Screening of 2A peptides for polycistronic gene expression in yeast

Tatiana M. Souza-Moreira, Clara Navarrete, Xin Chen, Cleslei F. Zanelli, Sandro R. Valentini, Maysa Furlan, Jens Nielsen, Anastasia Krivoruchko*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

A complexity of pathway expression in yeast compared to prokaryotes is the need for separate promoters and terminators for each gene expressed. Single transcript expression and separated protein production is possible via the use of 2A viral peptides, but detailed characterization to assess their suitability and applications is needed. The present work aimed to characterize multiple 2A peptide sequences to determine suitability for metabolic engineering applications in Saccharomyces cerevisiae. We screened 22 peptides placed between fluorescent protein sequences. Cleaving efficiency was calculated by western blot intensity of bands corresponding to the cleaved and uncleaved forms of the reporter. Three out of the 22 sequences showed high cleavage efficiency: 2A peptide from Equine rhinitis B virus (91%), Porcine teschovirus-1 (85%) and Operophtera brumata cypovirus-18 (83%). Furthermore, expression of the released protein was comparable to its monocistronic expression. As a proof-of-concept, the triterpene friedelin was successfully produced in the same yeast strain by expressing its synthase with the truncated form of HMG1 linked by the 2A peptide of ERBV-1, with production titers comparable to monocistronic expression (via separate promoters). These results suggest that these peptides could be suitable for expression and translation of multiple proteins in metabolic engineering applications in S. cerevisiae.
Original languageEnglish
Article numberfoy036
JournalFEMS Yeast Research
Volume18
Issue number5
Number of pages9
ISSN1567-1356
DOIs
Publication statusPublished - 2018

Keywords

  • 'Polycistronic'
  • 'Self-cleavage'
  • 'Stop-carry on'
  • ERBV-1 2A peptide
  • Multi-gene expression
  • Saccharomyces cerevisiae
  • Yeast metabolic engineering

Cite this

Souza-Moreira, T. M., Navarrete, C., Chen, X., Zanelli, C. F., Valentini, S. R., Furlan, M., ... Krivoruchko, A. (2018). Screening of 2A peptides for polycistronic gene expression in yeast. FEMS Yeast Research, 18(5), [foy036]. https://doi.org/10.1093/femsyr/foy036
Souza-Moreira, Tatiana M. ; Navarrete, Clara ; Chen, Xin ; Zanelli, Cleslei F. ; Valentini, Sandro R. ; Furlan, Maysa ; Nielsen, Jens ; Krivoruchko, Anastasia. / Screening of 2A peptides for polycistronic gene expression in yeast. In: FEMS Yeast Research. 2018 ; Vol. 18, No. 5.
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abstract = "A complexity of pathway expression in yeast compared to prokaryotes is the need for separate promoters and terminators for each gene expressed. Single transcript expression and separated protein production is possible via the use of 2A viral peptides, but detailed characterization to assess their suitability and applications is needed. The present work aimed to characterize multiple 2A peptide sequences to determine suitability for metabolic engineering applications in Saccharomyces cerevisiae. We screened 22 peptides placed between fluorescent protein sequences. Cleaving efficiency was calculated by western blot intensity of bands corresponding to the cleaved and uncleaved forms of the reporter. Three out of the 22 sequences showed high cleavage efficiency: 2A peptide from Equine rhinitis B virus (91{\%}), Porcine teschovirus-1 (85{\%}) and Operophtera brumata cypovirus-18 (83{\%}). Furthermore, expression of the released protein was comparable to its monocistronic expression. As a proof-of-concept, the triterpene friedelin was successfully produced in the same yeast strain by expressing its synthase with the truncated form of HMG1 linked by the 2A peptide of ERBV-1, with production titers comparable to monocistronic expression (via separate promoters). These results suggest that these peptides could be suitable for expression and translation of multiple proteins in metabolic engineering applications in S. cerevisiae.",
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author = "Souza-Moreira, {Tatiana M.} and Clara Navarrete and Xin Chen and Zanelli, {Cleslei F.} and Valentini, {Sandro R.} and Maysa Furlan and Jens Nielsen and Anastasia Krivoruchko",
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Souza-Moreira, TM, Navarrete, C, Chen, X, Zanelli, CF, Valentini, SR, Furlan, M, Nielsen, J & Krivoruchko, A 2018, 'Screening of 2A peptides for polycistronic gene expression in yeast', FEMS Yeast Research, vol. 18, no. 5, foy036. https://doi.org/10.1093/femsyr/foy036

Screening of 2A peptides for polycistronic gene expression in yeast. / Souza-Moreira, Tatiana M.; Navarrete, Clara; Chen, Xin; Zanelli, Cleslei F.; Valentini, Sandro R.; Furlan, Maysa; Nielsen, Jens; Krivoruchko, Anastasia.

