Methods for tuning the hydrophilic-lipophilic balance of extracellular glycolipids

Abstract

Non-naturally occurring PEFA producing microorganisms are provided that express heterologous gene(s) for production of PEFA with shorter average fatty acid carbon chain length or with altered hydrophilic-lipophilic balance. Non-naturally occurring microorganisms are also provided that have altered expression of endogenous gene(s) for degradation of fatty acids, such as b-oxidation. Polynucleotide constructs and methods of engineering the microorganisms for altered PEFA production are described. Methods of production of PEFA in the engineered microorganisms, and compositions that include PEFA produced in the microorganisms, are also provided.

Description

METHODS FOR TUNING THE HYDROPHILIC-LIPOPHILIC BALANCE OF EXTRACELLULAR GLYCOLIPIDS CROSS-REFERENCE TO RELATED APPLI CATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 616,181, filed on December 29, 2023, which is incorporated by reference herein in its entirety. FIELD OF THE INVENTION

[0002] The invention relates to genetically modified PEFA producing microorganism cells, in particular microorganisms containing heterologous enzymes for production of PEFA with shorter fatty acid moieties and / or altered expression of endogenous genes for degradation of fatty acids. BACKGROUND

[0003] Polyol lipid compounds have a variety of uses, including as surfactants. Surfactants (both petroleum and bio-based) are an example of compounds that can act as emulsifiers, detergents, dispersants, foam control agents, and wetting agents used in a variety of consumer applications, including agricultural, nutritional, cosmetic, veterinary, therapeutic, paint, ink, home care, and industrial applications. However, such surfactants can exhibit poor performance with regard to sustainability, bio-accumulation, eco-toxicity, and / or biodegradability. Therefore, there is a need for development of alternative surfactants and compositions for use in consumer and industrial products in these applications.

[0004] Several yeast species are attractive chassis strains for producing polyol lipids, such as polyol esters of fatty acids (PEFA), which have applications as surfactants. (See phylogeny in Garay et al., 2018.) Surfactants can be categorized by their hydrophilic-lipophilic balance (HLB), which is a ratio that describes the balance of hydrophilic and lipophilic moieties of a surfactant.

[0005] The hydrophilic-lipophilic balance of a surfactant is a numeric scale from 0 to 20 that characterizes the relative affinity of a surfactant for oil (low HLB) or water (high HLB). (Griffin, 1949) The HLB scale groups surfactants by their suitability for use as emulsifiers, wetting agents, or solubilizers. The HLB of non-ionic surfactants (such as PEFA) can be calculated based on either the structure of the molecule using the method of Griffin (Griffin, 1954), or experimentally using methods such as those reviewed in Nollet, et al., 2019.

[0006] A number of yeast species have been described that naturally produce and secrete PEFA. These natively produced PEFA are more soluble in oil than in water (e.g., have a low HLB), enabling uses as water in oil emulsifiers and wetting agents. In contrast, surfactants used in applications such as cleansing products require that the surfactant be water soluble (e.g., have a high HLB value). Thus, strains of yeast that could produce PEFA that are more soluble in water are (e.g., have a higher HLB value) are desirable to expand the number of applications that PEFA molecules can be used in. However, there are no known native yeast species that produce appreciable quantities of PEFA that have higher HLB values.

[0007] Natively produced PEFA have fatty acid moieties that are predominantly C16 and C18, although some strains produce small amounts (up to 5%) of C14 PEFA along with trace amounts of shorter chain lengths. It would be desirable to engineer PEFA producing microorganisms to produce PEFA with shorter carbon chain lengths, to render the PEFA more hydrophilic. It is desirable to produce shorter tailed PEFA from glucose by harnessing the native fatty acid synthesis capabilities of the host microorganism since this would avoid the need for hydrophobic substrates such as palm oil and coconut oil, which are not sustainable. Furthermore, medium and short chain fatty acids are preferentially targeted to the peroxisomes and mitochondria for degradation by beta oxidation pathways. These pathways can block the incorporation of medium-chain fatty acids from extracellular sources into glycolipids (Van Bogaert, I., et al., 2009). To enable efficient incorporation of shortened fatty acid moieties into PEFA molecules, it would be desirable to block or down- regulate these degradation pathways. BRIEF SUMMARY OF THE INVENTION

[0008] Non-naturally occurring microorganisms that contain modified metabolic pathways for production and / or degradation of polyol esters of fatty acids (PEFA) are provided, as well as methods and polynucleotide constructs for making the microorganisms and methods for using the microorganisms to produce PEFA in microbial culture.

[0009] In one aspect, a non-naturally occurring microorganism is provided which produces polyol esters of fatty acids (PEFA). The PEFA include: (i) a polyol head portion; and (ii) a fatty acid tail portion having a carbon chain length. The average carbon chain length of the fatty acid tail portion of the PEFA produced by the non-naturally occurring microorganism is altered in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived. In some embodiments, the fatty acid tail portion of the PEFA produced by the non-naturally occurring microorganism has an average carbon chain length that is shorter than the average carbon chain length of the PEFA produced by the parent microorganism. For example, the average carbon chain length of fatty acid tail portions of PEFA produced by the non-naturally occurring microorganism may be C8 to C18, C8 to C16, C8 to C14, C8 to C12, C8 to C10, C10 to C14, or C10 to C16.

[0010] In some embodiments, the hydrophilic lipophilic balance (HLB) of the PEFA produced by the non-naturally occurring microorganism is altered such that the PEFA are more hydrophilic than PEFA that are produced by the parent microorganism from which the non-naturally occurring microorganism is derived.

[0011] In some embodiments, the non-naturally occurring microorganism expresses a heterologous polynucleotide that includes a nucleotide coding sequence that encodes an enzyme or a polypeptide with an enzymatic activity in a biosynthetic pathway for production of PEFA with a preferred fatty acid carbon chain length. In some embodiments, the heterologous polynucleotide includes a nucleotide coding sequence that encodes a polypeptide that exhibits a thioesterase enzymatic activity or a thioesterase enzyme. In some embodiments, the heterologous polynucleotide includes a nucleotide coding sequence encodes a polypeptide that exhibits a thioesterase enzymatic activity or that encodes a thioesterase enzyme, fused to nucleotidesequence that encodes a polypeptide that exhibits a fatty acid synthase (e.g., type 1 fatty acid synthase) enzymatic activity or that encodes all or part of a fatty acid synthase enzyme (e.g., a type 1 fatty acid synthase enzyme) having fatty acid synthase activity. In some embodiments, the thioesterase enzyme or thioesterase enzyme activity increases the production of PEFA with fatty acid carbon chain length C8 to C14 in comparison to the parent microorganism from which the non- naturally occurring microorganism is derived. In some embodiments, the heterologous polynucleotide includes a nucleotide coding sequence that encodes all or a portion of a fatty acid synthase (e.g., type 1 fatty acid synthase) enzyme having fatty acid synthase activity, which has been modified to produce a greater portion of C8 to C14 acyl-CoA molecules than the fatty acid synthase encoded by the parent microorganism (e.g., produces predominantly C8 to C14 acyl-CoA molecules).

[0012] In some embodiments, the non-naturally occurring microorganism includes a heterologous polynucleotide with a nucleotide coding sequence that encodes the thioesterase enzyme UcFat1B (Uniprot Q41635) from Umbellularia californica (SEQ ID NO:1) or encodes a polypeptide having at least about 60%, 65%, 70%, 75%, 80%, 85%., 90%, 95%, 98%, or 99% sequence identity thereto and retaining the thioesterase enzymatic activity thereto, the thioesterase enzyme CvFat1B (Uniprot G3ESU9) from Cuphea viscosissima (SEQ ID NO:2) or encodes a polypeptide having at least about 60%, 65%, 70%, 75%, 80%, 85%., 90%, 95%, 98%, or 99% sequence identity thereto and retaining the thioesterase enzymatic activity thereto, the thioesterase enzyme FatB4 from Cinnamomum camphora (SEQ ID NO:3) or encodes a polypeptide having at least about 60%, 65%, 70%, 75%, 80%, 85%., 90%, 95%, 98%, or 99% sequence identity thereto and retaining the thioesterase enzymatic activity thereto, the thioesterase enzyme AcTesA from Acinetobacter baylyi (SEQ ID NO:4) (Zheng, et al., 2012) or encodes a polypeptide having at least about 60%, 65%, 70%, 75%, 80%, 85%., 90%, 95%, 98%, or 99% sequence identity thereto and retaining the thioesterase enzymatic activity thereto, or any of the thioesterases listed in U.S. Patent No. 10,648,043 or encodes a polypeptide having at least about 60%, 65%, 70%, 75%, 80%, 85%., 90%, 95%, 98%, or 99% sequence identity thereto and retaining the thioesterase enzymatic activity thereto.

[0013] In some embodiments, the non-naturally occurring microorganism includes a heterologous polynucleotide with a nucleotide coding sequence that encodes the type 1 fatty synthase whose sequence has been modified, for example, wherein residues in the ketoacyl synthase domain, the malonyl / palmitoyl transferase domain, and / or the acetyl-transferase domain of the fatty acid synthase is mutated relative to the sequence in the parent microorganism (e.g., the wild type sequence), in order to produce short and medium chain acyl-CoA molecules. (See, e.g., Rigoiun, 2017; Gajewski, 2017.)

[0014] In some embodiments, the non-naturally occurring microorganism includes a polynucleotide sequence that encodes a type 1 fatty synthase that produces short chain fatty acids. Such fatty acid synthases are frequently found in biosynthetic gene clusters involved in the production of secondary metabolites (e.g. sporothriolides, aflatoxin). (See, e.g., Tian et al., 2020)

[0015] In some embodiments, the non-naturally occurring microorganism includes a polynucleotide sequence wherein mutations are incorporated into a single polypeptide as the fusion of tetrafunctional fatty acid synthase subunit (FAS1) and trifunctional fatty acid synthase subunit (FAS2) (SEQ ID NO:5) (see, e.g., Wernig, 2020). In some embodiments, an extra copy of one or more endogenous FAS1 and / or FAS2 genes is expressed that contains mutations, for example, mutations that are homologous to the mutated residues described by Rigouin, 2017 and Gajewski, 2017. In some embodiments, the endogenous FAS1 and / or FAS2 genes are altered to encode the mutations. In some embodiments, these genes are modified by editing the endogenous sequence using CRISPR-based technologies, such as Cas9 or Cpf1 based editing.

[0016] In some embodiments, the nucleotide coding sequence in a heterologous polynucleotide that is expressed in a non-naturally occurring microorganism as described herein is codon optimized for expression in the non-naturally occurring (host) microorganism. For example, the nucleotide coding sequence may be codon optimized for expression in the host microorganism in accordance with the codon usage of 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.

[0017] In some embodiments of the non-naturally occurring microorganisms that express a heterologous polynucleotide as described herein, the non-naturally occurring microorganism also includes a knocked out endogenous gene or reduced expression of an endogenous gene that encodes an enzyme that catalyzes β-oxidation of fatty acids, thereby reducing or eliminating degradation of the fatty acids (e.g., shortened fatty acids) and / or acyl-CoA molecules produced by the organism. In one embodiment, the non-naturally occurring microorganism includes knocked out or reduced expression of endogenous multifunctional enzyme type 2 (MFE-2), wherein peroxisomal β-oxidation of fatty acids in the non-naturally occurring microorganism is eliminated or reduced in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived. In another embodiment, the non-naturally occurring microorganism includes knocked out or reduced expression of endogenous enoyl-CoA hydratase, wherein mitochondrial β-oxidation of fatty acids in the non-naturally occurring microorganism is eliminated or reduced in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived. In other embodiments, the non-naturally occurring microorganism includes knocked out or reduced expression of both endogenous MFE-2 and endogenous enoyl-CoA hydratase, wherein both peroxisomal and mitochondrial β-oxidation of fatty acids are eliminated or reduced in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived. One skilled in the art will recognize that other genes that participate in β-oxidation in the peroxisomes or mitochondria can knocked out or altered for reduced expression instead. To identify such genes, one can consult published functional genomics data that identify knock-outs that reduce the growth of the organism on hydrophobic substrates such as oleic acid. For example, Coradetti et al., 2018 provide such functional genomics data.

[0018] In another aspect, a non-naturally occurring microorganism is provided which produces polyol esters of fatty acids (PEFA), wherein the PEFA include: (i) a polyol head portion; and (ii) afatty acid tail portion having a carbon chain length, wherein the average carbon chain length of the fatty acid tail portion of the PEFA produced by the non-naturally occurring microorganism is altered in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived, and wherein the non-naturally occurring microorganism includes a knocked out endogenous gene or reduced expression of an endogenous gene that encodes an enzyme that catalyzes β-oxidation of fatty acids, thereby reducing or eliminating degradation of fatty acids. In one embodiment, the non-naturally occurring microorganism includes knocked out or reduced expression of endogenous multifunctional enzyme type 2 (MFE-2), wherein peroxisomal β-oxidation of fatty acids in the non-naturally occurring microorganism is eliminated or reduced in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived. In another embodiment, the non-naturally occurring microorganism includes knocked out or reduced expression of endogenous enoyl-CoA hydratase, wherein mitochondrial β-oxidation of fatty acids in the non-naturally occurring microorganism is eliminated or reduced in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived. In other embodiments, the non-naturally occurring microorganism includes knocked out or reduced expression of both endogenous MFE-2 and endogenous enoyl-CoA hydratase, wherein both peroxisomal and mitochondrial β-oxidation of fatty acids are eliminated or reduced in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived.

[0019] In another aspect, a microbial culture is provided, which includes: a non-naturally occurring microorganism as described herein or a consortium of two or more non-naturally occurring microorganisms as described herein; a culture medium; and at least about 1 g / L PEFA that is produced by the microorganism. In some embodiments, the PEFA is secreted by the microorganisms, and the culture medium includes the secreted PEFA.

[0020] In another aspect, a method is provided for production of PEFA, wherein the method includes: growing a non-naturally occurring microorganism as described herein or a consortium of two or more non-naturally occurring microorganisms as described herein in a culture medium under conditions that are suitable for growth of the microorganism(s) and production of biosynthetic products, including PEFA, wherein PEFA are produced by the microorganisms. In some embodiments, the PEFA are secreted into the culture medium, and the method further includes: recovering the PEFA product from the culture medium. In some embodiments, the PEFA are recovered from the culture medium without lysis of the microorganisms or extraction with an organic solvent. The PEFA may be denser than water, and recovery may include separation from the culture medium by centrifugation, continuous decanting, or passive settling. In some embodiments, at least about 1 g / L of the PEFA are secreted into the culture medium. The PEFA may have a density of about 1.00 g / mL to about 1.10 g / mL. The method may be performed as a batch, fed batch, or continuous process.

[0021] In another aspect, compositions are provided that are produced in any of the non-naturally occurring microorganisms described herein and in any of the microbial cultures or methods for production of PEFA that are described herein. In some embodiments, the composition is a surfactant.

[0022] In some embodiments of the non-naturally occurring microorganisms, microbial cultures, methods, or compositions described herein, the microorganism is from the subkingdom Dikarya. For example, the microorganism may be from the phylum Ascomycota or Basidiomycota, such as from the order Sporidiobolales. In certain embodiments, the microorganism is from the genus Rhodotorula, Rhodosporidiobolus, or Sporobolomyces. For example, the microorganism species may be 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 certain embodiments, the microorganism may be a strain selected from Rhodotorula babjevae strain NRRL Y-67018, Rhodotorula babjevae strain NRRL Y-67017, Rhodotorula babjevae strain UCDFST 68-916.1, Rhodotorula babjevae strain UCDFST 67-458, Rhodotorula babjevae strain UCDFST 05-736, Rhodotorula diobovata strain UCDFST 04-830, Rhodotorula diobovata strain NRRL Y-67015, Rhodotorula kratochvilovae strain NRRL Y-67016, Rhodotorula paludigena strain NRRL Y-67012, Rhodotorula paludigena strain UCDFST 82-646.2, Rhodotorula paludigena strain UCDFST 81-492, Rhodotorula aff. paludigena strain NRRL Y-67009, Rhodotorula sphaerocarpa strain NRRL Y-67010, Rhodotorula dairenensis strain NRRL Y-67011, Rhodosporidiobolus aff. colostri strain NRRL Y-67014, Rhodosporidiobolus aff. colostri NRRL Y-67014, Rhodosporidiobolus aff. nylandii strain NRRL Y-67013, Rhodosporidiobolus ruineniae NRRL Y-17302, Rhodotorula taiwanensis strain MD1149, and Rhdodotorula mucilaginosa strain 50-3-19 / 208. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A schematically shows the native biosynthetic pathway for PEFA production in fungal microorganisms. Figure 1B schematically shows an embodiment of an altered biosynthetic pathway for production of PEFA with altered fatty acid tail carbon chain length as described herein, with heterologous thioesterase enzyme and heterologous or altered type 1 fatty acid synthase enzyme, and elimination of β-oxidation.

[0024] Figure 2A schematically shows the native biosynthetic pathway for PEFA production in fungal microorganisms. Figure 2B schematically shows an embodiment of an altered biosynthetic pathway for production of PEFA with altered fatty acid tail carbon chain length as described herein, with heterologous PhaG enzyme and elimination of β-oxidation.

[0025] Figures 3A – 3C show a design schematic of replacing a segment of the genome with a cassette for the expression of a coding sequence of a thioesterase enzyme.3A. A plasmid containing DNA containing sequences homologous to a genomic region flanking an expression cassette.3B. Homologous recombination between linear DNA fragments produced from the plasmid and the genome of the organism.3C. The genomic sequence replaced by the expression cassette.

[0026] Figures 4A – 4C show a design schematic of replacing a segment of the genome with a cassette for changing the promoter of an endogenous gene.4A. A plasmid containing DNA containing sequences homologous to a genomic region flanking a new promoter and / or N-terminustag.4B. Homologous recombination between linear DNA fragments produced from the plasmid and the genome of the organism.4C. The genomic sequence replaced by the new promoter and / or tag.

[0027] Figures 5A – 5C show a design schematic of replacing a segment of the genome with a cassette for changing the terminator of an endogenous gene.5A. A plasmid containing DNA containing sequences homologous to a genomic region flanking a new terminator and / or a C- terminus tag.5B. Homologous recombination between linear DNA fragments produced from the plasmid and the genome of the organism.5C. The genomic sequence replaced by the new terminator and / or TAG.

[0028] Figures 6A-6B show a schematic of the modular assembly system for creating backbones and expression constructs.

[0029] Figure 7A-7D show activity of two promoters in Strain 1 measured by GFP fluorescence. 7A-7B. FITC fluorescence of promoters in YPD and nitrogen limited media.7C-7D. Transmitted light for same micrographs.

[0030] Figure 8 shows activity of three promoters in Strain 2 measured by GFP fluorescence measured in YPD and nitrogen limited media.

[0031] Figure 9 shows change in PEFA chain length as a result of expressing one of four thioesterases relative to wild-type in 24W plates for Strain 2.

[0032] Figure 10 shows change in MAN-PEFA as a result of expressing one of four thioesterases relative to wild-type in 24W plates for Strain 2.

[0033] Figure 11 shows change in PEFA chain length as a result of expressing one of two thioesterases or a D4ADH relative to wild-type in flasks for Strain 2.

[0034] Figure 12 shows change in PEFA chain length as a result of expressing one of two thioesterases relative to wild-type in 250ml flasks for Strain 1.

[0035] Figure 13 shows change in MAN-PEFA as a result of expressing one of two thioesterases relative to wild-type in flasks for Strain 1.

[0036] Figure 14 shows change in PEFA titer as a result of expressing either fused Saccharomyces cerevisiae FAS1 and FAS2 genes or the FAS1 / FAS2 fusion with the R1834K mutation.

[0037] Figure 15 shows change in PEFA chain length as a result of expressing Saccharomyces cerevisiae FAS1 and FAS2 genes or the FAS1 / FAS2 fusion with the R1834K mutation relative to wild-type in flasks for Strain 2.

[0038] Figure 16 shows the protein sequence alignment between the Saccharomyces cerevisiae enzyme Fas1p and the Rhodotorula Fas1, which encodes the alpha subunit of the fatty acid synthase in Rhodotorula babjevae 04-877 (Rhodotorula Strain 1 in the Examples). The residue R1834 in S. cerevisiae is highlighted. DETAILED DESCRIPTION

[0039] Non-naturally occurring PEFA producing microorganisms are provided herein, which have been engineered to express one or more heterologous enzyme in a biosynthetic pathway for PEFAproduction, and / or have been engineered for altered expression (e.g., knock out or silencing) of one or more endogenous gene that encodes a fatty acid degradation enzyme, e.g., β-oxidation of fatty acids. Non-naturally occurring microorganisms described herein may express heterologous thioesterase and / or fatty acid synthase (e.g., type 1 fatty acid synthase) enzymes for production of PEFA with shorter average fatty acid carbon chain length than the parent (e.g., wild-type) microorganism. Transformation methods and polynucleotide constructs for producing such microorganism strains are described. Methods of culturing engineered microorganisms as described herein, and producing PEFA in cultures of the microorganisms, are also described.

[0040] Unless defined otherwise 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 belongs. Singleton, et al., Dictionary of Microbiology and Molecular Biology, second ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this invention. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0041] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984; Current Protocols in Molecular Biology (F. M. 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).

[0042] Numeric ranges provided herein are inclusive of the numbers defining the range.

[0043] Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Definitions

[0044] “A,” “an” and “the” include plural references unless the context clearly dictates otherwise.

[0045] The term “about” is used herein to mean plus or minus ten percent (10%) of a value. For example, “about 100” refers to any number between 90 and 110.

[0046] An “acetylated 3-hydroxy fatty acid” refers to a fatty acid compound wherein the hydroxyl group at the third carbon from the acid end of the fatty acid is acetylated.

[0047] As used herein, the term “analogous sequence” refers to a polypeptide sequence within a protein that provides a similar function, tertiary structure, and / or conserved residues with respect to a reference protein. For example, in epitope regions that contain an alpha helix or a beta sheet structure, replacement amino acid(s) in an analogous sequence maintain the same structural element. In some embodiments, analogous sequences are provided that result in a variant enzyme exhibiting a similar or improved function with respect to the parent protein from which the variant is derived.

[0048] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open- ended language such as “comprising” can refer, in one embodiment, 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); etc.

[0049] The term “bioreactor” or “fermenter” refers to a closed or partially closed vessel in which cells are grown and maintained. The cells may be, but are not necessarily held in liquid suspension. In some embodiments, rather than being held in liquid suspension, cells may alternatively be grown and / or maintained in contact with, on, or within another non-liquid substrate including but not limited to a solid growth support material.

[0050] “Coding sequence” or “CDS” refers to a polynucleotide sequence that encodes the sequence of amino acids of a polypeptide.

[0051] As used herein, the term “consortium” refers to two or more microorganisms that are grown together in a microbial culture.

[0052] The term “culturing” refers to growing a population of cells, e.g., microbial cells, under suitable conditions for growth, in a liquid or solid medium.

[0053] The term “derived from” encompasses the terms “originated from,” “obtained from,” “obtainable from,” “isolated from,” and “created from,” and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to another specified material.

[0054] As used herein, the term “enzymatic activity” or the like refers to a catalytic activity of a polypeptide. Certain polypeptides have a single known catalytic activity while other polypeptides have multiple catalytic activities. For example, a Fatty Acyl-Acyl Carrier Protein Thioesterase (fatty acyl-ACP thioesterase or thioesterase) is a polypeptide that has a single enzymatic activity that catalyzes the release of a fatty acid moiety from an acyl carrier protein (ACP). Fatty Acid Synthase (FAS or FAS I) is a polypeptide that has multiple catalytic functions that synthesize fatty acids. FAS I catalyzes growth of a fatty acid chain by ketoreductase, dehydratase, enoyl reductase and thiosterase functionalities.

[0055] As used herein, the term “expression” refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

[0056] As used herein, “expression cassette” refers to a DNA construct containing a promoter, a coding sequence (e.g., a gene sequence), and a terminator capable of effecting the expression of the coding sequence in a host.

[0057] As used herein, “expression vector” refers to a DNA construct containing a DNA coding sequence (e.g., gene sequence) that is operably linked to one or more suitable control sequence(s) capable of effecting expression of the coding sequence in a host. Such control sequences include a promoter to affect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. The plasmid is the most commonly used form of expression vector. However, the invention is intended to include such other forms of expression vectors that serve equivalent functions and which are, or become, known in the art.

[0058] A “gene” refers to a DNA segment that is involved in producing a polypeptide and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons).

[0059] A “gene knockout” or “knockout” refers to loss of an endogenous gene function through genome editing, e.g., the use of genetic engineering to inactivate or remove one or more specific gene sequences from the host microorganism from which a non-naturally occurring microorganism is derived.

[0060] “Gene silencing” or “gene knockdown” refers to the regulation of gene expression, e.g., regulation of expression of an endogenous gene, in a cell to prevent the expression of a gene at the transcriptional or translational level.

[0061] The term “heterologous” or “exogenous,” with reference to a polynucleotide or protein, refers to a polynucleotide or protein that does not naturally occur in a specified cell, e.g., a host cell. It is intended that the term encompass proteins that are encoded by naturally occurring genes, mutated genes, and / or synthetic genes. In contrast, the term “homologous” or “endogenous” with reference to a polynucleotide or protein, refers to a polynucleotide or protein that occurs naturally in the cell.

[0062] As used herein, the term “hydrophilic-lipophilic balance” or “HLB” refers to the degree to which a surfactant is hydrophilic or lipophilic. Measurement of HLB can be determined according to methods known in the art (e.g., PCT Publication No. WO2018 / 148465).

[0063] As used herein, “homologous protein” or “homolog” refers to a protein that has similar function and / or structure as a reference protein. Homologs may be from evolutionarily related or unrelated species. In some embodiments, a homolog has a quaternary, tertiary and / or primary structure similar to that of a reference protein, thereby potentially allowing for replacement of a segment or fragment in the reference protein with an analogous segment or fragment from the homolog, with reduced disruptiveness of structure and / or function of the reference protein in comparison with replacement of the segment or fragment with a sequence from a non-homologous protein.

[0064] “Homologous recombination” refers to the outcome of a DNA metabolic process characterized by resection, strand invasion, DNA synthesis to repair the resected ends, andcrossing over. This process can result in a new polynucleotide containing genetic modification in which nucleotide sequences of a host organism are altered with an exogenous polynucleotide sequence using sequences in the exogenous polynucleotide that are homologous to the region targeted for alteration. The exogenous polynucleotide sequence encodes a gene product that can be similar or completely unrelated to the replaced endogenous nucleotide sequence. Homologous recombination can be used to remove, inhibit, alter or modify the expression levels of an endogenous polynucleotide, express a similar or related gene product of an endogenous polynucleotide, or express a gene product that is dissimilar or unrelated to the gene product of an endogenous gene.

[0065] As used herein, the term “host cell” or “parent cell,” used interchangeably herein, refers to a cell or cell line into which a recombinant expression vector for production of a polypeptide may be transfected for expression of the polypeptide. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell may be naturally occurring or non-naturally occurring. A host cell includes cells transfected or transformed in vivo with an expression vector.

[0066] The term “hydrophilic carbon source” refers to an organic compound that is soluble in water at concentrations greater than 1 g / L.

[0067] 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.

[0068] A “3-hydroxy fatty acid” refers to a fatty acid wherein the third carbon from the acid end of the compound is hydroxylated.

[0069] The term “introduced,” in the context of inserting a nucleic acid sequence into a cell, includes “transfection,” “transformation,” or “transduction” and refers to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed.

[0070] A “naturally occurring” or “wild type” microorganism refers to an unmodified microorganism or a microorganism as found in nature.

[0071] A “non-naturally occurring” microorganism refers to a microorganism that contains one or more mutations or that has been genetically modified in comparison to a naturally occurring microorganism from which it is derived.

[0072] The term “operably linked” refers to a juxtaposition or arrangement of specified elements that allows them to perform in concert to bring about an effect. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the coding sequence.

[0073] As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length and any three-dimensional structure and single- or multi-stranded (e.g., single-stranded, double-stranded, triple-helical, etc.), which contain deoxyribonucleotides, ribonucleotides, and / or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs. Because the genetic code is degenerate, more than onecodon may be used to encode a particular amino acid, and the present invention encompasses polynucleotides which encode a particular amino acid sequence. Any type of modified nucleotide or nucleotide analog may be used, so long as the polynucleotide retains the desired functionality under conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2’-O-Me, phosphorothioates, etc.). Labels may also be incorporated for purposes of detection or capture, for example, radioactive or nonradioactive labels or anchors, e.g., biotin. The term polynucleotide also includes peptide nucleic acids (PNA). Polynucleotides may be naturally occurring or non-naturally occurring. The terms “polynucleotide,” “nucleic acid,” and “oligonucleotide” are used herein interchangeably. Polynucleotides may contain RNA, DNA, or both, and / or modified forms and / or analogs thereof. A sequence of nucleotides may be interrupted by non-nucleotide components. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR’, CO or CH2 (“formacetal”), in which each R or R’ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (--O--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Polynucleotides may be linear or circular or contain a combination of linear and circular portions.

[0074] The term “polyol” refers to an organic moiety that contains at least two hydroxyl groups. Exemplary polyols include glycerol (3-carbon), erythritol ( 4-carbon), threitol ( 4-carbon), arabitol (5- carbon), xylitol (5-carbon), ribitol (5-carbon), mannitol (6-carbon), sorbitol (6-car-bon), galactitol (6- carbon), fucitol (6-carbon), iditol (6-car-bon), inositol (6-carbon; a cyclic sugar alcohol), volemitol (7- carbon), cyclitol, and valienol. In some embodiments, a polyol is represented by a general formula HOCH2(CHOH)pCH2OH wherein p is 0-5.

[0075] A “polyol ester of a fatty acid” or “PEFA” refers to an amphiphilic molecule composed of an acetylated(R)-3-hydroxy fatty acid esterified through the carboxyl end to a 5 or 6 carbon polyol, typically D-mannitol or D-arabitol, with varying degrees of acetylations.

[0076] A “polyol lipid” refers to a compound wherein a polyol moiety is covalently attached to a fatty acid.

[0077] As used herein, “polypeptide” refers to a composition comprised of amino acids and recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0078] As used herein, the term “preferred” in reference to enzymatic activity means the propensity of an enzyme to catalyze a particular reaction. Fatty acyl-ACP thioesterases catalyze the release of fatty acids from the acyl carrier protein (ACP). Certain thioesterases preferentially catalyze the release of fatty acids that are “mid-chain” in length, that is, fatty acids that are C8-C14, while other thioesterases catalyze the release of fatty acids that are “long-chain,” that is, fatty acids that are C16-C18. The mid-chain preferring thioesterases can also catalyze the release of long-chain fatty acids, but are less active in this catalytic activity. Similarly, the long-chain preferring thioesterases can also catalyze the release of mid-chain fatty acids, but are less active in this catalytic activity.

[0079] The term “producing” includes both the production of compounds intracellularly and extracellularly, which is to include the secretion of compounds from the cell.

[0080] “Productivity” refers to the amount of a substance produced by a microorganism per unit volume per unit time in a microbial fermentation process. For example, polyol lipid productivity may be expressed as grams of polyol lipid produced per liter of solution per hour.

[0081] A “promoter” refers to a regulatory sequence that is involved in binding RNA polymerase to initiate transcription of a gene. A promoter may be an inducible promoter or a constitutive promoter. An “inducible promoter” is a promoter that is active under environmental or developmental regulatory conditions.

[0082] The term “recombinant,” refers to genetic material (i.e., nucleic acids, the polypeptides they encode, and vectors and cells containing such polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to that 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 decreased or elevated level, expressing a gene conditionally or constitutively in a manner different from its natural expression profile, and the like. Generally recombinant nucleic acids, polypeptides, and cells based thereon, have been manipulated by man such that they are not identical to related nucleic acids, polypeptides, and cells found in nature.

[0083] The terms “recovered,” “isolated,” “purified,” and “separated” as used herein refer to a material (e.g., a protein, lipid, nucleic acid, or cell) that is removed from at least one component with which it is naturally associated, for example, at a concentration of at least 90% by weight, or at least 95% by weight, or at least 98% by weight, at least 99% by weight, or at least 99.5% by weight of the sample in which it is contained. For example, these terms may refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system.

[0084] The term “selective marker” or “selectable marker” refers to a gene capable of expression in a host cell that allows for ease of selection of those hosts containing an introduced nucleic acid or vector. Examples of selectable markers include but are not limited to resistance to antimicrobial substances (e.g., hygromycin, bleomycin, or chloramphenicol), genes that encode a protein that is detectable when expressed, or genes that confer a metabolic advantage, such as a nutritional advantage, on the host cell.

[0085] “Substantially free” means no more than about 10%, 5%, 1%, 0.5%, 0.2%, or 0.1% of a particular substance in a composition.

[0086] The phrases “substantially similar” and “substantially identical” in the context of at least two nucleic acids or polypeptides typically means a polynucleotide, polypeptide, or region or domain of a polypeptide that contains a sequence that has 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, in comparison with a reference (e.g., wild-type) polynucleotide, polypeptide, or region or domain of a polypeptide. A region or domain of a polypeptide may contain, for example, at least about 20, 50, 100, or 200 amino acids within a longer polypeptide sequence. Sequence identity may be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See, e.g., 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 analyses is publicly available through the National Center for Biotechnology Information. Also, databases may be searched using FASTA (Pearson, et al. (1988) Proc. Natl. Acad. Sci.85:2444- 2448.) In some embodiments, substantially identical polypeptides differ only by one or more conservative amino acid substitutions. In some embodiments, substantially identical polypeptides are immunologically cross-reactive. In some embodiments, substantially identical nucleic acid molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).

[0087] A “sugar alcohol” refers to an organic compound containing one hydroxyl group (–OH) attached to each carbon atom. Exemplary sugar alcohols include erythritol ( 4-carbon), threitol ( 4- carbon), arabitol (5-carbon), xylitol (5-carbon), ribitol (5-carbon), mannitol (6-carbon), sorbitol (6- carbon), galactitol (6-carbon), and fucitol (6-carbon).

[0088] “Titer” refers to an amount of a substance produced by a microorganism per unit volume in a microbial fermentation process. For example, polyol lipid titer may be expressed as grams of polyol lipid produced per liter of solution.

[0089] “Transfection” or “transformation” refers to the insertion of an exogenous polynucleotide into a host cell. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome. The term “transfecting” or “transfection” is intended to encompass all conventional techniques for introducing nucleic acid into host cells. Examples of transfection techniques include, but are not limited to, calcium phosphate precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, and microinjection.

[0090] As used herein, the terms “transformed,” “stably transformed,” and “transgenic” refer to a cell that has a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or as an episomal plasmid that is maintained through multiple generations.

[0091] “Under transcriptional control” is a term well understood in the art that indicates that transcription of a polynucleotide sequence depends on its being operably linked to an element which contributes to the initiation of, or promotes transcription.

[0092] “Under translational control” is a term well understood in the art that indicates a regulatory process which occurs after mRNA has been formed.

[0093] As used herein, a “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

[0094] Related (and derivative) proteins encompass “variant” proteins. Variant proteins differ from a parent protein and / or from one another by a small number of amino acid residues. In some embodiments, the number of different amino acid residues is any 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 additionally, variants may have a specified degree of sequence identity with a reference protein or nucleic acid, e.g., as determined using a sequence alignment tool, such as BLAST, ALIGN, and CLUSTAL (see, infra). For example, variant proteins or nucleic acid 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 a reference sequence.

[0095] “Yield” refers to amount of a product produced from a feed material (for example, sugar) relative to the total amount of the substance that would be produced if all of the feed substance were converted to product. For example, polyol lipid yield may be expressed as % of polyol lipid produced relative to a theoretical yield if 100% of the feed substance (for example, sugar) were converted to polyol lipid. Microorganisms

[0096] Non-naturally occurring microorganisms as described herein are derived from parent microorganisms (e.g., naturally occurring or wild-type microorganisms, or engineered, non-naturally occurring parent microorganisms) that produce PEFA. In some embodiments, the microorganisms produce PEFA in commercially relevant quantities in microbial culture without addition of hydrophobic substrates. In some embodiments, the parent microorganisms are fungal microorganisms, such as yeast microorganisms.

[0097] In some embodiments, the parent microorganisms are from subkingdom Dikarya. The microorganisms may be from the phylum Ascomycota or Basidiomycota. For example, the microorganisms may be in phylum Basidiomycota, class Microbotryomycetes, order Sporidiobolales, genus Rhodotorula, Rhodosporidiobolus, or Sporobolomyces. In various embodiments, microorganisms (e.g., basidiomycetous 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 certain embodiments, the microorganisms are a strain selected from Rhodotorula babjevae strain NRRL Y-67018, Rhodotorula babjevae strain NRRL Y-67017, Rhodotorula babjevae strain UCDFST 68-916.1, Rhodotorula babjevae strain UCDFST 67-458, Rhodotorula babjevae strain UCDFST 05-736,Rhodotorula diobovata strain UCDFST 04-830, Rhodotorula diobovata strain NRRL Y-67015, Rhodotorula kratochvilovae strain NRRL Y-67016, Rhodotorula paludigena strain NRRL Y-67012, Rhodotorula paludigena strain UCDFST 82-646.2, Rhodotorula paludigena strain UCDFST 81-492, Rhodotorula aff. paludigena strain NRRL Y-67009, Rhodotorula sphaerocarpa strain NRRL Y- 67010, Rhodotorula dairenensis strain NRRL Y-67011, Rhodosporidiobolus aff. colostri strain NRRL Y-67014, Rhodosporidiobolus aff. colostri NRRL Y-67014, Rhodosporidiobolus aff. nylandii strain NRRL Y-67013, Rhodosporidiobolus ruineniae NRRL Y-17302, Rhodotorula taiwanensis strain MD1149, and Rhdodotorula mucilaginosa strain 50-3-19 / 208.

[0098] Nonlimiting examples of microorganism species and strains that produce polyol lipids and that may be engineered in accordance with embodiments of the methods described herein are described in U.S. Patent Nos.10,196,663 and 11,401,539. Polyol esters of fatty acids

[0099] The microorganisms described herein naturally produce polyol esters of fatty acids (PEFA), and may be grown in a nutrient growth medium for production of the PEFA in microbial culture. In some embodiments, the microorganism secretes the PEFA into the nutrient growth medium. In some embodiments, the microorganism produces at least about 1 g / L PEFA when grown in the nutrient growth medium

[0100] PEFA are amphiphilic molecules that include a sugar alcohol, e.g., a D-mannitol and / or a D- arabitol, esterified to the carboxyl end of a 3-hydroxy fatty acyl moiety, which may or may not be acetylated. The non-esterified hydroxy groups of the sugar alcohol may or may not be acetylated as well. In varying embodiments, PEFA produced by a non-naturally occurring microorganism as described herein may be a mixture of similar compounds containing (R)-3-hydroxy fatty acyl moieties with varying carbon chain lengths, with average carbon chain length in the range of C8 to C18, C8 to C16, C8 to C14, C8 to C12, C8 to C10, C8 to C12, C10 to C12, C12 to C14, C12 to C18, C14 to C18, or C16 or C18. In varying embodiments, the (R)-3-hydroxy fatty acyl moieties can present varying degrees of unsaturation in the range of 0 to about 6, e.g., in the range of about 2 to about 5. In varying embodiments, the non-esterified hydroxy groups of the sugar alcohol can be esterified to acetyl groups. In various embodiments, the sugar alcohol is fully acetylated, partially acetylated, or non-acetylated.

[0101] In some embodiments, the PEFA sugar alcohol moiety includes a 5 carbon polyol (polyol head portion), which can be D-arabitol or L-arabitol. In some embodiments, the polyol lipid sugar alcohol moiety includes a 6 carbon polyol, which can be D-mannitol or L-mannitol. In some embodiments, the PEFA sugar alcohol moiety is attached or bound at the carboxyl end of the fatty acid (fatty acid tail portion). In some embodiments, the fatty acid is hydroxylated at position 4-3 from the carboxyl end. In some embodiments, the hydroxyl group in position 4-3 may or may not be acetylated.

[0102] In various embodiments, one or more PEFAs produced in a microorganism as described herein, and / or in a polyol lipid composition isolated and / or purified from a culture of the microorganisms is selected from: acetylated C6:03-hydroxy fatty acids esterified to D-arabitol with 4 acetylations; acetylated C6:03-hydroxy fatty acids esterified to D-arabitol with 3 acetylations; acetylated C6:03- hydroxy fatty acids esterified to D-arabitol with 2 acetylations; acetylated C6:03-hydroxy fatty acidsesterified to D-arabitol with 1 acetylation; acetylated C8:03-hydroxy fatty acids esterified to D-arabitol with 4 acetylations; acetylated C8:03-hydroxy fatty acids esterified to D-arabitol with 3 acetylations; acetylated C8:03-hydroxy fatty acids esterified to D-arabitol with 2 acetylations; acetylated C8:03- hydroxy fatty acids esterified to D-arabitol with 1 acetylation; acetylated C10:03-hydroxy fatty acids esterified to D-arabitol with 4 acetylations; acetylated C10:03-hydroxy fatty acids esterified to D-arabitol with 3 acetylations; acetylated C10:03-hydroxy fatty acids esterified to D-arabitol with 2 acetylations; acetylated C10:03-hydroxy fatty acids esterified to D-arabitol with 1 acetylation; acetylated C6:03- hydroxy fatty acids esterified to D-mannitol with 5 acetylations; acetylated C6:03-hydroxy fatty acids esterified to D-mannitol with 4 acetylations; acetylated C6:03-hydroxy fatty acids esterified to D- mannitol with 3 acetylations; acetylated C6:03-hydroxy fatty acids esterified to D-mannitol with 2 acetylations; acetylated C6:03-hydroxy fatty acids esterified to D-mannitol with 1 acetylation; acetylated C8:03-hydroxy fatty acids esterified to D-mannitol with 5 acetylations; acetylated C8:03-hydroxy fatty acids esterified to D-mannitol with 4 acetylations; acetylated C8:03-hydroxy fatty acids esterified to D- mannitol with 3 acetylations; acetylated C8:03-hydroxy fatty acids esterified to D-mannitol with 2 acetylations; acetylated C8:03-hydroxy fatty acids esterified to D-mannitol with 1 acetylation; acetylated C10:03-hydroxy fatty acids esterified to D-mannitol with 5 acetylations; acetylated C10:03-hydroxy fatty acids esterified to D-mannitol with 4 acetylations; acetylated C10:03-hydroxy fatty acids esterified to D-mannitol with 3 acetylations; acetylated C10:03-hydroxy fatty acids esterified to D-mannitol with 2 acetylations; acetylated C10:03-hydroxy fatty acids esterified to D-mannitol with 1 acetylation; acetylated C12:03-hydroxy fatty acid esterified to D-arabitol with 3 acetylations; acetylated C14:03- hydroxy fatty acid esterified to D-mannitol with 4 acetylations; acetylated C16:03-hydroxyfatty acid esterified to D-mannitol with 2 acetylations; acetylated C16:03-hydroxy fatty acid esterified to D-arabitol with 3 acetylations; acetylated C14:03-hydroxy fatty acid esterified to D-arabitol with 4 acetylations; acetylated C14:03-hydroxy fatty acid esterified to D-mannitol with 5 acetylations; acetylated C16:03- hydroxy fatty acid esterified to D-mannitol with 3 acetylations; acetylated C16:03-hydroxy fatty acid esterified to D-arabitol with 3 acetylations; acetylated C16:03-hydroxy fatty acid esterified to D-mannitol with 4 acetylations; acetylated C18:03-hydroxy fatty acid esterified to D-mannitol with 2 acetylations; acetylated C16:03-hydroxy fatty acid esterified to D-mannitol with 5 acetylations; acetylated C16:03- hydroxy fatty acid esterified to D-arabitol with 4 acetylations; acetylated C18:03-hydroxy fatty acid esterified to D-mannitol with 3 acetylations; acetylated C18:03-hydroxy fatty acid esterified to D- mannitol with 4 acetylations; acetylated C18:03-hydroxy fatty acid esterified to D-arabitol with 3 acetylations; acetylated C18:03-hydroxy fatty acid esterified to D-mannitol with 5 acetylations; acetylated C18:03-hydroxy fatty acid esterified to D-arabitol with 4 acetylations; acetylated C20:03- hydroxy fatty acid esterified to D-mannitol with 3 acetylations; and acetylated C20:03-hydroxy fatty acid esterified to D-mannitol with 4 acetylations. Metabolic pathway engineering

[0103] Microorganisms as described herein are engineered to produce PEFA with desired fatty acid tail carbon chain length and / or for reduced or eliminated degradation (e.g., β-oxidation) of fatty acids. The biosynthetic pathway for PEFA production is altered, through expression of heterologous enzyme(s) of the pathway or mutation of gene(s) that encode native enzyme(s) in the pathway, suchthat PEFA with desired fatty acid tail length are produced by the microorganism. Additionally, gene(s) that encode one or more enzyme(s) involved in fatty acid degradation, such as peroxisomal and / or mitochondrial β-oxidation of fatty acids, is altered, silenced, deleted, inactivated, or down-regulated, such that fatty acid degradation in the microorganism cells is reduced or eliminated, enabling the incorporation of fatty acids, e.g., shortened fatty acids, into PEFA.

