Systems, methods, processes, and microorganisms for the production of lipids from carbonaceous feedstock and industrial effluents

Abstract

The present invention provides inter alia, methods and processes for the high yield lipid production by development of new strains through a combination of Adapted Laboratory Evolution, Genetic Engineering and Bioprocess Engineering. Methods and processes of producing lipids through sequential steps, the method comprising the steps of a) providing a first microbial culture in an anaerobic bioreactor; b) exposing the first microbial culture in the anaerobic bioreactor to a source of carbon dioxide and hydrogen under conditions which allow microbial culture to produce carbonaceous feedstock; c) providing a second microbial culture in an aerobic bioreactor with carbonaceous feedstock under conditions in which the second microbial culture can produce a lipid; and; harvesting the lipid from step c).

Description

Attorney Docket No.11833-014WO1 ^ SYSTEMS, METHODS, PROCESSES, AND MICROORGANISMS FOR THE PRODUCTION OF LIPIDS FROM CARBONACEOUS FEEDSTOCK AND INDUSTRIAL EFFLUENTS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No.63 / 704,112, filed October 7, 2024, incorporated herein by reference in its entirety. SEQUENCE LISTING STATEMENT

[0002] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via Patent Center encoded as XML in UTF-8 text. The electronic document, created on October 2, 2025, is entitled “11833-014WO1_ST26.xml”, and is 167,770 bytes in size. FIELD

[0003] The present invention relates to methods of producing lipids from carbonaceous feedstock for use in sustainable fuels, palm oil replacements, lubricants, personal care products, and other sustainable chemicals. BACKGROUND

[0004] Sustainably produced lipids are desirable for a number of industries, for example those involving the sustainable production of nutrients, nutraceuticals, personal care and cosmetic products. As a further example, in pursuit of the Sustainable Aviation Fuel (SAF) Grand Challenge set by the US Department of Energy, it has become clear that feedstock will be the primary constraint to supply stated 2030 and 2050 goals for SAF production.2030 objectives include 3 billion gallons per year of SAF production. The use of hydrotreated Esters and Fatty Acids (HEFA) is the most commercially active SAF production method around the world, utilizing fats, oils, and greases to be transformed into diesel and jet fuel. This method requires 8.5 pounds per gallon of fuel produced, meaning 2030 objectives supplied solely by HEFA would require 25.5 billion pounds of feedstock. Not all fats, oils, and greases are made the same, however – they must be low carbon Intensity as well to produce desired emissions reductions and qualify for monetary incentives.

[0005] The United States alone imports almost 5 billion pounds of this low carbon intensity feedstock, primarily from Australia (tallow) and China (used cooking oil) and is on-track to have yet another record year in 2024 of imports of this highly desired feedstock. To help bridge this gap both in the United States and worldwide, novel means of providing ^Attorney Docket No.11833-014WO1 ^ sustainable lipid feedstock are required – to be used in the production of HEFA fuels, as well as other commodities such as sustainable chemicals and lubricants.

[0006] Further challenges are faced in the palm oil supply chain, such as environmental and sustainability issues including deforestation and loss of biodiversity. Given these challenges, sustainably produced lipids (produced via novel processes) are also desirable as a potential replacement for vegetable oil, including soybean oil, palm oil, coconut oil, cacao butter, etc. SUMMARY

[0007] The present invention relates to microorganisms and bioprocesses for the production of lipids, including oils, in a sustainable and efficient manner. In certain embodiments, the invention provides engineered microorganisms such as yeast, fungi, or bacteria, including specific engineered strains, which can be cultivated individually or as part of a microbial consortium. These engineered microorganisms may be maintained in culture, including high- density cultures with optical densities of 140 or greater, and in some embodiments the consortia are capable of increasing lipid productivity to more than 1.3 g / L / h when glycerol or a combination of glycerol and volatile fatty acids and carbohydrates are used as a carbon source.

[0008] The invention further provides methods for producing lipids using multi-stage bioprocesses that integrate anaerobic and aerobic microbial systems. In one embodiment, a first microbial culture is grown under anaerobic conditions in a bioreactor and is exposed to a source of carbon dioxide and hydrogen, resulting in the production of a carbonaceous feedstock. This feedstock which may include short chain fatty acids, formate, acetate, propionate, pyruvate, butyrate, acetic acid, lactate, methanol, ethanol, propanol, butanol, amino acids, glucose, xylose and sucrose, is subsequently transferred to a second bioreactor containing an aerobic microbial culture. Under aerobic conditions, the second microbial culture utilizes the carbonaceous feedstock to produce lipids, which can then be harvested. The carbon dioxide source may be supplied as a gas stream, such as syngas or biogenic carbon dioxide, or in the form of salts such as carbonate or bicarbonate produced by converting a portion of carbon dioxide into salts in the liquid phase prior to exposure to the first culture.

[0009] The microbial cultures employed in these processes may include consortia of anaerobic and aerobic microorganisms. The anaerobic consortium may comprise acetogenic microorganisms such as species from the Moorella, Sporomusa, or Acetobacterium genera, including Moorella thermoacetica, Sporomusa silvacetica, and Acetobacterium woodii. The aerobic consortium may include oleaginous fungi or bacteria, including yeasts from genera such as Cutaneotrichosporon, Yarrowia, Rhodotorula, Cryptococcus, Lipomyces, Debaryomyces, or Trichosporon, and species including Cutaneotrichosporon oleaginosus, ^Attorney Docket No.11833-014WO1 ^ Yarrowia lipolytica, Rhodotorula mucilaginosa, Rhodotorula glutinis, Cryptococcus curvatus, Debaryomyces hansenii, or Lipomyces starkeyi.

[0010] In certain embodiments, one or more of the microorganisms used in the anaerobic or aerobic stages are not naturally occurring and produce higher levels of carbonaceous feedstock or lipids than their naturally occurring counterparts. These strains may be genetically modified, subjected to directed evolution or adaptive laboratory evolution (ALE), or a combination of mutagenesis and genetic engineering. Engineered strains may demonstrate yield improvements of approximately 15–45% in either carbonaceous feedstock or lipid production. Such modifications may include the overexpression of enzymes such as malic enzyme (ME), diacylglycerol O-acyltransferases (DGA1 and DGA2), acetyl-CoA carboxylase, acetyl-CoA synthase, ATP-citrate lyase, NADPH-glyceraldehyde dehydrogenase, fatty acid synthase, or diacylglycerol O-acyltransferase, as well as the reduction or deletion of enzymes such as long-chain-fatty-acid-CoA ligase or multifunctional enzyme type-1. Additional genetic modifications may include overexpression of malonyl CoA-ACP transacylase, ^-ketoacyl-ACP synthase, ^-ketoacyl-ACP reductase, ^- hydroxybutyl-ACP dehydrogenase, enoyl-ACP reductase, and AMP deaminase, and / or suppression of multifunctional enzyme type-1 and peroxisome biogenesis factor 10. DNA sequences with high sequence identity (e.g., at least 95–99.9%) to SEQ ID NO: 6 or SEQ ID NO: 7 may also be incorporated to enhance production capabilities.

[0011] Following lipid production and harvesting, the lipids can be converted into fuel sources, such as sustainable aviation fuel, through hydrotreated esters and fatty acids (HEFA) processes. This may include steps of hydrodeoxygenation, cracking, and isomerization to yield usable fuel products. Lipids obtained by these methods may also serve as starting materials for a range of applications including bio-based fuels, lubricants, and personal care products.

[0012] The invention additionally provides methods for producing non-naturally occurring oleaginous microbes capable of enhanced lipid production. These methods may include exposing microbes to mutagenic conditions, such as Atmospheric and Room Temperature Plasma (ARTP) mutagenesis, followed by culturing, isolation, growth in nitrogen-limited media with glycerol, and selection based on lipid yield. Resulting strains can be confirmed as genetically distinct by sequencing and further characterized by optical density measurements or lipid quantification assays. These steps may be repeated iteratively to achieve optimized production strains.

[0013] Also provided is a bioreactor system for lipid production, comprising an anaerobic bioreactor containing a first microbial culture that produces a carbonaceous feedstock, and an aerobic bioreactor containing a second microbial culture that utilizes the feedstock to produce lipids. The system may employ microbial consortia in either stage and may ^Attorney Docket No.11833-014WO1 ^ incorporate engineered microorganisms with enhanced productivity. The method may employ a combination of ALE, genetic, metabolic and Bioprocess engineering with enhanced productivity.

[0014] Finally, the invention encompasses methods for producing lipids from a carbonaceous feedstock using aerobic microbial cultures alone, as well as lipids and fuels produced by any of the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The invention will now be more particularly described with reference to the following examples and figures, in which;

[0016] Figure 1 presents a flow diagram of an exemplary process for converting crude glycerol to oil.

[0017] Figure 2 presents a flow diagram of an exemplary process for converting CO2 and H2 to oil via acetate.

[0018] Figure 3 presents an exemplary process for converting CO2 and H2 to oil via acetate.

[0019] Figure 4A-B presents a comparison of growth rates between an evolved strain (ES) and a wild-type train (WT) of (A) C. oleaginosus and (B) Y. lipolytica.

[0020] Figure 5A-B presents a comparison of oil accumulation between an evolved strain (ES) and a wild-type train (WT) of (A) C. oleaginosus and (B) Y. lipolytica

[0021] Figure 6A-B presents a comparison of crude glycerol consumption between an evolved strain (ES) and a wild-type train (WT) of (A) C. oleaginosus and (B) Y. lipolytica.

[0022] Figure 7 presents the increase in oil productivity in evolved strains of C. oleaginosus after 4, 9 and 15 culture transfers, compared to their wild type (WT) counterpart. Biomass was measured by optical density (OD); oil content in cells was measured using Nile red dye relative fluorescent units (RFU).

[0023] Figure 8 shows an exemplary bioreactor setup and apparatus configuration.

[0024] Figure 9 shows A. woodii cell densities over time measured by OD600 and dry cell weight in bioreactor experiments CFF257, CFF265, and CFF267. Dry cell weight measurements were not yet taken in experiment CFF257, so only OD600data is shown to indicate cell density for that experiment.

[0025] Figure 10 shows measured acetate titers and measured cell densities over time in bioreactor experiments CFF257, CFF265, and CFF267.

[0026] Figure 11 shows instantaneous acetate production rates calculated by numerical differentiation and measured cell densities over time in bioreactor experiments CFF257, CFF265, and CFF267.

[0027] Figure 12 shows calculated average acetate production rates and measured cell densities over time in bioreactor experiments CFF257, CFF265, and CFF267. ^Attorney Docket No.11833-014WO1 ^

[0028] Figure 13 shows model-predicted partial growth associated acetate production over time with calculated acetate production rate and cell density over time in bioreactor experiment CFF267 after the draw and fill operation was performed. Production rate coefficients ^ and ^ were optimally calculated to be 11.7 and 2.3 respectively.

[0029] Figure 14 shows direct comparison of measured acetate titers over time in experiment CFF from the Sentia wine analyzer and HPLC analysis. Sentia analyzer results are scaled by a factor 1.28; the scaling factor was determined by least squares regression with the HPLC data.

[0030] Figure 15 shows repligen MicroKros module filtration efficiency and pressure drop across a range of total flow rates. Filtration efficiency is represented by the ratio between the retentate flow rate and filtrate flow rate at a given total flow rate.

[0031] Figure 16 shows the methodology flow chart used for mutant screening.

[0032] Figure 17 shows growth performance of selected mutants.

[0033] Figure 18 shows lipid accumulation in selected mutants.

[0034] Figure 19A-B compares the ALE-derived strain with the reference strain (C. oleaginosus ATCC 20509). Panel A: Microbial biomass production (dry cell weight) at 72 h and 96 h. Panel B: Lipid production at the same point.

[0035] Figure 20 shows that, prior to lipid accumulation, improved C. oleaginosus biomass titer through media optimization increases lipid productivity (Example 6). Solid circles- biomass titer on supplier A crude glycerol; open circle-biomass titer grown on supplier B crude glycerol; solid triangle-lipid productivity on supplier A crude glycerol; open triangle-lipid productivity on supplier B crude glycerol.

[0036] Figure 21 shows the use of acetic acid as co-substrate for high lipid productivity in Cutaneotrichosporon oleaginosus (Example 7). Open circles: Biomass growth on glucose and acetic acid; Green bars: Lipid productivity over fermentation time.

[0037] Figure 22 shows the mutant strain, designated as “Query,” was compared to the nucleotide sequence of the reference strain deposited in NCBI (SEQ ID NO: 12). A mutation was identified at nucleotide positions 747,416 within the gene COLE_00321 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819), as indicated by the arrow.

[0038] Figure 23 shows the mutant strain, designated as “Query,” was compared with the nucleotide sequence of the reference strain deposited in NCBI (SEQ ID NO: 15). A mutation was identified at nucleotide position 2,913,185 within the gene COLE_02845 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819), as indicated by the arrow.

[0039] Figure 24 shows the mutant strain, designated as “Query,” was compared with the nucleotide sequence of the reference strain deposited in NCBI (SEQ ID NO: 18). A mutation was identified at nucleotide position 1,506,518 within the gene COLE_06128 of the ^Attorney Docket No.11833-014WO1 ^ reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819), as indicated by the arrow.

[0040] Figure 25 shows the mutant strain, designated as “Query,” was compared with the nucleotide sequence of the reference strain deposited in NCBI (SEQ ID NO: 21). A mutation was identified at nucleotide position 493,871 within the gene COLE_06627 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819), as indicated by the arrow.

[0041] Figure 26 shows the mutant strain, designated as “Query,” was compared with the nucleotide sequence of the reference strain deposited in NCBI (SEQ ID NO: 24). A mutation was identified at nucleotide position 766,332 within the gene COLE_07529 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819), as indicated by the arrow.

[0042] Figure 27 shows the mutant strain, designated as “Query,” was compared with the nucleotide sequence of the reference strain deposited in NCBI. A mutation was identified at nucleotide position 2,185,025 downstream of the gene COLE_00879 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819), as indicated by the arrow. SEQ ID NO: 27 (query) and SEQ ID NO: 28 (Subject).

[0043] Figure 28: Increased lipid content in yeast as determined by the relative lipid content in cells normalized to biomass. ALE strain produced significantly higher lipid than the wild strain. DGA1 transformation using ALE strains as a chassis produced significantly higher lipid in comparison to ALE strains alone. CTRL strain1: Unmodified evolved strain used as chassis for modification of strains A3, A4, A5 and A9. CTRL strain 2: Non evolved strain Parental of CTRL strain 1. A3, A4, A5, A9: Transformed colonies of ALE and DGA-, selected from 12 different colonies after transformation. ^ DETAILED DESCRIPTION Definitions

[0044] By “microorganism” is meant any unicellular or multicellular microscopic organism, including but not limited to bacteria, fungi, yeast, archaea, and protozoa, whether naturally occurring, genetically modified, engineered, or mutated. The term encompasses wild-type strains, laboratory strains, industrial strains, synthetic organisms, and derivatives thereof.

[0045] By “culture” is meant a population of microorganisms maintained or propagated under controlled conditions in a nutrient medium, which may be liquid (suspension culture), solid (agar plates), or semi-solid. A culture may be batch, fed-batch, or continuous, and may be maintained under aerobic, anaerobic, or microaerophilic conditions.

[0046] By “consortium” is meant a population comprising two or more different strains, species, or genera of microorganisms co-cultivated or co-existing in a shared environment, where the organisms may interact metabolically, chemically, or physically to achieve a desired function such as enhanced product yield or conversion efficiency. ^Attorney Docket No.11833-014WO1 ^

[0047] By “lipid” is meant any organic compound that is hydrophobic or amphipathic and soluble in nonpolar solvents, including but not limited to oils, fats, waxes, triglycerides, free fatty acids, phospholipids, glycolipids, and sterols. Lipids produced by the invention may be intracellular or extracellular and may be harvested from cells or the culture medium.

[0048] By “oleaginous microorganism” is meant a microorganism capable of accumulating lipids to at least about 20% of its dry cell weight under appropriate cultivation conditions. Exemplary oleaginous microorganisms include certain species of yeasts, fungi, and bacteria known to produce and store significant quantities of triacylglycerols and related lipids.

[0049] By “cell lysis” is meant breaking open of a cell by any of the exemplary means: acid hydrolysis, sonication, milling, enzymatic, steam explosion.

[0050] By “anaerobic bioreactor” is meant a controlled fermentation vessel or cultivation system that operates in the substantial absence of molecular oxygen and is configured to support the growth and metabolism of anaerobic or facultatively anaerobic microorganisms.

[0051] By “aerobic bioreactor” is meant a cultivation system designed to provide sufficient oxygenation to support the metabolism of aerobic microorganisms, typically through sparging, agitation, or membrane aeration, and configured to enable microbial growth and product formation under aerobic conditions.

[0052] By “carbonaceous feedstock” is meant any organic or inorganic carbon-containing compound or mixture that can serve as a carbon source for microbial metabolism. This includes, but is not limited to, biomass. Exemplary carbonaceous feedstocks include, but are not limited to, formate, methanol, methane, acetate, acetic acid, ethanol, glycerol, butyrate, propionate, butanol, propanol, short-chain fatty acids, amino acids, pyruvate, lactate, glucose, xylose, sucrose, biomass hydrolysates, and industrial or biogenic carbon dioxide.

[0053] By “carbon source” is meant any compound containing carbon that is metabolizable by microorganisms to support growth, energy generation, or biosynthesis.

[0054] By “non-naturally occurring microorganism” is meant a microorganism that does not exist in nature in the same form and has been modified, selected, or evolved by human intervention to exhibit altered genotypic or phenotypic characteristics. Such microorganisms may be genetically engineered, chemically or physically mutagenized, subjected to directed evolution or adaptive laboratory evolution (ALE), or otherwise modified.

[0055] By “genetically modified microorganism” is meant a microorganism whose genome or genetic content has been intentionally altered by the insertion, deletion, substitution, or rearrangement of one or more nucleic acid sequences using recombinant DNA techniques, genome editing (e.g., CRISPR, TALENs, ZFNs), homologous recombination, or other molecular biology methods.

[0056] By “nucleic acid” is meant any polynucleotide molecule, whether composed of deoxyribonucleotides (DNA), ribonucleotides (RNA), analogs thereof (e.g., peptide nucleic ^Attorney Docket No.11833-014WO1 ^ acids (PNA), locked nucleic acids (LNA)), or mixtures thereof, and regardless of whether it is single-stranded, double-stranded, linear, circular, or branched. Nucleic acids may include coding sequences, non-coding sequences, regulatory elements, synthetic or engineered constructs, vectors, plasmids, and recombinant sequences. As used herein, “nucleic acid,” “nucleotide sequence,” “polynucleotide,” “gene,” and “coding sequence” may be used interchangeably, except where the context clearly dictates otherwise.

[0057] By “gene” is meant a nucleic acid sequence that encodes a functional product, which may be a protein, polypeptide, functional RNA (e.g., rRNA, tRNA, siRNA), or a combination thereof, and includes regulatory regions such as promoters, enhancers, terminators, untranslated regions, and introns.

[0058] By “protein” or “polypeptide” is meant a linear polymer of amino acids joined by peptide bonds, which may be naturally occurring, synthetic, recombinant, or engineered, and which may undergo post-translational modifications such as phosphorylation, glycosylation, or lipidation. The terms “protein,” “polypeptide,” and “peptide” may be used interchangeably, except where context indicates otherwise.

[0059] By “overexpression” is meant the production of a protein or enzyme in a microorganism at levels greater than those observed in the corresponding wild-type strain, achieved through genetic modification, promoter engineering, copy number increase, or other molecular techniques.

[0060] By “ablation” or “reduction of expression” is meant the partial or complete elimination or downregulation of a gene’s transcription or translation, resulting in decreased or absent protein production. This may be achieved through gene deletion, promoter attenuation, RNA interference, antisense expression, or CRISPR-based knockouts.

[0061] By “directed evolution” is meant a process for generating genetic diversity and selecting improved variants of microorganisms or enzymes by iterative cycles of mutagenesis and selection or screening under defined conditions.

[0062] By “adaptive laboratory evolution (ALE)” is meant a technique for evolving microorganisms by prolonged cultivation under defined selection pressures, resulting in spontaneous genetic adaptations that confer improved phenotypes such as increased tolerance, productivity, or metabolic efficiency.

[0063] By “mutagenesis” is meant any process that introduces heritable genetic changes into a microorganism, including but not limited to exposure to chemical mutagens, radiation, or physical methods such as Atmospheric and Room Temperature Plasma (ARTP).

[0064] By “Atmospheric and Room Temperature Plasma (ARTP)” is meant a non-thermal plasma technology generated under atmospheric pressure and near-ambient temperatures, producing reactive species capable of inducing mutations in microbial genomes without extensive cell lethality. ^Attorney Docket No.11833-014WO1 ^

[0065] By “metabolic engineering” is meant the modification of cellular metabolic pathways through genetic, enzymatic, regulatory, or process-level interventions to improve or redirect the production of desired metabolites such as lipids.

[0066] By “bioprocess engineering” is meant the design and optimization of biological production systems, including control of cultivation parameters (e.g., pH, temperature, pressure, agitation, dissolved gases, media formulation), bioreactor configuration, feeding strategies, and downstream processing, to maximize productivity and yield.

[0067] By “optical density (OD)” is meant a measure of microbial cell density in a culture, typically measured spectrophotometrically at a wavelength of 600 nm (OD600). An OD of at least 300 indicates a high-density culture.

[0068] By “fuel source” is meant a combustible composition derived from biological lipids, including biofuels such as biodiesel, renewable diesel, and sustainable aviation fuel (SAF).

[0069] By “hydrotreated esters and fatty acids (HEFA) process” is meant a chemical process for converting lipid feedstocks into hydrocarbon fuels, comprising catalytic hydrodeoxygenation followed by cracking and isomerization to yield hydrocarbon molecules suitable for use as transportation fuels.

[0070] By “hydrodeoxygenation” is meant a catalytic reaction that removes oxygen from lipid molecules, typically in the form of water, under a hydrogen atmosphere to yield long-chain hydrocarbons.

[0071] By “biogenic carbon dioxide” is meant carbon dioxide derived from biological sources such as fermentation, anaerobic digestion, or biomass combustion, as opposed to fossil- derived CO^.

[0072] By “syngas” is meant a mixture of carbon monoxide, carbon dioxide, and hydrogen produced from gasification of biomass or hydrocarbons and usable as a carbon and energy source for microbial conversion.

[0073] By “short-chain fatty acids (SCFAs)” is meant saturated or unsaturated fatty acids containing fewer than six carbon atoms, including but not limited to formic, acetic, propionic, butyric, and valeric acids.

[0074] By “biomass lysate” is meant a liquid fraction derived from the chemical, enzymatic, or mechanical breakdown of biomass material, containing soluble sugars, organic acids, amino acids, and other carbonaceous components suitable as feedstock.

[0075] By “nitrogen minimum medium (NMM)” is meant a defined culture medium containing limited nitrogen, used to promote lipid accumulation or to facilitate selection of strains with enhanced lipid productivity.

[0076] By “hydrocarbon fuel” is meant a mixture of aliphatic, aromatic, or cycloaliphatic hydrocarbons suitable for combustion in engines or turbines, derived from renewable or fossil sources. ^Attorney Docket No.11833-014WO1 ^

[0077] By “comprising” is meant “including but not limited to.” The term is intended to be open-ended and does not exclude the presence of additional elements, components, steps, or features beyond those specifically recited. Likewise, the terms “including,” “having,” and “containing” are used in an open and inclusive sense, meaning that other elements or steps may be present in addition to those explicitly recited.

[0078] By “consisting essentially of” is meant that the composition, process, or system includes the specified components or steps and those that do not materially affect the basic and novel characteristics of the invention.

[0079] By “consisting of” is meant that the composition, process, or system includes only the specified components or steps and no others. Processes for Lipid Production

[0080] The present invention relates to biotechnological methods for producing lipids from renewable carbon sources using engineered or selected microorganisms. Lipids— particularly triacylglycerols, fatty acids, and other neutral lipids—are essential industrial intermediates with broad applications, including use as feedstocks for biofuels, sustainable aviation fuel (SAF), lubricants, oleochemicals, surfactants, and personal care products. Microbial lipid production, often referred to as “single-cell oil” (SCO) production, offers a highly sustainable alternative to conventional petroleum-derived or plant-based oils, overcoming challenges such as land use, deforestation, and volatility in agricultural supply chains. By leveraging advances in metabolic engineering, bioprocess optimization, and systems biology, microbial platforms can convert diverse carbon sources—ranging from simple sugars to industrial waste streams—into high-value lipids with exceptional efficiency.

[0081] In general, microbial lipid production involves cultivating oleaginous microorganisms under conditions that promote lipid biosynthesis and accumulation. These organisms naturally store excess carbon in the form of neutral lipids when subjected to nutrient-limited, carbon-rich conditions. Modern biotechnological approaches have significantly enhanced this capability by modifying key metabolic pathways, optimizing enzyme expression, and improving substrate uptake. As a result, engineered strains can achieve high lipid production and can convert a wide variety of feedstocks—including sugars, organic acids, alcohols, syngas-derived intermediates, and even carbon dioxide—into valuable lipid products.

[0082] The processes described herein exploit these capabilities in both single-stage and multi-stage configurations. In some embodiments, a carbonaceous feedstock is supplied directly to an aerobic microbial culture capable of lipid synthesis. In other embodiments, a two-stage process is employed, wherein carbon dioxide and hydrogen are first converted to a soluble carbonaceous intermediate by anaerobic microorganisms, and the resulting feedstock is then used by aerobic organisms to produce lipids. These methods provide ^Attorney Docket No.11833-014WO1 ^ flexible, scalable, and carbon-efficient routes to lipid production that can be integrated with renewable energy, industrial waste streams, or carbon capture technologies, enabling a circular, low-carbon bioeconomy. I. Single-Stage Lipid Production Process Using Aerobic Microorganisms

[0083] In one aspect, the invention provides a method for the production of lipids using a single-stage bioconversion process in which a carbonaceous feedstock is metabolized by an aerobic microbial culture under controlled bioreactor conditions. The method comprises providing a carbonaceous feedstock to an aerobic microbial culture in a bioreactor under conditions in which the microbial culture is capable of producing a lipid, and subsequently harvesting the lipid from the culture.

[0084] The carbonaceous feedstock supplied to the culture may comprise any carbon source that can be assimilated by microorganisms and metabolized to yield lipids. Non- limiting examples include simple organic acids such as acetate and acetic acid; short-chain alcohols such as ethanol, methanol, propanol, and butanol; polyols such as glycerol; short- chain fatty acids such as propionate or butyrate; monosaccharides such as glucose and xylose; disaccharides such as sucrose; and more complex sources such as biomass lysates, lignocellulosic hydrolysates, or industrial and agricultural waste streams (e.g., corn syrup, food waste effluents). In certain embodiments, the carbon source may also include carbon dioxide, either directly assimilated by autotrophic organisms or indirectly converted to an assimilable form.

[0085] A further advantage of the present invention is that the process can be operated effectively using crude feedstocks, e.g. feedstocks obtained from industrial process having impurities present. For example, crude glycerol has the benefit of being widely available and at commodity scale, as well as being competitively priced compared to sugars as a starting material. Where glycerol is used in step (a), this is preferably crude glycerol.

[0086] Preferably, lipid production is performed under aerobic conditions. This step will generally be performed in a bioreactor (e.g. a bubble column) under aerobic conditions, and, generally, a constant temperature will be maintained in the bioreactor throughout the step. The carbonaceous feedstock may be mixed with water, a pH regulator (e.g. ammonium hydroxide, which is also a nitrogen source), and / or minerals (in order to promote growth and metabolism of the yeast, bacterium or fungus) prior to addition to the (preferably oleaginous) yeast, bacterium or fungus.

[0087] A filtration step can occur after production of a carbonaceous feedstock before lipid production. Such filtration has the purpose of concentrating lipid-rich yeast, bacterial or fungal biomass, in preparation for lipid production. Filtration may be performed using any suitable filtration means, e.g. a hollow-fiber cross-flow filtration module. ^Attorney Docket No.11833-014WO1 ^

[0088] The (preferably oleaginous) yeast, bacterium or fungus may be cultured such that lipid accumulates intracellularly and / or extracellularly. Generally, the lipid will accumulate intracellularly. As such, lipid production steps can comprise homogenization of biomass comprising cells of the yeast, bacterium or fungus, followed by extraction of the lipid.

