Identification of a novel metabolic engineering target for carotenoid production in Saccharomyces cerevisiae via ethanol-induced adaptive laboratory evolution

Abstract

Carotenoids are a large family of health-beneficial compounds that have been widely used in the food and nutraceutical industries. There have been extensive studies to engineer Saccharomyces cerevisiae for the production of carotenoids, which already gained high level. However, it was difficult to discover new targets that were relevant to the accumulation of carotenoids. Herein, a new, ethanol-induced adaptive laboratory evolution was applied to boost carotenoid accumulation in a carotenoid producer BL03-D-4, subsequently, an evolved strain M3 was obtained with a 5.1-fold increase in carotenoid yield. Through whole-genome resequencing and reverse engineering, loss-of-function mutation of phosphofructokinase 1 (PFK1) was revealed as the major cause of increased carotenoid yield. Transcriptome analysis was conducted to reveal the potential mechanisms for improved yield, and strengthening of gluconeogenesis and downregulation of cell wall-related genes were observed in M3. This study provided a classic case where the appropriate selective pressure could be employed to improve carotenoid yield using adaptive evolution and elucidated the causal mutation of evolved strain.

Keywords

Saccharomyces cerevisiae / Adaptive laboratory evolution / Carotenoid / PFK1 / Reverse engineering / Cell wall

References

[1] Alper H, Moxley J, Nevoigt E, Fink GR, Stephanopoulos G. Engineering yeast transcription machinery for improved ethanol tolerance and production. Science, 2006, 314(5805): 1565-1568.

[2] Chen Y, Xiao W, Wang Y, Liu H, Li X, Yuan Y. Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering. Microb Cell Fact, 2016, 15(1): 113.

[3] Dragosits M, Mattanovich D. Adaptive laboratory evolution—principles and applications for biotechnology. Microb Cell Fact, 2013, 12: 64.

[4] Godara A, Rodriguez MAG, Weatherston JD, Peabody GL, Wu HJ, Kao KC. Beneficial mutations for carotenoid production identified from laboratory-evolved Saccharomyces cerevisiae. J Ind Microbiol Biotechnol, 2019, 46(12): 1793-1804.

[5] Guo L, Pang Z, Gao C, Chen X, Liu L. Engineering microbial cell morphology and membrane homeostasis toward industrial applications. Curr Opin Biotechnol, 2020, 66: 18-26.

[6] Hollinshead WD, Rodriguez S, Martin HG, Wang G, Baidoo EE, Sale KL, Keasling JD, Mukhopadhyay A, Tang YJ. Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Delta pfk mutants. Biotechnol Biofuels, 2016, 9: 212.

[7] Hong J, Park SH, Kim S, Kim SW, Hahn JS. Efficient production of lycopene in Saccharomyces cerevisiae by enzyme engineering and increasing membrane flexibility and NAPDH production. Appl Microbiol Biotechnol, 2019, 103(1): 211-223.

[8] Jiang G, Yang Z, Wang Y, Yao M, Chen Y, Xiao W, Yuan Y. Enhanced astaxanthin production in yeast via combined mutagenesis and evolution. Biochem Eng J, 2020, 156: 107519.

[9] Jin J, Wang Y, Yao M, Gu X, Li B, Liu H, Ding M, Xiao W, Yuan Y. Astaxanthin overproduction in yeast by strain engineering and new gene target uncovering. Biotechnol Biofuels, 2018, 11: 230.

[10] Kim HS, Kim NR, Yang J, Choi W. Identification of novel genes responsible for ethanol and/or thermotolerance by transposon mutagenesis in Saccharomyces cerevisiae. Appl Microbiol Biotechnol, 2011, 91(4): 1159-1172.

[11] Kratky Z, Biely P, Bauer S. Mechanism of 2-deoxy-D-glucose inhibition of cell-wall polysaccharide and glycoprotein biosyntheses in Saccharomyces cerevisiae. Eur J Biochem, 1975, 54(2): 459-467.

[12] Kwak S, Yun EJ, Lane S, Oh EJ, Kim KH, Jin YS. Redirection of the glycolytic flux enhances isoprenoid production in Saccharomyces cerevisiae. Biotechnol J, 2020, 15(2): e1900173.

[13] Lam FH, Ghaderi A, Fink GR, Stephanopoulos G. Biofuels. Engineering alcohol tolerance in yeast. Science, 2014, 346(6205): 71-75.

[14] Lee S, Kim P. Current status and applications of adaptive laboratory evolution in industrial microorganisms. J Microbiol Biotechnol, 2020, 30(6): 793-803.

[15] Li X-E, Wang J-J, Phornsanthia S, Yin X, Li Q. Strengthening of cell wall structure enhances stress resistance and fermentation performance in lager yeast. J Am Soc Brew Chem, 2014, 72(2): 88-94.

[16] Ma M, Liu ZL. Mechanisms of ethanol tolerance in Saccharomyces cerevisiae. Appl Microbiol Biotechnol, 2010, 87(3): 829-845.

[17] Ma T, Shi B, Ye Z, Li X, Liu M, Chen Y, Xia J, Nielsen J, Deng Z, Liu T. Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene. Metab Eng, 2019, 52: 134-142.

[18] Minard KI, McAlister-Henn L. Sources of NADPH in yeast vary with carbon source. J Biol Chem, 2005, 280(48): 39890-39896.

