Evolutionary engineering of Pichia pastoris for α-farnesene biosynthesis from methanol

Abstract

α-Farnesene is a valuable sesquiterpene with wide applications in cosmetics, biofuels, and pharmaceuticals. Microbial production of α-farnesene from low-cost carbon sources represents a sustainable alternative to chemical synthesis. In this study, Pichia pastoris was engineered to efficiently produce α-farnesene using methanol as the sole carbon source. First, the native mevalonate pathway was systematically optimized by stepwise overexpression of ERG10, ERG13, a truncated HMG1, and key downstream enzymes, resulting in P. pastoris cfn3 with α-farnesene production up to 172.1 mg/L. Then, adaptive laboratory evolution was conducted to improve methanol tolerance, and the evolved strain P. pastoris evo13-4 grew more rapidly than that of P. pastoris cfn3. To further boost α-farnesene production, ARTP mutagenesis was used to generate numerous mutants for screening strains with high performance, among which P. pastoris ccg3-8 exhibited the highest production of α-farnesene up to 449.4 mg/L. Finally, in a 5-L bioreactor, α-farnesene production with the resulting strain P. pastoris ccg3-8 reached 3.28 g/L. This study presents an effective strategy for engineering P. pastoris for methanol-based biosynthesis of α-farnesene, providing a promising platform for the effective and sustainable production of isoprenoid compounds.

Keywords

Pichia pastoris / Α-farnesene / Metabolic engineering / Evolutionary engineering

References

[1] MIYAZAWAM, TAMURAN. Components of the essential oil from sprouts of Polygonum hydropiper L. (‘Benitade’)[J]. Flavour Fragr J, 2007, 22(3): 188-90.

[2] George K W, Alonso-Gutierrez J, Keasling JD et al. Isoprenoid drugs, biofuels, and chemicals–artemisinin, Farnesene, and beyond[J]. Adv Biochem Eng Biotechnol, 2015;148:355– 89. https://doi.org/10.1007/10_2014_288.

[3] Liu Y, Wang Z, Cui Z et al. Progress and perspectives for microbial production of farnesene[J]. Bioresour Technol, 2022;347:126682. https://doi.org/10.1016/j.biortech.

[4] HOLOPAINENJK. Proteinaceous elicitor from a secretion of egg-laying insect herbivore induces plant emission that attracts egg parasitoids[J]. Plant Cell Environ, 2022, 45(4): 1029-32.

[5] SMAGALA T G Christensene, Christison K M et al. Hydrocarbon renewable and synthetic diesel fuel blendstocks: composition and properties[J]. Energy & Fuels, 2013;27(1):237– 46. https://doi.org/10.1021/ef3012849.

[6] Lin J, Wang D, Chen X et al. An (E,E)-α-farnesene synthase gene of soybean has a role in defence against nematodes and is involved in synthesizing insect-induced volatiles[J]. Plant Biotechnol J, 2017;15(4):510– 9. https://doi.org/10.1111/pbi.12649.

[7] Yu Y, Lyu S, Chen D et al. Volatiles emitted at different flowering stages of jasminum sambac and expression of genes related to α-farnesene biosynthesis[J]. Molecules, 2017;22(4). https://doi.org/10.3390/molecules22040546.

[8] Wang C, Yoon S H, Jang H J et al. Metabolic engineering of Escherichia coli for α-farnesene production[J]. Metab Eng, 2011;13(6):648– 55. https://doi.org/10.1016/j.ymben.2011.08.001.

[9] Yang X, Nambou K, Wei L et al. Heterologous production of alpha-farnesene in metabolically engineered strains of Yarrowia lipolytica[J]. Bioresour Technol, 2016;216:1040– 8. https://doi.org/10.1016/j.biortech.

[10] Ye Z, Shi B, Huang Y et al. Revolution of vitamin E production by starting from microbial fermented farnesene to isophytol[J]. Innovation (Camb), 2022;3(3):100228. https://doi.org/10.1016/j.xinn.

[11] SANDOVAL CM, AYSONM, MOSSN, et al.. Use of pantothenate as a metabolic switch increases the genetic stability of Farnesene producing Saccharomyces cerevisiae[J]. Metab Eng, 2014, 25: 215-26.

[12] Yao P, You S, Qi W et al. Investigation of fermentation conditions of biodiesel by-products for high production of beta-farnesene by an engineered Escherichia coli[J]. Environ Sci Pollut Res Int, 2020;27(18):22758– 69. https://doi.org/10.1007/s11356-020-08893-z.

[13] You S, Chang H, Zhang C et al. Recycling strategy and repression elimination for lignocellulosic-based farnesene production with an engineered Escherichia coli[J]. J Agric Food Chem, 2019;67(35):9858– 67. https://doi.org/10.1021/acs.jafc.9b03907.

[14] Wang C, Yoon S H, Jang H J et al. Metabolic engineering of Escherichia coli for alpha-farnesene production[J]. Metab Eng, 2011;13(6):648– 55. https://doi.org/10.1016/j.ymben.2011.08.001.

