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. 2018 May 21;8(1):82.
doi: 10.1186/s13568-018-0613-4.

A study on the use of strain-specific and homologous promoters for heterologous expression in industrial Saccharomyces cerevisiae strains

Daniel Pereira de Paiva  1 , Tiago Benoliel Rocha  1 , Marciano Regis Rubini  1 , André Moraes Nicola  2 , Viviane Castelo Branco Reis  1 , Fernando Araripe Gonçalves Torres  1 , Lidia Maria Pepe de Moraes  3
Affiliations

A study on the use of strain-specific and homologous promoters for heterologous expression in industrial Saccharomyces cerevisiae strains

Daniel Pereira de Paiva et al. AMB Express. .

Abstract

Polymorphism is well known in Saccharomyces cerevisiae strains used for different industrial applications, however little is known about its effects on promoter efficiency. In order to test this, five different promoters derived from an industrial and a laboratory (S288c) strain were used to drive the expression of eGFP reporter gene in both cells. The ADH1 promoter (P ADH1 ) in particular, which showed more polymorphism among the promoters analyzed, also exhibited the highest differences in intracellular fluorescence production. This was further confirmed by Northern blot analysis. The same behavior was also observed when the gene coding for secreted α-amylase from Cryptococcus flavus was placed under the control of either P ADH1 . These results underline the importance of the careful choice of the source of the promoter to be used in industrial yeast strains for heterologous expression.

Keywords: Amylase; Gene expression; Industrial yeast; Promoter; Saccharomyces cerevisiae.

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Figures

Fig. 1
Fig. 1
Comparison of promoter strength in different S. cerevisiae hosts. Promoters from industrial strain JPU (white bars) and laboratory strain S288C (gray bars) driving the expression of eGFP in JPU (a) and CEN.PK2 (b) were analyzed by flow cytometry. Only EGFP positive JPU (c) and CEN.PK2 (d) cells were analyzed for intensity of intracellular fluorescence of EGFP in both cell strains (c, d). The asterisk symbol indicates promoters with significant difference (Two-way ANOVA, p ≤ 0.05) in mean fluorescence. The error bars represent the standard error of biological triplicates
Fig. 2
Fig. 2
Transcription analysis of eGFP gene under control of PADH1 and PPGK1 from JPU and S288C in S. cerevisiae JPU. Total yeast RNA was prepared, run in a 1.5% agarose/formaldehyde gel and transferred to a nitrocellulose membrane. The membrane was probed with the eGFP PCR product (a) and with the ZWF1 PCR product as loading control (b). Lanes: 1 non-transformed cell; 2 Y2JADH; 3 Y2SADH; 4 Y2JPGK; 5 Y2SPGK. The predicted size of eGFP mRNA is approximately 0.7 kb and SWF1 mRNA is ~ 1.5 kb
Fig. 3
Fig. 3
Amylolytic activity of yeast transformants in plate assays. S. cerevisiae JPU and CEN.PK2 were transformed with the indicated plasmids and plated in MD medium supplemented with 1% starch. After 24 h (JPU) and 48 h (CEN.PK2) of growth at 30 °C, the plates were stained with iodine vapor. The different growth time between strains was due to the slower growth rate of CEN.PK2 laboratory strain
Fig. 4
Fig. 4
Kinetics of α-amylase from C. flavus production by recombinant S. cerevisiae JPU and CEN.PK2 host strains. Amylase activity in JPU (a) and CEN.PK2 (b) strains, and growth curves of JPU (c) and CEN.PK2 (d) strains. The symbols represent: non-transformed cell (circle); Y2JADH-AMY1 (square); Y2SADH-AMY1 (triangle); Y2JPGK-AMY1 (inverted triangle); Y2SPGK-AMY1 (diamond). The error bars represent the standard error of biological and experimental triplicates
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References

    1. Abreu-Cavalheiro A, Monteiro G. Solving ethanol production problems with genetically modified yeast strains. Braz J Microbiol. 2013;44(3):665–671. doi: 10.1590/S1517-83822013000300001. - DOI - PMC - PubMed
    1. Akao T, Yashiro I, Hosoyama A, Kitagaki H, Horikawa H, Watanabe D, Akada R, Ando Y, Harashima S, Inoue T, Inoue Y, Kajiwara S, Kitamoto K, Kitamoto N, Kobayashi O, Kuhara S, Masubuchi T, Mizoguchi H, Nakao Y, Nakazato A, Namise M, Oba T, Ogata T, Ohta A, Sato M, Shibasaki S, Takatsume Y, Tanimoto S, Tsuboi H, Nishimura A, Yoda K, Ishikawa T, Iwashita K, Fujita N, Shimoil H. Whole-genome sequencing of sake yeast Saccharomyces cerevisiae Kyokai no. 7. DNA Res. 2011;18(6):423–434. doi: 10.1093/dnares/dsr029. - DOI - PMC - PubMed
    1. Argueso JL, Carazzolle MF, Mieczkowski PA, Duarte FM, Netto OVC, Missawa SK, Galzerani F, Costa GGL, Vidal RO, Noronha MF, Dominska M, Andrietta MGS, Andrietta SR, Cunha AF, Gomes LH, Tavares FCA, Alcarde AR, Dietrich FS, McCusker JH, Petes TD, Pereira GAG. Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production. Genome Res. 2009;19(12):2258–2270. doi: 10.1101/gr.091777.109. - DOI - PMC - PubMed
    1. Babrzadeh F, Jalili R, Wang C, Shokralla S, Pierce S, Robinson-Mosher A, Nyren P, Shafer RW, Basso LC, de Amorim HV, de Oliveira AJ, Davis RW, Ronaghi M, Gharizadeh B, Stambuk BU. Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1. Mol Genet Genomics. 2012;287(6):485–494. doi: 10.1007/s00438-012-0695-7. - DOI - PubMed
    1. Bae JH, Sung BH, Seo JW, Kim CH, Sohn JH. A novel fusion partner for enhanced secretion of recombinant proteins in Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2016;100(24):10453–10461. doi: 10.1007/s00253-016-7722-2. - DOI - PMC - PubMed

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