Rhodotorula toruloides as an industrial platform for food and chemical production using alternative feedstocks

Abstract

The present invention relates to a Rhodotorula toruloides strain that is enhanced through metabolic engineering and laboratory directed evolution for use in producing food and chemical production using alternative feedstocks. More particularly, the engineered strain which overexpresses Carnitine acetyltransferase (CAT) and / or transcription factor HAA1 genes, and the evolved strain have enhanced acetate utilization and / or fatty acid and / or lipid and / or biomass production compared to wildtype R. toruloides.

Description

[0001] RHODOTORULA TORULOIDES AS AN INDUSTRIAL PLATFORM FOR FOOD AND

[0002] CHEMICAL PRODUCTION USING ALTERNATIVE FEEDSTOCKS

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims priority of Singapore Patent Application No. 10202400461 Q filed on 21 February 2024, the contents of which are incorporated herein in their entirety by reference.

[0005] FIELD OF THE INVENTION

[0006] The present invention relates to a Rhodotorula toruloides strain that is enhanced through metabolic engineering and laboratory-directed evolution for use in producing food and chemical production using alternative feedstocks. More particularly, the engineered strain which overexpresses Carnitine acetyltransferase {CAT) and / or transcription factor HAA1 genes, and the evolved strain have enhanced acetate utilization and / or fatty acid and / or lipid and / or biomass production compared to wildtype R. toruloides.

[0007] BACKGROUND OF THE INVENTION

[0008] The global shift to sustainability has driven the exploration of alternative feedstocks beyond sugars for biomanufacturing [Zhang, C., et al., 2022], Single-cell oils (SCO) and single-cell protein (SCP) produced by microorganisms can be used as food ingredients. SCO is intracellular storage lipids composing mainly triacylglycerols (TAGs) [Chawla, K., et al., 2022;

[0009] Maina, S., et al., 2017; Mhlongo, S.I., et al., 2021 ; Ochsenreither, K., et al., 2016]. SCO can be used to replace edible oils that are traditionally found in the plant and animal kingdoms [Demir, M. and A.G. Gundes, 2020]. SCP is semi-purified proteins from the cells of microorganisms. It has high protein and vitamins contents and is increasingly regarded as a future food ingredient [Demir, M. and A.G. Gundes, 2020]. SCP has already been used in food products, such as meat substitute, food spreads, flavouring agents and food supplements [Jach, M.E., et al. 2022; Ritala, A., et al., 2017], SCP-based products with the brand name QUORN, PEKILO, MARMITE, VEGEMITE and CENOVIS have a long history of safe human consumption [Ritala, A., et al., 2017; Garcia Martinez, J.B., et al., 2022; Nyyssbla, A., et al., 2022], The advantages of SCO and SCP include high productivity, low CO2 and water footprints, and pesticide-free production [Mhlongo, S.I., et al., 2021 ; Ochsenreither, K., et al., 2016; Demir, M. and A.G. Gundes, 2020]. SCO and SCP production in the past have been relying on sugars as the feedstock except phototropic cultivation of microalga. The first generation (G1 ) feedstock is produced from sugar crops, such as sugarcane and sugar beet while the second generation (G2) feedstock is derived from the more abundant cellulose-rich biomass from forestry and agriculture [Bratosin, B.C., S. Darjan, and D.C. Vodnar 2021]. Driven by the Paris Agreement on climate change with the goal to limit global warming to less than 1.5°C, most countries in the world have pledged to achieve CO2 neutrality by mid-century. With the natural abundance and rapid advance in catalytic, biocatalytic and microbial electrochemical synthesis, 01 (CO2, CO, methane, methanol and formate) and C2 (acetate and ethanol) substrates are increasing hailed as an alternative feedstock for microbial fermentation [Zhang, C., et al., 2022; Jiang, W., et al., 2021 ; Fang, X., et al., 2020]. The utilization of these feedstocks will be more effective in reducing carbon emissions and supporting sustainability. Methanol can be utilized efficiently as the sole carbon source by a small number of methylotrophic yeast and bacteria [Espinosa, M.L, et al., 2020], For example, engineered Pichia pastohs allowed the production of free fatty acids and fatty alcohol from methanol, with a free fatty acid titre and yield of 23.4 g / L and 0.078 g / g, respectively [Cai, P., et al., 2022], Similarly, Ogataea polymorpha has been engineered to produce 15.9 g / L free fatty acid from sole methanol [Gao, J., et al., 2022], With lower toxicity and higher energy density, C2 feedstock may be a better next generation feedstock than methanol. In addition, acetate is an abundant byproduct in biomass hydrolysate. For example, 49.8 mM and 34.3 mM acetate were found in soybean hull and spruce acid hydrolysate [Hu, C., et al., 2009; Chen, L., et al., 2021], Therefore, biomass conversion rate to food and chemicals should be significantly improved if acetate can be efficiently utilized as the co-substrate.

[0010] Different methods have been applied to increase the production of lipids from acetate, including bioprocess engineering, substrate co-feeding and metabolic engineering. For example, metabolic engineering and co-substrate fermentation in Y. lipolytica has significantly increased lipid production from acetate by overexpressing acetyl-CoA synthetase (ACS), acetyl-CoA carboxylase (ACC1) and fatty acid synthase [Chen, L., et al., 2021], The lipid content was increased by 95% through acetate and glycerol co-substrate fermentation. Coupled metabolic engineering with metabolic control in fermentation, Y. lipolytica was able to attain 1 15 g / L of lipid content, 0.16 g / g lipid yield and 0.8 g / L / h of productivity [Xu, J., et al., 2017],

[0011] Rhodotorula toruloides is an emerging host for metabolic engineering and synthetic biology due to its high lipid and cell mass productivity [Zhao, Y., et al., 2021 ; Park, Y.-K., et al., 2018]. Although acetic acid is naturally produced by many microbes, its presence could be a limiting factor in microbial fermentation. For example, ethanol production strongly inhibits Pichia stipites growth at 83 mM [Gong, Z., et al., 2015]. Acetate had little effect on lipid production in R. toruloides up to 70 mM (4.2 g / L). In fact, acetate at 120 mM has been shown to improve cell performance and lipid content in R. toruloides Y4 [Hu, C., et al., 2009; Chen, L., et al., 2021 ], Microbes have diverse capacity to utilize acetate. For example, Cryptococcus curvatus, Trichosporon cutaneum AS 2.571 and T. fermentans CICC 1368 are much more efficient in utilizing acetate for lipid production than R. toruloides and Y. lipolitica [Gong, Z., et al., 2015].

[0012] There is a need to develop improved lipid production, fatty acid and / or cell mass profile in a microorganism.

[0013] SUMMARY OF THE INVENTION

[0014] R. toruloides C3 is a strain isolated in Singapore by Temasek Life Sciences Laboratory Limited. The strain C3 was used as the host for yeast genetic manipulation. It’s genome was sequenced and the nucleotide sequences of the contigs are set forth in SEQ ID NOs: 33-50. Lipid production in R. toruloides C3 was enhanced through metabolic engineering and laboratory directed evolution. The R. toruloides C3 genome encodes 4 candidate carnitine acetyltransferase (CAT) genes, CAT1, CAT2, CATS and CAT4, the overexpression of which led to enhanced acetate resistance and utilization. CAT1 encoding a peroxisome targeted enzyme was the most effective. Overexpression of I-IAA1, which encodes a bread yeast homolog of transcriptional activator, also significantly enhanced acetate utilization although CAT1 and HAA 1 showed no additive effect. Over-expression of CAT 1-4 genes also significantly increased PUFA content. In 2L-scale fed-batch fermentation using 3% acetate with 0.5% dextrose monohydrate as the co-carbon source, the CATf-overexpressing strain increased acetic acid consumption, biomass titer and lipid titer respectively. Maximal dry biomass titer reached 156.5 g / L, 22% higher than control and this was reached 24 hr earlier. Lipid contents were significantly higher than control over the course of fermentation, reaching a maximum of 19.8 g / L. There was a 99.5% increase of cumulative consumption of acetic acid. The biomass yield, however, was not increased by the overexpression of CAT1. At maximal biomass titer, biomass yield was 0.55 g / g acetic acid (at 216 hr), lower than 0.68 g / g observed with control at 240 hr, possibly due to the need for more substrate to produce lipid. The low yield was, in part, attributable to high glycerol generation. At maximal biomass titer, glycerol titer was 65.6 g / L and 48.3 g / L respectively for CAT1 -OE and control strain. Consistent with the flask culture data (Table 3), The average ALA content of samples from all time-points was 2.58% of total fatty acids for pKCL2 control strain, compared with 9% in CAT1 -OE strain, an increase of 249%. Deletion of Cat1 peroxisome targeting signal only marginally weakened the biomass and lipid production-promoting activities of Cat1 , suggesting it functions mainly by increasing the cytoplasmic pool of L-carnitine-CoA, which may lead to higher levels of cytoplasmic and mitochondria acetyl-CoA.