[15] Liu H, Chen S L, Xu J Z et al. Dual regulation of cytoplasm and peroxisomes for improved alpha-farnesene production in recombinant Pichia pastoris[J]. ACS Synth Biol, 2021;10(6):1563– 73. https://doi.org/10.1021/acssynbio.1c00186.

[16] Wang J, Jiang W, Liang C et al. Overproduction of alpha-farnesene in Saccharomyces cerevisiae by farnesene synthase screening and metabolic engineering[J]. J Agric Food Chem, 2021;69(10):3103– 13. https://doi.org/10.1021/acs.jafc.1c00008.

[17] Liu Y, Jiang X, Cui Z et al. Engineering the oleaginous yeast Yarrowia lipolytica for production of alpha-farnesene[J]. Biotechnol Biofuels, 2019;12:296. https://doi.org/10.1186/s13068-019-1636-z.

[18] PATEL S K S, JEON M S, GUPTA R K, et al.. Hierarchical macroporous particles for efficient whole-cell immobilization: application in bioconversion of greenhouse gases to methanol[J]. ACS Appl Mater Interfaces, 2019, 11(21): 18968-77.

[19] Tsao G T. Annual reports on fermentation processes[J]. Elsevier, 2014.

[20] LIJ, GAOJ, YEM, et al.. Engineering yeast for high-level production of β-farnesene from sole methanol[J]. Metab Eng, 2024, 85: 194-200.

[21] Wang J, Jiang W, Liang C et al. Overproduction of α-farnesene in Saccharomyces cerevisiae by farnesene synthase screening and metabolic engineering[J]. J Agric Food Chem, 2021;69(10):3103– 13. https://doi.org/10.1021/acs.jafc.1c00008.

[22] Liu Y, Jiang X, Cui Z et al. Engineering the oleaginous yeast Yarrowia lipolytica for production of α-farnesene[J]. Biotechnology for Biofuels, 2019;12(1):296. https://doi.org/10.1186/s13068-019-1636-z.

[23] LVJ, WANGY, ZHANGC, et al.. Highly efficient production of fames and β-farnesene from a two-stage biotransformation of waste cooking oils[J]. Energy Conv Manag, 2019, 199112001.

[24] GAOL, ZHANGK, SHENY, et al.. Engineering a versatile yeast platform for sesquiterpene production from glucose or methanol[J]. Biotechnol J, 2024, 1982400261.

[25] Lu S, Deng H, Zhou C et al. Enhancement of β-caryophyllene biosynthesis in Saccharomyces cerevisiae via synergistic evolution of β-caryophyllene synthase and engineering the chassis[J]. ACS Synthetic Biology, 2023;12(6):1696– 707. https://doi.org/10.1021/acssynbio.3c00024.

[26] Whitaker B D, Solomos T, Harrison D J. Quantification of α-farnesene and its conjugated trienol oxidation products from apple peel by C¹⁸-HPLC with UV detection[J]. J Agric Food Chem, 1997;45(3):760– 5. https://doi.org/10.1021/jf960780o.

[27] MAY, YANGH, CHENX, et al.. Significantly improving the yield of Recombinant proteins in Bacillus subtilis by a novel powerful mutagenesis tool (ARTP): alkaline α-amylase as a case study[J]. Protein Exp Purif, 2015, 114: 82-8.

[28] Tippmann S, Nielsen J, Khoomrung S. Improved quantification of farnesene during microbial production from Saccharomyces cerevisiae in two-liquid-phase fermentations[J]. Talanta, 2016;146:100– 6. https://doi.org/10.1016/j.talanta.

[29] Wang S, Wang Y, Yuan Q et al. Development of high methanol-tolerance Pichia pastoris based on iterative adaptive laboratory evolution[J]. Green Chemistry, 2023;25(21):8845– 57. https://doi.org/10.1039/D3GC02874G.

[30] Gao J, Li Y, Yu W et al. Rescuing yeast from cell death enables overproduction of fatty acids from sole methanol[J]. Nature Metabolism, 2022;4(7):932– 43. https://doi.org/10.1038/s42255-022-00601-0.

[31] WAKAYAMAK, YAMAGUCHIS, TAKEUCHIA, et al.. Regulation of intracellular formaldehyde toxicity during methanol metabolism of the Methylotrophic yeast Pichia methanolica[J]. J Biosci Bioeng, 2016, 122(5): 545-9.

[32] BERRIOSJ, THERON C WSTEELSS, et al.. Role of dissimilative pathway of Komagataella phaffii (Pichia pastoris): formaldehyde toxicity and energy metabolism[J]. Microorganisms, 2022, 1071466

[33] Xiao Y, Tan X et al. He Q,. Systematic metabolic engineering of Zymomonas mobilis for β-farnesene production[J]. Frontiers in Bioengineering and Biotechnology, 2024;12:01-13. https://doi.org/10.3389/fbioe.2024.1392556.

RIGHTS & PERMISSIONS

Jiangnan University