[0104] Fatty acid moieties are produced natively in fungi by the fatty acid synthase complex, by elongation of nascent acyl chains through sequential incorporation and reduction of carbon in the form of malonyl-CoA. (Fig.1A) Natively, fatty acid synthase (FAS) enzymes produce primarily C16, C18, and trace amounts of C14 fatty acids as esters to Coenzyme A (CoA) molecules, whereupon they are incorporated into various fatty acid containing molecules, including PEFA.

[0105] Thioesterases are enzymes that release free fatty acids (FFA) from an extending acyl chain bound acyl-carrier protein (ACP) domain of a FAS, by cleaving a thiol bond between the ACP and the acyl chain. Genetic diversity in thioesterases can be exploited to release fatty acids with a specific chain length. For example, the Umbellularia californica thioesterase UcFat1B (Uniprot Q41635) specifically releases C12 fatty acids and CvFat1B thioesterase from Cuphea viscosissima (G3ESU9) produces predominantly C8 fatty acids.

[0106] Hosts transformed with thioesterases have been used to produce shortened fatty acid moieties that have been incorporated into triacylglycerol (TAG) molecules and have also been used to produce other medium chain oleochemicals. In some embodiments, microorganisms as described herein are transformed with heterologous polynucleotide(s) that express one or more heterologous thioesterase (i.e., expression of one heterologous thioesterase or a mixture of two or more different heterologous thioesterases) to produce PEFA with preferred fatty acid carbon chain length that is different, e.g., shorter, than the average fatty acid carbon chain length produced by the parent (e.g., wild-type) microorganism.

[0107] Fungal type I fatty acid synthases are composed of either one or two peptides that each contain multiple catalytic domains, which assemble into a large complex. In order to improve the accessibility of Acyl-ACP to the catalytic activity of thioesterases, a thioesterase enzyme may be fused adjacent to the ACP domain in the polypeptide (Zhu, 2017). In some embodiments, microorganisms as described herein are transformed with heterologous polynucleotide(s) that express one or more chimeric heterologous thioesterase fused to one or more subunit of a fatty acid synthase (e.g., type I fatty acid synthase) (i.e., expression of one chimeric heterologous thioesterase / fatty acid synthase or a mixture of two or more different chimeric heterologous thioesterase / fatty acid synthases) to produce PEFA with preferred fatty acid carbon chain length that is different, e.g., shorter, than the average fatty acid carbon chain length produced by the parent (e.g., wild-type) microorganism. The heterologous polynucleotide may encode one or more catalytic domain(s) of the fatty acid synthase polypeptide (e.g., one or both subunits of the fatty acid synthase, or at least one catalytic domain of the fatty acid synthase), fused to a polynucleotide that encodes a heterologous thioesterase. In some embodiments, the thioesterase is introduced into the sequence of the native enzyme adjacent to the ACP domain.

[0108] In some embodiments, an endogenous gene sequence that encodes a fatty acid synthase in a microorganism as described herein is mutated such that fatty acids of preferred carbon chain length are released, such as, but not limited to, C8-C14 fatty acids. In one nonlimiting example, a residue homologous to R1834 in the malonyl / palmitoyl / transferase (MPT) domain of the Fas1p gene in Saccharomyces cerevisiae may be mutated to lysine (K) such as the residue R665 in the Fas2p homolog in R. babjevae 04-877 (“Strain 1” in the Examples). This sequence R665K is depicted in SEQ ID NO:62. In some embodiments, the gene expressing the modified form of the gene replaces the native version of the gene. In some embodiments, the gene expressing the modified form of the gene may is an intron containing gene. In some embodiments, the gene expressing the modified form of the gene is codon optimized for expression in the microorganism.

[0109] In some embodiments, the profile of PEFA in a PEFA-producing microorganism is modified by expressing one or more fatty acid synthase genes from another organism that produces a different distribution of acyl-CoA molecules than the PEFA-producing microorganism. In some embodiments, these acyl-CoA molecules are enriched for shorter chain fatty acyl-CoA molecules (e.g. C12, C14, C16). In some embodiments, these fatty acid synthase genes are derived from specialized fatty acid synthases found in secondary metabolite clusters that produce short chain acyl-CoA molecules. In some embodiments, these fatty acid synthase genes are derived from a species in the genus Hypoxylon. In some embodiments, these genes are Spo_FasA and Spo_FasB from Hypoxylon monticulosum (See e.g. Tian et al.2020) (SEQ ID NO:96 and SEQ ID NO:97).

[0110] In some embodiments, a microorganism as described herein is engineered for reduced degradation of fatty acids, such as reduced or eliminated β-oxidation of fatty acids, in conjunction with PEFA biosynthetic pathway modification(s) as described above. For example, one or more endogenous gene(s) that encode an enzyme that degrades fatty acids (e.g., β-oxidation of fatty acids) may be altered, silenced. deleted, inactivated, or down-regulated. For example, endogenous peroxisomal β-oxidation and / or mitochondrial of fatty acids may be reduced or eliminated in the microorganism. In some embodiments, endogenous multifunctional enzyme type 2 (MFE-2) peroxisomal β-oxidation of fatty acids is reduced or eliminated, in comparison to the parent (e.g., wild-type) microorganism. In some embodiments, endogenous enoyl-CoA hydratase mitochondrial β-oxidation of fatty acids is reduced or eliminated, in comparison to the parent (e.g., wild-type) microorganism. In some embodiments, MFE-2 peroxisomal β-oxidation of fatty acids and enoyl- CoA hydratase mitochondrial β-oxidation of fatty acids are reduced or eliminated, in comparison to the parent (e.g., wild-type) microorganism. Transformation of microorganisms

[0111] PEFA producing microorganisms as described herein may be transformed with a polynucleotide construct to produce a non-naturally occurring microorganism with altered expression of one or more endogenous gene(s) and / or expression of one or more heterologous gene(s). Typically, the polynucleotide construct is a linear DNA sequence to be integrated into the genome of the microorganism or a DNA plasmid capable of autonomous replication in the microorganism cells.

[0112] In some embodiments, the polynucleotide construct may contain a coding sequence for a heterologous thioesterase enzyme or polypeptide with thioesterase enzymatic activity. In other embodiments, the polynucleotide construct may contain a coding sequence for a heterologous thioesterase enzyme or a polypeptide with thioesterase enzymatic activity that is fused to a coding sequence for one or more subunit of a fatty acid synthase (e.g., type I fatty acid synthase) enzyme or a polypeptide with fatty acid synthase catalytic activity (e.g., at least one catalytic domain of the fatty acid synthase). In some embodiments, the polynucleotide construct may contain a coding sequence for one or more subunit of a fatty acid synthase (e.g., type I fatty acid synthase) enzyme or a polypeptide with fatty acid synthase catalytic activity (e.g., at least one catalytic domain of the fatty acid synthase), wherein the coding sequence has been modified such that the encoded polypeptide enzymatically produces at least about 1%, or any of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% more C8 to C14 acyl-CoA molecules than the wild-type enzyme.

[0113] The polynucleotide (e.g., DNA) may be introduced into the microorganism cells by a method such as electroporation, PEG-mediated transformation, protoplasting, or agrobacterium mediated mutagenesis. Transformed cells may be detected by the presence of a selectable marker such as antibiotic resistance, a visually observable marker such as absence of a carotenoid, and / or auxotrophy for a required metabolic product. Stable integrants may be identified by serial passaging on non-selective media followed by screening on selective media and / or by amplifying the junction between the genome and recombinant DNA (e.g., via polymerase chain reaction (PCR)). Transformants that have integrated DNA via homologous recombination as opposed to non-homologous end joining can be enriched by splitting the selectable marker into two fragments such that homologous recombination between the selectable marker fragments is required for a cell where the DNA has integrated to become resistant to the selectable marker.

[0114] Nonlimiting examples of selectable markers include an enzyme that provides antibiotic resistance (e.g., hygromycin, nourseothricin), a fluorescent or otherwise detectable (e.g., visually detectable) protein or other molecule, or an enzyme that produces a required metabolic protein.

[0115] A polynucleotide construct for genetic modification of PEFA producing microorganisms as described herein may include: a promoter; and a nucleotide coding sequence, wherein the promoter is capable of driving gene expression of the coding sequence in the microorganism. In some embodiments, a promoter in a polynucleotide construct described herein is inducible in the microorganism.

[0116] In some embodiments, a polynucleotide construct for producing a gene knockout (e.g., disruption or deletion of an endogenous gene sequence) in a PEFA producing host microorganism is provided, which includes: a promoter; a nucleotide sequence that is homologous to at least a portion of a targeted gene sequence in the genome of the microorganism; a nucleotide coding sequence that encodes a protein that provides a selectable marker; and a terminator, wherein the promoter and terminator sequences are capable of driving gene expression of the selectable marker coding sequence in the microorganism.

[0117] In some embodiments, a polynucleotide construct for expression of a heterologous gene in a PEFA producing host microorganism is provided, which includes: a promoter; a nucleotide coding sequence for a heterologous gene; and a nucleotide coding sequence that encodes a protein that provides a selectable marker, wherein the promoter is capable of driving gene expression of the heterologous gene and selectable marker coding sequences in the microorganism.

[0118] In some embodiments, a promoter for an endogenous gene may be replaced with a different promoter, and / or a 3’ untranslated region (UTR) and / or a 5’ UTR may be added to the endogenous gene sequence, thereby increasing, decreasing, or changing the regulation of expression of the endogenous gene or the regulation of the stability of mRNA that is 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 PEFA producing host microorganism is provided, which includes: a nucleotide sequence that is homologous to at least a portion of a targeted gene sequence in the genome of the microorganism; a promoter sequence; and a 3’ UTR, and / or a polynucleotide sequence that encodes 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 PEFA producing host microorganism is provided, which includes: a nucleotide sequence that is homologous to at least a portion of a targeted gene sequence in the genome of the microorganism; a promoter sequence; and a terminator sequence, a 5’ UTR, and / or a polynucleotide sequence that encodes a C-terminal tag.

[0119] In some embodiments, as shown schematically in Figures 4A-4C, DNA sequences are provided that are homologous to the endogenous gene to be tagged and / or promoter to be replaced Figure 4A. Transformation can be accomplished using a split marker system as shown in Figure 4B, yielding a gene with an altered promoter and / or peptide sequence as shown in Figure 4C, wherein the promoter of the target gene has been replaced by a new promoter and / or has been fused to an N-terminal tag.

[0120] In some embodiments, as shown schematically in Figures 5A – 5C, DNA sequences are provided that are homologous to the endogenous gene to be tagged and / or terminator to be replaced Figure 5A. Transformation can be accomplished using a split marker system as in Figure 5B, yielding a gene with an altered terminator and / or peptide sequence as shown in Figure 5C, wherein the terminator of the target gene has been replaced by a new terminator and / or has been fused to a C-terminal tag.

[0121] In some embodiments, a coding sequence of the polynucleotide construct (e.g., a coding sequence for a heterologous gene, and optionally for a selectable marker) is codon optimized for expression in the host microorganism. Nonlimiting examples of codon usage in a microorganism as described herein are provided in Table 1 and Table 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. Table 1: Codon usage table for Rhodotorula babjevaeResidue Codon Percent Residue Codon Percent Residue Codon Percent Ala GCA 9.3ATA 2.5 Thr ACA 5.7 Ile GCC 45.9 ATC 76.9 ACC 45.5 GCG 34.7 ATT 20.6 ACG 41.9 GCT 10.1 Leu CTA 2.4 ACT 6.9 Asp GAC 79.9 CTC 52.9 Trp TGG 100 GAT 20.1 CTG 26.7Tyr TAC 87.2Asn AAC 82.7 CTT 9.6 TAT 12.8 AAT 17.3 TTA 2 Val GTA 6.8 Arg AGA 3.6 TTG 6.4 GTC 53.1 AGG 8.4 Lys AAA 13 GTG 29.5 CGA 12 AAG 87 GTT 10.7 CGC 48.1 Met ATG 1.0 CGG 15.4 CCA 9.5 Pro CGT 12.5 CCC 33.8 Cys TGC 83.2 CCG 42.6 TGT 16.8 CCT 14.1 Gln CAA 15.6 Phe TTC 80.2 CAG 84.4 TTT 19.8 Glu GAA 18.4 Ser AGC 21.9 GAG 81.6 AGT 3.4 GGA 8.2 TCA 4.2 Gly GGC 61.9 TCC 18.1 GGG 15.7 TCG 45.4 GGT 14.3 TCT 7 His CAC 78.6 CAT 21.4 Table 2: Codon usage table for Rhodotorula toruloidesResidue Codon Percent Residue Codon Percent Residue Codon Percent Ala GCA 14ATA 3.3 Thr ACA 10.3 Ile GCC 32.5 ATC 80.1 ACC 34.4 GCG 37 ATT 16.6 ACG 41.4 GCT 16.5 Leu CTA 2.6 ACT 13.8 Asp GAC 80.7 CTC 55.5 Trp TGG 100 GAT 19.3 CTG 17.1Tyr TAC 85.9Asn AAC 84.8 CTT 11.4 TAT 14.1 AAT 15.2 TTA 0.8 Val GTA 4.3 Arg AGA 3.2 TTG 12.7 GTC 64.3 AGG 14.7 Lys AAA 12.2 GTG 17.5 CGA 14.3 AAG 87.8 GTT 13.9 CGC 41.6 Met ATG 1.0 CGG 17 CCA 11.2 Pro CGT 9.2 CCC 29.8 Cys TGC 81.9 CCG 35.2 TGT 18.1 CCT 23.8 Gln CAA 24.5 Phe TTC 82.6 CAG 75.5 TTT 17.4 Glu GAA 22.6 Ser AGC 17.1 GAG 77.4 AGT 5.5 GGA 19.8 TCA 7.1 Gly GGC 50.2 TCC 21.7 GGG 15.8 TCG 38.6 GGT 14.2 TCT 10.1 His CAC 77.8 CAT 22.2 Altered expression of endogenous genes

[0122] Genetically modified PEFA producing microorganisms are provided as described herein, wherein one or more endogenous gene or particular polynucleotide sequence is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, or up-regulated. In certain embodiments, a non-naturally occurring microorganism as described herein contains at least one knocked out (e.g., disrupted or deleted) endogenous gene and / or at least one endogenous gene for which expression is altered, in comparison to the naturally occurring microorganism or a parent microorganism from which the non-naturally occurring microorganism is derived.

[0123] In some embodiments, a microorganism as described herein contains at least one endogenous gene with altered expression or at least one knocked out gene that encodes an enzyme that degrades fatty acids, such as one or more enzyme that catalyzes β-oxidation of fatty acids, e.g., peroxisomal and / or mitochondrial β-oxidation of fatty acids.

[0124] In some embodiments, the microorganism contains at least one knocked out gene. For example, the endogenous gene may be knocked out or edited by homologous recombination, CRISPR, zinc-finger nuclease, or TALEN.

[0125] In some embodiments, the microorganism contains at least one silenced gene. When genes are silenced, their expression is reduced. In contrast, when genes are knocked out, they are completely erased from the organism's genome and thus have no expression. For example, expression of an endogenous gene may be silenced by RNA interference, RNA silencing, siRNA, antisense oligonucleotides, ribozymes, or a CRISPR-based method (e.g., dCas9 or Cas13).

[0126] In some embodiments, an endogenous gene sequence is fused to a sequence that encodes a peptide, in order to increase, decrease, or change the regulation of expression of the endogenous gene. For example, the peptide may be the auxin-inducible degron IAA7, an N- terminal fusion of ubiquitin, or the tobacco etch virus degron tab TDegF.

[0127] An endogenous gene that is involved in peroxisomal beta oxidation may be knocked out, silenced, or reduced in expression level, resulting in a microorganism with a lower level of beta oxidation of fatty acids than the naturally occurring microorganism or parent microorganism from which the non-naturally occurring microorganism is derived. For example, the endogenous gene that is involved in peroxisomal beta oxidation may encode a peroxisomal membrane E3 ubiquitin ligase (PEX10) (EC 2.3.2.27), a 3-hydoxyacyl-CoA dehydrogenase / enoyl-CoA hydratase enzyme (MFE1; FOX2) (EC 4.2.1.119), an acyl-CoA oxidase enzyme (POX1-6) (EC 1.3.3.6), a peroxisomal transport protein (PXA1), or a peroxisomal fatty acid acyl-CoA synthase enzyme (FAA2) (EC 2.3.1.86), or a homolog thereof.

[0128] An endogenous gene that is involved in mitochondrial beta oxidation may be knocked out, silenced, or reduced in expression level, resulting in a microorganism with a lower level of beta oxidation of fatty acids than the naturally occurring microorganism or parent microorganism from which the non-naturally occurring microorganism is derived. For example, the endogenous gene may encode an enoyl-CoA hydratase enzyme (ECHS1) (EC 4.2.1.17), or a homolog thereof.

[0129] In some embodiments, the non-naturally occurring microorganism includes a gene in which the promoter has been altered or replaced, in order to increase, decrease, or change the regulation of expression of the endogenous gene or the regulation of the stability of mRNA that is transcribed from the endogenous gene. For example, the promoter may be replaced (swapped) by homologous recombination, CRISPR, zinc-finger nuclease, or TALEN. In some embodiments, the endogenous gene is fused to a sequence that encodes a peptide, in order to increase, decrease, or change the regulation of expression of the endogenous gene, such as, but not limited to, the auxin-inducible degron IAA7, an N-terminal fusion of ubiquitin, or the tobacco etch virus degron tag TDegF.

[0130] In some embodiments, a non-naturally occurring microorganism that has been modified for altered expression of an endogenous gene, e.g., modified such that an endogenous gene that encodes an enzyme that degrades fatty acids, such as β-oxidation of fatty acids, wherein the endogenous fatty acid degradation enzyme encoding gene is partially, substantially, or completely deleted, silenced, inactivated, or down-regulated, produces and / or secretes PEFA of a different composition than the naturally occurring microorganism or parent microorganism from which it isderived. For example, the PEFA composition of the PEFA produced by the modified microorganism may include at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or may include 100%, PEFA with chain length less than or equal to C14 in comparison to the naturally occurring microorganism or the parent microorganism from which the non-naturally occurring microorganism is derived.

[0131] In some embodiments, a non-naturally occurring microorganism that has been modified for altered expression of an endogenous gene, as described herein, e.g., modified such that an endogenous gene or a particular polynucleotide sequence is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, or up-regulated, also expresses a heterologous selectable marker, such as an enzyme that provides antibiotic resistance (e.g., resistance to hygromycin or nourseothricin), a fluorescent or otherwise detectable (e.g., visually detectable) protein or other molecule, or an enzyme that produces a required metabolic product. Expression of heterologous genes

[0132] Non-naturally occurring PEFA producing microorganisms are provided which have been modified for expression of one or more heterologous gene sequence. The non-naturally occurring microorganisms include one or more exogenous polynucleotide(s) that encode and express the one or more heterologous gene(s), i.e., one or more heterologous nucleotide coding sequence.

[0133] In some embodiments, one or more heterologous thioesterase enzyme or heterologous polypeptide with thioesterase enzymatic activity may be expressed, thereby altering (e.g., shortening) the average fatty acid carbon chain length of PEFA produced in the microorganism and / or altering the hydrophilic-lipophilic balance (HLB) of the PEFA produced by the microorganism. In some embodiments, a heterologous thioesterase enzyme or polypeptide with thioesterase enzymatic activity, fused to a subunit of a fatty acid synthase, a complete fatty acid synthase enzyme, or a polypeptide with fatty acid synthase enzymatic activity, may be expressed, thereby altering (e.g., shortening) the average fatty acid carbon chain length of PEFA produced in the microorganism and / or altering the hydrophilic-lipophilic balance (HLB) of the PEFA produced by the microorganism. In some embodiments, a non-naturally occurring microorganism as described herein contains a coding sequence for a heterologous thioesterase enzyme or polypeptide with thioesterase enzymatic activity that is fused to a coding sequence for one or more subunit of a fatty acid synthase (e.g., type I fatty acid synthase) enzyme or a polypeptide with fatty acid synthase catalytic activity (e.g., at least one catalytic domain of the fatty acid synthase).

[0134] In some embodiments, a non-naturally occurring microorganism that has been modified for expression of one or more heterologous thioesterase or thioesterase / fatty acid synthase fusion protein, as described herein, produces and / or secretes PEFA with preferred average fatty acid carbon chain length, such as any of C8 to C18, C8 to C16, C8 to C14, C8 to C12, C8 to C10, C8 to C12, C10 to C12, C12 to C14, C12 to C18, C14 to C18, or C16 to C18.

[0135] For example, fatty acids with the preferred carbon chain length may be produced in the engineered microorganism at a level that is at least about 1%, 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% higher than fatty acids with the same carbon chain length that are produced in the parent (e.g., wild-type) microorganism. The titer of PEFA with the preferred average fatty acid carbon chain length produced by the non-naturally occurring microorganism can be greater than any of about 0.01, 0.1, 1g / L, 25g / L, 50g / L, 75g / L, 100g / L, 125g / L, 150g / L, 175g / L, 200g / L, 250g / L, 300g / L, 400g / L, or 500g / L. The titer of PEFA with the preferred average fatty acid carbon chain length produced by the non-naturally occurring microorganism can be about 50g / L to about 100g / L, about 100g / L to about 150g / L, about 150g / L to about 200g / L, about 200g / L to about 250g / L, about 250g / L to about 300g / L, about 300g / L to about 350g / L, about 350g / L to about 400g / L, about 400g / L to about 450g / L, about 450g / L to about 500g / 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. The PEFA composition of the PEFA produced by the modified microorganism may include at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or may include 100%, PEFA with chain length less than the PEFA produced by the naturally occurring microorganism or the parent microorganism from which the non-naturally occurring microorganism is derived. In some embodiments, the PEFA composition of the PEFA produced by the modified microorganism includes at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or 100%, PEFA with chain length less than C14 in comparison to the naturally occurring microorganism or the parent microorganism from which the non-naturally occurring microorganism is derived.

[0136] In some embodiments, a non-naturally occurring microorganism that has been modified for expression of a heterologous gene, as described herein, expresses a heterologous selectable marker, such as an enzyme that provides antibiotic resistance (e.g., resistance to hygromycin or nourseothricin), a fluorescent or otherwise detectable (e.g., visually detectable) protein or other molecule, or an enzyme that produces a required metabolic product. Methods of producing PEFA

[0137] Methods are provided for producing polyol esters of fatty acids (PEFA) in a non-naturally occurring microorganism as described herein. One or a consortium of two or more of the non- naturally occurring microorganisms may be grown (cultured) in an environment, such as a bioreactor, that contains a nutrient growth medium (culture medium), under conditions that are suitable for growth of the microorganisms and biosynthesis, including biosynthesis of PEFA by the microorganisms. The culture medium includes carbon source(s), nitrogen source(s), inorganic substances (e.g., inorganic salts), and any other substances required for the growth of the microorganism (e.g., vitamins, amino acids, etc.).

[0138] Typically, the PEFA are secreted by the microorganisms into the culture medium, and may be recovered from the medium. In some embodiments, PEFA are produced at a titer 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, 425 g / L, or 450 g / L, 475 g / L,or 500 g / L. The method may be performed as a batch, fed batch, or continuous process. In some embodiments, a “continuous” method is used, in which fresh medium is continuously added, while culture medium is continuously removed at the same rate, keeping the culture volume relatively constant.

[0139] Typically, PEFA produced by the non-naturally occurring microorganisms described herein have a density of at least about 1.00 g / mL, e.g., about 1.00 g / mL to about 1.10 g / mL. In some embodiments, the PEFA may be recovered (harvested) from the culture medium without lysis of the microorganism cells. In some embodiments, the PEFA may be recovered from the culture medium without extraction with an organic solvent. In some embodiments, the PEFA may be recovered from the culture medium using a mechanical separation technique, such as, but not limited to, centrifugation, decanting (e.g., continuous decanting), or passive settling.

[0140] Further provided is a bioreactor or other microbial culture environment which includes a non-naturally occurring PEFA producing microorganism as described herein in a nutrient growth medium. The bioreactor may be operated within a batch, fed batch, or continuous system.

[0141] In some embodiments, the microorganism culture includes one or more hydrophilic carbon source (e.g., a mono- or disaccharide, e.g., 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 phosphate monobasic (e.g., 7.0 g / L), sodium phosphate dibasic (e.g., 5.0 g / L), magnesium sulfate hexahydrate (e.g., 1.5 g / L), and micronutrient solution containing various salts (e.g., 10 mL / L ). In some embodiments, the microorganism culture includes an iron salt. Illustrative but nonlimiting iron salts that may be used include, without limitation, iron chloride (II) & (III), iron sulfate (II) & (III), iron nitrate (II) & (III), iron phosphate (II) & (III), iron fumarate, iron edetate, iron citrate, iron malate, iron oxalate, iron tartrate, iron succinate, and iron acetate, in their anhydrous or their hydrated states, and organometallic complexes thereof. For example, the culture medium may include an iron salt in a concentration in the range of about 0.001 mg / L to about 10 g / L, e.g., about 1 mg / L to about 0.5 g / L or, such as about 0.01 g / L to about 0.5 g / L. In some embodiments, the microorganism culture is supplemented with one or more hydrophilic carbon source (e.g., a mono- or di-saccharide, e.g., glucose and / or sucrose) at a concentration 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, one or more hydrophobic carbon sources is included, such as an oil, an alkane, a fatty acid, a fatty ester, or a mixture thereof.

[0142] The method may include culturing a population of non-naturally occurring microorganisms as described in a culture medium that includes one or more hydrophilic ( e.g., non-hydrophobic) carbon sources. In some embodiments, the culture does not include hydrophobic carbon sources. In other embodiments, the culture includes one or more hydrophilic (e.g., non-hydrophobic) carbon sources and one or more hydrophobic carbon sources. In certain embodiments, additional hydrophilic carbon source is added, e.g., during any stage of growth including exponential growth, and after the population of microorganism cells reaches stationary phase

[0143] In various embodiments, the volume of the microorganism 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.

[0144] In some embodiments, the culture contains less than about 2% (w / v) nitrogen, or about 0.005% (w / v) to about 2% (w / v) nitrogen. For example, the nitrogen source may be selected from ammonia, ammonium salt, nitrate, nitrite, nucleotides, nucleosides, proteins, peptides, amino acids, urea and its derivatives, and mixtures thereof. In some embodiments, the culture contains about 0.05% (w / v) ammonium chloride.

[0145] In varying embodiments, the culture contains one or more hydrophilic carbon source(s) at a concentration of about 0.2% (w / v) to about 70% (w / v), e.g., 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., PEFA), organic acids, esters, aldehydes, ketones, alcohols, waste streams, plant materials, lignocellulosic hydrolysates, industrial co-products, and mixtures thereof. The hydrophilic carbon source may be a monosaccharide, an oligosaccharide, or a polysaccharide which contains 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.

[0146] 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, e.g., using a nitrogen source that can be consumed or utilized by the microorganism cells for growth and production of bioproducts. In some embodiments, the microorganism culture is maintained at a temperature in the range of about 24° C. to about 30° C. PEFA products and compositions

[0147] Compositions that contain PEFA, produced in any of the non-naturally occurring microorganisms described herein or in any of the methods for culturing the microorganisms as described herein, are provided. The compositions may include any of the PEFA described herein, such as PEFA containing (R)-3-hydroxy fatty acyl moieties with varying carbon chain lengths, with average carbon chain length in the range of C8 to C18, C8 to C16, C8 to C14, C8 to C12, C8 to C10, C8 to C12, C10 to C12, C12 to C14, C12 to C18, C14 to C18, or C16 -C18. In varying embodiments, the (R)-3-hydroxy fatty acyl moieties can present varying degrees of unsaturation in the range of 0 to about 6, e.g., in the range of about 2 to about 5. In varying embodiments, the non-esterified hydroxy groups of the sugar alcohol can be esterified to acetyl groups. In various embodiments, the sugar alcohol is fully acetylated, partially acetylated, or non-acetylated.

[0148] In various embodiments, the PEFA containing compositions are selected from a cleanser, a detergent, a surfactant (e.g., for recovery of oil), a wetting agent, an antifoam agent, an emulsifier, an emollient, a dispersant (e.g., for cleanup of oil including spilled petroleum), a humectant, an antibacterial agent, an antiviral agent, an antifungal agent, a spermicide, an insecticide, a lubricant, an adhesive, a crystal modifier, an instantizer, a viscosity modifier, a mixing / blending aid, a release agent, a cream, a foam, a mousse, a lotion, a balm, and an ointment. In some embodiments, the PEFA composition is free or substantially free of any hydrophobic carbon source.

[0149] Derivatives of the PEFA described herein, and produced in the non-naturally occurring microorganisms and methods described herein, are also provided. For example, derivatives may involve modification of either the sugar alcohol (polyol head) or the lipid (fatty acid tail) moiety, or both, including hydroxylated fatty acids, D-mannitol, D-arabitol, and their derivatives. Nonlimiting examples of derivatives are described in PCT Application Nos. PCT / US23 / 67577, PCT / US23 / 67582, and PCT / US23 / 67583, all of which are incorporated by reference herein in their entireties.

[0150] The PEFA compositions find use in numerous applications, including without limitation, household and industrial cleansers and detergents, textiles, agrochemicals such as control of fungal and insect pests, food processing such as cleaning agents for fresh and frozen fruits, vegetables, meats and processed foods, food processing such as rheologic modifiers in food applications including doughs, pastas and emulsions such as mayonnaise, dressings, and syrups, food processing such as antiadherents, photochemicals, petroleum extraction such as release agents for fracking, construction materials such as lubricants and demolding agents for brick, ceramic, cement and concrete, mining such as adjuvants in the coal industry, pulp and paper industry, cosmetics such as creams, foams, mousses, balms, ointments, personal care formulations such as shampoos, body washes, conditioners, soaps, creams, skin treatments, and moisturizing agents, therapeutics such as ointments and creams, spermicides, antibacterial, antiviral and anticancer agents, leather auxiliary agents, fuel oil emulsification for improved atomization, yielding a more complete combustion, bioremediation of contaminated soils, groundwater and surface water, dust suppression in mines and quarries, release agents for asphalt truck beds, facilitation of castable aqueous emulsions for the manufacture of explosives, ingredient in metal working fluids, and oleochemicals such as biodiesel and platform chemicals. Additional uses of the PEFA compositions produced by the present methods are described, e.g., in U.S. Patent Publication Nos. 2012 / 0022241 and 2013 / 0072414, both of which are incorporated by reference herein in their entireties. EXAMPLES

[0151] The following examples are intended to illustrate, but not limit, the invention. Example 1. Cloning plasmid backbones for expression of heterologous genes at the CAR2 locus in Rhodotorula species

[0152] Rhodotorula babjevae 04-877 (“Strain 1”) and Rhodotorula sp. (“Strain 2”) were used for the experimental work described herein. These strains belong to different species in the genus Rhodotorula.

[0153] To construct a DNA construct to integrate constructs expressing heterologous genes for expression, DNA was designed such that the antibiotic resistance cassette pTUB2>nat is flanked by sequences homologous to a region inside or adjacent to the CAR2 locus. (Figs.3A – 3C) Fig.3A shows DNA containing sequences homologous to a genomic region flanking a selectable marker (in this case a nourseothricin resistance cassette pTUB2>nat). Fig.3B shows linear DNA with either half of the pTUB2>nat expression cassette combining by homologous recombination with thegenome, Fig 3B. shows integration of the heterologous DNA and / or removal of the endogenous sequence, accomplished by homologous recombination.

[0154] Two plasmids were prepared for each integration construct, with each plasmid containing one homology arm and ⅔ of the selection marker. Subsequently, PCR or restriction digest was used to create linear DNA from the plasmids suitable for transformation. Homologous recombination between the split region in the selectable marker selects for cells that have also recombined the transformation cassette with the genome. Fig.3C schematically shows genomic sequence replaced by the selection cassette.

[0155] Genome sequencing. For genome sequencing, the strains were struck out on yeast peptone dextrose (YPD) media, then a single colony was inoculated into 5ml of YPD and incubated at 25 °C overnight. The saturated culture was then back-diluted into 100ml of YPD media in a baffled flask and grown until log phase. Log phase cells were washed two times in distilled water (dH20).15 optical density (OD) units of log-phase cells were resuspended in 10ml 1 M sorbitol, 0.1 M sodium citrate, 0.01 M EDTA, and 0.03 M β-mercaptoethanol (SCEM), pH 5.8. Cells were then digested overnight with 100U / ml of lyticase at 30 °C with gentle shaking, then for 8 hours with 200U / ml of Zymolyase for 1 hour at 30 °C. Genomic DNA was then isolated using the MasterPureTMyeast gDNA kit.

[0156] The strains were sequenced using a combination of Nanopore and Illumina reads. The genome was assembled using Flye and Pilon, and gene predictions were done using the FunAnnotate pipeline v1.5.3.

[0157] Cloning backbones for CAR2 integration. Two plasmids were prepared for each gene to be integrated at CAR2, with each plasmid containing one homology arm and ⅔ of the selection marker (Figure 6A). Left plasmid backbones were designed with an SapI golden gate site to enable subsequent cloning of expression constructs (Figure 6A). To clone these plasmids, parts were pre- cloned such that each DNA module was flanked with BsaI sites such that when they are digested with BsaI they yield the DNA part flanked by sticky ends are appropriate to assemble the plasmid. (Figure 6A)

[0158] A kanamycin selectable backbone for Golden Gate assembly was created by splitting the kanamycin resistance gene into two separate plasmids such that one plasmid (Int1B KanR5 / ColE1; PL427; SEQ ID NO: 1) contained the 5’ sequence of the kanamycin resistance gene and a ColE1 origin of replication and the second plasmid (Int1A KanR3; SEQ ID NO: 5) contained the 3’ end of the kanamycin resistance gene. The plasmids included flanking primer binding sites P1 (SEQ ID NO: 35) and P4 (SEQ ID NO: 36) such that all plasmids constructed from these plasmids could be amplified with the same primers and flanked by I-SceI and I-CeuI sites so that large DNA could be released by digestion.

[0159] The plasmid Int1B KanR5 / ColE1 PL427 (SEQ ID NO: 1) was constructed by ordering a gene fragment containing the origin of replication and kanamycin resistance gene fragment flanked by the appropriate BsaI sites from Twist Biosciences (SEQ ID NO: 2). The fragment was ordered with Twist adapters included; these adapters were subsequently used for cloning. The plasmid pGGASelect, a commercially available plasmid from NEB, was amplified using oligonucleotidesOLIGO305 (SEQ ID NO: 3) and OLIGO306 (SEQ ID NO: 4), which introduced the Twist adapters to the linearized Chloramphenicol selectable marker from this backbone. The subsequent amplicons were assembled using a HiFi DNA assembly kit using the manufacturer’s instructions.

[0160] The sequence In1A KanR3 (SEQ ID NO: 5) was ordered from Twist Bioscience cloned into a chloramphenicol selectable backbone.

[0161] In order to improve the rate of homologous recombination, the nourseothricin selection marker was divided into two pieces on separate plasmids. The first fragment (Int9 NatR_L, SEQ ID NO: 6) contains the promoter and the 5’ sequence of the nourseothricin resistance gene and is cloned with the left homology arm for the gene targeted. The second fragment (Int2 NatR_R, SEQ ID NO: 7) contains the 3’ sequence of the resistance gene and the terminator and is cloned with the right homology arm for the gene targeted. The nourseothricin resistance genes had 400 base pairs (bp) of shared sequence, and thus homologous recombination between them yields a functional marker. These DNA sequences were ordered from Twist Biosciences as plasmids cloned into a chloramphenicol selectable backbone.

[0162] The sequence of the CAR2 gene was identified based on the annotations generated above and by TBLASTN using the CAR2 gene from R. toruloides as the query sequence (Uniprot A0A2Z6EYV3). A single high-quality BLAST hit was identified in each strain. Homology arms were designed to replace the full CAR2 coding sequence in Strain 2 and were designed to delete the first three exons of CAR2 in Strain 1. The CAR2 homology sequences were mutated to remove any SapI, BsaI, or BsmBI sites to enable use in the modular cloning system. The sequence of the homology arms for CAR2 disruption in Strain 1 with flanking BsaI sites enabling subsequent assembly are: Int2 Strain 1 CAR2 HOML (SEQ ID NO: 8), and Int9 Strain 1 CAR2 HOMR (SEQ ID NO: 9). For Strain 2, they are: Int2 Strain 2 CAR2 HOML (SEQ ID NO: 10) and Int9 Strain 2 CAR2 HOMR (SEQ ID NO: 11).

[0163] A spacer sequence (Int345678 spacer; SEQ ID NO: 12) was included in the right plasmid used to integrate genes at CAR2. An SapI dropout sequence (Int345678 SapI dropout; SEQ ID NO: 13) was included in the left plasmids. The sequences were ordered as plasmids cloned into a chloramphenicol selectable high copy backbone from Twist Biosciences.

[0164] All plasmids used for assemblies were miniprepped using standard methods and resuspended in water.

[0165] Two Golden Gate assemblies were performed to assemble the plasmids for integration of heterologous genes into Strain 1. The DNA fragments used to assemble the left plasmid (PL434; SEQ ID NO: 14) were: (1) Int1A KanR3 PL359 (SEQ ID NO: 5), (2) Int1B KanR5 / ColE1 PL427 Int2 Strain 1 CAR2 HOML (SEQ ID NO: 8), (4) Int345678 dropout (SEQ ID NO: L (SEQ ID NO: 6). The DNA fragments used to assemble the right plasmid 15) were: (1) Int1A (SEQ ID NO: 5), (2) Int1B (SEQ ID NO: 1), (3) Int2 NatR R Int345678 spacer (SEQ ID NO: 12), and (5) Int9 Strain 1 CAR2 HOMR (SEQ ID

[0166] A left plasmid targeting CAR2 in Strain 1 without an SapI dropout was also cloned. The DNA fragments used to assemble this plasmid (PL440, SEQ ID NO: 16) were 1) Int1A (SEQ ID NO:5), (2) Int1B (SEQ ID NO: 1), (3) Int2 Strain 1 CAR2 HOML (SEQ ID NO: 8), (4) Int345678 spacer (SEQ ID NO: 12), and (5) Int9 NatR L (SEQ ID NO: 6).

[0167] A left backbone containing SapI dropout sites for integration of heterologous genes at the CAR2 locus in Strain 2 was created by standard molecular cloning techniques. The sequence of the left plasmid is SEQ ID PL148 (SEQ ID NO: 17). Similarly, a plasmid with both right and left homology arms and the full nourseothricin-resistance cassette was produced, PL044, which is SEQ ID: 18.

[0168] Plasmid backbones suitable for integrating DNA in Strain 2 can be cloned as follows. The DNA fragments to assemble the left plasmid are (SEQ ID NO: 19): (1) Int1A (SEQ ID NO: 5), (2) Int1B (SEQ ID NO: 1), (3) Int2 Strain 2 CAR2 HOML (SEQ ID NO: 10), (4) Int345678 dropout (SEQ ID NO: 13), and (5) Int9 NatR L (SEQ ID NO: 6). The DNA fragments used to assemble the right plasmid (SEQ ID NO: 20) are: (1) Int1A (SEQ ID NO: 5), (2) Int1B (SEQ ID NO: 1), (3) Int2 NatR R (SEQ ID NO: 7), (4) Int345678 spacer (SEQ ID NO: 12), and (5) Int9 Strain 2 CAR2 HOMR (SEQ ID NO: 11). Example 2. Identification of strong constitutive promoters for gene expression in multiple species of Rhodotorula

[0169] Strong promoters that do not have perfect homology to the host genome are desirable because they minimize undesirable recombination with the host genome. We sought to identify promoters likely to be sufficient to drive expression of heterologous genes in a new host. Moreover, to be compatible with a modular assembly technique, certain mutations (e.g. removal of Type IIS sites) must be made to the promoters that could affect their function. Therefore, we tested a variety of promoters derived from several species of Rhodotorula to identify promoters that supported strong expression of GFP in the strain 1. Below, we report a sequence that supported expression of heterologous genes in both species.

[0170] The RPS3A homolog in Rhodotorula taiwanensis MD1149 was identified using BLAST with the RPS3A protein sequence from Rhodotorula toruloides IFO0880 v4 protein ID 10325 as the query (SEQ ID NO: 21) We extracted 1000bp of sequence upstream of the coding sequence. We randomly mutated each sequence to remove SapI, BsaI and BsmBI restriction sites from the promoter and terminator sequences. We then ordered this sequence synthesized by Twist Biosciences into a chloramphenicol selectable backbone. The sequence were ordered flanked with SapI restriction sites for subsequent assembly. ATG and TAG were used as the overhangs between the promoter / CDS and the CDS / terminator respectively, enabling a seamless assembly (Figure 6B). The sequence of the domesticated R. taiwanensis RPS3A promoter flanked by SapI sites is provided in SEQ ID NO: 22; PL040.

[0171] We reasoned that native promoters could provide even stronger expression in a native host. The TEF1 promoter has been shown to drive strong constitutive gene expression in Rhodotorula toruloides (Nora et al.2019). We identified the TEF1 homolog in Strain 2 using as a query the TEF1 protein sequence from Rhodotorula toruloides (SEQ ID NO: 23). We extracted 1000bp upstream of the translational elongation factor 1 (TEF1) protein from the genome of Strain 2 and mutated SapI,BsaI, and BsmBI sites as described above. The sequence of the domesticated Strain 2 TEF1 promoter flanked by SapI sites is provided in SEQ ID NO: 24; PL082.

[0172] Promoters that are only active during growth in production media can be used to create strains that express enzymes that produce molecules that are toxic to cell growth. In particular, promoters that are only induced in production media and not in media used for building strains or during seed trains are useful because they prevent expression of an enzyme until large scale fermentation, decreasing the metabolic burden on the cell and improving strain stability. We identified a promoter driving an amino acid transporter that was strongly upregulated in nitrogen limited conditions using RNA-seq in Strain 2. Briefly, total RNA was extracted from the cells grown in for 72h in either Media A (Garay et al.2018) or in Yeast Peptone Dextrose in 500ml flasks. Ribosomes were depleted and 250bp RNA-seq reads were generated using the Illumina platform. Reads were then mapped to the genome generated for Strain 2. We counted the reads that mapped to each predicted protein in the genome and identified genes with both a high total expression and a high fold-change between Media A and YPD. One of these promoters drives a predicted amino acid transporter. The sequence of the predicted protein is SEQ ID NO: 25. This gene has 78% identity to the Rhodotorula toruloides IFO0880 V4 protein ID 8962 (SEQ ID: 26), a predicted homolog to S. cerevisiae DIP5. We extracted 1000bp upstream of the predicted start codon of this gene and ordered it synthesized into a chloramphenicol selectable backbone from Twist as described above (PL076; SEQ ID NO: 27). Example 3. Verification of promoter function in Rhodotorula Strain 1 and Strain 2

[0173] To test the function of the promoters we identified, we cloned plasmids into the CAR2 integration backbones in which the promoters drove a 3x-FLAG-GFP coding sequence using an SapI golden-gate protocol. The gene product of the CAR2 locus, Car2p—a bifunctional lycopene cyclase / phytoene synthase—participates in the carotenoid biosynthesis pathway in the Rhodotorula genus. If it is disrupted, cells do not produce the red pigment that is typically seen in Rhodotorula colonies. Therefore, it provides a straightforward read-out for whether transforming DNA integrates at that locus via homologous recombination.

[0174] The GFP coding sequence was optimized in DNA Chisel using a codon usage table determined from the genome sequence of R. toruloides (Table 2), the constraint AvoidRareCodons(0.1,codon_usage_table=TABLE), and providing the argument method="use_best_codon" to the OptimizeCodons objective. In order to facilitate the synthesis of the coding sequences, we specified in DNA Chisel to avoid homopolymeric sequences greater than 6 base pairs (bp), and to remove the recognition sites for restriction enzymes for BsaI, BsmBI, SapI, and BbsI to facilitate modular assembly techniques.

[0175] A 3x-FLAG-tagged GFP coding sequence was ordered cloned into a chloramphenicol backbone flanked by SapI sites from Twist Bioscience (PL091; SEQ ID NO: 28). As a terminator, we used the RPS3A terminator from Rhodotorula toruloides IFO0880 V4 (PL024; SEQ ID NO: 29).

