Promoters for high-efficiency expression and proinsulin secretion in Saccharomyces cerevisiae

INTRODUCTION. The lack of systematic research comparing promoter activity in Saccharomyces cerevisiae limits approaches to improving the efficiency of recombinant human proinsulin biosynthesis. This study compared the activity of constitutive promoters under standardised technological conditions to identify the most productive regulatory elements for application in industrial strains.

AIM. This study aimed to compare the influence of TEF1, ADH2, ALD4, TDH3 (GPD), and TPI1 promoters on recombinant proinsulin expression in Saccharomyces cerevisiae and rank their effectiveness for selecting a highly productive promoter.

METERIALS AND METHODS. S. cerevisiae YS3 diploid strain obtained by haploid cells fusion was used for proinsulin expression. The vectors based on the pRS425 plasmid consisted of synthetic promoters (TPI1, TEF1, ADH2, ALD4, TDH3) and signal sequence. Plasmids were electroporated in the cell. Comparative expression analysis was performed by cultivating S. cerevisiae YS3 in flasks and bioreactors using YPD media. Proinsulin and ethanol concentration was measured using spectrophotometry, HPLC, and gas chromatography. The expression was confirmed using HPLC-MS. Statistical analysis included variance and correlation analysis.

RESULTS. Strains S. cerevisiae YS3/pF1145   (TPI1),   YS3/pF1157   (TEF1),   YS3/pF1199   (ADH2), YS3/pF1200 (ALD4), and YS3/pF1201 (TDH3) were obtained, then expressions of recombinant protein were compared. The strain YS3/pF1201 that had proinsulin expression controlled by the promoter TDH3 showed a maximum productivity of 15.51±0.57 mg/L. When fermented in laboratory bioreactors, the strain YS3/pF1201 achieved a productivity of 139.17 mg/L and a biomass of 154.5 mg/L after 72 hours of cultivation.

CONCLUSIONS. Analysis of experimental data ranked the promoters for proinsulin expression in S. cerevisiae cells by their effectiveness: TDH3≈ALD4>ADH2>TEF1>TPI1. During the fermentation, TDH3 promoter showed significantly higher productivity, which was 6.1 times more than in the classical strain with the TPI1 promoter for proinsulin expression.

1. Navarrete C, Jacobsen IH, Martínez JL, Procentese A. Cell factories for industrial production processes: Current issues and emerging solutions. Processes. 2020;8(7):768. https://doi.org/10.3390/pr8070768

2. Slathia PS, Sagrika Sharma E, Khan IA, et al. Recombinant production of therapeutic proteins. In: Singh DB, Tripathi T, eds. Protein-based terapeutics. Springer, Singapore; 2023. P. 101–29. https://doi.org/10.1007/978-981-19-8249-1_4

3. Gomes AR, Byregowda SM, Veeregowda BM, Balamurugan V. An overview of heterologous expression host systems for the production of recombinant proteins. Adv Anim Vet Sci. 2016;4(7):346–56. https://doi.org/10.14737/journal.aavs/2016/4.7.346.356

4. Celik E, Calık P. Production of recombinant proteins by yeast cells. Biotechnol Adv. 2012;30(5):1108–18. https://doi.org/10.1016/j.biotechadv.2011.09.011

5. Huang M, Wang G, Qin J, et al. Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production. Proc Natl Acad Sci USA. 2018;115(47):E11025–32. https://doi.org/10.1073/pnas.1809921115

6. Madhavan A, Arun KB, Sindhu R, et al. Customized yeast cell factories for biopharmaceuticals: from cell engineering to process scale up. Microb Cell Fact. 2021;20(1):124. https://doi.org/10.1186/s12934-021-01617-z

7. Zhao M, Ma J, Zhang L, Qi H. Engineering strategies for enhanced heterologous protein production by Saccharomyces cerevisiae. Microb Cell Fact. 2024;23:32. https://doi.org/10.1186/s12934-024-02299-z

8. Nielsen J. Production of biopharmaceutical proteins by yeast. Bioengineered. 2013;4(4):207–11. https://doi.org/10.4161/bioe.22856

9. Zepeda AB, Pessoa Jr A, Farías JG. Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast. Braz J Microbiol. 2018;49 Suppl 1:119–27. https://doi.org/10.1016/j.bjm.2018.03.010

10. Schenk J, Balazs K, Jungo C, et al. Influence of specific growth rate on specific productivity and glycosylation of a recombinant avidin produced by a Pichia pastoris Mut+ strain. Biotechnol Bioeng. 2008;99(2):368–77. https://doi.org/10.1002/bit.21565