[0089] The aerobic microbial culture comprises at least one oleaginous microorganism— organisms capable of accumulating lipids to more than 20% of their dry cell weight. These microbes may include bacteria or fungi, including yeast. Exemplary oleaginous yeasts suitable for use in the invention include members of the genera Cutaneotrichosporon, Yarrowia, Rhodotorula, Cryptococcus, Lipomyces, Debaryomyces, and Trichosporon. Exemplary species for the first bioreactor and conversion of CO2 to caribous intermediate molecules include, but are not limited to, Sporomusa ovata, Sporomusa paucivorans, Sporomusa rhizae, Sporomusa silvacetica, Sporomusa sphaeroides, Sporomusa termitida, Terrisporobacter glycolicus, Terrisporobacter mayombei, Treponema primitia, Calderihabitans maritimus, Carboxydothermus ferrireducens, Carboxydothermus hydrogenoformans, Carboxydothermus pertinax, Moorella glycerini, Moorella mulder, Moorella thermoacetica, Clostridium carboxidivorans, Clostridium coskatii, Clostridioides difficile, Clostridium drakei, Clostridium formicaceticum, Clostridium ljungdahlii, Clostridium magnum, Clostridium methoxybenzovorans, Clostridium ragsdalei, Clostridium scatologenes, Desulfotomaculum thermobenzoicum subsp, thermosyntrophicum; Eubacterium aggregans, Eubacterium limosum, Fuchsiella alkaliacetigena, Fuchsiella ferrireducens, Holophaga foetidad, Marvinbryantia formatexigens, Oxobacter pfennigii, Sporomusa acidovorans, Sporomusa aerivorans, Sporomusa malonica, Acetitomaculum ruminis, Acetoanaerobium noterae, Acetobacterium bakii, Acetobacterium carbinolicum, Acetobacterium dehalogenans, Acetobacterium fimetarium, Acetobacterium malicum, Acetobacterium paludosum, Acetobacterium tundrae, Acetobacterium wieringae, Acetobacterium woodii, Acetohalobium arabaticum, Acetonema longum, Alkalibaculum bacchi, Blautia coccoides, Blautia hydrogenotrophica, Blautia producta, Blautia schinkii, Butyribacterium methylotrophicum, Clostridium aceticum and Clostridium autoethanogenum. In some embodiments, the acetogenic microorganism is of the genera Moorella, Sporomusa or Acetobacterium. Preferably, the acetogenic microorganism is of the species Moorella thermoacetica, Sporomusa silvacetica and / or Acetobacterium woodii.

[0090] The microorganisms used in the present invention may be naturally occurring, but are preferably non-naturally occurring strains that have been modified to improve lipid productivity, substrate utilization range, or process robustness. Such microorganisms may be genetically modified through recombinant DNA technologies, mutagenized through directed evolution and / or adaptive laboratory evolution (ALE), or generated by combinations of these approaches. Non-naturally occurring microorganisms contemplated herein may ^Attorney Docket No.11833-014WO1 ^ produce lipids at yields 15–45% higher than their naturally occurring counterparts. Examples and more detail regarding these microbes is provided below.

[0091] In certain cases, these engineered microorganisms may be modified to overexpress one or more enzymes that promote lipid biosynthesis, including acetyl-CoA carboxylase, acetyl-CoA synthase, ATP-citrate lyase, NADPH-glyceraldehyde dehydrogenase, fatty acid synthase, and / or diacylglycerol O-acyltransferase. (e.g. ^2, ^3, ^4, ^5, ^6, ^7, ^8, ^9, ^10, ^11 or all 12 of the foregoing). Overexpression may result in enhanced metabolic flux toward lipid biosynthetic pathways and increased accumulation of triacylglycerols or other neutral lipids.

[0092] In additional embodiments, the microorganisms may be engineered to overexpress malonyl-CoA-ACP transacylase, ^-ketoacyl-ACP synthase, ^-ketoacyl-ACP reductase, ^- hydroxybutyl-ACP dehydrogenase, enoyl-ACP reductase, and / or AMP deaminase, enzymes (or any of the foregoing in combination with each other) that further facilitate fatty acid chain elongation and lipid accumulation. In certain embodiments, downregulation or ablation of one or more enzymes involved in lipid catabolism—such as long-chain-fatty-acid-CoA ligase, multifunctional enzyme type-1, or peroxisome biogenesis factor 10—can further enhance net lipid yield by reducing degradation pathways.

[0093] In some aspects, overexpression targets may also include key regulatory or structural genes such as Acetyl-CoA carboxylase (ACC1), acyl-CoA synthetase 1 / 2 (ACS1 / 2), ATP-citrate lyase 1 / 2 (ACL1 / 2), glyceraldehyde-3-phosphate dehydrogenase 1–3 (TDH1-3), fatty acid synthase subunit 1 / 2 (FAS1 / 2), diacylglycerol acyltransferase 2 (DGA2). Conversely, lipid catabolism may be reduced by downregulating or ablating genes such as FAA1 and MFE1. These genetic interventions can be applied individually or in combination, with two, three, four, or more modifications introduced into a single strain.

[0094] Following fermentation, lipids produced by the microbial culture may be recovered using conventional downstream processing techniques, including solvent extraction, mechanical disruption, centrifugation, and separation. In certain embodiments, the recovered lipid is an oil suitable for conversion to biofuels. The oil may be processed using a hydrotreated esters and fatty acids (HEFA) process, which may include hydrodeoxygenation, cracking, and isomerization steps to produce a renewable fuel such as sustainable aviation fuel. II. Two-Stage Bioconversion Process: CO^-to-Carbon Feedstock-to-Lipid

[0095] In another aspect, the invention provides a two-stage bioprocess for the production of lipids in which carbon dioxide and hydrogen are first converted into a carbonaceous feedstock by a first microbial culture under anaerobic conditions, and this feedstock is then converted into a lipid by a second microbial culture under aerobic conditions. This approach ^Attorney Docket No.11833-014WO1 ^ enables the direct utilization of carbon dioxide as a primary carbon input, supporting a highly sustainable and circular biomanufacturing process.

[0096] In the first stage of the process, a first microbial culture is provided in an anaerobic bioreactor and exposed to a source of carbon dioxide and hydrogen under conditions that allow the culture to metabolize these gases into a soluble carbonaceous feedstock. The carbon source may be supplied as a gas stream (e.g., syngas or biogenic CO^) or in the form of a carbonate or bicarbonate salt. In some embodiments, at least a portion of the CO^ is converted into a soluble salt prior to contact with the culture, thereby increasing gas-to- liquid mass transfer efficiency and enhancing microbial uptake.

[0097] The first microbial culture may comprise one or more anaerobic microorganisms capable of autotrophic growth and reductive assimilation of CO^, preferably acetogenic bacteria. Exemplary genera include Moorella, Sporomusa, and Acetobacterium, with species such as Moorella thermoacetica, Sporomusa silvacetica, and Acetobacterium woodii being particularly suitable. In certain embodiments, the first microbial culture comprises a consortium of two or more anaerobic species, which can enhance overall carbon conversion efficiency through complementary metabolic pathways.

[0098] The carbonaceous feedstock produced in the first bioreactor may include formate, acetate, methanol, ethanol, propionate, butyrate, short-chain fatty acids, pyruvate, lactate, or other reduced carbon compounds. These metabolites can be recovered directly from the culture medium and transferred to a second bioreactor containing an aerobic microbial culture.

[0099] In the second stage, the carbonaceous feedstock is supplied to an aerobic bioreactor containing a second microbial culture under conditions that promote lipid biosynthesis. As in the single-stage process, the aerobic culture comprises one or more oleaginous microorganisms, which may include bacteria, fungi, or yeasts such as Cutaneotrichosporon oleaginosus, Yarrowia lipolytica, or Rhodotorula mucilaginosa. The culture may be a pure culture or a consortium of multiple aerobic strains.

[0100] Similar to the single-stage embodiment, the microorganisms used in either the first or second bioreactors may be non-naturally occurring strains generated by genetic engineering, adaptive laboratory evolution, or directed evolution. Examples of these are given below. Such strains may yield 15–45% more carbonaceous feedstock or lipid, respectively, than their wild-type counterparts. Genetic modifications may include overexpression of enzymes such as malic enzyme (ME), DGA1 / DGA2, acetyl-CoA carboxylase, acetyl-CoA synthase, ATP-citrate lyase, NADPH-glyceraldehyde dehydrogenase, fatty acid synthase, and diacylglycerol O-acyltransferase, or downregulation of enzymes such as long-chain-fatty-acid-CoA ligase and multifunctional enzyme type-1. ^Attorney Docket No.11833-014WO1 ^

[0101] Further enhancements may involve overexpressing enzymes involved in fatty acid chain elongation, or downregulating ^-oxidation and peroxisomal lipid degradation pathways. As with the single-stage approach, two, three, four, or more genetic modifications can be combined in a single strain to maximize yield.

[0102] Following lipid accumulation in the second bioreactor, the lipids may be harvested and processed as described above. The final products include microbial oils suitable for use as bio-based feedstocks, chemicals, or fuels, including hydrocarbon fuels produced by HEFA or other catalytic upgrading processes. Bioreactor Systems

[0103] As described above, methods of the present invention can optionally utilize a two- stage bioreactor system designed to convert carbon dioxide into lipids via a sequential microbial process. The first stage comprises an anaerobic bioreactor housing a microbial culture capable of fixing carbon dioxide and hydrogen into a soluble carbonaceous feedstock. The second stage comprises an aerobic bioreactor, in which an oleaginous microbial culture converts the carbonaceous feedstock into lipids under conditions conducive to biosynthesis and accumulation. This sequential configuration decouples the gas-fixation and lipid-production steps, allowing each stage to be independently optimized for microbial growth, metabolic activity, and yield.

[0104] The anaerobic bioreactor is configured to maintain strict anoxic conditions and to support the growth and activity of acetogenic or other carbon-fixing microorganisms. The vessel is typically a stirred-tank or bubble column bioreactor constructed from stainless steel or other biocompatible materials and equipped with gas inlets for the controlled introduction of carbon dioxide and hydrogen. Gas flow rates, pressure, and mixing are adjusted to maximize gas-liquid mass transfer, ensuring sufficient substrate availability to the microbial culture. In certain embodiments, the reactor includes spargers or microbubble diffusers to enhance gas dissolution, as well as pH and redox control systems to maintain optimal metabolic conditions. Operating conditions may include temperatures from about 20 °C to 60 °C, pH values from about 4 to 8, and agitation sufficient to maintain homogeneity without disrupting cell viability. In some embodiments, the reactor is operated in a continuous or semi-continuous mode, allowing for steady production and withdrawal of carbonaceous intermediates such as volatile fatty acids, including formate, acetate, and / or ethanol.

[0105] The carbonaceous feedstock generated in the anaerobic stage is withdrawn from the reactor either continuously or in batches and is optionally subjected to conditioning prior to introduction into the aerobic stage. Conditioning steps may include filtration to remove microbial biomass, sterilization or pasteurization to prevent cross-contamination, concentration or dilution to achieve the desired substrate concentration, or pH adjustment to ^Attorney Docket No.11833-014WO1 ^ optimize compatibility with the aerobic culture. In certain embodiments, the feedstock is supplied directly to the second bioreactor without further processing, enabling an integrated and streamlined production system.

[0106] The aerobic bioreactor is configured to support robust growth and lipid biosynthesis by oleaginous microorganisms, including yeasts, fungi, or bacteria. This reactor is typically a stirred-tank, airlift, or packed-bed system designed to maximize oxygen transfer rates, which is a critical parameter for high biomass production. Aeration and agitation are carefully controlled to maintain dissolved oxygen levels above the minimum threshold required for biomass and lipid synthesis, often in the range of 5–40% air saturation. Temperature and pH conditions are selected based on the optimal growth parameters of the chosen organism, typically ranging from about 20 °C to 35 °C and pH 4.5 to 7.0. Nutrient composition is also tightly regulated; in particular, nitrogen or phosphorus limitation in the presence of excess carbon is frequently employed to trigger lipid accumulation. In certain embodiments, fed- batch or continuous operation is used to sustain high cell densities and maximize volumetric lipid productivity.

[0107] Integration of the two bioreactor stages may be achieved through a variety of process configurations. In some embodiments, the systems are physically separate but linked by a transfer line that allows the carbonaceous feedstock to flow from the anaerobic to the aerobic reactor under sterile conditions. In other embodiments, the reactors are part of a modular system with shared monitoring and control systems, allowing for automated adjustment of parameters based on real-time feedback from either stage. For example, gas flow to the anaerobic reactor may be modulated based on carbon consumption rates in the aerobic reactor, or feedstock transfer may be synchronized with biomass growth phase to maximize lipid yield. Additionally, in certain integrated designs, residual gases or byproducts from one stage can be recycled into the other, further enhancing process efficiency and reducing waste.

[0108] By separating the carbon fixation and lipid synthesis steps, each microbial population can be maintained under optimal conditions for its metabolic role, eliminating the need for compromise in environmental parameters. This modularity allows for independent scale-up of each stage and enables the system to accommodate fluctuations in gas supply, feedstock composition, or process demand. Moreover, the decoupled design facilitates the use of highly specialized microbial strains in each stage—such as acetogenic consortia for gas conversion and engineered oleaginous yeasts for lipid biosynthesis—resulting in improved overall carbon conversion efficiency, higher lipid titers, and reduced process costs. Lipids Produced by the Disclosed Processes ^Attorney Docket No.11833-014WO1 ^

[0109] The present invention provides lipids produced by the methods described herein. In some embodiments, the lipid is an oil produced by microbial biosynthesis from a carbonaceous feedstock as described above. The lipids produced by the inventive process are typically intracellularly accumulated by oleaginous microorganisms under nutrient-limiting and carbon-excess conditions and can be recovered from microbial biomass following cultivation. The resulting lipids may be present as triacylglycerols (TAGs), diacylglycerols (DAGs), monoacylglycerols (MAGs), free fatty acids, phospholipids, or combinations thereof. In certain embodiments, the lipid fraction comprises at least 40%, at least 50%, at least 70%, or at least 90% of the total dry weight of the harvested microbial biomass.

[0110] The lipid composition may vary depending on the microbial strain, feedstock, and process parameters used, but generally includes long-chain fatty acids (C14–C22), including saturated, monounsaturated, and polyunsaturated species. Representative fatty acids include, but are not limited to, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3). In certain embodiments, the lipid profile can be tailored through metabolic engineering or process control to enrich for particular classes of fatty acids suited for downstream applications. For example, increased expression of acetyl-CoA carboxylase and fatty acid synthase may bias production toward longer-chain, saturated fatty acids, whereas manipulation of desaturase activity can increase unsaturated lipid content.

[0111] The lipids obtained by the present methods are chemically and functionally comparable to those derived from plant or animal sources but are produced through a sustainable, non-agricultural biotechnological process. They may be directly utilized as raw materials in industrial formulations, including lubricants, surfactants, and polymer precursors. Additionally, the microbial oils can serve as renewable feedstocks for the production of a wide range of value-added chemicals, including fatty alcohols, wax esters, and biosurfactants. In some embodiments, the lipid is used as a direct palm oil replacement in food, cosmetic, or industrial applications, offering a sustainable alternative with reduced land and resource use. Fuel Sources Derived from Microbial Lipids

[0112] The present invention further provides fuel compositions derived from the lipids produced by the microbial methods disclosed herein. In some embodiments, the lipid obtained from the aerobic fermentation stage is subjected to chemical upgrading processes to yield hydrocarbon fuels suitable for use in transportation, aviation, and industrial applications. Such processes may include, but are not limited to, the hydrotreated esters and fatty acids (HEFA) pathway, in which microbial oils are converted into drop-in fuels compatible with existing infrastructure. ^Attorney Docket No.11833-014WO1 ^

[0113] In one embodiment, the lipid feedstock is subjected to hydrodeoxygenation, wherein the oxygen atoms present in the triglycerides or free fatty acids are removed as water, carbon monoxide, or carbon dioxide under hydrogenating conditions. This step typically employs a metal catalyst, such as NiMo, CoMo, or noble metals, under elevated temperature and pressure, producing a mixture of long-chain alkanes. Following hydrodeoxygenation, the hydrocarbon stream may be subjected to cracking, which cleaves longer chains into shorter, more volatile fractions suitable for use as diesel, jet fuel, or naphtha-range hydrocarbons. A subsequent isomerization step rearranges the molecular structure of the hydrocarbons to improve cold flow properties, oxidative stability, and other fuel characteristics.

[0114] The resulting fuel product is compositionally and functionally comparable to conventional petroleum-derived fuels but is produced from renewable carbon captured via microbial metabolism. In some embodiments, the fuel exhibits a high cetane number and energy density suitable for diesel engines, or a high flash point and thermal stability appropriate for aviation turbine fuel. The renewable fuels produced according to the present disclosure may be blended with or used as direct substitutes for fossil fuels, thereby reducing lifecycle greenhouse gas emissions and reliance on petroleum resources.

[0115] Moreover, the process allows for precise tailoring of the fuel product by adjusting upstream microbial lipid composition and downstream processing conditions. For instance, the fatty acid profile of the lipid feedstock may be optimized to yield a desired hydrocarbon distribution, or catalytic conditions may be modulated to favor specific chain lengths or branching patterns. In some embodiments, the microbial oil is converted into renewable jet fuel (SAF), renewable diesel, or bio-naphtha, each meeting relevant ASTM or EN specifications for commercial use. Thus, the invention provides not only a sustainable route to lipid production but also a fully integrated biotechnological platform for renewable fuel generation. Methods of Producing Non-Naturally Occurring Oleaginous Microorganisms

[0116] The present invention provides methods for generating non-naturally occurring oleaginous microorganisms capable of producing higher quantities of lipids than their naturally occurring counterparts. In general, the methods disclosed herein integrate targeted or random mutagenesis, selective cultivation, nutrients starvation, high-throughput screening, adaptive laboratory evolution (ALE), and downstream validation to create enhanced microbial strains optimized for lipid production under industrially relevant conditions. These methods may be applied to any oleaginous microorganism, including fungi, yeasts, or bacteria, and are particularly advantageous for strains such as Cutaneotrichosporon oleaginosus, Yarrowia lipolytica, Rhodotorula mucilaginosa, and other lipid-accumulating species. Example 6 provides detail on this method. ^Attorney Docket No.11833-014WO1 ^

[0117] In one embodiment, the method comprises exposing a parental oleaginous microbe to conditions that promote mutagenesis. Such mutagenesis introduces genetic diversity that serves as the substrate for phenotypic selection and improvement. In a preferred embodiment, mutagenesis is achieved using Atmospheric and Room Temperature Plasma (ARTP) technology. ARTP is a non-thermal plasma-based approach that induces random mutations while maintaining high cell viability, thereby enabling the creation of large, diverse mutant libraries. Exposure times can range from 10 seconds to 150 seconds or more, and may be applied in single or multiple rounds to maximize mutational diversity. Other mutagenesis approaches, including chemical mutagenesis (e.g., EMS, NTG), UV irradiation, or transposon-based mutagenesis, may also be employed alone or in combination with ARTP to further expand the diversity of the resulting population.

[0118] Following mutagenesis, the treated microbial population is cultured under conditions conducive to colony formation. In some embodiments, cells are plated on rich or defined agar media (e.g., YMPG) and incubated to permit the growth of discrete colonies. These colonies represent independent mutational events and form the basis for downstream screening. The colonies may then be isolated and transferred into multi-well plate cultures, where they are grown in media containing a carbon source such as crude glycerol. In one embodiment, initial screening is performed in 24-well plates containing YMP medium supplemented with 2% glycerol, followed by secondary cultivation in nitrogen-limited NMM medium containing 5% glycerol to induce lipid accumulation.

[0119] A key feature of the invention is the use of high-throughput screening methodologies to identify mutant strains with superior growth or lipid production phenotypes. In some embodiments, optical density (OD) measurements are used to assess growth kinetics, while lipid production is quantified using a fluorescence-based assay such as Nile Red staining. In this approach, cells are stained with Nile Red (e.g., 1 µg / mL in DMSO), and relative fluorescence units (RFU) are measured using a microplate reader. This screening strategy enables rapid and quantitative assessment of lipid content across hundreds or thousands of isolates. Control strains, including the wild-type parent and known high-producing mutants, may be included as benchmarks for comparative performance.

[0120] ALE may be performed in parallel with or subsequent to mutagenesis and initial screening, and involves serial passaging of microbial populations under selective pressure to enrich for desirable traits such as improved growth rate, carbon utilization efficiency, or lipid yield. In one example, non-isolated colonies are pooled and cultured in nitrogen-limited NMM medium with 5% glycerol. At defined intervals (e.g., 24, 48, and 72 hours), cultures are screened and the lipid-rich cells collected and used to seed subsequent passages. This process may be repeated for two or more rounds, with isolates from later passages demonstrating enhanced lipid accumulation relative to earlier generations. ^Attorney Docket No.11833-014WO1 ^

[0121] Following enrichment and screening, top-performing mutants are isolated for further characterization and validation. Such validation may include batch fermentation studies in controlled bioreactors to confirm lipid production under industrial conditions, plating on selective media to verify strain purity, and whole-genome sequencing to identify beneficial mutations associated with the improved phenotype. Sequencing also serves to confirm that the resulting strains are genetically distinct from their parental strains, providing molecular evidence of their non-natural origin.

[0122] In some embodiments, the workflow described herein is iterative, and steps (a) through (g) — mutagenesis, cultivation, isolation, screening, selection, and validation — can be repeated two, three, four, five, or more times to achieve progressive improvements in strain performance. Each successive round builds upon the genetic and phenotypic gains of the previous one, enabling the generation of highly optimized strains that exhibit significantly enhanced lipid yields compared to wild-type strains. In certain embodiments, engineered strains obtained through this process produce at least 15%, 25%, 35%, or even 45% more lipid than their unmodified counterparts under equivalent conditions.

[0123] The methods disclosed herein are not limited to the specific culture conditions or media compositions described. Variations in media formulation, carbon source, nitrogen limitation strategies, and screening parameters may be employed depending on the target organism and desired application. For instance, glycerol may be replaced with alternative feedstocks such as glucose, acetate, or biomass-derived sugars, and fluorescence-based screening can be complemented or replaced with gravimetric lipid quantification, GC-MS analysis of fatty acid methyl esters, or other analytical approaches.

[0124] Through this integrated approach — combining plasma-based mutagenesis, high- throughput screening, adaptive laboratory evolution, and iterative selection — the present invention enables the efficient creation of novel oleaginous microorganisms with superior lipid production capabilities. These engineered strains serve as foundational biocatalysts for sustainable biomanufacturing, providing renewable lipid feedstocks for fuels, chemicals, and materials while reducing dependence on agricultural or fossil-derived inputs. Engineered Microorganisms for Lipid Production from Carbonaceous Feedstocks

[0125] The present invention provides novel microorganisms that have been engineered, selected, or evolved to perform specific and enhanced metabolic functions for the bioconversion of carbon into lipids and other valuable bioproducts. These microorganisms differ fundamentally from their naturally occurring counterparts in that they comprise one or more genetic modifications, mutations, regulatory alterations, or engineered metabolic pathways that confer improved performance, productivity, or stability under industrial conditions. Such modifications may be introduced through recombinant DNA techniques, ^Attorney Docket No.11833-014WO1 ^ genome editing (e.g., CRISPR / Cas systems), adaptive laboratory evolution (ALE), directed evolution, or combinations thereof, and may include gene insertions, deletions, substitutions, overexpression, downregulation, or the introduction of heterologous sequences.

[0126] Any two, three, four, or more modifications described herein may be present within a single engineered strain, and in some embodiments, the microorganisms may be employed individually or as part of a synthetic or semi-synthetic consortium, wherein multiple strains cooperate metabolically to improve overall conversion efficiency.

[0127] The engineered microorganisms described herein fall into two primary functional classes. A first class comprises anaerobic microorganisms that utilize carbon dioxide and, optionally, hydrogen or other reductants to produce carbonaceous feedstocks, such as acetate, ethanol, or other soluble reduced carbon compounds. A second class comprises aerobic microorganisms that convert such carbonaceous feedstocks into lipids, including triacylglycerols and other neutral lipids, which can be used as precursors for fuels, oleochemicals, and other industrial products. The following sections describe each class in greater detail.

[0128] As used herein, the term “overexpression” refers to an increase in the level of expression of a gene, RNA transcript, or encoded protein in a microorganism relative to a corresponding wild-type or unmodified control strain of the same species, preferably a type strain. Overexpression may be measured at the level of mRNA (e.g., by quantitative RT- PCR or RNA-Seq), protein (e.g., by quantitative Western blotting, ELISA, or proteomic analysis), or enzymatic activity (e.g., by specific activity assays).

[0129] In some embodiments, “overexpression” refers to an increase in expression of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500% relative to the corresponding wild-type expression level. In other embodiments, “overexpression” refers to an increase in expression by at least 1.1- fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 50-fold or more relative to a wild-type or unmodified control strain.

[0130] As used herein, the terms “reduction of expression,” “downregulation,” or “downregulated expression” refer to a decrease in the level of expression of a gene, RNA transcript, or encoded protein in a microorganism relative to a corresponding wild-type or unmodified control strain of the same species, preferably a type strain. Reduction of expression may be measured at the level of mRNA (e.g., by quantitative RT-PCR or RNA- Seq), protein (e.g., by quantitative Western blotting, ELISA, or proteomic analysis), or enzymatic activity (e.g., by specific activity assays).

[0131] In some embodiments, reduction of expression refers to a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% relative to the corresponding wild-type expression level. In other embodiments, reduction of ^Attorney Docket No.11833-014WO1 ^ expression refers to a decrease in expression to 0.95-fold, 0.9-fold, 0.8-fold, 0.7-fold, 0.5- fold, 0.3-fold, 0.2-fold, 0.1-fold, or less relative to the expression level in a wild-type or unmodified control strain.

[0132] In certain embodiments, the reduction of expression is partial, meaning expression is decreased but not eliminated. In other embodiments, the reduction is substantial, meaning expression is decreased by at least 80%, 90%, or 95%. In yet other embodiments, reduction of expression results in near-complete silencing or ablation, where expression is decreased by at least 99% or is below the limit of detection.

[0133] Unless otherwise indicated, measurements of reduced or increased expression are taken under substantially comparable culture conditions (e.g., identical media composition, temperature, oxygenation, carbon source, and growth phase) to allow accurate comparison between engineered and wild-type strains. Reduction of expression or overexpression may be partial or substantial, transient or stable, constitutive or inducible, and may be achieved by any means known in the art. Engineered Anaerobic Microorganisms

[0134] In some embodiments, the invention provides engineered anaerobic microorganisms that are capable of utilizing a carbon dioxide source, optionally in the presence of hydrogen or other reducing equivalents, to produce one or more carbonaceous feedstocks. These microorganisms represent a distinct class of chassis organisms designed to convert waste or atmospheric CO^ into soluble metabolites such as acetate, ethanol, formate, butyrate, or other reduced carbon intermediates suitable for downstream biological conversion, including lipid biosynthesis. The engineered anaerobes described herein are distinct from their naturally occurring counterparts in that they have been modified, selected, or evolved to achieve superior growth, carbon utilization, product yield, or metabolic stability under industrial conditions.

[0135] Preferred anaerobic microorganisms include those from acetogenic genera such as Moorella, Sporomusa, Clostridium, and Acetobacterium, although other anaerobic or facultative species capable of CO^ fixation through the Wood–Ljungdahl pathway or alternative autotrophic routes are also contemplated. Exemplary species include, but are not limited to, Moorella thermoacetica, Sporomusa silvacetica, and Acetobacterium woodii. These organisms may be employed as individual strains or as part of a microbial consortium comprising two or more distinct anaerobic species, optionally with complementary metabolic functions. In such consortia, one strain may optimize CO^ fixation and acetyl-CoA generation while another enhances downstream conversion of intermediates, thereby improving overall carbon capture efficiency and feedstock production. ^Attorney Docket No.11833-014WO1 ^

[0136] In some embodiments, engineered microorganisms are constructed by incorporating heterologous genes, pathway modules, or regulatory sequences into the genome or by modifying endogenous genes to improve their catalytic efficiency or regulatory behavior.

[0137] The engineered anaerobic microorganisms described herein may be modified through genetic engineering, adaptive laboratory evolution (ALE), and / or directed evolution to enhance key metabolic traits. These include increased rates of CO^ uptake, improved tolerance to high CO^ or hydrogen partial pressures, enhanced resistance to product toxicity, and expanded substrate utilization profiles. In some embodiments, mutations introduced through ALE or directed evolution are combined with targeted genomic edits (e.g., promoter engineering, gene knock-ins, or knock-outs) to further optimize performance. Two, three, four, or more such modifications may be incorporated into a single strain to achieve synergistic effects.

[0138] In some embodiments, anaerobic strains are engineered to overexpress key enzymes involved in carbon fixation and downstream conversion, including but not limited to: carbon monoxide dehydrogenase, acetyl-CoA synthase, formate dehydrogenase, hydrogenase, phosphotransacetylase, acetate kinase, aldehyde dehydrogenase, and alcohol dehydrogenase. Overexpression of one or more of these enzymes can accelerate flux through the Wood–Ljungdahl pathway, thereby increasing the rate and yield of carbonaceous feedstock production. Overexpression may be constitutive or inducible and may be achieved by promoter replacement, increased gene copy number, or integration of strong heterologous regulatory sequences.

[0139] In additional embodiments, anaerobic strains are engineered to reduce or ablate expression of genes whose products negatively impact carbon fixation or redirect metabolic flux away from desired end products. Examples include genes encoding competing hydrogenases, acetate uptake transporters, or enzymes associated with by-product formation. Reducing or eliminating these functions can improve carbon and electron allocation toward desired feedstocks. Ablation can be achieved by targeted gene deletion, CRISPR / Cas-mediated knockout, insertional inactivation, or RNA interference-based approaches.