[0176] SapI reaction conditions. Cloning to assemble expression constructs (promoter, CDS, terminator) was performed using SapI by assembling a reaction containing 2µL of 10X ligase buffer,1.5µL of SapI enzyme (10U / µL), and 1.25µL of T4 ligase (400U / µL), along with 3nM of each DNA fragment or plasmid for a final volume of 20µL. The reaction was then cycled between 5 minutes at 37 °C and 5 minutes at 16 °C for 30 cycles. After this cycling, an additional 1.5µL of SapI was added to each reaction and incubated for 1h at 37C then heat inactivated at 60C. The resulting assembly was transformed into NEB 10-beta and sequence verified as above.

[0177] Cloning for Rhodotorula Strain 1 integration GFP sequences were cloned into the PL434 (SEQ ID NO: 14) left plasmid backbone. The SapI assembly contained the plasmids PL434, the RPS3A terminator (PL024; SEQ ID NO: 29), the GFP coding sequence (PL091; SEQ ID NO:28) and either the Rhodotorula taiwanensis RPS3A promoter (PL040; SEQ ID NO: 22) or the Rhodotorula Strain 2 TEF1 promoter (PL082; SEQ ID NO: 24). This generated the plasmids PL451 (SEQ ID NO: 30) and PL453 (SEQ ID NO: 31), respectively.

[0178] Cloning for Rhodotorula Strain 2 integration The actual plasmids expressing GFP used in this section were assembled into the PL044 backbone using standard techniques. In the following paragraph, we describe how to clone these plasmids using the modular assembly system.

[0179] Assembly uses the Strain 2 CAR2 SapI left backbone (SEQ ID NO: 19); the shared GFP coding sequence (PL091; SEQ ID NO: 28); the terminator sequence (PL024; SEQ ID NO: 29); and the promoter sequences for the Rhodotorula taiwanensis RPS3A promoter (PL040; SEQ ID NO 22), Rhodotorula Strain 2 TEF1 promoter (PL082; SEQ ID NO: 24), and the Rhodotorula Strain 2 IndProm1 PL076 (SEQ ID NO: 27) to produce plasmids, (SEQ ID NO: 32), (SEQ ID NO: 33), and (SEQ ID NO: 34), respectively.

[0180] Preparation of linear DNA for Strain 1. We produced linear DNA using primers P1 GGACCATCTGAATCATGCGC (SEQ ID NO: 35) and P4 GTCGTGAGTTCGTGTCATCC (SEQ ID NO: 36) using the SuperFi HotStart II polymerase from plasmid (SEQ ID NO: 30), (SEQ ID NO: 31). PCR Reactions were performed at 25µL reaction volume with 5ng of template, 0.5µM of primers, a 15 sec annealing time, a 60 °C annealing temperature, and 60s / kb of extension time. Approximately 400µL of polymerase chain reaction (PCR) was prepared per reaction. The PCR was checked for purity on a gel, then purified using a Zymo Clean and Concentrator 25 column and resuspended in water. DNA concentration was measured using spectrophotometry using a NanoDrop.

[0181] Preparation of linear DNA for Strain 2. We produced left linear DNA using primers P1 (SEQ ID NO: 35) and P4 (SEQ ID NO: 36) from plasmids (SEQ ID NO: 32), (SEQ ID NO: 33), (SEQ ID NO: 34) as above. We produced right linear DNA from PL044 as above except the primers used were TGACCAAGGTGTTCCCCGACGA (N3; SEQ ID NO: 37) / P4 (SEQ ID NO: 36).

[0182] Transformation of Strain 1. To transform cells using electroporation, a similar method was used as described in Coradetti et al., 2018.10ml of log-phase cells at OD 2 prepared as above were pelleted at 3500xg for 5 minutes at 4 °C. Cells were kept ice cold from this point on. Cells were resuspended in 1ml of 0.75M ice cold D-sorbitol per 10ml of original volume and transferred to microfuge tubes. Cells were washed 4x with 0.75M sorbitol by spinning down at 8000xg for 30 sec. After the final wash, 25µL of 0.75M sorbitol was added to resuspend the pellet. The pellet was then mixed with 5µL of water containing approximately 3µg of each fragment of transforming DNA and transferred to a 0.1cm ice cold cuvette. The cells were electroporated with a Bio Rad Gene Pulser IIat 1.5 kV, 200 ohms and 25 µF. Immediately after electroporation, cells were resuspended in 1ml of an ice cold mixture of 1:10.75M sorbitol / YPD. This mixture was transferred to 14ml cell culture tubes, placed in an incubator at 25 °C, 250rpm, and allowed to recover for 4h. Cells were pelleted, resuspended in 200µL of yeast extract-peptone-dextrose (YPD), and then plated on YPD agar with 100µg / ml nourseothricin. Plates were incubated for 3 days at 25 °C.

[0183] Transformation of Strain 2. The same protocol was used, except that cells were grown at 27 ˚C and selection was done on YPD with 50µg / ml nourseothricin.

[0184] Isolation of edited strains. The vectors used for expression of heterologous genes target integration into the CAR2 locus. Therefore, after transformation, white colonies were chosen to isolate strains that were produced by single integration events at the CAR2 locus. White colonies were immediately visible on plates in Strain 1, whereas carotenoid development in Strain 2 was triggered by incubation of transformation plates for 5 days at 4C. In each case, white colonies were restruck on YPD plates with the appropriate antibiotic concentration (100µg / ml or 50µg / ml nourseothricin) and grown for three days to isolate single colonies. These colonies were then grown for 24-48h at the appropriate growth temperature (25C or 27C) in YPD and frozen in 25% glycerol at -80C.

[0185] Verification of promoter for Strain 1. Seed cultures for three biological replicates of each strain transformed with the GFP expression constructs were prepared by inoculating 50µL of glycerol stocks into 3ml of YPD in 14ml culture tubes and grown for 48h at 25C. Ten microliters of this culture was then back-diluted into either YPD or Media A containing 50g / L glucose. The cultures were grown for 48h at 25C. Cells were then imaged under a 40X objective with both transmitted light and a FITC filter set. Fluorescence was visible for both promoters, with greater fluorescence for the R. taiwanensis RPS3A promoter and greater fluorescence in YPD than Media A. (Figure 7)

[0186] Verification of promoter activity for Strain 2. Strains were tested for their ability to produce GFP in 24W Deutz plates (Catalog number CR1424a) sealed with sandwich covers (Catalog number CR1224). Seed cultures were started by inoculating 50µL of glycerol stocks into 2.5ml of YPD and grown for 24h at 27C at 345rp. Main cultures were started by inoculating 50µL of seed culture into 2.45ml of YPD or a nitrogen limited media based on Media A from Garay et al., 2017. The media (Media A1) was modified to include 2.5g / L yeast extract, 0.5g / L of ammonium chloride, and 100g / L of glucose. GFP and optical density of the cultures was measured using a fluorescent plate reader at 24h and 72h.

[0187] We confirmed that the promoter identified as inducible in Strain 2 had low GFP expression, especially during early growth in YPD and that it was strongly upregulated in nitrogen limited media after 3 days of growth. The R. taiwanensis RPS3A promoter and the Rhodotorula Strain 1 TEF1 promoter showed strong fluorescence in both media types and at both time points.

[0188] Taken together, these data indicate that both the Rhodotorula Strain 2 TEF1 promoter and the R, taiwanensis RPS3A promoter are suitable for heterologous gene expression in multiple species of Rhodotorula. Furthermore, they demonstrate an inducible promoter suitable for use in Rhodotorula Strain 2. (Figure 8)Example 4. Decreasing average PEFA chain length using thioesterases in Strain 2

[0189] Thioesterases can be expressed in fungi to produce medium chain fatty acids which can then become activated to acyl-CoA molecules and incorporated into molecules such as tri-acyl glycerides (Rutter et al 2015; Xu et al.2016). To accomplish this, a thioesterase enzyme that catalyzes the production of a free fatty acid from an acyl carrier protein (ACP)-acyl thioester of the desired length can be overexpressed in the yeast, thus terminating the elongation of the fatty acid and producing shortened fatty acids.

[0190] Voelker et al. reported a thioesterase from Umbellularia californica (UcFatB2, Genbank: U17097) that produces mostly C12 fatty acids. In plants, this enzyme is targeted to the plastid. Removal of the peptide leader sequence targeting the enzyme to the plastid can reduce aggregation and improve expression of similar enzymes (Hernández, L., 2018). The UcFatB2 enzyme sequence from U. californica was downloaded from Genbank (SEQ ID NO: 38), and the plastid targeting sequence was identified and truncated (SEQ ID NO: 39).

[0191] Other plant thioesterases have also been identified. The protein sequence of a predicted dodecanoyl ACP-thioesterase from Cinnamomum camphora (CcFatB2b) (WO 2014 / 151904 A1) is (SEQ ID NO: 40) and the sequence with a truncated plastid targeting sequence is (SEQ ID NO: 41).

[0192] We designed DNA to express codon-optimized versions of the thioesterase proteins and as described above and ordered them synthesized from Twist in Chloramphenicol selectable plasmids flanked by SapI sites. The sequence of the insert of these plasmids are provided in (UcFatB2 SEQ ID NO: 42), (UcFatB2_trunc SEQ ID NO: 43), (CcFatB2b SEQ ID NO: 44), (CcFatB2b_trunc SEQ ID NO: 45.

[0193] Plasmids expressing the thioesterases were assembled with the PL148 backbone (SEQ ID NO: 17); the RPS3A terminator sequence (SEQ ID NO: 29); the Rhodotorula Strain 2 TEF1 promoter sequence (SEQ ID NO: 24) and the thioesterase coding sequence containing plasmids (SEQ ID NO: 42), (SEQ ID NO: 43), (SEQ ID NO: 44), (SEQ ID NO: 45) as described above. This produced the plasmids PL244 (SEQ ID NO: 46), PL248 (SEQ ID NO: 47), PL245 (SEQ ID NO: 48), PL247 (SEQ ID NO: 49), respectively.

[0194] Linear DNA was produced from the left plasmids using PCR with primers P1 and P4 and from PL040 using primers N2 and P4 as described above and was transformed into Strain 2 as described above. Six white colonies (referred to hereafter as “biological replicates”) were restruck on YPD+Nat50, then banked in glycerol stocks in 96W plates.

[0195] Screening thioesterase expressing strains in 24W plates. Six biological replicates were screened for their ability to alter the chain length of PEFA produced by Strain 2 in 24W Deutz plates (Enzyscreen, catalog number CR1424a) sealed with sandwich covers (Enzyscreen, catalog number CR1224). Seed cultures were started by inoculating 50µL of glycerol stocks into 2.5ml of YPD and grown for 24h at 27C at 345rpm. Main cultures were started by inoculating 125µL of seed culture into 2.35ml of Media A1 in the same plates. Strains were grown for 5 days.

[0196] PEFA was extracted from each well using a protocol adapted from Garay et al., 2017. Briefly, 2ml of well-mixed culture was transferred to a glass tube. Six ml of ethyl acetate were addedto the culture, vortexed for 30s, then allowed to settle for 30 minutes. The ethyl acetate supernatant was evaporated in pre-weighed borosilicate tubes to recover the glycolipids.

[0197] The average titer of PEFA extracted from the cultures was approximately 5g / L. PEFA composition was measured using LCMS as described in Cajka et al.2016. The total fraction of each species of PEFA was determined, and the total fraction of PEFA of a particular fatty acid chain length (across all acetylation levels and head groups) was determined for each sample and then normalized to the average fraction of that chain length in wild-type Strain 2.

[0198] Expression of thioesterases decreased the fraction of C18-PEFA relative to wild-type and increased the amount of C8- and C14-PEFA. Each of the strains expressing one of the four thioesterases contained 90% of the amount of C18-PEFA than wild-type. (Figure 9) These differences in C18-PEFA content were statistically significant (Wilcox t-test, p<0.01). Conversely, each of the four strains contained 6-11% higher levels of C14-PEFA. These differences were statistically significant (Wilcox t-test, p<0.01). Three of the four strains also produced more than double the amount of C8-PEFA than wild-type, with only the truncated version of UcaFatB2 not showing an increase (Wilcox t-test, p<0.01). The average PEFA chain length decreased by 0.18% for both strains relative to wild-type, and in each case this difference was statistically significant (Wilcox t-test, p<0.01). Thus, the total amount of shorter chain PEFA produced was approximately 0.09g / L. Taken together these data demonstrate a decrease in the average chain length of PEFA and an increase in the levels of specific short chain PEFA-species.

[0199] Surprisingly, the shortening of the average PEFA chain length was also accompanied by the production of greater PEFA with a mannitol head group (MAN-PEFA). (Figure 10) The strains expressing the thioesterases produce 10-75% more MAN-PEFA than wild-type.

[0200] Screening thioesterase expressing strains in flasks. The strains expressing the UcaFatB2 and CcFatB2 truncated thioesterases were further tested in flasks to determine if the change in PEFA chain lengths observed were sensitive to growth conditions. As a control to test if changes in PEFA chain length were due to either heterologous gene expression per se or integration at the CAR2 locus, we constructed a strain with a predicted D-arabitol 4-dehydrogenase (DA4DH)) from Rhodotorula toruloides driven by the same promoter in the same backbone as above. The protein sequence and the sequence of the codon optimized DA4DH gene sequence are SEQ ID NO: 50 and SEQ ID NO: 51, respectively. The DA4DH enzymatic activity is not expected to affect the chain length of PEFA. The strain containing expression of the D4ADH gene was constructed as described above.

[0201] Fifty microliters of glycerol stocks of four biological replicates of each thioesterase- expressing strain along with four replicates of the DA4DH expression strain and of Strain 2 were inoculated into 5ml of YPD in 50ml bioreactor tubes and grown for 16-24h at 27C. The seed cultures were then used to inoculate 250ml flasks containing 15ml of Media A1100 grams per liter of glucose at a 5% inoculum volume. Flasks were grown at 27°C and 250rpm of agitation. Consumption of glucose was monitored using a GlucCell device. After 70h of growth, glucose was exhausted in all shake flasks.

[0202] PEFA titer was measured using a protocol adapted from Garay et al., 2017. Briefly, three volumes of ethyl acetate were added to the flasks, stirred at 550rpm using a magnetic stir bar for 1 hour, allowed to settle for 30 minutes, and then 10ml of the ethyl acetate supernatant was removed into a pre-weighed borosilicate tube. The ethyl acetate was evaporated to measure the total titer of PEFA produced. Strains expressing the modified thioesterases produced approximately 10% lower titer of PEFA than the wild-type or DA4DH expressing strains.

[0203] The samples were analyzed using LCMS as above. Consistent with the results in plates, the amount of C18-PEFA decreased in both thioesterase expressing strains, whereas there was no significant difference in chain length for the DA4DH-expressing strains. (Figure 11) No significant increase in C14-PEFA was observed, but as in the plates, the strains produced more C8-PEFA than wild-type. Approximately 20% more MAN-PEFA was observed in strains expressing thioesterases than was observed in the wild-type strain or the strain expressing the DA4DH enzyme.

[0204] Expression of thioesterases in fungi can produce medium chain fatty acids, but the behavior of these enzymes in fungal systems is often different than when they are expressed in plants or bacteria, with less specificity in the chain length of fatty acids produced. Although in plants these thioesterases primarily release C12 fatty acids, expression in Rhodotorula Strain 2 increases C8- PEFA levels and decreases C18-PEFA levels relative to wild-type. Example 5. Decreasing average PEFA chain length using thioesterases in Strain 1

[0205] Given that thioesterase expression in Rhodotorula Strain 2 increases the production of shorter chain PEFA, we tested whether this strategy was applicable to other species of Rhodotorula.

[0206] Plasmids expressing the thioesterases were assembled with the PL434 backbone (SEQ ID NO: 14); the RPS3A terminator sequence (SEQ ID NO: 29); the R. taiwanensis RPS3A promoter sequence (SEQ ID NO: 22) and the thioesterase coding sequence containing plasmids (SEQ ID NO: 42), and (SEQ ID NO: 45) as described above. This produced the plasmids (PL465; SEQ ID NO: 52), (PL464; SEQ ID NO: 53) respectively. As a control to test if disruption of the CAR2 locus affected PEFA chain length, empty left plasmid PL440 (SEQ ID NO: 15) was used.

[0207] Linear DNA was produced from each plasmid using PCR with primers P1 and P4 and transformed into Strain 1 as described above. Six white colonies were restruck on YPD+Nat100 and grown for three days to isolate single colonies. These colonies were then grown for 24-48h at 25C in YPD and frozen in 25% glycerol at -80C.

[0208] Seed cultures were started for shake flask fermentation by inoculating 50µL of glycerol stocks for both with wild-type Rhodotorula Strain 1 and strains expressing the thioesterases into 5ml of YPD media and incubating for 16-24 hours at 25°C in bioreactor tubes.

[0209] An initial screen (using the same protocol as described below) identified two biological replicates from each transformation with a reduced amount of C16-PEFA relative to wild-type. To confirm these data, we chose one biological replicate from each transformation with reduced C16- PEFA and rescreened them with six technical replicates for each strain.

[0210] Six replicates of each thioesterase-expressing strain along with six replicates of the knockout strain and six replicates of wild-type Strain 1 in Media A as described in Garay et al., 2017 using 15ml of media with 50 grams per liter of glucose as a carbon source in 250ml glass shake flasks at 25°C and 250rpm of agitation. A 5% inoculum of the seed culture was used. Glucose consumption was monitored using a GlucCell device. After 90h of growth, glucose was exhausted in all shake flasks containing the wild-type or CAR2 knockout strains, and approximately 1.5g / L of glucose remained in the strains containing the thioesterases.

[0211] PEFA titer was measured as above. The average titer was 3.5 g / L. No significant difference in PEFA titer was observed between the strains tested.

[0212] PEFA composition was measured using LCMS as described above. Thioesterase expressing strains produced 8-10% less C18-PEFA than wild-type and approximately 5% more C16-PEFA than wild-type. Interestingly, these strains also produced less C10-PEFA and C8-PEFA than wild-type or the CAR2 knockout strain. (Figure 12) The average chain length decreased by 0.03% for both strains relative tow wild-type, and in each case this difference was statistically significant (Wilcox t-test, p<0.01). Thus, the total amount of shorter chain PEFA produced was approximately 0.010 g / L.

[0213] As in Strain 1, the shortening of the average PEFA chain length was also accompanied by the production of greater PEFA with a mannitol head group (MAN-PEFA). The thioesterase- expressing strains produced 15-20% more MAN-PEFA and the CAR2 knockout strain produced approximately 20% less MAN-PEFA. (Figure 13)

[0214] These data confirm that thioesterase expression can alter PEFA chain length in multiple species of Rhodotorula. Example 6. C8-PEFA production using heterologously expressed fatty acid synthase genes

[0215] Rhodotorula strain 2 was used for the work described herein.

[0216] The Saccharomyces cerevisiae fatty acid synthase can be functionally expressed as a single polypeptide— a fusion of the alpha and beta subunits, facilitating heterologous expression in a new host since the stoichiometry between the subunits is guaranteed (Wernig, 2020). Previous studies have identified mutations that lead to increased production of medium chain acyl-CoA esters. In particular, the mutation R1834K to FAS1 malonyl / palmitoyl transferase domain produces a larger proportion of C8 CoA esters (Gajewski et al.2017). The fusion protein containing the R1834K mutation (SEQ ID NO: 54) was codon optimized as above (SEQ ID NO:55). The codon optimized sequence of the Scer fusFAS R1834K protein was split into ten fragments using the NEB SplitSet tool and cloned into the pGGASelect backbone using the BsaI Master Mix kit from NEB using the manufacturers recommendations. The R1834K mutation (codon AAG) was reverted to the wildtype R (CGC) codon by designing primers using NEBaseChanger and the Q5 Site directed mutagenesis kit from NEB using the manufacturers recommendations. The protein sequence of the fused proteins without the R1834K mutation is SEQ ID NO: 56 and the codon optimized sequence is SEQ ID NO: 57. It was necessary to grow the E. coli containing these plasmids at 25 ˚C to isolatethe correct plasmid presumably due to either the size of the plasmid or the metabolic load of propagating large DNA on a high-copy backbone.

[0217] These coding sequences were cloned into the PL148 (SEQ ID NO 17) backbone with the Strain 2 IndProm1 (SEQ ID: 27) and the RPS3A terminator (SEQ ID: 29) and the Scer fusFAS R1834K (SEQ ID NO: 55) and Scer_fusFAS (SEQ ID NO: 57) as described above to produce plasmids PL214 (SEQ ID NO: 58) and PL281 (SEQ ID NO: 59) for the Scer FusFAS R1834K and Scer FusFAS coding sequences, respectively. Fifty milliliter cultures of E. coli containing these plasmids were midiprepped using a ZymoPure midiprep kit following the manufacturer’s instructions. The 25µg of plasmid was then double-digested with I-SceI and I-CeuI and purified with a Zymo Clean and Concentrator 25 column and eluted in water. These plasmids were transformed into Strain 2 along with right linear DNA prepared from PL044 as above.

[0218] The transformation efficiency was lower for these transformations, and very few white colonies were obtained. Therefore, we chose to screen colonies where the plasmids had integrated randomly into the genome (red colonies). Four biological replicates each strain were banked and tested in 250ml flasks using Media A1 as described in Example 4. PEFA was extracted using ethyl acetate, and the titer and profile was measured as above. Note that this experiment was performed at the same time as the one described above testing the thioesterases in Strain 2 in Example 4 and therefore contains the same data for the wild-type strain. The data for DA4DH strain is not analyzed in this section since the DA4DH edit is integrated at the CAR2 locus.

[0219] The titer of the PEFA produced by the strains expressing the Scer fusFAS R1834K coding sequence was approximately 10% lower than wild-type, whereas there was no significant difference in titer from the strain expressing the Scer fusFAS sequence. (Figure 14) The PEFA profile for each of the biological replicates expressing the Scer fusFAS R1834K gene contained approximately 50% more C8-PEFA than wild-type, whereas the strains containing the Scer fusFAS gene contained only 60% of the C8-PEFA as wild-type, with an overall enrichment in C18-PEFA. (Figure 15) These data demonstrate the C8-CoA produced by the Scer fusFAS R1834K gene can be incorporated into PEFA. The decreased titer for these strains suggest that the shorter chain acyl-CoA molecules produced are not as efficiently incorporated into PEFA as the acyl-CoA molecules produced by the Scer fusFAS gene, which presumably has a profile more similar to the native FAS system. The decrease in C8- C10- and C12- PEFA in strains containing the Scer fusFAS gene could reflect greater C18-CoA produced by this system than the native Strain 2 FAS system. Example 7. Replacement of native FAS genes with FAS sequences producing shorter chain acyl-CoA

[0220] The native FAS genes in Rhodotorula species are capable of supporting high flux to acyl- CoA; the FAS genes of PEFA producing Rhodotorula species may have evolved to coordinate with the PEFA pathway. Therefore, it can be desirable to express mutated forms of the native FAS synthase genes. Furthermore, in other species of oleaginous yeast, high titer of shorter chain fatty acids requires complete replacement of the wild-type FAS system with a system modified to produce shorter chain acyl-CoA (Rigouin et al.2018).

[0221] In our previous examples, we demonstrated that expressing a heterologous FAS at the CAR2 locus increases the production of C8-PEFA. The production of shorter chain PEFA was contingent on the R1834K mutation in the malonyl / palmitoyl transferase (MPT) domain, In Rhodotorula species, the MPT domain is located on the alpha subunit of the FAS synthase, the FAS2 gene (Zhu et al.2012). Therefore, replacing the wild-type FAS2 gene with one carrying mutations designed to increase the production of shorter chain acyl-CoA is expected to improve the overall titer of shorter chain PEFA. In the following example, we describe the design of DNA to replace the native FAS2 gene with modified FAS2 genes in the Strain 1.

[0222] We identified the Strain 1 FAS genes using BLAST using the Rhodotorula toruloides FAS protein sequences as queries (Uniprot M7XM89, alpha subunit, FAS2 and Uniprot M7WSW5, beta subunit FAS1). A single high-quality hit was identified for each subunit: (SEQ ID NO: 60) and (SEQ ID NO: 61) for FAS1 and FAS2, respectively. In S. cerevisiae, the MPT domain is located on the FAS1 gene. We aligned the Strain 1 FAS2 gene to the Saccharomyces cerevisiae FAS1 gene to identify the homologous residue to R1834 in Scer_FAS1 (Figure 16). The gene Strain 1 FAS2 with R665 mutated to K (SEQ ID NO:62) was codon optimized as above to generate (SEQ ID NO:63). The fragment was split into 10 fragments using the NEB Split Set tool and cloned into the pGGAselect backbone using BsaI with flanking SapI sites that reveal the overhangs ATG and TAG flanking the codon optimized coding sequence.

[0223] We designed constructs to replace the native FAS2 coding sequence with the coding sequence of a codon optimized Strain1 FAS2_R665K coding sequence. Due to the presence of an SapI restriction site in the 1000bp upstream of the FAS2 gene, we used a modified assembly protocol. The left plasmid was constructed using HiFi assembly. We designed primers to replace the left CAR2 homology arm with the left FAS2 homology arm. The primers ACACCACACACCTTAATTAAAAGTGAATAAAGCTCCACACA (SEQ ID NO: 64) and GCCGCTCTCGGCGTCGAGCGTGTGGCGCATGATTCAGATG (SEQ ID NO: 65) were used to amplify the 1000bp FAS2 homology arm from Strain 1 genomic DNA. The primers CATCTGAATCATGCGCCACACGCTCGACGCCGAGAGCGGC (SEQ ID NO: 66) and CTTTATTCACTTTTAATTAAGGTGTGTGGTGTACTCGGGGGTG (SEQ ID NO: 67) were used to linearize the PL440 backbone and introduce the appropriate overhangs. The backbone was digested with DpnI, then both PCR products were purified using a spin column. HiFi assembly was performed according to the manufacturer’s recommendations, and the plasmids were verified using Nanopore sequencing. The plasmid was constructed with an XbaI restriction site between the left homology arm and the beginning of the left half of the NatR marker to allow linearization of the backbone using restriction digest. This produced plasmid PL485 (SEQ ID NO: 68).

[0224] A right plasmid was constructed using BsaI assembly. The right FAS2 homology arm was amplified from the 04-877 genome using PCR with primers TGACGAGGTCTCCGCTAAGACGCAGCTCGTAGAGG (SEQ ID NO: 69) and TGACGAGGTCTCCGCGGAACTTGAGAAGGTCGACC (SEQ ID NO: 70), then cleaned up with a spin column. These primers add appropriate BsaI sites for modular assembly. The right plasmid was assembled using the plasmids PL359 Int1A (SEQ ID NO: 5), PL427 Int1B (SEQ ID NO: 1),PL374 Int2 NatR R (SEQ ID NO: 7), PL362 Spacer 345678 (SEQ ID NO: 12), and the PCR fragment generated for the right homology arm. This produced plasmid PL491, SEQ ID NO: 71.

[0225] The Rhobab1_FAS2_R665K gene was assembled along with the R. toruloides RPS3A (SEQ ID NO: 29) terminator into a backbone with an SapI dropout site flanked by 25bp sequences suitable for assembly into the PL485 backbone using HiFi assembly. These sequences in turn were flanked by XbaI sites such that the Strain 1 FAS2_R665K / terminator sequence could be cloned into the PL485 backbone using HiFi assembly after digestion of the plasmid with XbaI. Note that the final construct uses the native Strain 1 FAS2 promoter for expression. The SapI dropout plasmid was ordered synthesized from Twist in an ampicillin selectable high copy backbone (PL490; SEQ ID NO: 72). An SapI assembly reaction was performed with Strain 1 FAS2_R665K (SEQ ID NO: 62), the RPS3A terminator (SEQ ID NO: 29) and the SapI backbone (SEQ ID NO: 72). The sequence of this plasmid is provided in SEQ ID NO: 73.

[0226] Plasmids provided in (SEQ ID NO: 73) and (SEQ ID NO: 68) were midiprepped and digested with XbaI and gel purified using standard techniques. The fragments were assembled using the NEB HiFi kit according to the manufacturer’s instructions. The sequence of the assembled plasmid is provided in SEQ ID NO: 74.

[0227] For transformation, the right plasmid (SEQ ID NO: 71) was amplified using oligos P1 and P4 as described above. The left plasmid (SEQ ID NO: 74) was midiprepped and digested with I- CeuI / I-SceI to release a linear fragment. The digested plasmids were cleaned up and transformed into Strain 1 as described above.

[0228] To screen for colonies where the codon optimized Strain 1 FAS2_R665K gene replaced the native FAS2 gene, we designed primers to amplify ~1200bp amplicons across the left homology arm (GGTGGGACTACAAGACGGGCCT; SEQ ID NO: 75 / ACGGGGTCGAGGGGTTGTACTG; SEQ ID NO: 76) and the right homology arm (CGCACGTTCAGACCGTCTGGAC SEQ ID NO: 77 / GCTCACGCAGGAGGAGGAGTGA SEQ ID NO: 78), these primers will yield amplicons only if the Strain1 FAS2_R665K is present. We also designed primers to amplify a 500bp amplicon within the native Strain1 FAS2 gene (CAACTCGGACACGGGCAAGACC SEQ ID NO: 79 / ACGTAGTTGGACTCGACGCGGA SEQ ID NO: 80), these primers will yield amplicons only if the native gene is absent. Colonies from the selection plate were first restruck on YPD+Nat100, then grown 24-48h in YPD. Genomic DNA was extracted using the Zymo Quick DNA Fungal miniprep kit and used for screening.

[0229] Colonies with the native FAS2 gene replaced by the Strain 1 FAS2_R665K sequence were screened for increased production of shorter chain PEFA as described above. Example 8. Expression of heterologous fatty acid synthase containing with thioesterase domains

[0230] Thioesterase enzymes can be incorporated into type I fatty acid synthases adjacent to the acyl-carrier domain. The genes encoding Rhodotorula sp. and Aplanochytrium kerguelense FAS synthases have duplicated acyl-carrier domains (Zhu et al., 2017), and replacing one of these domains with a thioesterase can bring the thioesterase into contact more readily with the extendingacyl chain than expression of a thioesterase as a standalone enzyme. We retrieved the sequences (1) AKFAS – ACPII2TE (SEQ ID NO:81) and (2) RtFAS ACPII2TE AA (SEQ ID NO:82) (U.S. Patent No.10,648,043). These sequences encode the fatty acid synthase from Aplanochytrium kerguelense and the FAS2 gene from Rhodotorula toruloides, respectively, in which one of the acyl carrier protein domains was replaced by a truncated thioesterase from Acinetobacter baylyi (SEQ ID NO:83). The proteins were codon optimized as described above and expression cassettes with the R. taiwanensis RPS3A promoter and the R. toruloides RPS3A terminator were cloned into the Strain 1 CAR2 PL434 backbone as described in Example 5. The codon optimized sequences for these FASs are depicted SEQ ID Nos: 84, 85 for AKFAS, and RtFAS respectively. These genes were integrated at the CAR2 locus of Strain 1 as described above in Example 5. White colonies were picked from transformation plates and screened for the production of short chain PEFA. Example 9. Expression of a heterologous PhaG enzyme

[0231] In Pseudomonas, the enzyme PhaG catalyzes the transfer of three hydroxy acyl chains with primarily 6 to 12 carbons from acyl carrier proteins to CoA molecules to form 3-(R)-OH-CoA molecules. These medium chain 3-OH-Acyl-CoA molecules can be incorporated into PEFA. Several homologs were tested by Yan, 2022, including a PhaG homolog from Pseudomonas koreensis (PkPhaG) (SEQ ID NO:86), which was found to be the most active at producing medium chain oleochemicals in E. coli. The sequence was codon optimized as described above (SEQ ID NO: 87). An expression construct was cloned for integration into the Strain 1 CAR2 backbone PL434 with the R. taiwanensis RPS3A promoter and the R. toruloides RPS3A terminator as described in Example 5 (SEQ ID NO: 88). DNA was prepared for transformation using PCR, and colonies were screened for increases in C10-C12 PEFA. Example 10. Improving short chain acyl-CoA production by lowering beta oxidation and improving fatty acid activation

[0232] Functional genomics in Rhodotorula toruloides has shown that both mitochondrial and peroxisomal β-oxidation are important for growth of Rhodotorula sp. on hydrophobic substrates (Coradetti et al.2018). We identified genes involved in β-oxidation in Strain 1 by using BLAST to query predicted proteins in Strain 1 with the following proteins referenced using their JGI protein IDs: (1) ECHS1 enoyl-CoA hydratase (RTO4_14805), (2) FOX2 multifunctional beta-oxidation protein (RTO4_11362) In Strain 1, these genes are encoded by the proteins (1) SEQ ID NO:89, (2) SEQ ID NO:90.

[0233] Free fatty acids are activated to acyl-CoA by acyl-CoA synthases. In Saccharomyces, the FAA2 (Uniprot P39518) gene is targeted to peroxisomes, where it facilitates beta-oxidation of medium chain fatty acids. FAA2 can activate fatty acids of varying chain lengths, but it has the highest activity on medium chain fatty acids. The FAA2 protein sequence was retrieved from Uniprot, and the EKL peroxisomal targeting signal was removed from the C terminal of the protein sequence (SEQ ID NO: 91). The Rhodotorula codon-optimized sequence was ordered cloned from Twist Biosciences in a chloramphenicol-selectable plasmid (SEQ ID NO: 92).

[0234] Here we describe how to simultaneously disrupt either mitochondrial or peroxisomal β- oxidation while simultaneously expressing a thioesterase and a FAA enzyme to improve production of short chain PEFA. We designed homology arms for these loci that are compatible with the modular assembly system and constructed backbones targeting these loci in the same way as the backbone targeting expression constructs to the CAR2 locus. The left and right homology arms for ECHS1 are SEQ IDs NO: 93 and 94. The left and right homology arms for MFE1 are SEQ IDs NO: 95 and 96. The sequences was ordered cloned in Twist Biosciences in a chloramphenicol plasmids.

[0235] Right and left plasmid backbones containing SapI dropout sites for cloning expression constructs were constructed using BsaI as described above. Then, SapI was used to assemble the UcFatB2 thioesterase (SEQ ID NO: 42) into the left plasmid with the R. taiwanensis RPS3A promoter (SEQ ID NO: 22) and the R. toruloides RPS3A terminator (SEQ ID NO: 29) and the FAA2 sequence (SEQ ID NO: 92) into the right plasmid with the Strain2 TEF1 promoter (SEQ ID NO: 24) and R. toruloides RPS3A terminator (SEQ ID NO: 29), respectively.

[0236] DNA was produced from the left and right plasmids for each locus. Transformation was performed as described in Example 5. Colonies were screened using PCR spanning the junction of the edit with the genome as described in Example 5.

[0237] Although the foregoing invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced without departing from the spirit and scope of the invention, which is delineated in the appended claims. Therefore, the description should not be construed as limiting the scope of the invention.

