyol" refers to an organic moiety containing at least two hydroxyl groups. Exemplary polyols include glycerol (3-carbon), erythritol (4-carbon), threitol (4-carbon), arabinitol (5-carbon), xylitol (5-carbon), ribitol (5-carbon), mannitol (6-carbon), sorbitol (6-carbon), galactitol (6-carbon), fucitol (6-carbon), idutol (6-carbon), inositol (6-carbon; a cyclic sugar alcohol), vormiel (7-carbon), cyclic alcohols, and streptomycin. In some embodiments, the polyol is of the general formula HOCH2(CHOH). p CH2OH represents a value where p is 0-5.

[0084] "Fat fatty acid polyol ester" or "PEFA" refers to an amphiphilic molecule composed of acetylated (R)-3-hydroxy fatty acids esterified to 5- or 6-carbon polyols (usually d-mannitol or d-arabinitol) via carboxyl-terminal esterification, with varying degrees of acetylation.

[0085] "Polyol lipids" refers to compounds in which a polyol is partially covalently attached to a fatty acid.

[0086] As used herein, “peptide” refers to a composition consisting of amino acids and recognized by those skilled in the art as a protein. Conventional single-letter or three-letter codes for amino acid residues are used herein. The terms “peptide” and “protein” are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. These terms also include amino acid polymers that are naturally occurring or modified by intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as coupling with a labeled component. This definition also includes, for example, peptides containing one or more amino acid analogs (e.g., non-natural amino acids, etc.), and other modifications known in the art.

[0087] The term "production" includes the production of intracellular and extracellular compounds, including the secretion of compounds from cells.

[0088] "Productivity" refers to the amount of substance produced per unit volume per unit time during microbial fermentation. For example, the productivity of polyol lipids can be expressed as the number of grams of polyol lipids produced per liter of solution per hour.

[0089] A promoter is a regulatory sequence that participates in binding to RNA polymerase to initiate gene transcription. Promoters can be inducible or constitutive. An inducible promoter is a promoter that is active under environmental or developmental regulatory conditions.

[0090] The term "recombinant" refers to genetic material (i.e., nucleic acids, their encoded polypeptides, and vectors and cells containing such polynucleotides) that has been modified to alter their sequence or expression characteristics. Examples include mutating the coding sequence to produce an altered polypeptide, fusing a coding sequence with the coding sequence of another gene, placing a gene under the control of a different promoter, expressing a gene in a heterologous organism, expressing a gene at a reduced or increased level, conditionally or constitutively expressing a gene in a manner different from its natural expression profile, and so on. Typically, recombinant nucleic acids, polypeptides, and the cells based on them have been artificially manipulated to differ from their natural counterparts.

[0091] As used herein, the terms “recovered,” “separated,” “purified,” and “isolated” refer to material (e.g., proteins, lipids, nucleic acids, or cells) removed from at least one component in which it is naturally bound, for example, present in the sample in which it is contained at a concentration of at least 90% by weight, or at least 95% by weight, or at least 98% by weight, or at least 99% by weight, or at least 99.5% by weight. For example, these terms may refer to material that is substantially or essentially free of components (e.g., whole biological systems) that are normally present in their natural state.

[0092] The term "selective marker" or "selective marker" refers to a gene that can be expressed in a host cell, allowing for easy selection of hosts that contain an introduced nucleic acid or vector. Examples of selective markers include, but are not limited to, resistance to antimicrobial substances (e.g., hygromycin, bleomycin, or chloramphenicol), genes encoding proteins that are detectable upon expression, or genes that confer a metabolic advantage (e.g., nutritional advantage) on the host cell.

[0093] "Substantially free of" means that the content of a specific substance in the composition does not exceed about 10%, 5%, 1%, 0.5%, 0.2% or 0.1%.

[0094] In the context of at least two nucleic acids or polypeptides, the phrases "substantially similar" and "substantially identical" generally refer to a region or domain of a polynucleotide, polypeptide, or polypeptide that contains a sequence having at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity compared to a reference (e.g., wild-type) polynucleotide, polypeptide, or polypeptide region or domain. A polypeptide region or domain may, for example, contain at least about 20, 50, 100, or 200 amino acids within a longer polypeptide sequence. Sequence identity can be determined using known procedures (e.g., BLAST, ALIGN, and CLUSTAL) with standard parameters. (See, for example, Altshul, et al. (1990) J. Mol. Biol. 215:403-410; Henikoff, et al. (1989) Proc. Natl. Acad. Sci. 89:10915; Karin, et al. (1993) Proc. Natl. Acad. Sci. 90:5873; and Higgins, et al. (1988) Gene 73:237). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. Additionally, databases can be searched using FASTA (Pearson, et al. (1988) Proc. Natl. Acad. Sci. 85:2444-2448). In some embodiments, substantially identical polypeptides differ only by substitutions of one or more conserved amino acids. In some embodiments, substantially identical polypeptides exhibit immunological cross-reactivity. In some embodiments, substantially identical nucleic acid molecules hybridize to each other under stringent conditions (e.g., within a moderate to high stringency range).

[0095] "Sugar alcohol" refers to an organic compound in which a hydroxyl group (-OH) is attached to each carbon atom. Exemplary sugar alcohols include erythritol (4 carbons), threitol (4 carbons), arabinitol (5 carbons), xylitol (5 carbons), ribitol (5 carbons), mannitol (6 carbons), sorbitol (6 carbons), galactitol (6 carbons), and fucitol (6 carbons).

[0096] "Yield" refers to the amount of substance produced per unit volume during microbial fermentation. For example, the yield of polyol lipids can be expressed as the number of grams of polyol lipids produced per liter of solution.

[0097] “Transfection” or “transformation” refers to the insertion of exogenous polynucleotides into host cells. Exogenous polynucleotides can be maintained as non-integrating vectors (e.g., plasmids) or can be integrated into the host cell genome. The term “transfection” or “transfection” is intended to encompass all conventional techniques for introducing nucleic acids into host cells. Examples of transfection techniques include, but are not limited to, calcium phosphate precipitation, DEAE-glucan-mediated transfection, liposome transfection, electroporation, and microinjection.

[0098] As used herein, the terms “transformed,” “stable-transformed,” and “transgenic” refer to cells that have a non-natural (e.g., heterologous) nucleic acid sequence integrated into their genome or maintained as an addendum plasmid over multiple generations.

[0099] "Transcriptionally controlled" is a recognized term in the field, which means that the transcription of a polynucleotide sequence depends on its operative connection with elements that help initiate or promote transcription.

[0100] "Translation-controlled" is a recognized term in the field, referring to the regulatory processes that occur after mRNA formation.

[0101] As used in this article, "vector" refers to a multinucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, etc.

[0102] Related (and derived) proteins include “variant” proteins. Variant proteins differ from parent proteins and / or from each other by a small number of amino acid residues. In some embodiments, the number of different amino acid residues is any one of about 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, variants differ by about 1 to about 10 amino acids. Alternatively, or further, variants may have a specific degree of sequence identity with a reference protein or nucleic acid, for example, determined by using sequence alignment tools such as BLAST, ALIGN, and CLUSTAL (see below). For example, variant proteins or nucleic acids may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% amino acid sequence identity with the reference sequence.

[0103] "Yield" refers to the amount of product produced from a raw material (e.g., sugar) relative to the total amount of substance that would be produced if all the raw material were converted into the product. For example, polyol lipid yield can be expressed as the percentage of polyol lipid produced relative to the theoretical yield if 100% of the raw material (e.g., a carbon source such as sugar) were converted into polyol lipids. Non-limiting examples of carbon raw materials include glucose, xylose, and acetate.

[0104] microorganism

[0105] The non-naturally occurring microorganisms described herein are derived from parent microorganisms that produce polyol lipids (e.g., naturally occurring or wild-type microorganisms, or engineered non-naturally occurring parent microorganisms). In some embodiments, the microorganisms produce polyol lipids in commercially relevant quantities in microbial cultures without the addition of hydrophobic substrates. In some embodiments, the parent microorganisms are fungal microorganisms, such as yeast microorganisms.

[0106] In some implementations, the parent microorganism originates from the subkingdom of Dikaryotic Bacteria. This microorganism can belong to either the phylum Ascomycota or Basidiomycota. For example, it could belong to the phylum Basidiomycota, class Microsporidioides, order Sporoorder, genus *Rhodotorula*, genus *Rhodotorula*, or genus *Pyrodactylus*. In various embodiments, the microorganisms (e.g., basidiomycete yeast cells) are selected from Rhodotorula babjavae, Rhodotorula diobovata, Rhodotorula kratochvilovae, Rhodotorula graminis, Rhodotorula paludigena, Rhodotorula aff. paludigena, Rhodotorula sphaerocarpa, Rhodosporidiobolus aff. colostri, Rhodotorula dairenensis, Rhodosporidiobolus ruineniae, Rhodotorula taiwanensis, Rhodotorula muculaginosa, and Rhodosporidiobolus aff. (nylandii). In some embodiments, the microorganism is selected from the following strains: Rhodotorula bladderwynil strain NRRL Y-67018, Rhodotorula bladderwynil strain NRRL Y-67017, Rhodotorula bladderwynil strain UCDFST 68-916.1, Rhodotorula bladderwynil strain UCDFST 67-458, Rhodotorula bladderwynil strain UCDFST 05-736, Rhodotorula bladderwynil strain UCDFST 04-830, Rhodotorula diopside strain NRRL Y-67015, Rhodotorula kratochevilova strain NRRL Y-67016, Rhodotorula swampyra strain NRRL Y-67012, Rhodotorula swampyra strain UCDFST 82-646.2, Rhodotorula swampyra strain UCDFST 81-492, Rhodotorula similar to Rhodotorula swampyra strain NRRL Y-67009, Rhodotorula sacchariformis strain NRRL Y-67010, Rhodotorula dalianis strain NRRL Y-67011, NRRL Y-67014 (similar to Rhodotorula colostrum), NRRL Y-67013 (similar to Rhodotorula natans), NRRL Y-17302 (similar to Rhodotorula roximans), MD1149 (similar to Rhodotorula taiwanensis), and 50-3-19 / 208 (similar to Rhodotorula mucilaginosa).

[0107] Non-limiting examples of microbial species and strains that produce polyol lipids and can be engineered according to the methods described herein are described in U.S. Patent Nos. 10,196,663 and 11,401,539.

[0108] Polyol lipids

[0109] The microorganisms described herein naturally produce polyol lipids and can be grown in vegetative growth media to produce polyol lipids in microbial cultures. In some embodiments, the microorganisms secrete polyol lipids into the vegetative growth medium. In some embodiments, when grown in the vegetative growth medium, the microorganisms produce at least about 1 g / L of polyol lipids. In various embodiments, the microorganisms can produce one or more polyol lipids selected from: esterification of a 3-hydroxy fatty acid moiety of 6 to 24 carbon atoms to a sugar alcohol moiety; esterification of an acetylated 3-hydroxy fatty acyl moiety to a sugar alcohol moiety; esterification of a sugar alcohol moiety containing a non-esterified hydroxyl group to the carboxyl terminus of the 3-hydroxy fatty acyl moiety; esterification of a sugar alcohol moiety containing a mannitol or arabinitol backbone to the carboxyl terminus of the 3-hydroxy fatty acyl moiety; and fatty acid polyol esters (PEFAs).

[0110] In some embodiments, microorganisms produce one or more PEFAs. PEFAs are amphiphilic molecules comprising sugar alcohols, such as d-mannitol and / or d-arabinitol, esterified to the carboxyl terminus of a 3-hydroxy fatty acyl moiety, which may or may not be acetylated. The non-esterified hydroxyl group of the sugar alcohol may also be acetylated or unacetylated. In various embodiments, the microbially produced polyol lipids described herein may be mixtures of similar compounds comprising (R)-3-hydroxy fatty acyl moieties with different chain lengths (ranging from about 6 to about 24, about 6 to about 12, about 12 to about 24, about 10 to about 18, about 10 to about 24, or about 12 to about 20 carbons). In various embodiments, the (R)-3-hydroxy fatty acyl moieties may exhibit different degrees of unsaturation ranging from 0 to about 6 (e.g., from about 2 to about 5). In various embodiments, the non-esterified hydroxyl group of the sugar alcohol may be esterified to an acetyl group. In various embodiments, the sugar alcohol may be fully acetylated, partially acetylated, or unacetylated.

[0111] In some embodiments, the polyol lipid sugar alcohol moiety comprises a 5-carbon polyol, which may be d-arabinitol or l-arabinitol. In some embodiments, the polyol lipid sugar alcohol moiety comprises a 6-carbon polyol, which may be d-mannitol or l-mannitol. In some embodiments, the polyol lipid sugar alcohol moiety is attached to or bound to the carboxyl terminus of a fatty acid. In some embodiments, the fatty acid is hydroxylated at a position 4-3 from the carboxyl terminus. In some embodiments, the hydroxyl group at the 4-3 position may or may not be acetylated.

[0112] In various embodiments, one or more PEFAs produced in and / or isolated and / or purified from microbial cultures as described herein are selected from: acetylated C12:0 3-hydroxy fatty acid esterified to 3-acetylated d-arabinol; acetylated C14:0 3-hydroxy fatty acid esterified to 4-acetylated d-mannitol; acetylated C16:0 3-hydroxy fatty acid esterified to 2-acetylated d-mannitol; acetylated C16:0 3-hydroxy fatty acid esterified to 3-acetylated d-arabinol; acetylated C14:0 3-hydroxy fatty acid esterified to 4-acetylated d-arabinol; acetylated C14:0 3-hydroxy fatty acid esterified to 5-acetylated d-mannitol; acetylated C16:0 3-hydroxy fatty acid esterified to 3-acetylated d-mannitol; acetylated C16:0 3-hydroxy fatty acid esterified to 3-acetylated d-arabinol; acetylated C16:0 3-Hydroxy fatty acids esterified to d-mannitol with 4 acetylations; acetylated C18:0 3-Hydroxy fatty acids esterified to d-mannitol with 2 acetylations; acetylated C16:0 3-Hydroxy fatty acids esterified to d-mannitol with 5 acetylations; acetylated C16:0 3-Hydroxy fatty acids esterified to d-arabinol with 4 acetylations; acetylated C18:0 3-Hydroxy fatty acids esterified to d-mannitol with 3 acetylations; acetylated C18:0 3-Hydroxy fatty acids esterified to d-mannitol with 4 acetylations; acetylated C18:0 3-Hydroxy fatty acids esterified to d-arabinol with 3 acetylations; acetylated C18:0 3-Hydroxy fatty acids esterified to d-arabinol with 5 acetylations; acetylated C20:0 3-hydroxy fatty acids esterified to 3-acetylated d-mannitol; and acetylated C20:0 3-hydroxy fatty acids esterified to 4-acetylated d-mannitol.

[0113] Microbial transformation

[0114] The polyol lipid-producing microorganisms described in this article can be transformed with polynucleotide constructs to produce non-naturally occurring microorganisms with altered expression of one or more endogenous genes and / or one or more heterologous genes. Typically, the polynucleotide constructs are linear DNA sequences that will be integrated into the microbial genome, or DNA plasmids capable of autonomous replication within the microbial cell.

[0115] Polynucleotides (e.g., DNA) can be introduced into microbial cells via electroporation, PEG-mediated transformation, protoplast formation, or Agrobacterium-mediated mutagenesis. Transformed cells can be detected by the presence of selectable markers (e.g., antibiotic resistance), visible markers (e.g., lack of carotenoids), and / or auxotrophic responses to desired metabolites. Stable integrators can be identified by continuous passage on non-selective media followed by selection on selective media and / or by amplifying the junction between the genome and recombinant DNA (e.g., by polymerase chain reaction (PCR)). Transformants that integrate DNA via homologous recombination rather than non-homologous end ligation can be enriched by splitting the selectable marker into two fragments, such that homologous recombination between the selectable marker fragments is necessary for cell resistance to the selectable marker in cells where the DNA has been integrated.

[0116] Non-limiting examples of the choice of markers include enzymes that provide antibiotic resistance (e.g., hygromycin, norsinolate), fluorescent proteins, or enzymes that produce the desired metabolic protein.