11. Maury J, Kannan S, Jensen NB, et al. Glucose-dependent promoters for dynamic regulation of metabolic pathways. Front Bioeng Biotechnol. 2018;6:63. https://doi.org/10.3389/fbioe.2018.00063

12. Li S, Ma L, Fu W, et al. Programmable synthetic upstream activating sequence library for fine-tuning gene expression levels in Saccharomyces cerevisiae. ACS Synth Biol. 2022;11(3):1228–39. https://doi.org/10.1021/acssynbio.1c00511

13. Deng J, Wu Y, Zheng Z, et al. A synthetic promoter system for well-controlled protein expression with different carbon sources in Saccharomyces cerevisiae. Microb Cell Fact. 2021;20:202. https://doi.org/10.1186/s12934-021-01691-3

14. Liu R, Liu L, Li X, et al. Engineering yeast artificial core promoter with designated base motifs. Microb Cell Fact. 2020;19(1):38. https://doi.org/10.1186/s12934-020-01305-4

15. Tang H, Wu Y, Deng J, et al. Promoter architecture and promoter engineering in Saccharomyces cerevisiae. Metabolites. 2020;10(8):320. https://doi.org/10.3390/metabo10080320

16. He S, Zhang Z, Lu W. Natural promoters and promoter engineering strategies for metabolic regulation in Saccharomyces cerevisiae. J Ind Microbiol Biotechnol. 2023;50(1):kuac029 https://doi.org/10.1093/jimb/kuac029

17. Ottoz DS, Rudolf F. Constitutive and regulated promoters in yeast: how to design and make use of promoters in S. cerevisiae. In: Smolke C, Lee SY, Nielsen J, Stephanopoulos G, eds. Synthetic biology. Parts, devices and applications. Wiley-VCH Verlag GmbH & Co; 2018. P. 107–30. https://doi.org/10.1002/9783527688104.ch6

18. Myburgh MW, Schwerdtfeger KS, Cripwell RA, et al. Promoters and introns as key drivers for enhanced gene expression in Saccharomyces cerevisiae. In: Gadd GM, Sariaslani S, eds. Advances in applied microbiology. Academic Press; 2023. P. 1–29. https://doi.org/10.1016/bs.aambs.2023.07.002

19. Feng X, Marchisio MA. Saccharomyces cerevisiae promoter engineering before and during the synthetic biology era. Biology (Basel). 2021;10(6):504. https://doi.org/10.3390/biology10060504

20. Peng B, Williams TC, Henry M, et al. Controlling hetero­logous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities. Microb Cell Fact. 2015;14:91. https://doi.org/10.1186/s12934-015-0278-5

21. Partow S, Siewers V, Bjørn S, et al. Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast. 2010;27(11):955–64. https://doi.org/10.1002/yea.1806

22. Sun J, Shao Z, Zhao H, et al. Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae. Biotechnol Bioeng. 2012;109(8):2082–92. https://doi.org/10.1002/bit.24481

23. Scott EW, Allison HE, Baker HV. Characterization of TPI gene expression in isogeneic wild-type and gcr1-deletion mutant strains of Saccharomyces cerevisiae. Nucleic Acids Res. 1990;18(23):7099–107. https://doi.org/10.1093/nar/18.23.7099

24. Compagno C, Brambilla L, Capitanio D, et al. Alterations of the glucose metabolism in a triose phosphate isomerase-negative Saccharomyces cerevisiae mutant. Yeast. 2001;18(7):663–70. https://doi.org/10.1002/yea.715

25. Vignais ML, Huet J, Buhler JM, Sentenac A. Contacts between the factor TUF and RPG sequences. J Biol Chem. 1990;265(24):14669–74. https://doi.org/10.1016/S0021-9258(18)77354-7

26. Munshi R, Kandl K, Carr-Schmid A, et al. Overexpression of translation elongation factor 1A affects the organization and function of the actin cytoskeleton in yeast. Genetics. 2001;157(4):1425–36. https://doi.org/10.1093/genetics/157.4.1425

27. Bi X, Broach JR. UASrpg can function as a heterochromatin boundary element in yeast. Genes Dev. 1999;13(9):1089–101. https://doi.org/10.1101/gad.13.9.1089

28. Price VL, Taylor WE, Clevenger W, et al. Expression of heterologous proteins in Saccharomyces cerevisiae using the ADH2 promoter. Methods Enzymol. 1990;185:308–18. https://doi.org/10.1016/0076-6879(90)85027-l

