表面展示表达植酸酶的重组酿酒酵母构建及酒精发酵

Surface display of phytase on Saccharomyces cerevisiae for efficient bioethanol production from corn starch

摘要:

以淀粉为原料的同步糖化发酵是目前乙醇生产的主要途径之一。然而原料中含有的植酸不仅影响酒精发酵效率，而且也会导致环境中难以被植物吸收的磷含量的增加，加剧环境污染。将来源于大肠杆菌的植酸酶基因与酵母编码α-凝集素C端编码序列连接并置于α-因子分泌信号肽下游，构建植酸酶表面展示表达重组载体pMGK-AG-phy并转化工业酿酒酵母，成功获得了在细胞表面锚定表达植酸酶的重组菌PHY。重组酵母的植酸酶表达水平达到6.4 U/g (菌体湿重)，其最适温度为55 ℃，最适pH 4.0，在pH 3.5–4.5范围内具有较高的活性。以玉米粉为原料的同步糖化发酵实验表明，重组酵母PHY的生长速度高于出发菌株，同时酒精产量相较于出发菌株提高了3.7%。更为重要的是发酵后酒糟中植酸磷含量与对照相比降低了91%。构建的表面展示表达植酸酶的重组工业酿酒酵母能够有效降低植酸含量，提高了酒糟的利用价值，减少磷排放，对燃料酒精的环境友好生产具有重要的借鉴意义。

Abstract:

Production of bioethanol using starch as raw material has become a very prominent technology. However, phytate in the raw material not only decreases ethanol production efficiency, but also increases phosphorus discharge. In this study, to decrease phytate content in an ethanol fermentation process, Saccharomyces cerevisiae was engineered for heterologous expression of phytase on the cell surface. The phy gene encoding phytase gene was fused with the C-terminal-half region of α-agglutinin and then inserted downstream of the secretion signal gene, to produce a yeast surface-display expression vector pMGK-AG-phy, which was then transformed into S. cerevisiae. The recombinant yeast strain, PHY, successfully displayed phytase on the surface of cells producing 6.4 U/g wet cells and its properties were further characterized. The growth rate and ethanol production of the PHY strain were faster than the parent S. cerevisiae strain in the fermentation medium by simultaneous saccharification and fermentation. Moreover, the phytate concentration decreased by 91% in dry vinasse compared to the control. In summary, we constructed recombinant S. cerevisiae strain displaying phytase on the cell surface, which could effectively reduce the content of phytate, improve the utilization value of vinasse and reduce the discharge of phosphorus. The strain reported here represents a useful novel engineering platform for developing an environment-friendly system for bioethanol production from a corn substrate.