[0117] Genetically modified polynucleotide constructs for use in the polyol lipid production microorganisms described herein may include: a promoter; and a nucleotide coding sequence providing a selection marker, wherein the promoter is capable of driving gene expression of the coding sequence in the microorganism. In some embodiments, the promoter in the polynucleotide constructs described herein is inducible in the microorganism.

[0118] In some embodiments, a polynucleotide construct for generating gene knockout (e.g., disruption or deletion of an endogenous gene sequence) in a host microorganism producing polyol lipids is provided, comprising: a promoter; a nucleotide sequence homologous to at least a portion of a target gene sequence in the microbial genome; a nucleotide coding sequence encoding a protein providing a selection marker; and a terminator, wherein the promoter and terminator sequences are capable of driving gene expression of the selection marker coding sequence in the microorganism.

[0119] In some embodiments, a polynucleotide construct is provided for expressing a heterologous gene in a host microorganism producing polyol lipids. The polynucleotide construct comprises: a promoter; a nucleotide-coding sequence of the heterologous gene; and a nucleotide-coding sequence encoding a protein providing a selection marker, wherein the promoter is capable of driving gene expression of the coding sequence in the microorganism. The nucleotide-coding sequence of the heterologous gene may further include an amino acid sequence (“tag”) that facilitates the detection or recovery of the heterologous gene expression product. Non-limiting examples of such sequences include His tag sequences, such as a His tag sequence having six to nine consecutive histidine residues, a FLAG tag sequence having the amino acid sequence DYKDDDDK (SEQ ID NO: 1), and a Myc tag sequence having the amino acid sequence EQKLISEEDL (SEQ ID NO: 2).

[0120] In some embodiments, the promoter of an endogenous gene can be replaced by a different promoter, and / or a 3' untranslated region (UTR) and / or a 5' UTR can be added to the endogenous gene sequence, thereby increasing, decreasing, or altering the regulation of endogenous gene expression or the regulation of mRNA stability transcribed from the endogenous gene. For example, a polynucleotide construct for replacing a promoter and adding a 3' UTR and / or an N-terminal tag in a polyol lipid production host microorganism is provided, comprising: a nucleotide sequence homologous to at least a portion of a target gene sequence in the microbial genome; a promoter sequence; and a 3' UTR and / or a polynucleotide sequence encoding an N-terminal tag. In another example, a polynucleotide construct for replacing a promoter and adding a 5' UTR and / or a C-terminal tag in a polyol lipid production host microorganism is provided, comprising: a nucleotide sequence homologous to at least a portion of a target gene sequence in the microbial genome; a promoter sequence; and a terminator sequence, a 5' UTR, and / or a polynucleotide sequence encoding a C-terminal tag.

[0121] In some implementation schemes, such as Figures 9A-9B The diagram illustrates DNA sequences homologous to the endogenous gene to be labeled and / or the promoter to be replaced. Transformation can be performed using... Figure 9A The splitting marker system shown is completed, producing, as... Figure 9B The gene shown has an altered promoter and / or peptide sequence, wherein the promoter of the target gene has been replaced by a new promoter and / or fused to an N-terminal tag.

[0122] In some implementation schemes, such as Figures 10A-10B The diagram illustrates DNA sequences homologous to the endogenous gene to be labeled and / or the terminator to be replaced. Transformation can be performed using... Figure 10A The splitting marker system shown is completed, producing, as... Figure 10BThe gene shown has an altered terminator and / or peptide sequence, wherein the terminator of the target gene has been replaced by a new terminator and / or fused to a C-terminal tag.

[0123] In some embodiments, the coding sequence of the polynucleotide construct (e.g., the coding sequence of a selection marker or a heterologous gene) is codon-optimized for expression in the host microorganism. Non-limiting examples of codon usage in the microorganisms described herein are provided in Tables 1 and 2. In some embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the codons in the nucleotide coding sequence are the most common or second most common codons in the host microorganism.

[0124] Table 1: Codon Usage Table of Red Yeast Rice

[0125]

[0126] Table 2: Codon Usage Table of Rhodotorula buergerianum

[0127]

[0128] Alterations in endogenous gene expression

[0129] This document provides genetically modified polyol lipid-producing microorganisms in which one or more endogenous genes or specific polynucleotide sequences are partially, substantially, or completely deleted, silenced, inactivated, downregulated, or upregulated. In some embodiments, the non-naturally occurring microorganisms described herein contain at least one knocked-out (e.g., disrupted or deleted) endogenous gene and / or at least one endogenous gene whose expression is altered, compared to parental microorganisms derived from naturally occurring or non-naturally occurring microorganisms.

[0130] In some implementations, the microorganism contains at least one knocked-out gene. For example, endogenous genes can be knocked out or edited via homologous recombination, CRISPR, zinc finger nucleases, or TALEN.

[0131] In some implementations, the microorganism contains at least one silenced gene. When genes are silenced, their expression is reduced. Conversely, when genes are knocked out, they are completely absent from the organism's genome and therefore not expressed. For example, the expression of endogenous genes can be silenced by RNA interference, RNA silencing, siRNA, antisense oligonucleotides, ribozymes, or CRISPR-based methods (e.g., dCas9 or Cas13).

[0132] In some implementations, an endogenous gene sequence is fused with a sequence encoding a peptide to increase, decrease, or alter the expression regulation of the endogenous gene. For example, the peptide could be the auxin-inducible degrader IAA7, an N-terminal fusion of ubiquitin, or the tobacco etch virus degrader tab TDegF.

[0133] Endogenous genes in the triglyceride (TAG) production and / or storage biosynthetic pathway can be knocked out, silenced, or have their expression levels reduced, resulting in non-naturally occurring microorganisms producing at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% less TAG than naturally occurring or parental microorganisms from which the non-naturally occurring microorganisms are derived. For example, enzymes in the biosynthetic pathway of TAG production and / or storage can be acyltransferases (e.g., diacylglycerol acyltransferase (DGAT) (EC 2.3.1.20) or phospholipid:diacylglycerol acyltransferase (LRO1) (EC 2.3.1.158)), hydrolases (e.g., phosphatidic acid hydrolase (Pah1) (EC 3.1.3.4)), dehydrogenases (e.g., glycerol-3-phosphate dehydrogenase (GPD1 / 2) (EC: 1.1.1.8)), or seipin (SEl1), or homologs thereof.

[0134] Endogenous genes involved in the production enzymes of the carotenoid biosynthetic pathway can be knocked out, silenced, or downexpressed, leading to a reduction or elimination of carotenoid production by microorganisms. This can provide a visual marker for transformation (e.g., white or substantially white colonies). Furthermore, polyol lipids extracted from fermentation, such as PEFA, may be whiter due to reduced or eliminated carotenoid levels in the culture medium. For example, enzymes in the carotenoid biosynthetic pathway could be bifunctional lycopene cyclase / hydrophobic acid synthase (CAR2) (EC 2.5.1.32) or its homologs.

[0135] Endogenous genes encoding enzymes that produce essential metabolites can be knocked out, silenced, or downexpressed, resulting in microorganisms auxotrophic to that metabolite. For example, knocking out or silencing the gene encoding 5-phosphate decarboxylase (ODCase) (URA3) (EC4.1.1.23) or cytosine deaminase (FCY1) (EC 3.5.4.1), or its homologs, leads to auxotrophic microbes resistant to 5-fluoroorotic acid.

[0136] Endogenous genes encoding enzymes involved in DNA repair, such as Ku70, Ku80, or Lig4, or their homologs, can be knocked out, silenced, or have their expression levels reduced, thereby improving homologous recombination.

[0137] Endogenous genes involved in peroxisome β-oxidation can be knocked out, silenced, or have their expression levels reduced, resulting in lower levels of fatty acid β-oxidation in microorganisms than in naturally occurring microorganisms or parental microorganisms from which non-naturally occurring microorganisms originate. For example, endogenous genes involved in peroxisome β-oxidation may encode peroxisome membrane E3 ubiquitin ligase (PEX10) (EC 2.3.2.27), 3-hydroxyacyl-CoA dehydrogenase / enoyl-CoA hydratase (MFE1; FOX2) (EC 4.2.1.119), acyl-CoA oxidase (POX1-6) (EC 1.3.3.6), peroxisome transporter (PXA1), or peroxisome fatty acyl-CoA synthase (FAA2) (EC 2.3.1.86), or their homologs.

[0138] Endogenous genes involved in mitochondrial β-oxidation can be knocked out, silenced, or have their expression levels reduced, resulting in lower levels of fatty acid β-oxidation in microorganisms than in naturally occurring microorganisms or parent microorganisms from which non-naturally occurring microorganisms are derived. For example, endogenous genes may encode enoyl-CoA hydratase (ECHS1) (EC 4.2.1.17) or its homologs.

[0139] Endogenous genes involved in the degradation of polyol lipids (such as PEFA) can be knocked out, silenced, or downexpressed, thereby enabling microorganisms to produce higher levels of one or more polyol lipids than naturally occurring microorganisms or parental microorganisms from which non-natural microorganisms originate. For example, the endogenous gene may encode a secretory lipase that degrades polyol lipids, or a homolog thereof.

[0140] In some embodiments, the non-naturally occurring microorganism contains a gene in which the promoter has been altered or replaced to increase, decrease, or modify the regulation of endogenous gene expression or the regulation of mRNA stability transcribed from the endogenous gene. For example, the promoter can be replaced (exchanged) via homologous recombination, CRISPR, zinc finger nucleases, or TALEN. In some embodiments, the endogenous gene is fused with a sequence encoding a peptide to increase, decrease, or modify the regulation of endogenous gene expression, such as, but not limited to, the auxin-inducible degrader IAA7, the N-terminal fusion of ubiquitin, or the tobacco-etched viral degrader tag TDegF.

[0141] In some implementations, the endogenous genes with altered expression levels encode mannitol dehydrogenase (EC 1.1.1.255), mannitol-1-phosphate phosphatase (EC 3.1.3.22), mannitol-1-phosphate dehydrogenase (EC 1.1.1.17), d-xylulose kinase (EC 2.7.1.17), arabinitol dehydrogenase (EC 1.1.1.11), glucose-6-phosphate dehydrogenase (EC 1.1.1.363), 6-phosphogluconolactonease (EC 3.1.1.31), 6-phosphogluconolactonease (EC 1.1.1.44), phosphoglucose isomerase (EC 5.3.1.9), transaldolase (EC 2.2.1.2), transketolase (EC 2.2.1.1), triacylglycerol lipase (EC 3.1.1.3), PEFA synthase, and acetyltransferase (EC 5.3.1.9). 2.3.-), long-chain fatty acid α-hydroxylase, PEFA transporter, fatty acid synthase (EC 2.3.1.86), acetyl-CoA carboxylase (EC 6.4.1.2) or malate dehydrogenase (EC 1.1.1.37), or their homologs.

[0142] In some embodiments, non-naturally occurring microorganisms modified as described herein to alter endogenous gene expression—for example, modified to partially, substantially, or completely delete, silence, inactivate, downregulate, or upregulate endogenous genes or specific polynucleotide sequences—produce and / or secrete one or more polyol lipids (e.g., PEFA) at higher yields than naturally occurring microorganisms or parental microorganisms from which they are derived. For example, compared to naturally occurring microorganisms or parental microorganisms from which non-naturally occurring microorganisms are derived, polyol lipids can be increased by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 250%, 260%, 270%, 280%, 290%, or 300%, or higher. The yield of polyol lipids produced by non-naturally occurring microorganisms can be greater than any of about 1 g / L, 25 g / L, 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 250 g / L, 300 g / L, 400 g / L or 500 g / L. The yield of polyol lipids produced by non-naturally occurring microorganisms can be from about 50 g / L to about 100 g / L, about 100 g / L to about 150 g / L, about 150 g / L to about 200 g / L, about 200 g / L to about 250 g / L, about 250 g / L to about 300 g / L, about 300 g / L to about 350 g / L, about 350 g / L to about 400 g / L, about 400 g / L to about 450 g / L, about 450 g / L to about 500 g / L, about 50 g / L to about 200 g / L, about 50 g / L to about 250 g / L, about 100 g / L to about 300 g / L, about 150 g / L to about 400 g / L, about 250 g / L to about 500 g / L, or about 300 g / L to about 500 g / L.

[0143] In some implementations, non-naturally occurring microorganisms that have been modified, as described herein, to alter the expression of endogenous genes, for example, by partially, substantially, or completely deleting, silencing, inactivating, downregulating, or upregulating endogenous genes or specific polynucleotide sequences, produce one or more polyol lipids (e.g., PEFA) in higher quantities than naturally occurring microorganisms or their parental microorganisms. For example, compared with parental microorganisms derived from naturally occurring microorganisms or non-naturally occurring microorganisms, the production of polyol lipids can be increased by at least approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, and 470%. %, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990% or 1000%, or higher. The yield of polyol lipids produced by non-naturally occurring microorganisms can be greater than any one of about 0.1 g / g, 0.05 g / g, 0.1 g / g, 0.15 g / g, 0.2 g / g, 0.25 g / g, 0.3 g / g, 0.35 g / g, 0.4 g / g, 0.45 g / g, 0.5 g / g, 0.55 g / g, 0.6 g / g, 0.65 g / g, 0.7 g / g, or 0.75 g / g.The yield of polyol lipids produced by non-naturally occurring microorganisms can be any one of about 0.01 g / g to about 0.75 g / g, about 0.01 g / g to about 0.05 g / g, about 0.05 to about 0.1 g / g, about 0.1 to about 0.25 g / g, about 0.25 to about 0.5 g / g, about 0.5 g / g to about 0.75 g / g, about 0.01 g / g to about 0.1 g / g, about 0.05 g / g to about 0.25 g / g, about 0.1 g / g to about 0.5 g / g, about 0.25 g / g to about 0.75 g / g, or about 0.2 g / g to about 0.6 g / g.

[0144] In some embodiments, non-naturally occurring microorganisms that have been modified, as described herein, to alter the expression of endogenous genes, for example, by partially, substantially, or completely deleting, silencing, inactivating, downregulating, or upregulating endogenous genes or specific polynucleotide sequences, also express heterologous selection markers, such as enzymes that provide antibiotic resistance (e.g., resistance to hygromycin or norsinomycin), fluorescent proteins, or enzymes that produce desired metabolites.

[0145] Expression of heterologous genes

[0146] This provides non-naturally occurring polyol lipid-producing microorganisms that have been modified for the expression of one or more heterologous gene sequences. The non-naturally occurring microorganisms contain one or more exogenous polynucleotides encoding and expressing one or more heterologous genes (i.e., one or more heterologous nucleotide-coding sequences).

[0147] Expression of heterologous gene sequences can lead to increased TAG degradation, for example, the expression of heterotriacylglycerol lipase (TGL1) or its homologs.

[0148] Expression of heterologous gene sequences can increase the biosynthesis of polyol lipids (e.g., PEFA). For example, one or more heterologous PEFA synthases, acetyltransferases, long-chain fatty acid α-hydroxylases (e.g., Ahd1 from Ustilago maydis; Uniprot A0A0D1DT68), or PEFA transporters, or their homologs, can be expressed or overexpressed.

[0149] Expression of heterologous gene sequences can increase fatty acid synthesis. For example, one or more heterologous fatty acid synthases (e.g., FAS1 / 2, for example, from Rhodotorula buergerianum), acetyl-CoA carboxylase 1 (ACC1), or malic acid esterase, or homologs thereof, can be expressed.

[0150] In some embodiments, the microorganism expresses one or more gene sequences encoding triacylglycerol lipase, PEFA synthase, acetyltransferase, long-chain fatty acid α-hydroxylase, PEFA transporter, fatty acid synthase, acetyl-CoA carboxylase, mannitol dehydrogenase, mannitol-1-phosphate phosphatase, mannitol-1-phosphate dehydrogenase, d-xylulose kinase, arabinose dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphate gluconate lactonease, 6-phosphate gluconate dehydrogenase, phosphoglucose isomerase, transaldolase, transketolase, or malate dehydrogenase, or homologs thereof.

[0151] In some embodiments, the non-naturally occurring microorganisms, as described herein, are modified by expressing one or more heterologous genes and produce and / or secrete one or more polyol lipids (e.g., PEFA) in higher yields than naturally occurring microorganisms or their parental microorganisms.