[0015] An adaptive laboratory evolution-derived strain, designated C3_64B, was able to produce 99.63 g / L of biomass, with 13%-16% lipid in 2L fed-batch fermentation. Interestingly the evolved strains had a drastically increased a-linolenic acid content, from ~4% to 15%. In a semi-continuous fermentation process, C3 strain produced > 100 g / L dry cell mass with 44- 45% protein, ~ 10% lipid and 6.0-7.2 g / L / h productivity when glucose was used as the sole carbon source. Thus, R. toruloides is a promising host for SCP and SCO production, using either sugars or acetic acid as the feedstock.

[0016] Accordingly, in a first aspect the disclosure provides a genetically engineered R. toruloides cell, wherein the cell has been transformed by at least one polynucleotide molecule; the at least one polynucleotide molecule comprising a carnitine acetyltransferase CAT) selected from the group consisting of CAT1, CAT2, CAT3 and CAT4; and / or transcription factor HAA1 gene, operably linked to at least one promoter, wherein overexpression of CAT and / or HAA1 provides increased acetate utilization, fatty acid and / or lipid and / or biomass production compared to wildtype R. toruloides. In some embodiments the CAT gene is CAT1.

[0017] In some embodiments, the CAT and / or HAA1 genes are operably linked to a strong promoter to enhance the expression level of the genes and achieve a protein expression level that is at least 5 times higher, preferably 10, 100 or 100 times higher. In some embodiments the strong promoter is a constitutive promoter such as, for example selected from the group comprising translation-elongation factor 1 alpha (TEF1) and glycerol 3- phosphate dehydrogenase {GPD1).

[0018] In some embodiments, the TEF1 promoter and the GPD1 promoter are from R. graminis.

[0019] Preferably the promoters are from R. graminis WP1 strain. In some embodiments the TEF1 promoter comprises the nucleotide sequence set forth in SEQ ID NO: 1 1. In some embodiments the GPD1 promoter comprises the nucleotide sequence set forth in SEQ ID NO: 13. In some embodiments, the isolated genetically engineered Fl. toruloides cell has a genome sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, to the contig polynucleotide sequences set forth in SEQ ID NOs: 33-50. In some embodiments, the genetically engineered R. toruloides cell is genetically engineered R. toruloides 03 strain. In some embodiments the genome nucleotide sequence of R. toruloides C3 strain is set forth in SEQ ID NOs: 33-50.

[0020] In some embodiments, the CAT gene is CAT1 and encodes the amino acid sequence set forth in SEQ ID NO: 1.

[0021] In some embodiments, the HAA1 gene encodes the amino acid sequence set forth in SEQ ID NO: 3.

[0022] In some embodiments, the CAT1 gene comprises a polynucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, due to the degeneracy of the genetic code, to the polynucleotide sequence shown in SEQ ID NO: 2.

[0023] In some embodiments, the HAA1 gene comprises a polynucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, due to the degeneracy of the genetic code, to the polynucleotide sequence shown in SEQ ID NO: 4.

[0024] In a second aspect the disclosure provides an isolated directed-evolution-derived R. toruloides C3 strain having enhanced acetate utilization, decreased glycerol by-product accumulation, and stress resistance compared to wildtype R. toruloides. Preferably, in some embodiments the isolated directed-evolution-derived R. toruloides strain evolution is performed by adapting a culture of R. toruloides C3 against a gradual increase of acetate concentration in the culture medium.

[0025] In some embodiments, the evolved C3 strain can tolerate up to 6% acetic acid. In some embodiments, the evolved C3 strain can tolerate up to 6% acetic acid and up to 4% glycerol. In one embodiment, the evolved C3 strain is C3_64B strain.

[0026] In some embodiments, the evolved C3 strain can produce higher a-linolenic acid (ALA) content compared to genetically engineered CAT1 C3 strain because it can utilize glycerol as a carbon source.

[0027] In a third aspect the disclosure provides an isolated R. toruloides C3 cell, wherein the cell has a genome sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, to the polynucleotide sequences set forth in SEQ ID NOs: 33-50.

[0028] In a fourth aspect the disclosure provides a method of producing increased fatty acids, lipids and / or biomass by a genetically engineered R. toruloides cell, or by a directed-evolution- derived R. toruloides cell, comprising culturing a plurality of genetically engineered R. toruloides cells or directed-evolution-derived R. toruloides cells of any one of the embodiments described above in medium under conditions for fatty acids, lipid production and / or increased biomass, wherein the medium comprises acetate as carbon source, wherein said genetically engineered cell or directed-evolution-derived cell is capable of increased production of fatty acids, lipids and / or biomass compared to a strain of identical genome background or non-evolved R. toruloides cell.

[0029] In some embodiments, the R. toruloides cell is an R. toruloides C3 cell, preferably a CAT1 C3 cell, more preferably a C3_64B cell.

[0030] In some embodiments, the increased fatty acid is alpha-linolenic acid.

[0031] In some embodiments, the said method further comprises having glycerol in the medium.

[0032] In a fifth aspect the disclosure provides the use of a R. toruloides cell of any aspect of the invention in a method of producing enhanced lipid and / or biomass production compared to wildtype R. toruloides.

[0033] In some embodiments, the R. toruloides cell is an R. toruloides C3 cell, preferably a CAT1 C3 cell, more preferably a C3 64B cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Certain embodiments of the present disclosure are described more fully hereinafter with reference to the accompanying drawings.

[0035] Figure 1 shows microbial diversity on acetate utilization. RT1 , RT2 and C3: R. toruloides strains; WP1 : R. graminis WP1 ; GS1 15: Pichia pastohs (Komagataella phaffii); SSY: Saccharomyces cerevisiae. A single colony of each strain was inoculated into 5 mL YPD in 50 mL Falcon tubes and incubated at 28 °C overnight. After dilution to OD600 0.2, 1 mL diluted culture was inoculated into 49 mL acetate assimilation medium with 3% acetate in 250 mL conical flasks and cultured at 30 °C for 3 and 5 days.

[0036] Figure 2 shows optimization of medium for cell mass and lipid production for C3 strain, (a) Different medium compositions. The reported media were modified to have glucose substituted by 3% acetic acid as the sole carbon source, (b) Effect of different pH on biomass, (c) Medium with different nitrogen sources. Y1 : yeast extract 2.15 g / L, urea 2g / L; Y2: yeast extract 2.15 g / L, NH4CI 3.68 g / L ; Y3 : yeast extract 2.15 g / L, KNO36.63 g / L; Y4: yeast extract 4.3 g / L, urea 2 g / L; Y5: yeast extract 4.3 g / L, NH4CI 3.68 g / L; Y6: yeast extract 4.3 g / L, KNO3 6.63 g / L; Y7: yeast extract 6.45 g / L, urea 2g / L; Y8: yeast extract 6.45 g / L, NH4CI 3.68 g / L; Y9: yeast extract 6.45 g / L, KNO3 6.63 g / L. (d) Effect of acetate concentrations on biomass and lipid production. Error bars are the standard deviation of triplicates.

[0037] Figure 3 shows the performance of Wt C3 and the final adapted population, C3_64B was inoculated into media with various acetic acid to glycerol ratio. GAAM24 contains 2% acetate / 4% glycerol and so on. (a) OD 600 nm of strain C3 and C3_64B and (b) Acetate consumption of strain C3 and C3_64B.

