表面展示表达果胶酯酶的重组酿酒酵母构建及乙醇发酵

Surface display of pectinesterase on Saccharomyces cerevisiae for efficient bioethanol production from sweet potato starch

摘要:

[目的] 以淀粉为原料的乙醇发酵工艺仍然是当前燃料乙醇的主要生产方式。然而,一些原料中含有的果胶物质不仅降低了乙醇产率,而且会导致醪液粘度增大,从而会进一步影响传质和传热、增加设备负担等。构建能够自主降解果胶质的重组酿酒酵母并应用于燃料乙醇生产是值得探索的领域。[方法] 论文将来源于黑曲霉的果胶酯酶基因克隆于α因子信号肽下游并通过酵母α-凝集素C-端蛋白的介导构建了在细胞表面锚定表达果胶酯酶的重组酿酒酵母PE。[结果] 重组酵母的果胶酯酶表达水平达到2.6 U/g (菌体湿重),并进一步鉴定了重组果胶酯酶性质。以甘薯粉为原料的同步糖化发酵实验中,重组酵母PE的乙醇浓度和乙醇转化率分别达到95 g/L和88.1%,与出发菌株相比提高了2.2%。更重要的是,表面展示果胶酯酶能够显著降低发酵过程中的发酵液粘度。[结论] 通过在工业酿酒酵母表面展示表达果胶酯酶不仅能够提高糖化酶等的作用效果和酿酒酵母的代谢能力,而且能够显著降低乙醇生产过程中发酵液的粘度,将对工业规模乙醇生产在降低设备负担、节约能耗方面具有一定的潜在价值。

Abstract:

[Objective] Bioethanol is a new type of green energy, and predominantly produced currently from starch such as corn and sweet potato. However, pectin contained in the substrates will increase the viscosity of ethanol fermentation broth, which will affect the mass transfer and increase the burden of equipment. In this study, we expressed pectinesterase on the Saccharomyces cerevisiae cell surface and evaluated its effect on fermentation viscosity decrease. [Methods] Pectinesterase gene was fused with the C-terminal-half region of α-agglutinin and then inserted into the downstream of the secretion signal gene, to generate a yeast surface-display expression vector pMGK-AG-PE, which was then transformed into the industrial S. cerevisiae. [Results] Recombinant yeast strain PE successfully displayed pectinesterase on the surface of cells with 2.6 U/g wet cells. The recombinant enzyme performed the maximal activity at 60 ℃, pH 5.0, and this enzyme had high activity and stability from pH 4.0 to 5.5. In the simultaneous saccharification and fermentation of sweet potato, the ethanol production of the PE strain was 95 g/L. The viscosity of fermentation broth using the PE strain was lower than that of the parent strain, 120 mPa.s compared to 145 mPa.s after 12 h of fermentation. [Conclusion] Expressing pectinesterase on yeast cells surface decreased viscosity of fermentation broth, which is beneficial to starch degradation and ethanol production.