[0152] For example, compared to parental microorganisms derived from naturally occurring microorganisms or non-naturally occurring microorganisms, the yield of polyol lipids can be increased by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 250%, 260%, 270%, 280%, 290%, or 300%, or higher. The yield of polyol lipids produced by non-naturally occurring microorganisms can be greater than any one of about 1 g / L, 25 g / L, 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 250 g / L, 300 g / L, 400 g / L, or 500 g / L. The yield of polyol lipids produced by non-naturally occurring microorganisms can be about 50 g / L to about 100 g / L, about 100 g / L to about 150 g / L, about 150 g / L to about 200 g / L, about 200 g / L to about 250 g / L, about 250 g / L to about 300 g / L, about 300 g / L to about 350 g / L, about 350 g / L to about 400 g / L, about 400 g / L to about 450 g / L, about 450 g / L to about 500 g / L, about 50 g / L to about 200 g / L, about 50 g / L to about 250 g / L, about 100 g / L to about 300 g / L, about 150 g / L to about 400 g / L, about 250 g / L to about 500 g / L, or about 300 g / L to about 500 g / L.

[0153] In some embodiments, non-naturally occurring microorganisms, modified as described herein for the expression of heterologous genes, express heterologous selection markers, such as enzymes that provide antibiotic resistance (e.g., resistance to hygromycin or norsinomycin), fluorescent proteins, or enzymes that produce desired metabolites. Heterologous selection markers may be expressed alone or in combination with one or more heterologous genes (e.g., gene sequences encoding protein products, such as the enzymes described herein).

[0154] In some embodiments, non-naturally occurring microorganisms modified as described herein for expressing heterologous genes also express amino acid sequences (“tag” sequences) that are attached to the amino acid sequence of the gene expression product and facilitate the detection or recovery of the heterologous gene expression product. For example, the tag sequence may be a His tag sequence (e.g., six to nine consecutive histidine residues), a FLAG tag sequence having the amino acid sequence DYKDDDDK (SEQ ID NO: 1), or a Myc tag sequence having the amino acid sequence EQKLISEEDL (SEQ ID NO: 2).

[0155] Methods for producing polyol lipids

[0156] A method for producing polyol lipids from non-naturally occurring microorganisms described herein is provided. One or more non-naturally occurring microorganisms can be grown (cultured) in an environment (e.g., a bioreactor) containing a vegetative growth medium (culture medium) under conditions suitable for microbial growth and biosynthesis, including the biosynthesis of polyol lipids by the microorganisms. The culture medium contains a carbon source, a nitrogen source, inorganic substances (e.g., inorganic salts), and any other substances required for microbial growth (e.g., vitamins, amino acids, etc.).

[0157] Typically, one or more polyol lipids (e.g., selected from one or more of the following: esterification of a 3-hydroxy fatty acid moiety with a length of 6 to 24 carbon atoms to a sugar alcohol moiety; esterification of an acetylated 3-hydroxy fatty acyl moiety to a sugar alcohol moiety; esterification of a sugar alcohol moiety containing a non-esterified hydroxyl group to the carboxyl terminus of a 3-hydroxy fatty acyl moiety; esterification of a sugar alcohol moiety containing a mannitol or arabinitol backbone to the carboxyl terminus of a 3-hydroxy fatty acyl moiety; and fatty acid polyol esters (PEFA)) are secreted into the culture medium by microorganisms and can be recovered from the culture medium. In some embodiments, polyol lipids (e.g., but not limited to PEFA) are present at concentrations of at least about 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, and 425 g / L. Production is carried out at yields of 450 g / L, 475 g / L, or 500 g / L. The method can be performed as a batch, fed-batch, or continuous process. In some embodiments, a “continuous” method is used, wherein fresh culture medium is continuously added while the medium is continuously removed at the same or substantially the same rate, thereby maintaining a relatively constant culture volume.

[0158] Typically, the non-naturally occurring microbial-derived polyol lipids (e.g., but not limited to PEFA) described herein have a density of at least about 1.00 g / mL, for example, about 1.00 g / mL to about 1.10 g / mL. In some embodiments, polyol lipids can be recovered (harvested) from the culture medium without lysing the microbial cells. In some embodiments, polyol lipids can be recovered from the culture medium without using organic solvent extraction. In some embodiments, polyol lipids can be recovered from the culture medium using mechanical separation techniques (e.g., but not limited to centrifugation, decantation (e.g., continuous decantation), or passive sedimentation).

[0159] A bioreactor or other microbial culture environment is further provided, comprising the non-naturally occurring polyol lipid-producing microorganisms described herein in a vegetative growth medium. The bioreactor can be operated in a batch, fed-batch, or continuous system.

[0160] In some embodiments, the microbial culture comprises one or more hydrophilic carbon sources (e.g., monosaccharides or disaccharides, such as glucose and / or sucrose) (e.g., 30 g / L), yeast extract (e.g., 1.5 g / L), ammonium chloride (e.g., 0.5 g / L), potassium dihydrogen phosphate (e.g., 7.0 g / L), disodium hydrogen phosphate (e.g., 5.0 g / L), magnesium sulfate hexahydrate (e.g., 1.5 g / L), and micronutrient solutions containing various salts (e.g., 10 mL / L). In some embodiments, the microbial culture comprises iron salts. Illustrative but not limited to iron salts that may be used include, but are not limited to, ferric chloride (II) and (III), ferric sulfate (II) and (III), ferric nitrate (II) and (III), ferric phosphate (II) and (III), ferric fumarate, ferric edetate, ferric citrate, ferric malate, ferric oxalate, ferric tartrate, ferric succinate, and ferric acetate, in their anhydrous or hydrated states, and their organometallic complexes. For example, the culture medium may contain iron salts at concentrations ranging from about 0.001 mg / L to about 10 g / L, such as about 1 mg / L to about 0.5 g / L, or for example, about 0.01 g / L to about 0.5 g / L. In some embodiments, the microbial culture is supplemented with one or more hydrophilic carbon sources (e.g., monosaccharides or disaccharides, such as glucose and / or sucrose) at concentrations of at least about 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, or 300 g / L. In some embodiments, it contains one or more hydrophobic carbon sources, such as oils, alkanes, fatty acids, fatty esters, or mixtures thereof.

[0161] This method may include culturing a non-naturally occurring microbial community as described herein in a culture medium containing one or more hydrophilic (e.g., non-hydrophobic) carbon sources. In some embodiments, the culture medium does not contain a hydrophobic carbon source. In other embodiments, the culture medium contains one or more hydrophilic (e.g., non-hydrophobic) carbon sources and one or more hydrophobic carbon sources. In some embodiments, additional hydrophilic carbon sources are added, for example, at any stage of growth, including exponential growth, and after the microbial cell population has reached a stationary phase.

[0162] In various embodiments, the volume of the microbial culture medium is at least about 0.5 L, 1 L, 2 L, 3 L, 4 L, 5 L, 10 L, 25 L, 50 L, 75 L, 100 L, 250 L, 500 L, 1000 L or more.

[0163] In some embodiments, the culture medium contains less than about 2% (w / v) of nitrogen, or from about 0.005% (w / v) to about 2% (w / v) of nitrogen. For example, the nitrogen source may be selected from ammonia, ammonium salts, nitrates, nitrites, nucleotides, nucleosides, proteins, peptides, amino acids, urea and its derivatives, and mixtures thereof. In some embodiments, the culture medium contains about 0.05% (w / v) of ammonium chloride.

[0164] In various embodiments, the culture medium contains one or more hydrophilic carbon sources at a concentration of about 0.2% (w / v) to about 70% (w / v), for example, about 0.2% (w / v) to about 10%, 20%, 30%, 40%, 50%, or 60% (w / v). In various embodiments, the hydrophilic carbon source is selected from monosaccharides, oligosaccharides, polysaccharides, sugar alcohols, polyols, polyol lipids (e.g., fatty acid polyol esters (PEFA)), organic acids, esters, aldehydes, ketones, alcohols, waste streams, plant materials, lignocellulose hydrolysates, industrial by-products, and mixtures thereof. The hydrophilic carbon source can be a monosaccharide, oligosaccharide, or polysaccharide containing one or more sugar moieties, for example, selected from glucose, sucrose, xylose, galactose, rhamnose, arabinose, mannose, cellobiose, galacturonic acid, lactose, sophorose, glycerol, and mixtures thereof.

[0165] In some embodiments, the carbon-to-nitrogen ratio in the culture medium is about 5:1 to about 400:1, about 10:1 to about 200:1, about 20:1 to about 100:1, about 25:1 to about 75:1, about 30:1 to about 70:1, about 40:1 to about 60:1, or about 30:1 to about 40:1, for example, using a nitrogen source that can be used by microbial cells for growth and bioproduct production. In some embodiments, the microbial culture is maintained at a temperature range of about 24°C to about 27°C.

[0166] Polyol lipid products and compositions

[0167] Provided are polyol lipids or mixtures of two or more polyol lipids produced in any engineered microorganism described herein or in any microbial culture method described herein. In a related aspect, compositions incorporating polyol lipids produced in non-naturally occurring microorganisms as described herein are provided. The polyol lipids may include any polyol lipids described herein, such as those selected from: esterified 3-hydroxy fatty acid moieties of 6 to 24 carbon atoms to a sugar alcohol moieties; esterified acetylated 3-hydroxy fatty acyl moieties to a sugar alcohol moieties; esterified sugar alcohol moieties containing non-esterified hydroxyl groups to a carboxyl terminus of a 3-hydroxy fatty acyl moieties; esterified sugar alcohol moieties containing mannitol or arabinitol skeletons to a carboxyl terminus of a 3-hydroxy fatty acyl moieties; and fatty acid polyol esters (PEFAs).

[0168] In various embodiments, the polyol lipid-containing compositions are selected from detergents, washes, surfactants (e.g., for oil extraction), wetting agents, defoamers, emulsifiers, emollients, dispersants (e.g., for cleaning oil, including spills), humectants, antibacterial agents, antiviral agents, antifungal agents, spermicides, insecticides, lubricants, adhesives, crystal modifiers, instant solvents, viscosity modifiers, mixing / blending aids, release agents, creams, foams, mousses, lotions, lip balms, and ointments. In some embodiments, the polyol lipid compositions contain no or substantially no hydrophobic carbon sources.

[0169] Derivatives of the polyol lipids described herein, as well as derivatives produced by the non-naturally occurring microorganisms and methods described herein, are also provided. For example, derivatives may involve modification of the sugar alcohol or lipid moiety, or both, including hydroxylated fatty acids, d-mannitol, d-arabinitol, and derivatives thereof. Non-limiting examples of derivatives are described in PCT applications PCT / US23 / 67577, PCT / US23 / 67582, and PCT / US23 / 67583, all of which are incorporated herein by reference in their entirety.

[0170] Polyol lipid compositions can be used in a wide range of applications, including but not limited to household and industrial cleaners and detergents, textiles, agricultural chemicals (e.g., for controlling fungal and insect pests), food processing (e.g., cleaners for fresh and frozen fruits, vegetables, meats, and processed foods), food processing (e.g., rheology modifiers in food applications such as dough, pasta, and emulsions (e.g., mayonnaise, sauces, and syrups), food processing (e.g., anti-sticking agents), photochemicals, oil extraction (e.g., fracturing release agents), building materials (e.g., lubricants and release agents for bricks, ceramics, cement, and concrete), and mining (e.g., additives in the coal industry). Applications include pulp and paper industries, cosmetics (e.g., creams, foams, mousses, lip balms, ointments), personal care formulations (e.g., shampoos, shower gels, conditioners, soaps, creams, skin care and moisturizers), therapeutics (e.g., ointments and creams), spermicides, antibacterial agents, antiviral agents and anticancer agents, leather auxiliaries, fuel oil emulsification to improve atomization for more complete combustion, bioremediation of contaminated soils, groundwater and surface water, dust suppression in mines and quarries, release agents for asphalt truck bodies, promoting the use of castable water-based emulsions in the manufacture of explosives, components in metalworking fluids, and oleochemicals such as biodiesel and platform chemicals. Other non-limiting uses in engineered microorganisms and in polyol lipid compositions produced by this method are described, for example, in U.S. Patent Publications 2012 / 0022241 and 2013 / 0072414, both of which are incorporated herein by reference in their entirety.

[0171] The following examples are intended to illustrate, not limit, the invention.

[0172] Example

[0173] Example 1. Transformation of *Beggaria rubella* using antibiotic labeling

[0174] The experimental work described used the *Rhodotorula biceps* strain NRRL Y67018 (UCDFST 08-477). The cassette vector pTUB2>nat, containing the norsinoxin N-acetyltransferase (NAT) gene expressed by the *Rhodotorula buergerianum* promoter pTUB2, was adapted from the plasmid provided in Nora et al., 2019 (JPUB_013281). This sequence was mutagenized on a computer to remove the BsaI, BsmBI, and SapI sites and synthesized by order from Genewiz (SEQ ID NO: 3), and placed in a high-copy-count chloramphenicol select plasmid.

[0175] DNA for transformation was prepared by PCR amplification of the plasmid insert using SuperFi HotStart II polymerase with primers P1 GGACCATCTGAATCATGCGC (SEQ ID NO: 4) and P4 GTCGTGAGTTCGTGTCATCC (SEQ ID NO: 5). The reaction was performed in a 25 µL reaction volume containing 5 ng template, 0.5 µM primers, a 15 s annealing time, a 60°C annealing temperature, and a 60 s / kb extension time. Approximately 400 µL of polymerase chain reaction (PCR) was prepared for each reaction. PCR products were examined for purity on a gel, purified by isopropanol precipitation, and resuspended in water. DNA concentration was measured using a spectrophotometer (NanoDrop).

[0176] For cell transformation using electroporation, a method similar to that described in Coradetti et al., 2018, was employed. The strain was streaked onto yeast peptone glucose (YPD) medium, and single colonies were inoculated into 5 ml of YPD and incubated at 25°C for approximately 6 hours. The culture was then dediluted to 100 ml of YPD medium and cultured in a baffled flask to the logarithmic growth phase; under these conditions, the doubling time of the strain was approximately 2 hours. 10 ml of logarithmic growth phase cells (optical density (OD) of 2) were centrifuged at 3500 x g for 5 minutes at 4°C. From this point onward, the cells were kept ice-cold. The cells were resuspended in 1 ml of 0.75 M ice-cold d-sorbitol per 10 ml original volume and transferred to microcentrifuge tubes. The cells were washed 4x with 0.75 M sorbitol, centrifuged at 8000 x g for 30 seconds each time. After the final wash, the pellet was resuspended in 25 µL of 0.75 M sorbitol. The precipitate was then mixed with 5 µL of water containing approximately 2 µg of transforming DNA and transferred to a 0.1 cm ice-cold cuvette. Cells were electroporated using Bio Rad Gene Pulser II at 1.5 kV, 200 ohms, and 25 µF. Immediately after electroporation, cells were resuspended in 1 ml of ice-cold 1:1 0.75 M sorbitol / YPD mixture. This mixture was transferred to 14 ml cell culture tubes and incubated at 27°C and 250 rpm for 4 hours. The cell pellet was resuspended in 200 µL of yeast extract-peptone-glucose (YPD) and then plated on YPD agar containing 100 µg / ml noroside. Plates were incubated at 27°C for 3 days. This method yields approximately 250 colony-forming units (CFU) / µg of transforming DNA.

[0177] Red yeast rice was transformed using linear DNA encoding pTUB2>nat via the method described above. Water was used instead of the amplicon as a negative control. After recovery, the strain was serially diluted 1:10 and then spotted in 4 µL droplets onto YPD agar supplemented with different concentrations of noroside. Figure 1 At higher concentrations of noroside, there were significant differences between transformed cells and cells transformed without antibiotic selection markers. Based on this data, 100 µg / ml of noroside was determined to be sufficient for selection.