[0038] Figure 4 shows the effect of overexpression of various genes were over-expressed under the Rg TEF1 promoter in C3CK strain, a derivative of C3 containing the KU70 gene knockout and an inducible ere recombinase gene as described in Koh, C.M.J., Liu, Y., Moehninsi et al. (2014). Three different single colonies of each construct were inoculated into 5 ml YPD in 50 ml Falcon tube and incubated at 28-30 °C overnight. Cells were diluted to 0.05 OD600 in 50 ml medium in a 250 ml conical flask. A. Biomass of different strains cultured in AMP3-Y6 medium with 3% acetic acid at 28 °C for 5 days. B. Biomass of different strains cultured at 30 °C in AMP3-Y4.3 with 4% acetic acid ( ie 6.63 g / L KNO3, 5.7 g / L KH2PO4, 1 g / L MgSO4.7H2O , 0.015 g / L FeCI3.6H2O, 40 g / L acetic acid and 4.3 g / L yeast extract. The p values indicate the probability that an observed difference between control strain and overexpression strains. ** and * indicate p<0.01 ; 0.05, respectively. Figure 5 shows the effect of MgSCU level on dry cell mass. A single colony was inoculated into 5 mL YPD in 50 mL Falcon tube and incubated at 28 °C overnight. All of the 5 mL overnight culture was inoculated into 45 L YPD in 250 mL conical flasks and incubated for 24 hours at 30 °C. 5 mL of this subculture was inoculated into 45 mL of each different medium with different MgSC .TFfeO content in 250 mL conical flasks and incubated for 72 hours at 30 °C. To remove calcium precipitates, cell pellets were treated with 1 mL 1 M citric acid for 30 mins, and washed twice with distilled water before being dried at 65°C for 48 hours. The basic medium contained yeast extract 4.3 g / L, urea 2 g / L, KH2PO4 5.7 g / L, FeCl3.6H2O 0.015 g / L, acetic acid 30 g / L. All media were adjusted to pH 7.0 with NaOH.

[0039] Figure 6 shows the effect of medium pH on cell biomass production. A single colony of C3 was inoculated into 5 mL YPD in 50 mL Falcon tube and incubated at 28 °C overnight. All the 5 mL overnight culture was inoculated into 45 mL YPD in 250 mL conical flasks and incubated for 24 hours at 30 °C. 5 mL of this subculture was inoculated into 45 mL of APM2 medium of different pH value in 250 mL conical flasks and incubated for 72 hours at 30 °C. All the cell pellets were treated with 1 mL 1 M citric acid for 30 mins, washed twice with distilled water before being at 65°C for 48 hours.

[0040] Figure 7 shows the effect of acetate and glycerol concentrations. Acetate assimilation medium (AAM, pH 6.5) described by Gong et al. (2015) was used to culture strain C3 at various acetate and glycerol concentrations. Cell were cultured for 3 days at 30°C with agitation (210 rpm) in 250 mL flasks, (a) C3 was cultured in AAM medium with 0-3% acetic acid, (b) C3 was cultured in AAM medium with 1% acetic acid and various levels of glycerol.

[0041] Figure 8 shows the effects of co-substrates. Strain C3 seed culture (5 mL) prepared in YPD medium was inoculated into 50 mL APM2 basal medium 250 mL conical flasks. APM2 basal medium contained 10 g / L acetic acid and 25 g / L of respective co-substrate as the carbon source. Culture was re-inoculated into APM2 with no glucose but 10 g / L acetic acid only after Day 3, except for glucose setup. Upper line in each graph is the co-substrate level. Lower line in each graph represents acetate level.

[0042] Figure 9 shows a comparison of cell biomass and lipid production of different evolved isolates. C3 Wt and 22 individual colonies of 25thgeneration of the adapted population (C3_64) were cultured in 50 mL GJM3 in 250 mL conical flasks for 5 days at 30°C and 210 rpm.

[0043] Figure 10 shows the effects of overexpression of CAT1 , HAA1 , DGA1 on biomass (a) and lipid (b) production. Figure 11 shows the effect of overexpression of ACS1 . C3_pKC2.3GPD1_ACS1 : The ACS1 CDS was expressed using the RgGPDI promoter and terminator. C3_pKC2.3TEF1_ACS1 / MTS: the RtACSI ORF was fused with the mitochondria using the malate dehydrogenase 2 mitochondria targeting sequence, MAAATRQSSRLARSFSTSARAN (SEQ ID NO: 15) and overexpressed under the control of RgTEFI promoter and terminator. C3_pKC2 is the control vector. All constructs were integrated at the CAR2 locus by homologous recombination. Error bars indicate standard deviation of 3 biological replicates. Strains were cultured in AAM medium with 3% acetate for 5 days.

[0044] Figure 12 shows the effect of peroxisome targeting on CAT1 functions. The C-terminal PTS signal of CAT1 , AKL tripeptide, was deleted by PCR and expressed under the same RgTEFI promoter. 3 independent knock-in mutants at the CAR2 locus of C3CK strain were cultured in 50 ml AMP3-Y6 medium with 4% acetic acid at 28 °C for 5 days. *** indicate p value <0.001 between control vector pKCL2 and overexpression constructs. A. Cell density over 5 days; B. Dry Cell Mass; C. lipid content.

[0045] Figure 13 shows semi-continuous fed-batch fermentation. Wt C3 frozen stock at -80°C was inoculated to 5 mL APM2 medium and cultured for about 16 hr at 30°C. 100 mL APM2 medium of about 10 OD6oo units is prepared by culturing the overnight culture (1 mL) in APM2 medium in conical flasks. Seed culture was inoculated to 1 litre of APM2 medium at a dilution rate of 10% in a 2 L bioreactor and cultured at 30°C. pO2 was maintained at 30% by cascade control of agitation speed and air-flow. pH was maintained at 5.5 using HCI and NaOH. Glucose level was maintained at 40-60 g / L by adjusting the flow rate of the feeding solution, between 4-8 mL / hr. In the semi-continuous fed-batch phase, 700-1000 mL fermentation broth was removed when OD600 reached about 320 and replaced with 500- 800 mL Replacement Medium (CRM). This bleeding / feeding process was repeated about every 6 hours so as to maintain the cell density at 300-400 OD600 units. Biomass productivity and glucose consumption (glucose consumed per gram biomass) refer the transient value of over the two sampling times. (SI Te to confirm the volume for RM). The figures on top of the biomass titers indicate the protein contents of the respective time point, (a) Effect of nitrogen source and content in CM. The content of yeast extract (YE) and urea in the RM in each phase are in the respective window, (b) Semi-continuous fed-batch fermentation of Wt C3 strain. RM contained 17.2 g / L yeast extract and bleeding / feeding was repeated every 6 hours, (c) Effect of nitrogen source on lipid content.

[0046] Figure 14 shows fed-batch fermentation of CAT4 and C3-64B strains. 100 mL of Seed culture in YPD was inoculated into 1 L AMP3-Y6 medium with 3% acetic acid and supplemented with 5 g / L glucose, 0.1 g / L L-Histidine, 0.01 g / L; L-Aspartic acid; 10 mL vitamins of AAM in a 2 L bioreactor. The pH was adjusted to pH 7 using 5 M NaOH and pC>2 was maintained at 30%. pH during fermentation was maintained at 7.0 using glacial acetic acid. Feeding is started about 40 h at the flow rate so that glucose and acetic acid can be maintained at about 5 g / L and 30 g / L respectively. (A) Biomass titer; (B) Growth rate; (C) Biomass yield; (D) Lipid titer; (E) Lipid content; (F) Lipid yield.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] Further details of the invention will now be described with reference to the following nonlimiting examples. Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art.

[0049] A. Definitions

[0050] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0051] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of”. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases that require the presence of the named features / steps and permit the presence of other features / steps. However, such description should be construed as also describing compositions, mixtures, or processes as “consisting of” and “consisting essentially of’ the enumerated features / steps, which allows the presence of only the named features / steps, along with any impurities that might result therefrom, and excludes other features / steps.

[0052] The terms "amino acid" or "amino acid sequence," as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

[0053] As used herein, the terms “polypeptide”, “peptide” or “protein” refer to one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or peptide can comprise a plurality of chains noncovalently and / or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence. A “polypeptide”, “peptide” or “protein” can comprise one (termed “a monomer”) or a plurality (termed “a multimer”) of amino acid chains.

[0054] The terms “nucleotide”, “nucleic acid” or “nucleic acid sequence”, as used herein, refer to an oligonucleotide, polynucleotide, or any fragment thereof, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.

[0055] The term “operably linked” as used herein refers to a first molecule that can be joined to a second molecule, wherein the molecules are so arranged that the first molecule affects the function of the second molecule. The term “operably linked” includes the juxtaposition of two or more components (e.g., a promoter and another sequence) such that both components function normally and allow for the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, a promoter is operably linked to a polynucleotide molecule if the promoter modulates transcription of the polynucleotide molecule of interest in a cell. In additional embodiments, two portions of a transcription regulatory element are operably linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion. Two transcription regulatory elements may be operably linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to one another with no intervening nucleotides present.