[0140] The engineered anaerobes may also incorporate modifications that improve industrial robustness and scalability. For example, strains may be engineered to tolerate high dissolved gas concentrations, resist shear stress in stirred bioreactors, or maintain metabolic activity under nutrient-limited or pH-variable conditions. Stress tolerance genes, chaperones, and redox-balancing enzymes may be overexpressed or otherwise optimized to prolong culture longevity and productivity. Similarly, strains may be engineered to operate efficiently under non-sterile or semi-sterile conditions, reducing operational costs in large-scale CO^ conversion facilities. ^Attorney Docket No.11833-014WO1 ^

[0141] In some embodiments, anaerobic consortia are deliberately designed, with different engineered species each contributing specialized metabolic functions. For instance, a first engineered acetogen may convert CO^ and H^ primarily to acetate, while a second engineered strain converts acetate into ethanol or butyrate. A third organism in the consortium may enhance overall gas utilization by scavenging residual CO^ or H^. By tailoring the division of labor in such consortia, total carbon capture and conversion efficiency can be significantly improved beyond the capabilities of monocultures.

[0142] The engineered anaerobic microorganisms described herein are not limited to naturally occurring strains but may include synthetic or semi-synthetic organisms assembled from multiple species, genera, or synthetic biology components. These strains may further comprise combinations of engineered traits, including but not limited to: increased CO^ assimilation rate, enhanced acetyl-CoA flux, reduced competing pathways, improved tolerance to process conditions, and optimized secretion of carbonaceous products. Any two, three, four, or more such modifications described above may be combined within a single engineered strain to achieve desired performance targets. Engineered Aerobic Microorganisms

[0143] In other embodiments, the invention provides engineered aerobic microorganisms that are capable of converting carbonaceous feedstocks — such as acetate, formate, ethanol, methanol, short-chain fatty acids, sugars, or other reduced carbon molecules — into lipids. These microorganisms represent a second major class of engineered biocatalysts, optimized for lipid biosynthesis and accumulation under industrially relevant conditions. Engineered aerobic microorganisms described herein are distinct from their naturally occurring counterparts in that they exhibit increased lipid yield, productivity, metabolic efficiency, or robustness as a result of genetic modification, evolutionary selection, or a combination thereof.

[0144] Preferred aerobic microorganisms include oleaginous yeasts, fungi, and bacteria that naturally accumulate substantial amounts of triacylglycerols or other neutral lipids. Representative genera include Cutaneotrichosporon, Yarrowia, Rhodotorula, Cryptococcus, Lipomyces, Debaryomyces, Trichosporon, Aspergillus, Cunninghamella, Thermomyces, and Mortierella. Exemplary species include Cutaneotrichosporon oleaginosus, Yarrowia lipolytica, Rhodotorula mucilaginosa, Rhodotorula glutinis, Cryptococcus curvatus, Debaryomyces hansenii, Lipomyces starkeyi, Aspergillus niger, Cunninghamella echinulata, Thermomyces lanuginosus, and Mortierella isobellina. These microorganisms may be employed individually or as part of a consortium of two or more species, wherein each strain performs a complementary role in the conversion of feedstock into lipid. For example, one strain may specialize in acetate uptake, while another exhibits enhanced lipid accumulation, collectively improving overall yield and productivity. ^Attorney Docket No.11833-014WO1 ^

[0145] In certain embodiments, engineered aerobic microorganisms comprise a nucleic acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to a reference sequence described herein (e.g., SEQ ID NO: 1-12, 15, 18, 21, 24, and / or 27-41), or a sequence that hybridizes under stringent conditions to SEQ ID NO: 1-12, 15, 18, 21, 24, and / or 27-41. These criteria encompass natural variants, synthetic derivatives, and codon- optimized sequences designed to improve expression efficiency or metabolic regulation. Such microorganisms may be genetically modified, derived through adaptive laboratory evolution (ALE), subjected to directed evolution, or created through combinations of these approaches to achieve enhanced growth, carbon utilization, and lipid accumulation.

[0146] Engineered aerobic microorganisms may be modified to overexpress one or more enzymes that drive flux through lipid biosynthetic pathways. Non-limiting examples of such enzymes include acetyl-CoA carboxylase, acetyl-CoA synthase, ATP-citrate lyase, NADPH- glyceraldehyde dehydrogenase, fatty acid synthase, diacylglycerol O-acyltransferase (DGAT), malonyl-CoA–ACP transacylase, ^-ketoacyl-ACP synthase, ^-ketoacyl-ACP reductase, ^-hydroxybutyryl-ACP dehydrogenase, enoyl-ACP reductase, and AMP deaminase. Overexpression of any one of these enzymes can improve the rate of fatty acid synthesis, but in preferred embodiments, two, three, four, or more of these enzymes are simultaneously overexpressed in a single organism to produce a synergistic effect on lipid yield and productivity. Overexpression may be achieved by promoter engineering, copy number amplification, chromosomal integration of additional gene copies, or introduction of strong heterologous regulatory elements.

[0147] In some embodiments, engineered aerobic microorganisms are further modified to ablate or reduce expression of genes encoding enzymes that compete with lipid synthesis or catalyze lipid degradation. Examples include long-chain-fatty-acid-CoA ligase, multifunctional enzyme type-1, triacylglycerol lipase, and peroxisome biogenesis factor 10. Reduction or elimination of such functions redirects metabolic flux toward lipid accumulation and reduces catabolic losses. Ablation or downregulation may be accomplished by CRISPR / Cas-mediated genome editing, targeted deletion, insertional disruption, antisense inhibition, or RNA interference. Any two, three, four, or more of these modifications may be combined in a single strain for optimal performance.

[0148] In certain embodiments, engineered aerobic microorganisms are designed to overexpress key metabolic genes involved in lipid biosynthesis and energy metabolism. These include, without limitation, ME, DGA1, DGA2, ACC1, ACS1, ACS2, ACL1, ACL2, TDH1, TDH2, TDH3, FAS1, and FAS2. These genes regulate crucial steps in the conversion of acetyl-CoA to fatty acids and their subsequent assembly into triacylglycerols. Overexpression of multiple genes in combination — such as ACC1 with DGA2 and FAS1 / FAS2 — can substantially improve lipid yield. In certain embodiments, engineered ^Attorney Docket No.11833-014WO1 ^ strains exhibit a 15–45% or greater increase in lipid production compared to their wild-type counterparts.

[0149] In additional embodiments, engineered aerobic microorganisms are designed to reduce or ablate the expression of genes such as FAA1, FAA2, FAA3, FAA4, MFE1, TGL3, TGL4, TGL5, and PEX10, which are associated with fatty acid activation, ^-oxidation, or lipid turnover. Downregulating these genes increases net lipid retention and prolongs the accumulation phase. The terms “downregulation” and “ablation” are intended to include partial reductions in expression as well as near-complete or complete silencing, with expression levels reduced by at least 5%, 10%, 20%, 50%, 80%, 95%, or 99% relative to wild-type strains.

[0150] In some embodiments, aerobic strains are engineered for enhanced physiological robustness under industrial conditions. Genetic modifications may be introduced to improve tolerance to high carbon substrate concentrations, osmotic or oxidative stress, pH fluctuations, or accumulation of inhibitory metabolites. Additional modifications may enhance oxygen transfer efficiency, redox balance, or cofactor regeneration (e.g., NADPH supply), all of which contribute to improved lipid biosynthesis. Such traits are particularly advantageous for continuous or high-cell-density fermentation processes, where prolonged metabolic activity and culture stability are critical.

[0151] The engineered aerobic microorganisms described herein may also be incorporated into synthetic or semi-synthetic consortia comprising two or more engineered strains with complementary capabilities. For example, one strain may specialize in rapid acetate uptake, another in high triacylglycerol accumulation, and a third in secreting growth factors or removing inhibitory by-products. The use of engineered consortia allows division of metabolic labor and can significantly improve total lipid yield, process robustness, and scalability.

[0152] The aerobic microorganisms disclosed herein are not limited to naturally occurring isolates but include fully synthetic or hybrid organisms constructed through metabolic engineering, synthetic biology, or genome-scale design. These organisms may simultaneously incorporate multiple advantageous traits — including increased lipid synthesis, decreased lipid catabolism, enhanced substrate utilization, and improved process tolerance — and may be tailored for specific industrial applications such as biofuel production, sustainable aviation fuel synthesis, or production of oleochemicals. Any two, three, four, or more of the modifications described above may be present within a single engineered strain. EXAMPLES Example 1 – Conversion of crude glycerol into oil-rich biomass ^Attorney Docket No.11833-014WO1 ^

[0153] The process of converting crude glycerol into oil-rich biomass may be facilitated by a yeast in the presence of oxygen. The yeast is grown to accumulate the sustainable oil inside of its cell walls, after which it is broken open and extracted. The remaining biomass is dried and made ready to be sold as a sustainable protein in the animal feed market. A flow diagram of an exemplary process is shown in Figure 1, which is referenced throughout this example. Aerobic bioreactor (101)

[0154] The aerobic bioreactor takes glycerol as feed and grows an oil-rich microbe which is modeled with the following stoichiometry (Table 1). Table 1 – reactions and conversions in aerobic bioreactor (101)Biomass processing

[0155] The concentrated biomass slurry (2101) from the wet biomass storage tank (106) is pumped to a bead mill (109) for cell disruption. The homogenized biomass (3100) from the bead mill (109) is stored in another storage tank (110) and is then transferred (3101) to, and concentrated further, in a centrifuge (111) from 26.4% to 62% solids and removed at a flow rate of 0.7 gal / min (1.7 US tons / day) (4102) to be stored in a product slurry tank (112). The liquid phase from the centrifuge (4101) is removed at a rate of 0.4 gal / min and pumped to the recycle water tank.

[0156] Oil (5000) is then separated from protein-rich biomass (5001) – both are then stored until transported out of the facility. Dryer

[0157] The concentrated, homogenized biomass (4104) from the product slurry tank (112) is transferred to a dryer (113) where 0.56 US tons / day of water is evaporated and vented as steam (5100) to make oil-rich solid biomass (1.2 US tons / day) (5101). Oil (5000) is then separated from protein-rich biomass (5001) – both are then stored until transported out of the facility. Recycle water ^Attorney Docket No.11833-014WO1 ^

[0158] Wastewater streams (2102 and 4101) are combined and stored in the recycle water tank (107). The tank is sealed from atmosphere and kept under a nitrogen headspace (to avoid introduction of oxygen). The recycle water has an acetic acid concentration of 3.9% and a small purge stream (3100) of 0.18 gal / min (1 US tons / day) is used to avoid build-up of acetic acid in the system. Minerals Mineral requirements for microbial biomass production includes phosphorous, sulfur, potassium, iron, magnesium, calcium, zinc, manganese, copper and cobalt, to satisfy biomass constituation as demonstrated in table 2. Table 2 – Basis of mineral use by microbes from Popovic Microbe Elemental compositionSeed reactor

[0159] A smaller agitated seed reactor (114) of approximately 1500 gal working volume is used to grow the yeast inoculum (1107) for start-up of the larger bioreactor and as needed during the process. Example 2 – Exemplary conversion of CO2and H2to acetate

[0160] The process of converting CO2 and H2 to the precursors of Sustainable Aviation Fuel (SAF), lipids, consists of three major steps. First, CO2 and H2 are anaerobically converted into acetate by an acetogenic microbe; then yeast is grown aerobically on the acetate produced in the first step to accumulate the Sustainable Oil inside of its cell walls. In the third step, the yeast biomass is processed by breaking open the cells, drying, and extracting oil by high pressure. The remaining biomass is made ready to be used as a Sustainable Protein in the animal feed market. A flow diagram of an exemplary process is shown in Figure 2, which is referenced throughout this example. Anaerobic bioreactor (201)

[0161] The anaerobic bioreactor takes H2 and CO2 gases as feed and grows an acetic acid producing microbe which is modeled with the following stoichiometry (Table 3). Table 3 – reactions and conversions in anaerobic bioreactor (201)^Attorney Docket No.11833-014WO1 ^

[0162] The reactor is a bubble column (201) with a working liquid volume of 15,850 gallon (60 m3) with actual vessel diameter of 9 ft and height of 40 ft. An external liquid cooling loop (1111, 1112 and 1113) through a heat exchanger (202), using a pump (203), using cooling water (1114) is used to maintain the reactor temperature at 104oF. Liquid flows through the heat exchanger (202) at an average rate of 4100 gal / min with an inlet and outlet temperatures of 104.7oF and 93.4oF respectively.

[0163] A mix of gases, consisting of (1) fresh H2 (1002, 0.6 US tons / day), (2) fresh CO2 (1003, 6.4 US tons / day) and (3) unreacted gases from the outlet of the reactor (1001) compressed by compressor (204) from 43 psig to 60 psig to give high pressure unreacted gas (1004), are mixed in a gas mixer (205) and delivered to the bottom gas inlet of the reactor at 60 psig (1000). The flow rates of the fresh H2(1002) and CO2(1003) feeds are controlled to maintain a flow of 3640 ft3 / min and a composition of 90% H2 and 7.5% CO2 at the inlet of the reactor.

[0164] Liquid feed (1104) consisting of water (1100), ammonium hydroxide (1101), mineral salts (1102 and 1103) and recycled acetate (4103), combined in mixer (206) flows into the reactor at a rate of 18.5 gal / min (4.2 m3 / hr) corresponding to a dilution rate of 0.07 / hr (or residence time of 15 hr).

[0165] A hollow fiber crossflow filtration module (207) that permeates small molecules (acetate, water, nutrient, etc.) and retains cell biomass is used to concentrate and recycle cells (2101) to the reactor. Cell culture (1109) with biomass concentration of 2.3% (w / w) enters the filtration module (207) from the reactor (201) at a rate of 42.7 gal / min (228 US tons / day) and exits the module (2101) at 23.7 gal / min (116.5 US tons / day) with a biomass concentration of 4.5%. A part of this retentate (2102) is purged at a rate of 0.48 gal / min to prevent overloading of the reactor with cells. The permeate (2104) consisting of 7.9 US tons / day acetic acid (7%) is stored in an acetate storage tank (208).

[0166] Assumptions for the reactor (201) are in two ranges of production: (1) acetate productivity is 120 g / L / d or higher values. Aerobic bioreactor (209)

[0167] The aerobic bioreactor (209) takes acetic acid as feed and grows an oil-rich microbe which is modeled with the following stoichiometry (Table 4). Table 4 – reactions and conversions in aerobic bioreactor (209)^Attorney Docket No.11833-014WO1 ^

[0168] The reactor (209) is a bubble column with a working liquid volume of 15,850 gallon (60 m3) with actual vessel diameter of 9 ft and height of 40 ft. An external liquid cooling loop (1111, 1112 and 1113) through a heat exchanger (202), using a pump (203), using cooling water (114) is used to maintain the reactor temperature at 104oF. Liquid flows through the heat exchanger (202) at an average rate of 4100 gal / min with an inlet and outlet temperatures of 104.7oF and 83.6oF respectively.

[0169] The acetate stream (2105) from the storage tank (208) is mixed with the fresh ammonium hydroxide (3101) to feed the reactor (209) at a rate of about 19.2 gal / min (4.4 m3 / hr). The liquid outlet (3102) from the reactor is passed through another hollow-fiber cross-flow filtration module (210) to concentrate the oil-rich yeast biomass to process for oil. Cell culture (3102) with biomass concentration of 1% (with 60% oil) enters the filtration module (210) at a rate of 18.7 gal / min (107.6 US tons / day) and exits the filtration module (210) as a concentrated stream (4104) at 2.3 gal / min (11.7 US tons / day) with a biomass concentration of 8.9% and stored in a wet biomass storage tank (211). The permeate (4100) free of cell biomass is pumped to a recycle water tank (212) at a rate of 16.3 gal / min. An air compressor (213) takes air (1106) delivers compressed air (3000) to the bottom of the reactor at a rate of 1,190 ft3 / min (33.7 m3 / min) at 17 psig to supply the oxygen needed for the reaction. The unreacted air (3001) is vented from the reactor. Biomass processing

[0170] The concentrated biomass slurry (4105) from the wet biomass storage tank (211) is pumped to a bead mill (214) for cell disruption. The homogenized biomass (5100) from the bead mill (214) is stored in another tank (215) and is transferred to (5102) and then concentrated further in a centrifuge (216) from 8.9% to 31.2% solids and removed at a flow rate of 0.8 gal / min (3.1 US tons / day) (6100) to be stored in a product slurry tank (217). The liquid phase from the centrifuge (216) is removed at a rate of 1.5 gal / min and pumped via stream (6101) to the recycle water tank (212). Dryer (218)

[0171] The concentrated, homogenized biomass (6104) from the product slurry tank (217) is transferred to a dryer (218) where 1.8 US tons / day of water (7102) is evaporated to make oil-rich solid biomass (7101, 0.2 US tons / day).

[0172] Oil (8000) is then separated from protein-rich biomass (8001) – both are then stored until transported out of the facility. Recycle water

[0173] Wastewater Streams (4100 and 6101) are combined and stored in the recycle water tank (212). The tank (212) is sealed from atmosphere and kept under a nitrogen headspace ^Attorney Docket No.11833-014WO1 ^ (to avoid introduction of oxygen). The recycle water has an acetic acid concentration of 3.9% and a small purge stream (4101) of 0.18 gal / min (1 US tons / day) is used to avoid build-up of acetic acid in the system.

[0174] It is important to maintain low oxygen concentration in the reactor (201) liquid. It was found that the oxygen concentration in the feed media to the reactor (201) is 260 ^M (equal to air saturation) but drops to 20 ^M in the reactor due to equilibration with the gas phase (which has a concentration of 0.4% O2). Especially when grown in higher densities as they are capable of scavenging small amounts of oxygen. Therefore, no de-oxygenation equipment is included in the current process. Minerals

[0175] Mineral requirements for microbial biomass production includes phosphorous, sulfur, potassium, iron, magnesium, calcium, zinc, manganese, copper and cobalt. It is assumed that 50% of the total mineral feed is incorporated in the biomass and the rest is recycled (in recycled water). Fresh mineral feed quantities are calculated from compositions listed by Popovic – reference above (Table 5, below). For minerals in the model that are not listed in the stoichiometry (zinc, manganese, copper and cobalt), it was assumed that they are equal to the composition of iron. Table 5 – Basis of Mineral use by Microbes from PopovicSeed reactor

[0176] A smaller agitated seed reactor (114) of approximately 1500 gal working volume is used to grow the yeast inoculum (1107) for start-up of the larger bioreactors and as needed during the process. Example 3 – Microorganisms to convert glycerol or acetate or a combination of glycerol and acetate to single cell oil

[0177] Exemplary species which may be used include Cutaneotrichosporon oleaginosus, Yarrowia lipolytica, Rhodotorula mucilaginosa, Rhodotorula glutinis, Cryptococcus curvatus and / or Lipomyces starkeyi. Yeast cells might be subjected to Adapted Laboratory Evolution (ALE), Genetic and Metabolic engineering or a combination of ALE, Genetic, and Metabolic Engineering for improving thermotolerance and / or oil productivity, e.g. as detailed described in the following sections. ^Attorney Docket No.11833-014WO1 ^

[0178] Herein, two wild-type yeast strains, Cutaneotrichosporon oleaginosus and Yarrowia lipolytica, were cultured in base medium with waste or crude glycerol as the carbon source at 30°C. The yeasts were incubated for 96 to 120 hours to allow them to adapt to the new conditions. After this period, cells were selected based on physiological characteristics such as lower density and transferred to fresh media for each passage. After multiple cells generations, this iterative process resulted in the development of an evolved strain, which was then subjected to functional testing.

[0179] For developing thermotolerance, the wild-type strains were initially cultured in -NMM with waste or crude glycerol as the carbon source at an optimal temperature of 30°C. Subsequently, the temperature gradually increased after each 96 to 120-hour incubation period. The current evolved strain were able to tolerate 37°C. Since temperature impacts cell structure by affecting membrane lipids, several passages were occasionally conducted without raising the temperature to stabilize the adaptation.

[0180] Surprisingly, the evolved strain of C. oleaginosus was found to be tolerant of a fermentation at pH 3.

[0181] Figure 4 demonstrates benefits in terms of enhanced growth rates for the evolved strains (“ES”) of C. oleaginosus and Y. lipolytica, compared to wild-type strains (“WT”). Figure 5 demonstrates benefits in terms of enhanced oil accumulation (as measured by OD600) for the evolved strains (“ES”) of C. oleaginosus and Y. lipolytica, compared to wild- type strains (“WT”). An increase in oil accumulation of 15-40% is observed with the evolved strains compared to wild-type strains. Figure 6 demonstrates benefits in terms of enhanced glycerol consumption for evolved strains (“ES”) of C. oleaginosus and Y. lipolytica, compared to wild-type strains (“WT”). An increase in glycerol consumption 35-40% is observed with the evolved strains compared to wild-type strains. Figure 7 demonstrates the increase in oil productivity in ALE-evolved strains of C. oleaginosus compared to its wild type (WT) counterpart. Tests were performed using 2.5% crude glycerol as a carbon source. Increasing the number of culture transfers promoted the proliferation of more adapted strains with higher oil content (see, e.g. results for 4, 9 and 15 culture transfers).

[0182] Oil yields may be further increased by overexpressing various genes, via gene integration methods including, but not limited to Homologous Recombination (HR), and CISPR / Cas9 genome editing. Exemplary genes are shown in Table 6, which are responsible for fatty acid production via triglycerides. Table 6^Attorney Docket No.11833-014WO1 ^

[0183] Further exemplary genes are shown in Table 7, which are responsible for increasing pathway flux from glycerol to fatty acids. Table 7

[0184] Another approach is expression of combinations of genes from Table 6 and / or Table 7. For gene overexpression, inducible promoters under nitrogen starvation maximizes lipid production at nitrogen limiting conditions. Therefore, biomass production is not affected through inducible overexpression of lipid biosynthesis pathways. Additional modifications may be added to yeast strains to confer reduced viscosity and increased thermotolerance.

[0185] Oil yields are increased by knocking out various genes associated with the consumption of fatty acids, e.g. through CRISPR / Cas9 genome editing. Exemplary genes are shown in Table 8 – these genes may be targeted to minimize fatty acid consumption during cellular metabolism. Table 8^Attorney Docket No.11833-014WO1 ^ Example 4 - Development of new oleaginous strains through metabolic engineering and increasing carbon flux toward acetate and consequently lipid biosynthesis

[0186] A combinatorial gene expression approach was applied to increase the expression (relative to wild-type) of the following genes: acetyl CoA carboxylase (ACC), malonyl CoA- ACP trans acylase (MCA), ^-ketoacyl-ACP synthase, ^-ketoacyl-ACP reductase, ^-hydroxy butyl-ACP dehydrogenase, enoyl-ACP reductase, AMP deaminase (AMPD).

[0187] ACC pushes carbon flux from central carbon metabolism toward fatty acid precursors. AMPD inhibits the Krebs cycle to increase the accumulation of citrate. Consequently, citrate diverts toward the fatty acid synthesis pathway. Citric acid breaks down to Acetyl CoA by ACL1, 2. Then fatty acid synthesis will be triggered by carboxylation of acetyl CoA to malonyl CoA by ACC and in the presence of NADPH, which is generated by MAE or the pentose Phosphate pathway. MEA increases the NADPH supply.

[0188] ^-oxidation of fatty acids was also downregulated via targeting of the PEX10 and MFE genes. Example 5 – Fermentation Optimization of Autotrophic Acetic Acid Production Using Acetobacterium woodii Introduction

[0189] Acetic acid / acetate is a useful feedstock candidate for the production of lipids by oleaginous yeasts for industrial applications and by upcycling of industrial carbon waste. A strong reason for this is that acetate can be sustainably produced using carbon dioxide as a carbon source and hydrogen gas as an energy source by Acetobacteria . Acetobacterium woodii is one of the more well-studied acetobacteria (Ragsdale 2008). Other exemplary strains there are less comm on found in industrial application, including but are not limited to, Sporomusa ovata, Sporomusa paucivorans, Sporomusa rhizae, Sporomusa silvacetica, Sporomusa sphaeroides, Sporomusa termitida, Terrisporobacter glycolicus, Carboxydothermus ferrireducens, Carboxydothermus hydrogenoformans, Carboxydothermus pertinax, Moorella glycerini, Moorella mulder, Moorella thermoacetica, Acetitomaculum ruminis, Acetoanaerobium noterae, Acetobacterium bakii, Acetobacterium carbinolicum, Acetobacterium dehalogenans, Acetobacterium fimetarium, Acetobacterium malicum, Acetobacterium paludosum, Acetobacterium tundrae, Acetobacterium wieringae, Acetobacterium woodii, Acetohalobium arabaticum, Acetonema longum, Alkalibaculum bacchi, In some embodiments, the acetogenic microorganism is of the genera Moorella, Sporomusa or Acetobacterium. Preferably, the acetogenic microorganism is of the species Sporomusa silvacetica Moorella thermoacetica,Clostridium magnum, Acetobacterium woodii in a pure culture or a consortium. ^Attorney Docket No.11833-014WO1 ^ Methodology Long-Term Passaging and Maintenance of Acetogenic strains including A. woodii Cultures, Sporomusa silvacetica, and Moorella thermoacetica

[0190] A wild-type strain of A. woodii, Sporomusa silvacetica, and Moorella thermoacetica was acquired from the DSMZ culture collection, E.g., A. woodii DSM 1030 in lyophilized form. This lyophilizate was resuspended in heterotrophic and anoxic DSMZ 135 acetobacterium medium as recommended by DSMZ in an anaerobic chamber and passaged to anaerobic Balch tubes for further propagation of active cultures for experimental work and until a cryopreservation method could be developed and tested.

[0191] Active cultures were passaged in pairs in Balch tubes on a weekly basis with fresh medium addition mid-week and grown in a shaker incubator (New Brunswick Innova 44, orbit: 2”) at 30 °C. From these active cultures, pairs of backup cultures to be stored at 4 °C are created monthly. Seed cultures for experiments in serum bottles would also be propagated at 30 °C from these weekly active cultures. Old cultures are retained at room temperature until at least two more pairs active cultures had been passaged. Nearly all culture passages and liquid transfers were performed outside an anaerobic chamber via syringe after using a sterile anoxic gas to flush the syringe and needle. In this work, different acetogenic strains were used and various bio-process optimization methods applied, comprising conversion of CO2to a carbonate / bicarbonate substrate to enhance the mass transfer and CO2solubility of CO2in the liquid phase and enhance the productivity. Prior to or immediately after passaging of a culture, the new culture vessel(s) was pressurized to approximately 5 psig with 80 / 20 v / v% H2 / CO2. Acetate Production Bioreactor Experiments Bioreactor configuration

[0192] All acetate production experiments were conducted in a precision-controlled bioreactor. An Eppendorf BioFlo® 120 bioreactor controller system with a 2 L bioreactor vessel was used for anaerobic cultivation. The vessel was configured with two Rushton impellers, a microsparger, and no baffles. The vessel was also equipped with a temperature sensor, oxidation-reduction potential (ORP) sensor (Hamilton EasyFerm Plus Arc), pH sensor (Mettler Toledo InPro® 3253i), and polarographic dissolved oxygen (DO) sensor (Mettler Toledo InPro 6810). For temperature control of the bioreactor was achieved with a heating jacket, an internal cooling loop and a vapor condenser for reducing media evaporation. The cooling loop and condenser were supplied by a chiller set to 4 °C (Figure 8).

[0193] During operation, the bioreactor was sparged with anoxic gas. Hydrogen, carbon dioxide, and nitrogen gases each had their flow rates controlled by their own dedicated thermal mass flow controllers (TMFCs); Alicat TMFCs were used for hydrogen and carbon ^Attorney Docket No.11833-014WO1 ^ dioxide, and the TMFC in the BioFlo 120 controller were used for nitrogen gas. The gases were mixed downstream of the TMFCs and were passed through a Vici Metronics oxygen trap to remove additional trace of oxygen from the mixed gas. The mixed gas was passed through a sterile 0.2 µm filter and sparged into the agitated bioreactor. The exhaust gas was cooled in a condenser and passed through another filter before being directed to a stream selector supplying an SRI Instruments gas chromatograph equipped with a thermal conductivity detector (TCD) for CO2, H2 and N2 measurements. To prevent over pressurization of the bioreactor, a bleed check valve was attached to the exhaust gas line immediately prior to the stream selector. A feed bottle pressurized with nitrogen gas containing an anoxic base titrant supplied base to the bioreactor via controller peristaltic pumps to maintain a constant pH. Experiment setup, media preparation, and liquid transfers

[0194] Bioreactor inocula were grown in 1 L serum bottles with anoxic and reduced heterotrophic media. Filter-sterilized culture tube and bottle media was made anoxic by sparging the medium in a stoppered media or serum bottle with a 80 / 20 v / v% H2 / CO2 gas mixture at a flowrate between 0.5-2.0 SLM for 1-3 hours on shaker table followed by the addition of a reducing agent using an anoxic gas-flushed syringe. A CO2-containing gas mix was used to maintain a medium pH close to neutral, as the media used contained high concentrations of sodium bicarbonate; the stripping of carbon dioxide via sparging without carbon dioxide causes the media to become very alkaline. Prior to or after inoculation of seed cultures, the vessels are pressurized to approximately 5 psig with the 80 / 20 v / v% H2 / CO2 gas mixture to facilitate autotrophic growth.