[0178] Example 2. Design and assembly of DNA with CAR2 and DGA1 knockouts via cleavage marker transformation

[0179] To construct the DNA construct for gene knockout, the DNA was designed so that the antibiotic resistance cassette pTUB2>nat was flanked by sequences homologous to the inner regions of the gene coding sequence, thereby disrupting the gene sequence. Figure 2A-2CTo obtain homologous sequences (homologous arms), we sequenced the genome of strain 08-477. Figure 2A DNA containing sequences homologous to genomic regions flanking the selection marker (in this case, the norsinolate resistance cassette pTUB2>nat) is displayed. Figure 2B This demonstrates the integration of heterologous DNA and / or removal of endogenous sequences via homologous recombination. Two plasmids, each containing a homologous arm and a selection marker (⅔), were prepared for each gene to be knocked out. Subsequently, linear DNA suitable for transformation was prepared from the plasmids using PCR. Homologous recombination selection between splitting regions in the selection marker was also performed on the transformation cassette with the genomically recombined cells. Figure 2C The diagram illustrates the genome sequence that was replaced by the selection box.

[0180] Strain 04-877, used for genome sequencing, was obtained from the Pfaff Collection at the University of California, Davis. The strain was streaked onto yeast peptone glucose (YPD) medium, and single colonies were inoculated into 5 ml of YPD and incubated overnight at 25°C. The saturated culture was then reverse diluted to 100 ml of YPD medium in a shaker-equipped flask and grown to the logarithmic growth phase. Logarithmic-phase cells were washed twice with distilled water (dH2O). Logarithmic-phase cells with 15 optical density (OD) units were resuspended in 10 ml of 1 M sorbitol, 0.1 M sodium citrate, 0.01 M EDTA, and 0.03 M β-mercaptoethanol (SCEM), pH 5.8. The cells were then digested overnight for 8 hours with 100 U / ml lysozyme at 30°C with gentle shaking, followed by digestion for 1 hour with 200 U / ml Zymolyase at 30°C. Genomic DNA was then isolated using the MasterPure™ Yeast gDNA Kit.

[0181] The strain was sequenced using a combination of nanopore and Illumina sequencing. The genome was assembled using Flye and Pilon, and gene prediction was performed using the FunAnnotate pipeline v1.5.3.

[0182] Based on the annotations generated above and using the TBLASTN of the CAR2 gene from *Rhodotorula buergerianum* as the query sequence (Uniprot A0A2Z6EYV3), the sequence of the CAR2 gene was identified. A high-quality BLAST-predicted protein ID was identified for 04-877. The genomic DNA predicting CAR2 in this strain is provided in SEQ ID NO: 6.

[0183] The DGA1 gene sequence was identified using the DGA1 gene (protein ID 16460) (SEQ ID NO:7) from the genome of *Rhodotorula glutinis* IFO0880 V4. A high-quality BLAST hit was identified for 04-877. The genomic DNA predicted to encode DGA1 in this strain is provided in SEQ ID NO: 8.

[0184] DNA for knocking out any of the genes in 04-877 was prepared using a modular cloning system for Jinmen assembly. Each DNA module was flanked by a BsaI site, so that when digested with BsaI, it would produce a DNA portion flanked by sticky ends suitable for plasmid assembly. Jinmen assembly was performed using NEB's BsaI V2 assembly kit, following the manufacturer's instructions. Figures 3A-3B ).

[0185] A kanamycin selection backbone for Kinmen assembly was created by splitting the kanamycin resistance gene into two separate plasmids. One plasmid (Int1B KanR5 / ColE1; PL427) contains the 5' sequence of the kanamycin resistance gene and the ColE1 origin of replication, while the second plasmid (Int1A KanR3) contains the 3' end of the kanamycin resistance gene. These plasmids contain flanking primer binding sites P1 and P4, allowing all plasmids constructed from these plasmids to be amplified using the same primers.

[0186] Plasmid In1B KanR5 / ColE1 PL427 (SEQ ID NO: 9) was constructed from a gene fragment containing the origin of replication and a kanamycin resistance gene, flanked by a suitable BsaI site (SEQ ID NO: 10), ordered from Twist Biosciences. This fragment included Twist adapters; these adapters were subsequently used for cloning. The commercially available NEB plasmid pGGASelect was amplified using oligonucleotides OLIGO305 (SEQ ID NO: 11) and OLIGO306 (SEQ ID NO: 12), which introduce Twist adapters into the linear chloramphenicol selection markers of the backbone. Subsequent amplicons were assembled using a HiFi DNA Assembly Kit according to the manufacturer's instructions.

[0187] The sequence In1A KanR3 (SEQ ID NO: 13) was ordered from Twist Bioscience and cloned into the chloramphenicol selective backbone.

[0188] To improve the homologous recombination rate, the norotiline selection marker was divided into two parts, located on different plasmids. The first fragment (Int9 NatR_L, SEQ ID NO: 14) contains the promoter and the 5' sequence of the norotiline resistance gene, and is cloned with the left homologous arm of the target gene. The second fragment (Int2 NatR_R, SEQ ID NO: 15) contains the 3' sequence of the resistance gene and the terminator, and is cloned with the right homologous arm of the target gene. The norotiline resistance genes share a 400-base-pair (bp) sequence, thus homologous recombination between them produces a functional marker. These DNA sequences were ordered from TwistBiosciences as plasmids cloned into the chloramphenicol selection backbone.

[0189] Homologous arms targeting CAR2 and DGA1 were synthesized through random mutations, with mutations relative to the wild-type sequences to remove the SapI and BsaI cleavage sites. To avoid disrupting the function of adjacent genes, the homologous arms were designed so that any mutations required for domestication were applied only within the coding sequence of the gene targeted for disruption. The homologous arms were designed such that, in the event of successful homologous recombination, they removed at least two exons from the gene center. The homologous arm sequences in 04-877 used for CAR2 disruption (with flanking BsaI sites for subsequent assembly) are: Int2 04-877 CAR2 HOML (SEQ ID NO: 16) and Int9 04-877 CAR2 HOMR (SEQ ID NO: 17). For DGA1, they are: 04-877 DGA1 HOML (SEQ ID NO: 18) and 04-877 DGA1 HOMR (SEQ ID NO: 19).

[0190] Each plasmid assembly used to knock out DGA1 or CAR2 contains a spacer sequence (Int345678 spacer sequence) SEQ ID NO 20. These sequences were ordered from Twist Biosciences as plasmids cloned into a chloramphenicol-selective high-copy backbone.

[0191] All plasmids used for assembly were prepared in small quantities using standard methods and resuspended in water.

[0192] Two Kinmen reactions were performed to assemble plasmids for CAR2 gene knockout in 04-877. The DNA fragments used to assemble the left plasmid (PL440; SEQ ID NO: 21) were: (1) Int1A, (2) Int1B, (3) Int2 04-877CAR2 HOML, (4) Int345678 spacer sequence, and (5) Int9 NatR L. The DNA fragments used to assemble the right plasmid (PL436; SEQ ID NO: 22) were: (1) Int1A, (2) Int1B, (3) Int2 NatR R, (4) Int345678 spacer sequence, and (5) Int9 04-877 CAR2 HOMR.

[0193] Similarly, two Kinmen reactions were performed to assemble plasmids for DGA1 gene knockout. The DNA fragments used to assemble the left plasmid (PL439; SEQ ID NO: 23) were: (1) Int1A, (2) Int1B, (3) Int2 04-877DGA1 HOML, (4) Int345678 spacer sequence, and (5) Int9 NatR L. The DNA fragments used to assemble the right plasmid (PL435; SEQ ID NO: 24) were: (1) Int1A, (2) Int1B, (3) Int2 NatR R, (4) Int345678 spacer sequence, and (5) Int9 04-877DGA1 HOMR.

[0194] Example 3. DNA design for GFP expression

[0195] The coding sequences for heterologous expression were optimized in DNA Chisel using a codon usage table (Table 2) determined based on the Rhodotorula buergerianum genome sequence. To facilitate coding sequence synthesis, we specified avoidance of homopolymeric sequences longer than 6 base pairs (bp) in DNA Chisel and removed recognition sites for BsaI, BsmBI, SapI, and BbsI restriction enzymes to facilitate modular assembly techniques.

[0196] A 3x-FLAG-tagged GFP coding sequence was ordered from Twist Bioscience and cloned into a chloramphenicol-selective backbone with SapI sites on its flanks (PL091; SEQ ID NO: 25).

[0197] Strong promoters with no perfect homology to the host genome are ideal because they minimize undesirable recombination with the host genome. We sought to identify promoters that might be sufficient to drive the expression of a heterologous gene in a new host. Furthermore, to be compatible with modular assembly techniques, certain mutations must be made to the promoters (e.g., removal of the IIS morphology site), which could affect their function. Therefore, we tested a variety of promoters from several *Rhodozyma* species to identify a promoter that supports strong GFP expression in strain 04-877. Below, we report a sequence that supports the expression of a heterologous gene in this organism.

[0198] We extracted a 1000 bp sequence upstream of the small ribosomal subunit sequence RPS3A from *Rhodotorula taiwanensis* MD1149 (SEQ ID NO: 26). Each sequence was randomly mutated to remove the SapI, BsaI, and BsmBI restriction sites from the promoter and terminator sequences. These sequences were then ordered and synthesized into a chloramphenicol-selective backbone by Twist Biosciences. The ordered sequences were flanked by SapI restriction sites for subsequent assembly. ATG and TAG were used as overhangs between the promoter / CDS and CDS / terminator sequences, respectively, to achieve seamless assembly.

[0199] As a terminator, the RPS3A terminator (SEQ ID NO:27) from Rhodotorula glutinis IFO0880 V4 was used.

[0200] The GFP expression cassette was integrated into the CAR2 locus. When CAR2 was disrupted, white colonies formed, facilitating the recognition of DNA integration at the target site. The expression backbone was constructed in the same manner as PL440 (04-877 CAR2 KO left plasmid; SEQ ID NO: 21), except that a high-copy chloramphenicol selection plasmid containing the Int345678 SapI exfoliation sequence (SEQ ID: 28) was used instead of the Int345678 spacer sequence (SEQ ID NO: 20). The backbone (PL434 04-877CAR2 GFP left backbone) was constructed using plasmids (1) Int1A, (2) Int1B, (3) Int2 04-877 CAR2 HOML, (4) Int345678 Sap I exfoliation, and (5) Int9 NatRL to generate (PL434 04-877 CAR2 SapI exfoliation; SEQ ID NO: 29) Figures 4A-4B ).

[0201] The GFP expression vector was cloned into PL434 using the SapI-based Golden Gate reaction assembly.

[0202] The GFP-expressing plasmid was assembled with the PL434 backbone (SEQ ID NO: 29); the shared GFP coding sequence (SEQ ID NO: 25); the terminator sequence (SEQ ID NO: 27); and the promoter sequence (SEQ ID NO: 26) to generate PL451 (SEQ ID NO: 30). Cloning was performed using SapI, by assembling a reaction containing 2 µL of 10X ligase buffer, 1.5 µL of SapI-HF enzyme (10 U / µL), and 1.25 µL of T4 ligase (400 U / µL), with 3 nM of each DNA fragment, for a final volume of 20 µL. The reaction was then cycled 30 times between 5 min at 37 °C and 5 min at 16 °C. The resulting assembly was transformed into NEB10-beta and sequenced as described above.

[0203] Example 4. CAR2 knockout

[0204] DNA from PL440 (SEQ ID NO: 21) and PL436 (SEQ ID NO: 22) was prepared by PCR using SuperFi HotStart II polymerase to generate linear amplicones on the plasmid insert fragment, and was used to transform Red Bastard yeast 04-877. Primers P1 (SEQ ID NO: 4) and P4 (SEQ ID NO: 5) were used. Homologous recombination selection between dividing regions in the selection markers was also performed on the transformation cassette with the genomically recombined cells.

[0205] The gene product Car2p from the CAR2 locus, a bifunctional lycopene cyclase / hydrolycopene synthase, is involved in the carotenoid biosynthesis pathway in Rhodotorula rubrum. If it is disrupted, the cell will not produce the red pigment typically seen in Rhodotorula rubrum colonies. Therefore, it provides a direct indication of whether transforming DNA has integrated into this locus via homologous recombination. Furthermore, the carotenoids produced by yeast during fermentation are hydrophobic and thus isolated within the PEFA produced by the strain. This can result in an excessively dark color in the resulting PEFA. Therefore, it is desirable to produce PEFA from strains that do not exhibit this coloration.

[0206] Approximately 1.5 µg of linear DNA from plasmids targeting each CAR2 locus (04-877) was transformed into *Rhodotorula glutinis*, as follows: Figure 2A-2C As shown. We observed that out of approximately 100 colonies grown on the plate, about 10 were white (…). Figures 5A-5BWe re-stripe colonies on selective media and observed that the color phenotype was stable. To confirm that the DNA encoding the CAR2 locus had been successfully removed, we amplified the genomic DNA extracted from the CAR2 knockout strain using primers that amplify the DGA1 or CAR2 region. Figure 5C Primers used to amplify CAR2 in the CAR2 knockout strain (OLIGO496 ACCTCACCTCGACCCTGTACGC (SEQ ID NO: 31) and OLIGO497CGGGCGAGAGGAAGGAAGGTCA (SEQ ID NO: 32)) did not produce amplicons, while primers used for DGA1 (OLIGO502TCTTCGGCTACCACCCTCACGG (SEQ ID NO: 33) and OLIGO503 ACGCCGCACAGAGAGACGATCT (SEQ ID NO: 34)) produced amplicons for the CAR2 knockout strain. Figure 5D The obtained strain was stored in 20% glycerol at -80°C.

[0207] Example 5. Heterologous gene expression

[0208] Linear DNA for transforming *Rhodotorula baicalensis* 04-877 was produced by PCR from PL451 (SEQ ID NO: 30) using primers P1 (SEQ ID NO: 4) and P4 (SEQ ID: 5), as described above, and purified using a Zymo Clean and Concentrator 25 kit and eluted with deionized water. These plasmids contained the left two-thirds of the noroside resistance gene and the left homologous arm targeting the CAR2 locus. Linear DNA was also produced from PL436 (SEQ ID NO: 22) in the same manner. This plasmid contained the right two-thirds of the noroside resistance gene and the right homologous arm targeting the CAR2 locus. For each transformation, 1.5 µg of DNA from the left plasmid and 1.5 µg of DNA from the right plasmid were transformed as described above. After recovery, cells were plated on YPD supplemented with 100 µg / ml noroside and incubated at room temperature for approximately 72 hours, at which point white and red colonies were visible on the plate. White colonies generated from each transformation were streaked onto YPD+Nat100 medium, and then single colonies were inoculated into YPD and incubated at 25°C for 72 hours. GFP was observed using an Echo microscope with a FITC filter set at 40X magnification for 0.5 seconds. As a control, white colonies generated by transformations via PL440 and PL436, which disrupted the CAR2 locus but did not introduce GFP, were imaged. Figure 6Cells containing the GFP expression cassette showed green fluorescence, while the control did not.

[0209] Example 6. DGA1 knockout

[0210] DNA from plasmids PL439 (SEQ ID NO: 23) and PL435 (SEQ ID NO: 24) was amplified using primers P1 (SEQ ID NO: 4) and P4 (SEQ ID NO: 5) and purified as described above. Approximately 1.5 µg of each resulting linear fragment was transformed into *Rhodotorula glutinis* 04-877 as described above. To examine the integration of the antibiotic resistance marker at DGA1, three sets of primers were used: (1) OLIGO500 (SEQ ID NO: 35) and OLIGO514 (SEQ ID NO: 36), which amplified approximately 1.5 kb of sequence on the left homologous arm; (2) OLIGO502 (SEQ ID NO: 37) and OLIGO503 (SEQ ID NO: 38), which amplified approximately 0.5 kb of sequence within the homologous arm of the genomic DNA, which was removed after the antibiotic marker integrated into the DGA1 locus; and (3) OLIGO515 (SEQ ID NO: 39) and OLIGO505 (SEQ ID NO: 40), which amplified approximately 1.5 kb of sequence on the right homologous arm. Figure 8A ).

[0211] The proposed knockout strain was re-streaked onto YPD+Nat100 medium. A single colony from the re-streaked plate was inoculated into 3 ml of YPD and incubated for 24–48 hours. Genomic DNA (gDNA) was isolated using 2 ml of the culture via the ZymoQuick DNA Beading Kit, following the manufacturer's instructions. The gDNA was then screened using the primers and SuperFi Hotstart II polymerase described above, following the manufacturer's instructions. Several colonies were identified that showed positive amplicones on both left and right PCR, no amplification was observed in the DGA1 internal PCR, and positive amplification was observed at the CAR2 locus primers. Figure 7B The obtained strain was stored at -80°C in 20% glycerol.

