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Rescuing yeast from cell death enables overproduction of fatty acids from sole methanol

Abstract

Methanol is an ideal feedstock for biomanufacturing that can be beneficial for global carbon neutrality; however, the toxicity of methanol limits the efficiency of methanol metabolism toward biochemical production. We here show that engineering production of free fatty acids from sole methanol results in cell death with decreased cellular levels of phospholipids in the methylotrophic yeast Ogataeapolymorpha, and cell growth is restored by adaptive laboratory evolution. Whole-genome sequencing of the adapted strains reveals that inactivation of LPL1 (encoding a putative lipase) and IZH3 (encoding a membrane protein related to zinc metabolism) preserve cell survival by restoring phospholipid metabolism. Engineering the pentose phosphate pathway and gluconeogenesis enables high-level production of free fatty acid (15.9 g l−1) from sole methanol. Preventing methanol-associated toxicity underscores the link between lipid metabolism and methanol tolerance, which should contribute to enhancing methanol-based biomanufacturing.

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Fig. 1: Engineering fatty acid production from methanol resulted in cell death.
Fig. 2: Genome sequencing of evolved strains revealed two key mutations that restored cell growth of faa1Δ strain and enhanced tolerance of wild-type strain in methanol medium.
Fig. 3: Decreased phospholipids levels caused cell death via necrosis during fatty acid accumulation of faa1Δ strain in methanol.
Fig. 4: DEGs related to central metabolism and their effect on methanol utilization.
Fig. 5: Engineering O.polymorpha to improve FFA production from methanol.
Fig. 6: Fed-batch fermentation of strain HpFAM16u in bioreactor.

Data availability

The data that support the findings of this study are available within the article, Supplementary Information files and source data files linked to each figure. Source data are provided with this paper.

Code availability

No custom code was used in this paper. GC–MS and LC–MS data analysis were performed using published software, as indicated in Methods.

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Acknowledgements

The authors thank R. Chen from Naval Medical University in China for providing the method for extracting the total FFAs and the Energy Biotechnology Platform of Dalian Institute of Chemical Physics (DICP) for providing facility assistance. The authors thank AiMi Academic Services (www.aimieditor.com) for the English language editing. This study was financially supported by National Key Research and Development Program of China (2021YFC2100500 and 2021YFC2104200), the National Natural Science Foundation of China (21922812 and 21808216), DMTO research grant (DICP DMTO201701) and DICP innovation grant (DICP I202021 and DICP BioChE-X201801) from Dalian Institute of Chemical Physics, CAS.

Author information

Authors and Affiliations

Authors

Contributions

J.G. and Y.J.Z. conceived the study. J.G. and Y.L. designed and performed most of the experiments. Y.L. performed ALE experiments. W.Y. assisted with experimental performance. J.G. and Y.J.Z. wrote the manuscript.

Corresponding author

Correspondence to Yongjin J. Zhou.

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Competing interests

J.G., Y.L. and Y.J.Z. applied for a patent for protecting fatty acid production from methanol. The remaining authors declare no competing interests.

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Nature Metabolism thanks Rodrigo Ledesma-Amaro, Eun Yeol Lee and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary handling editor: Alfredo Giménez-Cassina in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Effects of extra nutrition, glucose and xylose on cell growth of faa1Δ strain in methanol medium.

a, Fermentations were conducted in complex medium (YPM) containing 20 g/L peptone, 10 g/L yeast extract and 10 g/L methanol. b-d, faa1Δ cells were cultivated in Delft minimal medium containing various ratios of glucose and methanol that were equal to the molar amount of 10 g/L methanol. e-i, Xylose promoted cell growth and methanol assimilation of faa1Δ strain due to enhanced Xu5P supply. faa1Δ cells were cultivated in minimal medium containing substrates that were equal amount of carbon atoms to 10 g/L methanol (M), including sole xylose (Xyl) and different ratios of mixed methanol and xylose of 7:3 (7 M:3X), 8:2 (8 M:2X), 9:1 (9 M:1X). In particular, the sole xylose of 3X, 2X and 1X were used as controls to illustrate the utilization of methanol for cell growth and FFA production in the presence of xylose. Cells cultivated in sole methanol (YPM) and mixed methanol and xylose, were collected to extract intracellular metabolites and Xu5P was determined by LC–MS/MS. Data are presented as mean ± s.e.m. (n = 3 biologically independent samples).