[0056] A vector can include one or more carnitine acetyltransferase (CAT) or HAA1 nucleic acid(s) in a form suitable for overexpression of the nucleic acid(s) in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence(s) to be expressed. The term "regulatory sequence" includes promoters, enhancers, ribosome binding sites and / or IRES elements, and other expression control elements (e.g., polyadenylation signals). The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides encoded by nucleic acids as described herein (e.g., CAT or HAA1 proteins). The recombinant expression vectors of the invention can be designed for over-expression of CAT or HAA1 proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in yeast (e.g., R. toruloides). Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

[0057] A person skilled in the art will appreciate that the present invention may be practiced without undue experimentation according to the methods given herein. The methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology textbooks.

[0058] EXAMPLES

[0059] The following examples are intended to exemplify the present disclosures and are not limitations of the claimed invention. All molecules, compositions, methods, assays, and results disclosed in the examples and other sections of the specification, figures, and claims form part of the disclosure of the invention.

[0060] Example 1

[0061] Materials and methods

[0062] 1.1 Strains, medium compositions

[0063] The strains used were R. toruloides ATCC 10657 and R. toruloides ATCC 10788 which were obtained from American Type Culture Collection (USA), R. gramminis strain WP1 which obtained from Fungal Genetics Stock Center (University of Missouri, USA) and R. toruloides C3 is a strain isolated in Singapore by Temasek Life Sciences Laboratory Limited. Yeast strains were maintained in YPD agar and medium at 28-30°C. Escherichia coli XL1 -BLUE competent cells were used for plasmid amplification and R. toruloides. The strain C3 was used as the host for yeast genetic manipulation.

[0064] Various media were used in this work. Acetate Assimilation Medium (AAM) has been described by Gong, Shen [Gong, Z., et al., 2015]. AAM is usually supplemented with 10 mL / L AAM-vitamins composed of 50 mg / L thiamine hydrochloride, 50 mg / L riboflavin, 50 mg / L nicotinic acid, 50 mg / L pantothenic acid, 10 mg / L pyridoxine hydrochloride, 20 mg / L biotin, 20 mg / L folic acid, 50 mg / L 4-aminobenzoic acid, 50 mg / L cyanocobalamin.

[0065] Glycerol acetate assimilation medium (GAAM12) contains 8.6 g / L yeast extract, 5.7 g / L KH2PO4, 5.7 g / L MgSO4.7H2O, 2 g / L NaCI, 80 mg / L FeSO4.7H2O, 10 g / L acetate, and 20 g / L glycerol. Different levels of acetate and glycerol were used in GAAM during the course of laboratory-directed evolution. For example, GAAM with 2% acetate and 4% glycerol was denoted as GAAM24.

[0066] The original APM medium contains 8.6 g / L yeast extract, 5.7 g / L KH2PO4, 5.7 g / L MgSO4.7H2O, 2 g / L NaCI, 80 mg / L FeSO4.7H2O, 10 g / L, 80 g / L glucose monohydrate. APM2 medium is same as AMP except 8.6 g / L yeast extract was reduced to 4.3 g / L and 2 g urea was supplemented as the inorganic nitrogen source. The Replacement Medium (RM) used semi-continuous fed-batch fermentation contains 17.2 g / L yeast extract, 5.7 g / L KH2PO4, 5.7 g / L MgSO4.7H2O, 2 g / L monosodium glutamate (MSG), 80 mg / L FeSO4.7H2O, 80 g / L glucose monohydrate, 15 g / L NH4CI. The Continuous Feeding Medium (CFM) for the semi-continuous fed-batch fermentation contains 800 g / L glucose monohydrate and 35 g / L NH4CL Acetate Feeding Medium (AFM), composed of 30 g / L acetic acid glacial, 5 g / L glucose, 100 g / L yeast extract, 20 g / L MgSO4.7H2O, 66 g / L KNO3, 150 mg / L FeSO4.7H20, 1 g / L L-Histidine, 100 mg / L L-aspartic acid and 10 mL AAM vitamin solution.

[0067] 1 .2 Shake-flask cultures

[0068] A single colony established on an agar plate was inoculated into 5 mL YPD in a 50 mL Falcon tube; incubated at 28°C overnight with agitation (210 rpm) before inoculated into 50 mL appropriate medium in a 250 mL conical flask. The culture usually started at 0.2 OD6oo and cultured at 28-30°C with agitation at 200 rpm for 3-5 days. Biomass, lipid content and acetate consumption were determined as described previously [Liu, Y., et al., 2021 ],

[0069] 1 .3 Plasmid construction and yeast transformation

[0070] The primers and plasmids used are listed in Table 1 and Table 2, respectively.

[0071] Table 1 : Primers used in the Examples

[0072] Primer Sequence (5’-3’) SEQ ID NO:

[0073] 3GPD1 ACS1 F caccacctttctccagctcgaaccATGGCCGACGTCGACTACAA 19

[0074] 3GPD1 ACS1 R ggtacggaggcgctggatatcTCACTCGGACGAGAGCTTG 20

[0075] 3TEF1 CAT1 F cgtcctcgccgccctgcagcacgccATGCACGCAGCCCGCAGAA 21

[0076] 3TEF1 CAT1 R aagaagcagctcgtgaggatatcTTACAGCTTGGCGTTGTCGGC 22

[0077] 3TEF1 CAT2F cgtcctcgccgccctgcagcacgccATGCTTGTTGCACGGCTG 23

[0078] 3TEF1 CAT2R aagaagcagctcgtgaggatatcTCACGCCTTCGCCTTCC 24

[0079] 3TEF1 CAT3F gtcctcgccgccctgcagcacgccATGCGCTCCCGCGCCGAGAC 25 3TEF1 CAT3R aagaagcagctcgtgaggatatcTCACAGCTCCACCGTCG 26

[0080] 3TEF1 CAT4F cgtcctcgccgccctgcagcacgccATGCTCCTGTCGCGCACCG 27

[0081] 3TEF1 CAT4R aagaagcagctcgtgaggatatcCTACAGCTTCGCCTTGGCC 28

[0082] 3TEF1 HAA1 F tcgccgccctgcagcacgccATGGTCCTCATCGACGGG 29

[0083] 3TEF1 HAA1 R aagaagcagctcgtgaggatatcTCAGCCGAAAGCGCCGCT 30

[0084] DGA1 F1 cctgcaatcatggccttaatGGACGGCTTGTTCTCTCC 31

[0085] DGA1 R1 tcgaccttctcaagcactagAACCAACTTTATCCAGCAGC 32

[0086] Table 2: Plasmids used in the Examples

[0087] Plasmid Genotype and purpose pKC2.RgGPD1 -ACS1 SpR, CAR2L-P3GPDi-ACS1-HygR-CAR2R, for overexpression of ACS1 pKC2.RgTEF1 -CAT1 SpR, CAR2L-P3TEFi-CAT1-HygR-CAR2R, for overexpression of CAT1 pKC2.RgTEF1 -CAT2 SpR, CAR2L-P3TEFi-CAT2-HygR-CAR2R, for overexpression of CAT2 pKC2.RgTEF1 -CAT3 SpR, CAR2L-P3TEFi-CAT3-HygR-CAR2R, for overexpression of CATS pKC2.RgTEF1 -CAT4 SpR, CAR2L-P3TEFi-CAT4-HygR-CAR2R, for overexpression of CAT4 pKC2.RgTEF1 -HAA1 SpR, CAR2L-P3TEFi-HAA1-HygR-CAR2R, for overexpression of HAA1 pKC2.RgGPD1 -DGA1 SpR, CAR2L-PGpoi-DGA1-HygR-CAR2R, for overexpression of DGA1

[0088] Acetyl-CoA synthetase CDS (ACS1) was amplified by RT-PCR using cDNA pool as the template and oligonucleotides 3GPD1ACS1 F and 3GPD1ACS1 R as the primers. Carnitine acetyltransferase 1 -carnitine acetyltransferase genes, CAT1, CAT2, CAT3 and CAT4, and transcriptional activator CDS (HAA1) were amplified using genomic DNA R. toruloides as the template. The CAT1-CAT4 CDSs were amplified using primers 3TEF1 CAT1 F / 3TEF1CAT1 R, 3TEF1 CAT2F / 3TEF1 CAT2R, 3TEF1 CAT3F / 3TEF1 CAT3R and 3TEF1CAT4F / 3TEF1 CAT4R, respectively. The HAA1 sequence was amplified using primers 3TEF1 HAA1 F / 3TEF1 HAA1 R. Amplified ACS1 PCR products was fused with pKCL2.RgGPD1 digested with Ncol and EcoRV by Gibson cloning to create plasmid pKC2.RgGPD1 ACS1 . Amplified CAT1-4 and HAA 1 PCR products was fused with pKCL2.RgTEF1 digested with Ncol and EcoRV by Gibson cloning to create plasmid pKC2.RgTEF1 CAT1 -CAT4 and pKC2.RgTEF1 HAA1 respectively. All plasmids were verified by sequencing. DGA1 was amplified from pE1 GLA2 [Liu, Y., et al., 2021 ] using primers DGA1 F1 and DGA1 R1 (Table 1 ) and fused with pKC2.RgTEF1CAT1 using Gibson assembly method.