[0195] The starting bioreactor medium was filter sterilized and transferred to the bioreactor in a laminar flow hood. The bioreactor was then sparged with the anoxic gas mix to be used in the experiment for several hours before a reducing agent is added to fully reduce the medium. Medium for mid-run addition is filter sterilized and aseptically transferred to a feed bottle with or without a pump tubing line. The bottle is then sparged through the dip tube and out the vent line with a 80 / 20 v / v% H2 / CO2 gas mixture. Following sparging, the bottle is connected to the bioreactor in an aseptic and anoxic manner and the medium is pumped in using either a peristaltic pump or nitrogen gas in the headspace. If using a pump, the bottle headspace need to be continually or intermittently supplied with more nitrogen gas to keep the bottle pressurized. Medium withdrawals were done by closing off the bioreactor exhaust valve to pressurize the vessel and opening the valve on the harvest or sample line directed to a broth capture bottle. Anoxic and aseptic transfers of seed cultures to the bioreactor via sterile tubing with a hypodermic needle piercing the bottle stopper and pressurizing the serum bottles with sterile nitrogen gas through another needle. Sample processing and data collection ^Attorney Docket No.11833-014WO1 ^

[0196] Samples were taken from the bioreactor via syringe through a harvest dip tube. A 2- 4 mL sample was withdrawn to flush the dip tube and another 1.5-2.0 mL sample was taken for processing

[0197] Biomass production and cell growth were quantified by multiple methods. Optical density measurements of whole broth samples were diluted as needed below 0.600 at a wavelength of 600 nm (OD600) using a Thermo Scientific GENESYS 50 spectrophotometer. Whole broth samples were also clarified by centrifugation and the resulting cell pellets were weighed and dried via lyophilization to obtain wet and dry cell weights. Lastly, an SBI Scientific Bioprocessing bioreactor cell growth quantifier (CGQ BioR) was used on the bioreactor to collect real-time absorbance data that could be correlated to cell density.

[0198] Acetate production was quantified by two separate methods. For instant data collection, a SentiaTMwine analyzer with acetic acid test strips was used. Clarified whole broth supernatant was diluted in DI water to a suitable range for the instrument. For accurate measurements, clarified supernatant would also be saved and frozen at -20 °C for HPLC analysis at the end of each executed fermentation run. The HPLC analysis would more accurately quantify acetate titers and allow for the measurement of other analytes of interest including ethanol, fructose, and others (Error! Reference source not found.).

[0199] The consumption rate of base titrant required to maintain a stable pH was quantified by measuring the weight of the base titrant feed bottle when sampling. This information also allows for the indirect determination of the rate of broth acidification.

[0200] The composition of fermenter exhaust gas was measured using an SRI Instruments GC-TCD. Filtered exhaust gas from the bioreactor condenser was routed to the GC. Nitrogen gas was added to the sparge gas mix to act as an inert tracer to allow for the direct comparison of peak areas associated with each gas due to changes in outlet gas flow rate due to consumption and production of gas phase components. Calculations

[0201] Biomass production of A. woodii was modeled to follow a logistic exponential growth kinetics until stationary phase was reached. Equation 1 was used to model cell growth and was rearranged to isolate the specific growth rate µ for estimation using numeric differentiation of the natural log of cell density over time.Equation 1. Exponential growth model for biomass production. ^t is the time interval of analysis, Xi and X are respective biomass concentrations at the start and end of the time interval, and µ is the specific growth rate. ^Attorney Docket No.11833-014WO1 ^

[0202] Numerical differentiation of acetate and biomass concentration to calculate production and growth rates was done by calculating and differentiating a Lagrange interpolation polynomial for three consecutive data points. The equation of this derivative is shown as Equation 2. With equally spaced points, the equation will simplify to the often- used 2nd-order numeric differentiation equations for forward, central and finite differences; however, using a Lagrange polynomial allows for numerical differentiation of data with unequally-spaced points albeit with some increase in error and reduced precision. This allowed for much greater flexibility regarding when samples could be taken and processed during experiments. Forward approximations were used for the first time point and time points directly following a large broth volume change event (e.g., a draw and fill operation, medium addition, etc.); backward approximations were used for the final time point and time points directly prior to a large broth volume change event; central approximations were used for all other evaluated time points.

[0203] The autotrophic production of acetate by A. woodii is possibly partially growth- associated. To determine this case senario, acetate production and biomass growth rates calculated using Equation 2 were used in Equation 3 to determine the growth and non- growth associated product formation coefficients. This was done by performing an iterative multivariable least squares regression to minimize the squared difference between the calculated acetate production rates from the experimental data and the total production rates predicted by the partial growth-associated product formation model.^Attorney Docket No.11833-014WO1 ^ Equation 3. Model of growth and non-growth associated product formation.5dP / dt is the instantaneous rate of change in product concentration (i.e., acetate production rate), and ^ and ^ are coefficients corresponding to growth and non-growth-associated product formation. A higher ^ / ^ ratio would indicate that product formation is more strongly associated with growth and a lower ^ / ^ ratio would indicate that product formation is less strongly associated with growth.

[0204] In addition to calculating instantaneous production rates, average production rates over the course of the full fermentation runs were also calculated using Equation 4. Initial time points were chosen to be the first samples in each fermentation run and the first samples taken after a significant broth volume change event like a draw and fill since such events drastically reduce the concentration of acetate and create less useful results. ^^^ ^ ^^^%^ "#$ ^ ^ ^%Equation 4. Average product formation rate over time. (dP / dt)avgis the average product formation rate, P is the product concentration at time point t, and P0is the product concentration at the nearest prior initial time point t0. Experiments executed

[0205] Three separate bioreactor experiments were executed withwoodii to produce acetate using carbon dioxide and hydrogen gas as carbon and energy sources. These experiments have been given the titles of CFF257, CFF265, and CFF267. Important process conditions and information for each experiment are listed in Error! Reference source not found.. For all bioreactor experiments, seed cultures were grown with either DSMZ135, DSMZ135-DWB001, or DSMZ13-DWB001a heterotrophic medium at 30°C in a shaker incubator until late log or early stationary phase was reached. In all cases where fresh medium was added to the bioreactor, the medium was filter sterilized and made anoxic via gas sparging prior to addition. If necessary, reducing agent was added to the bioreactor during or immediately after the medium transfer.

[0206] Experiment CFF257 was the first bioreactor conducted. In this experiment, 250 mL of seed culture was used to inoculate 1 L of DSMZ135-DWB003 mixotrophic medium at the beginning of the culture to promote the initial growth and gradually transitioned to autotrophic condition. This medium contained fructose and high concentrations of trace metals, yeast extract, and vitamins to promote initial heterotrophic cell growth and acetate production with a gradual transition to autotrophic growth and continued acetate production. The bioreactor was then operated in batch mode for six days before it was terminated. To manage and avoid foam formation, antifoam 50 % polypropylene glycol was added to the media. Agitation and gas sparge rates were manually increased stepwise within the first 48 hours of ^Attorney Docket No.11833-014WO1 ^ the run. The increase in agitation rate created excessive heat generation, raising the culture temperature 2-3 °C above the setpoint for a few hours. When the temperature rise was observed, active cooling of the vessel was quickly implemented.

[0207] Experiment CFF265 was the second bioreactor experiment conducted. This experiment was a biological replicate of CFF257 with some operational changes. 220 mL of seed culture was used to inoculate 1 L of DSMZ135-DWB003 mixotrophic medium. Agitation at 1200 rpm and sparge rates at 0.5VVM were kept constant throughout the entire run to improve consistency and reliability of real-time culture absorbance and exhaust gas composition data. Polypropylene glycol (50 v / v%) antifoam addition was added immediately following inoculation and continued with manual addition as-needed. An improved GC separation and peak integration method for the GC was also implemented in this experiment. After 6 days of operation, cellular activity and acetate production appeared to have declined significantly, so 10 mL of sterile 500 g / L fructose and 2 mL of 500x Wolin’s vitamin solution were added to the broth. One day later, 500 mL of broth was removed and 500 mL of autotrophic DSMZ135-DWB002 autotrophic medium was added to the bioreactor in a draw-and-fill operation. The next day, the run was terminated because no growth or acetate productivity was observed.

[0208] Experiment CFF267 was the third bioreactor experiment conducted. It was executed in two separate phases. In the first phase, approximately 1.1 L of seed culture was concentrated via tangential flow filtration to a volume of approximately 550 mL, though it was originally planned to concentrate it to a volume of 100 mL. The bioreactor was originally filled with 1 L of DSMZ135-DWB002 autotrophic medium. Prior to inoculation, 500 mL was removed and stored at 4 °C; this volume would be added to the bioreactor again after 3 days of operation post-inoculation, bringing the broth volume up to about 1.5 L. This medium had a lower trace metal, vitamin, and yeast extract concentration compared to the DSMZ135- DWB003 medium used in the prior two experiments due to the use of a higher initial cell density and the elimination of the initial heterotrophic growth period. After another 5 days of operation, 1400 mL of the 1.5 L of broth was withdrawn to leave approximately 100-150 mL remaining in the bioreactor, then 1000 mL of DSMZ135-DWB002b autotrophic medium was then added. This medium contained half the concentration of sodium bicarbonate than the prior media used. After hours of no discernable change in pH or ORP, 20 mL of another seed culture that had been prepared for the initial bioreactor inoculation was transferred to the reactor. The run was then terminated after another 7 days of operation. Bioreactor Cell Retention Module Testing

[0209] In order to achieve high acetate production rate, a perfusion bioreactor using a cell- retention modules needed to be implemented. Two different TFF modules were tested for use in a perfusion fermentation process. The first module tested was a Repligen MicroKros ^Attorney Docket No.11833-014WO1 ^ hollow fiber filter module (C02-P20U-05-N). This module contained nine mPES hollow fibers 20 cm in length with an inner diameter of 0.5 mm, an effective filtration area of 28 cm2, and a pore size of 0.2 µm. The second module tested was a Pall MinimateTMcassette filter module (OA030C12). This module contained OmegaTMmPES membranes with an effective filtration area of 50 cm2and a molecular weight cutoff (MWCO) of 30 kDa.

[0210] The modules were first tested abiotically to evaluate their performance characteristics. DI water was pumped through each of the modules at different rates using an INTLLAB peristaltic pump. The weight of water flow from the filtrate and retentate sides of the module over a set length of time was measured to determine the total water flow rate and the ratio between filtrate and retentate flows. A pressure gauge was also positioned immediately upstream of the module to measure the pressure drop across the length of the modules at different flow rates. Being a hollow fiber module, the Repligen TFF unit was tested with the process water on both the shell and the lumen side.

[0211] The Repligen MicroKros module was used for further biotic testing. The module was used to concentrate approximately 1.1 L of A. woodii seed culture for a bioreactor experiment to test it under experimental conditions. The module was autoclaved prior to use following the manufacturer’s directions as closely as possible given the cycle programming limitations of the usable autoclaves at the time. The module was connected to sterile tubing and flushed with anoxic gas before connecting to a seed culture bottle via syringe needles. Anoxic nitrogen gas was injected to the culture bottle to continue to flush the module and replenish the headspace volume as the liquid volume decreased over time. The culture bottle was then inverted and liquid whole broth was pumped through the lumen side of the module using a peristaltic pump. Retentate from the module was returned to original culture bottle for further concentration while the shell side filtrate was collected in another vessel. The process was operated for several hours until filtration performance had diminished to the point that further culture concentration would not be feasible. Results & Discussion Acetate Production Experiments

[0212] In Figures 9- 12, data from the three bioreactor experiments conducted are shown. Dry cell weight measurements were not taken in experiment CFF 257. As such, only OD600measurements from the experiment are plotted instead for that experiment. A summary of experimental conditions is defined in table 9.

[0213] All experiments were batch phase with occasional draw-and-fill operation for some experiments. Experiment CFF257 was the first attempt to replicate these published results. Fructose and additional yeast extract, trace metals, and vitamins were added to the fermentation medium to allow for a fast heterotrophic growth period to accumulate biomass ^Attorney Docket No.11833-014WO1 ^ during the beginning of the run and allow additional nutrients for a transition to autotrophic growth and acetate production.

[0214] Cell growth and biomass accumulation of Acetobacterium woodii were monitored in bioreactor experiments CFF257, CFF265, and CFF267 (Figure 9). A summary of experimental conditions is defined in table 9. Cell density was measured over time by optical density at 600 nm (OD&'') in all experiments, with dry cell weight (DCW) measurements available for CFF265 and CFF267. In CFF257, only OD&'' data were collected, which served as the primary indicator of biomass accumulation in that run.

[0215] Acetate production dynamics closely followed cell growth patterns across all bioreactor experiments (Figure 10). Acetate titers increased over time in parallel with rising cell densities, indicating growth-associated production behavior. Instantaneous acetate production rates, calculated by numerical differentiation, revealed distinct production phases corresponding to growth kinetics (Figure 11), while calculated average production rates provided a broader overview of acetate synthesis trends throughout the cultivation period (Figure 12).

[0216] In experiment CFF267, after a draw-and-fill operation was performed, modeling of partial growth-associated acetate production aligned closely with measured data (Figure 13). Production rate coefficients ^ and ^ were determined to be 11.7 and 2.3, respectively, reflecting both growth-associated and non-growth-associated contributions to acetate synthesis.

[0217] Analytical comparison of acetate measurements demonstrated strong agreement between Sentia wine analyzer data and high-performance liquid chromatography (HPLC) results (Figure 14). Sentia measurements were scaled by a factor of 1.28, determined by least-squares regression, to match HPLC titers, validating the analyzer’s use for rapid acetate quantification.

[0218] Finally, performance characterization of the Repligen MicroKros filtration module showed the relationship between filtration efficiency and total flow rate, as well as the corresponding pressure drop across the membrane (Figure 15). Filtration efficiency, defined as the ratio of retentate to filtrate flow rate, varied with total flow, providing critical data for optimizing downstream processing conditions. Table 9. CO2 and H2 conversion to acetate experimental condition.^Attorney Docket No.11833-014WO1 ^EXAMPLE 6: Enhanced Screening and Adaptive Laboratory Evolution of Cutaneotrichosporon oleaginosus for Improved Growth and Lipid Production

[0219] Disclosed herein is a pipeline to identify and evolve C. oleaginosus mutants with enhanced growth and lipid production traits. The approach combines mutagenesis (UV or ARTP), high-throughput screening, and long-term Adaptive Laboratory Evolution (ALE). This strategy maximizes genetic diversity while applying selective pressures to redirect carbon flux toward lipid biosynthesis on crude glycerol.^Cutaneotrichosporon oleaginosus (ATCC 20509; NCBI TaxID: 879819) was used in the following experiments. Mutagenesis Approaches: • UV Mutagenesis: Cells were exposed to UV irradiation for 0–5 minutes, introducing random mutations. • Atmospheric and Room Temperature Plasma (ARTP): To induce high genetic diversity through non-thermal plasma exposure while preserving cell viability. Exposure times ranged from 0 to 150 seconds across multiple intervals, with 24 agar plates (YMP) prepared for mutant recovery. High-Throughput Screening Pipeline: Single Colony Screening ^Attorney Docket No.11833-014WO1 ^ • Colony Selection and Inoculation: After mutagenesis distinct colonies were isolated and cultured in 24-well plates containing 800 µL YMP + 2% crude glycerol. • Incubation: 16 plates were incubated at 30°C and 700 RPM for 72 hours using an orbital plate shaker. • Secondary Culturing: A 5 µL aliquot from each well was transferred into fresh 24-well plates with nitrogen-limited NMM + 5% crude glycerol for another 72-hour growth phase. • Cryopreservation: Original YMP+2% crude glycerol cultures were archived at –80°C with 50% glycerol. Population-Based Screening: • Non-isolated colonies were enriched in NMM + 5% crude glycerol. • At 24, 48, and 72 hours, lipid-rich population was isolated by lipid content analysis and screening. • Two passages of this “plate wash” method was conducted before re-plating and re- screening colonies Long-term Adaptive Laboratory Evolution (ALE) • ALE consisted of 33 serial passages, each lasting 96 hours in nitrogen-limited media with crude glycerol as the carbon source. • After each cycle, lipid-rich cells were collected and transferred to fresh media. • This regime applied strong selective pressure for enhanced glycerol utilization and lipid accumulation Validation of Long-term Adaptive Laboratory Evolution (ALE) Selected mutants underwent: 1. Antibiotic-Selective Plating – to ensure clonal purity. 2. Batch Fermentation Trials – conducted in 125 mL baffled flasks with 25 mL NMM + 5% crude glycerol, inoculated to OD 1.0, and incubated at 30°C with agitation. Biomass and lipid production was accessed at 72h and 96hrs. 3. Whole Genome Sequencing – to identify mutations underpinning phenotypic improvements Media Compositions • NMM (Nitrogen Minimal Medium): Yeast extract 1.5 g / L, (NH()^SO( 0.25 g / L, MgSO(·7H^O 1.5 g / L, KH^PO( 7 g / L, Na^HPO(·2H^O 5 g / L, FeSO(·7H^O 0.08 g / L, ZnSO(·7H^O 0.01 g / L, CaCl^·2H^O 0.1 g / L, MnSO(·4H^O 0.1 g / L, CuSO(·5H^O 0.002 g / L, CoCl^·6H^O 0.002 g / L, crude glycerol 50 g / L. • YMP(G) Medium (for agar plates): Yeast extract 3 g / L, Malt extract 3 g / L, Peptone 5 g / L, Glucose 10 g / L. ^Attorney Docket No.11833-014WO1 ^

[0220] Fermentation trials comparing the ALE-derived strain with the reference strain C. oleaginosus ATCC 20509 demonstrated that overall biomass production (cell dry weight) was not significantly different between the two strains at either 72 h or 96 h of cultivation. Both strains reached comparable levels of growth under the tested conditions, indicating that the ALE process did not substantially alter general proliferation capacity.

[0221] In contrast, lipid accumulation showed clear and reproducible improvements in the ALE strain. At 72 h, the evolved strain exhibited a 9% increase in lipid content relative to the reference strain. By 96 h, the performance advantage increased to a 19% higher lipid accumulation. These results highlight that while biomass yield remained unchanged, the ALE strain preferentially redirected carbon flux toward lipid biosynthesis when grown on crude glycerol. Results of full genome sequencing of the ALE strain indicated 8 regions of mutations described in table 10.

[0222] The ability of the ALE strain to maintain growth while enhancing lipid accumulation represents a commercially relevant improvement, making it particularly suitable for industrial applications where high-value lipid yield is prioritized over biomass output.^

[0223] Increasing lipid content in yeast as a result of a combination of ALE and genetic modification as determined by the relative lipid content in cells normalized to biomass. ALE strain produced significantly higher lipid than the wild strain. Fig 28 demonstrates the results of DGA1 transformation using ALE strains as a chassis produced significantly higher lipid in comparison to ALE strains alone. A3, A4, A5, A9 were selected, transformed colonies of ALE and DGA-1 out of 12 colonies. CTRL strain 1: Unmodified evolved strain used as chassis for modification of strains A3, A4, A5 and A9. CTRL strain 2: Non evolved strain Parental of CTRL strain 1. A3, A4, A5, A9: Transformed colonies of ALE and DGA-, selected from 12 different colonies after transformation. Example 7: A method and process to increase overall lipid productivity of yeast on industrial crude glycerol as carbon source Introduction / Summary

[0224] A method for increasing the lipid productivity of Cutaneotrichosporon oleaginosus is described. This is accomplished through the strategic optimization of biomass growth prior to lipid accumulation which enables an improved lipid accumulation rate. The process includes: (1) a process for enhancing lipid productivity by adjusting nutrient conditions to promote biomass accumulation before lipid synthesis begins; (2) the use of specific cultivation parameters to delay lipid onset while maximizing cell growth; and (3) an optimized bioprocess workflow that increases total lipid output per unit time.

[0225] The reactors were seeded with 5 %(v / v) of inoculum in the bioreactor containing the initial media composition to commence the experiment. After 8-9 hours of fermentation, 2.7 g / L of nitrogen was added to prevent nitrogen limitation in order to maintain biomass growth ^Attorney Docket No.11833-014WO1 ^ (while limiting lipid accumulation). This additional nitrogen boots biomass in order to maintain the growth rate longer as compared to previous experiments with lower nitrogen. At 48 hours, the phosphorus content was doubled that the initial medium to prevent depletion or reaching limiting phosphorus concentrations during the lipid accumulation phase. This process was executed with crude glycerol from two different industrial scale biodiesel facilities denominated herein A and B (Figure 20). The whole cell biomass concentration achieved was 160 – 168 g / L when cultivated with A glycerol and 142 g / L with B crude glycerol after 72 hours. Lipid productivity analyzed between 42 to 72 hours of fermentation yields 1.2 – 1.3 g / L / h on A crude glycerol and 1.14 g / L / h on B crude glycerol. Example 8: Use of acetic acid as co-substrate for high lipid productivity in Cutaneotrichosporon oleaginosus

[0226] As described above, the biological production of acetate from CO2 and H2 is a fundamental methodology to this technology. This process produces acetate which is then used as sole source of substrate or amended with other organic carbon sources by yeast to produce biomass and oil. Since C. oleanginosus can accumulate high lipid content using acetic acid, the feasibility was assessed in the lab. C. oleaginosus was cultivated in 1L fermentation in a pH-stat. The initial media contained 30 g / L glucose and acetic acid addition with rises in pH. The culture grew to an OD600 of 140 in 44 hours achieving a productivity of 1.3 g / L / h. The results are shown in Figure 21. EXAMPLE 9: Mutations Unique to the ALE strain compared with the wild-type and reference strains. Table 10. Mutations Unique to the ALE strain compared with the wild-type and reference strains.^Attorney Docket No.11833-014WO1 ^^Attorney Docket No.11833-014WO1 ^EXAMPLE 10: Engineered DGA1

[0227] A hygromycin resistance cassette containing a recoded version of the DGA1 gene from C. oleaginosus was introduced into the wild-type strain, resulting in ectopic integration of the cassette. Twelve colonies from the transformation were selected, and their performance was evaluated using the Nile Red assay. One isolate, DGA1_Co_0_11, exhibited higher fluorescence than the wild-type control and was selected for further validation. ^Attorney Docket No.11833-014WO1 ^

[0228] The lipid content of isolate DGA1_Co_0_11 was assessed in flask cultures, showing a 60% increase in lipid titer compared to the wild-type strain after 72 hours. Integration of the cassette in DGA1_Co_0_11 was confirmed by colony PCR followed by NGS amplicon sequencing. Components of the cassette are shown in SEQ ID NOS: 27-41. Example 11: Engineering ME