[0212] Example 7. Evidence of reduced TAG and increased PEFA production in strain dga1

[0213] Seed culture for shake-flask fermentation was initiated by inoculating 50 µL of glycerol stock solution of wild-type Rhodotorula begivirida strain 04-877 and strain 04-877 dga1 into 5 ml of YPD medium and incubating in a bioreactor tube at 25°C for 16-24 hours.

[0214] As described by Gary et al. (2017), strain 04-877 dga1 was cultured together with wild-type 04-877 in medium A using 15 ml of medium with glucose as the carbon source at 50 g / L, in 250 ml glass shake flasks at 25°C and 250 rpm with stirring. A 5% seed culture was used for inoculation. Glucose consumption was monitored using a GlucCell device. After approximately 135 hours of growth, glucose was depleted in the shake flask containing the wild-type strain, but the shake flask containing the 04-877 dga1 knockout strain still had 12.5 g / L of glucose and was no longer being consumed. Micrographs of both strains were collected. The size of the TAG droplets visible in the micrographs was used as a guideline. Figure 8A The dga1 strain (04-877) showed significantly reduced TAG accumulation compared to the wild type. PEFA yield was measured using a protocol adapted from Garay et al. (2017). Briefly, three volumes of ethyl acetate were added to a flask, stirred at 550 rpm for 1 hour with a magnetic stir bar, allowed to stand for 30 minutes, and then 10 ml of the ethyl acetate supernatant was transferred to a pre-weighed borosilicate tube. The ethyl acetate was evaporated to measure the total PEFA yield produced. The wild type and the dga1 disruptive strain produced average yields of 2.74 g / L and 3.1 g / L of PEFA, respectively, and PEFA yields per gram of glucose of 0.055 g / g and 0.082 g / g, respectively. The dga1 disruptive strain produced approximately 49% more PEFA per gram of glucose; this difference was statistically significant (Welch two-sample t-test, p = 0.022). Figure 8B ).

[0215] Example 8. Candidate genes for the PEFA biosynthetic pathway

[0216] The EST1 protein sequence (Uniprot A0A385XI13; SEQ ID NO: 41) from *Aureobasidium melanogenum* was BLASTP queried against the predicted protein database of *Rhodotorula bergipani* 04-877 to search for homologs. A single protein (SEQ ID NO: 42) was returned with an E value of 1.48e-03. BLAST analysis against the Uniprot database identified homologs of this protein, which were annotated as "serine hydrolase FSH domain-containing protein" in *Rhodotorula gracilis* (A0A194S8T6, 88.1% identity) and *Rhodotorula diopside* (A0A5C5FMS2, 79.5% identity).

[0217] To locate additional candidate genes, the CLOCI algorithm described by Konkel et al. (2024) was applied, which identifies putative biosynthetic gene clusters based on unexpectedly conserved homolinear levels. Functional annotations within these clusters were then examined to identify clusters potentially involved in PEFA biosynthesis. Two putative biosynthetic clusters were identified whose PFAM domains are consistent with the proposed PEFA biosynthetic pathway described by Garay et al. (2018).

[0218] The first proposed cluster contains a single copy of fatty acid synthase α protein 04-877 (FAS2), a protein labeled “fatty acid hydroxylase” (protein ID: 2353) (SEQ ID NO: 43), and a protein labeled “sterol-binding protein (esterase)” (protein ID: 2354) (SEQ ID NO: 44). Other genes in the cluster are not obviously associated with PEFA synthesis. These genes may be involved in: (1) adding a 3-OH group to acyl-CoA derived from central fatty acid biosynthesis; and (2) forming ester bonds between the fatty acid tail and polyol head of PEFA, respectively.

[0219] The second cluster contains: (1) a gene annotated as mannitol dehydrogenase (protein ID: 5143) (SEQ ID NO: 45); (2) a gene annotated as glycosyltransferase (protein ID: 5152) (SEQ ID NO: 46); and (3) a gene annotated as choline / carnitine O-acyltransferase (protein ID: 5149) (SEQ ID NO: 47). These genes may be involved in: (1) the production of polyols; (2) the formation of ester bonds between polyols and fatty acid tails; and (3) the acetylation of 3-OH groups on the polyol head and / or fatty acid tail of PEFA.

[0220] Although the foregoing invention has been described in detail with reference to illustrations and embodiments for clarity of understanding, those skilled in the art will recognize that certain changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. Therefore, this specification should not be construed as limiting the scope of the invention.

[0221] All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety and to the same extent as each individual publication, patent or patent application is specifically and individually indicated as incorporated by reference.

[0222] Nucleotide and amino acid sequences

[0223] SEQ ID NO: 1

[0224] FLAG tag

[0225] DYKDDDDK

[0226] SEQ ID NO: 2

[0227] Myc tag

[0228] EQKLISEEDL

[0229] SEQ ID NO: 3

[0230] An insert from PL011 (pTUB2>nat); containing P1 and P4 binding sites.

[0231]

[0232] SEQ ID NO: 4

[0233] P1 synthetic primer

[0234] GGACCATCTGAATCATGCGC

[0235] SEQ ID NO: 5

[0236] P4 synthetic primer

[0237] GTCGTGAGTTCGTGTCATCC

[0238] SEQ ID NO: 6

[0239] 04-877 CAR2 gDNA, exons underlined

[0240] ATGGGTGGCTTCGACTACTGGCTCGT GTGCGTCCCTCTCTCTCTCTCTCTCCCTGCGCTCGCCGCAGCCGCACCGGCTGAGCCCTCGCTGACCCCTCGTCGTCGCCGTTGCTGCAG CCATGCGCGTTGGACGATCCCTCCCTCGGT CGCGCTGTGGCTCGTGTTCCGCAAGCTGCGGACCTGGAGGGACGTGTACAAGACGCTGTTCCTCATCACG GTCCGTCTCGAGCTCCCCCCGCGGCGAGACGGCCAAGGCTGACCTCCTCCCCTCTTTGCTCCGAGCAG ATCGCTGTCACG GTGCGCCGCTCTGCGCGCTCGAGACTAGGCGCACGCGCTCGGGAGAGCTGACCCTTGCCCCTCGCAG GCGACGATACCCTG GGATTCGTACCTCATCCGGAACCGC GTGCGTCCTCGTCCTCCTCCTCACCCAACCCTCCCGAGCCGCGACGGCGAGCTGACTCGCTCCCTCGCTCCGCTCGCAACAG ATTCTGGTCCTACCCGGACTCGTCCGTCGTCGGCCCGACCCTGTTCGC CATCCCCTACGAGGAGGTCTTCTTCTTCTTCGTCCAGACCTACCTCACCTCGACCCTGTACGCCGTCCTCACGCGCC CGATGTCCACGCGACCCTCCTCCCGCGCACGCCGAGCGAAGGGCGCACCGTCAAGTGGACCGGCACGGCGCTGCTG TGCGGCGTCTTTGCCCTGTCGTGGGCCAAGCTCGAGGAGGGCGGCGAGGGGACCTACCTCGCCCTCATCGTCGGCTG GGTCGCGCCGTTCCTCACCTTGCTCTG GTGCGTCCTCGCTCCTCTCATCGTCCCCTGCTCGAGGTGCCGAGCTGACTTTGGCTCTCGCAG GTGGGTCGCGTCTGAGCACATCTGCGCCATGCCGCGCGCGACGCTCCTCCTCGCCATCTTCGCC CCGACCGTCTTCCTGTGGGAGCTCGACGCGCGGCCTCTGCAGCGTGGCACCTGGGTCATCGAGCAGGGGACCAAGCT CGGGTGGGACTTCCGCGGGCTCGAGATCGA GTGCGTCCTCTCTCCCCTCCTCGCCCTCTCCTCGCTCGCCTCGCTGACTCTCCCTCGCCCGCTCGCAG GGAGGCCGTCTTCTTCCTCTTGACCAACGTCATGATCGTCTTTGGCATGGCCGCCT GGTGCGTGCGCCCTGCTGTAAGCTCGAGAGCGAGCGGCGCTGACCTTCCTTCCTCTCGCCCGCAG CGACCACTGCCT CGCCGTCCACGACCTGCGCTCGTATGACAAGGGCACCTCGTCCGTCTTCCCGGCGCTCCACCGAGTTCGGCCCAATCC TCGTCAACTCGCCCGACGCCAAGCAGGGGCAGCGCATCGACGACCTGCGCGCCGCCATCGAGATCCTGTCGGTCCAC TCGAAGAGCTTCTCGACGCGAGCATGGTCTTTGACGGTCGGCTGCGGCTCGACCTCCTCTCCTT GTGCGTCTCTCTCTCTCTCTCTCTCTCTGCATCTTGCTCACAAAGAGGAGCCCGAGCTGACTTGACTGTGCAG GTAGCGCTGGTGCGCGC GTCTGCGACGACCTCGTCGACAAGCCCTCGTCGGTCGCCGCCGCCGAGGCCAACATCGACAAGATCAAGTCGTGCCCT CGACCTCCTGTACCCTCGCGCGACGTCGACGCCGACCTCGCACCCCGTCGCCGTCTCGAACGACGCCATCGCCGCCG CCCTCCCCGGCTTGAGCGAGCCCGAGCGCGGCTCGTTCCGCCTCCTCGCCCTTTCCCCCATCACTCGCCCCCCCCTC GACGAGCTCCTCGCCGGCTTCCGCACCGACTTGTCGTTCCTCGCCTTCGCCGGCGAGAAGGAGAGCCCGGCTCGAG CACGTCGATCCCCCGAGCTGCCGATCAAGACGGACGCCGACCTGCTCGAGTACGCCAACAACGTCGCGTCGTCGG TCGCCGACCTGTGCGTGCAGCTCGTGTGGGCGCACTGCGCCTCGTCGGTGCCCGAGCCCGAGCAGCGCGCCATCCTC GCCGCCGCGCGCGAGATGGGCCAGGCGCTCCAGCTCGTCAACATCGCGCGCGACGTGCCGGCCGACCGCGACATCCA CCGCATCTACCTCCCCGGCCGGTCGCCCGAGGTCGCCGTCGAGGCCATGACGCCCGACCGGCGAGAGCTCCTGCGCC GCGCGCGCGCCATGGCGGCGCACAGCCGCGAGGCGATCGAGCGCCTGCCGCGCGAGGCGAGGGGCGGGATCCGCGCG GCGTGCGACGTGTACCTGTCGATCGGCGGGGCGGTCGAGCGCGCGCTCGACGAGGGGAGGGTGCACGAGCGCGCGAG GGTTGCAAAGGGGACGAGGGCGTGGAAGGCCTGGACGGCGTTGTGA

[0241] SEQ ID NO: 7

[0242] Protein sequence of Rhodosporidium toruloides DGA1

[0243] MGQQATLEELYTRSEISKIKFAPFGVPRSRRLQTFSVFAWTTALPILLGVFFLLCSFPPLWPAVIAYLTWVFFIDQAPTHGGRAQSWLRKSRIWVWFAGYYPVSLIKSADLPPDRKYVFGYHPHGVIGMGAIANFATDATGFSTLFPGLNPHLLTLQSNFKLPLYRELLLALGICSVSMKSCQNILRQGPGSALTIVVGGAAESLSAHPGTADLTLKRRKGFIKLAIRQGADLVPVFSFGENDIFGQLRNERGTRLYKLQKRFQGVFGFTLPLFYGRGLFNYNVGLMPYRHPIVSVVGRPISVQQKDHPTTADLEEVQARYIAELKRIWEDYKDAYAKSRTRELNIIA*

[0244] SEQ ID NO: 8

[0245] 04 - 877 DGA1 gDNA, exons underlined

[0246] ATGGGCGCACAAGAAGAGGTCGACTACGACCAGTCGGAGCACTCCAAGATTAA GTGCGCCCTCTCCCTCGTCCTCCTCCTCGCCCTTCCCCCACCTCGTCCCCGAGCACCCGGTCTCAGGCCGCACACAGCACGCCAGACCCTGCGCACCGTGCCAAAGCTGACCAGTACCTCGTCTGCGTCCTGTGCAG GTTCGTACCCTTTGTCGTCCCGCGGCATCGTCG CCTTCAGACCTTCTCGGTGTTCCTCTGGACGACGGCCCTCCCGCTCTCGCTCGGCATCTTCTGCATCCTCTG GTGCGCTCAAACTCGCTCGTCAAGCTCCAGCTACACTGTGGAGGCGCGGAACGCTGACTCTCGTACACCCTCGCAG CTCGTT CCCTCCCCTGTGGCCGCTCGTCATCGGCTACCTCACCTGGGTCTTCCTCATTGACCAGGCGCCGATGCGCGGCGGGC GGCCACAAGCGTGGCTGCGAAAGTCGCGAGTGTGGGAGTGGTTCGCCGGATACTACCCCGTCAG GTGCGCTCCACTTTGCCCTCCTCGTCGTCGCGAAGCCTCTCCTCGCAAACGAGCGCGAGCTGACCAGTCCTCCCTGGTCCTTCTCGGCGCTCAG CCTGATCAAG GTACGTCGAGCGCACGCCCTCTTCAACAAAGCACCGCCGGCCGCGGACGAGTTGTCCACGCCGCGCAGGATCTCCTAGCTGACCCTCTCTCCCTCGCCCTGCAG AGCGCCGACCTCCCGCCCGACCAGCGCTACGTCTTC GGCTACCACCCTCACGGCGTCATCGGCATGGGCGCCATCGCCAACTTTGGCACCGACGCGACCGGCTTCTCGCGCCT GTTCCCGGGCATCACGCCGCACCTCCTCACGCTCGCGAGCAACTTCCAGCTGCCGCTGTACCGCGAGCTCCTCCTCG GCCTCGGCATCTCGTCCGTCTCGATGAAGAGCTGCCAGAACATCCTGCGGCAAG GTGCGTCTCGCGCTCTCCCACCCTGCGTTCGACTCGACCGAGCCCCGGCTGACCCTCCCTCGACCCTGCGCGCAG GTCCTGGCTCGTCCATCACGATCGT CGTTGGCGGTGCAGCCGAGAGCCTGAGCGCGCACCCTGGCACCGCCGACCTGACGCTCAAGCGGCGCAAGGGCTTCA TCAAGCTCGCCATCCGCACCGGCGCCTCGCTCGTCCCCGTCTTTTCCTTTGGCGAGAACGAC GTGCGTCCCCTCTCTCTCTTGTAGCTGCCCTGAGATCGTCTCTCTGTGCGGCGTGCTGACCTCGTCGACGTGCGCGCAG ATCTTCAACCAGC TGTCGAACGAGCGAGGGACGCGCTTGTACAAGCTGCAGAAGCGGTTTCAGGCCGTCTTTGGCTTCACTTTGC GTGCGTCCTTGTCTCTCGCTCTCTCGCTACGACCCATCGAGTGAATCTGACGCTCGGCGACGTGCGCGCAG CCATCTTCTTC GGCCGCGGCCTGTTCAACT GTGCGTCGCTCGCCCTCGTCGATTCCTGCTCTCGACCGGCGCACTGACCCTCTCCTGCTTCCACGCTCGCAG ATAACATGGGCTTGATGCCTTACCGACACCCAATCGTCTCTGTCG GTGCGTCCACCTCTTCCTTCCTGCGCCGGCGCGCGAGAGACGGTCGAGCCTCCTCCTCGCTCAGGAGCTGACGTGCGCTCGGCCCACCTCCCTCTTGCAG TTGGCCGTCCGATCAAGGTCAAGCAGAAGGACCACCCCTCGACTGCCGACCTCGAGGAGGTGCAGGAGCGCT ACATCGCAGAGCTCAAGAG GTCCGTCCCCATACTCGAGCTCTCCCCCCTCGCCTCGCAGACCTCGGCGCTGACGCCTCGTCGCGCTCGTCCGCAG GATCTGGGAGGACTACAAGGAGGTGTACGCCAAGAGCCGCACCAAGGAGCTCACCATCA TTGCGTGA

[0247] SEQ ID NO: 9

[0248] PL427 Int1B KanR5 / ColE1 plasmid

[0249]

[0250] SEQ ID NO: 10

[0251] The Int1B KanR5 / ColE1 gene fragment is flanked by the BsaI site.