[0089] To explore the effects of double gene overexpression on lipid production, the DGA1 and HAA1 cassettes are released from the backbones by cutting with restriction enzyme Pad and Pmel. pKC2.3TEF1 CAT1 was digested with Spel, blunt-ended using T4 DNA polymerase and then digested with Pac1. The above two fragments were ligated using Quick Ligation and transformed into E. co / / XL1 -Blue. The T-DNA vectors were transformed into R toruloides strain C3 by Agrobacterium tumafaciens-medateb transformation (ATMT) as described previously [Liu, Y., et al., 2013; Liu, Y., et al., 2018]. Gene expression cassettes were site-specifically-integrated to the CAR2 locus (encoding phytoene synthase / lycopene cyclase) to eliminate the positional effect. Overexpression mutants were selected by albino phenotype and at least 3 biological replicates were applied in the assays [Liu, Y., et al., 2013; Liu, Y., et al., 2018].

[0090] 1.4 Laboratory directed evolution to enhance acetate utilization

[0091] Directed evolution of C3 strain for enhancing acetate utilization and stress resistance was performed by adapting the culture against a gradual increase of acetate concentration in the culture medium. A single colony of C3 was inoculated into 5 mL of YPD in a 50 mL Falcon tube and culture for 24 hours at 30°C with 210 rpm agitation. The whole 5 mL culture was inoculated into 45 mL of GAAM12 and cells were cultured at 30°C, 210 rpm for 5 days. The culture was sub cultured into fresh media of GAAM12 for 4 more times for a total of 5 cycles. This adaptation process was repeated with 4 subcultures in GAAM24 (2% acetate and 4% glycerol); 5 subcultures in GAAM34 (3% acetate and 4% glycerol), 5 subcultures in GAAM44 (4% acetate and 4% glycerol); GAAM54 (5% acetate and 4% glycerol); 5 subcultures in GAAM64 (6% acetate and 4% glycerol). The final culture was streaked on a YPD plate and 22 colonies were assessed for biomass growth, lipid content and fatty acid profile in shakeflask cultures. Strains were named as C3-64x, which x represents various pure isolates tested for fatty acid content and profiles.

[0092] 1 .5 Fed-batch fermentation

[0093] 2L-scale fermentation was done using the Biostat B Plus bioreactor system (Sartorius, Germany). Seed cultures were prepared by inoculating 1 mL pre-seed culture into 100 mL medium of interest in a 250 mL conical flask and cultured 28-30 °C for 40-48 hours with vigorous agitation. The pre-seed culture was made by inoculating a single colony into 5 mL YPD medium in a 50 mL Falcon tube and cultured at 28-30 °C with vigorous agitation for 18- 24 hours. Using acetate and glucose as the carbon source

[0094] For fermentation using acetate and glucose as the carbon sources, 100 mL of seed culture was inoculated aseptically into 1 L AMP3-Y6 with additional supplements (30 g / L acetic acid, 5 g / L glucose, 5.38 g / L yeast extract, 5.7 g / L KH2PO4, 2.0 g / L MgSO47H2O, 6.6 g / L KNO3, 15 mg / L FeSO4.7H20, 0.1 g / L L-Histidine, 10 mg / L L-Aspartic acid, neutralized to pH 7 with NaOH) in a 2L vessel. The pH of the culture was maintained at 7, using only 100% acetic acid. Base pump for the controller has been disabled. pO2was maintained at 30%, cascade- controlled with the stirrer (maximum 1200 rpm) and air-flow (maximum 3 L / min). At 40-48 hours after inoculation when growth rate of culture slowed down significantly, 100 mL of Acetate Feeding Medium (AFM, containing 30 g / L acetic acid, 5 g / L glucose monohydrate, 100 g / L yeast extract, 20 g / L MgSO4‘7H20, 66 g / L KNO3, 150 mg / L FeSO4*7H20, 1 g / L histidine, 0.1 g / L aspartic acid, 100 ml / L AAM vitamin stock solution) was pumped into the vessel at flow rate of 0.9 mL / min. This process was repeated every 40 to 48 hrs when acetic consumption levelling off. The levels of glucose and acetic acid were monitored by HPLC every 24 hr and maintained at 30 and 5 g / L respectively by topping up with stock solution of 80% glucose monohydrate solution and 50% acetic acid, respectively.

[0095] Using glucose as the carbon source

[0096] In glucose only fermentation, 100 mL of the seed culture was aseptically inoculated into 1 L of APM2 in a 2 L vessel. The pH of the culture was maintained at 5.5, using 3 M HCI and 25% NH4OH. The pC>2 was maintained at 30%, cascaded firstly by the stirrer (maximum 1200 rpm) and secondly by airflow (maximum 3 L / min). Continuous feeding was set at 4.17 mL / h for the first 24 hours to reach an OD6QO of 160 to 200 units. Glucose levels were monitored by HPLC and maintained at 40 to 60 g / L. The flow-rate of the continuous feeding medium was adjusted to 8.34 mL / h for the next 18-24 hours. At 40-48 hours after inoculation, OD6oo of the culture should reach at least 320 units. To maintain a high growth rate, approximately 20% of the culture volume was removed aseptically and topped up with the Replacement Medium. This “bleeding and feeding” process was repeated every 6 hours.

[0097] 1 .6 Analytical methods

[0098] Biomass and lipid content were determined as previously described [Liu, Y., et aL, 2018]. The concentration of sugars, acetate and glycerol were determined by HPLC as previously described [Liu, Y., et al., 2018], HPLC equipped with Animex HPX-87H column (Bio-Rad Laboratories, Inc., USA) and Refractive Index Detector were applied. The samples were analysed using 5 mM of sulfuric acid as a mobile phase and a flow rate of 0.7 mL / min. The column temperature was maintained at 50°C.

[0099] Fatty acid profiles were analysed using GCMS. A HP-88 fused silica capillary column (Agilent Technologies, Inc., USA) fitted in GCMS QP2010 Ultra (Shimadzu, USA) was used. Samples were run with helium as the carrier gas which was set with a pressure of 8.9 psi and flow rate of 16.0 mL / min. The GC column flow was set at 1.00 mL / min. Oven temperature started at 70°C, held for 1 minute, and increased 25°C per min to 200°C. After holding for 1 minute, the temperature was increased 5°C per minute to 230°C. After holding for 1 minute, the temperature was increased from 50°C to 250°C and held for 3 minutes. The total run time for each sample was 17.6 minutes.

[0100] Protein content was analysed by Dumas method. Elemental N of freeze-dried biomass was quantified using a Perkin Elmer Avio 500 Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES). Protein content was calculated from the nitrogen content using a conversion factor of 6.25 [Hayes, M., 2020].

[0101] Example 2

[0102] Identification of an elite acetate-utilizing yeast strain

[0103] Although R toruloides strains showed relatively poor performance in acetate utilization [Gong, Z., et al., 2015], we were intrigued if there were significant microbial strain diversity in acetate utilization. We cultured 4 Rhodotorula strains, one Sacharomyces cerevisiae and one Pichia pastoris strain in acetate assimilation medium with 3% acetic acid as the sole carbon source. R toruloides strain C3 showed a biomass titer that was close to the best acetate utilizing yeast species reported [Gong, Z., et al., 2015]. Its biomass titer was more than twice that of ATCC 10788 and 7 times that of strain ATCC 10657. Notably, all Rhodotorula strains showed much better growth in the medium than S. cerevisiae and P. pastoris. Therefore, strain C3 was selected for further studies.