[0229] A hygromycin resistance cassette containing a recoded version of the Malonic Enzyme gene from C. oleaginosus was introduced into the wild-type strain (Sequence ID NO.39). ^Attorney Docket No.11833-014WO1 ^ REFERENCES (1) ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^-Ljungdahl Pathway of CO2 Fixation. Biochim. Biophys. Acta - Proteins Proteomics 2008, 1784 (12), 1873–1898.. (2) ^^^^^^ ^^!^^^"^#^^^^^^^^^^$^^^^-Botz, D. Continuous Gas Fermentation by Acetobacterium Woodii in a Submerged Membrane Reactor with Full Cell Retention. J. Biotechnol.2015, 212, 11–18. (3) "^^#^^%^^"^^^^^^^^^^^^%^^^^^^&$^^^^^^^^^^^^^'^^^^(^^#^^^^^^"^^^^^^^(^^^^^^^^^)^^^^^^^ Am. J. Clin. Nutr.1972, 25 (12), 1318–1323. (4) &^^^*+,^-+^^.^^^ / 0^1^^-+^^"^^^^^^^2^^^^^^^^^^-M. R. The Historical Development of Cultivation Techniques for Methanogens and Other Strict Anaerobes and Their Application in Modern Microbiology. Microorganisms 2022, 10 (2), 412. (5) 3^^^^^^^&^^^^^^!^^^,^^4^^^^^Biochemical Engineering^^5^#^^^^6^7^^^^^^^8^^$9^^:;;<^ (6) 3^^^^^^^&^^7^^^3^^^^^#^^^^^=^^^((^^^^-^^!^^^^^^^^^^^^^^^^^^^^^^^^(^^^^#^$^(^^^^ Ultrafiltration Membrane Systems. J. Dairy Sci.1992, 75 (3), 718–724. (7) 8$^^^^^^^&^$^^^^%^^^>^^^^^,^#^^^7^^7^$^^^^^^^^^!^^2^^^^^!^^^^^^^^^(^"^^^^(^^^^^^^^^^ Membranes: A Mini-Review. Polymers 2021, 13 (6), 846. (8) "^^^^^ ^,^^^3^^^?^,^^^^^^^^$^^$^^^^!^^^^^^^^^^^@9^^^^^^^^^^4^^^^^^^^^^^^^^(^^^^^'(^ Polyethersulfone (Pes) Membrane Due to Chemical Cleaning. Sci. Rep.2019, 9 (1), 422. (9) .^^2^^^^^^=^-^^^,#^^A^^^8^^^^^^ / ^^^$^(^^^^^^^!^^^^^^^^^(^'7^"^2)^^^^^^7^$^^^^)#^ Proteins. Sep. Purif. Technol.2012, 94, 39–43. (10) 3^^^$,^^^^-^^^^^^.^^^$^^-^^^^^=^^^-Term Storage of Obligate Anaerobic Microorganisms in Glycerol. Appl. Biochem. Microbiol.2006, 42, 177–180. (11) ^)$)^^,^^^^&^^.^^^ / ^^^^^^"^^!^^^^^^^^^^^!^^3^^^^^^^^^^!^^-^^^^^^^^(^!^^)^^^ Monoxide: Rich Syngas or Waste Gases to Bioethanol. Biofuels Bioprod. Biorefining 2011, 5 (1), 93–114. (12) Beck, M. H. Biological Conversion of CO2 into the Platform Chemicals Lactate and 3- Hydroxypropionate Using Recombinant Strains of Acetbacterium Woodii, Ulm University, 2019. (13) .^$^^^^^(^^!^^^^^^ / ^^^^^^^^^8^^^^^^4^^^^^^^!^^^5^2^^^^5^^^.^-^,^^^^^^"^^^^^^^^=^^^3^^^^^- 8^B^)^^^^^^^^^^^^(^B^^^^^^^^^C$^^^^^^^^-^^"^^^)^^^^^^^^^#^^^^^^-^^^^^Acetobacterium Woodii as a Flexible and Robust Host for Formate-Based Bioproduction. Metab. Eng.2021, 68, 68– 85. (14) Grimalt-^^^2^^#^^^^^^DEF#,^^"^^^^^^2^,^9^$^^^^^^^^^^,^^^^^^^A^^ / ^^^8^-^^^^^&^^.^^ Cryopreservation and Fast Recovery of Enriched Syngas-Converting Microbial Communities. Water Res.2020, 177, 115747. (15) "^^^^^%^^^"B^^^^^^ / ^^^^#^^^^^^#^^^^^8^^^^^^^^^(^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Bacterium Acetobacterium Woodii. Environ. Microbiol.2021, 23 (11), 6953–6964.15739. ^Attorney Docket No.11833-014WO1 ^ (16) &^^^^^^^^^^"B^^^^^^ / ^^^8^^^^^^^^,^^8^^^^^^$2^4^9^^^^^^^^^(^^^^^^^^^7^^2^^^^^^)#^ the Acetogenic Bacterium Acetobacterium Woodii. J. Bacteriol.1989, 171 (10), 5473–5478. (17) 4^2^^^^^"^^^^^$^^^^-Botz, D. Reaction Engineering Analysis of Hydrogenotrophic Production of Acetic Acid by Acetobacterium Woodii. Biotechnol. Bioeng.2011, 108 (2), 470– 474. (18) 3^=!&^^^^^^^^^^!&@3^^5&^^^^^^5^..^^^^^^^^^^^^@=7^^^^^^^^^^^^^^)^^^^^^$2^^^^ New Genus of Hydrogen-Oxidizing, Carbon Dioxide-Reducing, Anaerobic Bacteria. Int. J. Syst. Evol. Microbiol.1977, 27 (4), 355–361. ^ ^Attorney Docket No.11833-014WO1 ^ SEQUENCES SEQ ID NO: 1 LOCUS YL_DGA2-_YALI0D07986g 1581 bp DNA linear 1 atggaagtcc gacgacgaaa aatcgacgtg ctcaaggccc agaaaaacgg ctacgaatcg 61 ggcccaccat ctcgacaatc gtcgcagccc tcctcaagag catcgtccag aacccgcaac 121 aaacactcct cgtccaccct gtcgctcagc ggactgacca tgaaagtcca gaagaaacct 181 gcgggacccc cggcgaactc caaaacgcca ttcctacaca tcaagcccgt gcacacgtgc 241 tgctccacat caatgctttc gcgcgattat gacggctcca accccagctt caagggcttc 301 aaaaacatcg gcatgatcat tctcattgtg ggaaatctac ggctcgcatt cgaaaactac 361 ctcaaatacg gcatttccaa cccgttcttc gaccccaaaa ttactccttc cgagtggcag 421 ctctcaggct tgctcatagt cgtggcctac gcacatatcc tcatggccta cgctattgag 481 agcgctgcca agctgctgtt cctctctagc aaacaccact acatggccgt ggggcttctg 541 cataccatga acactttgtc gtccatctcg ttgctgtcct acgtcgtcta ctactacctg 601 cccaaccccg tggcaggcac aatagtcgag tttgtggccg ttattctgtc tctcaaactc 661 gcctcatacg ccctcactaa ctcggatctc cgaaaagccg caattcatgc ccagaagctc 721 gacaagacgc aagacgataa cgaaaaggaa tccacctcgt cttcctcttc ttcagatgac 781 gcagagactt tggcagacat tgacgtcatt cctgcatact acgcacagct gccctacccc 841 cagaatgtga cgctgtcgaa cctgctgtac ttctggtttg ctcccacact ggtctaccag 901 cccgtgtacc ccaagacgga gcgtattcga cccaagcacg tgatccgaaa cctgtttgag 961 ctcgtctctc tgtgcatgct tattcagttt ctcatcttcc agtacgccta ccccatcatg 1021 cagtcgtgtc tggctctgtt cttccagccc aagctcgatt atgccaacat ctccgagcgc 1081 ctcatgaagt tggcctccgt gtctatgatg gtctggctca ttggattcta cgctttcttc 1141 cagaacggtc tcaatcttat tgccgagctc acctgttttg gaaacagaac cttctaccag 1201 cagtggtgga attcccgctc cattggccag tactggactc tatggaacaa gccagtcaac 1261 cagtacttta gacaccacgt ctacgtgcct cttctcgctc ggggcatgtc gcggttcaat 1321 gcgtcggtgg tggttttctt tttctccgcc gtcatccatg aactgcttgt cggcatcccc 1381 actcacaaca tcatcggagc cgccttcttc ggcatgatgt cgcaggtgcc tctgatcatg 1441 gctactgaga accttcagca tattaactcc tctctgggcc ccttccttgg caactgtgca 1501 ttctggttca cctttttcct gggacaaccc acttgtgcat tcctttatta tctggcttac 1561 aactacaagc agaaccagta g SEQ ID NO: 2 LOCUS YL_TGL4_-_(YALI0F10010g) 2451 bp DNA linear 1 atgttcacct ccagagtttc cgaagcaagc accaccaact ttatccggcc gacggcacgg 61 tctcacatcc actttttttt cgccttcatc gccgcaaccg tccaccaact gctgctcatg 121 ctctaccaac tgcttggaga cggctacctc aagtcgtttg tcgacacagg tatcacgctg ^Attorney Docket No.11833-014WO1 ^ 181 gcccaacagt cggggctttc gggtatcgtc aacgccttga cttcagaggc caaactgcgg 241 atcgataaac ggtccatcat caaaaagctg ctagaggacc aggaaaacgc cgagtcgtac 301 tttgactggc tcaaggcgtc cagcgaactc gactatctgc tcggcaacca ggaatggaag 361 gaaagagacg agtgtccagc ttacgattac gaatacgtcc gactccgatt ggacgaactg 421 agacacgccc gaaccaataa cgacaccacc cgactgcttt acctcgtgcg aacaacgtgg 481 agtagaaacc tcggcaacct cggagacgtc aagctctacc acaactcctt taccggaacc 541 aaacgactca tcgaagacta cattctggaa tgcgaactgg ctctcaacgc gctcctggca 601 gccggaaacg acaagatccc ggaccaggag ctgctcacgg agctgctcaa caccagaaag 661 gcatttggac gaactgccct tctgctgtcc ggcggaggat gtctcggtct gctccacacc 721 ggtgttctcc aggccctctc agacacatcg ctcttgcccc acgtcatatc gggttcgtcg 781 gcaggctcaa tcatggccgc gggactgtgc attcacaaag acgaagaaca cgaggctttc 841 atcaccgagc tcatggagcg agactttgac attttcgaag agtccggaaa cgaagacacg 901 gtgctcgaac gagtgtctcg aatgctcaaa catggatcgc tactcgacaa cagatatatg 961 caggacacta tgcgagaatt atttggcgac atgacctttc tggaggccta caaccggact 1021 cgccgtattc tcaacgttac ggtatcgtct gctggcatct acgaaatgcc tcgtttgctc 1081 aactacctga cggcccccaa cgtactcatt tggtcggctg tctgcgcctc ctgctcggtg 1141 cctctcattt tcaatgccta cactctgctt gaaaaggagc ccaaaacagg agctattcag 1201 acctggaacg cttcttcgct gcggttcatt gacggatccg tctatgccga cgtgcccatt 1261 gcgcgtctct cagaaatgtt caatgtaaat catttcattg tctcgcaggt aaaccctcac 1321 gttgctcctt tcctcaagct cacagaagac aaggccaacc cggactcggt cgacgaaatc 1381 tacacgctca agctttggca caacttcaag acgctggtca ccgacgaggt catgcaccag 1441 ttgcaggtgc tgtacgagtt tggcatcttc aagaacctgt gttcgaaaat gggaggcgta 1501 ctgtcccagc gatacaaggg agacatcaca atcctgcccc aggtccatct ctcagagctc SEQ ID NO: 3 LOCUS Kl_TGL3-_(optimized_in_Y.l) 1792 bp DNA linear UNA 20-JUN-2024 1 ggatcccaca atgttctacc agcgaattat gatgtggttt aagcgagtgt tcttttactt 61 cttttacctg ctcctgaaca acgtgcccaa gaccgtctgg gacatcctgc acgtggtcac 121 tgatatctac atgttctggt ggcgaaagtt tatggacaag gttcgacctc gatcccgaac 181 catgtaccga gaggccgtgg ctggactgta cggttgtgac gattacgagg agtggtacga 241 gaaggcctcg ctcgttgacg agctgaccgg tgtggatctg tggcgacgaa acttcttttc 301 taagcgattc gactttgagg ctgtcctgga gcagtacgcc gctctcatgg agtctctgga 361 ggaagacgat ttcgagaccg ttaagtctcg attcaccaac actggcccca ctatgctgcg 421 aaacttcgcc ggaattgtcg acaagcgact ctttaccaag tctctggttg gaactaagct 481 cctgatcgag cagtacctcg acaaggttgt ggataccctg tacctcctga ctgctcaccc 541 tgaggtcgtt cagcgaacct tctttcagcg atgcaagctc tccctgggag ctaccgctct 601 ggctctgcag ggcggatcgc tcttcggtct gtttcatctc ggcgtgctga agggactcct ^Attorney Docket No.11833-014WO1 ^ 661 ggaccgaaac ctcctgccca acatcattaa cggtacctct atgggcgctt gtgttgcctc 721 gatggcttgt tgcctctctg acggagagct ggaggatctc ctgactggta accgactcgt 781 ctccaccatt aagcgagaca ctgccctgat gaaggagtgc ggttacggca acatcgacga 841 gcacttcaac attggtaccc tggtggagaa cgtggtccat aacggctact ctaaggacgt 901 gtacctcttc atcaagttta ttcagaagtc cgtcatcgga gatctgacct tcgaggaagc 961 cttccagcga actggcaagg tcctcaacat cgttgtgcac cccaccaaca agaacatttg 1021 tcctaacctc ctgaactacg tcaccactcc caacgttctg atctcttccg ctattgactg 1081 ctctttcggc accaacacta tttccaagaa cacctggctc ctgggaaaga acatcgagaa 1141 caagattgtg gactacctcg atcgaaagga gccccactac caggagctga agttctccgc 1201 ccctcagtac gtccaggact cgtctcagct ggaggctcct tacacccgac tcactgagct 1261 gttcaacgtg aacaacttta ttgtctccct cgctcgacct tacctggccc ctctcgctct 1321 gaacgacctg aagcatgata ttcgaacctc ggagtactac tactacaagc gattccccta 1381 catcgaccct ggctcctact cgcccatgca gctgtcgaag gtgaccaagc tcgagcctct 1441 ggccttcaag tttaagtacc acctggagcg aaagatgaag catatcctca ctatggagct 1501 gaagcaccga gtcgagatta tggactctct gggactcctg tccaactgga tcaagcgaat 1561 cgctattgat gagaagaccc ctcgatctgc cactgaggtc gctattgttc ctcatatgaa 1621 ctctctctcc gtgtcgcgaa tcattgaggg tcgactggac aacattaact actggatgaa 1681 gtgtggccag gagtccacct ggcccgttat ctcgctcgtg aagacccgat gcgccgtcga 1741 gttcactctg gacgatatca ttaagggctt caagaagtct atctagccta gg SEQ ID NO: 4 LOCUS Yl_Faa1_-_(YALI0D17864) 2076 bp DNA linear 1 atggtcggat acacaatttc ctcaaagccc gtgtcggtgg aggtcggccc cgccaagcct 61 ggcgagactg ccccccgacg aaacgtcatt gccaaggacg cccctgtcgt cttccccgac 121 aacgactcgt ccctgaccac cgtctacaag ctgttcaaaa agtacgccga gatcaacagc 181 gagcgaaagg ccatgggatg gcgagacacc atcgacatcc acgtggagac caaacaggtg 241 accaaggtcg tggacggagt ggagaagaag gtgcccaagg aatggaagta ctttgagatg 301 ggcccttaca agtggctctc atacaaggag gcccttaagc tggtccatga ttatggagct 361 ggtcttcgac acctcggaat caagcccaag gagaagatgc acatttacgc ccagacctcc 421 caccgatgga tgctctctgg cctggcttct ctgtctcagg gtattcccat tgtcactgcc 481 tacgacactc ttggagagga gggtctcact cgatctctcc aggagaccaa ctcggtcatc 541 atgtttaccg acaaggctct gctgagctct ctcaaggtct ctctcaagaa gggcaccgat 601 ctgcgaatca tcatctacgg aggtgatctg acccccgacg acaagaaggc cggaaacacg 661 gagattgacg ccatcaagga gattgttcca gatatgaaga tctacaccat ggacgaggtt 721 gtcgctctcg gccgagaaca cccccacccc gtggaggagg tcgactatga ggacctggcc 781 ttcatcatgt acacctctgg ttctaccggt gtccccaagg gtgtggttct gcagcacaag 841 cagatcctcg cctctgtggc cggtgtcacc aagatcattg accgatctat catcggcaac ^Attorney Docket No.11833-014WO1 ^ 901 acagaccggc ttctcaactt cctgcccctc gcacacattt tcgagtttgt gttcgagatg 961 gtcaccttct ggtggggtgc ttctctgggt tacggaaccg tcaagaccat ttccgatctg 1021 tccatgaaga actgtaaggg agacattcga gagctcaagc ccaccatcat ggtcggcgtt 1081 cccgctgtct gggaacctat gcgaaagggt attcttggca agatcaagga gctgtctcct 1141 ctgatgcagc gggtcttctg ggcctcattt gccgccaagc agcgtctcga cgagaacgga 1201 ctccctggtg gatctatcct cgactcgctc attttcaaga aggtcaagga cgccactgga 1261 ggctgtctcc gatacgtgtg taacggaggt gctccagtat ctgtcgacac ccagaagttc 1321 atcaccactc tcatctgtcc catgctgatt ggatgcggtc tgaccgagac tacagccaac 1381 accaccatca tgtcgcctaa atcgtacgcc tttggcacca ttggtgagcc caccgccgcc 1441 gtgaccctca agctcattga cgtgcctgaa gccggctact tcgccgagaa caaccaggga 1501 gagctgtgca tcaagggcaa cgtcgtgatg aaggagtact acaagaacga ggaggagacc 1561 aagaaggcgt tctccgacga tggctatttc ctcaccggtg atattgccga gtggaccgcc 1621 aatggccagc tcagaatcat tgaccgacga aagaacctcg tcaagaccca gaacggagag 1681 tacattgctc tggagaagct cgagacacag taccgatcgt cgtcgtacgt ggccaacctg 1741 tgtgtgtacg ccgaccagaa ccgagtcaag cccattgctc tggtcattcc taacgagggc 1801 cccaccaaga agcttgccca gagcttgggc gtcgattctg acgactggga cgccgtctgt 1861 tccaacaaaa aggtggtcaa ggctgtgctc aaggacatgc tcgataccgg ccgatctctg 1921 ggtctgtccg gcattgagct gctgcaaggc attgtgttgc tgcctggcga gtggactcct 1981 cagaacagct acctgactgc tgcccagaag ctcaaccgaa agaagattgt ggatgataac 2041 aagaaggaaa ttgatgagtg ctacgagcag tcttag SEQ ID NO: 5 LOCUS YL_mfe1-_(YALI0E15378g) 2780 bp DNA linear 1 atgtctggag aactaagata cgacggaaag gtcgtcattg ttaccggtgc cggtggcggt 61 ctcggtaagg catacgccct tttctacggc tctcgaggag cctctgttgt tgtcaacgat 121 cttggtggcg acttcaaggg cgacggtgcc caggctggca gtggcaagcg agtgagtatc 181 attacaagcg cagcgaagcg aaacgaccca aaacgacacc acacagaagg ataaactaac 241 accaggttgc cgatgttgtc gtcgacgaga ttgtttccaa gggaggcaag gctgttgcta 301 actacgactc tgtcgagaac ggtgacaaga ttgtcgagac tgccgtcaag gcttttggct 361 ccgtccacat tgtcatcaac aacgccggta ttctccgaga tatttccttc aagaagatga 421 ccgacaagga ctgggatctt gtctacaagg tccacgtttt cggtgcctac aaggttaccc 481 gagctgcctg gccttacttc cgaaagcaga agtacggtcg agttatctct acctcttccg 541 ctgctggtct ttacggaaac ttcggccaga ccaactactc cgctgccaag ctcgccctgg 601 ttggtttcgg tgagactctc gccaaggagg gtgccaagta caacattact tccaacgtca 661 tcgctcctct tgctgcttcc cgaatgaccg agacagtcat gcccgaggat atcctcaagc 721 tcctcaagcc tgagtacgtt gttcctctgg tcggctacct cacccacgac tctgtcaccg 781 agtcttatgg tatttacgag gtcggtgctg gttacatggc taaaatccga tgggagcgag ^Attorney Docket No.11833-014WO1 ^ 841 gcaacggtgc tgttttcaag ggcgacgaca ctttcacccc gtctgctatt ctgaagcgat 901 gggatgaggt cacctctttt gagagcccca cctaccctaa cggccctgct gacttcttca 961 aatacgctga ggagtctgtt aagcgacccg agaaccccca gggacccacc gtctccttca 1021 aggaccaggt tgtcattgtc actggagccg gtgctggcat tggccgagct tactctcacc 1081 tccttgctaa gcttggtgcc aaggtcgttg ttaacgattt cggtaaccct cagaaggttg 1141 tcgatgaaat taaggccctc ggtggtatcg ccgtcgctga caagaacaac gtcatccacg 1201 gtgagaaggt tgttcagacc gctatcgacg ccttcggtgc tgtccacgcc gttgtcaaca 1261 acgctggtat tctccgagac aagtctttcg ccaacatgga tgatgagatg tggcagctga 1321 tctttgatgt ccacctcaac ggtacttact ccgttaccaa ggccgcgtgg ccccacttcc 1381 ttaagcagaa gtacggccgt gtcatcaaca ccacctcaac ttctggtatc tacggtaact 1441 tcggccaggc caactactct gccgccaagg ctggtatcct cggtttctcc cgagctcttg 1501 ctcgagaggg tgagaagtac aacattcttg tcaacaccat tgcccctaac gctggtactg 1561 ccatgactgc ttctgtcttc actgaggaga tgctcgagct cttcaagccc gatttcatcg 1621 cacccatcac cgtcctgctt gcttccgatc aggctcccgt caccggtgat ctgtttgaga 1681 ctggttctgc ttggatcgga cagactcgat ggcagcgagc tggtggtaag gccttcaaca 1741 ccaagaaggg tgtcaccccc gaaatggttc gagacagctg ggctaagatc gtcgacttcg 1801 atgatggtaa ctccacccat cccaccactc cctccgagtc tactactcag attcttgaga 1861 acatcttcaa cgtgcctgat gaggaggttg aggagactgc tctcgttgct ggtcccggtg 1921 gtcccggtat cctcaacaag gagggcgaac ctttcgacta cacttacact taccgagacc 1981 tcattcttta caaccttggt ctcggtgcca aggctaatga gctcaagtat gtcttcgagg 2041 gtgatgatga cttccagacc gtgcccactt tcggtgttat cccttacatg ggtggcctca 2101 tcactaccaa ctatggcgac ttcgttccta acttcaaccc tatgatgctt ctccacggtg 2161 agcagtacct tgaaatccga cagtggccta ttcctaccaa tgctacattg gagaacaagg 2221 ctaaggtcat cgatgtcgtt gacaagggca aggctgccct ccttgtcact gctaccacca 2281 ccacgaacaa ggagactggt gaggaggttt tctacaacga gtcttctctc ttcatccgag 2341 gctctggtgg tttcggtggt aagtctaccg gtactgaccg tggcgctgcc actgctgcca 2401 acaagccccc tgctcgagct cctgacttcg ttaaggagat caagatccag gaggaccagg 2461 ctgccattta ccgactttct ggtgattaca accctcttca catcgaccct gcttttgctg 2521 ctgttggtaa ctttgaccga cctattctcc acggtctctg ctcttttggt gtctccggta 2581 aggctcttta cgatcagttt ggtcctttca agaacgctaa ggtccgattt gctggtcacg 2641 tcttccctgg tgagaccctg aaggttgagg gctggaagga gggcaacaag gtcattttcc 2701 agaccaaggt tgttgagcga ggtactaccg ccatcagcaa tgccgccatt gagctcttcc 2761 ccaaggatgc taagctctaa ^Attorney Docket No.11833-014WO1 ^ SEQ ID NO: 6 ATCC 2050918S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 26S ribosomal RNA gene, partial sequence (including the D1D2 region) GTTTCCGTAGGTGAACCTGCGGAAGGATCATTAGTGAATTGCTCTTTGAGCGTTAACTA CATCCATCTACATCTGTGAACTGTTGATTGACTTCGGTCAATAACTTTTACAAACACTGT GTAATGAACGTCATGTTATTATAACAAAAATAACTTTCAACAACGGATCTCTTGGCTCTC GCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAA TCATCGAATCTTTGAACGCAACTTGCGCTCTCTGGTATTCCGGAGAGCATGCCTGTTTG AGTATCATGAAATCTCAACCATTAGGGTTTCTTAATGGCTTGGAATTGGGCGCTGCCAC TTGCCTGGCTCGCCTTAAAAGAGTTAGCGTGTTAAACTTGTCGTAAACTGGCGTAATAA GTTTCGCTGGTGGTAGACTTGTGAAGGACGCTTCTAATCGTCTTCGGACACTTCTTGAA CTCTGGTCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGG AAAAGAAACTAACAAGGATTCCCTTAGTAACGGCGAGTGAACCGGGAAAAGCTCAAATT TGTAATCTGGCTGTCTTCGATAGTCCGAGTTGTAATCTATAGACGTGTTTTCCGTGCTG GACCGTATCTAAGTCCCTTGGAACAGGGTATCAAAGAGGGTGACAATCCCGTGCTTGA TACGACCACCAGTGCTCTGTGATACACGTTCTACGAGTCGAGTTGTTTGGGAATGCAGC TCAAAATGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAAC AAGTACCGTGAGGGAAAGATGAAAAGCACTTTGGAAAGAGAGTTAAACAGTACGTGAAA TTGTTGAAAGGGAAACGATTGAAGTCAGTCGTGTTCTTCAGATTCAGCTGGTTCTTCCA GTCTACTTCTGTGGAACGGGTCAACATCAGTTTTGTCCGGTGGATAAAGGTAGTAGGAA TGTGACTCCCCCGGGAGTGTTATAGCCTATTATTGCATACACTGGGTGAGACTGAGGA CTGCAGCTCGCCTTTTGGCCGGTCTTCGGACACGTTCGAGCTTAGGATGTTGACATAAT GGCTTTAAACGAC SEQ ID NO: 7 ATCC 20509 D1D2 region of the 28S ribosomal RNA gene CATATCAATAAGCGGAGGAAAAGAAACTAACAAGGATTCCCTTAGTAACGGCGAGTGAA CCGGGAAAAGCTCAAATTTGTAATCTGGCTGTCTTCGATAGTCCGAGTTGTAATCTATA GACGTGTTTTCCGTGCTGGACCGTATCTAAGTCCCTTGGAACAGGGTATCAAAGAGGG TGACAATCCCGTGCTTGATACGACCACCAGTGCTCTGTGATACACGTTCTACGAGTCGA GTTGTTTGGGAATGCAGCTCAAAATGGGTGGTAAATTCCATCTAAAGCTAAATATTGGC GAGAGACCGATAGCGAACAAGTACCGTGAGGGAAAGATGAAAAGCACTTTGGAAAGAG AGTTAAACAGTACGTGAAATTGTTGAAAGGGAAACGATTGAAGTCAGTCGTGTTCTTCA GATTCAGCTGGTTCTTCCAGTCTACTTCTGTGGAACGGGTCAACATCAGTTTTGTCCGG TGGATAAAGGTAGTAGGAATGTGACTCCCCCGGGAGTGTTATAGCCTATTATTGCATAC ^Attorney Docket No.11833-014WO1 ^ ACTGGGTGAGACTGAGGACTGCAGCTCGCCTTTTGGCCGGTCTTCGGACACGTTCGAG CTTAGGATGTTGACATAATGGCTTTAAACGACCCGTC SEQ ID NO: 8 Debaryomyces hansenii ITS Forward primer NNNNNNNNNNNNNNNGTANGTGACCTGCGGAGGATCATTACAGTATTCTTTTTGCCAG CGCTTAATTGCGCGGCGAAAAAACCTTACACACAGTGTTTTTTGTTATTACAAGAACTTT TGCTTTGGTCTGGACTAGAAATAGTTTGGGCCAGAGGTTTACTGAACTAAACTTCAATAT TTATATTGAATTGTTATTTATTTAATTGTCAATTTGTTGATTAAATTCAAAAAATCTTCAAA ACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATA AGTAATATGAATTGCAGATTTTCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC TGGTATTCCAGAGGGCATGCCTGTTTGAGCGTCATTTCTCTCTCAAACCTTCGGGTTTG GTATTGAGTGATACTCTTAGTTGAACTAGGCGTTTGCTTGAAATGTATTGGCATGAGTG GTACTGGATAGTGCTATATGACTTTCAATGTATTAGGTTTATCCAACTCGTTGAATAGTT TAATGGTATATTTCTCGGTATTCTAGGCTCGGCCTTACAATATAACAAACAAGTTTGACC TCAAATCAGGTAGGATTACCCGCTGAACTTAAGCATATCANTAANNNGGNAGGAANNNN NNNNNNNNNGNNTTNNNNNNNNGNGGGGGGGNNNNNNGGGNGNNGNTGNNNNNNNN NNGGNNNNNNNNNNNNNNNNNNNTGNGNNNNNNNGGGGNGNNNNGNGTG SEQ ID NO: 9 Debaryomyces hansenii ITS reverse primer NNNNNNNNNNNNNNNNNNGNNTTGAGGTCAACTTGTTTGTTATATTGTAAGGCCGAGC CTAGAATACCGAGAAATATACCATTAAACTATTCAACGAGTTGGATAAACCTAATACATT GAAAGTCATATAGCACTATCCAGTACCACTCATGCCAATACATTTCAAGCAAACGCCTA GTTCAACTAAGAGTATCACTCAATACCAAACCCGAAGGTTTGAGAGAGAAATGACGCTC AAACAGGCATGCCCTCTGGAATACCAGAGGGCGCAATGTGCGTTCAAAGATTCGATGA TTCACGAAAATCTGCAATTCATATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCG AGAACCAAGAGATCCGTTGTTGAAAGTTTTGAAGATTTTTTGAATTTAATCAACAAATTG ACAATTAAATAAATAACAATTCAATATAAATATTGAAGTTTAGTTCAGTAAACCTCTGGCC CAAACTATTTCTAGTCCAGACCAAAGCAAAAGTTCTTGTAATAACAAAAAACACTGTGTG TAAGGTTTTTTCGCCGCGCAATTAAGCGCTGGCAAAAAGAATACTGTAATGATCCTTCC GCAGGTTCACCTACGGAAACCTTGTTACGACTTTTACTTCCTCTAANNN SEQ ID NO: 10 Rhodotorula mucilaginosa ITS Forward primer NNNNNNNNNNNNGNNNNNNTAGGTGACCTGCGGAGGATCATTAGTGAATATAGGACGT CCAACTTAACTTGGAGTCCGAACTCTCACTTTCTAACCCTGTGCATTTGTTTGGGATAGT ^Attorney Docket No.11833-014WO1 ^ AACTCTCGCAAGAGGGCGAACTCCTATTCACTTATAAACACAAAGTCTATGAATGTATTA AATTTTATAACAAAATAAAACTTTCAACAACGGATCTCTTGGCTCTCGCATCGATGAAGA ACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTT GAACGCACCTTGCGCTCCATGGTATTCCGTGGAGCATGCCTGTTTGAGTGTCATGAATA CTTCAACCCTCCTCTTTCTTAATGATTGAAGAGGTGTTTGGTTTCTGAGCGCTGCTGGC CTTTACGGTCTAGCTCGTTCGTAATGCATTAGCATCCGCAATCGAACTTCGGATTGACT TGGCGTAATAGACTATTCGCTGAGGAATTCTAATCTTCGGATTAGAGCCGGGTTGGGTT