[0252] GGTCTCAATTC TTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCA TCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATC TGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGAT AGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTT AATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGA CAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTT GTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTC CACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTG CAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAAC TGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTG TAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACC GGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAG CTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGA GAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCC TGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCG GAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCT TTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGC CACATGAGACC

[0253] SEQ ID NO: 11

[0254] OLIGO305 ChlorR Twist3 FWD

[0255] CTACTCTGGCGTCGATGAGGGAGTAGACTTCTTAATTAAGACGTCA

[0256] SEQ ID NO: 12

[0257] OLIGO306 ChlorR Twist5 REV

[0258] CGGTTGTAGTGAGGGCGGATTGTTCGAATTCGGATCCCTCGAGC

[0259] SEQ ID NO: 13

[0260] The Int1A KanR3 gene fragment is flanked by the BsaI site.

[0261] CGGTCTCACCGC GGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTA CAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTAT CAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGA TCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTT ATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTT GTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCC TGAGCGAGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACAC TGCCAGCGCATCAACAATATTTTCACCTGAATCAGGAT ATTCTGAGACCG

[0262] SEQ ID NO: 14

[0263] The Int9 NatR L gene fragment is flanked by the BsaI site.

[0264] CGGTCTCAGCTA GCGACGACGTGTGGGGCTACCGCTCGCTCGAGCCGACGCGGTGCTGCATCGCGTCGA TCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCGCCGGCGAGCCCGACG GCGCCCGAGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCT CGCGACGGCGCAGAAGCTGCTTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGT CGAGCGCAGTGACTGACTGTGGCTGTCACACGGGCCCTGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCT CGCGAACGGCAAGGAGGTCAAGATCTGGGCGCGGCGAGTGTGGACGCTCGAGCTGAGCCTGATCTGAGCCGTCGTCG TCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTCGCGATACCCCCCCTTTTCCCTCGCTCGTCTTG TTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGTGCGTCCAGCGAGGGCGA GAGAGAGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCC CCCCTCGCCCCATCTTCCACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCT AGACCCCGACTGACCCGTCCACCCACTCCCGCAAGCCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGT ACCGCACCAGTGTCCCGGGGGACGCCGAGGCCATCGAGGCACTGGATGGGTCCTTCACCACCGACACCGTCTTCCGC GTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCCCTGACCAAGGTGTTCCCCGACGA CGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTCGTCGCGTACGGGGACGACG GCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCC CCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGG GCACCTCTGGCTGGAGGTCACCAACGTTAACGCACCGGCTATCCACGC CCGCTGAGACCG

[0265] SEQ ID NO: 15

[0266] The Int2 NatR R gene fragment is flanked by the BsaI site.

[0267] CGGTCTCACACA TGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACG GCGACCCGGACTCCCGGACGTTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCC GGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTT GATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGTCACCAACGTTAACGCAC CGGCTATCCACGCGTACCGGCGGATGGGGTTCACCCTCTGCGGCCTGGACACCGCCCTGTACGACGGCACCGCCTCG GACGGCGAGCAGGCGCTCTACATGAGCATGCCCTGCCCCTAAGCTTATTCACCTGCACTCGTCCAACCTCGTCGTCG TCGTGTCCCCCTCCCCGTCCGTTTGCCCCTTCGTCTTTTGTCGTCGCCCGCCCGCCCGGCCCCCTCGAGCGGTTGCG GCCCTTACCCCCCACCCACACTACTTCTGACGTTCTTTCGTCACGTTCTCCGTCGGCTCTACCCCCCTGCCAAGTCA AAGGCTGGTTTCTCTCGTCGGCTCGGGCGCGCGCGAGATGGGCTCGAGAGTGAGAGCGAGAGGAGCAGCAGGCCGAG TTGGCTCGGCGGTCTGGGCTCAGGACTGGCGTCGCAGCATGGGCGTCGCACGTTCAGACCGTCTGGACGTGAGAGGC TCGGCTACTCGTTGACGACGAGGTCAGAGCGACCTTGCCGCCACGCAGTCGGCTCTACTTTTCCGCCGTCTCTCGTC GGCCTCACCCAGGCGCGGTGTGGCACTGCCTTTCTCGCGTCTCTCGCGAGAGCGCGCCTCTCGTCGAAAGCCAACCT CGCTCGCGAGA AGCGTGAGACCG

[0268] SEQ ID NO: 16

[0269] Int2 04-877 CAR2 HOML gene fragment, flanked by BsaI loci.

[0270] CGGTCTCACACA TCGAGGTGCTCCTGGCCGAGGCCGAGTGGCGCCTGGCGGTGAACGACGAGGTCCCGG TCTGCTGAGCGTGACGCGGTGGTGCGGGAGGGCGAGCGGCTCGTCGGCGAGAGCCGGTGGGCGGCGGGGCCTTGGTA TGCGCTGTGCAGCAAGTGCGTGAGGAGCAGCAAGGTGAGAGGTGCACAGAAGGAGAGAGCCCATGGCCGAAGGCATG GAGGCGATGATGCTGGTTCGGCGCACGCCGGCCAAAGTTGGCCTGCGTCGAGATCGGTCGCCAATTCGGCAGCTGCT GCGTCCAGTCGAAACCGCAACCTTAGCGTTGCCTTACCTTTTCGCAACCAGCCCGCTTGTAAGCCAGCGAGGCCGAG TTATGTACCGGCTCTGCCGATTCGAGCGGGTTCGAGTTGGAGCCCCCGCGCGCGCGGGCTCCGCGCTTCGCTCCGAG CTCCGAGGCCGCCGCGCGCGCGACAAGTCGCACATCCTCATCTCGCCCTCCTCACCGGCCACCGCTCGCTCGCTCGA TACCCCCTCTGTGCTCTCTGTTCCTCGCGTCTCGGCATGGGTGGCTTCGACTACTGGCTCGTGTGCGTCCCTCTCTC TCTCTCTCTCCCTGCGCTCGCCGCAGCCGCACCGGCTGAGCCCTCGCTGACCCCTCGTCGTCGCCGTTGCTGCAGCC ATGCGCGTTGGACGATCCCTCCCTCGGTCGCGCTGTGGCTCGTGTTCCGCAAGCTGCGGACCTGGAGGGACGTGTAC AAGACGCTGTTCCTCATCACGGTCCGTCTCGAGCTCCCCCCGCGGCGAGACGGCCAAGGCTGACCTCCTCCCCTCTT TGCTCCGAGCAGATCGCTGTCACGGTGCGCCGCTCTGCGCGCTCGAGACTAGGCGCACGCGCTCGGGAGAGCTGACC CTTGCCCCTCGCAGGCGACGATACCCTGGGATTCGTACCTCATCCGGAACCGCGTGCGTCCTCGTCCTCCTCCTCAC CCAACCCTCCCGAGCCGCG CTGAACTGGCCGATAATTGCAGACGAGCGTGAGACCG

[0271] SEQ ID NO: 17

[0272] Int9 04-877 CAR2 HOMR gene fragment, flanked by BsaI sites.

[0273] CGGTCTCAGCTA CATCTTGCTCACAAAGAGGAGCCCGAGCTGACTTGACTGTGCAGGTACGCCTGGTGC CGCGTCTGCGACGACCTCGTCGACAACGCCTCGTCGGTCGCCGCCGCCGAGGCCAACATCGACAAGATCAAGTCGTG CCTCGACCTCCTGTACCCTCGCGCGACGTCGACGCCGACCTCGCACCCCGTCGCCGTCTCGAACGACGCCATCGCCG CCGCCCTCCCCGGCTTGAGCGAGCCCGAGCGCGGCTCGTTCCGCCTCCTCGCCCTTCTCCCCATCACTCGCCCCCCC CTCGACGAGCTCCTCGCCGGCTTCCGCACCGACTTGTCGTTCCTCGCCTTCGCCGGCGAGAAGGAGAGCGCCGGCTC GAGCACGTCGATCCCCGCCGAGCTGCCGATCAAGACGGACGCCGACCTGCTCGAGTACGCCAACAACGTCGCGTCGT CGGTCGCCGACCTGTGCGTGCAGCTCGTGTGGGCGCACTGCGCCTCGTCGGTGCCCGAGCCCGAGCAGCGCGCCATC CTCGCCGCCGCGCGCGAGATGGGCCAGGCGCTCCAGCTCGTCAACATCGCGCGCGACGTGCCGGCCGACCGCGACAT CCACCGCATCTACCTCCCCGGCCGGTCGCCCGAGGTCGCCGTCGAGGCCATGACGCCCGACCGGCGAGAGCTCCTGC GCCGCGCGCGCGCCATGGCGGCGCACAGCCGCGAGGCGATCGAGCGCCTGCCGCGCGAGGCGAGGGGCGGGATCCGC GCGGCGTGCGACGTGTACCTGTCGATCGGCGGGGCGGTCGAGCGCGCGCTCGACGAGGGGAGGGTGCACGAGCGCGC GAGGGTTGCAAAGGGGACGAGGGCGTGGAAGGCCTGGACGGCGTTGTGAGGGCGGGCGTCGGTGCATAGGGGAGGAC TTGGGACGTAGACTAGACTGCGCAGCTCGTGTAAAGAGAGAAAGGATCCTGCGTGCTTCCCTCGTCGTGCTCGTCTC GCTCGAGCTCTCCGAGCTCT CCGCTGAGACCG

[0274] SEQ ID NO: 18

[0275] Int2 04-877 DGA1 HOML gene fragment, flanked by BsaI sites.

[0276] CGGTCTCACACA AGAGCGTGAGCGGTCGAAGGAGATGAGGACTCGACGAGGAGCTGATGAGAATCGCCG CCGAGACATGCGCTCCTGGCTCGCTCAAAGGCGTCTTCCGCTGAGCTCAGCCTACCGCTACAGAAGCTATGCACGGC GTAGCTCGCTCGGCGCTCGCTGGAGAAGACTCGCGCCTGCAGCTCCCCGCTTTTCGAGCACAACAGCTGCCTCTCGT TCGGGGTGCGCCTGGGTAGAGTCGCCTCCAGAACTGCGCGAGACTGGCCCAGCTGGAGAAGCACGCCGCAAGCCGAG GGCGACGAGACTGCGGCCGCGAGACGAGGCGCTGCCAGTCCCGTCCGCTCGGCGACTTGCTCGTCTCGCCTCACCAC ACGCCAGCATCCTCGCCTGGGGCCCAGGCTGCCCGACTCGCACGATGGGCGCACAAGAAGAGGTCGACTACGACCAG TCGGAGCACTCCAAGATTAAGTGCGCCCTCTCCCTCGTCCTCCTCCTCGCCCTTCCCCCACCTCGTCCCCGAGCACC CTGTCTCAGGCCGCACACAGCACGCCAGACCCTGCGCACCGTGCCAAAGCTGACCAGTACCTCGTCTGCGTCCTGTG CAGGTTCGTACCCTTTGTCGTCCCGCGGCATCGTCGCCTTCAGACCTTCTCGGTGTTCCTCTGGACGACGGCCCTCC CGCTCTCGCTCGGCATCTTCTGCATCCTCTGGTGCGCTCAAACTCGCTCGTCAAGCTCCAGCTACACTGTGGAGGCG CGGAACGCTGACTCTCGTACACCCTCGCAGCTCGTTCCCTCCCCTGTGGCCGCTCGTCATCGGCTACCTCACCTGGG TCTTCCTCATTGACCAGGCGCCGATGCGCGGCGGGCGGCCACAAGCGTGGCTGCGAAAGTCGCGAGTGTGGGAGTGG TTCGCCGGATACTACCCCGTCAGGTGCGCTCCACTTTGCCCTCCTCGTCGTCGCGAAGCCTCTCCTCGCAAACGAGC GCGAGCTGACCAGTCCTCCC CTGAACTGGCCGATAATTGCAGACGAGCGTGAGACCG

[0277] SEQ ID NO: 19

[0278] Int9 04-877 DGA1 HOMR gene fragment, flanked by BsaI sites.

[0279] CGGTCTCAGCTA TCGTCGATTCCTGCTCTCGACCGGCGCACTGACCCTCTCCTGCTTCCACGCTCGCAG ATAACATGGGCTTGATGCCTTACCGACACCCAATCGTCTCTGTCGGTGCGTCCACCTCTTCCTTCCTGCGCCGGCGC GCGAGAGACGGTCGAGCCTCCTCCTCGTCAGGAGCTGACGTGCGCTCGGCCCACCTCCCTCTTGCAGTTGGCCGTC CGATCAAGGTCAAGCAGAAGGACCACCCCTCGACTGCCGACCTCGAGGAGGTGCAGGAGCGCTACATCGCAGAGCTC AAGAGGTCCGTCCCCATACTCGAGCTTCCCCCCCTCGCCTCGCAGACCTCGGCGCTGACGCCTCGTCGCGTCGTCC GCAGGATCTGGGAGGACTACAAGGAGGTGTACGCCAAGAGCCGCACCAAGGAGCTCACCATCATTGCGTGACCGGCCG CCGCCGTCGGAAAAGTACCTACCCTCGCCGCTGCTCTCGCCTCGTCGCCGCCGTACCTCGCCCATCCTTCCGAGCGC CCTTTCACCTTCTGCGCCCCCTCCTCCGTACCGCGCTTCTGGCATGCAGCGAACTGTGTCCTCCCTCTTGCGGGCCGA GCGGCTCGCGGCGAGACGAGTTGTCGGTCGTGCGAGCTCTGGCGCTGCTTCTCGTGAGGGAGAGAGCCCTGAGAGCG CTCGTTCAGATGACAGCTGCTCGCGCCCTCGTCCGAGAAGCTCAGCGCGTCGCTCCCTTGCCGTCAGCGGGTCG GCCGGCGCGGGCGCCGGGAAGCCGCGGCCGGCCGGACGGCGCATTGATGAGGACAGCTCGCTCTCTCGCTTCCCAC CAGGCTGCGCTCCTCGTGCTTCCACGCTAGACCAGCCACCTGCTGCGCTCCCTCTGCTCATGACTGCTACGCGCGCCA TCGAGCTGGTGCCCTTCGATCCCGAGCGCCCGACCGAGGTCGACGAGCTCAAGCGCCAGCGCATTGTGAGCCCTCTT GCGCTCGCGAGGACCGCGCG CCGCTGAGACCG

[0280] SEQ ID NO: 20

[0281] The Int345678 spacer gene fragment is flanked by BsaI sites and filler sequences.

[0282] ACGGGGTCATCACGGCTCATCATGCGCCAAACAAATGTGTGCAATACACGCTCGGATGACTGCATGATGACCGCACTGACTGGGGACAGCAGATCCACCTAAGCCTGTGAGAGAAGCAGACACCCGACAGATCAAGGCAGTTACGGTCTCAAGCG AAGTGAATAAAGCTCCACACAGTCG GCTATGAGACCGGGTAGAGCCACAAACAGCCGGTACAAGCAACGATCTCCAGGACCATCTGAATCATGCGCGGATGACACGAACTCACGACGGGATCACAGACATTAACCCACAGTACAGACACTGCGACAACGTGGCAATTCGTCGCAATAC

[0283] SEQ ID NO: 21

[0284] PL440 04-877 CAR2 KO left plasmid

[0285]

[0286] SEQ ID NO: 22

[0287] PL436 04-877 CAR2 KO right plasmid

[0288]

[0289] SEQ ID NO: 23

[0290] PL439 04-877 DGA1 KO left plasmid

[0291]

[0292] SEQ ID NO: 24

[0293] PL435 04-877 DGA1 KO right plasmid

[0294]

[0295] SEQ ID NO: 25

[0296] The PL091 3x-FLAG-GFP gene fragment is flanked by SapI sites.

