Department of Microbiology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa.
FEMS Yeast Res. 2023 Jan 4;23. doi: 10.1093/femsyr/foad047.
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.
淀粉的综合生物加工（CBP）需要生产原淀粉降解酶的重组酿酒酵母菌株，并在一步中将产生的糖发酵成乙醇。在这项研究中，评估了天然酿酒酵母 COX4 和 RPS25A 启动子近端内含子，以增强淀粉酶基因（ateA、temA 或 temG_Opt）在酿酒酵母启动子（ENO1P、TEF1P、TDH3P 或 HXT7P）的控制下的表达。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生产有不同的影响：ENO1P-COX4i 和 TDH3P-RPS25Ai 是 AteA 的最佳启动子，其次是 HXT7P。后者也是 TemA 和 TemG 生产的最佳启动子，其次是 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高达 62%，与无内含子的启动子相比有显著提高。共表达α-淀粉酶和葡萄糖淀粉酶基因的菌株可从 20%（w/v）原淀粉中产生高达 56 g/L 的乙醇，与共表达 HXT7P-temG_Opt 的菌株相比，共表达 TDH3P-RPS25Ai-temG_Opt 的菌株观察到更高的碳转化率。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用于表达在更有效的用于原淀粉 CBP 的工业酵母菌株中。