[0238] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes and to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference. Nucleotide and Amino Acid Sequences SEQ ID NO:1 PL427; In1B KanR5 / ColE1 TGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCCAATCCGCCCTCACTACAACCGGGTCTCAATTCTTCTA ATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGC ATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGC ATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGT TGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTT ATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGC TGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT CTTCTTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGC TACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTC AAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA CCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTC GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCG ATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTG CTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGCCAC ATGAGACCCTACTCTGGCGTCGATGAGGGAGTAGACTTCTTAATTAAGACGTCAGAATTCTCGAGGCGGCCGCATGTGAGTCTCCCTATAGT GAGTCGTATTAATTTCGCGGGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGTAGCACCAGGCGTT TAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATG GAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGG GGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACAAAAAACATATTCTCAATA AACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCA TTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTC TTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCA TTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGC CCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAAAGTCTCATTTTCGCCAAAAGTTGTCATGACCAAAA TCCCTTAACG SEQ ID NO:2 Int1B KanR5 / ColE1 gene fragment flanked with BsaI sites GGTCTCAATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTT GATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCA GAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATAT AAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTAT GTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAA TAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTA GAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGT TTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTA GTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGT CGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACC TCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCC TTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCAT CTGAATCATGCGCCACATGAGACC SEQ ID NO:3 OLIGO305 ChlorR Twist3 FWD, synthetic primer CTACTCTGGCGTCGATGAGGGAGTAGACTTCTTAATTAAGACGTCA SEQ ID NO:4 OLIGO306 ChlorR Twist5 REV, synthetic primer CGGTTGTAGTGAGGGCGGATTGTTCGAATTCGGATCCCTCGAGC SEQ ID NO:5 Int1A KanR3 gene fragment, flanked by BsaI sites CGGTCTCACCGCGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGATT AGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAA GGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAA TTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTT TCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCG AGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATT TTCACCTGAATCAGGATATTCTGAGACCG SEQ ID NO:6 Int9 NatR L gene fragment, flanked by BsaI sites CGGTCTCAGCTAGCGACGACGTGTGGGGCTACCGCTCGCTCGAGCCGACGCGGTGCTGCATCGCGTCGATCGCGCTCGTCCCCCTCAAGACG GGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCGCCGGCGAGCCCGACGGCGCCCGAGGACGGCTTGGCGTCGGCGCAGGGCGACGA CGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCTCGCGACGGCGCAGAAGCTGCTTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCT CTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACTGACTGTGGCTGTCACACGGGCCCTGCAGCGCAAGGTGTGGTGGGACCACAGCAC CGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGGCGCGGCGAGTGTGGACGCTCGAGCTGAGCCTGATCTGAGCCGTCGTCGTCGTCG TCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTCGCGATACCCCCCCTTTTCCCTCGCTCGTCTTGTTTCCGTAGCTTGGTCCGGCT CTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGTGCGTCCAGCGAGGGCGAGAGAGAGACGGTCGACACGCGCCGCCGGTGTCGAGG TTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCCCCCCTCGCCCCATCTTCCACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAG TACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTGACCCGTCCACCCACTCCCGCAAGCCACGATGGCGGCCGCCACTCTTGACGACAC GGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGCCATCGAGGCACTGGATGGGTCCTTCACCACCGACACCGTCTTCCGCGTCA CCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCCCTGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCG GACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTC CGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGA CGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGTCACCAACGTTAACGCACCGGCTATCCACGCCCGCTGAGACCG SEQ ID NO:7 Int2 NatR R gene fragment, flanked by BsaI sites CGGTCTCACACATGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTC GTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGT CGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGC TGGAGGTCACCAACGTTAACGCACCGGCTATCCACGCGTACCGGCGGATGGGGTTCACCCTCTGCGGCCTGGACACCGCCCTGTACGACGGC ACCGCCTCGGACGGCGAGCAGGCGCTCTACATGAGCATGCCCTGCCCCTAAGCTTATTCACCTGCACTCGTCCAACCTCGTCGTCGTCGTGT CCCCCTCCCCGTCCGTTTGCCCCTTCGTCTTTTGTCGTCGCCCGCCCGCCCGGCCCCCTCGAGCGGTTGCGGCCCTTACCCCCCACCCACAC TACTTCTGACGTTCTTTCGTCACGTTCTCCGTCGGCTCTACCCCCCTGCCAAGTCAAAGGCTGGTTTCTCTCGTCGGCTCGGGCGCGCGCGA GATGGGCTCGAGAGTGAGAGCGAGAGGAGCAGCAGGCCGAGTTGGCTCGGCGGTCTGGGCTCAGGACTGGCGTCGCAGCATGGGCGTCGCAC GTTCAGACCGTCTGGACGTGAGAGGCTCGGCTACTCGTTGACGACGAGGTCAGAGCGACCTTGCCGCCACGCAGTCGGCTCTACTTTTCCGC CGTCTCTCGTCGGCCTCACCCAGGCGCGGTGTGGCACTGCCTTTCTCGCGTCTCTCGCGAGAGCGCGCCTCTCGTCGAAAGCCAACCTCGCT CGCGAGAAGCGTGAGACCG SEQ ID NO:8 Int2 Strain 1 CAR2 HOML gene fragment, flanked by BsaI sites CGGTCTCACACATCGAGGTGCTCCTGGCCGAGGCCGAGTGGCGCCTGGCGGTGAACGACGAGGTCCCGGTCTGCTGAGCGTGACGCGGTGGT GCGGGAGGGCGAGCGGCTCGTCGGCGAGAGCCGGTGGGCGGCGGGGCCTTGGTATGCGCTGTGCAGCAAGTGCGTGAGGAGCAGCAAGGTGAGAGGTGCACAGAAGGAGAGAGCCCATGGCCGAAGGCATGGAGGCGATGATGCTGGTTCGGCGCACGCCGGCCAAAGTTGGCCTGCGTCGAGA TCGGTCGCCAATTCGGCAGCTGCTGCGTCCAGTCGAAACCGCAACCTTAGCGTTGCCTTACCTTTTCGCAACCAGCCCGCTTGTAAGCCAGC GAGGCCGAGTTATGTACCGGCTCTGCCGATTCGAGCGGGTTCGAGTTGGAGCCCCCGCGCGCGCGGGCTCCGCGCTTCGCTCCGAGCTCCGA GGCCGCCGCGCGCGCGACAAGTCGCACATCCTCATCTCGCCCTCCTCACCGGCCACCGCTCGCTCGCTCGATACCCCCTCTGTGCTCTCTGT TCCTCGCGTCTCGGCATGGGTGGCTTCGACTACTGGCTCGTGTGCGTCCCTCTCTCTCTCTCTCTCCCTGCGCTCGCCGCAGCCGCACCGGC TGAGCCCTCGCTGACCCCTCGTCGTCGCCGTTGCTGCAGCCATGCGCGTTGGACGATCCCTCCCTCGGTCGCGCTGTGGCTCGTGTTCCGCA AGCTGCGGACCTGGAGGGACGTGTACAAGACGCTGTTCCTCATCACGGTCCGTCTCGAGCTCCCCCCGCGGCGAGACGGCCAAGGCTGACCT CCTCCCCTCTTTGCTCCGAGCAGATCGCTGTCACGGTGCGCCGCTCTGCGCGCTCGAGACTAGGCGCACGCGCTCGGGAGAGCTGACCCTTG CCCCTCGCAGGCGACGATACCCTGGGATTCGTACCTCATCCGGAACCGCGTGCGTCCTCGTCCTCCTCCTCACCCAACCCTCCCGAGCCGCG CTGAACTGGCCGATAATTGCAGACGAGCGTGAGACCG SEQ ID NO:9 Int9 Strain 1 CAR2 HOMR gene fragment, flanked by BsaI sites CGGTCTCAGCTACATCTTGCTCACAAAGAGGAGCCCGAGCTGACTTGACTGTGCAGGTACGCCTGGTGCCGCGTCTGCGACGACCTCGTCGA CAACGCCTCGTCGGTCGCCGCCGCCGAGGCCAACATCGACAAGATCAAGTCGTGCCTCGACCTCCTGTACCCTCGCGCGACGTCGACGCCGA CCTCGCACCCCGTCGCCGTCTCGAACGACGCCATCGCCGCCGCCCTCCCCGGCTTGAGCGAGCCCGAGCGCGGCTCGTTCCGCCTCCTCGCC CTTCTCCCCATCACTCGCCCCCCCCTCGACGAGCTCCTCGCCGGCTTCCGCACCGACTTGTCGTTCCTCGCCTTCGCCGGCGAGAAGGAGAG CGCCGGCTCGAGCACGTCGATCCCCGCCGAGCTGCCGATCAAGACGGACGCCGACCTGCTCGAGTACGCCAACAACGTCGCGTCGTCGGTCG CCGACCTGTGCGTGCAGCTCGTGTGGGCGCACTGCGCCTCGTCGGTGCCCGAGCCCGAGCAGCGCGCCATCCTCGCCGCCGCGCGCGAGATG GGCCAGGCGCTCCAGCTCGTCAACATCGCGCGCGACGTGCCGGCCGACCGCGACATCCACCGCATCTACCTCCCCGGCCGGTCGCCCGAGGT CGCCGTCGAGGCCATGACGCCCGACCGGCGAGAGCTCCTGCGCCGCGCGCGCGCCATGGCGGCGCACAGCCGCGAGGCGATCGAGCGCCTGC CGCGCGAGGCGAGGGGCGGGATCCGCGCGGCGTGCGACGTGTACCTGTCGATCGGCGGGGCGGTCGAGCGCGCGCTCGACGAGGGGAGGGTG CACGAGCGCGCGAGGGTTGCAAAGGGGACGAGGGCGTGGAAGGCCTGGACGGCGTTGTGAGGGCGGGCGTCGGTGCATAGGGGAGGACTTGG GACGTAGACTAGACTGCGCAGCTCGTGTAAAGAGAGAAAGGATCCTGCGTGCTTCCCTCGTCGTGCTCGTCTCGCTCGAGCTCTCCGAGCTC TCCGCTGAGACCG SEQ ID NO:10 Int2 Strain 2 CAR2 HOML, flanked by BsaI sites CGGTCTCACACAACCCTCTCCGCCCCGCGCAGCTCTCGCTCTCGGTCCACCTCCCCACCGGGTCCGACGTCGTCTTCGACCTCGACCCGCTC GCGCACCCAGTCGACCCGGCAGCGTCCTCGTTCCGCGTCCTGCAGCCCAAGATTGAGCTGAAGCTCAAGAAGCGCGACGGCGGCGTAAAGTG GAGCAAGATCGAGGGCGAGGATGAGGGTGTTGGCTCGTTCGGTGCGTCCATATGCCTTTTCTCAAGCTTCGAGGCGGGCTGTCGTGCCCCTT GTCTGCGTCTTCAAGAGCTGCGCGTGACGCGGGAAGTCGTCGCTCGCAGGTGCCGCGGCCGAAAAGGCTGCAACCCACGCGTACCCGTCCTC GTCGCGCAAGCGCCACGACTGGGAGAAGATCGTCAAGGACTCTGCCGAGGAGGATGAGCAGCTCCAGAAGGAGTTCTCCAAGGACCCGAACG CCGGCGGCGACAAGGCGCTCAATGAGCTCTTTCAGAAGCTCTACGCCGACGCGACCGACGACCAGCGCCGTGCCATGATCAAGAGCTACCAG GAGAGCAACGGCACGGCGCTCAGCACCGATTGGTCCGACGTCAGCAAGGTCTGTCCTTCTCTCGCTCTGGCCAACATACGGGACTGACCTGC AACGTCTTCTACCTCGCAGAAAAAGGTCGAGACGCGCCCGCCCGACTCGATGCTCGCCAAGAAGTGGGAGCAGTAGTCCTTGCGCGCCCGGC CTGTCATTCTGCCGTGTCTAGCAGACGGCGGCGTTCCGTCTGTGGTTTCTCTCGTGCTTGTTCTCGCACTGTCGCAACTTTACCCTCGCCTT CGTTCCTTTGCGCTCCCTCCTTTTCAGTGTAGCAATGCATACCTCTTCGTCGTTCGCCTCCTGCAAGCGGCCTCGTGAACAGAGTTTCGCAC CGAGGGGTGTCTACAGGGAAAGGAGCTATGCAAGTCTAGGTACAGCGCATTTGACAGAGTCTTTTGTAGCGAGAAACTCGACAACTGCCTCT CTGAACTGGCCGATAATTGCAGACGAGCGTGAGACCG SEQ ID NO:11 Int9 Strain 2 CAR2 HOMR, flanked by BsaI sites CGGTCTCAGCTACCCGCACGGCACAGAACACACGGCACAGCAACTGACAGAAACAACTCTCGGGTATCGGCAGCGGAACCAAGCCCCCGATC GTGATGAACGCATCGGACCGCCTCGCGCCAGCTCCCCCTGAACTCGGCTGCTCTCTTTCTCTTCTCGGGATGTGACAGACTGCTGGGCGCAT TACCGCTCTAGCGACCTACTCTAGAACAACACGGGGACAGAAAACCGGCACTCCGCAGGGAAGCCCTCTCGACAGGTATGCAACACGAGGAA CGGACCCGCGTCAGGAGATCGCTCCGCCTCGATAAACCCGCCGCGCTCTTTACTGCTTGAGGTACACGGCGAGGGCGAGGAAGAGTGCCGCG AGGAGTAGGAGGATCGAGACGCCGCCGGCCGCGTCGCGCACAGTGCGGTGCGTCGCCGCCTCGCTCGCCGAGACGTTCCACCGCGCGGGGAC AGGCACGCCCTGGTCCATCATGATCTGCGTCGTCGCCATCCTCGCGCCGGCGAGCACGATCGGGACGCCCGTGCCCGGGTGCGCCGACGCGC CGACAAAGTAGGCGTTCTTGATCGACGGGTGGCGCGTCTTGGGCCGGAACGAGAGCACGTTGAAAAAGTCGTGCGAGAGGCCGAGGATTGAC CCGCGGTGCAGGTTGAACTTGTCGCCCCACGTGATGGGCGTGTTGACCGTCTCGTGCGCGATCAGGTCGCGCAGGCCCGAGAGCCCGAGACG CCGCTCCACCTCGCCGATGACCTTGTTGCGTGTGTCCTCGACGACCTTGTCCCAGTCCGACGACGACGGAAGCGCGGCCGAGATATGCCCGA CCGGAACGAGGACGATGACAGCGTCCTTGTCCTCGGGAGCAGCTCTACGAGGCAGACCGGCGTCAGTCTTGCGCAGCTCGTACCTCGTTCTG CCAATAAACAACGTACGAAGGGTCATGCCGACTCGGGACGTTGACATAGAAGGAAGGTTCGCGCGGGATTTTGTGGTCGCGGAAGATCGAAT CCGCTGAGACCG SEQ ID NO:12 Int345678 spacer gene fragment, flanked by BsaI sites and stuffer sequence ACGGGGTCATCACGGCTCATCATGCGCCAAACAAATGTGTGCAATACACGCTCGGATGACTGCATGATGACCGCACTGACTGGGGACAGCAG ATCCACCTAAGCCTGTGAGAGAAGCAGACACCCGACAGATCAAGGCAGTTACGGTCTCAAGCGAAGTGAATAAAGCTCCACACAGTCGGCTA TGAGACCGGGTAGAGCCACAAACAGCCGGTACAAGCAACGATCTCCAGGACCATCTGAATCATGCGCGGATGACACGAACTCACGACGGCGA TCACAGACATTAACCCACAGTACAGACACTGCGACAACGTGGCAATTCGTCGCAATAC SEQ ID NO:13 Int345678 SapI dropout gene fragment, flanked by SapI sites CGGTCTCAAGCGCGATGAAGAGCTGGTAGAGCCACAAACAGCCGAAAGTGAAACGTGATTTCATGCGTCATTTTGAACATTTTGTAAATCTT ATTTAATAATGTGTGCGGCAATTCACATTTAATTTATGAATGTTTTCTTAACATCGCGGCAACTCAAGAAACGGCAGGTTCGGATCTTAGCT ACTAGAGAAAGAGGAGAAATACTAGATGCGTAAAGGCGAGGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCA ACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTG CCGGTTCCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTT CAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTG AAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAGGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTT AACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGT GCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTC TGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTAC AAATGACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATACCTGCAGGGGTACAAGCAACGATCTCCAGCTCTTCAAAAAAGTGAATAA AGCTCCACACAGTCGGCTATGAGACCG SEQ ID NO:14 PL434 Strain 1 CAR2 SapI dropout plasmid GTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCAT ATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCG ACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGG TGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAAC CGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGC AGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGT GAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCAT CTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCT GATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCG TTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGT AACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCT TAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTCTGCGCGTAATCTGCTGC TTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAG AGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGC TAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAG CGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAA ACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGG AAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGT GGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGCCACATCGAGGTGCTCCTGGCCGAGGCCGAGTGGCGCCTGG CGGTGAACGACGAGGTCCCGGTCTGCTGAGCGTGACGCGGTGGTGCGGGAGGGCGAGCGGCTCGTCGGCGAGAGCCGGTGGGCGGCGGGGCC TTGGTATGCGCTGTGCAGCAAGTGCGTGAGGAGCAGCAAGGTGAGAGGTGCACAGAAGGAGAGAGCCCATGGCCGAAGGCATGGAGGCGATG ATGCTGGTTCGGCGCACGCCGGCCAAAGTTGGCCTGCGTCGAGATCGGTCGCCAATTCGGCAGCTGCTGCGTCCAGTCGAAACCGCAACCTT AGCGTTGCCTTACCTTTTCGCAACCAGCCCGCTTGTAAGCCAGCGAGGCCGAGTTATGTACCGGCTCTGCCGATTCGAGCGGGTTCGAGTTG GAGCCCCCGCGCGCGCGGGCTCCGCGCTTCGCTCCGAGCTCCGAGGCCGCCGCGCGCGCGACAAGTCGCACATCCTCATCTCGCCCTCCTCA CCGGCCACCGCTCGCTCGCTCGATACCCCCTCTGTGCTCTCTGTTCCTCGCGTCTCGGCATGGGTGGCTTCGACTACTGGCTCGTGTGCGTC CCTCTCTCTCTCTCTCTCCCTGCGCTCGCCGCAGCCGCACCGGCTGAGCCCTCGCTGACCCCTCGTCGTCGCCGTTGCTGCAGCCATGCGCG TTGGACGATCCCTCCCTCGGTCGCGCTGTGGCTCGTGTTCCGCAAGCTGCGGACCTGGAGGGACGTGTACAAGACGCTGTTCCTCATCACGG TCCGTCTCGAGCTCCCCCCGCGGCGAGACGGCCAAGGCTGACCTCCTCCCCTCTTTGCTCCGAGCAGATCGCTGTCACGGTGCGCCGCTCTG CGCGCTCGAGACTAGGCGCACGCGCTCGGGAGAGCTGACCCTTGCCCCTCGCAGGCGACGATACCCTGGGATTCGTACCTCATCCGGAACCG CGTGCGTCCTCGTCCTCCTCCTCACCCAACCCTCCCGAGCCGCGCTGAACTGGCCGATAATTGCAGACGAGCGCGATGAAGAGCTGGTAGAG CCACAAACAGCCGAAAGTGAAACGTGATTTCATGCGTCATTTTGAACATTTTGTAAATCTTATTTAATAATGTGTGCGGCAATTCACATTTA ATTTATGAATGTTTTCTTAACATCGCGGCAACTCAAGAAACGGCAGGTTCGGATCTTAGCTACTAGAGAAAGAGGAGAAATACTAGATGCGT AAAGGCGAGGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGG TGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTTCCTTGGCCGACTCTGGTAACGACGC TGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAG GAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCT GAAAGGCATTGACTTTAAAGAGGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATA AACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACT CCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCA TATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAATGACCAGGCATCAAATAAAACGAAAGGC TCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTT TCTGCGTTTATACCTGCAGGGGTACAAGCAACGATCTCCAGCTCTTCAAAAAAGTGAATAAAGCTCCACACAGTCGGCTAGCGACGACGTGT GGGGCTACCGCTCGCTCGAGCCGACGCGGTGCTGCATCGCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGC GGCAGCGCGTCGGCGCCGGCGAGCCCGACGGCGCCCGAGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGT CAACACTCAGCAGCTCGCGACGGCGCAGAAGCTGCTTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGT CGAGCGCAGTGACTGACTGTGGCTGTCACACGGGCCCTGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGA GGTCAAGATCTGGGCGCGGCGAGTGTGGACGCTCGAGCTGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGT GCAGATTCCCCGGTCGCGATACCCCCCCTTTTCCCTCGCTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTA TCCAGCAGTTCGAGTGCGTCCAGCGAGGGCGAGAGAGAGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAG CGAGGCGCTCCTCCTCCCCCCTCGCCCCATCTTCCACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAG CTAGACCCCGACTGACCCGTCCACCCACTCCCGCAAGCCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGT CCCGGGGGACGCCGAGGCCATCGAGGCACTGGATGGGTCCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCC TGCGGGAGGTGCCGGTGGACCCGCCCCTGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCG GACTCCCGGACGTTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGT CGAGGACATCGAGGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCG CCGGGCACCTCTGGCTGGAGGTCACCAACGTTAACGCACCGGCTATCCACGCCCGCGGATGACACGAACTCACGACTAACTATAACGGTCCT AAGGTAGCGAACCAT SEQ ID NO:15 PL436 Strain 1 CAR2 KO Right plasmid CCGCGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGATTAGAAAAAC TCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAA CTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCT CGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACT TGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAA TACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTG AATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGC TTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTT CAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCAT ATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTT ATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCG TAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTT GTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCG TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAA GTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCA CCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACC ATCTGAATCATGCGCCACATGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCG GACGTTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACA TCGAGGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCAC CTCTGGCTGGAGGTCACCAACGTTAACGCACCGGCTATCCACGCGTACCGGCGGATGGGGTTCACCCTCTGCGGCCTGGACACCGCCCTGTA CGACGGCACCGCCTCGGACGGCGAGCAGGCGCTCTACATGAGCATGCCCTGCCCCTAAGCTTATTCACCTGCACTCGTCCAACCTCGTCGTC GTCGTGTCCCCCTCCCCGTCCGTTTGCCCCTTCGTCTTTTGTCGTCGCCCGCCCGCCCGGCCCCCTCGAGCGGTTGCGGCCCTTACCCCCCA CCCACACTACTTCTGACGTTCTTTCGTCACGTTCTCCGTCGGCTCTACCCCCCTGCCAAGTCAAAGGCTGGTTTCTCTCGTCGGCTCGGGCG CGCGCGAGATGGGCTCGAGAGTGAGAGCGAGAGGAGCAGCAGGCCGAGTTGGCTCGGCGGTCTGGGCTCAGGACTGGCGTCGCAGCATGGGC GTCGCACGTTCAGACCGTCTGGACGTGAGAGGCTCGGCTACTCGTTGACGACGAGGTCAGAGCGACCTTGCCGCCACGCAGTCGGCTCTACT TTTCCGCCGTCTCTCGTCGGCCTCACCCAGGCGCGGTGTGGCACTGCCTTTCTCGCGTCTCTCGCGAGAGCGCGCCTCTCGTCGAAAGCCAA CCTCGCTCGCGAGAAGCGAAGTGAATAAAGCTCCACACAGTCGGCTACATCTTGCTCACAAAGAGGAGCCCGAGCTGACTTGACTGTGCAGG TACGCCTGGTGCCGCGTCTGCGACGACCTCGTCGACAACGCCTCGTCGGTCGCCGCCGCCGAGGCCAACATCGACAAGATCAAGTCGTGCCT CGACCTCCTGTACCCTCGCGCGACGTCGACGCCGACCTCGCACCCCGTCGCCGTCTCGAACGACGCCATCGCCGCCGCCCTCCCCGGCTTGA GCGAGCCCGAGCGCGGCTCGTTCCGCCTCCTCGCCCTTCTCCCCATCACTCGCCCCCCCCTCGACGAGCTCCTCGCCGGCTTCCGCACCGAC TTGTCGTTCCTCGCCTTCGCCGGCGAGAAGGAGAGCGCCGGCTCGAGCACGTCGATCCCCGCCGAGCTGCCGATCAAGACGGACGCCGACCT GCTCGAGTACGCCAACAACGTCGCGTCGTCGGTCGCCGACCTGTGCGTGCAGCTCGTGTGGGCGCACTGCGCCTCGTCGGTGCCCGAGCCCG AGCAGCGCGCCATCCTCGCCGCCGCGCGCGAGATGGGCCAGGCGCTCCAGCTCGTCAACATCGCGCGCGACGTGCCGGCCGACCGCGACATC CACCGCATCTACCTCCCCGGCCGGTCGCCCGAGGTCGCCGTCGAGGCCATGACGCCCGACCGGCGAGAGCTCCTGCGCCGCGCGCGCGCCAT GGCGGCGCACAGCCGCGAGGCGATCGAGCGCCTGCCGCGCGAGGCGAGGGGCGGGATCCGCGCGGCGTGCGACGTGTACCTGTCGATCGGCG GGGCGGTCGAGCGCGCGCTCGACGAGGGGAGGGTGCACGAGCGCGCGAGGGTTGCAAAGGGGACGAGGGCGTGGAAGGCCTGGACGGCGTTG TGAGGGCGGGCGTCGGTGCATAGGGGAGGACTTGGGACGTAGACTAGACTGCGCAGCTCGTGTAAAGAGAGAAAGGATCCTGCGTGCTTCCC TCGTCGTGCTCGTCTCGCTCGAGCTCTCCGAGCTCT SEQ ID NO:16 PL440 Strain 1 CAR2 KO Left plasmid GGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCAT CGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCA CCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTC AAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTT CAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAATACG CGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATC AGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGA TGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGA AACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAA ATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGT AAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATA AATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGA AAAGATCAAAGGATCTTCTTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTT GCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGT TAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCG TGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGG TAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTT TTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCT GAATCATGCGCCACATCGAGGTGCTCCTGGCCGAGGCCGAGTGGCGCCTGGCGGTGAACGACGAGGTCCCGGTCTGCTGAGCGTGACGCGGT GGTGCGGGAGGGCGAGCGGCTCGTCGGCGAGAGCCGGTGGGCGGCGGGGCCTTGGTATGCGCTGTGCAGCAAGTGCGTGAGGAGCAGCAAGG TGAGAGGTGCACAGAAGGAGAGAGCCCATGGCCGAAGGCATGGAGGCGATGATGCTGGTTCGGCGCACGCCGGCCAAAGTTGGCCTGCGTCG AGATCGGTCGCCAATTCGGCAGCTGCTGCGTCCAGTCGAAACCGCAACCTTAGCGTTGCCTTACCTTTTCGCAACCAGCCCGCTTGTAAGCC AGCGAGGCCGAGTTATGTACCGGCTCTGCCGATTCGAGCGGGTTCGAGTTGGAGCCCCCGCGCGCGCGGGCTCCGCGCTTCGCTCCGAGCTC CGAGGCCGCCGCGCGCGCGACAAGTCGCACATCCTCATCTCGCCCTCCTCACCGGCCACCGCTCGCTCGCTCGATACCCCCTCTGTGCTCTC TGTTCCTCGCGTCTCGGCATGGGTGGCTTCGACTACTGGCTCGTGTGCGTCCCTCTCTCTCTCTCTCTCCCTGCGCTCGCCGCAGCCGCACC GGCTGAGCCCTCGCTGACCCCTCGTCGTCGCCGTTGCTGCAGCCATGCGCGTTGGACGATCCCTCCCTCGGTCGCGCTGTGGCTCGTGTTCC GCAAGCTGCGGACCTGGAGGGACGTGTACAAGACGCTGTTCCTCATCACGGTCCGTCTCGAGCTCCCCCCGCGGCGAGACGGCCAAGGCTGA CCTCCTCCCCTCTTTGCTCCGAGCAGATCGCTGTCACGGTGCGCCGCTCTGCGCGCTCGAGACTAGGCGCACGCGCTCGGGAGAGCTGACCC TTGCCCCTCGCAGGCGACGATACCCTGGGATTCGTACCTCATCCGGAACCGCGTGCGTCCTCGTCCTCCTCCTCACCCAACCCTCCCGAGCC GCGCTGAACTGGCCGATAATTGCAGACGAGCGAAGTGAATAAAGCTCCACACAGTCGGCTAGCGACGACGTGTGGGGCTACCGCTCGCTCGA GCCGACGCGGTGCTGCATCGCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCGCCGG CGAGCCCGACGGCGCCCGAGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCTCGCG ACGGCGCAGAAGCTGCTTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACTGACTG TGGCTGTCACACGGGCCCTGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGGCGCGG CGAGTGTGGACGCTCGAGCTGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTCGCGA TACCCCCCCTTTTCCCTCGCTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGTGCGT CCAGCGAGGGCGAGAGAGAGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCCCC CCTCGCCCCATCTTCCACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTGACCCG TCCACCCACTCCCGCAAGCCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGCC ATCGAGGCACTGGATGGGTCCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGA CCCGCCCCTGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCC CCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGA GGTCACCAACGTTAACGCACCGGCTATCCACGCCCGC SEQ ID NO:17 PL148 - Strain 2 CAR2 L SapI dropout plasmid TTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGCCTGAACT GGCCGATAATTGCAGACGACCCTCTCCGCCCCGCGCAGCTCTCGCTCTCGGTCCACCTCCCCACCGGGTCCGACGTCGTCTTCGACCTCGAC CCGCTCGCGCACCCAGTCGACCCGGCAGCGTCCTCGTTCCGCGTCCTGCAGCCCAAGATTGAGCTGAAGCTCAAGAAGCGCGACGGCGGCGT AAAGTGGAGCAAGATCGAGGGCGAGGATGAGGGTGTTGGCTCGTTCGGTGCGTCCATATGCCTTTTCTCAAGCTTCGAGGCGGGCTGTCGTG CCCCTTGTCTGCGTCTTCAAGAGCTGCGCGTGACGCGGGAAGTCGTCGCTCGCAGGTGCCGCGGCCGAAAAGGCTGCAACCCACGCGTACCC GTCCTCGTCGCGCAAGCGCCACGACTGGGAGAAGATCGTCAAGGACTCTGCCGAGGAGGATGAGCAGCTCCAGAAGGAGTTCTCCAAGGACC CGAACGCCGGCGGCGACAAGGCGCTCAATGAGCTCTTTCAGAAGCTCTACGCCGACGCGACCGACGACCAGCGCCGTGCCATGATCAAGAGC TACCAGGAGAGCAACGGCACGGCGCTCAGCACCGATTGGTCCGACGTCAGCAAGGTCTGTCCTTCTCTCGCTCTGGCCAACATACGGGACTG ACCTGCAACGTCTTCTACCTCGCAGAAAAAGGTCGAGACGCGCCCGCCCGACTCGATGCTCGCCAAGAAGTGGGAGCAGTAGTCCTTGCGCG CCCGGCCTGTCATTCTGCCGTGTCTAGCAGACGGCGGCGTTCCGTCTGTGGTTTCTCTCGTGCTTGTTCTCGCACTGTCGCAACTTTACCCT CGCCTTCGTTCCTTTGCGCTCCCTCCTTTTCAGTGTAGCAATGCATACCTCTTCGTCGTTCGCCTCCTGCAAGCGGCCTCGTGAACAGAGTT TCGCACCGAGGGGTGTCTACAGGGAAAGGAGCTATGCAAGTCTAGGTACAGCGCATTTGACAGAGTCTTTTGTAGCGAGAAACTCGACAACT GCCTCTCCAAACCAGATGTCAACACAGCTACACTGCGATGAAGAGCTGGTAGAGCCACAAACAGCCGAAAGTGAAACGTGATTTCATGCGTC ATTTTGAACATTTTGTAAATCTTATTTAATAATGTGTGCGGCAATTCACATTTAATTTATGAATGTTTTCTTAACATCGCGGCAACTCAAGA AACGGCAGGTTCGGATCTTAGCTACTAGAGAAAGAGGAGAAATACTAGATGCGTAAAGGCGAGGAGCTGTTCACTGGTGTCGTCCCTATTCT GGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGT TCATCTGTACTACTGGTAAACTGCCGGTTCCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGAC CATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAA AACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAGGACGGCAATATCCTGG GCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGC CACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCA CTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCA CGCATGGTATGGATGAACTGTACAAATGACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTT TGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATACCTGCAGGGGTACAAGCAACGATCTC CAGCTCTTCAAAAAGTAAGCGCACACACTGGCTTAAGATGACGCGACGACGTGTGGGGCTACCGCTCGCTCGAGCCGACGCGGTGCTGCATC GCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCGCCGGCGAGCCCGACGGCGCCCGA GGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCTCGCGACGGCGCAGAAGCTGCTTC TCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACTGACTGTGGCTGTCACACGGGCCCT GCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGGCGCGGCGAGTGTGGACGCTCGAGC TGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTCGCGATACCCCCCCTTTTCCCTCG CTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGTGCGTCCAGCGAGGGCGAGAGAGA GACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCCCCCCTCGCCCCATCTTCCACC TCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTGACCCGTCCACCCACTCCCGCAAGC CACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGCCATCGAGGCACTGGATGGGT CCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCCCTGACCAAGGTG TTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTCGTCGCGTACGGGGACGACGGCGA CCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCCCCGGAGCACCGGGGGCACG GGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGTCACCAACGTTAACGCA CCGGCTATCCACGCGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGA TTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATG AAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATT AATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTC TTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAG CGAGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATA TTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACG GATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTT TGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCAT TTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGT ATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGT GGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCG TCAGACCCCGTAGAAAAGATCAAAGGATCTTC SEQ ID NO:18 PL044, plasmid encoding NatR flanked by Strain 2 CAR2 homology arms TTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGCCTGAACT GGCCGATAATTGCAGACGACCCTCTCCGCCCCGCGCAGCTCTCGCTCTCGGTCCACCTCCCCACCGGGTCCGACGTCGTCTTCGACCTCGAC CCGCTCGCGCACCCAGTCGACCCGGCAGCGTCCTCGTTCCGCGTCCTGCAGCCCAAGATTGAGCTGAAGCTCAAGAAGCGCGACGGCGGCGTAAAGTGGAGCAAGATCGAGGGCGAGGATGAGGGTGTTGGCTCGTTCGGTGCGTCCATATGCCTTTTCTCAAGCTTCGAGGCGGGCTGTCGTG CCCCTTGTCTGCGTCTTGAAGAGCTGCGCGTGACGCGGGAAGTCGTCGCTCGCAGGTGCCGCGGCCGAAAAGGCTGCAACCCACGCGTACCC GTCCTCGTCGCGCAAGCGCCACGACTGGGAGAAGATCGTCAAGGACTCTGCCGAGGAGGATGAGCAGCTCCAGAAGGAGTTCTCCAAGGACC CGAACGCCGGCGGCGACAAGGCGCTCAATGAGCTCTTCCAGAAGCTCTACGCCGACGCGACCGACGACCAGCGCCGTGCCATGATCAAGAGC TACCAGGAGAGCAACGGCACGGCGCTCAGCACCGATTGGTCCGACGTCAGCAAGGTCTGTCCTTCTCTCGCTCTGGCCAACATACGGGACTG ACCTGCAACGTCTTCTACCTCGCAGAAAAAGGTCGAGACGCGCCCGCCCGACTCGATGCTCGCCAAGAAGTGGGAGCAGTAGTCCTTGCGCG CCCGGCCTGTCATTCTGCCGTGTCTAGCAGACGGCGGCGTTCCGTCTGTGGTTTCTCTCGTGCTTGTTCTCGCACTGTCGCAACTTTACCCT CGCCTTCGTTCCTTTGCGCTCCCTCCTTTTCAGTGTAGCAATGCATACCTCTTCGTCGTTCGCCTCCTGCAAGCGGCCTCGTGAACAGAGTT TCGCACCGAGGGGTGTCTACAGGGAAAGGAGCTATGCAAGTCTAGGTACAGCGCATTTGACAGAGTCTTTTGTAGCGAGAAACTCGACAACT GCCTCTCACACACTGGCTTAAGATGACGCGACGACGTGTGGGGCTACCGCTCGCTCGAGCCGACGCGGTGCTGCATCGCGTCGATCGCGCTC GTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCGCCGGCGAGCCCGACGGCGCCCGAGGACGGCTTGGCGTC GGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCTCGCGACGGCGCAGAAGCTGCTTCTCTTCTGGCGCAAGC CTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACTGACTGTGGCTGTCACACGGGCCCTGCAGCGCAAGGTGTG GTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGGCGCGGCGAGTGTGGACGCTCGAGCTGAGCCTGATCTGAG CCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTCGCGATACCCCCCCTTTTCCCTCGCTCGTCTTGTTTCCG TAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGTGCGTCCAGCGAGGGCGAGAGAGAGACGGTCGACACGCG CCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCCCCCCTCGCCCCATCTTCCACCTCGCCCCTCTCCCTC TAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTGACCCGTCCACCCACTCCCGCAAGCCACGATGGCGGCCGC CACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGCCATCGAGGCACTGGATGGGTCCTTCACCACCGACA CCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCCCTGACCAAGGTGTTCCCCGACGACGAA TCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGT GGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGT TGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGTCACCAACGTTAACGCACCGGCTATCCACGCG TACCGGCGGATGGGGTTCACCCTCTGCGGCCTGGACACCGCCCTGTACGACGGCACCGCCTCGGACGGCGAGCAGGCGCTCTACATGAGCAT GCCCTGCCCCTAAGCTTATTCACCTGCACTCGTCCAACCTCGTCGTCGTCGTGTCCCCCTCCCCGTCCGTTTGCCCCTTCGTCTTTTGTCGT CGCCCGCCCGCCCGGCCCCCTCGAGCGGTTGCGGCCCTTACCCCCCACCCACACTACTTCTGACGTTCTTTCGTCACGTTCTCCGTCGGCTC TACCCCCCTGCCAAGTCAAAGGCTGGTTTCTCTCGTCGGCTCGGGCGCGCGCGAGATGGGCTCGAGAGTGAGAGCGAGAGGAGCAGCAGGCC GAGTTGGCTCGGCGGTCTGGGCTCAGGACTGGCGTCGCAGCATGGGCGTCGCACGTTCAGACCGTCTGGACGTGAGAGGCTCGGCTACTCGT TGACGACGAGGTCAGAGCGACCTTGCCGCCACGCAGTCGGCTCTACTTTTCCGCCGTCTCTCGTCGGCCTCACCCAGGCGCGGTGTGGCACT GCCTTTCTCGCGTCTCTCGCGAGAGCGCGCCTCTCGTCGAAAGCCAACCTCGCTCGCGAGAGGTAGACTACCCATGAGTCACAATGCCCGCA CGGCACAGAACACACGGCACAGCAACTGACAGAAACAACTCTCGGGTATCGGCAGCGGAACCAAGCCCCCGATCGTGATGAACGCATCGGAC CGCCTCGCGCCAGCTCCCCCTGAACTCGGCTGCTCTCTTTCTCTTCTCGGGATGTGACAGACTGCTGGGCGCATTACCGCTCTAGCGACCTA CTCTAGAACAACACGGGGACAGAAAACCGGCACTCCGCAGGGAAGCCCTCTCGACAGGTATGCAACACGAGGAACGGACCCGCGTCAGGAGA TCGCTCCGCCTCGATAAACCCGCCGCGCTCTTTACTGCTTGAGGTACACGGCGAGGGCGAGGAAGAGTGCCGCGAGGAGTAGGAGGATCGAG ACGCCGCCGGCCGCGTCGCGCACAGTGCGGTGCGTCGCCGCCTCGCTCGCCGAGACGTTCCACCGCGCGGGGACAGGCACGCCCTGGTCCAT CATGATCTGCGTCGTCGCCATCCTCGCGCCGGCGAGCACGATCGGGACGCCCGTGCCCGGGTGCGCCGACGCGCCGACAAAGTAGGCGTTCT TGATCGACGGGTGGCGCGTCTTGGGCCGGAACGAGAGCACGTTGAAAAAGTCGTGCGAGAGGCCGAGGATTGACCCGCGGTGCAGGTTGAAC TTGTCGCCCCACGTGATGGGCGTGTTGACCGTCTCGTGCGCGATCAGGTCGCGCAGGCCCGAGAGCCCGAGACGCCGCTCCACCTCGCCGAT GACCTTGTTGCGTGTGTCCTCGACGACCTTGTCCCAGTCCGACGACGACGGAAGCGCGGCCGAGATATGCCCGACCGGAACGAGGACGATGA CAGCGTCCTTGTCCTCGGGAGCAGCTCTACGAGGCAGACCGGCGTCAGTCTTGCGCAGCTCGTACCTCGTTCTGCCAATAAACAACGTACGA AGGGTCATGCCGACTCGGGACGTTGACATAGAAGGAAGGTTCGCGCGGGATTTTGTGGTCGCGGAAGATCGAATAAGTGAATAAAGCTCCAC ACAGTCGGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGATTAGAAA AACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGA AAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCC CCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAG ACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCG AAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCAC CTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAA TGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATG TTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACC CATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTG TTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTG TTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACC CCGTAGAAAAGATCAAAGGATCTTC SEQ ID NO:19 PL493, Strain 2 CAR2 SapI dropout plasmid GGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCAT CGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCA CCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTC AAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTT CAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAATACG CGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATC AGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGA TGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGA AACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAA ATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGT AAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATA AATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGA AAAGATCAAAGGATCTTCTTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTT GCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGT TAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCG TGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGG TAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTT TTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGCCACAACCCTCTCCGCCCCGCGCAGCTCTCGCTCTCGGTCCACCTCCCCACCGGGTCCGACGTCGTCTTCGACCTCGACCCG CTCGCGCACCCAGTCGACCCGGCAGCGTCCTCGTTCCGCGTCCTGCAGCCCAAGATTGAGCTGAAGCTCAAGAAGCGCGACGGCGGCGTAAA GTGGAGCAAGATCGAGGGCGAGGATGAGGGTGTTGGCTCGTTCGGTGCGTCCATATGCCTTTTCTCAAGCTTCGAGGCGGGCTGTCGTGCCC CTTGTCTGCGTCTTCAAGAGCTGCGCGTGACGCGGGAAGTCGTCGCTCGCAGGTGCCGCGGCCGAAAAGGCTGCAACCCACGCGTACCCGTC CTCGTCGCGCAAGCGCCACGACTGGGAGAAGATCGTCAAGGACTCTGCCGAGGAGGATGAGCAGCTCCAGAAGGAGTTCTCCAAGGACCCGA ACGCCGGCGGCGACAAGGCGCTCAATGAGCTCTTTCAGAAGCTCTACGCCGACGCGACCGACGACCAGCGCCGTGCCATGATCAAGAGCTAC CAGGAGAGCAACGGCACGGCGCTCAGCACCGATTGGTCCGACGTCAGCAAGGTCTGTCCTTCTCTCGCTCTGGCCAACATACGGGACTGACC TGCAACGTCTTCTACCTCGCAGAAAAAGGTCGAGACGCGCCCGCCCGACTCGATGCTCGCCAAGAAGTGGGAGCAGTAGTCCTTGCGCGCCC GGCCTGTCATTCTGCCGTGTCTAGCAGACGGCGGCGTTCCGTCTGTGGTTTCTCTCGTGCTTGTTCTCGCACTGTCGCAACTTTACCCTCGC CTTCGTTCCTTTGCGCTCCCTCCTTTTCAGTGTAGCAATGCATACCTCTTCGTCGTTCGCCTCCTGCAAGCGGCCTCGTGAACAGAGTTTCG CACCGAGGGGTGTCTACAGGGAAAGGAGCTATGCAAGTCTAGGTACAGCGCATTTGACAGAGTCTTTTGTAGCGAGAAACTCGACAACTGCC TCTCTGAACTGGCCGATAATTGCAGACGAGCGCGATGAAGAGCTGGTAGAGCCACAAACAGCCGAAAGTGAAACGTGATTTCATGCGTCATT TTGAACATTTTGTAAATCTTATTTAATAATGTGTGCGGCAATTCACATTTAATTTATGAATGTTTTCTTAACATCGCGGCAACTCAAGAAAC GGCAGGTTCGGATCTTAGCTACTAGAGAAAGAGGAGAAATACTAGATGCGTAAAGGCGAGGAGCTGTTCACTGGTGTCGTCCCTATTCTGGT GGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCA TCTGTACTACTGGTAAACTGCCGGTTCCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCAT ATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAAC GCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAGGACGGCAATATCCTGGGCC ATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCAC AACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTA TCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGC ATGGTATGGATGAACTGTACAAATGACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGT CGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATACCTGCAGGGGTACAAGCAACGATCTCCAG CTCTTCAAAAAAGTGAATAAAGCTCCACACAGTCGGCTAGCGACGACGTGTGGGGCTACCGCTCGCTCGAGCCGACGCGGTGCTGCATCGCG TCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCGCCGGCGAGCCCGACGGCGCCCGAGGA CGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCTCGCGACGGCGCAGAAGCTGCTTCTCT TCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACTGACTGTGGCTGTCACACGGGCCCTGCA GCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGGCGCGGCGAGTGTGGACGCTCGAGCTGA GCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTCGCGATACCCCCCCTTTTCCCTCGCTC GTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGTGCGTCCAGCGAGGGCGAGAGAGAGAC GGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCCCCCCTCGCCCCATCTTCCACCTCG CCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTGACCCGTCCACCCACTCCCGCAAGCCAC GATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGCCATCGAGGCACTGGATGGGTCCT TCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCCCTGACCAAGGTGTTC CCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTCGTCGCGTACGGGGACGACGGCGACCT GGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCCCCGGAGCACCGGGGGCACGGGG TCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGTCACCAACGTTAACGCACCG GCTATCCACGCCCGC SEQ ID NO:20 PL494, Strain 1 CAR2 KO Left plasmid GGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCAT CGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCA CCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTC AAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTT CAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAATACG CGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATC AGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGA TGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGA AACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAA ATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGT AAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATA AATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGA AAAGATCAAAGGATCTTCTTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTT GCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGT TAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCG TGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGG TAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTT TTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCT GAATCATGCGCCACATGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACG TTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGA GGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCT GGCTGGAGGTCACCAACGTTAACGCACCGGCTATCCACGCGTACCGGCGGATGGGGTTCACCCTCTGCGGCCTGGACACCGCCCTGTACGAC GGCACCGCCTCGGACGGCGAGCAGGCGCTCTACATGAGCATGCCCTGCCCCTAAGCTTATTCACCTGCACTCGTCCAACCTCGTCGTCGTCG TGTCCCCCTCCCCGTCCGTTTGCCCCTTCGTCTTTTGTCGTCGCCCGCCCGCCCGGCCCCCTCGAGCGGTTGCGGCCCTTACCCCCCACCCA CACTACTTCTGACGTTCTTTCGTCACGTTCTCCGTCGGCTCTACCCCCCTGCCAAGTCAAAGGCTGGTTTCTCTCGTCGGCTCGGGCGCGCG CGAGATGGGCTCGAGAGTGAGAGCGAGAGGAGCAGCAGGCCGAGTTGGCTCGGCGGTCTGGGCTCAGGACTGGCGTCGCAGCATGGGCGTCG CACGTTCAGACCGTCTGGACGTGAGAGGCTCGGCTACTCGTTGACGACGAGGTCAGAGCGACCTTGCCGCCACGCAGTCGGCTCTACTTTTC CGCCGTCTCTCGTCGGCCTCACCCAGGCGCGGTGTGGCACTGCCTTTCTCGCGTCTCTCGCGAGAGCGCGCCTCTCGTCGAAAGCCAACCTC GCTCGCGAGAAGCGAAGTGAATAAAGCTCCACACAGTCGGCTACCCGCACGGCACAGAACACACGGCACAGCAACTGACAGAAACAACTCTC GGGTATCGGCAGCGGAACCAAGCCCCCGATCGTGATGAACGCATCGGACCGCCTCGCGCCAGCTCCCCCTGAACTCGGCTGCTCTCTTTCTC TTCTCGGGATGTGACAGACTGCTGGGCGCATTACCGCTCTAGCGACCTACTCTAGAACAACACGGGGACAGAAAACCGGCACTCCGCAGGGA AGCCCTCTCGACAGGTATGCAACACGAGGAACGGACCCGCGTCAGGAGATCGCTCCGCCTCGATAAACCCGCCGCGCTCTTTACTGCTTGAG GTACACGGCGAGGGCGAGGAAGAGTGCCGCGAGGAGTAGGAGGATCGAGACGCCGCCGGCCGCGTCGCGCACAGTGCGGTGCGTCGCCGCCT CGCTCGCCGAGACGTTCCACCGCGCGGGGACAGGCACGCCCTGGTCCATCATGATCTGCGTCGTCGCCATCCTCGCGCCGGCGAGCACGATCGGGACGCCCGTGCCCGGGTGCGCCGACGCGCCGACAAAGTAGGCGTTCTTGATCGACGGGTGGCGCGTCTTGGGCCGGAACGAGAGCACGTT GAAAAAGTCGTGCGAGAGGCCGAGGATTGACCCGCGGTGCAGGTTGAACTTGTCGCCCCACGTGATGGGCGTGTTGACCGTCTCGTGCGCGA TCAGGTCGCGCAGGCCCGAGAGCCCGAGACGCCGCTCCACCTCGCCGATGACCTTGTTGCGTGTGTCCTCGACGACCTTGTCCCAGTCCGAC GACGACGGAAGCGCGGCCGAGATATGCCCGACCGGAACGAGGACGATGACAGCGTCCTTGTCCTCGGGAGCAGCTCTACGAGGCAGACCGGC GTCAGTCTTGCGCAGCTCGTACCTCGTTCTGCCAATAAACAACGTACGAAGGGTCATGCCGACTCGGGACGTTGACATAGAAGGAAGGTTCG CGCGGGATTTTGTGGTCGCGGAAGATCGAATCCGC SEQ ID NO:21 R. toruloides RPS3A AA sequence, protein ID 10325 MAVGKNKRLSKGKKGIKKRVVDPFTRKDWYDLKAPSMFDTRNVGKTLVNRSSGLKNANDSLKGRIFELSLGDLNKDDENTFRKIRLRVDEVQ GKNCLTNFHGMDFTSDKLRSLVRKWQTLIEAQLDVKTTDGYLVRLFTIGFTKRRPNQVKKTTYAQSAQIREIRRKMFDIMQREASSCDLKQL VQKLIPEVIGREIEKASQSIYPLQNCYVRKVKIMKQPKFDVGKLLELHSSSTEEEVGQKVVREFKEPEVLASV SEQ ID NO:22 PL040, R taiwanensis RPS3A promoter gene fragment, flanked by SapI sites GCTCTTCACGACGCTAGCAGCTCGTGGCGCCATAAAAGCGTGCGAGTCACACGACCCGACTCGGCAGCAGAGCCTTAGGGTCGCGAGCACCG TGACCATGCGTAGAGCCTAGGGGCGTGCGGGACAATGTCGGTCGTGCATTGACGTGTGCTGCTGGACGCAAGAAGGCTGATGTTGGACCGTC GAGGCGTCAGGCGCTGTGGGCGGGGTAAGGTGCTAGGGCGTGTGCACCAGGGAGAGGGCGGCGAGCGCGGAGGGGAGCGGTGGCGGCTGTGA GCCAAAGTCAAGCAAGCTATCGTGTGTGTTCACCAGAAGACAGCACGTTACAGCGTAGAGGGATACGGTGAGGAAGGCGCGCGCAAAAAGCG GGTGCAGAGTCGAGGCCGGCGTCGCCGACACGCGGACGAGCGACCGACCGACTTGGTGTCGAGCGCGGCAACCTCGCTCTTGTCCCTCGCAC CCTCGCCGCGTCTTCACGCTCGCCCTTACCCAACATGGGCAAGTCATTCTCACCTATACTATCGTCGCAGTCGACCCTCCGCCCTCCACCCT AGGCCCTCGCACGACGCGGCGGTGTCGGGAGGTGTCGCCGCTTCGAGCTGTGCCCAGTCGACCCGCTCGCAATTTTCGCCCGCCGACTCGAC CCCGCCCGCTTCGCCCTGAGCACCGCCTGACCGGCCCTACACTCGATGAGGGGGATTTAGAGTGTACTGGCGGTATCGTGCACGCGCCTGGG TGGGCGGGAAGGTTGGGTCGGCGGGTCGGCTAGGTCGAGCTCGGCAGGCACGGGTCGGCGGTCACGCGCCGTTTTCAGTCGCTCGAAGGGCT GCAGGGTACCGCCAGGCTCGGATCTGTCTCGTATGACACGGATGGCGGCCGCGCAGCGGGTCAGGCGTCGCCAGCTCGAGCGGTACACGCGT CGCCCTCCGACCCCAGCAAACCTACCCTCGTGCATCGAAATCGCGGGGAACCTGGCGCTAACCCGGCCTCTGCTCCTGTCCTACAGCTATGT GAAGAGCPGHRDFIKNMITGTSQADCAILIIAAGTGEFEAGISKDGQTREHALLAFTLGVRQLIVAINKMDTTKYSEARYEEIIKETSNFIKKVGFNPK GVPFVPISGWHGDNMIEATTNMPWYKGWKKETKSGEVTGKTLLDAIDAIEPPSRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGTIKAGMVV TFAPSNVTTEVKSVEMHHEQLEAGLPGDNVGFNVKNVSVKDIRRGNVCGDSKNDPPKEAASFKAQVIVMNHPGQIGNGYAPVLDCHTAHIAC KFDTLLEKIDRRSGKSVEDLPKFIKSGDAAIVKMVPSKPMCVESFAEYPPLGRFAVRDMRQTVAVGVIKAVEKTDGKGGKVTKSAEKAAGKK K SEQ ID NO:24 PL082, Strain 2 TEF1 promoter, flanked by SapI sites GCTCTTCACGAGAAGAAGCGACTGCATTCGTCGAGGAAGGTGATTCGTACCGAGCTTTGGCATCCGCCGATAGAGTCAACGTTCGTCAGTCT TACAGTGCGGCGAGGCGGTGCACAAAAGCCGGCCAGCGCCGGCAGAACCCGACTTTCGACGCCGCGAGGGCAACCTTCCCAAGCAGAGGGCG CGCACGCAAGTGCACAGAGTACCGCAGGGCATGCAGAGAGCATGCCAGACTTTGCTGCAGCGCGAGTACGCTTGCCAGCATGCATCACCGGG CGATGCAGCGGGATACTCATAGGGAAGACGCGCGACGGGGCAGCAAGCGATTTGGCGGGTCTAGAGACTAAGCGAGTCCGATCCCCTTGGAT CGGGATATTACGGGTGCACGAGGAGAACCCGCGTTCTAGCGTTGTGCCGCCTTGAGGGCGCCACCCAAAGCACGCACGCAGATGGCCCTGGT CTGCGGCTCCCCAGCGCTGGTTTCGATGGCTGGCGAGAGGGTATTTCCGGTTGCTCGTATTGACAGTCTCGTCTATGTGGGTAACGAGCGGC GTCGAGGCGTCGAGGCAGAAAAGGGAGCGAAGGCAGGAAACGAGCACCAGTCGACGCCAGCCTCCTTGAGCGTCGCCGCTCCCAGCAGTTCA GAGGCAGCGGAAGCCAGCAGGAGTGAGTGCAAGAGCCATGCAGAGAGGTGGGCAACATGAAGAAGTGCCGTGAGGTGGTGAGGTGGTGAGGT GGTGAGGTGAGGCGGTCGGTGAGGGAGCAGAGCAGGAGAGGAGGAGGGGGCAAGGAGACTAAGCGATGAAACCCGGGGTCGGGGCGCAGGCT GGGAGTGATCGAGGTGAGCTTGCAGGGGCAGTGGCCCCCGTGGCAGTCGCAGCGAGGCGCAGGGCGCGCGCGGCAAGTCGGCCCAAGCTCGA CGTGCGCGCGTGCCCGCCCCCGGCGCCGTGCTCGCCGCCTCTTCTCTTCTCTCCACCACTCGGTCCTCCAACTACTCAAACCTCGCACACCA TGTGAAGAGC SEQ ID NO:25 Strain 2 predicted amino acid transporter AA sequence MSASPDLYNDPEKGKATTFATSAEKVESGGNGVVLRGLDADAHEGVVHRNLKARHLQMIALGGCIGTGLFVGSGAALAGGGPLGLWLGYSIM GLVVGTMMIALGEMTTLYPVSGAFVHYSSRFLDPAVGFALGYNYWYSYAITLPTEITAAALVINFWRDDINVAVWITVFYVVICSFNFFGVR AYGEAEFWFSVLKIITILGLILLGIIITAGGVPGTEPIGFRYWHDPGPFQQDNGIPGAKGRFLAFWTTLVQGAFSYLGTEIVALTAGEAQNP RRNVPKAIKRTFFRILLFYVIGTFVIGLIVSPNDPNLTNADGVNASPWVIAIQNAGIQGLPSVINAAVLLSAFSAGNSDLYASSRTLYGLAC SGQAPAFFRRCTKQGLPVYCVIATALVGLLAYMNVSTGSTTAFNYLSNLSTITGLITWACINLSYIRFYHGCKRAGIDRKTFPYRAPFQPYA SYIGLVLIILVLFFNGFNVFLEGNWDTADFIISYITIVIFIVLYCGWKLFKRTKYVRLENMDFDTGRRELDAMADEEAANFKPATTWYGKIW DWIM SEQ ID NO:26 R. toruloides protein ID RHTO4_8962 MATSRDHLADVELGDIKGGSPTLNRTNSLSKHDGNGVVLRGLDGDSPDGSVHRNLKARHLQMIALGGTIGTGLFVGAGGALATGGPLGIWLG YTIMGFTVGTMMVALGEMTTLYPVSGAFTHYTARFVDPAAGFALGWNYWYSYAITLPTEVTAAALVIQYWRDDINVAVWITIFLVVICCFNF LGVRAYGEAEFWFSLMKIITILGLILLGIIITAGGVPGTDPIGFRFWRNPGPFQQENGIPGTKGRFLAFWTVLVQAAFSYLGTEIVALTAGE AENPRRNVPKAIRRVFYRILFFYVIGTFVMGLIVSPNDPNLTNADGVNASPWVIAIKNAGIKGLPSVINTVVLLSAFSAGNSDLYASSRTLY GLACDGKAPAIFRRCTKNGLPIYCLILTALVGLLAYMNVSTGSTTAFNYLSNLSSITGVITWLCICVSYIRFYHGAKAHGLDRNDFPYKAPL QPWASYWGAFFFFIVIIFNGYTIFLDGNWSTADFIVAYVTCWVFIVLYVFWKLFKRTKFVRIENMDFDTGRRELDQIAEDEAARYKAPTTWY GRLWDAIM SEQ ID NO:27 PL076, Strain 2 IndProm1 promoter GCTCTTCACGAATGGACACTGTAGCTCCTTTTCGAGGCAAAACAGAAATTCTCGTACTCTGAATCTTGCGGAAGCGGGCTGATCGCAATCGC CGGCTCGCCTTCTAACCAAACTGACGTGATTGAGGTGGCGGCTCGGTTTTTGAAATGAGCTTGGCTATGCTATCGCGTCGTCGTCTTCTTGT CGCAAAGCCGCGAAGTCGAGCGGGTGACTGGAAGCAGTGCGTTCGCTTCCTCCCTGCTCTACCTTGCTTCGACTCCGTGATGCAGGACTGATCTGCTTACTGCTTGCAGTGCTCTTGGCTGCTATCTTGACTGTGCTCAAGCTGCGGCGCTCGCCTCAGATGCCCGTCTGCACCGCTTCGGCAC GCCGCCGCCGACGATCCCAGGCAACGGAAGATAAGAGCAAGCAGATCTAGCAATACGGCTTCGTGCTGCTATGCGGTCATCTGTTCGGGCTC TTGTATCGGATGCTGCGGGCCCGCAAGCCGATGGGTCCCCTCAGCTGCTTGCCGGACTCCATTTCTAGCTCGATAGCGCTCTCACCTCGCTT CGCCTCGTCTAACACGTCCTGTTCAATCCGGATGCAGCTATTGAGCCTCCGACACTTTTCAAGTGATGCGGCGCGCGCTGTCCACCTCGTTC AGGCTCAAAATCTCGGGTTCCTAAAACAGCTCGTCTAACGGACAGACCAAATTTGGTCGTTCTCCTGGCCGACCGACGTCGCGCCTTGCGGA CGTAGAGATATCGCAAGGCACGGGCCGCGACAAGGAGCTTTTGCGGAGACATGCTGGCCTTGTGGCTCGAATCGCCCTCTTCCTCTCCTTTC CCCCCTCGCCTCCCCTCGCTGGCCTCGGCTTCCGTTCTCCCTCCCGCCATGTGAAGAGC SEQ ID NO:28 PL0913x-FLAG-GFP gene fragment, flanked by SapI sites GCTCTTCAATGGACTACAAGGACGACGACGACAAGGACTACAAAGACGATGACGACAAAGACTATAAAGACGACGATGACAAGGGCTCGGTG TCGAAGGGCGAGGAGCTCTTTACCGGCGTCGTCCCGATCCTCGTCGAGTTGGACGGCGACGTCAACGGCCACAAGTTCTCGGTTTCGGGCGA GGGCGAGGGAGATGCCACCTACGGCAAGCTCACCCTCAAGTTCATCTGCACCACCGGCAAGTTGCCGGTCCCGTGGCCGACGCTCGTCACCA CCCTCACCTACGGAGTCCAGTGCTTCTCGCGCTACCCGGACCACATGAAGCAGCACGACTTCTTTAAGTCGGCCATGCCGGAGGGCTACGTC CAGGAGCGCACCATCTTCTTCAAGGATGACGGCAACTACAAGACCCGCGCCGAGGTCAAGTTTGAGGGCGACACCCTCGTCAACCGCATCGA GCTCAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTCGGCCACAAACTCGAGTACAACTACAACTCGCACAACGTCTACATCATGGCGG ACAAGCAGAAGAACGGCATCAAGGTCAACTTCAAGATCCGCCACAACATCGAGGACGGCTCGGTCCAGCTCGCCGACCACTACCAGCAGAAC ACCCCGATCGGAGATGGCCCCGTCCTCTTGCCGGACAACCACTACCTCTCGACCCAGTCGGCCCTCTCGAAGGACCCGAACGAGAAGCGCGA CCACATGGTCCTCCTCGAATTCGTCACCGCCGCCGGCATCACCCTCGGCATGGACGAGCTCTACAAGTAGTGAAGAGC SEQ ID NO:29 PL024 R. toruloides RPS3A terminator gene fragment, flanked by SapI sites GCTCTTCATAGGCGGAGCAGCGACGGACGGACCTTGTGCGTTGTGTTTGTAGGGAGGGCGAGTGGACCCGCAATGCGAGGGAAACTCTTTCT CTTCCTTCGTGCGTCCTGCCCGAGAGACTTGCGAGTCGAAGTTGACGAGGAGAGGGTTGGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGCTC GCTCGCTGCTTCGGCTGCTTCTCGACGTGCTCTAGTCGATGTTGAGTGTACTGCTACATCCCACGAAGAAGGGAAGCTCGCACGGCCGTGAG ACTGCTCTCTTCCCTCGACCGCATCGCTGGCCATCCCTCTCCACGCTTGCCGCTTCGACTGCTTCTCAACGTGCACAATTCGCTGCTTGAAC AATTGATGCTGCCGCCTACGTCGAGCGCAAGCGACCCTCACATGATTGCGAGCGAGAGTTCTCGCGTTCTCCCCAGTCCCGCCGTCGACAAC GCCGCACGCATGTTGAGATCTAACGCAATTACACCTATACGATCCTTGAATAAATGAAGAGC SEQ ID NO:30 PL451 Strain 1 CAR2 left with pRhota_RPS3A>3x-FLAG-GFP expression plasmid CCGCGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGATTAGAAAAAC TCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAA CTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCT CGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACT TGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAA TACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTG AATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGC TTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTT CAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCAT ATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTT ATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTG AATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCG TAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTT GTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCG TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAA GTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCA CCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACC ATCTGAATCATGCGCCACATCGAGGTGCTCCTGGCCGAGGCCGAGTGGCGCCTGGCGGTGAACGACGAGGTCCCGGTCTGCTGAGCGTGACG CGGTGGTGCGGGAGGGCGAGCGGCTCGTCGGCGAGAGCCGGTGGGCGGCGGGGCCTTGGTATGCGCTGTGCAGCAAGTGCGTGAGGAGCAGC AAGGTGAGAGGTGCACAGAAGGAGAGAGCCCATGGCCGAAGGCATGGAGGCGATGATGCTGGTTCGGCGCACGCCGGCCAAAGTTGGCCTGC GTCGAGATCGGTCGCCAATTCGGCAGCTGCTGCGTCCAGTCGAAACCGCAACCTTAGCGTTGCCTTACCTTTTCGCAACCAGCCCGCTTGTA AGCCAGCGAGGCCGAGTTATGTACCGGCTCTGCCGATTCGAGCGGGTTCGAGTTGGAGCCCCCGCGCGCGCGGGCTCCGCGCTTCGCTCCGA GCTCCGAGGCCGCCGCGCGCGCGACAAGTCGCACATCCTCATCTCGCCCTCCTCACCGGCCACCGCTCGCTCGCTCGATACCCCCTCTGTGC TCTCTGTTCCTCGCGTCTCGGCATGGGTGGCTTCGACTACTGGCTCGTGTGCGTCCCTCTCTCTCTCTCTCTCCCTGCGCTCGCCGCAGCCG CACCGGCTGAGCCCTCGCTGACCCCTCGTCGTCGCCGTTGCTGCAGCCATGCGCGTTGGACGATCCCTCCCTCGGTCGCGCTGTGGCTCGTG TTCCGCAAGCTGCGGACCTGGAGGGACGTGTACAAGACGCTGTTCCTCATCACGGTCCGTCTCGAGCTCCCCCCGCGGCGAGACGGCCAAGG CTGACCTCCTCCCCTCTTTGCTCCGAGCAGATCGCTGTCACGGTGCGCCGCTCTGCGCGCTCGAGACTAGGCGCACGCGCTCGGGAGAGCTG ACCCTTGCCCCTCGCAGGCGACGATACCCTGGGATTCGTACCTCATCCGGAACCGCGTGCGTCCTCGTCCTCCTCCTCACCCAACCCTCCCG AGCCGCGCTGAACTGGCCGATAATTGCAGACGAGCGCGACGCTAGCAGCTCGTGGCGCCATAAAAGCGTGCGAGTCACACGACCCGACTCGG CAGCAGAGCCTTAGGGTCGCGAGCACCGTGACCATGCGTAGAGCCTAGGGGCGTGCGGGACAATGTCGGTCGTGCATTGACGTGTGCTGCTG GACGCAAGAAGGCTGATGTTGGACCGTCGAGGCGTCAGGCGCTGTGGGCGGGGTAAGGTGCTAGGGCGTGTGCACCAGGGAGAGGGCGGCGA GCGCGGAGGGGAGCGGTGGCGGCTGTGAGCCAAAGTCAAGCAAGCTATCGTGTGTGTTCACCAGAAGACAGCACGTTACAGCGTAGAGGGAT ACGGTGAGGAAGGCGCGCGCAAAAAGCGGGTGCAGAGTCGAGGCCGGCGTCGCCGACACGCGGACGAGCGACCGACCGACTTGGTGTCGAGC GCGGCAACCTCGCTCTTGTCCCTCGCACCCTCGCCGCGTCTTCACGCTCGCCCTTACCCAACATGGGCAAGTCATTCTCACCTATACTATCG TCGCAGTCGACCCTCCGCCCTCCACCCTAGGCCCTCGCACGACGCGGCGGTGTCGGGAGGTGTCGCCGCTTCGAGCTGTGCCCAGTCGACCC GCTCGCAATTTTCGCCCGCCGACTCGACCCCGCCCGCTTCGCCCTGAGCACCGCCTGACCGGCCCTACACTCGATGAGGGGGATTTAGAGTG TACTGGCGGTATCGTGCACGCGCCTGGGTGGGCGGGAAGGTTGGGTCGGCGGGTCGGCTAGGTCGAGCTCGGCAGGCACGGGTCGGCGGTCA CGCGCCGTTTTCAGTCGCTCGAAGGGCTGCAGGGTACCGCCAGGCTCGGATCTGTCTCGTATGACACGGATGGCGGCCGCGCAGCGGGTCAG GCGTCGCCAGCTCGAGCGGTACACGCGTCGCCCTCCGACCCCAGCAAACCTACCCTCGTGCATCGAAATCGCGGGGAACCTGGCGCTAACCC GGCCTCTGCTCCTGTCCTACAGCTATGGACTACAAGGACGACGACGACAAGGACTACAAAGACGATGACGACAAAGACTATAAAGACGACGA TGACAAGGGCTCGGTGTCGAAGGGCGAGGAGCTCTTTACCGGCGTCGTCCCGATCCTCGTCGAGTTGGACGGCGACGTCAACGGCCACAAGT TCTCGGTTTCGGGCGAGGGCGAGGGAGATGCCACCTACGGCAAGCTCACCCTCAAGTTCATCTGCACCACCGGCAAGTTGCCGGTCCCGTGG CCGACGCTCGTCACCACCCTCACCTACGGAGTCCAGTGCTTCTCGCGCTACCCGGACCACATGAAGCAGCACGACTTCTTTAAGTCGGCCATGCCGGAGGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGATGACGGCAACTACAAGACCCGCGCCGAGGTCAAGTTTGAGGGCGACACCC TCGTCAACCGCATCGAGCTCAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTCGGCCACAAACTCGAGTACAACTACAACTCGCACAAC GTCTACATCATGGCGGACAAGCAGAAGAACGGCATCAAGGTCAACTTCAAGATCCGCCACAACATCGAGGACGGCTCGGTCCAGCTCGCCGA CCACTACCAGCAGAACACCCCGATCGGAGATGGCCCCGTCCTCTTGCCGGACAACCACTACCTCTCGACCCAGTCGGCCCTCTCGAAGGACC CGAACGAGAAGCGCGACCACATGGTCCTCCTCGAATTCGTCACCGCCGCCGGCATCACCCTCGGCATGGACGAGCTCTACAAGTAGGCGGAG CAGCGACGGACGGACCTTGTGCGTTGTGTTTGTAGGGAGGGCGAGTGGACCCGCAATGCGAGGGAAACTCTTTCTCTTCCTTCGTGCGTCCT GCCCGAGAGACTTGCGAGTCGAAGTTGACGAGGAGAGGGTTGGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGCTCGCTCGCTGCTTCGGCTG CTTCTCGACGTGCTCTAGTCGATGTTGAGTGTACTGCTACATCCCACGAAGAAGGGAAGCTCGCACGGCCGTGAGACTGCTCTCTTCCCTCG ACCGCATCGCTGGCCATCCCTCTCCACGCTTGCCGCTTCGACTGCTTCTCAACGTGCACAATTCGCTGCTTGAACAATTGATGCTGCCGCCT ACGTCGAGCGCAAGCGACCCTCACATGATTGCGAGCGAGAGTTCTCGCGTTCTCCCCAGTCCCGCCGTCGACAACGCCGCACGCATGTTGAG ATCTAACGCAATTACACCTATACGATCCTTGAATAAAAAGTGAATAAAGCTCCACACAGTCGGCTAGCGACGACGTGTGGGGCTACCGCTCG CTCGAGCCGACGCGGTGCTGCATCGCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGC GCCGGCGAGCCCGACGGCGCCCGAGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGC TCGCGACGGCGCAGAAGCTGCTTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACT GACTGTGGCTGTCACACGGGCCCTGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGG CGCGGCGAGTGTGGACGCTCGAGCTGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGT CGCGATACCCCCCCTTTTCCCTCGCTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAG TGCGTCCAGCGAGGGCGAGAGAGAGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCC TCCCCCCTCGCCCCATCTTCCACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTG ACCCGTCCACCCACTCCCGCAAGCCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCG AGGCCATCGAGGCACTGGATGGGTCCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCG GTGGACCCGCCCCTGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTT CGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGG TCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGG CTGGAGGTCACCAACGTTAACGCACCGGCTATCCACGC SEQ ID NO:31 PL453 Strain 1 CAR2 L Str2_pTEF1>GFP CCGCGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGATTAGAAAAAC TCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAA CTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCT CGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACT TGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAA TACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTG AATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGC TTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTT CAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCAT ATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTT ATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTG AATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCG TAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTT GTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCG TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAA GTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCA CCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACC ATCTGAATCATGCGCCACATCGAGGTGCTCCTGGCCGAGGCCGAGTGGCGCCTGGCGGTGAACGACGAGGTCCCGGTCTGCTGAGCGTGACG CGGTGGTGCGGGAGGGCGAGCGGCTCGTCGGCGAGAGCCGGTGGGCGGCGGGGCCTTGGTATGCGCTGTGCAGCAAGTGCGTGAGGAGCAGC AAGGTGAGAGGTGCACAGAAGGAGAGAGCCCATGGCCGAAGGCATGGAGGCGATGATGCTGGTTCGGCGCACGCCGGCCAAAGTTGGCCTGC GTCGAGATCGGTCGCCAATTCGGCAGCTGCTGCGTCCAGTCGAAACCGCAACCTTAGCGTTGCCTTACCTTTTCGCAACCAGCCCGCTTGTA AGCCAGCGAGGCCGAGTTATGTACCGGCTCTGCCGATTCGAGCGGGTTCGAGTTGGAGCCCCCGCGCGCGCGGGCTCCGCGCTTCGCTCCGA GCTCCGAGGCCGCCGCGCGCGCGACAAGTCGCACATCCTCATCTCGCCCTCCTCACCGGCCACCGCTCGCTCGCTCGATACCCCCTCTGTGC TCTCTGTTCCTCGCGTCTCGGCATGGGTGGCTTCGACTACTGGCTCGTGTGCGTCCCTCTCTCTCTCTCTCTCCCTGCGCTCGCCGCAGCCG CACCGGCTGAGCCCTCGCTGACCCCTCGTCGTCGCCGTTGCTGCAGCCATGCGCGTTGGACGATCCCTCCCTCGGTCGCGCTGTGGCTCGTG TTCCGCAAGCTGCGGACCTGGAGGGACGTGTACAAGACGCTGTTCCTCATCACGGTCCGTCTCGAGCTCCCCCCGCGGCGAGACGGCCAAGG CTGACCTCCTCCCCTCTTTGCTCCGAGCAGATCGCTGTCACGGTGCGCCGCTCTGCGCGCTCGAGACTAGGCGCACGCGCTCGGGAGAGCTG ACCCTTGCCCCTCGCAGGCGACGATACCCTGGGATTCGTACCTCATCCGGAACCGCGTGCGTCCTCGTCCTCCTCCTCACCCAACCCTCCCG AGCCGCGCTGAACTGGCCGATAATTGCAGACGAGCGCGAGAAGAAGCGACTGCATTCGTCGAGGAAGGTGATTCGTACCGAGCTTTGGCATC CGCCGATAGAGTCAACGTTCGTCAGTCTTACAGTGCGGCGAGGCGGTGCACAAAAGCCGGCCAGCGCCGGCAGAACCCGACTTTCGACGCCG CGAGGGCAACCTTCCCAAGCAGAGGGCGCGCACGCAAGTGCACAGAGTACCGCAGGGCATGCAGAGAGCATGCCAGACTTTGCTGCAGCGCG AGTACGCTTGCCAGCATGCATCACCGGGCGATGCAGCGGGATACTCATAGGGAAGACGCGCGACGGGGCAGCAAGCGATTTGGCGGGTCTAG AGACTAAGCGAGTCCGATCCCCTTGGATCGGGATATTACGGGTGCACGAGGAGAACCCGCGTTCTAGCGTTGTGCCGCCTTGAGGGCGCCAC CCAAAGCACGCACGCAGATGGCCCTGGTCTGCGGCTCCCCAGCGCTGGTTTCGATGGCTGGCGAGAGGGTATTTCCGGTTGCTCGTATTGAC AGTCTCGTCTATGTGGGTAACGAGCGGCGTCGAGGCGTCGAGGCAGAAAAGGGAGCGAAGGCAGGAAACGAGCACCAGTCGACGCCAGCCTC CTTGAGCGTCGCCGCTCCCAGCAGTTCAGAGGCAGCGGAAGCCAGCAGGAGTGAGTGCAAGAGCCATGCAGAGAGGTGGGCAACATGAAGAA GTGCCGTGAGGTGGTGAGGTGGTGAGGTGGTGAGGTGAGGCGGTCGGTGAGGGAGCAGAGCAGGAGAGGAGGAGGGGGCAAGGAGACTAAGC GATGAAACCCGGGGTCGGGGCGCAGGCTGGGAGTGATCGAGGTGAGCTTGCAGGGGCAGTGGCCCCCGTGGCAGTCGCAGCGAGGCGCAGGG CGCGCGCGGCAAGTCGGCCCAAGCTCGACGTGCGCGCGTGCCCGCCCCCGGCGCCGTGCTCGCCGCCTCTTCTCTTCTCTCCACCACTCGGT CCTCCAACTACTCAAACCTCGCACACCATGGACTACAAGGACGACGACGACAAGGACTACAAAGACGATGACGACAAAGACTATAAAGACGA CGATGACAAGGGCTCGGTGTCGAAGGGCGAGGAGCTCTTTACCGGCGTCGTCCCGATCCTCGTCGAGTTGGACGGCGACGTCAACGGCCACA AGTTCTCGGTTTCGGGCGAGGGCGAGGGAGATGCCACCTACGGCAAGCTCACCCTCAAGTTCATCTGCACCACCGGCAAGTTGCCGGTCCCG TGGCCGACGCTCGTCACCACCCTCACCTACGGAGTCCAGTGCTTCTCGCGCTACCCGGACCACATGAAGCAGCACGACTTCTTTAAGTCGGC CATGCCGGAGGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGATGACGGCAACTACAAGACCCGCGCCGAGGTCAAGTTTGAGGGCGACA CCCTCGTCAACCGCATCGAGCTCAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTCGGCCACAAACTCGAGTACAACTACAACTCGCAC AACGTCTACATCATGGCGGACAAGCAGAAGAACGGCATCAAGGTCAACTTCAAGATCCGCCACAACATCGAGGACGGCTCGGTCCAGCTCGCCGACCACTACCAGCAGAACACCCCGATCGGAGATGGCCCCGTCCTCTTGCCGGACAACCACTACCTCTCGACCCAGTCGGCCCTCTCGAAGG ACCCGAACGAGAAGCGCGACCACATGGTCCTCCTCGAATTCGTCACCGCCGCCGGCATCACCCTCGGCATGGACGAGCTCTACAAGTAGGCG GAGCAGCGACGGACGGACCTTGTGCGTTGTGTTTGTAGGGAGGGCGAGTGGACCCGCAATGCGAGGGAAACTCTTTCTCTTCCTTCGTGCGT CCTGCCCGAGAGACTTGCGAGTCGAAGTTGACGAGGAGAGGGTTGGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGCTCGCTCGCTGCTTCGG CTGCTTCTCGACGTGCTCTAGTCGATGTTGAGTGTACTGCTACATCCCACGAAGAAGGGAAGCTCGCACGGCCGTGAGACTGCTCTCTTCCC TCGACCGCATCGCTGGCCATCCCTCTCCACGCTTGCCGCTTCGACTGCTTCTCAACGTGCACAATTCGCTGCTTGAACAATTGATGCTGCCG CCTACGTCGAGCGCAAGCGACCCTCACATGATTGCGAGCGAGAGTTCTCGCGTTCTCCCCAGTCCCGCCGTCGACAACGCCGCACGCATGTT GAGATCTAACGCAATTACACCTATACGATCCTTGAATAAAAAGTGAATAAAGCTCCACACAGTCGGCTAGCGACGACGTGTGGGGCTACCGC TCGCTCGAGCCGACGCGGTGCTGCATCGCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTC GGCGCCGGCGAGCCCGACGGCGCCCGAGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGC AGCTCGCGACGGCGCAGAAGCTGCTTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTG ACTGACTGTGGCTGTCACACGGGCCCTGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCT GGGCGCGGCGAGTGTGGACGCTCGAGCTGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCC GGTCGCGATACCCCCCCTTTTCCCTCGCTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTC GAGTGCGTCCAGCGAGGGCGAGAGAGAGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCC TCCTCCCCCCTCGCCCCATCTTCCACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGA CTGACCCGTCCACCCACTCCCGCAAGCCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACG CCGAGGCCATCGAGGCACTGGATGGGTCCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTG CCGGTGGACCCGCCCCTGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGAC GTTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCG AGGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTC TGGCTGGAGGTCACCAACGTTAACGCACCGGCTATCCACGC SEQ ID NO:32 Strain 2 CAR2 L Rhota_pRPS3A>GFP TTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGCCTGAACT GGCCGATAATTGCAGACGACCCTCTCCGCCCCGCGCAGCTCTCGCTCTCGGTCCACCTCCCCACCGGGTCCGACGTCGTCTTCGACCTCGAC CCGCTCGCGCACCCAGTCGACCCGGCAGCGTCCTCGTTCCGCGTCCTGCAGCCCAAGATTGAGCTGAAGCTCAAGAAGCGCGACGGCGGCGT AAAGTGGAGCAAGATCGAGGGCGAGGATGAGGGTGTTGGCTCGTTCGGTGCGTCCATATGCCTTTTCTCAAGCTTCGAGGCGGGCTGTCGTG CCCCTTGTCTGCGTCTTCAAGAGCTGCGCGTGACGCGGGAAGTCGTCGCTCGCAGGTGCCGCGGCCGAAAAGGCTGCAACCCACGCGTACCC GTCCTCGTCGCGCAAGCGCCACGACTGGGAGAAGATCGTCAAGGACTCTGCCGAGGAGGATGAGCAGCTCCAGAAGGAGTTCTCCAAGGACC CGAACGCCGGCGGCGACAAGGCGCTCAATGAGCTCTTTCAGAAGCTCTACGCCGACGCGACCGACGACCAGCGCCGTGCCATGATCAAGAGC TACCAGGAGAGCAACGGCACGGCGCTCAGCACCGATTGGTCCGACGTCAGCAAGGTCTGTCCTTCTCTCGCTCTGGCCAACATACGGGACTG ACCTGCAACGTCTTCTACCTCGCAGAAAAAGGTCGAGACGCGCCCGCCCGACTCGATGCTCGCCAAGAAGTGGGAGCAGTAGTCCTTGCGCG CCCGGCCTGTCATTCTGCCGTGTCTAGCAGACGGCGGCGTTCCGTCTGTGGTTTCTCTCGTGCTTGTTCTCGCACTGTCGCAACTTTACCCT CGCCTTCGTTCCTTTGCGCTCCCTCCTTTTCAGTGTAGCAATGCATACCTCTTCGTCGTTCGCCTCCTGCAAGCGGCCTCGTGAACAGAGTT TCGCACCGAGGGGTGTCTACAGGGAAAGGAGCTATGCAAGTCTAGGTACAGCGCATTTGACAGAGTCTTTTGTAGCGAGAAACTCGACAACT GCCTCTCCAAACCAGATGTCAACACAGCTACACTGCGACGCTAGCAGCTCGTGGCGCCATAAAAGCGTGCGAGTCACACGACCCGACTCGGC AGCAGAGCCTTAGGGTCGCGAGCACCGTGACCATGCGTAGAGCCTAGGGGCGTGCGGGACAATGTCGGTCGTGCATTGACGTGTGCTGCTGG ACGCAAGAAGGCTGATGTTGGACCGTCGAGGCGTCAGGCGCTGTGGGCGGGGTAAGGTGCTAGGGCGTGTGCACCAGGGAGAGGGCGGCGAG CGCGGAGGGGAGCGGTGGCGGCTGTGAGCCAAAGTCAAGCAAGCTATCGTGTGTGTTCACCAGAAGACAGCACGTTACAGCGTAGAGGGATA CGGTGAGGAAGGCGCGCGCAAAAAGCGGGTGCAGAGTCGAGGCCGGCGTCGCCGACACGCGGACGAGCGACCGACCGACTTGGTGTCGAGCG CGGCAACCTCGCTCTTGTCCCTCGCACCCTCGCCGCGTCTTCACGCTCGCCCTTACCCAACATGGGCAAGTCATTCTCACCTATACTATCGT CGCAGTCGACCCTCCGCCCTCCACCCTAGGCCCTCGCACGACGCGGCGGTGTCGGGAGGTGTCGCCGCTTCGAGCTGTGCCCAGTCGACCCG CTCGCAATTTTCGCCCGCCGACTCGACCCCGCCCGCTTCGCCCTGAGCACCGCCTGACCGGCCCTACACTCGATGAGGGGGATTTAGAGTGT ACTGGCGGTATCGTGCACGCGCCTGGGTGGGCGGGAAGGTTGGGTCGGCGGGTCGGCTAGGTCGAGCTCGGCAGGCACGGGTCGGCGGTCAC GCGCCGTTTTCAGTCGCTCGAAGGGCTGCAGGGTACCGCCAGGCTCGGATCTGTCTCGTATGACACGGATGGCGGCCGCGCAGCGGGTCAGG CGTCGCCAGCTCGAGCGGTACACGCGTCGCCCTCCGACCCCAGCAAACCTACCCTCGTGCATCGAAATCGCGGGGAACCTGGCGCTAACCCG GCCTCTGCTCCTGTCCTACAGCTATGGACTACAAGGACGACGACGACAAGGACTACAAAGACGATGACGACAAAGACTATAAAGACGACGAT GACAAGGGCTCGGTGTCGAAGGGCGAGGAGCTCTTTACCGGCGTCGTCCCGATCCTCGTCGAGTTGGACGGCGACGTCAACGGCCACAAGTT CTCGGTTTCGGGCGAGGGCGAGGGAGATGCCACCTACGGCAAGCTCACCCTCAAGTTCATCTGCACCACCGGCAAGTTGCCGGTCCCGTGGC CGACGCTCGTCACCACCCTCACCTACGGAGTCCAGTGCTTCTCGCGCTACCCGGACCACATGAAGCAGCACGACTTCTTTAAGTCGGCCATG CCGGAGGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGATGACGGCAACTACAAGACCCGCGCCGAGGTCAAGTTTGAGGGCGACACCCT CGTCAACCGCATCGAGCTCAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTCGGCCACAAACTCGAGTACAACTACAACTCGCACAACG TCTACATCATGGCGGACAAGCAGAAGAACGGCATCAAGGTCAACTTCAAGATCCGCCACAACATCGAGGACGGCTCGGTCCAGCTCGCCGAC CACTACCAGCAGAACACCCCGATCGGAGATGGCCCCGTCCTCTTGCCGGACAACCACTACCTCTCGACCCAGTCGGCCCTCTCGAAGGACCC GAACGAGAAGCGCGACCACATGGTCCTCCTCGAATTCGTCACCGCCGCCGGCATCACCCTCGGCATGGACGAGCTCTACAAGTAGGCGGAGC AGCGACGGACGGACCTTGTGCGTTGTGTTTGTAGGGAGGGCGAGTGGACCCGCAATGCGAGGGAAACTCTTTCTCTTCCTTCGTGCGTCCTG CCCGAGAGACTTGCGAGTCGAAGTTGACGAGGAGAGGGTTGGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGCTCGCTCGCTGCTTCGGCTGC TTCTCGACGTGCTCTAGTCGATGTTGAGTGTACTGCTACATCCCACGAAGAAGGGAAGCTCGCACGGCCGTGAGACTGCTCTCTTCCCTCGA CCGCATCGCTGGCCATCCCTCTCCACGCTTGCCGCTTCGACTGCTTCTCAACGTGCACAATTCGCTGCTTGAACAATTGATGCTGCCGCCTA CGTCGAGCGCAAGCGACCCTCACATGATTGCGAGCGAGAGTTCTCGCGTTCTCCCCAGTCCCGCCGTCGACAACGCCGCACGCATGTTGAGA TCTAACGCAATTACACCTATACGATCCTTGAATAAAAGTAAGCGCACACACTGGCTTAAGATGACGCGACGACGTGTGGGGCTACCGCTCGC TCGAGCCGACGCGGTGCTGCATCGCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCG CCGGCGAGCCCGACGGCGCCCGAGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCT CGCGACGGCGCAGAAGCTGCTTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACTG ACTGTGGCTGTCACACGGGCCCTGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGGC GCGGCGAGTGTGGACGCTCGAGCTGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTC GCGATACCCCCCCTTTTCCCTCGCTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGT GCGTCCAGCGAGGGCGAGAGAGAGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCCCCCCTCGCCCCATCTTCCACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTGA CCCGTCCACCCACTCCCGCAAGCCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGA GGCCATCGAGGCACTGGATGGGTCCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGG TGGACCCGCCCCTGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTC GTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGT CGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGC TGGAGGTCACCAACGTTAACGCACCGGCTATCCACGCGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGT TACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTT TTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTC GTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAG AATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTT ATTCATTCGTGATTGCGCCTGAGCGAGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGA ACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGT AACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGT AACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATT GCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGA ATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACA TCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAAC GTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC SEQ ID NO:33 Strain 2 CAR2 L Str2_pTEF1>GFP TTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGCCTGAACT GGCCGATAATTGCAGACGACCCTCTCCGCCCCGCGCAGCTCTCGCTCTCGGTCCACCTCCCCACCGGGTCCGACGTCGTCTTCGACCTCGAC CCGCTCGCGCACCCAGTCGACCCGGCAGCGTCCTCGTTCCGCGTCCTGCAGCCCAAGATTGAGCTGAAGCTCAAGAAGCGCGACGGCGGCGT AAAGTGGAGCAAGATCGAGGGCGAGGATGAGGGTGTTGGCTCGTTCGGTGCGTCCATATGCCTTTTCTCAAGCTTCGAGGCGGGCTGTCGTG CCCCTTGTCTGCGTCTTCAAGAGCTGCGCGTGACGCGGGAAGTCGTCGCTCGCAGGTGCCGCGGCCGAAAAGGCTGCAACCCACGCGTACCC GTCCTCGTCGCGCAAGCGCCACGACTGGGAGAAGATCGTCAAGGACTCTGCCGAGGAGGATGAGCAGCTCCAGAAGGAGTTCTCCAAGGACC CGAACGCCGGCGGCGACAAGGCGCTCAATGAGCTCTTTCAGAAGCTCTACGCCGACGCGACCGACGACCAGCGCCGTGCCATGATCAAGAGC TACCAGGAGAGCAACGGCACGGCGCTCAGCACCGATTGGTCCGACGTCAGCAAGGTCTGTCCTTCTCTCGCTCTGGCCAACATACGGGACTG ACCTGCAACGTCTTCTACCTCGCAGAAAAAGGTCGAGACGCGCCCGCCCGACTCGATGCTCGCCAAGAAGTGGGAGCAGTAGTCCTTGCGCG CCCGGCCTGTCATTCTGCCGTGTCTAGCAGACGGCGGCGTTCCGTCTGTGGTTTCTCTCGTGCTTGTTCTCGCACTGTCGCAACTTTACCCT CGCCTTCGTTCCTTTGCGCTCCCTCCTTTTCAGTGTAGCAATGCATACCTCTTCGTCGTTCGCCTCCTGCAAGCGGCCTCGTGAACAGAGTT TCGCACCGAGGGGTGTCTACAGGGAAAGGAGCTATGCAAGTCTAGGTACAGCGCATTTGACAGAGTCTTTTGTAGCGAGAAACTCGACAACT GCCTCTCCAAACCAGATGTCAACACAGCTACACTGCGAGAAGAAGCGACTGCATTCGTCGAGGAAGGTGATTCGTACCGAGCTTTGGCATCC GCCGATAGAGTCAACGTTCGTCAGTCTTACAGTGCGGCGAGGCGGTGCACAAAAGCCGGCCAGCGCCGGCAGAACCCGACTTTCGACGCCGC GAGGGCAACCTTCCCAAGCAGAGGGCGCGCACGCAAGTGCACAGAGTACCGCAGGGCATGCAGAGAGCATGCCAGACTTTGCTGCAGCGCGA GTACGCTTGCCAGCATGCATCACCGGGCGATGCAGCGGGATACTCATAGGGAAGACGCGCGACGGGGCAGCAAGCGATTTGGCGGGTCTAGA GACTAAGCGAGTCCGATCCCCTTGGATCGGGATATTACGGGTGCACGAGGAGAACCCGCGTTCTAGCGTTGTGCCGCCTTGAGGGCGCCACC CAAAGCACGCACGCAGATGGCCCTGGTCTGCGGCTCCCCAGCGCTGGTTTCGATGGCTGGCGAGAGGGTATTTCCGGTTGCTCGTATTGACA GTCTCGTCTATGTGGGTAACGAGCGGCGTCGAGGCGTCGAGGCAGAAAAGGGAGCGAAGGCAGGAAACGAGCACCAGTCGACGCCAGCCTCC TTGAGCGTCGCCGCTCCCAGCAGTTCAGAGGCAGCGGAAGCCAGCAGGAGTGAGTGCAAGAGCCATGCAGAGAGGTGGGCAACATGAAGAAG TGCCGTGAGGTGGTGAGGTGGTGAGGTGGTGAGGTGAGGCGGTCGGTGAGGGAGCAGAGCAGGAGAGGAGGAGGGGGCAAGGAGACTAAGCG ATGAAACCCGGGGTCGGGGCGCAGGCTGGGAGTGATCGAGGTGAGCTTGCAGGGGCAGTGGCCCCCGTGGCAGTCGCAGCGAGGCGCAGGGC GCGCGCGGCAAGTCGGCCCAAGCTCGACGTGCGCGCGTGCCCGCCCCCGGCGCCGTGCTCGCCGCCTCTTCTCTTCTCTCCACCACTCGGTC CTCCAACTACTCAAACCTCGCACACCATGGACTACAAGGACGACGACGACAAGGACTACAAAGACGATGACGACAAAGACTATAAAGACGAC GATGACAAGGGCTCGGTGTCGAAGGGCGAGGAGCTCTTTACCGGCGTCGTCCCGATCCTCGTCGAGTTGGACGGCGACGTCAACGGCCACAA GTTCTCGGTTTCGGGCGAGGGCGAGGGAGATGCCACCTACGGCAAGCTCACCCTCAAGTTCATCTGCACCACCGGCAAGTTGCCGGTCCCGT GGCCGACGCTCGTCACCACCCTCACCTACGGAGTCCAGTGCTTCTCGCGCTACCCGGACCACATGAAGCAGCACGACTTCTTTAAGTCGGCC ATGCCGGAGGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGATGACGGCAACTACAAGACCCGCGCCGAGGTCAAGTTTGAGGGCGACAC CCTCGTCAACCGCATCGAGCTCAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTCGGCCACAAACTCGAGTACAACTACAACTCGCACA ACGTCTACATCATGGCGGACAAGCAGAAGAACGGCATCAAGGTCAACTTCAAGATCCGCCACAACATCGAGGACGGCTCGGTCCAGCTCGCC GACCACTACCAGCAGAACACCCCGATCGGAGATGGCCCCGTCCTCTTGCCGGACAACCACTACCTCTCGACCCAGTCGGCCCTCTCGAAGGA CCCGAACGAGAAGCGCGACCACATGGTCCTCCTCGAATTCGTCACCGCCGCCGGCATCACCCTCGGCATGGACGAGCTCTACAAGTAGGCGG AGCAGCGACGGACGGACCTTGTGCGTTGTGTTTGTAGGGAGGGCGAGTGGACCCGCAATGCGAGGGAAACTCTTTCTCTTCCTTCGTGCGTC CTGCCCGAGAGACTTGCGAGTCGAAGTTGACGAGGAGAGGGTTGGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGCTCGCTCGCTGCTTCGGC TGCTTCTCGACGTGCTCTAGTCGATGTTGAGTGTACTGCTACATCCCACGAAGAAGGGAAGCTCGCACGGCCGTGAGACTGCTCTCTTCCCT CGACCGCATCGCTGGCCATCCCTCTCCACGCTTGCCGCTTCGACTGCTTCTCAACGTGCACAATTCGCTGCTTGAACAATTGATGCTGCCGC CTACGTCGAGCGCAAGCGACCCTCACATGATTGCGAGCGAGAGTTCTCGCGTTCTCCCCAGTCCCGCCGTCGACAACGCCGCACGCATGTTG AGATCTAACGCAATTACACCTATACGATCCTTGAATAAAAGTAAGCGCACACACTGGCTTAAGATGACGCGACGACGTGTGGGGCTACCGCT CGCTCGAGCCGACGCGGTGCTGCATCGCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCG GCGCCGGCGAGCCCGACGGCGCCCGAGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCA GCTCGCGACGGCGCAGAAGCTGCTTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGA CTGACTGTGGCTGTCACACGGGCCCTGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTG GGCGCGGCGAGTGTGGACGCTCGAGCTGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCG GTCGCGATACCCCCCCTTTTCCCTCGCTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCG AGTGCGTCCAGCGAGGGCGAGAGAGAGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCT CCTCCCCCCTCGCCCCATCTTCCACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGAC TGACCCGTCCACCCACTCCCGCAAGCCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGC CGAGGCCATCGAGGCACTGGATGGGTCCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCCCTGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACG TTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGA GGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCT GGCTGGAGGTCACCAACGTTAACGCACCGGCTATCCACGCGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATG TGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATA TTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGA CTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGT GAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACC GTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCA GGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTG AGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATC TGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTG ATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGT TGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTA ACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC SEQ ID NO:34 Strain 2 CAR2 L Str2_pInd1>GFP TTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGCCTGAACT GGCCGATAATTGCAGACGACCCTCTCCGCCCCGCGCAGCTCTCGCTCTCGGTCCACCTCCCCACCGGGTCCGACGTCGTCTTCGACCTCGAC