[0297] GCTCTTCA ATGGACTACAAGGACGACGACGACACAAGGACTACAAAGACGATGACGACAAAGACTATAAAG ACGACGATGACAAGGGCTCGGTGTCGAAGGGCGAGGAGCTCTTTACCGGCGTCGTCCCGATCCTCGTCGAGTTGGAC GGCGACGTCAACGGCCACAAGTTCTCGGTTTCGGGCGAGGGCGAGGGAGATGCCACCTACGGCAAGCTCACCCTCAA GTTCATCTGCACCACCGGCAAGTTGCCGGTCCCGTGGCCGACGCTCGTCACCACCCTCACCTACGGAGTCCAGTGCT TCTCGCGCTACCCGGACCACATGAAGCAGCACGACTTCTTTAAGTCGGCCATGCCGGAGGGCTACGTCCAGGAGCGC ACCATCTTCTTCAAGGATGACGGCAACTACAAGACCCGCGCCGAGGTCAAGTTTGAGGGCGACACCCTCGTCAACCG CATCGAGCTCAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTCGGCCACAAACTCGAGTACAACTACAACTCGC ACAACGTCTACATCATGGCGGACAAGCAGAAGAACGGCATCAAGGTCAACTTCAAGATCCGCCACAACATCGAGGAC GGCTCGGTCCAGCTCGCCGACCACTACCAGCAGAACACCCCGATCGGAGATGGCCCCGTCCTCTTGCCGGACAACCA CTACCTCTCGACCCAGTCGGCCCTCTCGAAGGACCCGAACGAGAAGCGCGACCACATGGTCCTCCTCGAATTCGTCA CCGCCGCCGGCATCACCCTCGGCATGGACGAGCTCTACAAGTAG TGAAGAGC

[0298] SEQ ID NO: 26

[0299] The RPS3A promoter gene fragment of *Rhodotorula fusiforme* is flanked by a SapI site.

[0300] GCTCTTCACGA CGCTAGCAGCTCGTGGCGCCATAAAAGCGTGCGAGTCACACGACCCGACTCGGCAGCA GAGCCTTAGGGTCGCGAGCACCGTGACCATGCGTAGAGCCTAGGGGCGTGCGGGACAATGTCGGTCGTGCATTGACG TGTGCTGCTGGACGCAAGAAGGCTGATGTTGGACCGTCGAGGCGTCAGGCGCTGTGGGCGGGGTAAGGTGCTAGGGC GTGTGCACCAGGGAGAGGGCGGCGAGCGCGGAGGGGAGCGGTGGCGGCTGTGAGCCAAAGTCAAGCAAGCTATCGTG TGTGTTCACCAGAAGACAGCACGTTACAGCGTAGAGGGATACGGTGAGGAAGGCGCGCGCAAAAAGCGGGTGCAGAG TCGAGGCCGGCGTCGCCGACACGCGGACGAGCGACCGACCGACTTGGTGTCGAGCGCGGCAACCTCGCTCTTGTCCC TCGCACCCTCGCCGCGTCTTCACGCTCGCCCTTACCCAACATGGGCAAGTCATTCTCACCTATACTATCGTCGCAGT CGACCCTCCGCCCTCCACCCTAGGCCCTCGCACGACGCGGCGGTGTCGGGAGGTGTCGCCGCTTCGAGCTGTGCCCA GTCGACCCGCTCGCAATTTTCGCCCGCCGACTCGACCCCGCCCGCTTCGCCCTGAGCACCGCCTGACCGGCCCTACA CTCGATGAGGGGGATTTAGAGTGTACTGGCGGTATCGTGCACGCGCCTGGGTGGGCGGGAAGGTTGGGTCGGCGGGT CGGCTAGGTCGAGCTCGGCAGGCACGGGTCGGCGGTCACGCGCCGTTTTCAGTCGCTCGAAGGGCTGCAGGGTACCG CCAGGCTCGGATCTGTCTCGTATGACACGGATGGCGGCCGCGCAGCGGGTCAGGCGTCGCCAGCTCGAGCGGTACAC GCGTCGCCCTCCGACCCCAGCAAACCTACCCTCGTGCATCGAAATCGCGGGGAACCTGGCGCTAACCCGGCCTCTGC TCCTGTCCTACAGCT ATGTGAAGAGC

[0301] SEQ ID NO: 27

[0302] The PL024 RPS3A terminator gene fragment is flanked by the SapI site.

[0303] GCTCTTCATAG GCGGAGCAGCGACGGACGGACCTTGTGCGTTGTGTTTGTAGGGAGGGCGAGTGGACCC GCAATGCGAGGGAAACTCTTTCTCTTCCTTCGTGCGTCCTGCCCGAGAGACTTGCGAGTCGAAGTTGACGAGGAGAG GGTTGGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGCTCGCTCGCTGCTTCGGCTGCTTCTCGACGTGCTCTAGTCGA TGTTGAGTGTACTGCTACATCCCACGAAGAAGGGAAGCTCGCACGGCCGTGAGACTGCTCTCTTCCCTCGACCGCAT CGCTGGCCATCCCTCTCCACGCTTGCCGCTTCGACTGCTTCTCAACGTGCACAATTCGCTGCTTGAACAATTGATGC TGCCGCCTACGTCGAGCGCAAGCGACCCTCACATGATTGCGAGCGAGAGTTCTCGCGTTCTCCCCAGTCCCGCCGTC GACAACGCCGCACGCATGTTGAGATCTAACGCAATTACACCTATACGATCCTTGAAT AAATGAAGAGC

[0304] SEQ ID NO: 28

[0305] Int345678 is a SapI exfoliated gene fragment, flanked by SapI loci.

[0306] CGGTCTCAAGCG CGATGAAGAGCTGGTAGAGCCACAAACAGCCGAAAGTGAAACGTGATTTCATGCGTC ATTTTGAACATTTTGTAAATCTTATTTAATAATGTGTGCGGCAATTCACATTTAATTTATGAATGTTTTCTTAACAT CGCGGCAACTCAAGAAACGGCAGGTTCGGATCTTAGCTACTAGAGAAAGAGGAGAAATACTAGATGCGTAAAGGCGA GGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTG GCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTTCCT TGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGA CTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAA CGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAGGAC GGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAA TGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAA ACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGAT CCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACT GTACAAATGACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTC GGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATACCTGCAGGGGTACAA GCAACGATCTCCAGCTCTTCAAAAAAGTGAATAAAGCTCCACACAGTCG GCTATGAGACCG

[0307] SEQ ID NO: 29

[0308] PL434 04-877 CAR2 SapI exfoliated plasmid

[0309]

[0310] SEQ ID NO: 30

[0311] PL451 04-877 CAR2 on the left, carrying the pRhota_RPS3A>3x-FLAG-GFP expression plasmid

[0312]

[0313] SEQ ID NO: 31

[0314] OLIGO496

[0315] ACCTCACCTCGACCCTGTACGC

[0316] SEQ ID NO: 32

[0317] OLIGO497

[0318] CGGGCGAGAGGAAGGAAGGTCA

[0319] SEQ ID NO: 33

[0320] OLIGO502

[0321] TCTTCGGCTACCACCCTCACGG

[0322] SEQ ID NO: 34

[0323] OLIGO503

[0324] ACGCCGCACAGAGAGACGATCT

[0325] SEQ ID NO: 35

[0326] OLIGO500, for synthesizing oligonucleotides

[0327] GATGTAGGGGGCCGTCTCAGCT

[0328] SEQ ID NO: 36

[0329] OLIGO514, for the synthesis of oligonucleotides

[0330] CCTGCACCTCGACAACGACGAC

[0331] SEQ ID NO: 37

[0332] OLIGO502, for the synthesis of oligonucleotides

[0333] TCTTCGGCTACCACCCTCACGG

[0334] SEQ ID NO: 38

[0335] OLIGO503, for the synthesis of oligonucleotides

[0336] ACGCCGCACAGAGAGACGATCT

[0337] SEQ ID NO: 39

[0338] OLIGO515, for the synthesis of oligonucleotides

[0339] ACTCGTCCAACCTCGTCGTCGT

[0340] SEQ ID NO: 40

[0341] OLIGO505, for the synthesis of oligonucleotides

[0342] AGACCTCGACCTTGTCCGAGCC

[0343] SEQ ID NO: 41

[0344] EST1 from black-budding short-stemmed mold

[0345] MKFLCLHGAYGNIPAFRVQLGPMIDALEFDGSASFEFIQGRVSVVPPPGFESYFGPAPHYRFLKDRGEAEASAVEGIRVFPEAETPEDALRALMPRDDMEIGFADARGLALEQLYEALDADPEISGIIGYSEGASAAATLV YDEQERMRSEGYVPRIKAAIFFMGWPALTAENVPALSDEVENLLEVPSLHIVGANDPYKDGALALYNIFDDNMAVFFDSAGGHTIPRHGKLLQELVSAVKDLIADVPEDNSADLLAPAVTGKRLDSAVDIPNLSFLKNEDA

[0346] SEQ ID NO: 42

[0347] 04-877 Esterase Presumed

[0348] MLNRKARVLCLHGFAQNSRVLGAKLAQAERIFDGHVELVYVDAPNILLKPTMAYDNSPDEPYAPKDEPRSWWHALSNDGFEDPREVRPLSLVRTVLETQGPFDGVWGFSQGAATAAILCALVARPWLHPAFSSTPSPGVAWPPAPFKFAVFCSGYLPLDRRCESWFDSPVNTPALHVLGRSDVVAPNERTLANVPRFSNSRVEWHEGGHYIPRKPHFAHMFKDFILSKTFPVEQPPLFPLFAPSSGVSRLASPFDSPRTSSSPFPAEDPLMRSPVTVMPLSPALH

[0349] SEQ ID NO: 43

[0350] Protein ID: 2353, putative fatty acid hydroxylase

[0351] MDIALDFFDEYVGDKLYSYLDNPLAKFAPQLGLSASLANASSSSPSFASLDLSSAQAAASSVAAYLAAVPGLSSLPRDNMLRQAISLYGITYVGIFVLYFSVATASYYLIFDKRMEQHPRFLKNQVAKEIAFSLEAFPMLDLLTLPWFVGDVRGYSQLYDSIAEGPFGDKGGVLPWLYMAFSAALFLWFTDFAIYWVHRWLHLPFLYKRLHKPHHKWIIPTPFASHAFHPVDGYFQSVPYHMACYMFPIHKYMFIGLFSFVNLWSIFIHDSDMLCGHPLEHYINGPAHHTLHHLYFTCNYGQYFTWADKAFSSFRVPAKDDDPLVAILSGRTNNHATVSAPAPLATGPAPAKLDIATPSPSPTLADAPSALLRTAAQAVVAPEMKRVDSGLGSETELGLAGSARSSGASSSSASSSGDEGASEKKKAVPAAAATGAAARPRREGLRARK

[0352] SEQ ID NO: 44

[0353] Protein ID: 2354, putative esterase

[0354] MSIAESNIPSTKLFDLIKEGLEGMSEKEKKDNIKKVNGIFEMNVKSGSNEATYTIDLKKEGKVYKGPAKPKADVTISLQDDVFQQLADGKINGQKAFMSGKLKVRGNIMLATKLDTVLKGAQKKANL

[0355] SEQ ID NO: 45

[0356] Protein ID: 5143, putative mannitol dehydrogenase

[0357] MSFDDLQAQPADPAVLARSLLDKISSPASGDDELPPRRYLVALAGIPGSGKSTFSAKLLDHLNSPSSSDPSSPPAVILPMDGFHRTQAELAADPDPELSFRRRGAPFTFAPDKLLDLVRRLRTDERLTAPAFSHADKDPVEGAIAIEPHHRIVVVEGNYLLLDEPVWTDVSREMDERWFYRVPEDVALRRIIKRHVQAGLAQDEQAAEAKALENDVLNGRHCVAHMLRPDRILEEIDHTLPSFLGRASSSPSDGDFPRPTPQLSDSVMDLFSMKGRVSAITGGGSGIGWATAEAIAEAGGDVAIIGRRNQDAKAEELSKRFNIRCKAYQCDATDAEAIEKTIDAVGNDFGRLDCFVANAGGGVPGSIDENYALDSWKQTMDVNLNSTFWCARACAKWFKKAAESGVKASFIATTSISARIVNVPYDQPAYNSSKAGVVHFCRSIARDWRNFARVNCISPGFFDTAMGPSDAEVEKVVERLSVFGRPGNVKELKAAYLYLASNASSYTTGSDILVDGGYCLT

[0358] SEQ ID NO: 46

[0359] Protein ID: 5152, putative glycosyltransferase

[0360] MPSRRPPPPPLDHPSLAANYRCGSTGSPAASLASTSVGPAPLDASKGRFFLDSYGRRLLLHGANVSGLDKLPTEPNGFTHLDMGDAYFDGASISFVGRPWPLEESHEHLSRLRNWGLTFIRLVVTWEALEHSGPGEHDQAYIAYLHALVSLFPQYGIRCYVDAHQDVWSRHAGGSGAPLWTLSLVGFDVRNMKATGAAHAHNLHLEPHDPPPNVWPSGVTKLAAATAATVFWGGDVFAPKRRVRRRLHKGEWGQGGGDDEEVGLQTFLQESMCEAFGVLADRLRDLEGVMGFEVMNEPHKGYIELLSPYAWDFTADLAIGYFPSAVQSWALGSGHSVLVPHYAPSFPVTAVTHHVLLVPPQQRSVWLSPSSPAFDALPTSTTGCLWAEHGVWQWDAQRGEVGEGVVLKQEYFRRFPADVVLEGCVEMEGGEGGRKGRREQGEEVSWYRDFYFPFVRKFSARIRRAPNPPTWFTFVEPMPNEFCPTYPTVSRPRGMIYAPHWYDLQTLFEKKLGFMTANVQGLSRGMFLLKALYFGRKGLKKNYAIQISTILRHAYRQLGETPIVLGETGVPFDLNQKAAFSTKDFQWQERMLDGICSAIGEAGLSNFNLWNYNALNNDEWGDSWNGENFSWFSQSDVTPEALKAAEDGGEAARLNVGARALDAVQRPYACKTAGIPLRTSFDLHTRRFHLSYLNPIPPSHPFAAAVPAVAQQDAEPDAPPLVGAECRARETEVFLPARRYGEAARSGRLRVQLRDGDGEWRYDEELQTLYVLHTNTTPGFVHSLTVTVLGPSDRPSPRAWYEPPAALLDALALDAVWVHLVLVVGIGGYMGWWLLGQVWRGEGIAQGWGAWGGSGPVPSFEEL

[0361] SEQ ID NO: 47

[0362] Protein ID: 5149, putative choline / carnitine O-acyltransferase

[0363] MPSRPVTVASTPINSFPAGKTFQGQGLLPKLPVPPLEDTLARYLKALEALQSPAEHERTKKAVKDFQEHEGPELHHRLKEYAAGRASYIEEWWTESYLSHSESVVLSLNPFFILEDDPTPARGNQLMRATSLILASLSFIHDLRTGQLEPDQVRGTPLDMSQYQKLFGTARIPTRTGCELRIDSESRHIVVLCRGQFYWFEVLDSKHRPALTERALLSNLTAIVADAGRTPPNQVAQSALGVLSTERRATWASCRAGLMKNEGNSMCLDVIDKALFVVALDDTSPETASEMCNNMLCGTYKLEKGVQVGTCTNRWYDKLQIIVAANGAAGVNFEHSGVDGHTVLRYVADVYTELILRFARSINPAAAGLFKAKLSPWAKGGPGKKSAPADAVKDGEPEEIDVAPKKLEWIMTPELKMAVRFAETRLSDLICQNEAVALEFDVYGKNAITQHGFSPDAFVQMAYQAAYFSLYGRTECVYEPAMTKAFLHGRTEAVRSVTPESVAFVKTFCSEASPREKIDALRKACKAHTDLTRQCSKGLGQDRILYAMYCLAQQAREKRQRALSNGDASSSSSETESSDEADEVSVKIAERIPELFKDPGYAQLGHSTLSTSNCGNVSLRLFGFGPVVGDGLGIGYIIKEDSISVVASSKHRQTQRYLDALQAYFVEVHRLLKQLHQEATRKPNSRFVDHFAGEIDAKTGQPITHLHADTRRAEDELSYSDGYGFYGALTQDVEKALSDRHGAAAKEGGTAAAKRRGIVPGTRIRTVEL

[0364] References

[0365]

[0366]

[0367]

Claims

1. A non-naturally occurring polyol lipid production microorganism, said non-naturally occurring microorganism comprising: (i) At least one knocked-out endogenous gene; (ii) Compared to the naturally occurring microorganism from which the aforementioned non-naturally occurring microorganism is derived, there is an alteration in the expression of at least one endogenous gene; and / or (iii) At least one expressed heterologous polynucleotide.