Source data

Extended Data Fig. 2 Enhancing the supply of xylulose 5-phosphate and NADPH in faa1Δ strain.

a, Scheme of the rational design for the enhancement of Xu5P and NADPH, by enhancing gluconeogenesis, the pentose phosphate pathway and IDP2. b, Cell growth of strains with engineered NADPH related genes. Data are presented as mean ± s.e.m. (2 biologically independent samples were adopted).c, Overexpressing the Xu5P supply related genes TKL1, TALs, RPE, RKIs slightly increased the final amount of cell biomass but failed to fully restore growth in methanol medium. Data are presented as mean ± s.e.m. (3 biologically independent samples were adopted). For all experiments, strains were pre-cultured in YPD for 16~18 h and then washed twice with Delft medium and subsequently transferred into Delft medium with the initial OD600 of 0.2. Strains were cultivated at 37oC, 220 rpm for biomass analysis.

Source data

Extended Data Fig. 3 Adaptive laboratory evolution (ALE) of faa1Δ strain enabled cell growth and FFA production in sole methanol.

a, Procedure of ALE. Three independent colonies of HpFA01 were cultivated in minimal medium with a gradually reduced ratio of glucose and methanol. If the OD600 achieved up to 5~6 within 48 h, the evolved strains were transferred to a new medium, until the strains were able to grow normally in medium containing 10 g/L methanol as the sole carbon source. Final evolved strains were spotted on YPD plate for further characterization. b, Procedure of ALE that was illustrated by cell growth. Final OD600 before every transfer was measured and although distinguished processes had been gone through, all three groups finally achieved both cell growth and FFA production in minimal medium containing methanol as the sole carbon source. Five, or six colonies of each group were picked up for FFA production in minimal medium with 10 g/L methanol. Strains were pre-cultured in YPD for 16~18 h and then washed twice with minimal medium, which was subsequently transferred into fermentative media with the initial OD600 of 0.2. Strains were cultivated at 37oC, 220 rpm for analysis of growth curve (c-e), fatty acid titers (f-h) and fatty acid compositions (i). Data are presented as mean ± s.e.m. (n = 3 biologically independent samples).

Source data

Extended Data Fig. 4 13C labelling analysis shows that FFA were derived from the sole methanol.

Pre-cultured strain HpFAM01 was inoculated in minimal media containing 5 g/L 13C labelled methanol and 12C methanol, respectively. After 72 h of cultivation, the FFA products were analyzed by GC-MS. Total 5 types of FFAs were mainly detected, among which the mass spectrum of C16 and C1:-1 were displayed. The distinguished molecular and fragment ion peaks demonstrate that FFAs were derived from methanol.

Extended Data Fig. 5 Proportion of unsaturated fatty acids was increased in glucose medium because of enhanced expression of fatty acid desaturases.

a, FFA composition in Delft minimal medium containing methanol (Delft-MeOH) and glucose (Delft-Glc) as the sole carbon source. Cells were cultured at 37oC, 220 rpm for 96 h to measure FFA production for calculating saturated FFAs (C16 and C18), Δ9 single unsaturated FFAs (C16:1 and C18:1) and Δ9, Δ12 polyunsaturated FFAs (C18:2 and C18:3). b, Relative expression of gene OLE1, which encodes Δ9 desaturase. c, Relative expression of gene FAD2, which encodes Δ12 desaturase. At 48 h (methanol) or 24 h (glucose), cells were harvested to perform qPCR experiments to determine the relative expression levels of gene OLE1 and FAD2. Actin was selected as the reference and the 2-∆∆CT method was used to normalize the data. Data are presented as mean ± s.e.m. (n = 2 of a, 3 of b-c biologically independent samples).

Source data

Extended Data Fig. 6 FFA overproduction from methanol resulted in formaldehyde accumulation.

Cell growth (a) and methanol consumption (b) of wild-type, faa1Δ and evolved strains were detected in YPM medium and the corresponding formaldehyde accumulation was illustrated in Fig. 1g. c-d, Enhancing formaldehyde assimilation by overexpressed gene DASs improved cell growth and FFA production of faa1Δ strain in YPM medium. e-h, Lethal dose of formaldehyde in wild-type and FFA producing strains. e, cell growth (OD600) of wild-type under different formaldehyde concentrations at 24 h and 48 h; f, PI positive cells (%) of wild-type under different formaldehyde concentrations at 24 h and 48 h; g, cell growth (OD600) of evolved strain under different formaldehyde concentrations at 24 h and 48 h; h, PI positive cells (%) of evolved strain under different formaldehyde concentrations at 24 h and 48 h. Strains were pre-cultured in YPD for 16 - 18 h and then transferred into minimal medium containing 0.0, 1.0, 5.0, 10, 30, and 50 mM formaldehyde with an initial OD600 of 0.2. Strains were cultivated at 37 oC, 220 rpm, and OD600 and PI positive cells were detected at 24 h and 48 h, respectively. Data are presented as mean ± s.e.m. (n = 3 of a-d, or 2 of e-h biologically independent samples).

