Promoter-proximal introns impact recombinant amylase expression in Saccharomyces cerevisiae.

IF 2.4 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

FEMS yeast research Pub Date : 2023-01-04 DOI:10.1093/femsyr/foad047

Kirstie S Schwerdtfeger, Marthinus W Myburgh, Willem H van Zyl, Marinda Viljoen-Bloom

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Abstract

Consolidated bioprocessing (CBP) of starch requires recombinant Saccharomyces cerevisiae strains that produce raw starch-degrading enzymes and ferment the resultant sugars to ethanol in a single step. In this study, the native S. cerevisiae COX4 and RPS25A promoter-proximal introns were evaluated for enhanced expression of amylase genes (ateA, temA or temG_Opt) under the control of an S. cerevisiae promoter (ENO1P, TEF1P, TDH3P, or HXT7P). The results showed that different promoters and promoter-intron combinations differentially affected recombinant amylase production: ENO1P-COX4i and TDH3P-RPS25Ai were the best promoters for AteA, followed closely by HXT7P. The latter was also the best promoter for TemA and TemG production, followed closely by TDH3P-RPS25Ai for both these enzymes. Introducing promoter-proximal introns increased amylase activity up to 62% in Y294[ENO-COX-AteA] and Y294[TDH3-RPS-TemA], a significant improvement relative to the intron-less promoters. Strains co-expressing both an α-amylase and glucoamylase genes yielded up to 56 g/L ethanol from 20% w/v raw starch, with a higher carbon conversion observed with strains co-expressing TDH3P-RPS25Ai-temG_Opt than HXT7P-temG_Opt. The study showed that promoter-proximal introns can enhance amylase activity in S. cerevisiae and suggest that these alternative cassettes may also be considered for expression in more efficient ethanol-producing industrial yeast strains for raw starch CBP.

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启动子近端内含子影响酿酒酵母中重组淀粉酶的表达。

淀粉的联合生物加工（CBP）需要重组酿酒酵母菌株，该菌株能产生原淀粉降解酶，并在一步中将所得糖发酵成乙醇。在本研究中，评估了天然酿酒酵母COX4和RPS25A启动子近端内含子在酿酒酵母启动子（ENO1P、TEF1P、TDH3P或HXT7P）的控制下淀粉酶基因（ateA、temA或temG_Opt）的增强表达。结果表明，不同的启动子和启动子-内含子组合对重组淀粉酶的产生有不同的影响：ENO1P-COX4i和TDH3P-RPS25Ai是AteA的最佳启动子，其次是HXT7P。后者也是产生TemA和TemG的最佳启动子，其次是这两种酶的TDH3P-RPS25Ai。在Y294[ENO-COX-AteA]和Y294[TDH3-RPS-TemA]中引入启动子近端内含子可将淀粉酶活性提高至62%，这与无内含子的启动子相比有显著改善。共表达α-淀粉酶和葡糖淀粉酶基因的菌株从20%w/v的生淀粉中产生高达56g/L的乙醇，在共表达TDH3P-RPS25Ai-temG_Opt的菌株中观察到的碳转化率高于HXT7P-temG_Opt。研究表明，启动子近端内含子可以增强酿酒酵母中的淀粉酶活性，并表明这些替代盒也可以考虑在更有效的乙醇生产工业酵母菌株中表达生淀粉CBP。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

FEMS yeast research 生物-生物工程与应用微生物

CiteScore

5.70

自引率

6.20%

发文量

审稿时长

1 months

期刊介绍： FEMS Yeast Research offers efficient publication of high-quality original Research Articles, Mini-reviews, Letters to the Editor, Perspectives and Commentaries that express current opinions. The journal will select for publication only those manuscripts deemed to be of major relevance to the field and generally will not consider articles that are largely descriptive without insights on underlying mechanism or biology. Submissions on any yeast species are welcome provided they report results within the scope outlined below and are of significance to the yeast field.