[0104] Medium optimization for acetate utilization

[0105] Several medium compositions reported for yeast lipid production were modified, with acetic acid replacing glucose as the carbon source. As shown in Figure 2a, dry cell biomass was the highest when cultured in the modified APM medium followed by B2001 medium [Bhosale, P. and R.V. Gadre, 2001]. The former yielded 2.6 times as much cell biomass as the similarly substituted Y4 medium [Li, Y., Z. Zhao, and F. Bai, 2007]. The modified Yeast Nitrogen Base (YNB) medium yielded the lowest cell biomass. Therefore, the APM composition was selected for further optimization due to its potential cost advantage over others. As this medium contains 5.7 g / L MgSCh7H2O, which formed a precipitate with acetate, we were intrigued to see if this could be reduced. Cell mass production was marginally enhanced when MgSO47H2O as reduced to 0.5-2% (Fig. 5). Subsequently, the effect of medium pH was investigated by culturing C3 cells in this modified APM medium, hereafter referred to as APM3 which is composed of 6.5 g / L yeast extract, 5.7 g / L KH2PO4, 1 g / L MgSO47H2O, 0.015 g / L FeCl36H2O, 30 g / L acetic acid and pH adjusted to 7.0 using NaOH. A preliminary scanning of pH effect showed that C3 preferred neutral pH conditions (Fig. 6). This was confirmed in another set of experiments with medium pH set between 6.5 and 8.5. The results showed that pH 7.0 was the best for cell biomass production (Figure 2b). Therefore, the neutralized APM3 medium (pH 7.0, 3% acetic acid) was selected for further studies. This result is different from Y. lipolitica, which was found to grow better at alkaline pH conditions [Gao, R., et al., 2020].

[0106] The C:N ratio and nitrogen source are important factors affecting cell growth and lipid accumulation [Lopes, H.J.S., et al., 2020; Weng, L.-C., et al., 2014]. To determine the optimal nitrogen source for strain C3 growth, the optimized APM3 composition described above was partially substituted with different levels of urea, NH4CI and KNO3. As shown in Figure 2c, the highest biomass (10.0 g / L) and lipid (4.3 g / L) production were observed in medium with 6.63 g / L KNO3 in combination with 6.545 g / L yeast extract. With the same concentration of yeast extract in the medium, both the biomass and lipid production in the medium with 6.63 g / L KNO3 was more than twice as much as that in the medium with 2 g / L urea or 3.77 g / L NH4CL In particular, the biomass of strain C3 grown in medium with 6.6 g / L KNO3 in combination with 6.55 g / L yeast extract was about five times as much as that grown in medium with either 2 g / L urea or 3.77 g / L NH4CI in combination with 6.55 g / L yeast extract. Similarly, lipid production in medium with 6.63 g / L KNO3 in combination with 6.45 g / L yeast extract was more than four times as much as that grown in medium with either 2 g / L urea or 3.68 g / L NH4CI in combination with 6.55 g / L yeast extract. Biomass production was positively correlated with yeast extract input although lipid titer is only about 10% higher if yeast extract was increased by 50%, from 4.3 g / L (AMP3-Y6) to 6.5 g / L (AMP3-Y9) (Fig. 2c).

[0107] Lipid production is an energy intensive bioprocess, demanding a high level of carbon source. To see if a higher level of acetate can be tolerated in C3 cell culture, the strain was cultured in media with five different concentrations of acetic acid, from 3% to 7% (w / v). Although slightly more biomass and lipid were produced with 4% acetic acid than with 3% acetic acid in the 5-day culture, biomass was significantly higher with 3% acetate in the 3-day culture, suggesting faster cell growth in 3% acetate or below (Fig. 2d). Cell growth was severely inhibited when acetate was above 5%.

[0108] In preferred embodiments the R toruloides C3 strain is grown in AMP3-Y6 medium pH 7 (4.3 g / L yeast extract, 6.63 g / L KNO3, 5.7 g / L KH2PO4, 1 g / L MgSO47H2O, 0.015 g / L FeCI36H2O, 30 g / L acetic acid, pH 7.0).

[0109] Supplementation of co-substrate is an effective strategy to enhance acetate utilization [Chen, L., et al., 2021 ], Acetate assimilation medium (AAM) described previously was tested in some of our early experiments. In this medium, C3 strain grew poorly when acetate was added at 2% or 3% (w / v). Glycerol at 2% (w / v) significantly enhanced biomass production although it became inhibitory at higher concentrations (Fig. 7). The positive effect of glycerol on acetate utilization was confirmed in the APM2 medium composition that was supplemented with glycerol as acetate was fully depleted on day 3. It took a long time to fully consume acetate when other co-substrates were used (Fig. 8). Furthermore, we tested the effects of amino acids and trace elements in the AMP3-Y6 formulation. Overall, the supplementation had negligible effect except for L-histidine (1 g / L) and L-aspartic acid (0.1 g / L) (data not shown).

[0110] EXAMPLE 3

[0111] Directed evolution to enhance acetate and glycerol utilization

[0112] To see whether C3 strain could be enhanced for acetate utilization by laboratory-directed evolution, the strain was cultured in increasing concentration of acetate. Starting in GAAM with 1 % acetate and 2% glycerol as co-substrate, referred to as GAAM 12 medium, acetate concentration was increased stepwise after 5 cycles adaptation in any given medium. The carbon source was increased from 1% acetic acid with 2% glycerol to 6% acetic acid with 4% glycerol in a total of 25 cycles of cultures in 5 sets of media. We compared the growth of the 25thgeneration to the Wt C3 strain in different acetic acid concentrations. Significant improvement in cell optical density and acetate consumption were observed in all four media tested (Fig. 3). When glycerol was kept at 4%, acetate consumption was increased by 32.9%, 85.1 %, 25.2% and 174.3% in medium with 2%, 3%, 4%, 6% acetic acid, respectively. The cell optical densities were also increased significantly. Notably, the fastest cell growth was obtained with 2-3% acetic acid loading. We analyzed the cell growth and lipid production of 22 isolates of the 25thgeneration in a glucose based medium that we normally used for lipid production [Liu, Y., et al., 2021 ], Most strains grew slower in this glucose-based medium, with lower cell biomass and lipid content in the 5-day culture (Fig. 9). Surprisingly, most strains have 4-6 fold higher alpha-linolenic acid (ALA) content, an omega-3 fatty acid that is nutritionally valuable. ALA reached more than 15% of total fatty acids in the cells in fed-batch fermentations.

[0113] EXAMPLE 4

[0114] Metabolic engineering to enhance lipid production from acetate

[0115] Acetyl-CoA is a metabolic intermediate that is central for many biochemical reactions, including pathways for protein, carbohydrate, lipid and sterol biosynthesis. Over-expression of acetyl-CoA synthetase (ACS) enhances iso-butanol production from acetate in E. coli as it enhances acetyl-CoA production by directly coupling acetate to CoA at the cost of one ATP [Song, H.S., et aL, 2018], In S. cerevisiae, overexpression of transcription factor Haa1 enhances acetate resistance and utilization [Swinnen, S., et al., 2017], Acetate kinasephosphotransacetylase (ACK-PTA) makes the 2nd route for acetate utilization [Kiefer, D., et al., 2021], Carnitine acetyltransferase (CAT) shuttles acetyl units between cytoplasm, mitochondria and peroxisomes by reversibly converting acetyl-CoA to acetyl-carnitine Hynes Michael, J., et aL, 2011 ; Rong, L., et al., 2022], Most eukaryotic cells have multiple isoforms of this enzyme, suggesting the vital role this enzyme plays. The S. cerevisiae genome encodes three CAT genes but only CAT2 contributes to stress resistance to small organic acids and oxidative agents [Franken, J., et al., 2008]. By BLAST search of the R. toruloides transcriptomes using the S. cerevisiae CAT as the queries, four different CAT homologs and one obvious homolog for ACS1 and HAA1 were identified CAT1, SEQ ID NO: 2; CAT2, SEQ ID NO: 6; CAT3, SEQ ID NO: 8; CAT4, SEQ ID NO: 10; ACS1, SEQ ID NO: 16; HAA1, SEQ ID NO: 4).