CCGCTCGCGCACCCAGTCGACCCGGCAGCGTCCTCGTTCCGCGTCCTGCAGCCCAAGATTGAGCTGAAGCTCAAGAAGCGCGACGGCGGCGT AAAGTGGAGCAAGATCGAGGGCGAGGATGAGGGTGTTGGCTCGTTCGGTGCGTCCATATGCCTTTTCTCAAGCTTCGAGGCGGGCTGTCGTG CCCCTTGTCTGCGTCTTCAAGAGCTGCGCGTGACGCGGGAAGTCGTCGCTCGCAGGTGCCGCGGCCGAAAAGGCTGCAACCCACGCGTACCC GTCCTCGTCGCGCAAGCGCCACGACTGGGAGAAGATCGTCAAGGACTCTGCCGAGGAGGATGAGCAGCTCCAGAAGGAGTTCTCCAAGGACC CGAACGCCGGCGGCGACAAGGCGCTCAATGAGCTCTTTCAGAAGCTCTACGCCGACGCGACCGACGACCAGCGCCGTGCCATGATCAAGAGC TACCAGGAGAGCAACGGCACGGCGCTCAGCACCGATTGGTCCGACGTCAGCAAGGTCTGTCCTTCTCTCGCTCTGGCCAACATACGGGACTG ACCTGCAACGTCTTCTACCTCGCAGAAAAAGGTCGAGACGCGCCCGCCCGACTCGATGCTCGCCAAGAAGTGGGAGCAGTAGTCCTTGCGCG CCCGGCCTGTCATTCTGCCGTGTCTAGCAGACGGCGGCGTTCCGTCTGTGGTTTCTCTCGTGCTTGTTCTCGCACTGTCGCAACTTTACCCT CGCCTTCGTTCCTTTGCGCTCCCTCCTTTTCAGTGTAGCAATGCATACCTCTTCGTCGTTCGCCTCCTGCAAGCGGCCTCGTGAACAGAGTT TCGCACCGAGGGGTGTCTACAGGGAAAGGAGCTATGCAAGTCTAGGTACAGCGCATTTGACAGAGTCTTTTGTAGCGAGAAACTCGACAACT GCCTCTCCAAACCAGATGTCAACACAGCTACACTGCGAATGGACACTGTAGCTCCTTTTCGAGGCAAAACAGAAATTCTCGTACTCTGAATC TTGCGGAAGCGGGCTGATCGCAATCGCCGGCTCGCCTTCTAACCAAACTGACGTGATTGAGGTGGCGGCTCGGTTTTTGAAATGAGCTTGGC TATGCTATCGCGTCGTCGTCTTCTTGTCGCAAAGCCGCGAAGTCGAGCGGGTGACTGGAAGCAGTGCGTTCGCTTCCTCCCTGCTCTACCTT GCTTCGACTCCGTGATGCAGGACTGATCTGCTTACTGCTTGCAGTGCTCTTGGCTGCTATCTTGACTGTGCTCAAGCTGCGGCGCTCGCCTC AGATGCCCGTCTGCACCGCTTCGGCACGCCGCCGCCGACGATCCCAGGCAACGGAAGATAAGAGCAAGCAGATCTAGCAATACGGCTTCGTG CTGCTATGCGGTCATCTGTTCGGGCTCTTGTATCGGATGCTGCGGGCCCGCAAGCCGATGGGTCCCCTCAGCTGCTTGCCGGACTCCATTTC TAGCTCGATAGCGCTCTCACCTCGCTTCGCCTCGTCTAACACGTCCTGTTCAATCCGGATGCAGCTATTGAGCCTCCGACACTTTTCAAGTG ATGCGGCGCGCGCTGTCCACCTCGTTCAGGCTCAAAATCTCGGGTTCCTAAAACAGCTCGTCTAACGGACAGACCAAATTTGGTCGTTCTCC TGGCCGACCGACGTCGCGCCTTGCGGACGTAGAGATATCGCAAGGCACGGGCCGCGACAAGGAGCTTTTGCGGAGACATGCTGGCCTTGTGG CTCGAATCGCCCTCTTCCTCTCCTTTCCCCCCTCGCCTCCCCTCGCTGGCCTCGGCTTCCGTTCTCCCTCCCGCCATGGACTACAAGGACGA CGACGACAAGGACTACAAAGACGATGACGACAAAGACTATAAAGACGACGATGACAAGGGCTCGGTGTCGAAGGGCGAGGAGCTCTTTACCG GCGTCGTCCCGATCCTCGTCGAGTTGGACGGCGACGTCAACGGCCACAAGTTCTCGGTTTCGGGCGAGGGCGAGGGAGATGCCACCTACGGC AAGCTCACCCTCAAGTTCATCTGCACCACCGGCAAGTTGCCGGTCCCGTGGCCGACGCTCGTCACCACCCTCACCTACGGAGTCCAGTGCTT CTCGCGCTACCCGGACCACATGAAGCAGCACGACTTCTTTAAGTCGGCCATGCCGGAGGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGG ATGACGGCAACTACAAGACCCGCGCCGAGGTCAAGTTTGAGGGCGACACCCTCGTCAACCGCATCGAGCTCAAGGGCATCGACTTCAAGGAG GACGGCAACATCCTCGGCCACAAACTCGAGTACAACTACAACTCGCACAACGTCTACATCATGGCGGACAAGCAGAAGAACGGCATCAAGGT CAACTTCAAGATCCGCCACAACATCGAGGACGGCTCGGTCCAGCTCGCCGACCACTACCAGCAGAACACCCCGATCGGAGATGGCCCCGTCC TCTTGCCGGACAACCACTACCTCTCGACCCAGTCGGCCCTCTCGAAGGACCCGAACGAGAAGCGCGACCACATGGTCCTCCTCGAATTCGTC ACCGCCGCCGGCATCACCCTCGGCATGGACGAGCTCTACAAGTAGGCGGAGCAGCGACGGACGGACCTTGTGCGTTGTGTTTGTAGGGAGGG CGAGTGGACCCGCAATGCGAGGGAAACTCTTTCTCTTCCTTCGTGCGTCCTGCCCGAGAGACTTGCGAGTCGAAGTTGACGAGGAGAGGGTT GGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGCTCGCTCGCTGCTTCGGCTGCTTCTCGACGTGCTCTAGTCGATGTTGAGTGTACTGCTACA TCCCACGAAGAAGGGAAGCTCGCACGGCCGTGAGACTGCTCTCTTCCCTCGACCGCATCGCTGGCCATCCCTCTCCACGCTTGCCGCTTCGA CTGCTTCTCAACGTGCACAATTCGCTGCTTGAACAATTGATGCTGCCGCCTACGTCGAGCGCAAGCGACCCTCACATGATTGCGAGCGAGAG TTCTCGCGTTCTCCCCAGTCCCGCCGTCGACAACGCCGCACGCATGTTGAGATCTAACGCAATTACACCTATACGATCCTTGAATAAAAGTA AGCGCACACACTGGCTTAAGATGACGCGACGACGTGTGGGGCTACCGCTCGCTCGAGCCGACGCGGTGCTGCATCGCGTCGATCGCGCTCGT CCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCGCCGGCGAGCCCGACGGCGCCCGAGGACGGCTTGGCGTCGG CGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCTCGCGACGGCGCAGAAGCTGCTTCTCTTCTGGCGCAAGCCT GCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACTGACTGTGGCTGTCACACGGGCCCTGCAGCGCAAGGTGTGGT GGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGGCGCGGCGAGTGTGGACGCTCGAGCTGAGCCTGATCTGAGCC GTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTCGCGATACCCCCCCTTTTCCCTCGCTCGTCTTGTTTCCGTA GCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGTGCGTCCAGCGAGGGCGAGAGAGAGACGGTCGACACGCGCC GCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCCCCCCTCGCCCCATCTTCCACCTCGCCCCTCTCCCTCTA GTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTGACCCGTCCACCCACTCCCGCAAGCCACGATGGCGGCCGCCA CTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGCCATCGAGGCACTGGATGGGTCCTTCACCACCGACACC GTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCCCTGACCAAGGTGTTCCCCGACGACGAATC GGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGG TCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTG ATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGTCACCAACGTTAACGCACCGGCTATCCACGCGGA TGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCG AGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAA AATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAA CAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAATACGCGA TCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGG ATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGG TCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAAC AACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATC AGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAG CAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAAT CGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAA GATCAAAGGATCTTC SEQ ID NO:35 P1 Synthetic primer GGACCATCTGAATCATGCGC SEQ ID NO:36 P4 Synthetic primer GTCGTGAGTTCGTGTCATCC SEQ ID NO:37 Primer N3 TGACCAAGGTGTTCCCCGACGA SEQ ID NO:38 UcFatB2 full-length AA sequence MVTTSLASAFFSMKAVMLAPDGSGIKPRSSGLQVRAGKEQNSCKMINGTKVKDTEGLKGRSTLHGWSMPLELITTIFSAAEKQWTNLVSKPP QLLDDHLGLHGLVFRRTFAIRCSEVGPDRSTSIVAVMNYLQEAACNHAESLGLLGDGFGETLEMSRRDLIWVVRRTHVVVERYPAWGDTVEV EAWIGAAGNIGMRRHFLVRDCKTGHILARCTSVSVMMNMRTRRLSKIPQEVRGEIDPLFIEKFAVKEGEIKKLQKFNDSTADYIQGGWTPRW NDLDVNQHVNNIKYVGWIFKSVPDSIYENHHLSSITLEYRRECTRGRALQSLTTVCGGSSEAGIICEHLLQLEDGSEVLRGRTDWRPKRTDS FEGISERFPQQEPHN SEQ ID NO:39 UcFatB2 leader truncated AA sequence MDTEGLKGRSTLHGWSMPLELITTIFSAAEKQWTNLVSKPPQLLDDHLGLHGLVFRRTFAIRCSEVGPDRSTSIVAVMNYLQEAACNHAESL GLLGDGFGETLEMSRRDLIWVVRRTHVVVERYPAWGDTVEVEAWIGAAGNIGMRRHFLVRDCKTGHILARCTSVSVMMNMRTRRLSKIPQEV RGEIDPLFIEKFAVKEGEIKKLQKFNDSTADYIQGGWTPRWNDLDVNQHVNNIKYVGWIFKSVPDSIYENHHLSSITLEYRRECTRGRALQS LTTVCGGSSEAGIICEHLLQLEDGSEVLRGRTDWRPKRTDSFEGISERFPQQEPHN SEQ ID NO:40 CcFatB2b full-length AA sequence MVTTSLASAYFSMKAVMLAPDGRGIKPRSSGLQVRAGNERNSCKVINGTKVKDTEGLKGCSTLQGQSMLDDHFGLHGLVFRRTFAIRCYEVG PDRSTSIMAVMNHLQEAARNHAESLGLLGDGFGETLEMSKRDLIWWRRTHVAVERYPAWGDTVEVEAWVGASGNTGMRRDFLVRDCKTGHIL TRCTSVSVMMNMRTRRLSKIPQEVRAEIDPLFIEKVAVKEGEIKKLQKLNDSTADYIQGGWTPRWNDLDWQHVNNIIYVGWIFKSVPDSISE NHHLSSITLEYRRECTRGNKLQSLTTVCGGSSEAGIICEHLLQLEDGSEVLRARTEWRPKHTDSFQGISERFPQQEPHK SEQ ID NO:41 CcFatB2b leader truncated AA sequence MLDDHFGLHGLVFRRTFAIRCYEVGPDRSTSIMAVMNHLQEAARNHAESLGLLGDGFGETLEMSKRDLIWWRRTHVAVERYPAWGDTVEVEA WVGASGNTGMRRDFLVRDCKTGHILTRCTSVSVMMNMRTRRLSKIPQEVRAEIDPLFIEKVAVKEGEIKKLQKLNDSTADYIQGGWTPRWND LDWQHVNNIIYVGWIFKSVPDSISENHHLSSITLEYRRECTRGNKLQSLTTVCGGSSEAGIICEHLLQLEDGSEVLRARTEWRPKHTDSFQG ISERFPQQEPHK SEQ ID NO:42 UcFatB2 full-length CDS flanked by SapI sites GCTCTTCAATGGTCACCACCTCGCTCGCCTCGGCCTTCTTTTCGATGAAGGCCGTCATGCTCGCCCCGGACGGCTCGGGCATCAAGCCGCGA TCGTCGGGCCTCCAAGTTCGAGCTGGCAAGGAGCAGAACTCGTGCAAGATGATCAACGGCACCAAGGTCAAGGACACCGAGGGCCTCAAGGG CCGCTCGACCCTCCACGGCTGGTCGATGCCGCTCGAGCTCATCACCACGATCTTCAGCGCCGCTGAGAAGCAGTGGACCAACCTCGTCAGCA AGCCGCCGCAGCTCCTCGACGACCACCTCGGCCTCCACGGACTCGTTTTCCGCCGCACCTTTGCCATCCGCTGCTCGGAAGTCGGACCTGAC CGATCGACTTCGATCGTCGCCGTCATGAACTACCTCCAGGAGGCCGCCTGCAACCACGCCGAGTCGCTCGGCCTCTTGGGAGATGGCTTCGG CGAGACTCTCGAGATGTCGCGCCGCGACCTCATCTGGGTTGTCCGACGAACCCACGTCGTCGTCGAGCGCTACCCGGCCTGGGGAGACACTG TCGAGGTCGAGGCTTGGATTGGAGCCGCCGGCAACATCGGCATGCGACGACACTTCCTCGTCCGCGACTGCAAGACCGGCCACATCCTCGCC CGCTGCACCTCGGTTTCGGTCATGATGAACATGCGCACCCGCCGCCTCTCGAAGATCCCGCAGGAGGTCCGCGGCGAGATCGACCCGTTGTT CATCGAGAAGTTCGCCGTCAAGGAGGGCGAGATCAAGAAGCTCCAGAAGTTCAACGACTCGACCGCCGACTACATCCAGGGCGGCTGGACCC CGCGCTGGAACGACTTGGACGTCAACCAGCACGTCAACAACATCAAGTACGTCGGCTGGATCTTCAAGTCGGTCCCGGACTCGATCTACGAG AACCACCACTTGTCGTCGATCACCCTCGAGTACCGCCGCGAGTGCACCCGCGGCCGCGCCCTCCAGTCGCTCACTACCGTCTGCGGAGGATC GTCTGAAGCTGGAATCATCTGCGAGCACCTCCTCCAGTTGGAGGACGGCTCCGAGGTCCTCCGCGGCCGGACCGACTGGCGACCGAAGCGCA CCGACTCGTTCGAAGGCATTTCTGAACGCTTCCCGCAGCAGGAACCTCACAACTAGTGAAGAGC SEQ ID NO:43 UcFatB2 leader truncated CDS flanked by SapI sites GCTCTTCAATGGACACCGAGGGCCTCAAGGGCCGCTCGACCCTCCACGGCTGGTCGATGCCGCTCGAGCTCATCACCACGATCTTCTCGGCC GCCGAGAAGCAGTGGACTAACCTCGTTTCGAAGCCGCCGCAGCTCCTCGACGACCACCTCGGCCTCCACGGACTCGTCTTTCGCCGCACCTT CGCCATCCGCTGCTCGGAGGTTGGACCTGATCGATCGACTTCGATCGTCGCCGTCATGAACTACCTCCAGGAGGCCGCCTGCAACCACGCCG AGTCGCTCGGCCTCTTGGGAGATGGCTTCGGCGAGACTCTCGAGATGTCGCGCCGCGACCTCATCTGGGTTGTCCGACGAACCCACGTCGTC GTCGAGCGCTACCCGGCCTGGGGAGACACTGTCGAGGTCGAGGCTTGGATTGGAGCCGCCGGCAACATCGGCATGCGACGACACTTCCTCGTCCGCGACTGCAAGACCGGCCACATCCTCGCCCGCTGCACCTCGGTTTCGGTCATGATGAACATGCGCACCCGCCGCCTCTCGAAGATCCCGC AGGAGGTCCGCGGCGAGATCGACCCGCTCTTTATCGAGAAGTTCGCCGTCAAGGAGGGCGAGATCAAGAAGCTCCAGAAGTTCAACGACTCG ACCGCCGACTACATCCAGGGCGGCTGGACCCCGCGCTGGAACGACTTGGACGTCAACCAGCACGTCAACAACATCAAGTACGTCGGCTGGAT CTTCAAGTCGGTCCCGGACTCGATCTACGAAAACCACCACCTCTCGTCGATCACCCTCGAGTACCGCCGCGAGTGCACCCGCGGCCGCGCCC TCCAGTCGCTCACTACCGTCTGCGGAGGATCTTCTGAAGCTGGAATCATCTGCGAGCACCTCCTCCAGCTCGAGGACGGCTCGGAAGTCCTC CGCGGCCGGACCGACTGGCGCCCGAAGCGCACCGACTCGTTTGAAGGCATTTCTGAACGCTTCCCGCAGCAGGAGCCTCACAACTAGTGAAG AGC SEQ ID NO:44 CcFatB2b full-length CDS flanked by SapI sites GCTCTTCAATGGTCACCACCTCGCTCGCCTCGGCCTACTTCTCGATGAAGGCGGTCATGCTCGCCCCGGACGGCCGCGGCATCAAGCCGCGA TCGTCGGGCTTACAAGTACGAGCTGGCAACGAGCGCAACTCGTGCAAGGTCATCAACGGCACCAAGGTCAAGGACACCGAGGGCCTCAAGGG CTGCTCGACCCTTCAGGGCCAGTCGATGCTCGACGACCACTTCGGCCTCCACGGCCTCGTCTTTCGCCGCACCTTCGCTATCCGGTGCTACG AGGTCGGCCCGGATCGCTCGACTTCGATCATGGCCGTCATGAACCACCTCCAGGAGGCCGCCCGCAACCACGCCGAGTCGCTCGGCTTACTC GGAGATGGCTTCGGCGAGACTCTCGAGATGTCGAAGCGCGACCTCATCTGGTGGCGCCGAACCCACGTCGCCGTCGAGCGCTACCCGGCTTG GGGAGACACCGTTGAGGTCGAGGCTTGGGTTGGTGCTTCTGGAAACACCGGCATGCGACGCGACTTCCTCGTCCGCGACTGCAAGACCGGCC ACATCCTCACCCGCTGCACCTCGGTTTCGGTCATGATGAACATGCGCACCCGCCGCCTCTCGAAGATCCCGCAGGAGGTCCGCGCCGAGATC GACCCGCTCTTTATCGAGAAGGTCGCCGTCAAGGAGGGCGAGATCAAGAAGCTCCAGAAGCTCAACGACTCGACCGCCGACTACATCCAGGG CGGCTGGACCCCGCGCTGGAACGACCTCGACTGGCAGCACGTCAACAACATCATCTACGTCGGCTGGATCTTCAAGTCGGTCCCGGACTCGA TCTCCGAGAACCATCACCTCTCGTCGATCACCCTCGAGTACCGCCGCGAGTGCACCCGCGGCAACAAGCTCCAGTCGCTCACCACCGTCTGC GGCGGATCGTCGGAGGCTGGAATCATCTGCGAGCACCTCCTCCAGTTGGAGGACGGCTCGGAGGTCCTCCGCGCTCGAACCGAATGGCGACC GAAGCACACCGACTCGTTCCAGGGCATCTCGGAGCGCTTCCCGCAGCAGGAACCTCACAAGTAGTGAAGAGC SEQ ID NO:45 CcFatB2b leader truncated CDS flanked by SapI sites GCTCTTCAATGCTCGACGACCACTTCGGCCTCCACGGCCTCGTCTTTCGCCGCACCTTCGCCATCCGGTGCTACGAGGTCGGCCCGGACCGC TCGACCTCGATTATGGCTGTCATGAACCACCTCCAGGAGGCCGCCCGCAACCATGCCGAGTCGCTCGGCTTGCTCGGAGATGGCTTCGGCGA GACTCTCGAGATGTCGAAGCGCGACCTCATCTGGTGGCGCCGAACCCACGTCGCCGTCGAGCGCTACCCGGCTTGGGGAGACACCGTTGAGG TCGAGGCTTGGGTTGGAGCTTCTGGAAACACCGGCATGCGACGCGACTTCCTCGTCCGCGACTGCAAGACCGGCCACATCCTCACCCGCTGC ACCTCGGTTTCGGTCATGATGAACATGCGCACCCGCCGCCTCTCGAAGATCCCGCAGGAGGTCCGCGCCGAGATCGACCCGTTGTTCATCGA GAAGGTCGCCGTCAAGGAGGGCGAGATCAAGAAGTTGCAGAAGCTCAACGACTCGACCGCCGACTACATCCAGGGCGGCTGGACCCCGCGCT GGAACGACTTGGACTGGCAGCACGTCAACAACATCATCTACGTCGGCTGGATCTTCAAGTCGGTCCCGGACTCGATCTCCGAGAACCACCAT CTCTCGTCGATCACCCTCGAGTACCGCCGCGAGTGCACCCGCGGCAACAAGCTCCAGTCGCTCACCACCGTCTGCGGCGGATCGTCGGAGGC TGGAATCATCTGCGAGCACCTCCTCCAGTTGGAGGACGGCTCGGAGGTCCTCCGCGCTCGAACCGAATGGCGACCGAAGCACACCGACTCGT TCCAGGGCATCTCGGAGCGCTTCCCGCAGCAGGAACCTCACAAGTAGTGAAGAGC SEQ ID NO:46 Strain 2 CAR2 L Str2_pTEF1>UcFatB2 full-length plasmid TTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGCCTGAACT GGCCGATAATTGCAGACGACCCTCTCCGCCCCGCGCAGCTCTCGCTCTCGGTCCACCTCCCCACCGGGTCCGACGTCGTCTTCGACCTCGAC CCGCTCGCGCACCCAGTCGACCCGGCAGCGTCCTCGTTCCGCGTCCTGCAGCCCAAGATTGAGCTGAAGCTCAAGAAGCGCGACGGCGGCGT AAAGTGGAGCAAGATCGAGGGCGAGGATGAGGGTGTTGGCTCGTTCGGTGCGTCCATATGCCTTTTCTCAAGCTTCGAGGCGGGCTGTCGTG CCCCTTGTCTGCGTCTTCAAGAGCTGCGCGTGACGCGGGAAGTCGTCGCTCGCAGGTGCCGCGGCCGAAAAGGCTGCAACCCACGCGTACCC GTCCTCGTCGCGCAAGCGCCACGACTGGGAGAAGATCGTCAAGGACTCTGCCGAGGAGGATGAGCAGCTCCAGAAGGAGTTCTCCAAGGACC CGAACGCCGGCGGCGACAAGGCGCTCAATGAGCTCTTTCAGAAGCTCTACGCCGACGCGACCGACGACCAGCGCCGTGCCATGATCAAGAGC TACCAGGAGAGCAACGGCACGGCGCTCAGCACCGATTGGTCCGACGTCAGCAAGGTCTGTCCTTCTCTCGCTCTGGCCAACATACGGGACTG ACCTGCAACGTCTTCTACCTCGCAGAAAAAGGTCGAGACGCGCCCGCCCGACTCGATGCTCGCCAAGAAGTGGGAGCAGTAGTCCTTGCGCG CCCGGCCTGTCATTCTGCCGTGTCTAGCAGACGGCGGCGTTCCGTCTGTGGTTTCTCTCGTGCTTGTTCTCGCACTGTCGCAACTTTACCCT CGCCTTCGTTCCTTTGCGCTCCCTCCTTTTCAGTGTAGCAATGCATACCTCTTCGTCGTTCGCCTCCTGCAAGCGGCCTCGTGAACAGAGTT TCGCACCGAGGGGTGTCTACAGGGAAAGGAGCTATGCAAGTCTAGGTACAGCGCATTTGACAGAGTCTTTTGTAGCGAGAAACTCGACAACT GCCTCTCCAAACCAGATGTCAACACAGCTACACTGCGAGAAGAAGCGACTGCATTCGTCGAGGAAGGTGATTCGTACCGAGCTTTGGCATCC GCCGATAGAGTCAACGTTCGTCAGTCTTACAGTGCGGCGAGGCGGTGCACAAAAGCCGGCCAGCGCCGGCAGAACCCGACTTTCGACGCCGC GAGGGCAACCTTCCCAAGCAGAGGGCGCGCACGCAAGTGCACAGAGTACCGCAGGGCATGCAGAGAGCATGCCAGACTTTGCTGCAGCGCGA GTACGCTTGCCAGCATGCATCACCGGGCGATGCAGCGGGATACTCATAGGGAAGACGCGCGACGGGGCAGCAAGCGATTTGGCGGGTCTAGA GACTAAGCGAGTCCGATCCCCTTGGATCGGGATATTACGGGTGCACGAGGAGAACCCGCGTTCTAGCGTTGTGCCGCCTTGAGGGCGCCACC CAAAGCACGCACGCAGATGGCCCTGGTCTGCGGCTCCCCAGCGCTGGTTTCGATGGCTGGCGAGAGGGTATTTCCGGTTGCTCGTATTGACA GTCTCGTCTATGTGGGTAACGAGCGGCGTCGAGGCGTCGAGGCAGAAAAGGGAGCGAAGGCAGGAAACGAGCACCAGTCGACGCCAGCCTCC TTGAGCGTCGCCGCTCCCAGCAGTTCAGAGGCAGCGGAAGCCAGCAGGAGTGAGTGCAAGAGCCATGCAGAGAGGTGGGCAACATGAAGAAG TGCCGTGAGGTGGTGAGGTGGTGAGGTGGTGAGGTGAGGCGGTCGGTGAGGGAGCAGAGCAGGAGAGGAGGAGGGGGCAAGGAGACTAAGCG ATGAAACCCGGGGTCGGGGCGCAGGCTGGGAGTGATCGAGGTGAGCTTGCAGGGGCAGTGGCCCCCGTGGCAGTCGCAGCGAGGCGCAGGGC GCGCGCGGCAAGTCGGCCCAAGCTCGACGTGCGCGCGTGCCCGCCCCCGGCGCCGTGCTCGCCGCCTCTTCTCTTCTCTCCACCACTCGGTC CTCCAACTACTCAAACCTCGCACACCATGGTCACCACCTCGCTCGCCTCGGCCTTCTTTTCGATGAAGGCCGTCATGCTCGCCCCGGACGGC TCGGGCATCAAGCCGCGATCGTCGGGCCTCCAAGTTCGAGCTGGCAAGGAGCAGAACTCGTGCAAGATGATCAACGGCACCAAGGTCAAGGA CACCGAGGGCCTCAAGGGCCGCTCGACCCTCCACGGCTGGTCGATGCCGCTCGAGCTCATCACCACGATCTTCAGCGCCGCTGAGAAGCAGT GGACCAACCTCGTCAGCAAGCCGCCGCAGCTCCTCGACGACCACCTCGGCCTCCACGGACTCGTTTTCCGCCGCACCTTTGCCATCCGCTGC TCGGAAGTCGGACCTGACCGATCGACTTCGATCGTCGCCGTCATGAACTACCTCCAGGAGGCCGCCTGCAACCACGCCGAGTCGCTCGGCCT CTTGGGAGATGGCTTCGGCGAGACTCTCGAGATGTCGCGCCGCGACCTCATCTGGGTTGTCCGACGAACCCACGTCGTCGTCGAGCGCTACC CGGCCTGGGGAGACACTGTCGAGGTCGAGGCTTGGATTGGAGCCGCCGGCAACATCGGCATGCGACGACACTTCCTCGTCCGCGACTGCAAG ACCGGCCACATCCTCGCCCGCTGCACCTCGGTTTCGGTCATGATGAACATGCGCACCCGCCGCCTCTCGAAGATCCCGCAGGAGGTCCGCGG CGAGATCGACCCGTTGTTCATCGAGAAGTTCGCCGTCAAGGAGGGCGAGATCAAGAAGCTCCAGAAGTTCAACGACTCGACCGCCGACTACATCCAGGGCGGCTGGACCCCGCGCTGGAACGACTTGGACGTCAACCAGCACGTCAACAACATCAAGTACGTCGGCTGGATCTTCAAGTCGGTC CCGGACTCGATCTACGAGAACCACCACTTGTCGTCGATCACCCTCGAGTACCGCCGCGAGTGCACCCGCGGCCGCGCCCTCCAGTCGCTCAC TACCGTCTGCGGAGGATCGTCTGAAGCTGGAATCATCTGCGAGCACCTCCTCCAGTTGGAGGACGGCTCCGAGGTCCTCCGCGGCCGGACCG ACTGGCGACCGAAGCGCACCGACTCGTTCGAAGGCATTTCTGAACGCTTCCCGCAGCAGGAACCTCACAACTAGGCGGAGCAGCGACGGACG GACCTTGTGCGTTGTGTTTGTAGGGAGGGCGAGTGGACCCGCAATGCGAGGGAAACTCTTTCTCTTCCTTCGTGCGTCCTGCCCGAGAGACT TGCGAGTCGAAGTTGACGAGGAGAGGGTTGGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGCTCGCTCGCTGCTTCGGCTGCTTCTCGACGTG CTCTAGTCGATGTTGAGTGTACTGCTACATCCCACGAAGAAGGGAAGCTCGCACGGCCGTGAGACTGCTCTCTTCCCTCGACCGCATCGCTG GCCATCCCTCTCCACGCTTGCCGCTTCGACTGCTTCTCAACGTGCACAATTCGCTGCTTGAACAATTGATGCTGCCGCCTACGTCGAGCGCA AGCGACCCTCACATGATTGCGAGCGAGAGTTCTCGCGTTCTCCCCAGTCCCGCCGTCGACAACGCCGCACGCATGTTGAGATCTAACGCAAT TACACCTATACGATCCTTGAATAAAAGTAAGCGCACACACTGGCTTAAGATGACGCGACGACGTGTGGGGCTACCGCTCGCTCGAGCCGACG CGGTGCTGCATCGCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCGCCGGCGAGCCC GACGGCGCCCGAGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCTCGCGACGGCGC AGAAGCTGCTTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACTGACTGTGGCTGT CACACGGGCCCTGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGGCGCGGCGAGTGT GGACGCTCGAGCTGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTCGCGATACCCCC CCTTTTCCCTCGCTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGTGCGTCCAGCGA GGGCGAGAGAGAGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCCCCCCTCGCC CCATCTTCCACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTGACCCGTCCACCC ACTCCCGCAAGCCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGCCATCGAGG CACTGGATGGGTCCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCC CTGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTCGTCGCGTACGG GGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCCCCGGAGC ACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGTCACC AACGTTAACGCACCGGCTATCCACGCGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATT AACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCC GTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCA ATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAG CTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTG ATTGCGCCTGAGCGAGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGC GCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATC ATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGG CAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTA TCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCAT AACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTT GAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCG TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC SEQ ID NO:47 Strain 2 CAR2 L Str2_pTEF1>UcFatB2 leader truncated plasmid TTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGCCTGAACT GGCCGATAATTGCAGACGACCCTCTCCGCCCCGCGCAGCTCTCGCTCTCGGTCCACCTCCCCACCGGGTCCGACGTCGTCTTCGACCTCGAC CCGCTCGCGCACCCAGTCGACCCGGCAGCGTCCTCGTTCCGCGTCCTGCAGCCCAAGATTGAGCTGAAGCTCAAGAAGCGCGACGGCGGCGT AAAGTGGAGCAAGATCGAGGGCGAGGATGAGGGTGTTGGCTCGTTCGGTGCGTCCATATGCCTTTTCTCAAGCTTCGAGGCGGGCTGTCGTG CCCCTTGTCTGCGTCTTCAAGAGCTGCGCGTGACGCGGGAAGTCGTCGCTCGCAGGTGCCGCGGCCGAAAAGGCTGCAACCCACGCGTACCC GTCCTCGTCGCGCAAGCGCCACGACTGGGAGAAGATCGTCAAGGACTCTGCCGAGGAGGATGAGCAGCTCCAGAAGGAGTTCTCCAAGGACC CGAACGCCGGCGGCGACAAGGCGCTCAATGAGCTCTTTCAGAAGCTCTACGCCGACGCGACCGACGACCAGCGCCGTGCCATGATCAAGAGC TACCAGGAGAGCAACGGCACGGCGCTCAGCACCGATTGGTCCGACGTCAGCAAGGTCTGTCCTTCTCTCGCTCTGGCCAACATACGGGACTG ACCTGCAACGTCTTCTACCTCGCAGAAAAAGGTCGAGACGCGCCCGCCCGACTCGATGCTCGCCAAGAAGTGGGAGCAGTAGTCCTTGCGCG CCCGGCCTGTCATTCTGCCGTGTCTAGCAGACGGCGGCGTTCCGTCTGTGGTTTCTCTCGTGCTTGTTCTCGCACTGTCGCAACTTTACCCT CGCCTTCGTTCCTTTGCGCTCCCTCCTTTTCAGTGTAGCAATGCATACCTCTTCGTCGTTCGCCTCCTGCAAGCGGCCTCGTGAACAGAGTT TCGCACCGAGGGGTGTCTACAGGGAAAGGAGCTATGCAAGTCTAGGTACAGCGCATTTGACAGAGTCTTTTGTAGCGAGAAACTCGACAACT GCCTCTCCAAACCAGATGTCAACACAGCTACACTGCGAGAAGAAGCGACTGCATTCGTCGAGGAAGGTGATTCGTACCGAGCTTTGGCATCC GCCGATAGAGTCAACGTTCGTCAGTCTTACAGTGCGGCGAGGCGGTGCACAAAAGCCGGCCAGCGCCGGCAGAACCCGACTTTCGACGCCGC GAGGGCAACCTTCCCAAGCAGAGGGCGCGCACGCAAGTGCACAGAGTACCGCAGGGCATGCAGAGAGCATGCCAGACTTTGCTGCAGCGCGA GTACGCTTGCCAGCATGCATCACCGGGCGATGCAGCGGGATACTCATAGGGAAGACGCGCGACGGGGCAGCAAGCGATTTGGCGGGTCTAGA GACTAAGCGAGTCCGATCCCCTTGGATCGGGATATTACGGGTGCACGAGGAGAACCCGCGTTCTAGCGTTGTGCCGCCTTGAGGGCGCCACC CAAAGCACGCACGCAGATGGCCCTGGTCTGCGGCTCCCCAGCGCTGGTTTCGATGGCTGGCGAGAGGGTATTTCCGGTTGCTCGTATTGACA GTCTCGTCTATGTGGGTAACGAGCGGCGTCGAGGCGTCGAGGCAGAAAAGGGAGCGAAGGCAGGAAACGAGCACCAGTCGACGCCAGCCTCC TTGAGCGTCGCCGCTCCCAGCAGTTCAGAGGCAGCGGAAGCCAGCAGGAGTGAGTGCAAGAGCCATGCAGAGAGGTGGGCAACATGAAGAAG TGCCGTGAGGTGGTGAGGTGGTGAGGTGGTGAGGTGAGGCGGTCGGTGAGGGAGCAGAGCAGGAGAGGAGGAGGGGGCAAGGAGACTAAGCG ATGAAACCCGGGGTCGGGGCGCAGGCTGGGAGTGATCGAGGTGAGCTTGCAGGGGCAGTGGCCCCCGTGGCAGTCGCAGCGAGGCGCAGGGC GCGCGCGGCAAGTCGGCCCAAGCTCGACGTGCGCGCGTGCCCGCCCCCGGCGCCGTGCTCGCCGCCTCTTCTCTTCTCTCCACCACTCGGTC CTCCAACTACTCAAACCTCGCACACCATGGACACCGAGGGCCTCAAGGGCCGCTCGACCCTCCACGGCTGGTCGATGCCGCTCGAGCTCATC ACCACGATCTTCTCGGCCGCCGAGAAGCAGTGGACTAACCTCGTTTCGAAGCCGCCGCAGCTCCTCGACGACCACCTCGGCCTCCACGGACT CGTCTTTCGCCGCACCTTCGCCATCCGCTGCTCGGAGGTTGGACCTGATCGATCGACTTCGATCGTCGCCGTCATGAACTACCTCCAGGAGG CCGCCTGCAACCACGCCGAGTCGCTCGGCCTCTTGGGAGATGGCTTCGGCGAGACTCTCGAGATGTCGCGCCGCGACCTCATCTGGGTTGTC CGACGAACCCACGTCGTCGTCGAGCGCTACCCGGCCTGGGGAGACACTGTCGAGGTCGAGGCTTGGATTGGAGCCGCCGGCAACATCGGCAT GCGACGACACTTCCTCGTCCGCGACTGCAAGACCGGCCACATCCTCGCCCGCTGCACCTCGGTTTCGGTCATGATGAACATGCGCACCCGCC GCCTCTCGAAGATCCCGCAGGAGGTCCGCGGCGAGATCGACCCGCTCTTTATCGAGAAGTTCGCCGTCAAGGAGGGCGAGATCAAGAAGCTC CAGAAGTTCAACGACTCGACCGCCGACTACATCCAGGGCGGCTGGACCCCGCGCTGGAACGACTTGGACGTCAACCAGCACGTCAACAACATCAAGTACGTCGGCTGGATCTTCAAGTCGGTCCCGGACTCGATCTACGAAAACCACCACCTCTCGTCGATCACCCTCGAGTACCGCCGCGAGT GCACCCGCGGCCGCGCCCTCCAGTCGCTCACTACCGTCTGCGGAGGATCTTCTGAAGCTGGAATCATCTGCGAGCACCTCCTCCAGCTCGAG GACGGCTCGGAAGTCCTCCGCGGCCGGACCGACTGGCGCCCGAAGCGCACCGACTCGTTTGAAGGCATTTCTGAACGCTTCCCGCAGCAGGA GCCTCACAACTAGGCGGAGCAGCGACGGACGGACCTTGTGCGTTGTGTTTGTAGGGAGGGCGAGTGGACCCGCAATGCGAGGGAAACTCTTT CTCTTCCTTCGTGCGTCCTGCCCGAGAGACTTGCGAGTCGAAGTTGACGAGGAGAGGGTTGGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGC TCGCTCGCTGCTTCGGCTGCTTCTCGACGTGCTCTAGTCGATGTTGAGTGTACTGCTACATCCCACGAAGAAGGGAAGCTCGCACGGCCGTG AGACTGCTCTCTTCCCTCGACCGCATCGCTGGCCATCCCTCTCCACGCTTGCCGCTTCGACTGCTTCTCAACGTGCACAATTCGCTGCTTGA ACAATTGATGCTGCCGCCTACGTCGAGCGCAAGCGACCCTCACATGATTGCGAGCGAGAGTTCTCGCGTTCTCCCCAGTCCCGCCGTCGACA ACGCCGCACGCATGTTGAGATCTAACGCAATTACACCTATACGATCCTTGAATAAAAGTAAGCGCACACACTGGCTTAAGATGACGCGACGA CGTGTGGGGCTACCGCTCGCTCGAGCCGACGCGGTGCTGCATCGCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGA TCGGCGGCAGCGCGTCGGCGCCGGCGAGCCCGACGGCGCCCGAGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTC CAGGTCAACACTCAGCAGCTCGCGACGGCGCAGAAGCTGCTTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCC TGTGTCGAGCGCAGTGACTGACTGTGGCTGTCACACGGGCCCTGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGC AAGGAGGTCAAGATCTGGGCGCGGCGAGTGTGGACGCTCGAGCTGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGC GGCGTGCAGATTCCCCGGTCGCGATACCCCCCCTTTTCCCTCGCTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCG TCGTATCCAGCAGTTCGAGTGCGTCCAGCGAGGGCGAGAGAGAGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGAC GAGAGCGAGGCGCTCCTCCTCCCCCCTCGCCCCATCTTCCACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACG CCAAGCTAGACCCCGACTGACCCGTCCACCCACTCCCGCAAGCCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACC AGTGTCCCGGGGGACGCCGAGGCCATCGAGGCACTGGATGGGTCCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTT CACCCTGCGGGAGGTGCCGGTGGACCCGCCCCTGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCG ACCCGGACTCCCGGACGTTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTG ACCGTCGAGGACATCGAGGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCG GGGCGCCGGGCACCTCTGGCTGGAGGTCACCAACGTTAACGCACCGGCTATCCACGCGGATGACACGAACTCACGACTAACTATAACGGTCC TAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCA GGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTA TCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAG TGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCA CTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAAT CGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCC CGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTT AGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCG ATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGG AGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTT TTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGAT CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC SEQ ID NO:48 Strain 2 CAR2 L Str2_pTEF1>CcFatB2b full-length plasmid TTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGCCTGAACT GGCCGATAATTGCAGACGACCCTCTCCGCCCCGCGCAGCTCTCGCTCTCGGTCCACCTCCCCACCGGGTCCGACGTCGTCTTCGACCTCGAC CCGCTCGCGCACCCAGTCGACCCGGCAGCGTCCTCGTTCCGCGTCCTGCAGCCCAAGATTGAGCTGAAGCTCAAGAAGCGCGACGGCGGCGT AAAGTGGAGCAAGATCGAGGGCGAGGATGAGGGTGTTGGCTCGTTCGGTGCGTCCATATGCCTTTTCTCAAGCTTCGAGGCGGGCTGTCGTG CCCCTTGTCTGCGTCTTCAAGAGCTGCGCGTGACGCGGGAAGTCGTCGCTCGCAGGTGCCGCGGCCGAAAAGGCTGCAACCCACGCGTACCC GTCCTCGTCGCGCAAGCGCCACGACTGGGAGAAGATCGTCAAGGACTCTGCCGAGGAGGATGAGCAGCTCCAGAAGGAGTTCTCCAAGGACC CGAACGCCGGCGGCGACAAGGCGCTCAATGAGCTCTTTCAGAAGCTCTACGCCGACGCGACCGACGACCAGCGCCGTGCCATGATCAAGAGC TACCAGGAGAGCAACGGCACGGCGCTCAGCACCGATTGGTCCGACGTCAGCAAGGTCTGTCCTTCTCTCGCTCTGGCCAACATACGGGACTG ACCTGCAACGTCTTCTACCTCGCAGAAAAAGGTCGAGACGCGCCCGCCCGACTCGATGCTCGCCAAGAAGTGGGAGCAGTAGTCCTTGCGCG CCCGGCCTGTCATTCTGCCGTGTCTAGCAGACGGCGGCGTTCCGTCTGTGGTTTCTCTCGTGCTTGTTCTCGCACTGTCGCAACTTTACCCT CGCCTTCGTTCCTTTGCGCTCCCTCCTTTTCAGTGTAGCAATGCATACCTCTTCGTCGTTCGCCTCCTGCAAGCGGCCTCGTGAACAGAGTT TCGCACCGAGGGGTGTCTACAGGGAAAGGAGCTATGCAAGTCTAGGTACAGCGCATTTGACAGAGTCTTTTGTAGCGAGAAACTCGACAACT GCCTCTCCAAACCAGATGTCAACACAGCTACACTGCGAGAAGAAGCGACTGCATTCGTCGAGGAAGGTGATTCGTACCGAGCTTTGGCATCC GCCGATAGAGTCAACGTTCGTCAGTCTTACAGTGCGGCGAGGCGGTGCACAAAAGCCGGCCAGCGCCGGCAGAACCCGACTTTCGACGCCGC GAGGGCAACCTTCCCAAGCAGAGGGCGCGCACGCAAGTGCACAGAGTACCGCAGGGCATGCAGAGAGCATGCCAGACTTTGCTGCAGCGCGA GTACGCTTGCCAGCATGCATCACCGGGCGATGCAGCGGGATACTCATAGGGAAGACGCGCGACGGGGCAGCAAGCGATTTGGCGGGTCTAGA GACTAAGCGAGTCCGATCCCCTTGGATCGGGATATTACGGGTGCACGAGGAGAACCCGCGTTCTAGCGTTGTGCCGCCTTGAGGGCGCCACC CAAAGCACGCACGCAGATGGCCCTGGTCTGCGGCTCCCCAGCGCTGGTTTCGATGGCTGGCGAGAGGGTATTTCCGGTTGCTCGTATTGACA GTCTCGTCTATGTGGGTAACGAGCGGCGTCGAGGCGTCGAGGCAGAAAAGGGAGCGAAGGCAGGAAACGAGCACCAGTCGACGCCAGCCTCC TTGAGCGTCGCCGCTCCCAGCAGTTCAGAGGCAGCGGAAGCCAGCAGGAGTGAGTGCAAGAGCCATGCAGAGAGGTGGGCAACATGAAGAAG TGCCGTGAGGTGGTGAGGTGGTGAGGTGGTGAGGTGAGGCGGTCGGTGAGGGAGCAGAGCAGGAGAGGAGGAGGGGGCAAGGAGACTAAGCG ATGAAACCCGGGGTCGGGGCGCAGGCTGGGAGTGATCGAGGTGAGCTTGCAGGGGCAGTGGCCCCCGTGGCAGTCGCAGCGAGGCGCAGGGC GCGCGCGGCAAGTCGGCCCAAGCTCGACGTGCGCGCGTGCCCGCCCCCGGCGCCGTGCTCGCCGCCTCTTCTCTTCTCTCCACCACTCGGTC CTCCAACTACTCAAACCTCGCACACCATGGTCACCACCTCGCTCGCCTCGGCCTACTTCTCGATGAAGGCGGTCATGCTCGCCCCGGACGGC CGCGGCATCAAGCCGCGATCGTCGGGCTTACAAGTACGAGCTGGCAACGAGCGCAACTCGTGCAAGGTCATCAACGGCACCAAGGTCAAGGA CACCGAGGGCCTCAAGGGCTGCTCGACCCTTCAGGGCCAGTCGATGCTCGACGACCACTTCGGCCTCCACGGCCTCGTCTTTCGCCGCACCT TCGCTATCCGGTGCTACGAGGTCGGCCCGGATCGCTCGACTTCGATCATGGCCGTCATGAACCACCTCCAGGAGGCCGCCCGCAACCACGCC GAGTCGCTCGGCTTACTCGGAGATGGCTTCGGCGAGACTCTCGAGATGTCGAAGCGCGACCTCATCTGGTGGCGCCGAACCCACGTCGCCGT CGAGCGCTACCCGGCTTGGGGAGACACCGTTGAGGTCGAGGCTTGGGTTGGTGCTTCTGGAAACACCGGCATGCGACGCGACTTCCTCGTCC GCGACTGCAAGACCGGCCACATCCTCACCCGCTGCACCTCGGTTTCGGTCATGATGAACATGCGCACCCGCCGCCTCTCGAAGATCCCGCAG GAGGTCCGCGCCGAGATCGACCCGCTCTTTATCGAGAAGGTCGCCGTCAAGGAGGGCGAGATCAAGAAGCTCCAGAAGCTCAACGACTCGAC CGCCGACTACATCCAGGGCGGCTGGACCCCGCGCTGGAACGACCTCGACTGGCAGCACGTCAACAACATCATCTACGTCGGCTGGATCTTCAAGTCGGTCCCGGACTCGATCTCCGAGAACCATCACCTCTCGTCGATCACCCTCGAGTACCGCCGCGAGTGCACCCGCGGCAACAAGCTCCAG TCGCTCACCACCGTCTGCGGCGGATCGTCGGAGGCTGGAATCATCTGCGAGCACCTCCTCCAGTTGGAGGACGGCTCGGAGGTCCTCCGCGC TCGAACCGAATGGCGACCGAAGCACACCGACTCGTTCCAGGGCATCTCGGAGCGCTTCCCGCAGCAGGAACCTCACAAGTAGGCGGAGCAGC GACGGACGGACCTTGTGCGTTGTGTTTGTAGGGAGGGCGAGTGGACCCGCAATGCGAGGGAAACTCTTTCTCTTCCTTCGTGCGTCCTGCCC GAGAGACTTGCGAGTCGAAGTTGACGAGGAGAGGGTTGGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGCTCGCTCGCTGCTTCGGCTGCTTC TCGACGTGCTCTAGTCGATGTTGAGTGTACTGCTACATCCCACGAAGAAGGGAAGCTCGCACGGCCGTGAGACTGCTCTCTTCCCTCGACCG CATCGCTGGCCATCCCTCTCCACGCTTGCCGCTTCGACTGCTTCTCAACGTGCACAATTCGCTGCTTGAACAATTGATGCTGCCGCCTACGT CGAGCGCAAGCGACCCTCACATGATTGCGAGCGAGAGTTCTCGCGTTCTCCCCAGTCCCGCCGTCGACAACGCCGCACGCATGTTGAGATCT AACGCAATTACACCTATACGATCCTTGAATAAAAGTAAGCGCACACACTGGCTTAAGATGACGCGACGACGTGTGGGGCTACCGCTCGCTCG AGCCGACGCGGTGCTGCATCGCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCGCCG GCGAGCCCGACGGCGCCCGAGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCTCGC GACGGCGCAGAAGCTGCTTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACTGACT GTGGCTGTCACACGGGCCCTGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGGCGCG GCGAGTGTGGACGCTCGAGCTGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTCGCG ATACCCCCCCTTTTCCCTCGCTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGTGCG TCCAGCGAGGGCGAGAGAGAGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCCC CCCTCGCCCCATCTTCCACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTGACCC GTCCACCCACTCCCGCAAGCCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGC CATCGAGGCACTGGATGGGTCCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGG ACCCGCCCCTGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTCGTC GCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGC CCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGG AGGTCACCAACGTTAACGCACCGGCTATCCACGCGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTAC AACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTG AAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTC CAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAAT GGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATT CATTCGTGATTGCGCCTGAGCGAGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACA CTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAAC CATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAAC ATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCC CGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATA TGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCA GAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTG AGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC SEQ ID NO:49 Strain 2 CAR2 L Str2_pTEF1>CcFatB2b leader truncated plasmid TTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACCATCTGAATCATGCGCCTGAACT GGCCGATAATTGCAGACGACCCTCTCCGCCCCGCGCAGCTCTCGCTCTCGGTCCACCTCCCCACCGGGTCCGACGTCGTCTTCGACCTCGAC CCGCTCGCGCACCCAGTCGACCCGGCAGCGTCCTCGTTCCGCGTCCTGCAGCCCAAGATTGAGCTGAAGCTCAAGAAGCGCGACGGCGGCGT AAAGTGGAGCAAGATCGAGGGCGAGGATGAGGGTGTTGGCTCGTTCGGTGCGTCCATATGCCTTTTCTCAAGCTTCGAGGCGGGCTGTCGTG CCCCTTGTCTGCGTCTTCAAGAGCTGCGCGTGACGCGGGAAGTCGTCGCTCGCAGGTGCCGCGGCCGAAAAGGCTGCAACCCACGCGTACCC GTCCTCGTCGCGCAAGCGCCACGACTGGGAGAAGATCGTCAAGGACTCTGCCGAGGAGGATGAGCAGCTCCAGAAGGAGTTCTCCAAGGACC CGAACGCCGGCGGCGACAAGGCGCTCAATGAGCTCTTTCAGAAGCTCTACGCCGACGCGACCGACGACCAGCGCCGTGCCATGATCAAGAGC TACCAGGAGAGCAACGGCACGGCGCTCAGCACCGATTGGTCCGACGTCAGCAAGGTCTGTCCTTCTCTCGCTCTGGCCAACATACGGGACTG ACCTGCAACGTCTTCTACCTCGCAGAAAAAGGTCGAGACGCGCCCGCCCGACTCGATGCTCGCCAAGAAGTGGGAGCAGTAGTCCTTGCGCG CCCGGCCTGTCATTCTGCCGTGTCTAGCAGACGGCGGCGTTCCGTCTGTGGTTTCTCTCGTGCTTGTTCTCGCACTGTCGCAACTTTACCCT CGCCTTCGTTCCTTTGCGCTCCCTCCTTTTCAGTGTAGCAATGCATACCTCTTCGTCGTTCGCCTCCTGCAAGCGGCCTCGTGAACAGAGTT TCGCACCGAGGGGTGTCTACAGGGAAAGGAGCTATGCAAGTCTAGGTACAGCGCATTTGACAGAGTCTTTTGTAGCGAGAAACTCGACAACT GCCTCTCCAAACCAGATGTCAACACAGCTACACTGCGAGAAGAAGCGACTGCATTCGTCGAGGAAGGTGATTCGTACCGAGCTTTGGCATCC GCCGATAGAGTCAACGTTCGTCAGTCTTACAGTGCGGCGAGGCGGTGCACAAAAGCCGGCCAGCGCCGGCAGAACCCGACTTTCGACGCCGC GAGGGCAACCTTCCCAAGCAGAGGGCGCGCACGCAAGTGCACAGAGTACCGCAGGGCATGCAGAGAGCATGCCAGACTTTGCTGCAGCGCGA GTACGCTTGCCAGCATGCATCACCGGGCGATGCAGCGGGATACTCATAGGGAAGACGCGCGACGGGGCAGCAAGCGATTTGGCGGGTCTAGA GACTAAGCGAGTCCGATCCCCTTGGATCGGGATATTACGGGTGCACGAGGAGAACCCGCGTTCTAGCGTTGTGCCGCCTTGAGGGCGCCACC CAAAGCACGCACGCAGATGGCCCTGGTCTGCGGCTCCCCAGCGCTGGTTTCGATGGCTGGCGAGAGGGTATTTCCGGTTGCTCGTATTGACA GTCTCGTCTATGTGGGTAACGAGCGGCGTCGAGGCGTCGAGGCAGAAAAGGGAGCGAAGGCAGGAAACGAGCACCAGTCGACGCCAGCCTCC TTGAGCGTCGCCGCTCCCAGCAGTTCAGAGGCAGCGGAAGCCAGCAGGAGTGAGTGCAAGAGCCATGCAGAGAGGTGGGCAACATGAAGAAG TGCCGTGAGGTGGTGAGGTGGTGAGGTGGTGAGGTGAGGCGGTCGGTGAGGGAGCAGAGCAGGAGAGGAGGAGGGGGCAAGGAGACTAAGCG ATGAAACCCGGGGTCGGGGCGCAGGCTGGGAGTGATCGAGGTGAGCTTGCAGGGGCAGTGGCCCCCGTGGCAGTCGCAGCGAGGCGCAGGGC GCGCGCGGCAAGTCGGCCCAAGCTCGACGTGCGCGCGTGCCCGCCCCCGGCGCCGTGCTCGCCGCCTCTTCTCTTCTCTCCACCACTCGGTC CTCCAACTACTCAAACCTCGCACACCATGCTCGACGACCACTTCGGCCTCCACGGCCTCGTCTTTCGCCGCACCTTCGCCATCCGGTGCTAC GAGGTCGGCCCGGACCGCTCGACCTCGATTATGGCTGTCATGAACCACCTCCAGGAGGCCGCCCGCAACCATGCCGAGTCGCTCGGCTTGCT CGGAGATGGCTTCGGCGAGACTCTCGAGATGTCGAAGCGCGACCTCATCTGGTGGCGCCGAACCCACGTCGCCGTCGAGCGCTACCCGGCTT GGGGAGACACCGTTGAGGTCGAGGCTTGGGTTGGAGCTTCTGGAAACACCGGCATGCGACGCGACTTCCTCGTCCGCGACTGCAAGACCGGC CACATCCTCACCCGCTGCACCTCGGTTTCGGTCATGATGAACATGCGCACCCGCCGCCTCTCGAAGATCCCGCAGGAGGTCCGCGCCGAGAT CGACCCGTTGTTCATCGAGAAGGTCGCCGTCAAGGAGGGCGAGATCAAGAAGTTGCAGAAGCTCAACGACTCGACCGCCGACTACATCCAGG GCGGCTGGACCCCGCGCTGGAACGACTTGGACTGGCAGCACGTCAACAACATCATCTACGTCGGCTGGATCTTCAAGTCGGTCCCGGACTCG ATCTCCGAGAACCACCATCTCTCGTCGATCACCCTCGAGTACCGCCGCGAGTGCACCCGCGGCAACAAGCTCCAGTCGCTCACCACCGTCTG CGGCGGATCGTCGGAGGCTGGAATCATCTGCGAGCACCTCCTCCAGTTGGAGGACGGCTCGGAGGTCCTCCGCGCTCGAACCGAATGGCGACCGAAGCACACCGACTCGTTCCAGGGCATCTCGGAGCGCTTCCCGCAGCAGGAACCTCACAAGTAGGCGGAGCAGCGACGGACGGACCTTGTG CGTTGTGTTTGTAGGGAGGGCGAGTGGACCCGCAATGCGAGGGAAACTCTTTCTCTTCCTTCGTGCGTCCTGCCCGAGAGACTTGCGAGTCG AAGTTGACGAGGAGAGGGTTGGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGCTCGCTCGCTGCTTCGGCTGCTTCTCGACGTGCTCTAGTCG ATGTTGAGTGTACTGCTACATCCCACGAAGAAGGGAAGCTCGCACGGCCGTGAGACTGCTCTCTTCCCTCGACCGCATCGCTGGCCATCCCT CTCCACGCTTGCCGCTTCGACTGCTTCTCAACGTGCACAATTCGCTGCTTGAACAATTGATGCTGCCGCCTACGTCGAGCGCAAGCGACCCT CACATGATTGCGAGCGAGAGTTCTCGCGTTCTCCCCAGTCCCGCCGTCGACAACGCCGCACGCATGTTGAGATCTAACGCAATTACACCTAT ACGATCCTTGAATAAAAGTAAGCGCACACACTGGCTTAAGATGACGCGACGACGTGTGGGGCTACCGCTCGCTCGAGCCGACGCGGTGCTGC ATCGCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCGCCGGCGAGCCCGACGGCGCC CGAGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCTCGCGACGGCGCAGAAGCTGC TTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACTGACTGTGGCTGTCACACGGGC CCTGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGGCGCGGCGAGTGTGGACGCTCG AGCTGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTCGCGATACCCCCCCTTTTCCC TCGCTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGTGCGTCCAGCGAGGGCGAGAG AGAGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCCCCCCTCGCCCCATCTTCC ACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTGACCCGTCCACCCACTCCCGCA AGCCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGCCATCGAGGCACTGGATG GGTCCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCCCTGACCAAG GTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTCGTCGCGTACGGGGACGACGG CGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCCCCGGAGCACCGGGGGC ACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGTCACCAACGTTAAC GCACCGGCTATCCACGCGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTC TGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTA ATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCT ATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCAT TTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCT GAGCGAGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACA ATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGT ACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTAC CTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCC CATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCT TGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAA CGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGA GCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC SEQ ID NO:50 R. toruloides DA4DH AA sequence MSHPSDGLPRPTPQLPESMLEMLSMKGKVSIVTGGSGGIGFAAAEGLAEMGGDIALAYRSAEGMDKRAEDLAKKFGVKVKAYKCDVADYDEV TKLVNDVKSDFGRVDVFIANAGMGGSGRIKDLSLEQWRKIQAVNYDSVFYAMKAVGPIFEAQGSGSFIATTSISAHIVNVPLDQSAYNASKA AVVHLCKSVARDWRLFARVNTISPGFFDTAMGAAPEVADTVYRYSVLGRQGDPKELKGAFLLLASGAGSYITGSDYLVDGGYTLS SEQ ID NO:51 R. toruloides DA4DH coding sequence flanked by SapI sites GCTCTTCAATGTCGCACCCGTCGGACGGCCTCCCGCGCCCGACCCCGCAGCTCCCGGAGTCGATGCTCGAGATGCTCTCGATGAAGGGCAAG GTTTCGATCGTTACTGGAGGCTCGGGAGGCATCGGCTTCGCCGCCGCCGAGGGCCTCGCCGAGATGGGCGGCGACATTGCCTTGGCCTATCG ATCTGCTGAAGGCATGGACAAACGAGCCGAGGACCTCGCTAAGAAGTTCGGCGTCAAGGTCAAGGCCTACAAGTGCGACGTCGCCGACTACG ACGAGGTCACCAAGCTCGTCAACGACGTCAAGTCGGACTTCGGCCGCGTCGACGTTTTCATTGCCAACGCCGGCATGGGCGGCTCGGGCCGC ATCAAGGACCTCTCGCTCGAGCAGTGGCGCAAGATCCAGGCCGTCAACTACGACTCGGTTTTCTACGCCATGAAGGCCGTCGGCCCGATCTT CGAGGCCCAGGGCTCGGGCTCGTTCATTGCGACCACCTCGATCTCGGCCCACATCGTCAACGTCCCGCTCGACCAGTCTGCTTACAACGCCT CGAAGGCCGCCGTCGTCCACCTCTGCAAGTCGGTCGCCCGCGACTGGCGACTCTTCGCCCGAGTCAACACCATCTCGCCGGGCTTCTTCGAC ACTGCCATGGGAGCCGCCCCTGAGGTCGCTGACACTGTCTACCGCTACTCCGTCCTCGGCCGCCAGGGCGATCCTAAGGAGCTCAAGGGCGC CTTCCTCCTCTTGGCTTCGGGCGCCGGCTCTTACATCACCGGCTCGGATTACCTCGTCGACGGCGGATACACCCTCTCGTAGTAGTGAAGAG C SEQ ID NO:52 Strain 1 CAR2 L Rta_pRPS3A>UcFatB2 full-length plasmid CCGCGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGATTAGAAAAAC TCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAA CTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCT CGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACT TGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAA TACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTG AATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGC TTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTT CAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCAT ATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTT ATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTG AATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCG TAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTT GTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCG TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAA GTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCA CCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACC ATCTGAATCATGCGCCACATCGAGGTGCTCCTGGCCGAGGCCGAGTGGCGCCTGGCGGTGAACGACGAGGTCCCGGTCTGCTGAGCGTGACG CGGTGGTGCGGGAGGGCGAGCGGCTCGTCGGCGAGAGCCGGTGGGCGGCGGGGCCTTGGTATGCGCTGTGCAGCAAGTGCGTGAGGAGCAGCAAGGTGAGAGGTGCACAGAAGGAGAGAGCCCATGGCCGAAGGCATGGAGGCGATGATGCTGGTTCGGCGCACGCCGGCCAAAGTTGGCCTGC GTCGAGATCGGTCGCCAATTCGGCAGCTGCTGCGTCCAGTCGAAACCGCAACCTTAGCGTTGCCTTACCTTTTCGCAACCAGCCCGCTTGTA AGCCAGCGAGGCCGAGTTATGTACCGGCTCTGCCGATTCGAGCGGGTTCGAGTTGGAGCCCCCGCGCGCGCGGGCTCCGCGCTTCGCTCCGA GCTCCGAGGCCGCCGCGCGCGCGACAAGTCGCACATCCTCATCTCGCCCTCCTCACCGGCCACCGCTCGCTCGCTCGATACCCCCTCTGTGC TCTCTGTTCCTCGCGTCTCGGCATGGGTGGCTTCGACTACTGGCTCGTGTGCGTCCCTCTCTCTCTCTCTCTCCCTGCGCTCGCCGCAGCCG CACCGGCTGAGCCCTCGCTGACCCCTCGTCGTCGCCGTTGCTGCAGCCATGCGCGTTGGACGATCCCTCCCTCGGTCGCGCTGTGGCTCGTG TTCCGCAAGCTGCGGACCTGGAGGGACGTGTACAAGACGCTGTTCCTCATCACGGTCCGTCTCGAGCTCCCCCCGCGGCGAGACGGCCAAGG CTGACCTCCTCCCCTCTTTGCTCCGAGCAGATCGCTGTCACGGTGCGCCGCTCTGCGCGCTCGAGACTAGGCGCACGCGCTCGGGAGAGCTG ACCCTTGCCCCTCGCAGGCGACGATACCCTGGGATTCGTACCTCATCCGGAACCGCGTGCGTCCTCGTCCTCCTCCTCACCCAACCCTCCCG AGCCGCGCTGAACTGGCCGATAATTGCAGACGAGCGCGACGCTAGCAGCTCGTGGCGCCATAAAAGCGTGCGAGTCACACGACCCGACTCGG CAGCAGAGCCTTAGGGTCGCGAGCACCGTGACCATGCGTAGAGCCTAGGGGCGTGCGGGACAATGTCGGTCGTGCATTGACGTGTGCTGCTG GACGCAAGAAGGCTGATGTTGGACCGTCGAGGCGTCAGGCGCTGTGGGCGGGGTAAGGTGCTAGGGCGTGTGCACCAGGGAGAGGGCGGCGA GCGCGGAGGGGAGCGGTGGCGGCTGTGAGCCAAAGTCAAGCAAGCTATCGTGTGTGTTCACCAGAAGACAGCACGTTACAGCGTAGAGGGAT ACGGTGAGGAAGGCGCGCGCAAAAAGCGGGTGCAGAGTCGAGGCCGGCGTCGCCGACACGCGGACGAGCGACCGACCGACTTGGTGTCGAGC GCGGCAACCTCGCTCTTGTCCCTCGCACCCTCGCCGCGTCTTCACGCTCGCCCTTACCCAACATGGGCAAGTCATTCTCACCTATACTATCG TCGCAGTCGACCCTCCGCCCTCCACCCTAGGCCCTCGCACGACGCGGCGGTGTCGGGAGGTGTCGCCGCTTCGAGCTGTGCCCAGTCGACCC GCTCGCAATTTTCGCCCGCCGACTCGACCCCGCCCGCTTCGCCCTGAGCACCGCCTGACCGGCCCTACACTCGATGAGGGGGATTTAGAGTG TACTGGCGGTATCGTGCACGCGCCTGGGTGGGCGGGAAGGTTGGGTCGGCGGGTCGGCTAGGTCGAGCTCGGCAGGCACGGGTCGGCGGTCA CGCGCCGTTTTCAGTCGCTCGAAGGGCTGCAGGGTACCGCCAGGCTCGGATCTGTCTCGTATGACACGGATGGCGGCCGCGCAGCGGGTCAG GCGTCGCCAGCTCGAGCGGTACACGCGTCGCCCTCCGACCCCAGCAAACCTACCCTCGTGCATCGAAATCGCGGGGAACCTGGCGCTAACCC GGCCTCTGCTCCTGTCCTACAGCTATGGTCACCACCTCGCTCGCCTCGGCCTTCTTTTCGATGAAGGCCGTCATGCTCGCCCCGGACGGCTC GGGCATCAAGCCGCGATCGTCGGGCCTCCAAGTTCGAGCTGGCAAGGAGCAGAACTCGTGCAAGATGATCAACGGCACCAAGGTCAAGGACA CCGAGGGCCTCAAGGGCCGCTCGACCCTCCACGGCTGGTCGATGCCGCTCGAGCTCATCACCACGATCTTCAGCGCCGCTGAGAAGCAGTGG ACCAACCTCGTCAGCAAGCCGCCGCAGCTCCTCGACGACCACCTCGGCCTCCACGGACTCGTTTTCCGCCGCACCTTTGCCATCCGCTGCTC GGAAGTCGGACCTGACCGATCGACTTCGATCGTCGCCGTCATGAACTACCTCCAGGAGGCCGCCTGCAACCACGCCGAGTCGCTCGGCCTCT TGGGAGATGGCTTCGGCGAGACTCTCGAGATGTCGCGCCGCGACCTCATCTGGGTTGTCCGACGAACCCACGTCGTCGTCGAGCGCTACCCG GCCTGGGGAGACACTGTCGAGGTCGAGGCTTGGATTGGAGCCGCCGGCAACATCGGCATGCGACGACACTTCCTCGTCCGCGACTGCAAGAC CGGCCACATCCTCGCCCGCTGCACCTCGGTTTCGGTCATGATGAACATGCGCACCCGCCGCCTCTCGAAGATCCCGCAGGAGGTCCGCGGCG AGATCGACCCGTTGTTCATCGAGAAGTTCGCCGTCAAGGAGGGCGAGATCAAGAAGCTCCAGAAGTTCAACGACTCGACCGCCGACTACATC CAGGGCGGCTGGACCCCGCGCTGGAACGACTTGGACGTCAACCAGCACGTCAACAACATCAAGTACGTCGGCTGGATCTTCAAGTCGGTCCC GGACTCGATCTACGAGAACCACCACTTGTCGTCGATCACCCTCGAGTACCGCCGCGAGTGCACCCGCGGCCGCGCCCTCCAGTCGCTCACTA CCGTCTGCGGAGGATCGTCTGAAGCTGGAATCATCTGCGAGCACCTCCTCCAGTTGGAGGACGGCTCCGAGGTCCTCCGCGGCCGGACCGAC TGGCGACCGAAGCGCACCGACTCGTTCGAAGGCATTTCTGAACGCTTCCCGCAGCAGGAACCTCACAACTAGGCGGAGCAGCGACGGACGGA CCTTGTGCGTTGTGTTTGTAGGGAGGGCGAGTGGACCCGCAATGCGAGGGAAACTCTTTCTCTTCCTTCGTGCGTCCTGCCCGAGAGACTTG CGAGTCGAAGTTGACGAGGAGAGGGTTGGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGCTCGCTCGCTGCTTCGGCTGCTTCTCGACGTGCT CTAGTCGATGTTGAGTGTACTGCTACATCCCACGAAGAAGGGAAGCTCGCACGGCCGTGAGACTGCTCTCTTCCCTCGACCGCATCGCTGGC CATCCCTCTCCACGCTTGCCGCTTCGACTGCTTCTCAACGTGCACAATTCGCTGCTTGAACAATTGATGCTGCCGCCTACGTCGAGCGCAAG CGACCCTCACATGATTGCGAGCGAGAGTTCTCGCGTTCTCCCCAGTCCCGCCGTCGACAACGCCGCACGCATGTTGAGATCTAACGCAATTA CACCTATACGATCCTTGAATAAAAAGTGAATAAAGCTCCACACAGTCGGCTAGCGACGACGTGTGGGGCTACCGCTCGCTCGAGCCGACGCG GTGCTGCATCGCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCGCCGGCGAGCCCGA CGGCGCCCGAGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCTCGCGACGGCGCAG AAGCTGCTTCTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACTGACTGTGGCTGTCA CACGGGCCCTGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGGCGCGGCGAGTGTGG ACGCTCGAGCTGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTCGCGATACCCCCCC TTTTCCCTCGCTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGTGCGTCCAGCGAGG GCGAGAGAGAGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCCCCCCTCGCCCC ATCTTCCACCTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTGACCCGTCCACCCAC TCCCGCAAGCCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGCCATCGAGGCA CTGGATGGGTCCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCCCT GACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTCGTCGCGTACGGGG ACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCCCCGGAGCAC CGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGTCACCAA CGTTAACGCACCGGCTATCCACGC SEQ ID NO:53 Strain 1 CAR2 L Rta_pRPS3A>CcFatB2b leader truncated plasmid CCGCGGATGACACGAACTCACGACTAACTATAACGGTCCTAAGGTAGCGAACCATGTGTTACAACCAATTAACCAATTCTGATTAGAAAAAC TCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAA CTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCT CGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACT TGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAA TACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTG AATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGC TTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTT CAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCAT ATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTT ATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTG AATAAATCGAACTTTTGCTGAGTTGAAGGATCAGAGGATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCG TAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTT GTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCG TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAA GTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCA CCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGGAGTAGGGATAACAGGGTAATGGACC ATCTGAATCATGCGCCACATCGAGGTGCTCCTGGCCGAGGCCGAGTGGCGCCTGGCGGTGAACGACGAGGTCCCGGTCTGCTGAGCGTGACGCGGTGGTGCGGGAGGGCGAGCGGCTCGTCGGCGAGAGCCGGTGGGCGGCGGGGCCTTGGTATGCGCTGTGCAGCAAGTGCGTGAGGAGCAGC AAGGTGAGAGGTGCACAGAAGGAGAGAGCCCATGGCCGAAGGCATGGAGGCGATGATGCTGGTTCGGCGCACGCCGGCCAAAGTTGGCCTGC GTCGAGATCGGTCGCCAATTCGGCAGCTGCTGCGTCCAGTCGAAACCGCAACCTTAGCGTTGCCTTACCTTTTCGCAACCAGCCCGCTTGTA AGCCAGCGAGGCCGAGTTATGTACCGGCTCTGCCGATTCGAGCGGGTTCGAGTTGGAGCCCCCGCGCGCGCGGGCTCCGCGCTTCGCTCCGA GCTCCGAGGCCGCCGCGCGCGCGACAAGTCGCACATCCTCATCTCGCCCTCCTCACCGGCCACCGCTCGCTCGCTCGATACCCCCTCTGTGC TCTCTGTTCCTCGCGTCTCGGCATGGGTGGCTTCGACTACTGGCTCGTGTGCGTCCCTCTCTCTCTCTCTCTCCCTGCGCTCGCCGCAGCCG CACCGGCTGAGCCCTCGCTGACCCCTCGTCGTCGCCGTTGCTGCAGCCATGCGCGTTGGACGATCCCTCCCTCGGTCGCGCTGTGGCTCGTG TTCCGCAAGCTGCGGACCTGGAGGGACGTGTACAAGACGCTGTTCCTCATCACGGTCCGTCTCGAGCTCCCCCCGCGGCGAGACGGCCAAGG CTGACCTCCTCCCCTCTTTGCTCCGAGCAGATCGCTGTCACGGTGCGCCGCTCTGCGCGCTCGAGACTAGGCGCACGCGCTCGGGAGAGCTG ACCCTTGCCCCTCGCAGGCGACGATACCCTGGGATTCGTACCTCATCCGGAACCGCGTGCGTCCTCGTCCTCCTCCTCACCCAACCCTCCCG AGCCGCGCTGAACTGGCCGATAATTGCAGACGAGCGCGACGCTAGCAGCTCGTGGCGCCATAAAAGCGTGCGAGTCACACGACCCGACTCGG CAGCAGAGCCTTAGGGTCGCGAGCACCGTGACCATGCGTAGAGCCTAGGGGCGTGCGGGACAATGTCGGTCGTGCATTGACGTGTGCTGCTG GACGCAAGAAGGCTGATGTTGGACCGTCGAGGCGTCAGGCGCTGTGGGCGGGGTAAGGTGCTAGGGCGTGTGCACCAGGGAGAGGGCGGCGA GCGCGGAGGGGAGCGGTGGCGGCTGTGAGCCAAAGTCAAGCAAGCTATCGTGTGTGTTCACCAGAAGACAGCACGTTACAGCGTAGAGGGAT ACGGTGAGGAAGGCGCGCGCAAAAAGCGGGTGCAGAGTCGAGGCCGGCGTCGCCGACACGCGGACGAGCGACCGACCGACTTGGTGTCGAGC GCGGCAACCTCGCTCTTGTCCCTCGCACCCTCGCCGCGTCTTCACGCTCGCCCTTACCCAACATGGGCAAGTCATTCTCACCTATACTATCG TCGCAGTCGACCCTCCGCCCTCCACCCTAGGCCCTCGCACGACGCGGCGGTGTCGGGAGGTGTCGCCGCTTCGAGCTGTGCCCAGTCGACCC GCTCGCAATTTTCGCCCGCCGACTCGACCCCGCCCGCTTCGCCCTGAGCACCGCCTGACCGGCCCTACACTCGATGAGGGGGATTTAGAGTG TACTGGCGGTATCGTGCACGCGCCTGGGTGGGCGGGAAGGTTGGGTCGGCGGGTCGGCTAGGTCGAGCTCGGCAGGCACGGGTCGGCGGTCA CGCGCCGTTTTCAGTCGCTCGAAGGGCTGCAGGGTACCGCCAGGCTCGGATCTGTCTCGTATGACACGGATGGCGGCCGCGCAGCGGGTCAG GCGTCGCCAGCTCGAGCGGTACACGCGTCGCCCTCCGACCCCAGCAAACCTACCCTCGTGCATCGAAATCGCGGGGAACCTGGCGCTAACCC GGCCTCTGCTCCTGTCCTACAGCTATGCTCGACGACCACTTCGGCCTCCACGGCCTCGTCTTTCGCCGCACCTTCGCCATCCGGTGCTACGA GGTCGGCCCGGACCGCTCGACCTCGATTATGGCTGTCATGAACCACCTCCAGGAGGCCGCCCGCAACCATGCCGAGTCGCTCGGCTTGCTCG GAGATGGCTTCGGCGAGACTCTCGAGATGTCGAAGCGCGACCTCATCTGGTGGCGCCGAACCCACGTCGCCGTCGAGCGCTACCCGGCTTGG GGAGACACCGTTGAGGTCGAGGCTTGGGTTGGAGCTTCTGGAAACACCGGCATGCGACGCGACTTCCTCGTCCGCGACTGCAAGACCGGCCA CATCCTCACCCGCTGCACCTCGGTTTCGGTCATGATGAACATGCGCACCCGCCGCCTCTCGAAGATCCCGCAGGAGGTCCGCGCCGAGATCG ACCCGTTGTTCATCGAGAAGGTCGCCGTCAAGGAGGGCGAGATCAAGAAGTTGCAGAAGCTCAACGACTCGACCGCCGACTACATCCAGGGC GGCTGGACCCCGCGCTGGAACGACTTGGACTGGCAGCACGTCAACAACATCATCTACGTCGGCTGGATCTTCAAGTCGGTCCCGGACTCGAT CTCCGAGAACCACCATCTCTCGTCGATCACCCTCGAGTACCGCCGCGAGTGCACCCGCGGCAACAAGCTCCAGTCGCTCACCACCGTCTGCG GCGGATCGTCGGAGGCTGGAATCATCTGCGAGCACCTCCTCCAGTTGGAGGACGGCTCGGAGGTCCTCCGCGCTCGAACCGAATGGCGACCG AAGCACACCGACTCGTTCCAGGGCATCTCGGAGCGCTTCCCGCAGCAGGAACCTCACAAGTAGGCGGAGCAGCGACGGACGGACCTTGTGCG TTGTGTTTGTAGGGAGGGCGAGTGGACCCGCAATGCGAGGGAAACTCTTTCTCTTCCTTCGTGCGTCCTGCCCGAGAGACTTGCGAGTCGAA GTTGACGAGGAGAGGGTTGGTCTGCCGGCTGCTCGCGCCCGTCGTTGGGCTCGCTCGCTGCTTCGGCTGCTTCTCGACGTGCTCTAGTCGAT GTTGAGTGTACTGCTACATCCCACGAAGAAGGGAAGCTCGCACGGCCGTGAGACTGCTCTCTTCCCTCGACCGCATCGCTGGCCATCCCTCT CCACGCTTGCCGCTTCGACTGCTTCTCAACGTGCACAATTCGCTGCTTGAACAATTGATGCTGCCGCCTACGTCGAGCGCAAGCGACCCTCA CATGATTGCGAGCGAGAGTTCTCGCGTTCTCCCCAGTCCCGCCGTCGACAACGCCGCACGCATGTTGAGATCTAACGCAATTACACCTATAC GATCCTTGAATAAAAAGTGAATAAAGCTCCACACAGTCGGCTAGCGACGACGTGTGGGGCTACCGCTCGCTCGAGCCGACGCGGTGCTGCAT CGCGTCGATCGCGCTCGTCCCCCTCAAGACGGGCATCGTGCACCCGCCGATCGGCGGCAGCGCGTCGGCGCCGGCGAGCCCGACGGCGCCCG AGGACGGCTTGGCGTCGGCGCAGGGCGACGACGGCGGGGCGGGCGGCGTCCAGGTCAACACTCAGCAGCTCGCGACGGCGCAGAAGCTGCTT CTCTTCTGGCGCAAGCCTGCGGTGCGTCCCTCTCGTTCCTCTCGGGTCCCTGTGTCGAGCGCAGTGACTGACTGTGGCTGTCACACGGGCCC TGCAGCGCAAGGTGTGGTGGGACCACAGCACCGAGACGCTCGCGAACGGCAAGGAGGTCAAGATCTGGGCGCGGCGAGTGTGGACGCTCGAG CTGAGCCTGATCTGAGCCGTCGTCGTCGTCGTCGTTGTCGAGGTGCAGGCGGCGTGCAGATTCCCCGGTCGCGATACCCCCCCTTTTCCCTC GCTCGTCTTGTTTCCGTAGCTTGGTCCGGCTCTCTTCTTGTACATACCCGTCGTATCCAGCAGTTCGAGTGCGTCCAGCGAGGGCGAGAGAG AGACGGTCGACACGCGCCGCCGGTGTCGAGGTTCTCGACTTGGCCGCGACGAGAGCGAGGCGCTCCTCCTCCCCCCTCGCCCCATCTTCCAC CTCGCCCCTCTCCCTCTAGTCTTCTTGTGAGTACTCGAGGCTGCTCTACGCCAAGCTAGACCCCGACTGACCCGTCCACCCACTCCCGCAAG CCACGATGGCGGCCGCCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGCCATCGAGGCACTGGATGGG TCCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCCCTGACCAAGGT GTTCCCCGACGACGAATCGGACGACGAATCGGACGCCGGGGAGGACGGCGACCCGGACTCCCGGACGTTCGTCGCGTACGGGGACGACGGCG ACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCCCCGGAGCACCGGGGGCAC GGGGTCGGGCGCGCGTTGATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGTCACCAACGTTAACGC ACCGGCTATCCACGC SEQ ID NO:54 Scer fusFAS R1834K AA sequence MDAYSTRPLTLSHGSLEHVLLVPTASFFIASQLQEQFNKILPEPTEGFAADDEPTTPAELVGKFLGYVSSLVEPSKVGQFDQVLNLCLTEFE NCYLEGNDIHALAAKLLQENDTTLVKTKELIKNYITARIMAKRPFDKKSNSALFRAVGEGNAQLVAIFGGQGNTDDYFDELRDLYQTYHVLV GDLIKFSAETLSELIRTTLDAEKVFTQGLNILEWLENPSNTPDKDYLLSIPISCPLIGVIQLAHYVVTAKLLGFTPGELRSYLKGATGHSQG LVTAVAIAETDSWESFFVSVRKAITVLFFIGVRCYEAYPNTSLPPSILEDSLENNEGVPSPMLSISNLTQEQVQDYVNKTNSHLPAGKQVEI SLVNGAKNLVVSGPPQSLYGLNLTLRKAKAPSGLDQSRIPFSERKLKFSNRFLPVASPFHSHLLVPASDLINKDLVKNNVSFNAKDIQIPVY DTFDGSDLRVLSGSISERIVDCIIRLPVKWETTTQFKATHILDFGPGGASGLGVLTHRNKDGTGVRVIVAGTLDINPDDDYGFKQEIFDVTS NGLKKNPNWLEEYHPKLIKNKSGKIFVETKFSKLIGRPPLLVPGMTPCTVSPDFVAATTNAGYTIELAGGGYFSAAGMTAAIDSVVSQIEKG STFGINLIYVNPFMLQWGIPLIKELRSKGYPIQFLTIGAGVPSLEVASEYIETLGLKYLGLKPGSIDAISQVINIAKAHPNFPIALQWTGGR GGGHHSFEDAHTPMLQMYSKIRRHPNIMLIFGSGFGSADDTYPYLTGEWSTKFDYPPMPFDGFLFGSRVMIAKEVKTSPDAKKCIAACTGVP DDKWEQTYKKPTGGIVTVRSEMGEPIHKIATRGVMLWKEFDETIFNLPKNKLVPTLEAKRDYIISRLNADFQKPWFATVNGQARDLATMTYE EVAKRLVELMFIRSTNSWFDVTWRTFTGDFLRRVEERFTKSKTLSLIQSYSLLDKPDEAIEKVFNAYPAAREQFLNAQDIDHFLSMCQNPMQ KPVPFVPVLDRRFEIFFKKDSLWQSEHLEAVVDQDVQRTCILHGPVAAQFTKVIDEPIKSIMDGIHDGHIKKLLHQYYGDDESKIPAVEYFG GESPVDVQSQVDSSSVSEDSAVFKATSSTDEESWFKALAGSEINWRHASFLCSFITQDKMFVSNPIRKVFKPSQGMVVEISNGNTSSKTVVT LSEPVQGELKPTVILKLLKENIIQMEMIENRTMDGKPVSLPLLYNFNPDNGFAPISEVMEDRNQRIKEMYWKLWIDEPFNLDFDPRDVIKGK DFEITAKEVYDFTHAVGNNCEDFVSRPDRTMLAPMDFAIVVGWRAIIKAIFPNTVDGDLLKLVHLSNGYKMIPGAKPLQVGDVVSTTAVIES VVNQPTGKIVDVVGTLSRNGKPVMEVTSSFFYRGNYTDFENTFQKTVEPVYQMHIKTSKDIAVLRSKEWFQLDDEDFDLLNKTLTFETETEV TFKNANIFSSVKCFGPIKVELPTKETVEIGIVDYEAGASHGNPVVDFLKRNGSTLEQKVNLENPIPIAVLDSYTPSTNEPYARVSGDLNPIH VSRHFASYANLPGTITHGMFSSASVRALIENWAADSVSSRVRGYTCQFVDMVLPNTALKTSIQHVGMINGRKLIKFETRNEDDVVVLTGEAE IEQPVTTFVFTGQGSQEQGMGMDLYKTSKAAQDVWNRADNHFKDTYGFSILDIVINNPVNLTIHFGGEKGKRIRENYSAMIFETIVDGKLKT EKIFKEINEHSTSYTFRSEKGLLSATQFTQPALTLMEKAAFEDLKSKGLIPADATFAGHSLGEYAALASLADVMSIESLVEVVFYKGMTMQV AVPRDELGRSNYGMIAINPGRVAASFSQEALQYVVERVGKRTGWLVEIVNYNVENQQYVAAGDLRALDTVTNVLNFIKLQKIDIIELQKSLS LEEVEGHLFEIIDEASKKSAVKPRPLKLERGFACIPLVGISVPFHSTYLMNGVKPFKSFLKKNIIKENVKVARLAGKYIPNLTAKPFQVTKE YFQDVYDLTGSEPIKEIIDNWEKDEWGSPEVEQELAHILLTELLAYQFASPVRWIETQDVFLKDFNTERVVEIGPSPTLAGMAQRTLKNKYESYDAALSLHREILCYSKDAKEIYYTPDPSELAAKEEPAKEEAPAPTPAASAPAPAAAAPAPVAAAAPAAAAAEIADEPVKASLLLHVLVAHK LKKSLDSIPMSKTIKDLVGGKSTVQNEILGDLGKEFGTTPEKPEETPLEELAETFQDTFSGALGKQSSSLLSRLISSKMPGGFTITVARKYL QTRWGLPSGRQDGVLLVALSNEPAARLGSEADAKAFLDSMAQKYASIVGVDLSSAASASGAAGAGAAAGAAMIDAGALEEITKDHKVLARQQ LQVLARYLKMDLDNGERKFLKEKDTVAELQAQLDYLNAELGEFFVNGVATSFSRKKARTFDSSWNWAKQSLLSLYFEIIHGVLKNVDREVVS EAINIMNRSNDALIKFMEYHISNTDETKGENYQLVKTLGEQLIENCKQVLDVDPVYKDVAKPTGPKTAIDKNGNITYSEEPREKVRKLSQYV QEMALGGPITKESQPTIEEDLTRVYKAISAQADKQDISSSTRVEFEKLYSDLMKFLESSKEIDPSQTTQLAGMDVEDALDKDSTKEVASLPN KSTISKTVSSTIPRETIPFLHLRKKTPAGDWKYDRQLSSLFLDGLEKAAFNGVTFKDKYVLITGAGKGSIGAEVLQGLLQGGAKVVVTTSRF SKQVTDYYQSIYAKYGAKGSTLIVVPFNQGSKQDVEALIEFIYDTEKNGGLGWDLDAIIPFAAIPEQGIELEHIDSKSEFAHRIMLTNILRM MGCVKKQKSARGIETRPAQVILPMSPNHGTFGGDGMYSESKLSLETLFNRWHSESWANQLTVCGAIIGWTRGTGLMSANNIIAEGIEKMGVR TFSQKEMAFNLLGLLTPEVVELCQKSPVMADLNGGLQFVPELKEFTAKLRKELVETSEVRKAVSIETALEHKVVNGNSADAAYAQVEIQPRA NIQLDFPELKPYKQVKQIAPAELEGLLDLERVIVVTGFAEVGPWGSARTRWEMEAFGEFSLEGCVEMAWIMGFISYHNGNLKGRPYTGWVDS KTKEPVDDKDVKAKYETSILEHSGIRLIEPELFNGYNPEKKEMIQEVIVEEDLEPFEASKETAEQFKHQHGDKVDIFEIPETGEYSVKLLKG ATLYIPKALRFDRLVAGQIPTGWNAKTYGISDDIISQVDPITLFVLVSVVEAFIASGITDPYEMYKYVHVSEVGNCSGSGMGGVSALRGMFK DRFKDEPVQNDILQESFINTMSAWVNMLLISSSGPIKTPVGACATSVESVDIGVETILSGKARICIVGGYDDFQEEGSFEFGNMKATSNTLE EFEHGRTPAEMSRPATTTRNGFMEAQGAGIQIIMQADLALKMGVPIYGIVAMAATATDKIGRSVPAPGKGILTTAREHHSSVKYASPNLNMK YRKRQLVTREAQIKDWVENELEALKLEAEEIPSEDQNEFLLERTREIHNEAESQLRAAQQQWGNDFYKRDPRIAPLRGALATYGLTIDDLGV ASFHGTSTKANDKNESATINEMMKHLGRSEGNPVIGVFQKFLTGHPKGAAGAWMMNGALQILNSGIIPGNRNADNVDKILEQFEYVLYPSKT LKTDGVRAVSITSFGFGQKGGQAIVVHPDYLYGAITEDRYNEYVAKVSAREKSAYKFFHNGMIYNKLFVSKEHAPYTDELEEDVYLDPLARV SKDKKSGSLTFNSKNIQSKDSYINANTIETAKMIENMTKEKVSNGGVGVDVELITSINVENDTFIERNFTPQEIEYCSAQPSVQSSFAGTWS AKEAVFKSLGVKSLGGGAALKDIEIVRVNKNAPAVELHGNAKKAAEEAGVTDVKVSISHDDLQAVAVAVSTKK SEQ ID NO:55 Scer fusFAS R1834K CDS flanked by SapI sites GCTCTTCAATGGACGCCTACTCGACCCGCCCCCTCACCCTCTCGCACGGCTCCCTCGAGCACGTCCTCCTTGTCCCGACCGCCTCGTTCTTC ATAGCCTCGCAACTCCAAGAGCAGTTCAACAAGATCCTCCCGGAGCCGACCGAGGGATTCGCCGCTGACGACGAGCCGACCACCCCGGCCGA GCTCGTCGGAAAGTTCCTCGGCTACGTTTCGAGCTTGGTTGAGCCTTCGAAGGTCGGCCAGTTCGACCAGGTCCTCAACCTCTGCCTCACCG AGTTCGAGAACTGCTACCTCGAGGGCAACGACATCCACGCCCTCGCCGCCAAGCTTCTCCAGGAGAACGACACCACCCTCGTCAAGACTAAG GAGCTCATCAAGAACTACATCACCGCCCGCATCATGGCCAAGCGCCCGTTCGACAAGAAGTCCAACTCGGCCCTCTTCCGCGCCGTCGGCGA GGGTAACGCGCAGCTCGTCGCCATCTTTGGCGGCCAGGGAAACACTGACGACTACTTCGACGAGTTGCGCGACCTCTACCAGACCTACCACG TCCTCGTCGGCGACCTTATCAAGTTTTCGGCGGAGACTCTCTCGGAGCTCATTCGCACCACCTTGGACGCCGAGAAAGTTTTCACCCAGGGC CTCAACATTCTCGAGTGGCTCGAGAACCCGTCGAACACCCCGGACAAGGACTACCTCCTCTCGATCCCGATCTCGTGCCCGCTCATCGGCGT CATCCAGCTCGCCCACTACGTCGTCACCGCGAAGCTCCTCGGCTTCACCCCGGGCGAGTTGCGTTCGTACTTGAAGGGAGCCACCGGCCACT CTCAGGGCTTGGTCACCGCCGTTGCTATCGCCGAAACCGACTCGTGGGAGTCGTTCTTCGTGTCCGTCCGCAAGGCCATCACCGTCCTCTTC TTCATCGGGGTCCGCTGCTACGAGGCCTACCCGAACACCTCGCTCCCACCGTCGATCCTCGAAGATTCGTTGGAGAACAACGAGGGAGTCCC GTCGCCGATGCTCTCGATCAGCAACCTCACCCAGGAGCAAGTCCAGGACTACGTCAACAAGACCAACTCGCACCTCCCGGCCGGAAAGCAGG TCGAGATCTCGCTTGTCAACGGCGCGAAGAATCTCGTCGTGTCGGGACCGCCGCAGTCGCTCTACGGCTTGAACCTCACGCTCCGAAAGGCC AAGGCCCCGTCGGGCCTCGATCAGTCACGCATCCCGTTCTCTGAGCGAAAGCTTAAGTTCAGCAACCGCTTCCTCCCGGTGGCCTCGCCGTT CCACTCGCATCTCTTGGTCCCGGCCTCGGACCTCATCAACAAGGACCTCGTTAAGAACAACGTCAGCTTCAACGCTAAGGACATCCAGATCC CGGTCTACGACACCTTTGACGGCTCGGACTTGCGCGTCCTCTCGGGCTCGATCTCCGAGCGGATCGTCGACTGCATCATTCGCCTCCCCGTC AAGTGGGAGACAACCACCCAATTCAAGGCCACCCATATCCTCGACTTCGGCCCGGGCGGCGCCTCGGGCCTTGGAGTCCTCACCCATCGGAA CAAAGACGGCACCGGAGTAAGGGTAATTGTAGCCGGCACCCTCGACATCAACCCGGACGACGACTACGGCTTCAAGCAGGAGATCTTCGACG TCACCTCGAACGGCCTTAAGAAAAACCCGAACTGGTTGGAAGAGTACCACCCCAAGCTTATCAAGAATAAGTCCGGCAAGATCTTCGTCGAA ACGAAGTTCTCCAAGCTCATCGGTCGCCCGCCGCTCCTCGTTCCGGGCATGACCCCGTGCACCGTGTCGCCGGACTTCGTCGCCGCCACCAC TAATGCAGGCTACACAATCGAGTTGGCTGGAGGCGGATACTTCTCGGCCGCCGGCATGACCGCCGCCATCGACTCGGTCGTTTCGCAAATTG AGAAGGGCTCGACGTTCGGCATCAACCTCATTTACGTCAACCCGTTCATGCTCCAGTGGGGCATCCCCCTCATCAAGGAGTTGCGCTCCAAG GGCTACCCCATCCAGTTCCTCACCATCGGCGCTGGCGTCCCGTCGTTGGAGGTCGCTTCGGAGTACATCGAAACGCTCGGCCTCAAGTACCT CGGACTCAAGCCGGGCTCCATCGACGCCATTTCGCAGGTCATCAACATTGCCAAGGCGCACCCGAACTTCCCGATCGCCCTCCAGTGGACCG GCGGACGCGGCGGTGGACACCACTCGTTCGAGGACGCTCACACCCCGATGTTACAGATGTACTCGAAAATTCGCCGGCATCCGAACATTATG CTTATCTTCGGCTCGGGCTTCGGCTCCGCCGACGACACCTACCCGTACCTCACCGGCGAGTGGTCGACCAAGTTTGACTACCCGCCGATGCC GTTCGACGGTTTCCTCTTCGGATCGCGCGTCATGATCGCCAAGGAGGTCAAGACCTCGCCGGACGCGAAGAAGTGCATTGCCGCCTGCACCG GCGTCCCTGACGACAAGTGGGAACAGACCTACAAGAAGCCGACCGGCGGCATCGTCACCGTCCGGTCCGAGATGGGCGAACCTATCCACAAG ATTGCCACCCGCGGCGTCATGCTCTGGAAGGAGTTCGACGAGACTATCTTCAACCTCCCGAAGAACAAGCTCGTCCCCACCCTCGAGGCCAA GCGGGACTACATTATCTCCCGCCTCAACGCCGACTTCCAGAAGCCGTGGTTCGCCACCGTCAACGGCCAGGCCCGCGACCTCGCCACCATGA CGTACGAGGAGGTCGCCAAACGACTCGTCGAGCTCATGTTCATCCGCTCGACCAACTCCTGGTTCGACGTTACCTGGCGCACCTTCACGGGA GACTTCCTCCGCCGGGTCGAGGAGCGCTTCACCAAGTCCAAGACCCTCTCGTTGATCCAGTCGTATTCCTTGCTCGACAAGCCGGACGAGGC CATCGAGAAGGTCTTTAACGCCTACCCGGCCGCGCGGGAACAGTTCCTTAACGCCCAGGACATCGACCACTTCCTCTCCATGTGCCAGAACC CGATGCAAAAGCCGGTCCCGTTTGTCCCGGTCCTCGACCGCCGCTTCGAAATCTTCTTCAAGAAGGACTCGCTCTGGCAGTCGGAGCACCTC GAGGCGGTTGTCGACCAGGACGTCCAGCGCACCTGCATCCTCCACGGCCCGGTCGCCGCCCAATTCACTAAGGTCATCGACGAGCCCATCAA GTCGATCATGGACGGCATCCACGACGGCCACATTAAGAAGCTCTTGCACCAGTACTACGGAGATGACGAGAGCAAGATCCCGGCCGTCGAGT ACTTTGGCGGAGAATCGCCTGTCGACGTCCAGTCGCAAGTTGACTCGTCCTCGGTATCGGAGGACTCGGCCGTCTTTAAGGCCACCTCGTCC ACCGACGAAGAGTCGTGGTTCAAGGCCCTCGCGGGATCGGAGATCAACTGGCGCCACGCCTCCTTCCTCTGCAGCTTTATCACGCAGGACAA AATGTTTGTCTCGAACCCCATCCGCAAGGTTTTCAAGCCCTCGCAGGGAATGGTCGTCGAAATCTCGAATGGCAACACCTCCTCGAAAACGG TCGTCACCCTCAGCGAGCCCGTCCAGGGTGAGCTCAAGCCCACCGTCATCTTGAAGTTGCTCAAGGAGAACATTATCCAGATGGAAATGATC GAGAACCGCACCATGGACGGAAAGCCGGTGTCGCTCCCGTTGCTCTACAACTTTAACCCGGACAACGGCTTCGCCCCGATCAGCGAGGTCAT GGAGGACCGCAACCAGCGCATTAAGGAGATGTACTGGAAGCTCTGGATCGATGAGCCCTTCAACTTGGACTTCGACCCGCGGGACGTCATCA AGGGCAAGGACTTCGAGATCACCGCTAAGGAGGTCTACGATTTCACCCACGCCGTCGGAAACAACTGCGAGGACTTCGTATCGCGCCCGGAC CGCACGATGCTCGCCCCCATGGACTTCGCAATCGTCGTCGGATGGCGCGCGATCATCAAGGCCATTTTCCCGAACACGGTCGACGGCGATCT CCTCAAGCTCGTTCACCTCTCCAACGGTTACAAGATGATCCCGGGCGCCAAGCCACTCCAGGTCGGAGATGTCGTCAGCACCACCGCCGTCA TCGAGTCCGTCGTCAACCAACCGACCGGCAAAATCGTCGACGTCGTCGGTACCCTCTCTCGCAACGGCAAGCCGGTTATGGAGGTCACCTCG TCGTTCTTTTACCGGGGCAACTACACCGACTTCGAAAACACCTTCCAAAAGACTGTCGAGCCGGTCTACCAGATGCATATCAAGACCAGCAA GGATATCGCCGTCCTCCGCAGCAAGGAGTGGTTCCAGCTTGACGATGAGGACTTTGACCTCTTGAACAAGACGCTCACCTTCGAAACCGAGA CTGAGGTCACGTTCAAGAACGCGAACATCTTCTCGTCGGTCAAGTGCTTCGGCCCTATCAAGGTCGAGCTCCCCACCAAGGAAACGGTCGAG ATTGGCATCGTCGATTACGAGGCCGGCGCCTCCCACGGCAACCCAGTCGTCGACTTCCTCAAGCGCAACGGCTCGACGCTCGAGCAAAAGGT CAACCTCGAGAATCCGATCCCCATTGCCGTCCTCGACTCTTACACGCCGTCCACCAACGAGCCGTACGCCCGCGTTTCCGGCGACCTCAACC CGATCCACGTTTCGCGCCACTTCGCCTCGTACGCCAACCTCCCTGGCACTATCACCCACGGCATGTTCTCGTCCGCCTCGGTCCGCGCCCTC ATCGAGAATTGGGCCGCCGACTCGGTTAGCTCGCGCGTCCGAGGATACACCTGCCAGTTCGTCGACATGGTCTTGCCGAACACCGCGCTCAA GACTTCGATTCAGCACGTCGGCATGATCAACGGCCGGAAGCTCATTAAGTTCGAAACGCGCAACGAGGACGATGTGGTTGTCCTCACCGGGGAGGCCGAAATCGAGCAGCCGGTCACGACCTTCGTCTTTACCGGCCAGGGCTCGCAAGAGCAGGGCATGGGCATGGACCTCTACAAAACGTCG AAGGCCGCGCAAGACGTCTGGAACCGCGCCGACAACCACTTTAAGGACACGTACGGCTTCTCGATCTTGGACATCGTCATTAACAACCCGGT CAACTTGACCATCCACTTCGGCGGTGAGAAGGGAAAGCGCATCCGCGAAAACTACTCGGCCATGATCTTCGAGACTATTGTCGACGGCAAGC TCAAGACCGAGAAGATCTTCAAGGAGATTAACGAGCACTCGACCTCGTACACCTTCCGCTCCGAGAAGGGATTGCTCTCGGCCACGCAGTTC ACCCAGCCGGCCCTCACCTTGATGGAGAAGGCCGCCTTCGAGGACCTCAAGTCGAAGGGCCTCATCCCGGCGGATGCCACCTTCGCCGGCCA TTCGTTGGGCGAGTACGCCGCCCTCGCATCATTGGCTGACGTCATGTCGATCGAGTCCCTTGTCGAGGTCGTTTTCTACAAGGGCATGACGA TGCAGGTCGCGGTCCCGCGAGATGAGTTGGGTCGGTCGAACTACGGCATGATTGCCATCAACCCCGGACGCGTCGCCGCGAGCTTCTCGCAG GAGGCGCTCCAATACGTCGTCGAGCGGGTCGGCAAGCGCACCGGATGGTTGGTCGAAATCGTCAATTACAACGTCGAGAACCAGCAGTACGT CGCCGCTGGCGACCTTCGCGCCCTCGACACCGTCACCAACGTCTTGAACTTCATCAAGCTCCAGAAGATCGACATCATCGAGTTGCAGAAGT CGCTCTCGCTCGAGGAAGTTGAGGGCCACCTCTTCGAGATTATCGACGAGGCCTCCAAGAAGTCGGCCGTCAAGCCCCGCCCGCTTAAGTTG GAGCGCGGCTTCGCGTGCATCCCCTTGGTCGGCATCTCGGTCCCCTTCCATTCGACCTACCTCATGAACGGCGTCAAGCCGTTCAAGTCGTT CCTCAAAAAGAATATCATCAAAGAGAACGTCAAGGTCGCCCGGTTGGCGGGCAAGTACATCCCGAACTTGACCGCCAAGCCCTTCCAGGTCA CGAAGGAGTACTTTCAGGACGTGTACGACCTCACGGGCTCGGAGCCGATCAAGGAAATCATCGACAACTGGGAGAAGGACGAGTGGGGCTCG CCGGAGGTCGAACAAGAGCTCGCGCACATCCTCCTCACCGAACTCCTCGCCTACCAGTTCGCCTCGCCTGTCCGCTGGATCGAGACTCAGGA CGTCTTTCTCAAGGACTTTAACACCGAGCGCGTTGTCGAAATTGGCCCGTCGCCCACCTTGGCCGGCATGGCCCAGCGCACGCTCAAGAACA AATACGAATCGTACGACGCCGCCTTGTCGCTCCACCGCGAGATCCTCTGCTACTCGAAGGACGCCAAGGAAATTTACTACACGCCGGACCCG TCGGAGCTCGCCGCGAAGGAGGAGCCGGCCAAAGAAGAAGCACCTGCTCCTACACCAGCAGCTTCTGCTCCTGCACCAGCTGCAGCTGCCCC GGCTCCGGTCGCAGCTGCCGCTCCAGCAGCAGCTGCAGCAGAGATTGCTGACGAACCGGTCAAGGCCTCTCTCTTGCTCCACGTCTTGGTCG CCCACAAGCTTAAGAAGTCGTTGGACTCCATCCCGATGTCGAAAACGATTAAGGACTTGGTCGGAGGCAAGTCGACCGTCCAGAACGAGATC TTGGGCGACCTCGGAAAGGAGTTTGGCACCACCCCCGAGAAGCCCGAAGAAACCCCGTTGGAGGAGCTCGCGGAAACCTTCCAGGACACCTT CTCGGGCGCCCTCGGCAAGCAATCCTCGTCCCTCCTCAGCCGCTTGATCTCGTCCAAGATGCCGGGCGGCTTCACGATCACCGTCGCCCGCA AGTACCTTCAGACCCGCTGGGGCCTCCCGTCTGGACGGCAGGATGGCGTCCTCTTGGTTGCCTTGTCTAACGAGCCCGCGGCTCGATTGGGC TCGGAAGCCGACGCAAAAGCCTTCCTCGACAGCATGGCGCAGAAGTACGCCTCGATTGTCGGAGTCGACCTCTCGTCGGCCGCCTCCGCCTC TGGCGCTGCAGGAGCTGGAGCTGCTGCAGGTGCTGCTATGATCGACGCTGGAGCATTGGAGGAGATCACCAAGGACCACAAGGTCCTCGCCC GGCAGCAGTTGCAGGTCTTGGCCCGCTACCTCAAGATGGACCTCGATAACGGCGAGCGCAAGTTTCTCAAAGAAAAGGACACCGTCGCGGAG CTTCAGGCCCAGTTGGACTACTTGAACGCGGAGCTCGGAGAGTTCTTCGTCAACGGAGTCGCCACCTCCTTCAGCCGCAAGAAGGCCCGCAC CTTCGACTCGTCTTGGAACTGGGCCAAGCAGTCGTTGCTCTCCCTCTACTTTGAGATCATCCACGGCGTCCTTAAGAACGTGGACCGCGAAG TCGTGTCCGAGGCGATCAACATTATGAACCGCTCGAACGACGCGTTGATCAAATTCATGGAGTACCACATCTCGAACACGGACGAAACCAAG GGCGAGAACTACCAACTCGTAAAGACGCTCGGCGAGCAGCTCATCGAAAACTGCAAGCAGGTCCTCGATGTCGACCCGGTCTATAAGGACGT CGCCAAGCCTACCGGCCCCAAGACCGCCATTGACAAGAACGGCAACATCACCTACTCGGAGGAGCCCCGCGAAAAGGTCCGCAAGCTCTCGC AGTACGTGCAGGAAATGGCCCTCGGCGGACCGATCACCAAAGAGTCGCAGCCGACCATCGAGGAGGACTTGACCCGCGTCTACAAGGCGATC TCGGCCCAGGCCGACAAGCAGGACATTTCTTCGTCCACCCGCGTCGAGTTCGAAAAGCTCTACTCGGACCTGATGAAATTCCTCGAGTCGTC CAAGGAGATCGACCCCTCGCAGACCACGCAATTGGCCGGAATGGACGTTGAGGACGCCTTGGACAAGGATTCCACGAAGGAAGTCGCTTCCC TCCCGAACAAGTCGACTATCTCCAAGACGGTTTCGTCGACTATTCCGCGCGAAACCATCCCGTTTCTCCACCTCCGGAAAAAGACCCCCGCC GGCGACTGGAAGTACGACCGCCAGCTCTCGTCGCTCTTTTTGGACGGCCTCGAGAAAGCCGCCTTCAACGGCGTTACGTTCAAGGACAAGTA CGTCCTCATCACCGGCGCGGGCAAGGGCTCCATTGGCGCCGAGGTCCTCCAGGGCTTGCTCCAGGGCGGCGCGAAAGTCGTGGTCACGACTT CTCGATTCTCGAAGCAGGTCACCGACTACTACCAGTCGATCTACGCCAAGTACGGCGCCAAGGGTTCCACCTTGATCGTCGTCCCGTTCAAT CAAGGCTCGAAGCAAGATGTCGAAGCCCTCATTGAGTTCATTTACGACACGGAGAAGAACGGCGGCCTCGGCTGGGACTTGGACGCGATCAT TCCGTTCGCCGCCATTCCGGAGCAGGGAATCGAGCTCGAGCACATCGACTCCAAGTCCGAATTCGCCCACCGGATCATGCTCACCAACATCC TCCGCATGATGGGCTGCGTCAAGAAGCAGAAGTCCGCCCGCGGCATCGAAACCCGCCCGGCCCAGGTCATCCTCCCCATGTCGCCGAACCAT GGCACCTTCGGAGGAGATGGAATGTACTCGGAGTCCAAGCTGTCGCTCGAAACCCTCTTCAACCGCTGGCACTCGGAATCGTGGGCCAACCA GCTCACCGTCTGCGGCGCCATCATCGGCTGGACCCGCGGCACTGGCCTCATGTCGGCCAACAACATCATTGCGGAGGGCATTGAGAAGATGG GAGTCCGCACGTTCTCGCAGAAGGAGATGGCGTTCAACCTCTTGGGCCTCCTCACGCCGGAAGTCGTCGAATTGTGCCAAAAGTCCCCCGTC ATGGCGGACCTCAACGGAGGCTTGCAATTCGTCCCGGAGCTCAAGGAATTCACGGCCAAGTTGCGCAAGGAACTCGTTGAGACATCGGAGGT CCGGAAGGCGGTTTCGATTGAGACTGCCCTCGAACACAAGGTCGTCAACGGTAACTCGGCCGACGCCGCGTACGCCCAGGTGGAGATCCAGC CGCGCGCTAACATCCAGTTGGATTTCCCGGAGTTGAAGCCCTACAAGCAAGTCAAGCAGATCGCCCCGGCGGAACTCGAGGGCCTCCTTGAC TTGGAACGCGTCATCGTCGTCACTGGCTTCGCCGAGGTGGGCCCGTGGGGATCTGCTAGGACTAGGTGGGAAATGGAGGCCTTCGGAGAATT TTCGTTGGAAGGCTGCGTCGAGATGGCTTGGATCATGGGCTTCATCTCGTACCACAACGGCAACCTCAAGGGCCGCCCCTACACGGGCTGGG TCGACTCGAAAACCAAGGAACCGGTCGACGACAAGGACGTCAAGGCGAAGTACGAGACTTCCATCCTCGAACATTCGGGCATCCGGCTCATT GAGCCGGAGTTGTTCAATGGCTACAACCCGGAAAAGAAAGAGATGATCCAGGAGGTCATCGTGGAGGAGGATTTGGAGCCGTTTGAGGCCTC GAAGGAGACTGCCGAGCAATTCAAGCATCAGCACGGCGACAAGGTCGACATTTTCGAGATCCCGGAGACTGGCGAGTACTCGGTTAAGCTCC TCAAGGGTGCCACCCTCTACATCCCCAAGGCCTTGCGCTTCGACCGCTTGGTCGCGGGACAGATCCCGACCGGCTGGAACGCTAAGACGTAC GGCATCAGCGACGACATCATTTCCCAGGTCGACCCGATCACCCTCTTCGTCTTGGTTTCCGTCGTCGAAGCCTTCATTGCCTCGGGCATCAC CGACCCGTACGAAATGTACAAGTACGTTCACGTTTCGGAGGTCGGCAACTGCTCCGGCTCGGGAATGGGCGGCGTTTCGGCCCTCCGCGGCA TGTTTAAGGACCGCTTCAAGGACGAACCTGTCCAGAATGACATCCTTCAGGAGTCCTTCATCAACACGATGTCGGCTTGGGTCAACATGCTC TTGATCTCTTCCTCGGGCCCGATCAAGACCCCGGTCGGCGCCTGCGCTACCTCGGTCGAGAGCGTCGACATTGGCGTCGAAACCATTCTCTC GGGCAAGGCCCGGATCTGCATTGTCGGCGGCTACGACGATTTCCAGGAGGAGGGCTCGTTCGAATTCGGTAACATGAAGGCCACGTCCAACA CCCTCGAAGAGTTCGAGCATGGCCGCACCCCGGCTGAGATGTCGCGCCCCGCCACGACGACCCGCAACGGATTCATGGAAGCCCAGGGAGCT GGCATCCAGATTATCATGCAGGCCGACCTCGCGCTCAAAATGGGCGTCCCGATCTACGGCATCGTTGCCATGGCCGCCACCGCCACCGACAA GATCGGCCGCTCGGTTCCTGCTCCTGGTAAGGGCATCCTCACCACCGCTCGCGAGCATCACTCGTCGGTTAAGTACGCGTCCCCCAACCTCA ACATGAAGTACCGCAAGCGCCAGCTCGTCACCCGGGAGGCTCAGATCAAGGACTGGGTCGAGAACGAGCTCGAAGCGCTCAAGTTGGAGGCT GAAGAGATCCCGTCGGAAGATCAGAACGAGTTCTTGCTCGAACGCACCCGCGAAATCCACAACGAGGCCGAGTCGCAGTTGCGCGCCGCGCA GCAGCAGTGGGGTAACGACTTTTACAAGCGCGACCCGCGCATTGCCCCGCTCAGGGGAGCTTTGGCTACCTACGGCCTCACCATTGACGACT TGGGCGTCGCCTCGTTTCACGGCACCTCGACCAAGGCCAACGACAAGAACGAGTCGGCCACCATCAACGAAATGATGAAGCACCTTGGTCGC TCGGAGGGCAACCCCGTCATCGGAGTCTTTCAGAAGTTCTTGACGGGCCACCCGAAGGGCGCGGCGGGTGCCTGGATGATGAACGGAGCCCT CCAGATCCTCAACTCGGGCATTATCCCGGGCAACCGCAACGCGGACAACGTCGACAAGATTTTGGAGCAGTTCGAATACGTCCTCTACCCGA GCAAGACTCTCAAGACGGACGGCGTCCGCGCGGTTTCCATCACCTCGTTCGGCTTCGGCCAGAAGGGTGGCCAGGCCATTGTCGTCCATCCG GACTACCTTTACGGAGCCATCACGGAGGACCGGTACAACGAGTACGTTGCCAAGGTTTCGGCTCGCGAGAAGTCTGCCTACAAGTTCTTCCA CAACGGTATGATCTACAACAAGTTGTTCGTTAGCAAGGAACACGCCCCGTACACCGATGAACTCGAAGAAGATGTCTACCTCGACCCCCTCG CCCGAGTCTCCAAGGACAAAAAGTCGGGATCGCTCACTTTCAACTCTAAGAACATCCAGAGCAAGGACTCGTACATCAACGCCAACACCATT GAAACCGCGAAGATGATCGAAAACATGACGAAGGAGAAGGTCAGCAACGGTGGCGTCGGAGTTGACGTCGAGTTGATCACCTCGATCAACGT CGAAAATGACACCTTCATCGAGCGGAACTTCACCCCTCAGGAGATTGAGTACTGCTCGGCCCAACCGTCGGTCCAATCGTCCTTCGCTGGTA CCTGGTCGGCGAAGGAAGCCGTTTTCAAGAGCTTGGGAGTCAAGTCTCTCGGCGGCGGCGCGGCCCTCAAGGACATTGAGATCGTTCGCGTC AACAAAAACGCCCCGGCTGTCGAGCTTCACGGCAACGCCAAGAAGGCTGCCGAGGAGGCCGGAGTCACCGACGTCAAAGTCTCCATCTCGCA CGACGACTTGCAGGCCGTCGCTGTCGCCGTTTCGACTAAGAAGTAGTGAAGAGC SEQ ID NO:56 Scer fusFAS AA sequenceMDAYSTRPLTLSHGSLEHVLLVPTASFFIASQLQEQFNKILPEPTEGFAADDEPTTPAELVGKFLGYVSSLVEPSKVGQFDQVLNLCLTEFE NCYLEGNDIHALAAKLLQENDTTLVKTKELIKNYITARIMAKRPFDKKSNSALFRAVGEGNAQLVAIFGGQGNTDDYFDELRDLYQTYHVLV GDLIKFSAETLSELIRTTLDAEKVFTQGLNILEWLENPSNTPDKDYLLSIPISCPLIGVIQLAHYVVTAKLLGFTPGELRSYLKGATGHSQG LVTAVAIAETDSWESFFVSVRKAITVLFFIGVRCYEAYPNTSLPPSILEDSLENNEGVPSPMLSISNLTQEQVQDYVNKTNSHLPAGKQVEI SLVNGAKNLVVSGPPQSLYGLNLTLRKAKAPSGLDQSRIPFSERKLKFSNRFLPVASPFHSHLLVPASDLINKDLVKNNVSFNAKDIQIPVY DTFDGSDLRVLSGSISERIVDCIIRLPVKWETTTQFKATHILDFGPGGASGLGVLTHRNKDGTGVRVIVAGTLDINPDDDYGFKQEIFDVTS NGLKKNPNWLEEYHPKLIKNKSGKIFVETKFSKLIGRPPLLVPGMTPCTVSPDFVAATTNAGYTIELAGGGYFSAAGMTAAIDSVVSQIEKG STFGINLIYVNPFMLQWGIPLIKELRSKGYPIQFLTIGAGVPSLEVASEYIETLGLKYLGLKPGSIDAISQVINIAKAHPNFPIALQWTGGR GGGHHSFEDAHTPMLQMYSKIRRHPNIMLIFGSGFGSADDTYPYLTGEWSTKFDYPPMPFDGFLFGSRVMIAKEVKTSPDAKKCIAACTGVP DDKWEQTYKKPTGGIVTVRSEMGEPIHKIATRGVMLWKEFDETIFNLPKNKLVPTLEAKRDYIISRLNADFQKPWFATVNGQARDLATMTYE EVAKRLVELMFIRSTNSWFDVTWRTFTGDFLRRVEERFTKSKTLSLIQSYSLLDKPDEAIEKVFNAYPAAREQFLNAQDIDHFLSMCQNPMQ KPVPFVPVLDRRFEIFFKKDSLWQSEHLEAVVDQDVQRTCILHGPVAAQFTKVIDEPIKSIMDGIHDGHIKKLLHQYYGDDESKIPAVEYFG GESPVDVQSQVDSSSVSEDSAVFKATSSTDEESWFKALAGSEINWRHASFLCSFITQDKMFVSNPIRKVFKPSQGMVVEISNGNTSSKTVVT LSEPVQGELKPTVILKLLKENIIQMEMIENRTMDGKPVSLPLLYNFNPDNGFAPISEVMEDRNQRIKEMYWKLWIDEPFNLDFDPRDVIKGK DFEITAKEVYDFTHAVGNNCEDFVSRPDRTMLAPMDFAIVVGWRAIIKAIFPNTVDGDLLKLVHLSNGYKMIPGAKPLQVGDVVSTTAVIES VVNQPTGKIVDVVGTLSRNGKPVMEVTSSFFYRGNYTDFENTFQKTVEPVYQMHIKTSKDIAVLRSKEWFQLDDEDFDLLNKTLTFETETEV TFKNANIFSSVKCFGPIKVELPTKETVEIGIVDYEAGASHGNPVVDFLKRNGSTLEQKVNLENPIPIAVLDSYTPSTNEPYARVSGDLNPIH VSRHFASYANLPGTITHGMFSSASVRALIENWAADSVSSRVRGYTCQFVDMVLPNTALKTSIQHVGMINGRKLIKFETRNEDDVVVLTGEAE IEQPVTTFVFTGQGSQEQGMGMDLYKTSKAAQDVWNRADNHFKDTYGFSILDIVINNPVNLTIHFGGEKGKRIRENYSAMIFETIVDGKLKT EKIFKEINEHSTSYTFRSEKGLLSATQFTQPALTLMEKAAFEDLKSKGLIPADATFAGHSLGEYAALASLADVMSIESLVEVVFYRGMTMQV AVPRDELGRSNYGMIAINPGRVAASFSQEALQYVVERVGKRTGWLVEIVNYNVENQQYVAAGDLRALDTVTNVLNFIKLQKIDIIELQKSLS LEEVEGHLFEIIDEASKKSAVKPRPLKLERGFACIPLVGISVPFHSTYLMNGVKPFKSFLKKNIIKENVKVARLAGKYIPNLTAKPFQVTKE YFQDVYDLTGSEPIKEIIDNWEKDEWGSPEVEQELAHILLTELLAYQFASPVRWIETQDVFLKDFNTERVVEIGPSPTLAGMAQRTLKNKYE SYDAALSLHREILCYSKDAKEIYYTPDPSELAAKEEPAKEEAPAPTPAASAPAPAAAAPAPVAAAAPAAAAAEIADEPVKASLLLHVLVAHK LKKSLDSIPMSKTIKDLVGGKSTVQNEILGDLGKEFGTTPEKPEETPLEELAETFQDTFSGALGKQSSSLLSRLISSKMPGGFTITVARKYL QTRWGLPSGRQDGVLLVALSNEPAARLGSEADAKAFLDSMAQKYASIVGVDLSSAASASGAAGAGAAAGAAMIDAGALEEITKDHKVLARQQ LQVLARYLKMDLDNGERKFLKEKDTVAELQAQLDYLNAELGEFFVNGVATSFSRKKARTFDSSWNWAKQSLLSLYFEIIHGVLKNVDREVVS EAINIMNRSNDALIKFMEYHISNTDETKGENYQLVKTLGEQLIENCKQVLDVDPVYKDVAKPTGPKTAIDKNGNITYSEEPREKVRKLSQYV QEMALGGPITKESQPTIEEDLTRVYKAISAQADKQDISSSTRVEFEKLYSDLMKFLESSKEIDPSQTTQLAGMDVEDALDKDSTKEVASLPN KSTISKTVSSTIPRETIPFLHLRKKTPAGDWKYDRQLSSLFLDGLEKAAFNGVTFKDKYVLITGAGKGSIGAEVLQGLLQGGAKVVVTTSRF SKQVTDYYQSIYAKYGAKGSTLIVVPFNQGSKQDVEALIEFIYDTEKNGGLGWDLDAIIPFAAIPEQGIELEHIDSKSEFAHRIMLTNILRM MGCVKKQKSARGIETRPAQVILPMSPNHGTFGGDGMYSESKLSLETLFNRWHSESWANQLTVCGAIIGWTRGTGLMSANNIIAEGIEKMGVR TFSQKEMAFNLLGLLTPEVVELCQKSPVMADLNGGLQFVPELKEFTAKLRKELVETSEVRKAVSIETALEHKVVNGNSADAAYAQVEIQPRA NIQLDFPELKPYKQVKQIAPAELEGLLDLERVIVVTGFAEVGPWGSARTRWEMEAFGEFSLEGCVEMAWIMGFISYHNGNLKGRPYTGWVDS KTKEPVDDKDVKAKYETSILEHSGIRLIEPELFNGYNPEKKEMIQEVIVEEDLEPFEASKETAEQFKHQHGDKVDIFEIPETGEYSVKLLKG ATLYIPKALRFDRLVAGQIPTGWNAKTYGISDDIISQVDPITLFVLVSVVEAFIASGITDPYEMYKYVHVSEVGNCSGSGMGGVSALRGMFK DRFKDEPVQNDILQESFINTMSAWVNMLLISSSGPIKTPVGACATSVESVDIGVETILSGKARICIVGGYDDFQEEGSFEFGNMKATSNTLE EFEHGRTPAEMSRPATTTRNGFMEAQGAGIQIIMQADLALKMGVPIYGIVAMAATATDKIGRSVPAPGKGILTTAREHHSSVKYASPNLNMK YRKRQLVTREAQIKDWVENELEALKLEAEEIPSEDQNEFLLERTREIHNEAESQLRAAQQQWGNDFYKRDPRIAPLRGALATYGLTIDDLGV ASFHGTSTKANDKNESATINEMMKHLGRSEGNPVIGVFQKFLTGHPKGAAGAWMMNGALQILNSGIIPGNRNADNVDKILEQFEYVLYPSKT LKTDGVRAVSITSFGFGQKGGQAIVVHPDYLYGAITEDRYNEYVAKVSAREKSAYKFFHNGMIYNKLFVSKEHAPYTDELEEDVYLDPLARV SKDKKSGSLTFNSKNIQSKDSYINANTIETAKMIENMTKEKVSNG...