In: FEMS Yeast Research, Vol. 18, No. 5, foy036, 2018.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Screening of 2A peptides for polycistronic gene expression in yeast

AU - Souza-Moreira, Tatiana M.

AU - Navarrete, Clara

AU - Chen, Xin

AU - Zanelli, Cleslei F.

AU - Valentini, Sandro R.

AU - Furlan, Maysa

AU - Nielsen, Jens

AU - Krivoruchko, Anastasia

PY - 2018

Y1 - 2018

N2 - A complexity of pathway expression in yeast compared to prokaryotes is the need for separate promoters and terminators for each gene expressed. Single transcript expression and separated protein production is possible via the use of 2A viral peptides, but detailed characterization to assess their suitability and applications is needed. The present work aimed to characterize multiple 2A peptide sequences to determine suitability for metabolic engineering applications in Saccharomyces cerevisiae. We screened 22 peptides placed between fluorescent protein sequences. Cleaving efficiency was calculated by western blot intensity of bands corresponding to the cleaved and uncleaved forms of the reporter. Three out of the 22 sequences showed high cleavage efficiency: 2A peptide from Equine rhinitis B virus (91%), Porcine teschovirus-1 (85%) and Operophtera brumata cypovirus-18 (83%). Furthermore, expression of the released protein was comparable to its monocistronic expression. As a proof-of-concept, the triterpene friedelin was successfully produced in the same yeast strain by expressing its synthase with the truncated form of HMG1 linked by the 2A peptide of ERBV-1, with production titers comparable to monocistronic expression (via separate promoters). These results suggest that these peptides could be suitable for expression and translation of multiple proteins in metabolic engineering applications in S. cerevisiae.

AB - A complexity of pathway expression in yeast compared to prokaryotes is the need for separate promoters and terminators for each gene expressed. Single transcript expression and separated protein production is possible via the use of 2A viral peptides, but detailed characterization to assess their suitability and applications is needed. The present work aimed to characterize multiple 2A peptide sequences to determine suitability for metabolic engineering applications in Saccharomyces cerevisiae. We screened 22 peptides placed between fluorescent protein sequences. Cleaving efficiency was calculated by western blot intensity of bands corresponding to the cleaved and uncleaved forms of the reporter. Three out of the 22 sequences showed high cleavage efficiency: 2A peptide from Equine rhinitis B virus (91%), Porcine teschovirus-1 (85%) and Operophtera brumata cypovirus-18 (83%). Furthermore, expression of the released protein was comparable to its monocistronic expression. As a proof-of-concept, the triterpene friedelin was successfully produced in the same yeast strain by expressing its synthase with the truncated form of HMG1 linked by the 2A peptide of ERBV-1, with production titers comparable to monocistronic expression (via separate promoters). These results suggest that these peptides could be suitable for expression and translation of multiple proteins in metabolic engineering applications in S. cerevisiae.

KW - 'Polycistronic'

KW - 'Self-cleavage'

KW - 'Stop-carry on'

KW - ERBV-1 2A peptide

KW - Multi-gene expression

KW - Saccharomyces cerevisiae

KW - Yeast metabolic engineering

U2 - 10.1093/femsyr/foy036

DO - 10.1093/femsyr/foy036

M3 - Journal article

VL - 18

JO - F E M S Yeast Research

JF - F E M S Yeast Research

SN - 1567-1356

IS - 5

M1 - foy036

ER -

Souza-Moreira TM, Navarrete C, Chen X, Zanelli CF, Valentini SR, Furlan M et al. Screening of 2A peptides for polycistronic gene expression in yeast. FEMS Yeast Research. 2018;18(5). foy036. https://doi.org/10.1093/femsyr/foy036