[0116] To enhance acetate utilization in R. toruloides, the homologs for CAT, ACS1 and HAA 1 were individually over-expressed by fusing the CDS between the R GPDI promoter or the introncontaining Rg TEF1 promoter of R. graminis WP1 strain and the respective transcriptional terminator [Liu, Y., et al., 2021 ; Liu, Y., et al., 2016]. The constructs were site-specifically inserted at the CAR2 locus. Little improvement in cell growth was observed with most constructs if the resulted strains were cultured in the optimized acetate media, such as the APM3-Y6 with 3% acetic acid (Fig. 4a). Clear enhancement of cell growth was observed when acetic acid was raised to 4-5% and when the nutrient poor AAM medium was used (Fig. 4b). Overexpression of CAT1 showed the strongest enhancing effect, followed by HAA 1 while CAT3 showed the weakest effect in flask cultures. Cell biomass and lipid production were also improved by the overexpression of CAT1 and HAA1 although no additive effect was observed for the two genes (Fig. 10). Overexpression of CAT1 and HAA1 led to 7.2% and 9.1% improvement in biomass titer; and 14.5% and 5.1 % improvement in lipid content, respectively. Thus, CAT1 and HAA 1 were effective targets for enhancing acetate utilization in R. toruloides. The differences in the activity of the four CAT enzymes is not clear at present.

[0117] Similar to a previous report for Y. lipolitica, DGA1 overexpression significantly enhanced cell growth. ACS1 overexpression had weak effect on cell growth on acetate, be it targeted to cytoplasm or mitochondria. In AAM medium with 3% acetate, both Wt and ACS1- overexpressing cells showed a lag phase of growth of about 24 hours and only marginal improvement in ODeoo nm was observed in the ACSf-overexpressing cells at the end of 5 days culture. On the other hand, over-expressing the ACS1 variant with a mitochondria transit peptide of the malate dehydrogenase 2 (MDH2) (MAAATRQSSRLARSFSTSARAN; SEQ ID NO: 15) effectively relieved the lag phase. Surprisingly, the OD6oo of this strain levelled off much earlier and the final cell density ended up significantly lower than Wt (Fig. 11 ). This data suggest that ACS1 overexpression is not an effective strategy to enhance acetate utilization in R. toruloides.

[0118] Surprisingly, overexpression of the CAT genes resulted in significantly higher content of C18:2 and C18:3 (ALA) (Table 3).

[0119] Table 3: Effect of CAT overexpression on lipid composition

[0120] Note: Three different colonies of each construct were inoculated into 5 ml YPD in 50 ml Falcon tube and incubated at 28°C overnight. Cells were diluted to 0.05 OD600 in 50 ml AMP3-Y6 medium with 3% acetic acid in 250 ml conical flask and cultured for 5 days at 30°C. CAT1 , CAT2, CAT3 and CAT4 refer to C3CK strain inserted with the respective CAT gene fused with the RgTEF promoter and knocked in at the CAR2 locus. pKCL2 is the strain with control construct. p<0.05; p<0.01

[0121] The average ALA content of samples from all time-points was 2.58% of total fatty acids for pKCL2 control strain, compared with 9% in CAT1-OE strain, an increase of 249%.

[0122] An in silica analysis of subcellular targeting using the TargetP - 2.0 software program suggests that CAT1 , CAT2 and CAT3 is peroxisomal, mitrochondrial and cytoplasmic, respectively, while CAT4 would be dual-targeted to peroxisomes and mitrochondria. Thus, CAT4 is the homolog for the Candida albicans CTN2 or the Aspergillus nidulans ACL) J, which is dual targeted to peroxisomes and mitochondria due to the presence of a N-terminal MTS and a C-terminal PTS1 [Hynes Michael, J., et al., 201 1]. Our work suggests, for the first time, that overexpression of CAT, particularly the peroxisomal isozyme, is an effective strategy to balance acetyl-CoA levels in different cellular compartments, leading to enhanced acetate utilization and lipid production in R. toruloides. In addition, acetate resistance and metabolism are regulated by the conserved HAA1 transcriptional factor in R. toruloides.

[0123] To see if peroxisome targeting is important for the observed CAT activity (Fig. 4), the PTS of CAT1 was deleted. The CAT1 -AAKL marginally lost its activity in cell growth promotion in acetate medium. Both CAT and CAT1 -AAKL overexpression significantly increased cell mass and lipid content (Fig. 12). This suggests CAT1 exerts its function both in cytoplasm and peroxisome. The weak effect of PTS deletion may suggest the peroxisomal and mitochondrial CAT activities encoded by other 3 homologs are strong enough to mask the CAT1 mutation.

[0124] Our work suggests, for the first time, that overexpression of CAT, particularly the peroxisome or mitochondria targeted isozymes like CAT1 and CAT4, is an effective strategy to balance acetyl-CoA levels in different cellular compartments, leading to enhanced acetate metabolism, cell growth and lipid production in R. toruloides. In addition, acetate resistance and metabolism are regulated by the conserved HAA1 transcriptional factor in R. toruloides.

[0125] In preferred embodiments, R. toruloides is genetically engineered to overexpress CAT1 and / or HAA1 genes for enhanced acetate utilization and lipid productions compared to wildtype R. toruloides. In some embodiments, the genes are overexpressed under the influence of a promoter such as Rg TEF / or RgGPDI. More preferably, R. toruloides C3 strain is genetically engineered. Fed-batch fermentation

[0126] Studies on R. toruloides have been focusing on lipid production due to its high lipid productivity and lipid content. Like most oleaginous microorganisms, nitrogen starvation is a condition for inducing lipid biosynthesis [Ren, H.-Y., et al., 2013; Donzella, S., et al., 2019]. With the increasing interest to use SCP for food and animal feed applications [Jach, M.E., et al., 2022], we investigated the possibility of SCP production from R. toruloides C3 strain. After a series of process optimization, dry cell mass reached 98.3 g / L in 44 hours from an initial cell density of about 1 OD5oo unit. Cell mass productivity was 3.1 g / L / h in the first 20 h and slowed to 1.7 g / L / h in the next 24 h (Fig. 13a). To improve SCP productivity, semi- continuous fermentation mode [Yuan, Q., et al., 2022; Carlozzi, P. and E. Touloupakis, 2021 ] was investigated. We found nitrogen type and content were crucial for cell mass productivity. Cell biomass titre kept decreasing when the Replacement Medium contained 10.6 g / L yeast extract while increasing the yeast extract to 13.0 g / L reversed the downward trend. Inclusion of urea (at 4.3-6.9 g / L) further increased the biomass titre. However, urea was less effective in maintaining the high biomass productivity and dry cell mass titre. Dry cell mass productivity could be maintained above 6 g / L / *h when 17.2 g / L yeast extract was used in the RM (Fig. 13b). Under such conditions, dry cell mass titres were above 100 g / L and glucose consumption per gram biomass produced could be significantly reduced, from about 2.5 g / g to less than 2 g / g. The average lipid and protein content of cells ranged from 10% - 15% and 40% - 53%, respectively (Figs. 13 and 14). Fermentation results of other strains are summarized in Table 4.

[0127] Table 4: Comparison of fermentation parameters of various yeasts and carbon source

[0128] Note:

[0129] 1. The average lipid or biomass yield refer to the average product produced per gram of substrate (glucose or acetic acid) consumed. In this study, the input for glucose, which is about 14.7% of total substrate used, has not been included in the calculation.

[0130] 2. The maximal lipid or biomass yield refers to maximal value between any two sampling points (about 24 hours).

[0131] 3. Polg strain overexpressing ACC1 and DGA1.

[0132] 4. NA: not applicable

[0133] Summary

[0134] SCO and SCP produced from microorganisms are increasingly being regarded as alternative food staples for the future. Cell biomass and lipid yields (g / L) and productivities (g / L / h-1) are strongly dependent on microbial genetics, culture medium composition and environmental conditions [Reihani, S.F.S. and K. Khosravi-Darani, 2019]. Filamentous F. venenatum was reported to have a biomass yield of about 0.35 g / g using date biomass-derived sugar as the feedstock [Hosseini, S., et al., 2009]. The biomass yield of Crabtree positive S. cerevisiae is much lower (0.12 g / g) [Reihani, S.F.S. and K. Khosravi-Darani, 2019]. Crabtree negative yeasts, such as Kluyveromyces fragilis and Candida utilis, have a strong advantage for biomass and SCP production. Biomass yield of 0.74 g / g and 0.65 g / g, respectively, was reported using cheese whey and defatted rice polishing as the feedstock [Reihani, S.F.S. and K. Khosravi-Darani, 2019].