AAAGGAAGCTTCTAATCAGAATGTCTACATTTTAAGATTAGATCTCAAATCAGGTAGGAC TACCCGCTGAACTTANNNNNNNNANNGGNNGNGNNNNNANAN SEQ ID NO: 11 Rhodotorula mucilaginosa ITS Reverse primer NNNNNNNNNNNNNNNNACCTGANTTGAGANCTAATCTTANATGTAGACNTTCTGATTAG AAGCTTCCTTTAACCCAACCCGGCTCTAATCCGAAGATTAGAATTCCTCAGCGAATAGT CTATTACGCCAAGTCAATCCGAAGTTCGATTGCGGATGCTAATGCATTACGAACGAGCT AGACCGTAAAGGCCAGCAGCGCTCAGAAACCAAACACCTCTTCAATCATTAAGAAAGAG GAGGGTTGAAGTATTCATGACACTCAAACAGGCATGCTCCACGGAATACCATGGAGCG CAAGGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCA TTTCGCTGCGTTCTTCATCGATGCGAGAGCCAAGAGATCCGTTGTTGAAAGTTTTATTTT GTTATAAAATTTAATACATTCATAGACTTTGTGTTTATAAGTGAAATAGGAGTTCGCCCTC TTGCGAGAGTTACTATCCCAAACAAATGCACAGGGTTAGAAAGTGAGAGTTCGGACTCC AAGTTAAGTTGGACGTCCTATATTCACTAATGATCCTTCCGCAGGTTCACCTACGGAAA CCTTGTTACGACTTTTACTNNNNN Sequence ID: NW_027072170.1 | COLE_00321 The identified mutation at nucleotide position 747,416 within the gene COLE_00321 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819) results in a codon change from GAA (encoding glutamic acid, E) to TAA (a stop codon), thereby introducing a premature termination of translation. Gene of reference strain: SEQ ID NO: 12 >XM_018424836.1 Cutaneotrichosporon oleaginosus uncharacterized protein (COLE_00321) ATGCCTTACGCAGAGCCCACCTCTCCAACCTCGCCGCTCTCGCCGCTGGGGGATGCG CGACCTAGGTATGCACGGCGACACTCGCACAACACCTCTCTCGAAGTTCGCCCCACCA CCAGTCCCATGCCTGGACTTCCTCGCCGTGCCACATCGTCCGCTTTGACGACAACGAC GAACACTCATTCTCACCGCAAGGGCACCCTCTCCAACTCTCCTCGGACAGCAGCCAAC ^Attorney Docket No.11833-014WO1 ^ TCTGCTGCTCACCGTGAGGCTCTCGACGCCATGCGCACCGTGGAGCCACTCCAGTCG CCTCCTGCCAAGGGCTCCGACTATAGCATCCTGGGACTTCGAATTGACAAGGCCACTC CTTCTAGCACCTCTCGAGCCGCCAAACCTCCAGCTCTAGCTCCTTCAACGCCTCCAGTC TCCCATTCCACGTCCCATTTGCTCCAGCGGCAGAAGCGCGTCTCGTCCTCATCCTCGT CTTCATCACTTCCCTCACCAACCGAGCCACCCGAGCCGCATAGCCCCGTCAACTCCAC GCGTCCATCGGACCGAATGGTGCGCAAGAAGTCGGGCGAGATGGTCAAGCCCGCTCT CAAGAACCGCTCTCTATCCACTCCTGACCTCGGCCGTGGTGGGAAGGAGGATCTGTGG GCAAAGTCAAAGTCATCGCACAACTTGCGCGAAAGGACAAAGAGTGTGCGCTTCGACG AATGTGGGCTTGAATCTGTCGTTCACTTCGCGCGCGGTCAGCGGCCCGACGCACTGAT CAAGGGGCCGGACGGCGAGGATGATCAGGAACACGACCTGTCAGATTCTGTACACTTC CGCACACGCCGCAGCGGGGTTCCGCGTGGGGCGGGCGTCGCTCAGACGGAGATCCA GATCGACACATGCAGTGCCGTGCCCCGCGTGCGCCTCGACTTTGGCCCTGGTACATAT GGTCTGCTCAACAACGAGTATGTTGTCCTCGAGCGCATCGAGATGAGCCAGCCTTTGG GGCTGCGTGGATCGGTGCTCGTTCGCAACATCGCCTTTGAGAAGTGGGTGGCCGTGC GCTTCACCCTGGACGACTGGCAGACGGTTTCGGAGGTATCGGCGTCGCACGTTTCGCA CATCCCCGCTGGAACCACCGGCGACGAGGGCTGGGACCGTTTCACCTTCAACATTAAG CTCGAGGATTATCGCCGGAAAATTGAGAACCGCTCGTTCATGTTCTGCGTCCGGTACAA TGCCAACGGGGGCGAGTGGTGGGATTCGAACGGCGGCCAGAACTATCACATCTCGTT CAAGCAGCAGGCGAAGAGCCGCCAGCGTCCTACTTCGCAGTACTGGAGCGGAAGTCT CACGCACGGAACTGCATCGGCGGAGACTACCACTCTCCCTCGGCGCAACGGACCCCG CAACTGGTCGTTCCCCAAGTCGACCGTGCAGGCCATCGTTGCTCCGCCTGAGCGCCC CGATTCACCAGCACTCTCCCCACCGCCACAGGGCGCATTCCGCGCGCCCTCTGCACC CGACGTGCATTCGCACCTGCGCCTCCAGAAATATTGCGCGCCTTCGCCGCCCCAGTCA CCCCCCAAGACACTTCCATCCGCCGTGGGAGGAGGGCTTGTGCCCATTGTTCTGCCCC TGGCCCCTGAGCGGGCACGTGCCGTTCCTCCGTCGCCCATGACCCTTGTTCATGGCC GCCCGGCCACGACTTGGGTTCCCACCGAGTTCACTGTCAACGCGGAGGACGAAGTGT TTACAACGACATCGGAAGAGTCGTCAGAGGTCGACAGCGTCGGCGAAAGCACGCCGA CTGCGGCCGGTGCGCGCTCGCCAGAGATGCAGCCCATCATTGACGGTGCCACATTCG TCACGAGCCCCGTCACGAGTCCAGTGACGGCTAGAGCCGAGGACAAGGGTCGCAGCA ACATACAGCGCAACACGTCCGTCTCGGGGGATCTACACTCTCTGGCAAGTGTAGACTC GAGCGTCGGTCTCATGACTCCCCCGAGCAGCAATCTCTCCTCTCCGCCAACTCCAACG ACGCTCCTCCCTCCTGAATCCCCTACCACCTCCATGTCGACTGGAGACTCGTCTCCTGT CAACACGTGCAGCTCGGTCACGCCCGACGACAATGTCGATGGCGGCCGTCTAGGCGG TACTCTGAACGCTGCAACGTACCAGGAGTTTCTCGAGAGGTTCTGCTTCTTCAAGTCGC CCCCTACCACCGCCATGGCCCTCGAGGAGCAGTACACCGGGTCCTACTTCCCGCCCG TGACTGGCAGCAGCTACTACAGCGGCTTTGGCTTGGGCAGCACAACGCCGCCCGGTG CGATAACGCCGACGTTTGACAGATTCAACATGGCGCGCGCCACCACGCCCACAAACGC ^Attorney Docket No.11833-014WO1 ^ GAGTGCGACCTCGGGCTCGAGCTCGACCCCGCGCGCGTCGCCAACGCGCTCGCCGG CCCCGAACACGTTCCCGCTCCCGTGGACGCCCGAGGACCCGGCGCTGAAGATGACCG CGGCGTAA Protein on reference strain: SEQ ID NO: 13 >XP_018277645.1 uncharacterized protein COLE_00321 [Cutaneotrichosporon oleaginosus] MPYAEPTSPTSPLSPLGDARPRYARRHSHNTSLEVRPTTSPMPGLPRRATSSALTTTTNTH SHRKGTLSNSPRTAANSAAHREALDAMRTVEPLQSPPAKGSDYSILGLRIDKATPSSTSRA AKPPALAPSTPPVSHSTSHLLQRQKRVSSSSSSSSLPSPTEPPEPHSPVNSTRPSDRMVRK KSGEMVKPALKNRSLSTPDLGRGGKEDLWAKSKSSHNLRERTKSVRFDECGLESVVHFAR GQRPDALIKGPDGEDDQEHDLSDSVHFRTRRSGVPRGAGVAQTEIQIDTCSAVPRVRLDF GPGTYGLLNNEYVVLERIEMSQPLGLRGSVLVRNIAFEKWVAVRFTLDDWQTVSEVSASHV SHIPAGTTGDEGWDRFTFNIKLEDYRRKIENRSFMFCVRYNANGGEWWDSNGGQNYHISF KQQAKSRQRPTSQYWSGSLTHGTASAETTTLPRRNGPRNWSFPKSTVQAIVAPPERPDSP ALSPPPQGAFRAPSAPDVHSHLRLQKYCAPSPPQSPPKTLPSAVGGGLVPIVLPLAPERAR AVPPSPMTLVHGRPATTWVPTEFTVNAEDEVFTTTSEESSEVDSVGESTPTAAGARSPEM QPIIDGATFVTSPVTSPVTARAEDKGRSNIQRNTSVSGDLHSLASVDSSVGLMTPPSSNLSS PPTPTTLLPPESPTTSMSTGDSSPVNTCSSVTPDDNVDGGRLGGTLNAATYQEFLERFCFF KSPPTTAMALEEQYTGSYFPPVTGSSYYSGFGLGSTTPPGAITPTFDRFNMARATTPTNAS ATSGSSSTPRASPTRSPAPNTFPLPWTPEDPALKMTAA Protein on Mutant strain: SEQ ID NO : 14 >XP_018277645.1 uncharacterized protein COLE_00321 [CutaneotrichosporonSequence ID: NW_027072171.1 | COLE_02845 The identified mutation at nucleotide position 2,913,185 within the gene COLE_02845 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819) results in a codon change from GCG (encoding alanine, A) to GTG (encoding valine, V). Gene of reference strain: SEQ ID NO: 15 >XM_018421476.1 Cutaneotrichosporon oleaginosus uncharacterized protein (COLE_02845) ATGACCAGCGACCACCCAATTACCCAAGTGGCTTGTCCCTCCGCCGCCGATGCTGCCG ATCCCTATCCTTCAATCCTCTTGTCCACTTTCACATCTTTGCTCGCCATCGTCCATCTCG ^Attorney Docket No.11833-014WO1 ^ TCCGCTCTCATCTTTTATTAGCTGACTCCTTCTCGAATCCTCCCCCGGATCCCGCCCTC CCCGTGTCATTACTTGTCGCCTGGCTCTTCCCGCCCCCCGTCGCTGAGCGATCTCCTC TCTCCTCCCTGTTATCGTCACCGCTGTTTGGCCTCCTCCCCCCTCCACCTACCATCACC GACTTTCCATCCACACGCAATGTCCACTCAGCCTCTTCTCCAGCGCACCGCGAAGAAG GTGAGCTGCTGCACCGAGCTCGTCAAGTGTACACTGGTGCTAACTACCGCCTTTCCCC GGCCTTGACCCACCCCCTCTCTACTTCTGAACCACGCCCCCCAAAACCCTTTCCCCCA CCTTCCTTACTCGGCCTCCTGCTCGGCCAACCGACACAGCGCATCGCCCTCCCGGTTC GCGTCGAGCCCAAGGTATTCTTTGCCAACGAGCGTACCTTCCTCTCATGGCTCCACTTC GCCGTCGTTCTTGGTGGTCTTGCCGTGGGCCTCCTTAACTTCGGCGACAAGATCGGAC GCATCTCTGCCGCCATGTACACTGTGATCGCGGTCGGTGTCATGGTTTACGCGTTGTC AGTGTACCAGCGCCGTGCGCGCGCTATTAGGACACGCTCTGGAGCGCCGTACGACGA CCGTCTTGGTCCTACTATCCTCTGTGTATTCCTGCTTGCTGCGATCACGACCAACTTCA TCCTTCGCGCTGTTTACGAGTAG Protein on reference strain: SEQ ID NO: 16 >XP_018277116.1 uncharacterized protein COLE_02845 [Cutaneotrichosporon oleaginosus] MTSDHPITQVACPSAADAADPYPSILLSTFTSLLAIVHLVRSHLLLADSFSNPPPDPALPVSLL VAWLFPPPVAERSPLSSLLSSPLFGLLPPPPTITDFPSTRNVHSASSPAHREEGELLHRARQ VYTGANYRLSPALTHPLSTSEPRPPKPFPPPSLLGLLLGQPTQRIALPVRVEPKVFFANERT FLSWLHFAVVLGGLAVGLLNFGDKIGRISAAMYTVIAVGVMVYALSVYQRRARAIRTRSGAP YDDRLGPTILCVFLLAAITTNFILRAVYE Protein on mutant strain: SEQ ID NO: 17 >XP_018277116.1 uncharacterized protein COLE_02845 [Cutaneotrichosporon oleaginosus] MTSDHPITQVACPSAADAADPYPSILLSTFTSLLAIVHLVRSHLLLADSFSNPPPDPALPVSLL VAWLFPPPVAERSPLSSLLSSPLFGLLPPPPTITDFPSTRNVHSASSPAHREEGELLHRARQ VYTGANYRLSPALTHPLSTSEPRPPKPFPPPSLLGLLLGQPTQRIALPVRVEPKVFFANERT FLSWLHFAVVLGGLAVGLLNFGDKIGRISAAMYTVIAVGVMVYVLSVYQRRARAIRTRSGAP YDDRLGPTILCVFLLAAITTNFILRAVYE Sequence ID: NW_027072174.1 | COLE_06128 The identified mutation at nucleotide position 1,506,518 within the gene COLE_06128 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819) results in a codon change from CGC (encoding arginine, R) to CAC (encoding histidine, H), thereby leading to an amino acid substitution from arginine to histidine in the encoded polypeptide. ^Attorney Docket No.11833-014WO1 ^ Gene of reference strain: SEQ ID NO: 18 >XM_018426202.1 Cutaneotrichosporon oleaginosus uncharacterized protein (COLE_06128) ATGGACGCTGCGTCCCTTCATCCACCGGAGGGCCCGTCCCCATTCGAGGCGCACCTC CTCAACCTCCTCAGCACGTTCGAGCAGAATCCCTCTCTCGCTTATCAGACCGTCCCCG CTGCCAAGACTCCGTCCCAGCAGGCCATTGAGCGCGGCATCCTCGCCCTCGCCCAGC GCGCCTCAAATTACGCGTCTTCCCCCCCGTCTACCGCCCGCCGCGCTCTTGCTGATTC TCATCTGCCCCATACGCCGCCTTCCGCTGAGGCGCGCACGCCTACCTGTCCGTCATGC TCCACGCCGATCCCCCCGCCTTCTGCTACCCCCACGTCACCCTTCGCCTCCCTCGAAC TCTCGTCGCCCCGCCAGTCGTGGTCCAACGGCGTCGCCGACACTGGCATGAGCGCTG AGAAGGAATTGGAATTGCTCAAGGCTCAGGTTCAGGATATTGCTCGCGTTTGCAAGGC CGTCGCTACCGGTGATCTCTCTCAGAAGATTATCGTCCCGGTGAAAGGCCAGGCAATG ACGGAACTCAAGGATATTATCAACTCCATGGTCGACCGGCTCAAAACATTCGCCACCGA GGTCGAGCGCGTCTCGCTCGAAGTCGGTACTCAGGGCAAACTCGGCGGGCAGGCTGT TGTCGAAGGTGTCGAGGGCACATGGCGCGAGCTCACAAACGTTGTCAACCGTCTTGCA GCAAATCTCACCAATCAGGTGCGCAGCATCGCCAAGGTTACAAAGGCCGTGGCGCGTG GCGATCTGAGCGAAACCATTACTGTCGTCGCCAGCGGCGAGATCGCGGAACTGGCCA CCACGGTTAACGGAATGGTGTATAGTCTACGATTACTAGCAGACGAAGTCAGTCGTGTA TCTCTCGAGGTCGGTTCGCAAGGAAAGTTGGGTGGTCAAGCGCATGTACCCAATGTCG AAGGCGTATGGAAAGATCTCACAGTGAACGTCAATCGCATGTGTCACTCTCTTACAACT CAAGTACGGTCGATCGGCAGCGTCACAACTGCAGTAGCAAGGGGTGACCTGTCCAAGA CAATTGAGATTGAGGCTGAAGGCGAAATGGCCGTGCTCAAAGACACGGTCAACTCCAT GGTGAAGCAGCTCACCATCTTTGCCGGGGAAGTCACCCGTGTAGCTCTCGAAGTCGGG ACACAAGGAATCTTGGGGGGCCAGGCTGTGGTCGATGGGGTGGAAGGGGTCTGGGCA GACCTCACCACGAACGTCAACAAAATGGCTCGTAATCTTACCGAACAAGTGCGAGAGAT TGCGGAAGTCACCAAAGCGGTCGCCCGTGGTGACCTCACTAAAACGGTAAACGCCGAT GTCCAGGGCGAGATTCTCGAGCTCAAGCTCACTGTCAACGGAATGGTGGCCCAGCTGA ACGTGTTTGCGGCTGAGGTCACGCGAGTGTCCCTTGAAGTCGGTACGGAAGGCAAGCT CGGCGGTCAGGCCCTGGTCCCCAACGTCGAGGGCACCTGGAAGATTCTCACGGACAA CGTCAACTTGATGGCGCTTAACCTCACCACGCAGGTCCGCTCCGTTGCGGAAGTTACA ACTGCCGTGGCAGCGGGTGACCTGTCCAAGAAGATTACTGTCGAGGCCTTTGGGGAG ATCGCTCAACTCAAGAACACTGTCAATGCCATGGTGGACTCCCTTCGCTCGTTCTCGTC CGAAGTCACTCGTGTTGCCCGTGAAGTCGGTACCGATGGTCGCCTCGGTGGCCAGGC CCATGTACCCGGCGTGGCCGGGACCTGGAAGGACCTTACAGACTGTGTCAATGTCATG GCCGCCAACCTCACCGTCCAGGTGCGAACCATCGCCCACGCCACTACCGCTGTAGCG AGAGGTGATCTCACTCAAAAGGTCACTGGTGTGACTGTATCTGGAGAGATTCTCGACCT CGTCAACACCATCAACAACATGATTGACCAGCTGGCCATCTTCGCTGCCGAGGTTACG ^Attorney Docket No.11833-014WO1 ^ CGGGTTGCGCGCGAGGTCGGTACAGAGGGCAAGTTGGGTGTCCAAGCAGAGGTAGAG AACATCGAGGGGACATGGCAGGAGATCACCTCCAACGTCAACACCATGGCATCCAACC TTACATCGCAAGTGCGCGCGTTTGCTCAGATTTCGGCCGCTGCCACCGACGGTGACTT TACACGCTTCATCACCGTCGAGGCGTCGGGCGAGATGGATAGTCTCAAGACCAAAATC AACCAAATGGTGTACAATCTGCGCGAGTCGATTGAGAAGAACACGAGTGCGCGTCAGC AGGCAGAGCTTGCCAACCGCTCCAAGTCCGAGTTCCTCGCCAACATGTCGCACGAGAT CCGCACGCCCATGAACGGTATCATCGGCATGACTGTCCTTACACTTGAGAGCGAATTG ACTCGCCAGCAGCGCGAGAATCTGATGATTGTCTCGAGCTTGGCGCAATCGCTGCTCA CGATCATCGACGACATTCTCGACATTTCCAAAATCGAAGCTGGTCGCATGACCATGGAA CAGATTCCCTTCTCGCTCCGGTTGGCTGTCTTCAGCGTCCTCAAGACGTTGTGCGTCAA GGCATCACAGAACAAGCTAGACCTCATCTTCGATATCGACCCCACGATACCTGATCAGC TCATCGGTGATCCGCTTCGTCTCCGTCAGGTTATCACCAACCTCATCGGCAATGCGGTC AAGTTTACCACCGAGGGCCAAGTCGCCCTTTCCTGTCGTGTCAAGGCGAGGCGGGATG CCACAGTCGAGCTGCAATTCTGCGTGGCCGACACGGGCATTGGTATCAAGCAGGACAA GCTGGACGTCATCTTCGATACCTTCGCGCAAGCCGATGGCTCTACGACGCGCAAGTAC GGAGGCACGGGTCTTGGGTTGACCATCTCAAAGCGCCTGGTCAGCCTCATGAACGGC AACCTGTGGGTCGAGTCTGAATTTGGCGAGGGTAGTCGGTTCTACTTTACGATGACGA CCGAAACAACGACGACCTTGCGCGAGCAAGTCGCCGAACGGCTGGCGCCGTGGGCTG GACGGTCGGTGCTCTTCATCGACACGCTGAACGACAACACGGGGGTTAGCGAAATGCT GCAAAGCTTGCACCTCAAACCAACCGTGATTCACTCGCCAAACGCCGTTTGGGATCTCA AGCAAGCCCCAGAAGGGTTCCCACACTTTGACACGATGATCGTCGACTCGCTCGCGGC AGCCGAGAAGATTCGCACCATCGAACACCTGCGATACATCCCTATCGTGCTGCTCGCC CCGTCCAACAAGCCGAGCGGGCCCGACAACCCCTCTTTCATCGACCTGGACGAGACG CGGCGAAAGCTGCTGAACCTCCCGTCTCCCACTGAGCAGATACTCTCACCAGTACCAG TCAAGGACTGCCTTGACATGGGCATCAACACCTACTACACCACGCCACTCAACCTGCA GGAGCTCAGCAATGCAATCCTCCCTGCGCTTGAGTCTCACCAGGTTCAGCCAGGTGAC ACCGTCAAAGATACTGTCCTCAACATCTTGCTCGCAGAGGACAATGTGGTCAACCAGAA GCTTGCGGTCAAGCTCCTGGAAGTTGCAGGCCACAAGATCGAGGTGGCGGACAATGG CGAGATTGCGATCGATAAATACAAGCGTCGACAGGCGGAGCGTAAGCCATTTGACGTG ATTCTCATGGATGTGTCTATGCCTGTCATGGGGGGCATGGAGGCGACAGGGCTAATCC GCGAGTATGAGGCGTTCAACGGGGTCATGGCGACGCCCATTATCGCATTGACGGCTCA CGCGATGATAGGAGACAAAGAGCGGTGTTTGGCGGCCGGAATGACCGCATACGTGAC CAAGCCGTTACGGCGCGGCGACCTCCTGCAAGCTATCTCCAAGGTCCTACAGGCAAAA CCGCAGACGTCGCAGGCATTGTACCTCCCCGAAGCAACGCTCGACGACCCGGACCGG ACGCTCGTACTCGCGAGCCGCTGA Protein on reference strain: SEQ ID NO: 19 ^Attorney Docket No.11833-014WO1 ^ >XP_018277238.1 uncharacterized protein COLE_06128 [Cutaneotrichosporon oleaginosus] MDAASLHPPEGPSPFEAHLLNLLSTFEQNPSLAYQTVPAAKTPSQQAIERGILALAQRASNY ASSPPSTARRALADSHLPHTPPSAEARTPTCPSCSTPIPPPSATPTSPFASLELSSPRQSWS NGVADTGMSAEKELELLKAQVQDIARVCKAVATGDLSQKIIVPVKGQAMTELKDIINSMVDR LKTFATEVERVSLEVGTQGKLGGQAVVEGVEGTWRELTNVVNRLAANLTNQVRSIAKVTKA VARGDLSETITVVASGEIAELATTVNGMVYSLRLLADEVSRVSLEVGSQGKLGGQAHVPNV EGVWKDLTVNVNRMCHSLTTQVRSIGSVTTAVARGDLSKTIEIEAEGEMAVLKDTVNSMVK QLTIFAGEVTRVALEVGTQGILGGQAVVDGVEGVWADLTTNVNKMARNLTEQVREIAEVTK AVARGDLTKTVNADVQGEILELKLTVNGMVAQLNVFAAEVTRVSLEVGTEGKLGGQALVPN VEGTWKILTDNVNLMALNLTTQVRSVAEVTTAVAAGDLSKKITVEAFGEIAQLKNTVNAMVD SLRSFSSEVTRVAREVGTDGRLGGQAHVPGVAGTWKDLTDCVNVMAANLTVQVRTIAHAT TAVARGDLTQKVTGVTVSGEILDLVNTINNMIDQLAIFAAEVTRVAREVGTEGKLGVQAEVE NIEGTWQEITSNVNTMASNLTSQVRAFAQISAAATDGDFTRFITVEASGEMDSLKTKINQMV YNLRESIEKNTSARQQAELANRSKSEFLANMSHEIRTPMNGIIGMTVLTLESELTRQQRENL MIVSSLAQSLLTIIDDILDISKIEAGRMTMEQIPFSLRLAVFSVLKTLCVKASQNKLDLIFDIDPTI PDQLIGDPLRLRQVITNLIGNAVKFTTEGQVALSCRVKARRDATVELQFCVADTGIGIKQDKL DVIFDTFAQADGSTTRKYGGTGLGLTISKRLVSLMNGNLWVESEFGEGSRFYFTMTTETTT TLREQVAERLAPWAGRSVLFIDTLNDNTGVSEMLQSLHLKPTVIHSPNAVWDLKQAPEGFP HFDTMIVDSLAAAEKIRTIEHLRYIPIVLLAPSNKPSGPDNPSFIDLDETRRKLLNLPSPTEQIL SPVPVKDCLDMGINTYYTTPLNLQELSNAILPALESHQVQPGDTVKDTVLNILLAEDNVVNQ KLAVKLLEVAGHKIEVADNGEIAIDKYKRRQAERKPFDVILMDVSMPVMGGMEATGLIREYE AFNGVMATPIIALTAHAMIGDKERCLAAGMTAYVTKPLRRGDLLQAISKVLQAKPQTSQALY LPEATLDDPDRTLVLASR Protein on mutant strain: SEQ ID NO: 20 >XP_018277238.1 uncharacterized protein COLE_06128 [Cutaneotrichosporon oleaginosus] MDAASLHPPEGPSPFEAHLLNLLSTFEQNPSLAYQTVPAAKTPSQQAIERGILALAQRASNY ASSPPSTARRALADSHLPHTPPSAEARTPTCPSCSTPIPPPSATPTSPFASLELSSPRQSWS NGVADTGMSAEKELELLKAQVQDIARVCKAVATGDLSQKIIVPVKGQAMTELKDIINSMVDR LKTFATEVERVSLEVGTQGKLGGQAVVEGVEGTWRELTNVVNRLAANLTNQVRSIAKVTKA VARGDLSETITVVASGEIAELATTVNGMVYSLRLLADEVSRVSLEVGSQGKLGGQAHVPNV EGVWKDLTVNVNRMCHSLTTQVRSIGSVTTAVARGDLSKTIEIEAEGEMAVLKDTVNSMVK QLTIFAGEVTRVALEVGTQGILGGQAVVDGVEGVWADLTTNVNKMARNLTEQVREIAEVTK AVARGDLTKTVNADVQGEILELKLTVNGMVAQLNVFAAEVTRVSLEVGTEGKLGGQALVPN VEGTWKILTDNVNLMALNLTTQVRSVAEVTTAVAAGDLSKKITVEAFGEIAQLKNTVNAMVD ^Attorney Docket No.11833-014WO1 ^ SLRSFSSEVTRVAREVGTDGRLGGQAHVPGVAGTWKDLTDCVNVMAANLTVQVRTIAHAT TAVARGDLTQKVTGVTVSGEILDLVNTINNMIDQLAIFAAEVTRVAREVGTEGKLGVQAEVE NIEGTWQEITSNVNTMASNLTSQVRAFAQISAAATDGDFTRFITVEASGEMDSLKTKINQMV YNLRESIEKNTSARQQAELANRSKSEFLANMSHEIRTPMNGIIGMTVLTLESELTRQQRENL MIVSSLAQSLLTIIDDILDISKIEAGRMTMEQIPFSLRLAVFSVLKTLCVKASQNKLDLIFDIDPTI PDQLIGDPLRLRQVITNLIGNAVKFTTEGQVALSCRVKARRDATVELQFCVADTGIGIKQDKL DVIFDTFAQADGSTTRKYGGTGLGLTISKRLVSLMNGNLWVESEFGEGSRFYFTMTTETTT TLREQVAERLAPWAGRSVLFIDTLNDNTGVSEMLQSLHLKPTVIHSPNAVWDLKQAPEGFP HFDTMIVDSLAAAEKIRTIEHLRYIPIVLLAPSNKPSGPDNPSFIDLDETRRKLLNLPSPTEQIL SPVPVKDCLDMGINTYYTTPLNLQELSNAILPALESHQVQPGDTVKDTVLNILLAEDNVVNQ KLAVKLLEVAGHKIEVADNGEIAIDKYKRRQAERKPFDVILMDVSMPVMGGMEATGLIREYE AFNGVMATPIIALTAHAMIGDKERCLAAGMTAYVTKPLRHGDLLQAISKVLQAKPQTSQALY LPEATLDDPDRTLVLASR Sequence ID: NW_027072175.1 | COLE_06627 The identified mutation at nucleotide position 493,871 within the gene COLE_06627 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819) results in a codon change from AGC (encoding serine, S) to AAC (encoding asparagine, N), thereby causing an amino acid substitution from serine to asparagine in the encoded polypeptide. Gene of reference strain: SEQ ID NO: 21 >XM_018420541.1 Cutaneotrichosporon oleaginosus uncharacterized protein (COLE_06627) ATGCTTTTAACGCGCCTTCTGGCCGCTTCGGCGGCCCTCTCGTCGCTCGTCAAGGCGC AGACCAAGTCCGATGTCGACCTCATCCTCGAGCGCCGCCGTATTGACATGGCCAGCTT CACTACCCCAGCCGTTATGGCCAACGTCTCAATGTGGCTCGAGAGTCAAAGTGACGAG GGTATCTGGGCAGACGTCGACTACAGTCTTGGCTGCGCCGCTCGGCGCGCGAATTGG CCGATTCAGTTGCACTGGATTCGTGTCATTGCTTTAGCTTCGGCGTGGTCGGGCCTCAA CCCCTCCGCACCGGTTGAGTATCACGGCAATGAGGCGTTCCTTGCTGGTGCCCTCAAG GGCATGGACTGGTGGTTTGCGCGTGACTACACCAATCCCGGTTGTACCGCGGAAGGC GGCAAGCCCAGGCCTTGCCCTTGCGGAACGCCTGGCACCTGGAATCAGAACTGGTTC GGGAACGTGATTCTGATTCCTCAGCTCCTATCAACCGGATGTCTACTTGTGATGCCGGC CACACTCACGGACTTGCAGAGGGAGAAGTGCTTCAGCATCCCAAACCGCGCATGGGA GCTCCGCGATCTCAATAATCCGACTTTCGGGGTACTGACCGGTGCCAACATGGTCAAC GTTATGCAAAACTCGATCTCGATGGCACTGTTTGCCAACAACGTGACGATTGTCGAGGA AGCATTCTCCCGGGCGATGGGGTCTTATTTGTTCGCCGACGCACCAGCACAAGACGGC ACACACCGAGACGGCTCGTTCCTTCAGCACGATGGGATCCTTTACAATGGAAATTATGG ^Attorney Docket No.11833-014WO1 ^ GAAAGACGCCCTCAACGCGTTCATCCAGCTGGAGGGCGAGGCGATTGGCACCGCCTT TGCCGCCAACGACACTGTGCGCGAGGCCGTCGCGACCTTTATCCGCGGAAGCGAGTG GATGATTTACGCCGACCAGAGCACCAGGCGGCTGCACTGGGACTTCAATACGATCGGG CGCTTCCTCGCGTTCCACACGAGTGACCTTCAGGCGTCTGCCGACATCAATTTTAACAC AAGCAGGCTCGCAACTGCCGTCGCCGACTTTACTGGCGCCAATGACTTGTCGAGTACC GTCAGGCGTCTTCAGAGCAACGGTTCAAAGCCGCTTACGGGCAATCACGCCTTCTATG CTGCTGATTACATGGTTCATCGTCGTAACAACTACGTGCTGGCCAACAAGATGATCTCC TCTCGCTCGATTAACACTGAGTACGTGAACTCGGCGAATCCTTACGGCTTCCTCCTCGG CCAAGGCACCTTGTTCTCGTACGTGACTGGCAACGAGTACAAGGACATTCAGGCAGGC TGGGATTGGCACCTGATCCCCGGCACAACGTCAATTCTGCGTGCCGCTCCGCTGCGTT CCGACCGGGTGGAGTTCATGGGGAAGCTCAGCTACGTCGGAGTCGTGAGCAATGGCG AGTTCGGCGCCGGCGCCATGGACTACCTCGACCCTGCAGACGGCTCGTTAGCATTCC GCAAGGCCTGGTTCTTCTTTGAGGACAGCGTGCTCGTGACCACTACGAGCGTCGTCGT GAATAGGACCGCTGCAGCCAGCGATGCTCCGGTTATCACCACACTCGATCAGCGCACT GCCGCCGATGGCACACATGTAGCTGTCGATGGCAATGCTGTCGACATCCAGGACCAGC GTAACATCACAGGCTCGACGCTCCTGTACGCAGGCAACGGCTACCTTTCTCATGGGAC GCCGTTTAACCTGACCCTTTCAGCCGGTGCGCGCACCGGCAACTGGTCGGCCATCTCG ACGTCGGCGGCCGGCGTAAAGACTGTCGACATCTTCTCTGCGTACACGACTGTCCCCT CCACCGCGTACTCTTACGAGTTTTTCCCTGACACCAACGCCCACCGGCTGCGCCGCGA ACGCCGACGCCCTACCACCACGCCCCTGGACCTCGGCGGCGTCATCGGCGCAGCCTC GGGGCGAAATCATCTCGCCCTAATTTTCTGGCCCGGAGCGCCGCTGACGGCGACCGT CCCCCACCGTCGTTGGGATCTGGAGGTCACGGTCGACACGCCTGTTGCAGTGTTGTTC ACGACGGGCCGACGCCGCAGGATCGGGCGTGTCCTCATGGTCACTGTCTCCGACCCA AGTCAGGCGCACACAGGAGTGAGGATCACGGTCAGGAGTGCAGCCAGGCGCCTCCGC TGCGGTGAAGGGACTTGCACGGAAACGGACCGCGGCGTGGTTCTCGACCTTGTCCTG CCCACTGGGGGTTTAGGCGGCAGCAGCGTGAGCGTGGACATTCCGTACCGTTAA Protein of reference strain: SEQ ID NO: 22 >XP_018275666.1 uncharacterized protein COLE_06627 [Cutaneotrichosporon oleaginosus] MLLTRLLAASAALSSLVKAQTKSDVDLILERRRIDMASFTTPAVMANVSMWLESQSDEGIWA DVDYSLGCAARRANWPIQLHWIRVIALASAWSGLNPSAPVEYHGNEAFLAGALKGMDWWF ARDYTNPGCTAEGGKPRPCPCGTPGTWNQNWFGNVILIPQLLSTGCLLVMPATLTDLQRE KCFSIPNRAWELRDLNNPTFGVLTGANMVNVMQNSISMALFANNVTIVEEAFSRAMGSYLF ADAPAQDGTHRDGSFLQHDGILYNGNYGKDALNAFIQLEGEAIGTAFAANDTVREAVATFIR GSEWMIYADQSTRRLHWDFNTIGRFLAFHTSDLQASADINFNTSRLATAVADFTGANDLSS TVRRLQSNGSKPLTGNHAFYAADYMVHRRNNYVLANKMISSRSINTEYVNSANPYGFLLGQ ^Attorney Docket No.11833-014WO1 ^ GTLFSYVTGNEYKDIQAGWDWHLIPGTTSILRAAPLRSDRVEFMGKLSYVGVVSNGEFGAG AMDYLDPADGSLAFRKAWFFFEDSVLVTTTSVVVNRTAAASDAPVITTLDQRTAADGTHVA VDGNAVDIQDQRNITGSTLLYAGNGYLSHGTPFNLTLSAGARTGNWSAISTSAAGVKTVDIF SAYTTVPSTAYSYEFFPDTNAHRLRRERRRPTTTPLDLGGVIGAASGRNHLALIFWPGAPLT ATVPHRRWDLEVTVDTPVAVLFTTGRRRRIGRVLMVTVSDPSQAHTGVRITVRSAARRLRC GEGTCTETDRGVVLDLVLPTGGLGGSSVSVDIPYR Protein of Mutant strain: SEQ ID NO: 23 >XP_018275666.1 uncharacterized protein COLE_06627 [Cutaneotrichosporon oleaginosus] MLLTRLLAASAALSSLVKAQTKSDVDLILERRRIDMANFTTPAVMANVSMWLESQSDEGIWA DVDYSLGCAARRANWPIQLHWIRVIALASAWSGLNPSAPVEYHGNEAFLAGALKGMDWWF ARDYTNPGCTAEGGKPRPCPCGTPGTWNQNWFGNVILIPQLLSTGCLLVMPATLTDLQRE KCFSIPNRAWELRDLNNPTFGVLTGANMVNVMQNSISMALFANNVTIVEEAFSRAMGSYLF ADAPAQDGTHRDGSFLQHDGILYNGNYGKDALNAFIQLEGEAIGTAFAANDTVREAVATFIR GSEWMIYADQSTRRLHWDFNTIGRFLAFHTSDLQASADINFNTSRLATAVADFTGANDLSS TVRRLQSNGSKPLTGNHAFYAADYMVHRRNNYVLANKMISSRSINTEYVNSANPYGFLLGQ GTLFSYVTGNEYKDIQAGWDWHLIPGTTSILRAAPLRSDRVEFMGKLSYVGVVSNGEFGAG AMDYLDPADGSLAFRKAWFFFEDSVLVTTTSVVVNRTAAASDAPVITTLDQRTAADGTHVA VDGNAVDIQDQRNITGSTLLYAGNGYLSHGTPFNLTLSAGARTGNWSAISTSAAGVKTVDIF SAYTTVPSTAYSYEFFPDTNAHRLRRERRRPTTTPLDLGGVIGAASGRNHLALIFWPGAPLT ATVPHRRWDLEVTVDTPVAVLFTTGRRRRIGRVLMVTVSDPSQAHTGVRITVRSAARRLRC GEGTCTETDRGVVLDLVLPTGGLGGSSVSVDIPYR Sequence ID: NW_027072176.1 | COLE_07529 The identified mutation at nucleotide position 766,332 within the gene COLE_07529 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819) results in a codon change from CAC (Histidine, H) to CGC (Arginine, R), thereby causing an amino acid substitution from serine to asparagine in the encoded polypeptide. Gene