Cripwell Rosemary A, Rose Shaunita H, Favaro Lorenzo, van Zyl Willem H
1Department of Microbiology, Stellenbosch University, Private Bag X1, Matieland, 7602 South Africa.
2Department of Agronomy Food Natural resources Animals and Environment (DAFNAE), Università di Padova, Agripolis, Viale dell'Università 16, 35020 Legnaro, Padova Italy.
Biotechnol Biofuels. 2019 Aug 20;12:201. doi: 10.1186/s13068-019-1541-5. eCollection 2019.
Consolidated bioprocessing (CBP) combines enzyme production, saccharification and fermentation into a one-step process. This strategy represents a promising alternative for economic ethanol production from starchy biomass with the use of amylolytic industrial yeast strains.
Recombinant Y294 laboratory strains simultaneously expressing an α-amylase and glucoamylase gene were screened to identify the best enzyme combination for raw starch hydrolysis. The codon optimised glucoamylase encoding gene () and the native α-amylase encoding gene () were selected for expression in two industrial yeast strains, namely Ethanol Red™ (hereafter referred to as the ER) and M2n. Two δ-integration gene cassettes were constructed to allow for the simultaneous multiple integrations of the and genes into the yeasts' genomes. During the fermentation of 200 g l raw corn starch, the amylolytic industrial strains were able to ferment raw corn starch to ethanol in a single step with high ethanol yields. After 192 h at 30 °C, the ER T12 and M2n T1 strains (containing integrated and gene cassettes) produced 89.35 and 98.13 g l ethanol, respectively, corresponding to estimated carbon conversions of 87 and 94%, respectively. The addition of a commercial granular starch enzyme cocktail in combination with the amylolytic yeast allowed for a 90% reduction in exogenous enzyme dosage, compared to the conventional simultaneous saccharification and fermentation (SSF) control experiment with the parental industrial host strains.
A novel amylolytic enzyme combination has been produced by two industrial strains. These recombinant strains represent potential drop-in CBP yeast substitutes for the existing conventional and raw starch fermentation processes.
整合生物加工（CBP）将酶生产、糖化和发酵整合为一个一步过程。该策略是利用淀粉分解工业酵母菌株从淀粉生物质经济生产乙醇的一种有前景的替代方法。
筛选了同时表达α-淀粉酶和糖化酶基因的重组Y294实验室菌株，以确定用于生淀粉水解的最佳酶组合。选择密码子优化的糖化酶编码基因（）和天然α-淀粉酶编码基因（）在两种工业酵母菌株中表达，即乙醇红™（以下简称ER）和M2n。构建了两个δ-整合基因盒，以允许和基因同时多次整合到酵母基因组中。在200 g l生玉米淀粉的发酵过程中，淀粉分解工业菌株能够一步将生玉米淀粉发酵为乙醇，乙醇产量高。在30°C下培养192小时后，ER T12和M2n T1菌株（含有整合的和基因盒）分别产生了89.35和98.13 g l乙醇，估计碳转化率分别为87%和94%。与使用亲本工业宿主菌株的传统同步糖化发酵（SSF）对照实验相比，添加商业颗粒淀粉酶混合物与淀粉分解酵母相结合可使外源酶用量减少90%。
两种工业菌株产生了一种新型淀粉分解酶组合。这些重组菌株代表了现有传统和生淀粉发酵过程中潜在的直接替代CBP酵母。