[0135] R toruloides is a Crabtree negative oleaginous yeast that can utilize hexose, pentose, glycerol and a variety of fatty acids as the carbon source [Sitepu, I.R., et al., 2014; van der Hoek, S.A., et al., 2022], Our biomass and lipid productivity data suggest that the land productivity of a microbial cell factory based on R. toruloides would be thousands more productive than land crop when glucose is used as the feedstock. Therefore, harnessing the natural high productivity of R. toruloides could contribute significantly to enhance food security.

[0136] In contrast to a previous report regarding R. toruloides acetate utilization [Gong, Z., et al., 2015], we showed that R. toruloides strain C3 was able to utilize acetate as efficiently as other microbes, such as Trichosporon cutaneum, Y. lipolitica and Crytocococcus curvatus [Gong, Z., et al., 2015; Gao, R., et al., 2020]. Acetate is one of the promising future feedstocks for SCO and SCP production owing to its natural abundance, low toxicity and technical feasibility of production using CO2 and H2. Furthermore, acetate utilization in R. toruloides was enhanced through metabolic engineering and directed evolution. Amongst the six genes tested, CAT1 was the most effective in enhancing acetate utilization, leading to significantly better biomass and lipid production. In addition, the transcriptional factor Haa1 was equally effective. Our approach is different from previous reports in Y. lipolytica, which targeted acetyl-CoA synthetase (ACS), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Overexpression of the 3 genes led to 25.7% increase in lipid content when using acetate and glycerol co-substrate in fed-batch fermentation, achieving a maximum OD6OO of 29.2 and a lipid content of 41.7% [Chen, L., et al., 2021],

[0137] R. toruloides appears to require significantly different conditions for optimal growth. The effect of pH on lipid production by R. glutinis CGMCC 2.703 (re-named as R. toruloides) has been studied. The optimal pH for cell growth is 6.6 while pH 7.0 was found optimal for lipid production. Biomass and lipid titre of 67.2 g / L and 29.3 g / L was achieved, respectively, in pH-stat fed-batch fermentation of acetate. The biomass yield, lipid titer, and lipid content was increased to 68.2 g / L, 35.8 g / L, and 52.5%, respectively, using a two-stage pH regulation culture strategy [Zhang, W., et al., 2019]. Our results with R. toruloides C3 are quite similar to R. glutinis CGMCC 2.703, preferring neutral pH while Y. lipolytica prefers slightly alkaline conditions (pH 8-9). Notably, R. toruloides C3 had superior lipid yield from acetate. Maximal lipid yield (ie, substrate conversion rate) was about 0.3 g / g although the current average lipid yield was only 0.13 (Table 4; Fig. 14).

[0138] Dry cell mass for the CATTover-expressing strain was 156.5 g / L, much better than that of R. glutinis CGMCC 2.703 and Y. lipolytica also (37 g / L in small scale fed-batch fermentation) (Gao et al., 2020). This lipid yield is lower than the 0.16 g / g reported in the Y. lipolytica strain overexpressing ACC1 and diacylglycerol acyltransferase 1 (DGA1) [Xu, J., et al., 2017], We noticed that glycerol was produced as a significant by-product, in both the CAT4 and C3-64 strains (Fig. 14G). This suggests that certain steps in the coupling of mono / di-acylglycerol with acy-CoA were blocked. Overexpression of DGA1 was not effective in enhancing growth or lipid production in acetate medium (data not shown). The limiting step for TAG synthesis remains unknown at present.

[0139] We have demonstrated that laboratory-directed evolution using acetate and glycerol as cosubstrate is able to increase acetate resistance; enhance utilization and decrease the glycerol by-product accumulation. Strain C3-64B produces high ALA content compared to Wt C3 strain and CAT1 -overexpression strain because strain C3-64B could utilize glycerol as a carbon source. Unexpectedly, this adaptation strategy was able to increase ALA content. In 2L scale fermentation, ALA level was raised from about 4% up to 17.33% of total fatty acid when acetate was used as the main carbon source. Furthermore, the fatty acid profile can be improved by culturing R. toruloides cells under increasing acetate-glycerol ratio. Notably, a-linolenic acid content could be significantly increased using this approach.

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Claims

CLAIMS1. A genetically engineered R. toruloides cell, wherein the cell has been transformed by at least one polynucleotide molecule; the at least one polynucleotide molecule comprising a carnitine acetyltransferase CAT) selected from the group consisting of CAT1, CAT2, CAT3 and CAT4; and / or transcription factor HAA 1 genes, operably linked to at least one promoter, wherein overexpression of CAT and / or HAA1 provides increased acetate utilization and / or fatty acid and / or lipid and / or biomass production compared to wildtype R. toruloides.

2. The genetically engineered R. toruloides cell of claim 1 , wherein the CAT and / or HAA1 genes are operably linked to a constitutive promoter, such as a promoter selected from the group comprising TEF1 and GPD1, to achieve a protein expression level that is at least 5 times higher, preferably 10, 100 or 100 times higher.

3. The genetically engineered R. toruloides cell of claim 1 or 2, wherein the cell is R. toruloides C3 strain.

4. The genetically engineered R. toruloides cell of any one of claims 1 to 3, wherein the CAT gene is CAT1 and encodes the amino acid sequence set forth in SEQ ID NO: 1 .

5. The isolated genetically engineered R. toruloides cell of any one of claims 1 to 4, wherein the HAA 1 gene encodes the amino acid sequence set forth in SEQ ID NO: 3.

6. The genetically engineered R. toruloides cell of any one of claims 1 to 5, wherein the CAT1 gene comprises a polynucleotide sequence having at least 80% sequence identity, due to the degeneracy of the genetic code, to the polynucleotide sequence set forth in SEQ ID NO: 2.

7. The genetically engineered R. toruloides cell of any one of claims 1 to 5, wherein the HAA1 gene comprises a polynucleotide sequence having at least 80% sequence identity, due to the degeneracy of the genetic code, to the polynucleotide sequence set forth in SEQ ID NO: 4.

8. The genetically engineered R. toruloides cell of any one of claims 2 to 7, wherein the TEF1 promoter and the GPD1 promoter are from R. graminis.

9. The genetically engineered R. toruloides cell of claim 8, wherein the promoters are from R. graminis WP1 strain.

10. An isolated directed-evolution-derived R. toruloides C3 strain having enhanced acetate utilization, decreased glycerol by-product accumulation, and stress resistance compared to wildtype R. toruloides, wherein said evolution was performed by adapting a culture of R. toruloides against a gradual increase of acetate concentrations in culture medium.

11. The isolated directed-evolution-derived R. toruloides C3 strain of claim 10, wherein the evolved C3 strain can tolerate up to 6% acetic acid.

12. The isolated directed-evolution-derived R. toruloides C3 strain of claim 10, wherein the evolved C3 strain can tolerate up to 6% acetic acid and 4% glycerol.

13. The isolated directed-evolution-derived R. toruloides C3 strain of any one of claims 10 to12, wherein the evolved C3 strain can produce higher a-linolenic acid (ALA) content compared to genetically engineered CAT1 over-expressing C3 strain.

14. The isolated directed-evolution-derived R. toruloides C3 strain of any one of claims 10 to13, wherein the evolved C3 strain is C3 64B.

15. An isolated R. toruloides 03 cell, wherein the cell has a genome contig sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, to the contig polynucleotide sequences set forth in SEQ ID NOs: 33-50.

16. A method of producing increased fatty acids, lipids and / or biomass by a R. toruloides cell, comprising culturing a plurality of R. toruloides cells of any one of claims 1 to 15 in medium under conditions for fatty acid, lipid production and / or increased biomass, wherein the medium comprises acetate as carbon source.

17. The method according to claim 16, further comprising glycerol in the medium.

18. The method according to claim 16 or 17, wherein the R. toruloides cell is an R. toruloides CAT1 over-expressing C3 cell, more preferably a C3 64B cell.

19. The method according to any one of claims 15 to 18, wherein the increased fatty acids, lipids and / or biomass is relative to a strain of identical genome background or nonevolved R. toruloides cell.

20. The method according to any one of claims 15 to 19, wherein the increased fatty acid is alpha-linolenic acid.

21. The use of an R. toruloides cell of any one of claims 1 to 14 in a method of producing enhanced fatty acid, lipid and / or biomass production compared to wildtype R. toruloides.

22. The use according to claim 21 , wherein the R. toruloides cell is an R. toruloides C3 cell, preferably a CAT1 over-expressing C3 cell, more preferably a C3 64B cell.

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