29. Saint-Prix F, Bönquist L, Dequin S. Functional analysis of the ALD gene family of Saccharomyces cerevisiae during anaerobic growth on glucose: the NADP+-dependent Ald6p and Ald5p isoforms play a major role in acetate formation. Microbiology (Reading). 2004;150(Pt 7):2209–20. https://doi.org/10.1099/mic.0.26999-0

30. Navarro-Aviño JP, Prasad R, Miralles VJ, et al. A proposal for nomenclature of aldehyde dehydrogenases in Saccharomyces cerevisiae and characterization of the stress-inducible ALD2 and ALD3 genes. Yeast. 1999;15(10A):829–42. https://doi.org/10.1002/(sici)1097-0061(199907)15:10a<829::Aid-yea423>3.0.Co;2-9

31. Kjeldsen T, Balschmidt P, Diers I, et al. Expression of insulin in yeast: the importance of molecular adaptation for secretion and conversion. Biotechnol Genet Eng Rev. 2001;18:89–121. https://doi.org/10.1080/02648725.2001.10648010

32. Redden H, Alper HS. The development and characterization of synthetic minimal yeast promoters. Nat Commun. 2015;6:7810. https://doi.org/10.1038/ncomms8810

33. Yagi S, Yagi K, Fukuoka J, Suzuki M. The UAS of the yeast GAPDH promoter consists of multiple general functional elements including RAP1 and GRF2 binding sites. J Vet Med Sci. 1994;56(2):235–44. https://doi.org/10.1292/jvms.56.235

34. Hitzeman RA, Chen CY, Dowbenko DJ, et al. Use of hetero­logous and homologous signal sequences for secretion of heterologous proteins from yeast. Methods Enzymol. 1990;185:421–40. https://doi.org/10.1016/0076-6879(90)85037-o

35. Kaishima M, Ishii J, Matsuno T, et al. Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity. Sci Rep. 2016;6:35932. https://doi.org/10.1038/srep35932

36. Liu Z, Tyo KE, Martínez JL, et al. Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae. Biotechnol Bioeng. 2012;109(5):1259–68. https://doi.org/10.1002/bit.24409

37. Myburgh MW, Rose SH, Viljoen-Bloom M. Evaluating and engineering Saccharomyces cerevisiae promoters for increased amylase expression and bioethanol production from raw starch. FEMS Yeast Res. 2020;20(6):foaa047. https://doi.org/10.1093/femsyr/foaa047

38. Reider Apel A, d’Espaux L, Wehrs M, et al. A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic Acids Res. 2017;45(1):496–508. https://doi.org/10.1093/nar/gkw1023

39. Zhang J, Cai Y, Du G, et al. Evaluation and application of constitutive promoters for cutinase production by Saccharomyces cerevisiae. J Microbiol. 2017;55:538–44. https://doi.org/10.1007/s12275-017-6514-4

40. Kjeldsen T, Andersen AS, Hubálek F, et al. Molecular engineering of insulin for recombinant expression in yeast. Trends Biotechnol. 2024;42(4):464–78. https://doi.org/10.1016/j.tibtech.2023.09.012

41. Khasanshina ZR, Lunev IS, Filipenko AA, et al. Optimization of cultivation condition for the Saccharomyces cerevisiae strain producing proinsulin. Russian Journal of Biotechnology. 2022;38(3):35–48 (In Russ.). https://doi.org/10.56304/S0234275822030036

42. Kjeldsen T. Yeast secretory expression of insulin precursors. Appl Microbiol Biotechnol. 2000;54(3):277–86. https://doi.org/10.1007/s002530000402

43. Kazemi Seresht A, Nørgaard P, Palmqvist EA, et al. Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers. Appl Microbiol Biotechnol. 2013;97(9):3939–48. https://doi.org/10.1007/s00253-012-4263-1

44. Andersen AS, Diers I. Production of heterologous polypeptides in yeast. 2005. US patent US6861237B2. https://patents.google.com/patent/US6861237B2/en

45. Grey M, Brendel M. A ten-minute protocol for transforming Saccharomyces cerevisiae by electroporation. Curr Genet. 1992;22(4):335–6. https://doi.org/10.1007/bf00317931

46. Corbacho I, Teixidó F, Velázquez R, et al. Standard YPD, even supplemented with extra nutrients, does not always compensate growth defects of Saccharomyces cerevisiae auxotrophic strains. Antonie Van Leeuwenhoek. 2011;99(3):591–600. https://doi.org/10.1007/s10482-010-9530-5

47. Marty MT, Baldwin AJ, Marklund EG, et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal Chem. 2015;87(8):4370–6. https://doi.org/10.1021/acs.analchem.5b00140