[19] Mo W, Wang M, Zhan R, Yu Y, He Y, Lu H. Kluyveromyces marxianus developing ethanol tolerance during adaptive evolution with significant improvements of multiple pathways. Biotechnol Biofuels, 2019, 12(1): 63.

[20] Mussagy CU, Winterburn J, Santos-Ebinuma VC, Pereira JFB. Production and extraction of carotenoids produced by microorganisms. Appl Microbiol Biotechnol, 2019, 103(3): 1095-1114.

[21] Reyes LH, Gomez JM, Kao KC. Improving carotenoids production in yeast via adaptive laboratory evolution. Metab Eng, 2014, 21: 26-33.

[22] Saini RK, Keum YS. Microbial platforms to produce commercially vital carotenoids at industrial scale: an updated review of critical issues. J Ind Microbiol Biotechnol, 2019, 46(5): 657-674.

[23] Sandberg TE, Salazar MJ, Weng LL, Palsson BO, Feist AM. The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology. Metab Eng, 2019, 56: 1-16.

[24] Shi B, Ma T, Ye Z, Li X, Huang Y, Zhou Z, Ding Y, Deng Z, Liu T. Systematic metabolic engineering of Saccharomyces cerevisiae for lycopene overproduction. J Agric Food Chem, 2019, 67(40): 11148-11157.

[25] Snoek T, Picca Nicolino M, Van den Bremt S, Mertens S, Saels V, Verplaetse A, Steensels J, Verstrepen KJ. Large-scale robot-assisted genome shuffling yields industrial Saccharomyces cerevisiae yeasts with increased ethanol tolerance. Biotechnol Biofuels, 2015, 8: 32.

[26] Su B, Li A, Deng MR, Zhu H. Transcriptome analysis reveals a promotion of carotenoid production by copper ions in recombinant Saccharomyces cerevisiae. Microorganisms, 2021

[27] Su B, Song D, Yang F, Zhu H. Engineering a growth-phase-dependent biosynthetic pathway for carotenoid production in Saccharomyces cerevisiae. J Ind Microbiol Biotechnol, 2020, 47(4–5): 383-393.

[28] Su B, Song D, Zhu H. Metabolic engineering of Saccharomyces cerevisiae for enhanced carotenoid production from xylose-glucose mixtures. Front Bioeng Biotechnol, 2020, 8: 435.

[29] Tripodi F, Nicastro R, Reghellin V, Coccetti PR. Post-translational modifications on yeast carbon metabolism: regulatory mechanisms beyond transcriptional control. Biochim Biophys Acta, 2015, 1580(4): 620-627.

[30] Voordeckers K, Kominek J, Das A, Espinosa-Cantu A, De Maeyer D, Arslan A, Van Pee M, van der Zande E, Meert W, Yang Y, Zhu B, Marchal K, DeLuna A, Van Noort V, Jelier R, Verstrepen KJ. Adaptation to high ethanol reveals complex evolutionary pathways. PLoS Genet, 2015, 11(11): e1005635.

[31] Wang S, Sun X, Yuan Q. Strategies for enhancing microbial tolerance to inhibitors for biofuel production: a review. Bioresour Technol, 2018, 258: 302-309.

[32] Wang C, Zhao S, Shao X, Park JB, Jeong SH, Park HJ, Kwak WJ, Wei G, Kim SW. Challenges and tackles in metabolic engineering for microbial production of carotenoids. Microb Cell Fact, 2019, 18(1): 55.

[33] Xie W, Ye L, Lv X, Xu H, Yu H. Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae. Metab Eng, 2015, 28: 8-18.

[34] Xu W, Yao J, Liu L, Ma X, Li W, Sun X, Wang Y. Improving squalene production by enhancing the NADPH/NADP(+) ratio, modifying the isoprenoid-feeding module and blocking the menaquinone pathway in Escherichia coli. Biotechnol Biofuels, 2019, 12: 68.

[35] Yang Y, Xia Y, Hu W, Tao L, Ni L, Yu J, Ai L. Membrane Fluidity of Saccharomyces cerevisiae from Huangjiu (Chinese Rice Wine) Is Variably Regulated by OLE1 To Offset the Disruptive Effect of Ethanol. Appl Environ Microbiol, 2019

[36] Zampar GG, Kummel A, Ewald J, Jol S, Niebel B, Picotti P, Aebersold R, Sauer U, Zamboni N, Heinemann M. Temporal system-level organization of the switch from glycolytic to gluconeogenic operation in yeast. Mol Syst Biol, 2013, 9: 651.

[37] Zhao X, Shi F, Zhan W. Overexpression of ZWF1 and POS5 improves carotenoid biosynthesis in recombinant Saccharomyces cerevisiae. Lett Appl Microbiol, 2015, 61(4): 354-360.

[38] Zhu Z, Zhang J, Ji X, Fang Z, Wu Z, Chen J, Du G. Evolutionary engineering of industrial microorganisms-strategies and applications. Appl Microbiol Biotechnol, 2018, 102(11): 4615-4627.

Funding

Key-Area Research and Development Program of Guangdong Province(2018B020206001)

Science and Technology Planning Project of Guangdong Province (CN)(2016A010105013, 2019B030316017)