of reference strain: SEQ ID NO: 24 >XM_018425779.2 Cutaneotrichosporon oleaginosus uncharacterized protein (COLE_07529) ATGACGGCCGTGTCGCCCAAGCCGTCGTCCCCCGGCACACCGATCGTAGACTGGGAT GAGGCCCAGGTCAACGCGTACTTTGTCAGCTTGGGACTCAAGCAGTATGAGAGTGTAA TTTACGAACATGGTATCACTGGCGAGGTGCTGTGCGCTCTCGATAACGAAACGCTCGT CGACCTTGGTATGATGTCACTTGGGCACCGGCTCAACGTCTTGCGCGCTGTCTTTGAG ^Attorney Docket No.11833-014WO1 ^ CTCAAGAAGGAGCAAGGCGTAGAGATAGGCGAAGACGACTGGCGGCCACAAGAAGAA GTAGCGCTTGAGGCGATGGAGACGGCTGCTACGATCGACCGGATGTGGAGCCTTGTG TTGGATCAACAGGAACGCCTGCAGTCTCTGGAGAAGGAGCAGATACGATTAATGCGCG CCCTGACAGAAAATGGCATACAACTCCCTCCACCCCCACCAACTCTTGTCGAAGTGTCC GGCACACCAACAGCCGGTATGGGCGCCTACTTTGGCTCGCCACCACCAACGTCACAC GGCAAGCTTCAGACAAGTAAGCCGCCTCTCGCCAGACTCGCAAGCGGGACAGTCGTG CGGTCTGGGTCTAGATCACAGCGCAATGACGGCGCGTCAACAGCACCATTACCCTCCA GCCCTACACCTCCGGTGGAACCAGCACCTGCGCCCTCGGGCGGATCATCGCACGGCG CGCGCGACGCGGCCCACTCGGCAGCCAGGAGTTTCCGCGTTACAATGGAGGATCCGT GCTGGAAGGTGCTTCCTGCAGCGCTCAAGAAGTACAAGATCAACGATGACTGGAAGAT GTACGCGCTCTTCATCTGCTATGGCAACACGGAGAGATGTCTGAGTTATGACGAGAAG CCGTTGTTGCTGTTCCAAAAGCTCAAGGAGGGCGGGCAGCGACCAGTGTTCATGCTGC GGCACATTCGCGACATCAAGTCGCCTATCGCTGTCGCACAGCAGAAGCAGGCGATCAA GCTCGGACTCCCGGCCAACTCGACTGCAAATCTCTTGCCCCAGCTTGACGCACCATCA CCGTCACCCACGAAGGCGACCAGATCTCCAGGAGCCGCAAAGAGCGAATCTGGGGGG CGGACGCCGGGGGATGGGACATTCCCAGAATTGCCATCACCTGGTATGCGCGACCCC GACGCTGCGACCATTGCATCCAAGGATTTCAAGGAGAATAGCTCTGCTAGCTCCAGTAT ACAACCGCGCACACCAGTCACTGGTACGCTCATGGACAAGGACGGCAAGGTGCACAA GGTCACATACGCGATCAGCATCTATCCGTATATCGCAGACCGACAGGACGAGTTCGAT GTCGCTGTCGGCGCTGCCTTCGTGGTCATGTCCAAGGCCAAGGGGTGGTGGTTCGTT CTCAAGGACACGGAGGGGAGAGGCAACATCAGTTCGGAGACGACCAAAAGCGCCTGG GTCCCCGCCGGCTGCCTACTAGAACTCACAGCGCCGATCGCGAGCATTTCACCACAAT CGCCAGGCAGCATTCCAGGGCGTGCACCCATCCCGCCTGCCAACATTATGAGCTCGA GCTATCCGGGCGTGGTGCTTATGAACCACACGAGCAAAGACACGCACGAGCTCACGAT CAAGGAGGGCGAGAAGGTGCGCGTGTACAAGAAGTACTGCCACTGGAGTTATTCAATC AAGGAGGACACGGGCGAGCGTGGCTGGGTGCCCGCATGGTTTGTTGGCAAGCTCACG GGCGCGGGCGAGCGAGAGGCGGCCGCGCCCTCGGCGAGTGGGAGCGCCCCCGTCG CCGGTGCAGGCGCAGGCGGAAGTGGCGAGGCACCGCCGCCATACCAAAAGAGCAAG GAGGAGGGCGAGAAGAAGGACAAGGACGAGCAGCAGGGCGGCGTTTAA Protein of reference strain: SEQ ID NO: 25 >XP_018278542.1 uncharacterized protein COLE_07529 [Cutaneotrichosporon oleaginosus] MTAVSPKPSSPGTPIVDWDEAQVNAYFVSLGLKQYESVIYEHGITGEVLCALDNETLVDLG MMSLGHRLNVLRAVFELKKEQGVEIGEDDWRPQEEVALEAMETAATIDRMWSLVLDQQER LQSLEKEQIRLMRALTENGIQLPPPPPTLVEVSGTPTAGMGAYFGSPPPTSHGKLQTSKPPL ARLASGTVVRSGSRSQRNDGASTAPLPSSPTPPVEPAPAPSGGSSHGARDAAHSAARSFR ^Attorney Docket No.11833-014WO1 ^ VTMEDPCWKVLPAALKKYKINDDWKMYALFICYGNTERCLSYDEKPLLLFQKLKEGGQRPV FMLRHIRDIKSPIAVAQQKQAIKLGLPANSTANLLPQLDAPSPSPTKATRSPGAAKSESGGR TPGDGTFPELPSPGMRDPDAATIASKDFKENSSASSSIQPRTPVTGTLMDKDGKVHKVTYAI SIYPYIADRQDEFDVAVGAAFVVMSKAKGWWFVLKDTEGRGNISSETTKSAWVPAGCLLEL TAPIASISPQSPGSIPGRAPIPPANIMSSSYPGVVLMNHTSKDTHELTIKEGEKVRVYKKYCH WSYSIKEDTGERGWVPAWFVGKLTGAGEREAAAPSASGSAPVAGAGAGGSGEAPPPYQK SKEEGEKKDKDEQQGGV Protein of mutant strain: SEQ ID NO: 26 >XP_018278542.1 uncharacterized protein COLE_07529 [Cutaneotrichosporon oleaginosus] MTAVSPKPSSPGTPIVDWDEAQVNAYFVSLGLKQYESVIYEHGITGEVLCALDNETLVDLG MMSLGHRLNVLRAVFELKKEQGVEIGEDDWRPQEEVALEAMETAATIDRMWSLVLDQQER LQSLEKEQIRLMRALTENGIQLPPPPPTLVEVSGTPTAGMGAYFGSPPPTSHGKLQTSKPPL ARLASGTVVRSGSRSQRNDGASTAPLPSSPTPPVEPAPAPSGGSSHGARDAAHSAARSFR VTMEDPCWKVLPAALKKYKINDDWKMYALFICYGNTERCLSYDEKPLLLFQKLKEGGQRPV FMLRHIRDIKSPIAVAQQKQAIKLGLPANSTANLLPQLDAPSPSPTKATRSPGAAKSESGGR TPGDGTFPELPSPGMRDPDAATIASKDFKENSSASSSIQPRTPVTGTLMDKDGKVHKVTYAI SIYPYIADRQDEFDVAVGAAFVVMSKAKGWWFVLKDTEGRGNISSETTKSAWVPAGCLLEL TAPIASISPQSPGSIPGRAPIPPANIMSSSYPGVVLMNHTSKDTRELTIKEGEKVRVYKKYCH WSYSIKEDTGERGWVPAWFVGKLTGAGEREAAAPSASGSAPVAGAGAGGSGEAPPPYQK SKEEGEKKDKDEQQGGV FURTHER STRAINS WITH MUTATIONS: Sequence ID: NW_027072170.1 | downstream of COLE_00879 The identified mutation at nucleotide position 2,185,025 downstream of the gene COLE_00879 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819) corresponds to a base substitution from adenine (A) to guanine (G). This can be seen in Figure 27, and is represented by SEQ ID NO: 27 (query) and SEQ ID NO: 28 (Subject). Sequence ID: NW_027072174.1 | downstream of COLE_05948 The identified mutation at nucleotide position 1,055,259 downstream of the gene COLE_05948 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819) corresponds to a base substitution from thymine (T) to cytosine (C). This can be seen in Figure 28, and is represented by SEQ ID NO: 29 (query) and SEQ ID NO: 30 (Subject). Sequence ID: NW_027072174.1 | downstream of COLE_05601 ^Attorney Docket No.11833-014WO1 ^ The identified mutation at nucleotide position 212,800 downstream of the gene COLE_05601 of the reference strain (Cutaneotrichosporon oleaginosus, taxid: 879819) corresponds to a base substitution from thymine (T) to adenine (A). This can be seen in Figure 29, and is represented by SEQ ID NO: 31 (query) and SEQ ID NO: 32 (Subject). Engineered DGA1 >ACS Acetyl-coA synthase (SEQ ID NO: 27) ATGGCTGAGCAGAAGACTGTCCGCCACGTCGAGATCGCAGACACCCACGAGGGTCAC CAGCGCACCTACCCCCCTCCTGCCCGTCTCCAGGGCAAGGACGGTCGTCCCAAGCCC CACATTGGCCCAGACTTTGCCGCCTACAAGGCTGAGTGGGAGAAGTCGGTCGGCCCA GACTCGGACAAGTGGTGGGCCGAGAAGGCTCGCGACTGCCTGACCTGGTTCTCCGAC TTTAAGACTGTCCGCGCCGGTGGCTTTGCCGACGGCGACATCCAGTGGTTCCCTGAGG GCACGCTCAACGCCGCCTACAACTGCGTTGACCGCCACTACTACAAGAACCCCGACAA GGTCGCCATCATCTACGAGGCCGACGAGCCCTCGGACTCGCGTGAGATCACCTACCG TGAGCTCTTCCACGAGGTCTGCCGTGTCGCCAATGTCCTCCGCTCGTGGGGTGTCAAG AAGGGCGACGCCGTCTCGGTCTACCTCCCCATGACCTGGCAGGCTGTCGTTGCGTTCC TCGCCTGTGCCCGTATCGGTGCCGTCCACTCGGCTGTCTTTGCCGGCTTCTCCGCCGA GTCGCTCCGTGACCGTGTCAACGACTGCGAGTGCAAGGTCCTCATCACCACCGACGAG GGTCGCCGTGGTGGCAAGACCATTGCCACCAAGGCGATCGTTGACGCCGCCCTCGCC CAGTGCCCGGGTGTCGAGAAGGTCCTCGTCCTCCGCCGCACCGGCTCCAAGGTCCCC ATGACCGAGGGCCGTGACTTCTGGTGGGACGAGGAGGCTGAGCGCCTCCCCACCTAC TCCCCCTGCGAGCACATGAACTCGGAGGACCCCCTCTTCATCCTCTACACCTCGGGCT CGACTGGCAAGCCCAAGGGTGTCGTCCACTCCACCGCCGGCTACCTCCTCGGTGGCT ACCTTACCGTCAAGTACGTCTTCGACGTGCACCCGGACGACCGCTTCGCCTGCATGGC CGACGTCGGATGGATTACCGGCCACACCTACATCGTCTACGGCCCTCTCTGCCTGGGT GTCACCACCACCGTCTTCGAGTCGACCCCCGTGTACCCCACCGCCTCGCGCTACTGG GACGCAGTGGACAAGTGGAAGCTTACCCAGCTCTACACTGCTCCTACCGCCATCCGTC TGCTGCGCCGCATGGGCGAGGACGCCGTCAAGAACCACGACCTCTCGTCGCTCCGCG TTCTCGGCACGGTCGGTGAGCCGATCAACCCCGAGGCGTGGCACTGGTACAACGAGC ACGCTGGCCGTGGCCAGTGCGCCATCGTCGACACGTACTGGATGACCGAAACCGGGT CGCACATGGTCACCCCCATCCCCGGTGCCATTGCCACCAAGCCCGGATCGGCCACCTT CCCCTTCTTCGGTGTCGACGTAGACATCCTCGACCCCACCAACGGCGCTGTCCTCAAG GGCGACGACGTCGAGGGTGTCCTCGCCGCCAAGCGCCCTTGGCCCGCTATCGCCCGT ACCGTCTTCCGCGACCACAAGCGCTACCTCGAGACCTACATGAAGCCGTACCCCGGCT ACTTCTTCTTCGGCGACGGTGCCGCCCGCGACGAGGACGGCTACATCTGGATCAAGG GCCGTGTCGACGACGTCATCAACGTCTCCGGCCACCGCCTCTCGACCGCCGAGGTCG AGTCCGCCCTCATCCTCCACAAGGGTGTCTCTGAGACGGCCGTCGTCGGCTGTCCCGA ^Attorney Docket No.11833-014WO1 ^ CGAGCTCACTGGCCAGGCCGTCTACGCCTTTGTGCAGATGAAGCCCGAGTTTGACATC AAGGCCACCTCGATCGACGCCCTCACCAAGGAACTCGCCATCCAGGTCCGCAAGGTCA TTGGTCCTGTGAGTTTCGGTTCGCCGCCCCCAAGCGCATCTTCCTCATCACCGACCTC CCCAAGACGCGCTCGGGCAAGATTATGCGCCGCATCCTCCGCAAGATCTGCTCCAAGG AGGCCGACGCCCTCGGCGACCTCTCGTCCATGGAGAAGCCCGAGGTCGTCGACCAGC TCACCGAGCAGGTCTACACCGCCATGGGCTGGAAGTAAACTCGCGTTGCGCAGCGAC GCGAGGTCTAAAGATGGCGGCTCCCGTTGA >CoDGA1_recode_0 Diacylglycerol O-acyltransferase (SEQ ID NO: 28) ATGTCGACCCCCCTCGTGCCAGTTGTTCCACTTTCCCCAGCTGAAGACGGTGCCAAGA GCCCCGTTGATCATGCACCTGCACCAACTCCATCGCCCGAAAGCTCGAGCCCTCGTTC CCCTCTGGTTGCGCTCAAGCATGACAAGCCTCTGTCGCGCTCGTCGTCGCTTCTCGAG CTCCTGCATCTGAAGGACGTCCACATTCCGACTCCACCGCCCGTGAAGTTTGCACCGC TCGTTGTTCCGCGTCATCGCCGTCTCCAAACAGCAGTGGTGGCTACATGGAGCGTCAT GATTCCCATCTGCCTGACCGTCTTCTACGGCTCGCTTTTCTTCAGCAAGTGGCTGCGCC CGCTTGCGGTCATCTACGCCGTCTGGTTCATTGTGGTCGATCGCGCTTGGCGGCACAA AGGTGGCCGCCGTAAAGATTGGGTTCGGCGTTCCGCCTTCTGGCGCTACTTCGCCGAC TACTACCCTATCACGACCGTGAAGGAGGCAGATCTCCCGGCGGACCGGAAGTATGTCT TCGCCTACCATCCCCACGGGATCATCAGCATGGGGGCGGCGTGTACATTTGCCACTGA GGCTACGGGCTTCAGCAGCCTCTTTCCTGGCGTCACGTGCCATCTCCTCACCCTCGAC GCCAACTTCTGGATCCCGCTCTACCGCGACATTCTCATGGGTATGGGTCTGGCGTCGG TGTCCAAGCGTTCGTGCCGCAGCATCCTCAAAATGGGGCGCAGCATCTGCATCGTCAT CGGCGGTGCGTCCGAATCCCTCTATGCCTACCCTGGCACGAACAACCTTACGCTGAAG AAGCGCCTCGGCTTCATTAAGATCGCTATCCGGGAGGGCGCGAACCTTGTGCCCGTCT ACGGCTTTGGTGAGAACGACATCTACGAGCTTCTGCCCAATGAGAAGGGCACCATGAC GTACAAGTTCCAGAAGTGGTTCCAGGGCACATTCGGGTTCACGGTCCCCTTCTTTCATG GTCGCGGTGTTTTCACCTACAACTACGGCCTGATGCCGCATCGCCGGCCAGTTACCGT TGTTGTGGGTGCACCTATTCCCGTCAAGCAGATCGAGAAACCCACGGACGAGGAGGTG CAGGCGGTGCACAATCAATACATCGAGGCCCTCCAGGCGCTCTGGGACAAGCACAAG GACGAGTATGCGAAGGACCGCAAGTCGGAGCTGAAGCTGGTCGCCTAG >COLE_03742_60S_bidir_terminator Bidirectional terminator (SEQ ID NO: 29) GGGTCTAGAGGTGTATGTTGGTCGGATGTATAGCATTTCCCTACTGGGAGATGGGGCA GGATGGTGTGGTGGTGAGGGGCTTGATGAGCGGTGAGAGACGTTGGACTGATGACGC GCCGTTCTACGGGAGGGCCGACCGCACCGACAACAGTTACTGAAAGCGAAGTAGCCT ATTGTTCACTGCAACAAGCATCTGGCTGGAGGGCTGCAAACTTCCGTCTTGCCCGCCA GTTGTACGTGGCTTTGGGATACCAGTGCAATCTCCACTATACATTGTGTTCCATCCATC AGACTGGCTAA >COLE_05137p Promoter (SEQ ID NO : 30) ^Attorney Docket No.11833-014WO1 ^ CCCGCTTTGTCTCATGCATGGTCCAAGCTCCCCCATATTGGATGCGAGTCACAGCAACA TAGCGTGGAGGCTGCGCTGAAGGTCTGCGGTAGGAGGCAGATAGGTGTAGTCTGCAG ATCTGCAGATGTGGTGGGTAACGGGAAGTAATGGAACTGGCCTGGTTCCTAGATCCTA GATGCGCCAGCGTGATGGGTGGTTAGCGGATGGCGGAAGCACAAAGAGGCTCTGCGC CGAGCATGCCGACGATCCGACGTAGATCTAAAGGGCTCACCTCGAAGAGTCATTTATC GATGGTGCCTGGCGAATCCGTTTGGCACTCAGTCCAATAGGAGGTGGGATTGTCGCTC GGCATGCCCTCCCACATGTGACATGTGCGGAAGTGGGGGGAGACGTCGTTGCTTTGAT CGGCTATAAAACGAAGACATCCCTCTTCACAAGTGTCGTGAAGTGCTTTAGCATATCCT GTCTCCTCCTTGTCCCACGTATCTCGACACCTCACC >ENOp Promoter (SEQ ID NO: 31) CAACAACGCGCCCTTCAGGTGCGCGCGGGCGCACCGAGGCACACAGATTGTGTTGCG GCTCGGCAATGCGAGTTCGTTGTCATTGGCTGTTGATGCCATTGCCGAATCACAACGA GGGGTCCCCGCCCAAAATTAATACGTGTCAAATAAATGTATGCCATTGCCGCTTCCTCC CCTGAGTTGGGAAGGAGGGGCGGACCCCCGAATGTCCCGATGACTTTTGGCCCTGTT GTCGAGTTCCGTTCCCAGCCATCCAACCAGCGGGCAGCACACTCGCACCCGCCAGCA GCGAGGACTGTCAAGTCGCGCGCCCGCACGCGCCCCAAGCTCGCCCACCCAAGCCAC GTTGACTTCCCATGACATGACATACAGCCTATGACATTCTCCCCCGTTCTCCATCCTTC CTCCTCCGCCCTCCGCTTCCTCTTTCACTTCCTCTCTTTCTCTCATTCTCTCTCTCCACC TCTCCTCTCCTCACCACCTCCAACTCTCATCCAAAA >fabB ^-ketoacyl-ACP synthase (SEQ ID NO: 32) ATGACAAGAGTAGTGGTGACAGGACTTGGTCTGGTCAGTCCCCTCGGAGTCGGAGTAA AACAGGCATGGAACCGACTTATTGCTGGAAAATCTGGAATTGTGGCTCTCGACGAACG GTTCGACGCTTTATCCTGCCGAGTAGGAGGAGTGGTCCCTCAGGACGAATGGTCTGAA GCTTCATCTGCCAACCTCGCAGCTGGAGAGATCAAACGGGTACCTCTGTTTGCCCAGT ATGCCATGGTGGCTGCCCAGGAGGCTGTCAACGACAGCAAATTGCTAGCTGACGCAG GGGAGTCAGCTAAACACACAGATATTGGATGTTCGATTGGTACTGGAATCGCCAACATG GCTGAACTGGCAGATACAGTCACAGCTTTCAACAACAAGGGTCCCCGAGGTATTTCAC CACTATTTGTTCCCCGAATCATTGCTAACATGGGTGCGGGACACGTCAGTATGCGGTTC GGTCTCCAAGGACCCAATCACTCTGTATCCACTGCCTGTGCCACTGGAGCTCACAGCA TAGGAGATGCAGCCAACTTCATTCGACTGGGATACGCGAAGGCTATGATTGCCGGATC AACCGAGGCCTGTATGCACCCAATTGCACTTGGTGGATTTGCACGGGCCAAGAGTGTG GCTACCAAGTGGAACGACTCCCCCTCTGAAGCATCCAGACCCTTTGATAAAGATCGAG GAGGATTTGTTATGGGCGAGGGATCTGCTGTTCTTGTCCTAGAGGAATATGAACACGC CGTTGCCCGTGGTGCTCCCATTTACGCTGAAGTGTGTGGTTACGGTCTTTCTGGAGAT GCTCATCACATTACTGCCCCTCCTGATGATGGAAACGGGGCCTATCGAGCCATGAAAC AGGCTCTCAAGCAGGCCAATGTTTCTGCCGTCAAGATCGATTATGTTAACGCCCATGCC ACTTCCACCCCTCTTGGTGACGCGGCTGAAAATGCTGCTCTGACTCAACTCTTTGAGGG ^Attorney Docket No.11833-014WO1 ^ CAGAAACCTGTCTGATATCGCCGTTTCTTCCACCAAGGGTGCCATTGGCCATCTTCTGG GTGGAGCTGGATCTGTGGAGGCTCTGTTCACCATCAAGGCTCTCGAAACTGGAGACTT GCCTCCCACTCTCAACCTCAATGAGCTTTCTGACCCTGATGTCTTCAAGTTCAACTTTGT GCCCAAGGAGACTCAGCACCGTGATGTCCGGTATGCACTCAACAACAGTTTCGGATTC GGAGGCACCAACTCCAGTCTTCTTTTTGGTAAGGTATAG >fabD Malonyl CoA-ACP transacylase (SEQ ID NO: 33) ATGTCTTTGCGAGCAGCATTCTTCCCCGGACAGGGACTGCAGAGACCCCGAATGCTGC AGAAACTGTGGCAACAATCGGCCATAAGAGAAACGCTCAAACCGGCACTGGATCAGCT GAAGACCGAGGTCCAGGAGGTGCTACTGGGCAACAGCAGTCTGGGCGAAGACGAACA AATGAAGCTGATTACACAGACACAAAACGCCCAGCCGGGGATCCTTCTCTCGTCTTACG CCAGCTTCCTAGCCAATCAGGGCGCCAGCAAAAACTACTCCCATCTGCTGGGCCACTC CCTCGGAGAGTACTCGGCTCTGGTGTGTGGAGGCTACCTGACTCTCGAGCAGGGAGT GCAATTGGTCCAGAAGCGGGGCTACTACATGCAACGAGCCAACGACGAGAGTGGCAA GGAAACAGCCATGGTAGCATTGTTGTTGCATCGCACGGTTGACACCAACCGGTTGGTC CAAGCCTGCGAGGGCTGGAACGGAGCCAATGTGGCCAATATCAACAGCAGCGGCCAG GTGGTTCTCAGTGGCGAACGGTCCGCCATCAACGAGCTAGTTGCGCATCTCAAAGCCT CCAAACTGCGGGTTCTGAAGGCAATCCCCCTGAACGTTTCAGCGCCGTTCCATTCCGC CATCATGGCTCCGGTGGTGTCCGAGCTCGATGCGGTTTGTCAGCAGCTCAAGATCCGT CCCGATTTCAGCGCTGACAAGCCGCAGGTGGTATCCAACGCCTCGGGCAAGCCGTTC GGCTCTGCCGACGAGATGTACGAGTCCATGATCGGGTCTCCAGTGCATACAGTCAATT GGCTGGCTAGTGTGGAATACCTAAAACACGTCGGAGTGGTAGAATCGGACGGCTACGG TCCTGGATGTGTCGACATTGGCAAGATGGTGCTGCCAGGAGAGAGATATGACTAA >fabG ^-ketoacyl-ACP reductase (SEQ ID NO: 34) ATGTTCTTGAGACCGCTACAGAACTCCCAGAGAGTGATAAACCCACTCGTTCGAAAGTA CTCAATATCCGCGTCTTCTCTCTCTGGAAAAACCGCTCTGGTGACCGGCGGTTCGGGA GGAATCGGGCTAGTCATTGCTAAGAAGCTGGCAGCAAACGGAGCTCGAGTGATCCTGC TTGCTAGAGATGAAACCAAGTTGAATGGAGCTCTGGAGGAGCTGACACACACTCTTAAG GATGAGCAGACACAAAGGGATATCACACAGACCGCCCACAGCACGATATCTTACGACA TTGCTAAAGCAACGACACCACCAGAAATCGACTTCAAGATGGTAGATCTGCTCGTCAAC TGTGCCGGAGTCACGCAAACATCGCTGCTTATGACCACCAAAAACATTGACCAGATCAT CGGCACAAATCTCGCCGGAGCCATTAAAATGAGCCAGTATGCCATGCGTCCGTGGATG AAACGAAAGTCGGGCTGTATTGTCAACATCTCCTCGGTTTTGGGATTACGTGGCCTTAC AGGCGGGTCTACGGTCTACAGTGCAGCCAAGGCTGGTCTTGTGGGCTTCACGAAGGC TCTCGCGGTCGAAGTGGGCGCTAGAGGTATCCGTGTCAATTGCGTGTGTCCTGGACTG GTCGAGACGGAAATGACACAGAACGTGACTGTCCAGAATGGGTTTGCGACACCTCTTC AGGGCATGGGAAAGGATAATTACGTATCTGCTGACTCGGTGGCCGACGCTGTCCTCTA ^Attorney Docket No.11833-014WO1 ^ CCTTGCTGCTAGTGAGGAGCAGACCGGAAGCATTCTCACCATAGACAAGGGCTTGTCT GCGGTATAG >GAPDH Glyceraldehyde dehydrogenase (SEQ ID NO: 35) ATGGCTACCAAGGTCGGCATCAACGGTTTCGGTCGCATTGGCCGCATCGTGCTCCGCA ACGCCCTCGAGCACGGCGACATTGACGTCGTTGCCATCAACGACCCCTTCATTGACCT CGACTACATGGTCTACATGTTCAAGTACGACTCGACCCACGGCCGCTTCAAGGGCAAG GTTGAGACCAAGGACGGCAAGCTCGTCATTGAGGGCAAGCCCATCTCGGTCTTCGGC GAGCGTGACCCCGCTGCCATCCCTTGGGGCAAGGCCGGCGCCGACTACGTCGTCGAG TCGACCGGTGTCTTCACCACCATTGAGAAGGCCTCGCTCCACCTCAAGGGTGGTGCCA AGAAGGTCATCATCTCGGCCCCTTCGGCTGACGCCCCCATGTTCGTTTGCGGTGTCAA CCTCGAGGCTTACAAGCCCGAGTACGAGGTCATCTCGAACGCCTCGTGCACCACCAAC TGCCTTGCGCCCCTTGCCAAGGTCATCCACGACAACTTCGGCATCGTCGAGGGTCTCA TGACCACCGTCCACGCCACCACCGCCACCCAGAAGACCGTTGACGGCCCTTCGCACA AGGACTGGCGTGGCGGCCGTGGCGCTGCCGCCAACATCATTCCCTCGTCGACCGGTG CCGCCAAGGCCGTCGGCAAGGTCATCCCCTCGCTCAACGGCAAGCTCACCGGCATGT CGTTCCGTATCCCCACCTCGGACGTTTCGGTCGTTGACCTTGTCGTCCGTCTTGAGAA GGGCGCCTCGTACGACCAGATCAAGGAGGTCATCAAGAAGGCCTCGGAGGGCGAGCT CAAGGGCATCCTCGGCTACACTGAGGACGAGGTTGTCTCGACCGACTTCCTCGGCTCG ACCGAGTCGTCGGTCTTTGACGCCAAGGCGGGTATCCCCCTCAACGACAAGTTCGTCA AGCTCATCTCGTGGTACGACAACGAGTACGGCTACTCGCGCCGCGTCTGCGACCTCAT CGCCTACGTTGCCAAGAAGGACAAGTCGGCGTGA >GAPDHp Promoter (SEQ ID NO: 36) CAGCCAGCAGCCAGGCTCACCCTACGGCGTCACCAGTCGCCCTCGCTCATGGCTCAC TCCTCATACCCAGCCAAACCCAGCCCATGCCAGCCACGGCCAGCCAGCCAGTCATTCT GGTCCGTTCCATTCCCATCGATCCCTCCCCCCAGGCCCCCAACGTGTTCCGTTTCCGA CCCCCACCCCGGCCTCCTCCTCCGCCCCCCCCCCCTCCGCCCTCTCCCCCCCCATTC CAAGCCGAACCCACAACCCCATTTAACCGCGACCATCCTCGCCTCTTCCTCTCTCTCCT TCTCTCTCTTTCTCAACCACCTCCTCTTCTCAAAACTATTCCCCTCCTCCCAAAAATCAA CTTGATCAAC >hygR_-_HygR Hygromycin resistance gene (SEQ ID NO: 37) ATGGACCGCTCGGGCAAGCCCGAGCTGACGGCGACCTCTGTGGAGAAATTTCTCATCG AGAAGTTCGACAGCGTGAGCGACCTCATGCAGCTCAGTGAAGGAGAGGAGTCACGGG CATTCTCGTTTGACGTGGGCGGCCGGGGTTACGTTCTTCGCGTCAACTCATGTGCTGA CGGCTTCTACAAGGACCGGTATGTCTACCGGCACTTCGCGTCTGCTGCCTTACCCATC CCGGAGGTGCTGGACATTGGAGAGTTCTCCGAGTCGTTGACCTACTGTATTAGCCGCC GCGCCCAGGGCGTCACACTCCAGGATCTTCCTGAAACCGAATTGCCAGCTGTTTTGCA GCCCGTGGCAGAAGCGATGGACGCGATCGCTGCTGCAGATCTGTCACAGACGTCCGG ^Attorney Docket No.11833-014WO1 ^ TTTCGGTCCCTTTGGCCCGCAAGGGATTGGCCAATACACGACGTGGCGCGACTTCATC TGCGCGATTGCTGACCCGCACGTGTACCACTGGCAGACCGTCATGGACGACACAGTCT CAGCCTCTGTCGCCCAGGCACTGGATGAGCTCATGCTCTGGGCTGAGGATTGCCCAGA GGTCCGCCACCTGGTACATGCCGATTTCGGCAGCAACAACGTGCTCACCGACAACGGC CGCATTACAGCGGTCATCGACTGGTCCGAGGCGATGTTTGGAGACTCGCAGTACGAGG TCGCCAACATCTTCTTCTGGCGCCCCTGGTTGGCCTGCATGGAGCAGCAGACGCGCTA TTTCGAACGTCGGCACCCTGAATTGGCGGGGTCACCGCGTTTACGCGCTTACATGCTG CGCATCGGCTTAGACCAGCTCTACCAGTCCTTGGTAGACGGCAACTTCGACGACGCTG CCTGGGCTCAAGGCCGTTGTGATGCCATTGTTCGCTCAGGTGCCGGCACTGTTGGGC GCACTCAGATTGCACGTCGTTCTGCCGCAGTCTGGACGGATGGGTGCGTAGAGGTCCT TGCTGATAGCGGCAATCGTCGTCCAAGTACCCGCCCTCGCGCCAAGGAGTGA >kanR_-_aph G418 resistance gene (SEQ ID NO: 38) ATGATTGAACAGGACGGCTTGCACGCTGGATCCCCCGCAGCTTGGGTGGAGCGATTGT TCGGTTACGATTGGGCTCAGCAGACTATCGGATGCTCTGACGCCGCCGTGTTTCGACT CTCTGCCCAAGGCCGACCTGTGCTCTTCGTGAAGACCGATCTTTCCGGCGCTTTGAAC GAGCTGCAGGACGAGGCCGCCCGACTCTCATGGCTGGCAACCACTGGCGTGCCTTGT GCTGCTGTTCTCGATGTCGTCACAGAGGCCGGACGTGATTGGCTTTTGCTGGGAGAGG TTCCTGGTCAGGATTTGCTCTCTTCCCATCTCGCCCCCGCTGAGAAGGTCAGCATTATG GCTGATGCCATGCGACGACTCCACACACTTGACCCAGCCACTTGCCCCTTTGATCACC AGGCCAAGCATCGGATTGAACGAGCACGAACTCGAATGGAGGCCGGCCTGGTGGACC AGGACGATCTGGACGAGGAACATCAAGGCCTCGCCCCTGCAGAGCTCTTCGCCCGAC TCAAGGCTCGAATGCCCGACGGCGAGGACCTTGTGGTCACACATGGCGATGCCTGCC TCCCCAATATCATGGTCGAGAACGGACGATTTTCAGGTTTCATCGACTGTGGTCGACTG GGTGTGGCTGACCGATACCAGGACATTGCCCTTGCCACTCGAGACATTGCCGAGGAGC TGGGTGGTGAGTGGGCCGACCGATTTTTGGTTCTGTATGGAATCGCTGCGCCAGATTC CCAACGAATCGCCTTCTATCGATTGCTGGACGAGTTCTTCTAG >ME2 Malic enzyme (SEQ ID NO: 39) ATGGTGACCTACGACTTTAAGGAGAAGACCCCCATGCCCATCCGGACGAATCTGCGCG GCTCTGCCCTGCTAAATACGCCGAGTCTGAACAAGGGTGCAGGCTTCACGCGCGAAGA GCGCAGCATCTTCGGTCTTGAGGGCTTCCTCCCGTATGACGTGCACACTCTCGAGAAG CAGTGCGACCGAGCGTACAACCAGTTGAAGCGCCAGCCGACGCCGCTGCTGCAACAT GTATTCCTCGCCTCGTTGCGAGACCAGAACACCGTGCTGTTCTACCGCTTGCTGCAGG ATCATCTCAAAGAGCTGCTTGGCATCATCTACACGCCCACAGCTGGCGAGGCTGTTGC GCAGTACTCGAACCTCTTCCGCCGACCGGTGGGCTGCTACCTATCGTTCCCCAACCGT GATGGGATGCGAGCGCAGCTCGAGGCGCACCTGCAAAACATCAACATCGTGGCGGAT GTGGCTCGTCACGACGAGACGACCGCAGAGAGCATTGACCTGATCGTGGTCACAGAC GGCGAGGCTATTCTTGGGATCGGAGACTACGGCGTCGGTGGTATCACCATCAGTACCT ^Attorney Docket No.11833-014WO1 ^ CTAAATCGGCCCTGTACACTCTCGGCGCGGGTATCAACCCGAACCGCATCCTCCCCGT CGTGCTGGATGTCGGGACTAACAATCCCATTCTGTTCGCGAGCGACCTCTACATGGGT TGGAAGCGGACGCGGATGCGTGGCAAGAACTACGATGACTTCGTGGAAACCTTCATTA ATCACGCCCGGGACCTGTTCCCCAATGCGGTCATCCACTTCGAGGACTTTGGCCTGAA CAACGCTTACCGTCTTCTGGAGAAGTACCGGAACAAGTTCGCAATGTTCAACGACGACA TTCAGGGCACAGGCGCCGTGACGCTGGCGGCGATCATCGCTGCGCTGAAAGTGTCTG GCGGCGGAGCGCTCCAAGACCAGCGCATAGTGATCTACGGCGCGGGTTCGGCGGGC ATGGGCGTCGCTGACGCGATCCGAGAAGGCATGGAGGTGCAGGATAAGCTGGGGACG GCGGATGCGAGCCAGCGCTTCTGGGCCGTCGATCGAAACGGCCTCCTGCTCGAGAGC ATGGCGCCCGCCCTCCGCGGCAACCAGGTGCGGTATGCGCGCCCGGACGACGAGAT CAAGACGTGGGCGCTCGAGAACCCCGAGCTGCCGAGTATGGCGCAGTTGCTTGACGT GGTGCGGAACGTCAAGCCGACAATTTTGATCGGGACGTCGACCGCGACGGGGGCGTT CACAGAGGAGGTCGTGCGCGAGATGGCCAAGCATGTCGAGCGGCCCATTATCATGCC CCTCAGCAACCCCACGTCTCTTTGCGAAGTTGATCCTCAGGACGCCATCACGTGGACA AACGGCAAGGCGCTCGTAGCCACCGGCTCGCCTTTCTCGCCCGTCAAGCTGCCTGAC GGACATGAGTACCATGTCGCCCAGACGAACAATGCGATCGTTTATCCGTCGCTTGCGG CGGGCGCGATCCTCGCGCGCGCAAACACCATCTCTCCGCGCATGCTCATGGCAGGCG TGGACGGGTTGTCCGAGCTCTCGCCTGCGCTCGATGACCCCTCCCAGGCTCTCCTACC GTCGCTCGACAACATTCGTGCCGTCAGCGTGCATGTCACGGCAGCGGTTATTCGTGCA GCCGTCAAGGAGGAGAACGCGAAAAACGAGCTCGTGATCAAAATCGCCGAGGATAAAG GCGAGATGAGCCTCGAGGACTATATCAAGGCGCGGATGTGGGATCCGGTATACCGCC CGCTCGAGTTGGTGGATTAG >PPI_promoter Promoter (SEQ ID NO: 40) CACTATCCCCCCACCGCCCCCTCCCCACCACTTCCTCCTCCTTCCCCATCGCTGTCCC AGCGGCTTATACTCACCTAGCTTTGCGGTCTCGACTTGCCCCAACACACGTTCCCTCCT CTTAATAACCCCCATACTCTCACTCTCCTTCATTACA >TEF1p Promoter (SEQ ID NO: 41) ATTTTGTGGCGGCGCCTGGGCGGGGCATGGACCGGGCGGAAGATTGCAACTGGCGAG CTGCTCGGCAGGGTCCGCTCATACCGCTCGCTCAGCTCCCTCTCTCCTTCCTTCCTTCT TTCCTTCCCCTCCTGCCCTCCTGGCATCAAATTAACGTGAGGGGATCGGATAAACATTG CGCCAAATCACCCCGTACGGTGAGCAAGCCGACGTCAGGCTGCCACACAAGGGCCCT CTAGCGGCGTTATAGACCCCGCCACACGTCATAACACCATAACGCCGTGACTGGGCTT TGAGTAGACAAAGGCAATTCAGGGTCTGTTGGAGGGGCGAGCTCCAGTGCCAGCGCA CTGTAAAATCCGCGTGCGATCCGCTTTCTCTCTTCACTCCCACCCTCTCTCTCTCCTCC AAAAAACAGTGAGTAGCACCAACGACAACCATGGCAATGGTGGTCGCCAAAGAGGCAA AGACAGCGAGCGCACTGCGACCGACAACGACCTGCCCGTCATCGACTCGAACAACCT GCAGCACCATCGCTCTCGTCATCGCCGCGCGCCACTTGCCCTGCACTCCTTCCCCCCA ^Attorney Docket No.11833-014WO1 ^ CCCCTCACCATCCCATCCTTTCCTCTCGACCATGCAAGGCCACGTGCCTCGAGTGCAT GCGCTCTTGGCGATGCTCGCGCATTGGTATCGTGGTCGTCTGCATCGTCGTCAAACAG ACGGAGCACGACGACCGTGACGAATTGGATGGCACAAAGGGTCCATGTCAAATTTGAC CTCCCGCTTCCACCTTCAACCTTCCTCCATCCAACATCCTCCGCCTTTGACTCGCAATG CGCATGCTGTCTTTGTCTCCCCACTCCCACCATTGACTGCCACTCTCAGAGAATGTCGA CTAACATTGCAGACAAAACCTATTCACC ^

