Development of conditional cell lysis mutants of Saccharomyces cerevisiae as production hosts by modulating OCH1 and CHS3 expression
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The traditional yeast Saccharomyces cerevisiae has been widely used as a host for the production of recombinant proteins and metabolites with industrial potential. However, its thick and rigid cell wall presents problems for the effective recovery of products. In this study, we modulated the expression of ScOCH1, encoding the α-1,6-mannosyltransferase responsible for outer chain biosynthesis of N-glycans, and ScCHS3, encoding the chitin synthase III required for synthesis of the majority of cell wall chitin, by exploiting the repressible ScMET3 promoter. The conditional single mutants PMET3-OCH1 and PMET3-CHS3 and the double mutant PMET3-OCH1/PMET3-CHS3 showed comparable growth to the wild-type strain under normal conditions but exhibited increased sensitivity to temperature and cell wall-disturbing agents in the presence of methionine. Such conditional growth defects were fully recovered by supplementation with 1 M sorbitol. The osmotic lysis of the conditional mutants cultivated with methionine was sufficient to release the intracellularly expressed recombinant protein, nodavirus capsid protein, with up to 60% efficiency, compared to lysis by glass bead breakage. These mutant strains also showed approximately three-fold-enhanced secretion of a recombinant extracellular glycoprotein, Saccharomycopsis fibuligera β-glucosidase, with markedly reduced hypermannosylation, particularly in the PMET3-OCH1 mutants. Furthermore, a substantial increase of extracellular glutathione production, up to four-fold, was achieved with the conditional mutant yeast cells. Together, our data support that the conditional cell wall lysis mutants constructed based on the modulation of ScOCH1 and ScCHS3 expression would likely be useful hosts for the improved recovery of proteins and metabolites with industrial application.
KeywordsSaccharomyces cerevisiae Conditional mutant α-1,6-Mannosyltransferase Chitin synthase III MET3 promoter
This work was supported by grants from the National Research Foundation of Korea (NRF), NRF-2013M3A6A8073554 (Global Frontier Program for the Intelligent Synthetic Biology), NRF-2017M3C1B5019295 (STEAM Research Project), and NRF-2018R1025077 (Advanced Research Center Program). Van-Trinh Luu is a recipient of CASSY fellowship from Chung-Ang University.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Amberg D, Burke D, Strathern J (2005) Methods in yeast genetics. In: Cold Spring Harbor laboratory course manual, vol 230. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Berghem LE, Pettersson LG (1974) The mechanism of enzymatic cellulose degradation. FEBS J 46:295–305. https://doi.org/10.1111/j.1432-1033.1974.tb03621.x Google Scholar
- Choi YR, Kim HJ, Lee JY, Kang HA, Kim H-J (2013) Chromatographically-purified capsid proteins of red-spotted grouper nervous necrosis virus expressed in Saccharomyces cerevisiae form virus-like particles. Protein Expr Purif 89:162–168. https://doi.org/10.1016/j.pep.2013.03.007 CrossRefGoogle Scholar
- Choo JH, Hong CP, Lim JY, Seo J-A, Kim Y-S, Lee DW, Park S-G, Lee GW, Carroll E, Lee Y-W (2016) Whole-genome de novo sequencing, combined with RNA-Seq analysis, reveals unique genome and physiological features of the amylolytic yeast Saccharomycopsis fibuligera and its interspecies hybrid. Biotechnol Biofuels 9:246. https://doi.org/10.1186/s13068-016-0653-4 CrossRefGoogle Scholar