Source data

Extended Data Fig. 7 Lipidomics analysis of wild-type, faa1Δ and evolved strains.

a, Relative contents of lipids per gram dry cell weight (gDCW), including phospholipids, sphingolipids, neutral lipids, and other lipids. LPC, Lysophosphatidylcholine; PC, Phosphatidylcholine; PE, Phosphatidylethanolamine; PG, Phosphatidylglycerol; PI, Phosphatidylinositol; PS, Phosphatidylserine; CL, Cardiolipin; LPE, Lysophosphatidylethanolamine; Cer, Ceramides; Hex1Cer, Simple Glc series; SPH, Sphingosine; DG, Diglyceride; TG, Triglyceride; AcCa, Acyl carnitine; AEA, N-Acylethanolamine; OAHFA, OAcyl-(gamma-hydroxy) fatty acid; Co, Coenzyme. b, Composition of phosphatidylcholine (PC) with different chain lengths and saturation. Data are presented as mean ± s.e.m. (n = 3 biologically independent samples).

Source data

Extended Data Fig. 8 Transcriptome sequencing analysis of differentially expressed genes.

a, Venn diagram displaying numbers of upregulated (red) and downregulated (green) genes among the three groups; most genes were downregulated. b, Box plot displaying the average expression levels (RPKM values) between the reference strain HpFA01 and the three groups of evolved strains. The expression of evolved strains was significantly lower than expression in the reference strain (p < 0.001). Box boundaries are the 25th and 75th percentiles, the horizontal line across the box is the median, and the whiskers indicate the minimum and maximum values. c, Differentially expressed genes (DEGs) were annotated by Gene Ontology (GO), demonstrating a similar expression pattern among three groups. d, Differentially expressed genes (DEGs) were annotated by KEGG pathways, and Group II and III were distinguished with Group I, especially on pathways related to carbohydrate metabolism and lipid metabolism (Statistics analysis was conducted by two-paired t-test, *p < 0.05, **p < 0.01). Data are presented as mean ± s.e.m. (n = 3 biologically independent samples).

Source data

Extended Data Fig. 9 Effects of gene FBP1, RPE, and TAL1 on cell growth and FFA production in faa1Δ strain in methanol medium.

a, Genotype of engineered strains. According to transcriptional analysis, most genes were downregulated except for genes such as FBP1, RPE, and TAL1. For these three genes, coupled with disrupted lpl1 and izh3, were tested in faa1Δ strain. Engineered strains was cultivated in minimal methanol medium to detect the cell growth curve (b) and FFA production (c). Genes FBP1, RPE, and TAL1 failed to restore cell growth and FFA production without the double deletion of genes LPL1 and IZH3. Data are presented as mean ± s.e.m. (n = 3 biologically independent samples).

Source data

Extended Data Fig. 10 Engineering evolved strain to improve FFA production from methanol by manipulating genes related to supply of acetyl-CoA and NADPH.

a, The final biomass (OD600) of engineered strains. b, The final FFA titer of engineered strains. Evolved strain HpFAM02 (HpFAM01, ura3Δ, leu1.1:: HpLEU2) was used as the control strain. To increase the acetyl-CoA supply, a modified β-oxidation (pex10Δ), citrate lysis (MmACL), and PDH complex mutant from Escherichia coli were introduced. To increase the supply of NADPH, an enhanced gluconeogenesis (FBP1) and oxidative stage of PPP (ZWF1) was achieved. The introduction of PDHm did not cause any significant difference in growth and FFA production. Overexpression of MmACL and FBP1-ZWF1 increased the fatty acid titers by 10% and 30%, respectively. Data are presented as mean ± s.e.m. (n = 3 biologically independent samples). c, Proportion of unsaturated FFAs increased in a ScIDP2-expressing strain. d, Intracellular NADPH/NADP+ in strains with, without ScIDP2 overexpression. e, The expression level of genes that encode fatty acid desaturase (Δ9, Δ12, Δ15) in strains with, without ScIDP2 overexpression. Data are presented as mean ± s.e.m. (n = 3 biologically independent samples). f, Electron transfer in the process of desaturation of FFAs.

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Gao, J., Li, Y., Yu, W. et al. Rescuing yeast from cell death enables overproduction of fatty acids from sole methanol. Nat Metab 4, 932–943 (2022). https://doi.org/10.1038/s42255-022-00601-0

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