Claims

Attorney Docket No.11833-014WO1 ^ CLAIMS 1. A method and process of producing lipids through sequential dual steps, the method comprising the steps of: a. Providing a first microbial culture in an anaerobic bioreactor; b. Exposing the first microbial culture in the anaerobic bioreactor to a source of carbon dioxide and hydrogen under conditions which allow for the microbial culture to produce carbonaceous feedstock; c. Removing the carbonaceous feedstock from the first bioreactor; d. Providing a second microbial culture in an aerobic bioreactor; e. Exposing the second microbial culture in the aerobic bioreactor to the carbonaceous feedstock of step c) under conditions in which the second microbial culture can produce a lipid; and f. Harvesting the lipid from step e).

2. The method of claim 1, wherein the carbonaceous feedstock comprises volatile fatty acids, including formate, actetate, propionate, butyrate, short chain fatty acids such lactate, alcohols including methanol, ethanol, glycerol, crude glycerol, propanol, butanol, amino acids and sugars including glucose, xylose and sucrose.

3. The method of claim 1 or 2, wherein the first microbial culture comprises a consortium of at least two different strains of anaerobic microorganisms.

4. The method of any one of claims 1-3, wherein the first microbial culture comprises at least one acetogenic microorganism.

5. The method of claim 4, wherein the acetogenic microorganism is from Sporomusa, Moorella, or Acetobacterium genus.

6. The method of claim 5, wherein the microorganism comprises Sporomusa silvacetica, Moorella thermoacetica, or Acetobacterium woodii.

7. The method of any one of claims 1-6, wherein the source of carbon dioxide is a salt or gas stream.

8. The method of claim 7, wherein the salt is carbonate or bicarbonate, further wherein the salt is created by converting at least a portion of carbon dioxide into salt in liquid phase prior exposure to the first microbial culture. ^Attorney Docket No.11833-014WO1 ^ 9. The method of claim 7, wherein the gas stream comprises syngas or biogenic carbon dioxide.

10. The method of claim any one of claims 1-9, wherein the second microbial culture comprises a pure culture and / or a consortium of at least two different strains of aerobic microorganisms.

11. The method of any one of claims 1-10, wherein the second microbial culture comprises an oleaginous bacterium or fungus, wherein the fungus can be yeast.

12. The method of claim 11, wherein the yeast is of the genus Cutaneotrichosporon, Yarrowia, Rhodotorula, Cryptococcus, Lipomyces, Debaryomyces or Trichosporon.

13. The method of claim 12, wherein the yeast is of the species Cutaneotrichosporon oleaginosus, Yarrowia lipolytica, Rhodotorula mucilaginosa, Rhodotorula glutinis, Cryptococcus curvatus, Debaromyces hansenii or Lipomyces starkeyi.

14. The method of any one of claims 1-13, wherein at least one microorganism from the first microbial culture is not naturally occurring, and further wherein the microorganism produces more carbonaceous feedstock than its naturally occurring counterpart.

15. The method of any one of claims 1-14, wherein at least one microorganism from the second microbial culture is not naturally occurring, and further wherein the microorganism produces more lipid than its naturally occurring counterpart.

16. The method of any one of claims 14- 15, wherein the non-naturally occurring microorganism is genetically modified.

17. The method of any one of claims 14-15, wherein the non-naturally occurring organism has been mutated by directed evolution or adaptive laboratory evolution (ALE).

18. The method of any one of claims 14-15, wherein the non-naturally occurring organism has been modified for overexpression by promoter engineering, copy number amplification, chromosomal integration of additional gene copies, and / or introduction of strong heterologous regulatory elements.

19. The method of any one of claim 14-18, wherein the non-naturally occurring first microorganism results in a 15%-45% higher yield in carbonaceous feedstock.

20. The method of any one of claim 14-19, wherein the non-naturally occurring second microorganism results in a 15%-45% higher yield in lipid. ^Attorney Docket No.11833-014WO1 ^ 21. The method of any of claims 1-20, wherein at least one microorganism from the second microbial culture comprises a DNA sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO: 6-11, or comprises a DNA sequence according to SEQ ID NO: 6-11.

22. The method of any of claims 1-20, wherein at least one microorganism from the second microbial culture comprises a DNA sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO: 12-27, or comprises a DNA sequence according to SEQ ID NO: 12-27.

23. The method of any one of claims 14-22, wherein the non-naturally occurring organism has been engineered to overexpress any one of, or various combination of (i)-(vi): (i) ME (Malic Enzyme) (ii) DGA1 and / or DGA2 (iii) Acetyl-coA carboxylase (iv) Acetyl-coA synthase (v) ATP-citrate lyase (vi) NADPH-glyceraldehyde dehydrogenase (vii) Fatty acid synthase (viii) diacylglycerol O-acyltransferase.

24. The method of any one of claims 14-23, wherein the microorganism has been engineered to ablate or reduce expression of long-chain-fatty-acid-CoA ligase and / or multifunctional enzyme type-1.

25. The method of any one of claims 14-24, wherein the microorganism has been engineered to overexpress malonyl CoA-ACP transacylase, ^-ketoacyl-ACP synthase, ^- ketoacyl-ACP reductase, ^-hydroxybutyl-ACP dehydrogenase, enoyl-ACP reductase and AMP deaminase, and / or to ablate the expression of multifunctional enzyme type-1 and peroxisome biogenesis factor 10.

26. The method of any one of claims 14-25, wherein the non-naturally occurring organism has been engineered to overexpress any one of, or any combination of (i)-(vi): (i) ACC1 (ii) ACS1 and / or ACS2 (iii) ACL1 and / or ACL2 (iv) TDH1, TDH2 and / or TDH3 (v) FAS1 and / or FAS2. ^Attorney Docket No.11833-014WO1 ^ 27. The method of any one of claims 14-26, wherein the microorganism has been engineered to ablate or reduce expression of FAA1 and / or MFE1.

28. The method of any one of claims 1-27, wherein the lipid is an oil.

29. The method of any one of claims 1-26, wherein, after the lipid is harvested in step f, it is converted to a fuel source.

30. The method of claim 29, wherein conversion to a fuel source comprises using a hydrotreated esters and fatty acids (HEFA) process.

31. The method of claim 30, wherein the conversion is via the steps (i) hydrodeoxygenation of the lipid, followed by (ii) cracking and (iii) isomerization, to yield a fuel source.

32. A lipid obtained by the method of any one of claims 1-31.

33. A fuel source obtained by the method of any one of claims 29-31.

34. A method of producing a lipid, the method comprising: a. providing a carbonaceous feedstock to an aerobic microbial culture in bioreactor under conditions in which the aerobic microbial culture can produce a lipid, wherein the aerobic microbial culture comprises at least one oleaginous microbe; and b. harvesting the lipid produced in step a).

35. The method of claim 34, wherein the carbonaceous feedstock comprises acetate, acetic acid, glycerol, short-chain fatty acids, glucose, biomass lysate, xylose, sucrose, corn, corn syrup, waste from agriculture, lignocellulosic biomass waste, waste from the food industry and / or CO2.

36. The method of any one of claims 34-35, wherein the aerobic microbial culture comprises a consortium of at least two different strains of aerobic microorganisms.

37. The method of any one of claims 34-36, wherein the oleaginous microbe comprises a bacterium or fungus, wherein the fungus can be yeast.

38. The method of claim 37, wherein the yeast is of the genus Cutaneotrichosporon, Yarrowia, Rhodotorula, Cryptococcus, Lipomyces, Debaryomyces or Trichosporon. ^Attorney Docket No.11833-014WO1 ^ 39. The method of claim 38, wherein the yeast is of the species Cutaneotrichosporon oleaginosus, Yarrowia lipolytica, Rhodotorula mucilaginosa, Rhodotorula glutinis, Cryptococcus curvatus, Debaromyces hansenii or Lipomyces starkeyi.

40. The method of any one of claims 34-39, wherein at least one microorganism from the aerobic microbial culture is not naturally occurring, and further wherein the microorganism produces more lipid than its naturally occurring counterpart.

41. The method of claim 40, wherein the non-naturally occurring microorganism is genetically modified.

42. The method of any one of claim 40 or 41, wherein the non-naturally occurring organism has been mutated by directed evolution or adaptive laboratory evolution (ALE).

43. The method of any one of claim 40, wherein the non-naturally occurring organism has been mutated by directed evolution or ALE, and at least one genetic modification.

44. The method of any one of claim 40-43, wherein the non-naturally occurring aerobic microorganism results in a 15%-45% higher yield in lipid.

45. The method of any of claims 40-44, wherein at least one aerobic microorganism comprises a DNA sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO: 6-11, or comprises a DNA sequence according to SEQ ID NOs: 6-11.

46. The method of any of claims 40-44, wherein at least one aerobic microorganism comprises a DNA sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NOs: 12-27, or comprises a DNA sequence according to SEQ ID NO: 12-27.

47. The method of any one of claims 40-46, wherein the non-naturally occurring organism has been engineered to overexpress any one of, or any combination of SEQ ID NO 27- 41 including genes (i)-(viii): (i) Diacyl glycerol acyltransferase (DGA1) (ii) Diacyl glycerol acyltransferase (DGA2) (iii) Malic Enzyme (ME) (iv) Acetyl-coA carboxylase (v) Acetyl-coA synthase (vi) ATP-citrate lyase (vii) NADPH-glyceraldehyde dehydrogenase ^Attorney Docket No.11833-014WO1 ^ (viii) Fatty acid synthase (ix) diacylglycerol O-acyltransferase.

48. The method of any one of claims 40-47, wherein the microorganism has been engineered to ablate or reduce expression of long-chain-fatty-acid-CoA ligase and / or multifunctional enzyme type-1.

49. The method of any one of claims 40-48, wherein the microorganism has been engineered to overexpress malonyl CoA-ACP transacylase, ^-ketoacyl-ACP synthase, ^- ketoacyl-ACP reductase, ^-hydroxybutyl-ACP dehydrogenase, enoyl-ACP reductase and AMP deaminase, and / or to ablate the expression of multifunctional enzyme type-1 and peroxisome biogenesis factor 10.

50. The method of any one of claims 40-49, wherein the non-naturally occurring organism has been engineered to overexpress any one of, or any combination of (i)-(vi): (i) ACC1 (ii) ACS1 and / or ACS2 (iii) ACL1 and / or ACL2 (iv) TDH1, TDH2 and / or TDH3 (v) FAS1 and / or FAS2 (vi) DGA2.

51. The method of any one of claims 40-50, wherein the microorganism has been engineered to ablate or reduce expression of FAA1 and / or MFE1.

52. The method of any one of claims 40-51, wherein the lipid is an oil or fatty acids from carbon chain length in the range of C12- C20.

53. The method of any one of claims 40-52, wherein, after the lipid is harvested in step f, it is converted to a fuel source.

54. The method of claim 53, wherein conversion to a fuel source comprises using a hydrotreated esters and fatty acids (HEFA) process.

55. The method of claim 54, wherein the conversion is via the steps (i) hydrodeoxygenation of the lipid, followed by (ii) cracking and (iii) isomerization, to yield a fuel source.

56. A lipid obtained by the method of any one of claims 34-55. ^Attorney Docket No.11833-014WO1 ^ 57. A fuel source obtained by the method of any one of claims 53-56.

58. A method of producing a non-naturally occurring oleaginous microbe capable of producing a higher quantity of lipid than its naturally occurring counterpart, the method comprising the steps of: a. Exposing an oleaginous microbe to conditions which promote mutagenesis; b. Culturing the microbe of step a); c. Isolating microbes from a distinct colony resulting from step b); d. Incubating the microbe in a nutrient broth comprising glycerol and / or a carbon source; e. Centrifuging the culture of step d); f. Isolating high lipid content cells; and g. Determining that the resulting microbe produces higher a quantity of lipid than a naturally occurring counterpart.

59. The method of claim 58, wherein the conditions which promote mutagenesis comprise Atmospheric and Room Temperature Plasma (ATRP) mutagenesis.

60. The method of claim 58 or 59, wherein the microbe is a fungus or bacteria, wherein the fungus can be a yeast.

61. The method of any one of claims 58-60, wherein the microbe is sequenced to a system for producing a lipid, the system comprising: a. An anaerobic bioreactor comprising a first microbial culture, wherein the first microbial culture produces a carbonaceous feedstock; and b. An aerobic bioreactor comprising a second microbial culture, wherein the second microbial culture uses the carbonaceous feedstock produced by the first bioreactor to produce a lipid.

62. The system of claim 61, wherein the carbonaceous feedstock comprises formate, methanol, methane, acetate, acetic acid, ethanol, glycerol, butyrate, propionate, butanol, propanol, short-chain fatty acids, amino acids, pyruvate, lactate, glucose, xylose, or sucrose.

63. The system of claim 61 or 62, wherein the first microbial culture comprises a consortium of at least two different strains of anaerobic microorganisms.

64. The system of any one of claims 62-63, wherein the second microbial culture comprises a consortium of at least two different strains of aerobic microorganisms. ^Attorney Docket No.11833-014WO1 ^ 65. The system of any one of claims 62-64, wherein the second microbial culture comprises an oleaginous yeast, bacterium, or fungus.

66. The system of any one of claims 62-65, wherein at least one microorganism from the first microbial culture is not naturally occurring, and further wherein the microorganism produces more carbonaceous feedstock than its naturally occurring counterpart.

67. The system of any one of claims 62-66, wherein at least one microorganism from the second microbial culture is not naturally occurring, and further wherein the microorganism produces more lipid than its naturally occurring counterpart.

68. The system of any one of claims 62-67, wherein the non-naturally occurring microorganism is genetically modified.

69. The system of any one of claims 62-68, wherein the non-naturally occurring organism has been mutated by directed evolution or adaptive laboratory evolution (ALE).

70. The system of any one of claims 62-69, wherein the non-naturally occurring organism has been developed by a combination of adaptive laboratory evolution (SEQ ID NO 14- 26) and genetic modification including the over expression of lipid biosynthesis genes (SEQ ID NO27- 41), or a combination thereof. ^

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