- Fernández E, Toledo JR, Mansur M, Sánchez O, Gil DF, González-González Y, Lamazares E, Fernández Y, Parra F, Farnós O (2015) Secretion and assembly of calicivirus-like particles in high-cell-density yeast fermentations: strategies based on a recombinant non-specific BPTI-Kunitz-type protease inhibitor. Appl Microbiol Biotechnol 99:3875–3886. https://doi.org/10.1007/s00253-014-6171-z CrossRefGoogle Scholar
- Gmeiner C, Saadati A, Maresch D, Krasteva S, Frank M, Altmann F, Herwig C, Spadiut O (2015) Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase. Microb Cell Factories 14(1):1. https://doi.org/10.1186/s12934-014-0183-3 CrossRefGoogle Scholar
- Grabek-Lejko D, Kurylenko OO, Sibirny VA, Ubiyvovk VM, Penninckx M, Sibirny AA (2011) Alcoholic fermentation by wild-type Hansenula polymorpha and Saccharomyces cerevisiae versus recombinant strains with an elevated level of intracellular glutathione. J Ind Microbiol Biotechnol 38:1853–1859. https://doi.org/10.1007/s10295-011-0974-z CrossRefGoogle Scholar
- Gurgu L, Lafraya Á, Polaina J, Marín-Navarro J (2011) Fermentation of cellobiose to ethanol by industrial Saccharomyces strains carrying the β-glucosidase gene (BGL1) from Saccharomycopsis fibuligera. Bioresour Technol 102:5229–5236. https://doi.org/10.1016/j.biortech.2011.01.062 CrossRefGoogle Scholar
- Marini NJ, Meldrum E, Buehrer B, Hubberstey AV, Stone DE, Traynor Kaplan A, Reed SI (1996) A pathway in the yeast cell division cycle linking protein kinase C (Pkc1) to activation of Cdc28 at START. EMBO J 15:3040–3052 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC450245 CrossRefGoogle Scholar
- Rozkov A, Enfors S-O (1999) Stabilization of a proteolytically sensitive cytoplasmic recombinant protein during transition to downstream processing. Biotechnol Bioeng 62:730–738. https://doi.org/10.1002/(SICI)1097-0290(19990320)62:6%3C730::AID-BIT12%3E3.0.CO;2-Q CrossRefGoogle Scholar
- Tang H, Hou J, Shen Y, Xu L, Yang H, Fang X, Bao X (2013) High β-glucosidase secretion in Saccharomyces cerevisiae improves the efficiency of cellulase hydrolysis and ethanol production in simultaneous saccharification and fermentation. J Microbiol Biotechnol 23:1577–1585. https://doi.org/10.4014/jmb.1305.05011 CrossRefGoogle Scholar
- Tang H, Wang S, Wang J, Song M, Xu M, Zhang M, Shen Y, Hou J, Bao X (2016) N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae. Sci Rep 6. https://doi.org/10.1038/srep25654
- Thiéry R, Cozien J, Cabon J, Lamour F, Baud M, Schneemann A (2006) Induction of a protective immune response against viral nervous necrosis in the European sea bass Dicentrarchus labrax by using betanodavirus virus-like particles. J Virol 80:10201–10207. https://doi.org/10.1128/JVI.01098-06 CrossRefGoogle Scholar
- Ye J, Ly J, Watts K, Hsu A, Walker A, McLaughlin K, Berdichevsky M, Prinz B, Sean Kersey D, d'Anjou M (2011) Optimization of a glycoengineered Pichia pastoris cultivation process for commercial antibody production. Biotechnol Prog 27:1744–1750. https://doi.org/10.1002/btpr.695 CrossRefGoogle Scholar
- Yurkiv M, Kurylenko O, Vasylyshyn R, Dmytruk K, Fickers P, Sibirny A (2018) Gene of the transcriptional activator MET4 is involved in regulation of glutathione biosynthesis in the methylotrophic yeast Ogataea (Hansenula) polymorpha. FEMS Yeast Res 18:foy004. https://doi.org/10.1093/femsyr/foy004 CrossRefGoogle Scholar
- Zhang N, Gardner DC, Oliver SG, Stateva LI (1999b) Genetically controlled cell lysis in the yeast Saccharomyces cerevisiae. Biotechnol Bioeng 64:607–615. https://doi.org/10.1002/(SICI)1097-0290(19990905)64:5%3C607::AID-BIT11%3E3.0.CO;2-0 CrossRefGoogle Scholar