2. The non-naturally occurring microorganism according to claim 1, wherein the microorganism produces one or more polyol lipids selected from the group consisting of: esterification of a 3-hydroxy fatty acid moiety of 6 to 24 carbon atoms to a sugar alcohol moiety; esterification of an acetylated 3-hydroxy fatty acyl moiety to a sugar alcohol moiety; esterification of a sugar alcohol moiety containing a non-esterified hydroxyl group to the carboxyl terminus of the 3-hydroxy fatty acyl moiety; esterification of a sugar alcohol moiety containing a mannitol or arabinitol backbone to the carboxyl terminus of the 3-hydroxy fatty acyl moiety; and fatty acid polyol esters (PEFAs).

3. The non-naturally occurring microorganism according to claim 2, wherein the polyol lipid is PEFA.

4. The non-naturally occurring microorganism according to claim 1, wherein the yield of at least one polyol lipid produced by the non-naturally occurring microorganism is increased compared to that of the naturally occurring microorganism from which the non-naturally occurring microorganism is derived.

5. The non-naturally occurring microorganism according to claim 4, wherein the yield of at least one polyol lipid is 50 g / L to 100 g / L, 100 g / L to 150 g / L, 150 g / L to 200 g / L, 200 g / L to 250 g / L, 250 g / L to 300 g / L, 300 g / L to 350 g / L, 350 g / L to 400 g / L, 400 g / L to 450 g / L, or 450 g / L to 500 g / L.

6. The non-naturally occurring microorganism according to claim 1, wherein the microorganism originates from the subkingdom of binucleate bacteria.

7. The non-naturally occurring microorganism according to claim 6, wherein the microorganism originates from the phylum Ascomycota or Basidiomycota.

8. The non-naturally occurring microorganism according to claim 7, wherein the microorganism is from the order Cyclostomales.

9. The non-naturally occurring microorganism according to claim 8, wherein the microorganism is from a genera selected from the group consisting of: Rhodotorula, Rhodotorula, and Rhodotorula.

10. The non-naturally occurring microorganism according to claim 9, wherein the microorganism is a species selected from the group consisting of: *Rhodotorula biciliae*, *Rhodotorula diodactyla*, *Rhodotorula kratochevilova*, *Rhodotorula granatum*, *Rhodotorula swamp*, *Rhodotorula similar to ... colostrum*, *Rhodotorula dalianense*, *Rhodotorula rouxii*, *Rhodotorula taiwanense*, *Rhodotorula mucilaginosa*, and *Rhodotorula natans*.

11. The non-naturally occurring microorganism according to claim 10, wherein the microorganism is a strain selected from the group consisting of: *Rhodotorula basilicum* strain NRRL Y-67018, *Rhodotorula basilicum* strain NRRL Y-67017, *Rhodotorula basilicum* strain UCDFST 68-916.1, *Rhodotorula basilicum* strain UCDFST 67-458, *Rhodotorula basilicum* strain UCDFST05-736, *Rhodotorula basilicum* strain UCDFST 04-830, *Rhodotorula diopside* strain NRRL Y-67015, *Rhodotorula kratochvirova* strain NRRL Y-67016, *Rhodotorula sphaeroides* strain NRRL Y-67012, *Rhodotorula sphaeroides* strain UCDFST82-646.2, *Rhodotorula sphaeroides* strain UCDFST 81-492, *Rhodotorula sphaeroides* strain NRRL Y-67009, *Rhodotorula sphaeroides* strain NRRL Y-67010, and *Rhodotorula daliani* strain NRRL Y-67011, NRRL Y-67014 (similar to Rhodotorula colostrum), NRRL Y-67013 (similar to Rhodotorula natans), NRRL Y-17302 (similar to Rhodotorula roximans), MD1149 (similar to Rhodotorula taiwanensis), and 50-3-19 / 208 (similar to Rhodotorula mucilaginosa).

12. The non-naturally occurring microorganism according to claim 1, wherein the expression level of the endogenous gene is altered compared to the expression level of the endogenous gene in the naturally occurring microorganism from which the non-naturally occurring microorganism is derived.

13. The non-naturally occurring microorganism of claim 12, wherein the promoter of the endogenous gene is replaced with a different promoter, and / or wherein a 3' untranslated region (UTR) and / or a 5' UTR are added, thereby increasing, decreasing or altering the expression regulation of the endogenous gene or the stability regulation of mRNA transcribed from the endogenous gene.

14. The non-naturally occurring microorganism according to claim 12, wherein the promoter of the endogenous gene is replaced by homologous recombination, CRISPR, zinc finger nuclease or TALEN.

15. The non-naturally occurring microorganism of claim 12, wherein the endogenous gene is fused with a peptide to increase, decrease, or alter the expression regulation of the endogenous gene.

16. The non-naturally occurring microorganism according to claim 15, wherein the peptide is the auxin-induced degrader IAA7, the N-terminal fusion of ubiquitin, or the tobacco etched virus degrader tag TDegF.

17. The non-naturally occurring microorganism of claim 12, wherein the endogenous gene is knocked out, silenced, or its expression level is reduced.

18. The non-naturally occurring microorganism according to claim 12 or 17, wherein the endogenous gene is knocked out or edited by homologous recombination, CRISPR, zinc finger nuclease or TALEN.

19. The non-naturally occurring microorganism according to claim 12 or 17, wherein the endogenous gene is silenced by RNA interference, RNA silencing, antisense oligonucleotides, ribozymes, or a CRISPR-based method.

20. The non-naturally occurring microorganism according to claim 19, wherein the CRISPR-based method is selected from dCas9 and Cas13.

21. The non-naturally occurring microorganism of claim 17, wherein the endogenous gene that is knocked out, silenced, or expressed at a reduced level encodes an enzyme in the triglyceride (TAG) production biosynthetic pathway, wherein the microorganism produces at least about 10% less TAG than the naturally occurring microorganism from which the non-naturally occurring microorganism is derived.

22. The non-naturally occurring microorganism according to claim 21, wherein the enzyme in the TAG production biosynthetic pathway is an acyltransferase.

23. The non-naturally occurring microorganism according to claim 22, wherein the acyltransferase is a diacylglycerol acyltransferase.

24. The non-naturally occurring microorganism of claim 17, wherein the knocked-out, silenced, or reduced-expression endogenous gene encodes an enzyme in the biosynthetic pathway for carotenoid production.

25. The non-naturally occurring microorganism according to claim 24, wherein the enzyme in the carotenoid production biosynthetic pathway is a bifunctional lycopene cyclase / hydrolycopene synthase.

26. The non-naturally occurring microorganism according to claim 17, wherein the knocked-out, silenced, or reduced-expression endogenous gene encodes orotidine 5-phosphate decarboxylase, cytosine deaminase, Ku70 homolog, Lig4 homolog, 3-hydroxyacyl-CoA dehydrogenase / enoyl-CoA hydratase, acyl-CoA oxidase, peroxisome transporter, peroxisome fatty acyl-CoA synthase, secretory lipase, enoyl-CoA hydratase, phosphatidic acid hydrolase, glycerol-3-phosphate dehydrogenase, phospholipid:diacylglycerol acyltransferase, lipid droplet-coated protein-like protein, or seipin transmembrane protein.

27. The non-naturally occurring microorganism according to any one of claims 13 to 16, wherein the endogenous gene with altered expression levels encodes an enzyme, mannitol-1-phosphate phosphatase, mannitol-1-phosphate dehydrogenase, d-xylulose kinase, arabinose dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphate gluconate lactonease, 6-phosphate gluconate dehydrogenase, phosphoglucose isomerase, transaldolase, transketoolase, triglyceride lipase, PEFA synthase, acetyltransferase, long-chain fatty acid α-hydroxylase, PEFA transporter, fatty acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, or malate dehydrogenase.

28. A non-naturally occurring microorganism according to any one of claims 1 to 11, wherein the microorganism expresses a heteropolynucleotide encoding a protein providing a selectable marker.

29. The non-naturally occurring microorganism of claim 28, wherein the selection marker is an enzyme that provides antibiotic resistance, a fluorescent protein, or an enzyme that produces the desired metabolite.

30. The non-naturally occurring microorganism according to any one of claims 1 to 11, wherein the microorganism comprises an altered expression of an endogenous gene in the polyol lipid production biosynthesis pathway or a heteropolynucleotide encoding and expressing an enzyme in the polyol lipid production biosynthesis pathway.

31. A non-naturally occurring microorganism according to any one of claims 1 to 11, wherein the microorganism expresses a heteropolynucleotide encoding a triglyceride lipase, PEFA synthase, acetyltransferase, long-chain fatty acid α-hydroxylase, PEFA transporter, fatty acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, mannitol dehydrogenase, mannitol-1-phosphate phosphatase, mannitol-1-phosphate dehydrogenase, d-xylulose kinase, arabinose dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphate gluconate lactonease, 6-phosphate gluconate dehydrogenase, phosphoglucose isomerase, transaldolase, transketolase, or malate dehydrogenase.

32. A microbial culture comprising: a combination of one or more non-naturally occurring microorganisms according to claim 1; a culture medium; and at least about 1 g / L of polyol lipids produced by said microorganisms.

33. The microbial culture of claim 32, wherein the microorganism secretes the polyol lipids into the culture medium.

34. A polynucleotide construct for generating a gene knockout in a host microorganism producing polyol lipids, the polynucleotide construct comprising: a promoter; a nucleotide sequence homologous to at least a portion of a target gene sequence in the genome of the microorganism; a nucleotide coding sequence encoding a protein providing a selection marker; and a terminator, wherein the promoter and terminator sequences are capable of driving gene expression of the coding sequence in the microorganism.

35. A polynucleotide construct for expressing a heterologous gene in a host microorganism producing polyol lipids, the polynucleotide construct comprising: a promoter; a nucleotide coding sequence of the heterologous gene; and a nucleotide coding sequence encoding a protein providing a selection marker, wherein the promoter is capable of driving gene expression of the coding sequence in the microorganism.

36. The polynucleotide construct of claim 34 or 35, wherein the nucleotide coding sequence is codon-optimized for expression in the host microorganism.

37. The polynucleotide construct according to claim 34 or 35, wherein the nucleotide coding sequence is codon-optimized for expression in the host microorganism according to the codons in Table 1 or Table 2.

38. The polynucleotide construct of claim 37, wherein at least about 50% of the codons in the nucleotide coding sequence are the most common or second most common codons in the host microorganism.

39. The polynucleotide construct according to claim 34 or 35, wherein the promoter is inducible in the host microorganism.

40. The polynucleotide construct according to claim 34 or 35, wherein the selection marker is an enzyme that provides antibiotic resistance, a fluorescent protein, or an enzyme that produces the desired metabolite.

41. The polynucleotide construct of claim 35, wherein the nucleotide coding sequence of the heterologous gene further encodes an amino acid sequence that facilitates the detection or recovery of the expression product of the heterologous gene.

42. The polynucleotide construct of claim 41, wherein the amino acid sequence facilitating detection or recovery comprises a His tag sequence containing six to nine consecutive histidine residues, a FLAG tag sequence containing the amino acid sequence DYKDDDDK (SEQ ID NO: 1), or a Myc tag sequence containing the amino acid sequence EQKLISEEDL (SEQ ID NO: 2).

43. A polynucleotide construct for replacing a promoter and adding a 3' UTR and / or an N-terminal tag in a host microorganism for producing polyol lipids, the polynucleotide construct comprising: a nucleotide sequence homologous to at least a portion of a target gene sequence in the genome of the microorganism; a sequence promoter; and a 3' UTR, and / or a polynucleotide sequence encoding an N-terminal tag.

44. A polynucleotide construct for replacing a promoter and adding a 5' UTR and / or a C-terminal tag in a host microorganism for producing polyol lipids, the polynucleotide construct comprising: a nucleotide sequence homologous to at least a portion of a target gene sequence in the genome of the microorganism; a promoter sequence; and a terminator sequence, a 5' UTR, and / or a polynucleotide sequence encoding a C-terminal tag.

45. A recombinant polyol lipid production microorganism, wherein the microorganism has been transformed with a polynucleotide construct according to any one of claims 34, 35 or 44.

46. ​​A method for producing non-naturally occurring polyol lipid-producing microorganisms containing at least one knocked-out gene, the method comprising: The polynucleotide construct according to claim 34 is used to transform a host microorganism for the production of polyol lipids, wherein the nucleotide sequence homologous to at least a portion of a target gene sequence in the genome of the microorganism disrupts the gene sequence through homologous recombination, wherein the disrupted gene is inactivated or deleted in the microorganism.

47. A method for producing non-naturally occurring polyol lipids containing at least one expressed heterologous polynucleotide by microorganisms, the method comprising: The host microorganism for the production of polyol lipids is transformed using the polynucleotide construct according to claim 35, wherein the nucleotide coding sequence of the heterologous gene is expressed in the microorganism.

48. A method for producing polyol lipids, the method comprising: One or more combinations of the microorganisms according to claim 1 are cultured in a culture medium under certain conditions suitable for the growth of the microorganisms and the production of biosynthetic products, wherein the microorganisms produce polyol lipid products and secrete them into the culture medium.

49. The method of claim 48, further comprising: The polyol lipid product is recovered from the culture medium.

50. The method of claim 49, wherein the polyol lipids are recovered from the culture medium without lysing the microorganisms or extracting with an organic solvent.

51. The method according to claim 49 or 50, wherein the density of the polyol lipid is greater than that of water, and the recovery of the polyol lipid comprises separation from the culture medium by centrifugation, continuous decantation, or passive sedimentation.

52. The method of claim 48, wherein at least about 1 g / L of the polyol lipid is secreted into the culture medium.

53. The method of claim 48, wherein the polyol lipid has a density of about 1.00 g / mL to about 1.10 g / mL.

54. The method of claim 48, wherein the method is performed in a batch, replenished batch, or continuous process.

55. The method of claim 48, wherein the polyol lipid is selected from the group consisting of: esterification of a 3-hydroxy fatty acid moiety of 6 to 24 carbon atoms to a sugar alcohol moiety; esterification of an acetylated 3-hydroxy fatty acyl moiety to a sugar alcohol moiety; esterification of a sugar alcohol moiety containing a non-esterified hydroxyl group to the carboxyl terminus of the 3-hydroxy fatty acyl moiety; esterification of a sugar alcohol moiety containing a mannitol or arabinitol backbone to the carboxyl terminus of the 3-hydroxy fatty acyl moiety; and fatty acid polyol esters (PEFAs).

56. The method of claim 55, wherein the polyol lipid comprises PEFA.

57. A polyol lipid produced by the method according to claim 48.

58. The polyol lipid of claim 57, wherein the polyol lipid is selected from the group consisting of: esterification of a 3-hydroxy fatty acid moiety of 6 to 24 carbon atoms to a sugar alcohol moiety; esterification of an acetylated 3-hydroxy fatty acyl moiety to a sugar alcohol moiety; esterification of a sugar alcohol moiety containing a non-esterified hydroxyl group to the carboxyl terminus of the 3-hydroxy fatty acyl moiety; esterification of a sugar alcohol moiety containing a mannitol or arabinitol backbone to the carboxyl terminus of the 3-hydroxy fatty acyl moiety; and fatty acid polyol esters (PEFAs).

59. The polyol lipid according to claim 58, wherein the polyol lipid is PEFA.

60. The non-naturally occurring microorganism according to claim 1, wherein the yield / gram carbon source of at least one polyol lipid produced by the non-naturally occurring microorganism is increased compared to that of the naturally occurring microorganism from which the non-naturally occurring microorganism is derived.

61. The non-naturally occurring microorganism according to claim 60, wherein the yield of at least one polyol lipid per gram of glucose as a carbon source is from about 0.01 g / g to about 0.75 g / g.

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