Claims

CLAIMS We claim:

1. A non-naturally occurring microorganism, wherein the microorganism produces polyol esters of fatty acids (PEFA), said PEFA comprising: (i) a polyol head portion; and (ii) a fatty acid tail portion having a carbon chain length, wherein the average carbon chain length of the fatty acid tail portion of the PEFA produced by the non-naturally occurring microorganism is altered in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived.

2. The non-naturally occurring microorganism according to claim 1, wherein the fatty acid tail portion of the PEFA has an average carbon chain length that is shorter than the average carbon chain length of the PEFA produced by the parent microorganism.

3. The non-naturally occurring microorganism according to claim 1, wherein the hydrophilic-lipophilic balance (HLB) of the PEFA produced by the non-naturally occurring microorganism is altered such that the PEFA are more hydrophilic than PEFA that are produced by the parent microorganism from which the non-naturally occurring microorganism is derived.

4. The non-naturally occurring microorganism according to claim 1 , wherein the average carbon chain length of fatty acid tail portions of PEFA produced by the non-naturally occurring microorganism is C8 to C18, C8 to C16, C8 to C14, C8 to C12, or C8 to C10.

5. The non-naturally occurring microorganism according to claim 4, wherein the average carbon chain length of fatty acid tail portions of PEFA produced by the non-naturally occurring microorganism is C8 to C14, C10 to C14, C12 to C16, or C10 to C18.

6. The non-naturally occurring microorganism according to claim 3, wherein the fatty acid tail portions of the PEFA comprise at least about 10% C8-C12, at least about 20% C8-C12, at least about 30% C8-C12, at least about 40% C8-C12, at least about 50% C8-C12, at least about 60% C8-C12, at least about 70% C8-C12, at least about 80% C8-C12, at least about 90% C8-C12, at least about 10% C10-C14, at least about 20% C10-C14, at least about 30% C10-C14, at least about 40% C10-C14, at least about 50% C10-C14, at least about 60% C10-C14, at least about 70% C10- C14, at least about 80% C10-C14, at least about 90% C10-C14, at least about 10% C12-C16, at least about 20% C12-C16, at least about 30% C12-C16, at least about 40% C12-C16, at least about 50% C12-C16, at least about 60% C12-C16, at least about 70% C12-C16, at least about 80% C12- C16, or at least about 90% C12-C16.

7. The non-naturally occurring microorganism according to claim 1, wherein the HLB value of the PEFA is about 6 to about 10, about 10 to about 14, about 14 to about 18, or about 16 to about 20.

8. The non-naturally occurring microorganism according to claim 1, wherein the non-naturally occurring microorganism comprises an expressed heterologous polynucleotide that comprises a nucleotide coding sequence that encodes an enzyme in a biosynthetic pathway for production of PEFA with preferred fatty acid carbon chain lengths.

9. The non-naturally occurring microorganism according to claim 8, wherein the nucleotide coding sequence encodes a polypeptide that has thioesterase enzymatic activity.

10. The non-naturally occurring microorganism according to claim 9, wherein the nucleotide coding sequence encodes a thioesterase enzyme.

11. The non-naturally occurring microorganism according to claim 9, wherein the nucleotide coding sequence encodes a type 1 fatty acid synthase enzyme.

12. The non-naturally occurring microorganism according to claim 10, wherein the nucleotide coding sequence encodes a thioesterase enzyme fused to the coding sequence of all or part of a type 1 fatty acid synthase enzyme.

13. The non-naturally occurring microorganism according to claim 8, wherein the nucleotide coding sequence encodes all or a portion of a type 1 fatty acid synthase enzyme that has been modified to produce a greater amount of C8 to C14 acyl-CoA molecules in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived.

14. The non-naturally occurring microorganism according to any of claim 9, wherein the thioesterase enzyme activity increases the production of PEFA with fatty acid carbon chain length C8 to C14 in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived.

15. The non-naturally occurring microorganism according to claim 11, wherein the nucleotide coding sequence encodes the thioesterase enzyme UcFat1B (Uniprot Q41635) from Umbellularia californica (SEQ ID NO:1), the thioesterase enzyme CvFat1B (Uniprot G3ESU9) from Cuphea viscosissima (SEQ ID NO:2), or the thioesterase enzyme FatB4 from Cinnamomum camphora (SEQ ID NO:3), or a polypeptide with at least about 60% sequence identity thereof that comprises thioesterase enzyme activity.

16. The non-naturally occurring microorganism according to claim 9, wherein the nucleotide coding sequence encodes the all or part of a type 1 fatty acid synthase, wherein a ketoacyl synthase domain, a malonyl / palmitoyl transferase domain, and / or an acetyl-transferase domain of the fatty acid synthase is mutated relative to the wild type sequence.

17. The non-naturally occurring microorganism according to claim 8, wherein the nucleotide coding sequence is codon optimized for expression in the host microorganism.

18. The non-naturally occurring microorganism according to claim 17, wherein the nucleotide coding sequence is codon optimized for expression in the host microorganism in accordance with the codon usage of Table 1 or Table 2.

19. The non-naturally occurring microorganism according to claim 18, 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.

20. The non-naturally occurring microorganism according to claim 1, wherein the non-naturally occurring microorganism comprises a knocked out endogenous gene or reduced expression of an endogenous gene that encodes an enzyme that catalyzes b-oxidation of fatty acids.

21. The non-naturally occurring microorganism according to claim 20, comprising knocked out or reduced expression of endogenous multifunctional enzyme type 2 (MFE-2), wherein peroxisomal b- oxidation of fatty acids in the non-naturally occurring microorganism is eliminated or reduced in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived.

22. The non-naturally occurring microorganism according to claim 20, comprising knocked out or reduced expression of endogenous enoyl-CoA hydratase, wherein mitochondrial b-oxidation of fatty acids in the non-naturally occurring microorganism is eliminated or reduced in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived.

23. A non-naturally occurring microorganism, wherein the microorganism produces polyol esters of fatty acids (PEFA), said PEFA comprising: (i) a polyol head portion; and (ii) a fatty acid tail portion having a carbon chain length, wherein the average carbon chain length of the fatty acid tail portion of the PEFA produced by the non-naturally occurring microorganism is altered in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived, wherein the non-naturally occurring microorganism comprises a knocked out endogenous gene or reduced expression of an endogenous gene that encodes an enzyme that catalyzes b-oxidation of fatty acids.

24. The non-naturally occurring microorganism according to claim 23, comprising knocked out or reduced expression of endogenous multifunctional enzyme type 2 (MFE-2), wherein peroxisomal b- oxidation of fatty acids in the non-naturally occurring microorganism is eliminated or reduced in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived.

25. The non-naturally occurring microorganism according to claim 23, comprising knocked out or reduced expression of endogenous enoyl-CoA hydratase, wherein mitochondrial b-oxidation of fatty acids in the non-naturally occurring microorganism is eliminated or reduced in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived.

26. The non-naturally occurring microorganism according to claim 23, wherein the non-naturally occurring microorganism comprises an expressed heterologous polynucleotide that comprises a nucleotide coding sequence that encodes an enzyme in a biosynthetic pathway for production of PEFA with preferred fatty acid carbon chain lengths.

27. The non-naturally occurring microorganism according to any of claim 26, wherein the nucleotide coding sequence encodes a polypeptide that has thioesterase enzymatic activity.

28. The non-naturally occurring microorganism according to claim 27, wherein the nucleotide coding sequence encodes a thioesterase enzyme.

29. The non-naturally occurring microorganism according to claim 27, wherein the nucleotide coding sequence encodes a type 1 fatty acid synthase enzyme.

30. The non-naturally occurring microorganism according to claim 28, wherein the nucleotide coding sequence encodes a thioesterase enzyme fused to the coding sequence of all or part of a type 1 fatty acid synthase enzyme.

31. The non-naturally occurring microorganism according to claim 27, wherein the thioesterase enzyme activity increases the production of PEFA with fatty acid carbon chain length C8 to C14 in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived .

32. The non-naturally occurring microorganism according to claim 26, wherein the nucleotide coding sequence encodes all or a portion of a type 1 fatty acid synthase enzyme that has been modified to produce a greater amount of C8 to C14 acyl-CoA molecules in comparison to a parent microorganism from which the non-naturally occurring microorganism is derived.

33. The non-naturally occurring microorganism according to any of claims 1 to 32, wherein the microorganism is from the subkingdom Dikarya.

34. The non-naturally occurring microorganism according to claim 33, wherein the microorganism is from the phylum Ascomycota or Basidiomycota.

35. The non-naturally occurring microorganism according to claim 34, wherein the microorganism is from the order Sporidiobolales.

36. The non-naturally occurring microorganism according to claim 35, wherein the microorganism is from a genus selected from the group consisting of: Rhodotorula, Rhodosporidiobolus, and Sporobolomyces.

37. The non-naturally occurring microorganism according to claim 36, wherein the microorganism is a species selected from the group consisting of: 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.

38. The non-naturally occurring microorganism according to claim 37, wherein the microorganism is a strain selected from the group consisting of: Rhodotorula babjevae strain NRRL Y-67018, Rhodotorula babjevae strain NRRL Y-67017, Rhodotorula babjevae strain UCDFST 68-916.1, Rhodotorula babjevae strain UCDFST 67-458, Rhodotorula babjevae strain UCDFST 05-736, Rhodotorula diobovata strain UCDFST 04-830, Rhodotorula diobovata strain NRRL Y-67015, Rhodotorula kratochvilovae strain NRRL Y-67016, Rhodotorula paludigena strain NRRL Y-67012, Rhodotorula paludigena strain UCDFST 82-646.2, Rhodotorula paludigena strain UCDFST 81-492, Rhodotorula aff. paludigena strain NRRL Y-67009, Rhodotorula sphaerocarpa strain NRRL Y- 67010, Rhodotorula dairenensis strain NRRL Y-67011, Rhodosporidiobolus aff. colostri strain NRRL Y-67014, Rhodosporidiobolus aff. colostri NRRL Y-67014, Rhodosporidiobolus aff. nylandii strain NRRL Y-67013, Rhodosporidiobolus ruineniae NRRL Y-17302, Rhodotorula taiwanensis strain MD1149, and Rhdodotorula mucilaginosa strain 50-3-19 / 208.

39. A microbial culture, said culture comprising: one or a consortium of two or more non-naturally occurring microorganism(s) according to any of claims 1 to 32; a culture medium; and at least about 1 g / L PEFA that is produced by the microorganism.

40. A microbial culture according to claim 39, wherein the microorganism(s) secrete the PEFA into the culture medium.

41. A method for producing PEFA, said method comprising: growing one or a consortium of two or more microorganisms according to any of claims 1 to 32 in a culture medium under conditions that are suitable for growth of the microorganism(s) and production of biosynthetic products, wherein PEFA are produced by the microorganisms.

42. The method according to claim 41, wherein the PEFA are secreted into the culture medium, and the method further comprises: recovering the PEFA product from the culture medium.

43. The method according to claim 42, wherein the PEFA are denser than water, and recovery of the PEFA comprises separation from the culture medium by centrifugation, continuous decanting, or passive settling.

44. The method according to any of claim 42, wherein at least about 1 g / L of the PEFA are secreted into the culture medium.

45. The method according to claim 41, wherein the PEFA comprise a density of about 1.00 g / mL to about 1.10 g / mL.

46. The method according to claim 42, wherein the method is performed as a batch, fed batch, or continuous process.

47. A composition comprising PEFA that are produced according to the method of claim 41.

48. The composition according to claim 47, wherein the composition is a surfactant.

Images