菊粉酶基因在酿酒酵母中的表达及乙醇发酵

Ethanol fermentation from Jerusalem artichoke tubers by a genetically-modified Saccharomyces cerevisiae strain capable of secreting inulinase

摘要:

以乙醇耐受力较强的酿酒酵母为受体菌，构建了能够分泌菊粉酶的基因工程菌并进行了菊芋粉的生料发酵。首先，以马克斯克鲁维酵母Kluyveromyces marxianus中的基因组DNA为模板，PCR扩增菊粉酶编码基因inu，分别使用菊粉酶自身启动子和酵母磷酸甘油激酶 (Phosphoglycerate kinase，pgk) 启动子，构建重组表达质粒HO/p-inu和HO/pgk-inu。经NotⅠ线性化后，采用电击法转化酿酒酵母工业菌株Saccharomyces cerevisiae 6525，分别得到含菊

Abstract:

Ethanol fermentation from Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae strains expressing the inulinase gene (inu) from Kluyveromyces marxianus was investigated. The inu native and pgk promoters were used to drive the expression of the inu gene, and the inulinase was expressed as an extracellular enzyme. All positive clones (confirmed by PCR) were able to express inulinase as measured by enzyme activity in the culture supernatant, among which two clones HI6/6 and HPI6/3 were selected, and their inulinase activity and ethanol fermentation performance were compared with their wild type. The inulinase activities of 86 and 23.8 U/mL were achieved, which were 4.6-fold and 1.5-fold higher than that of the wild type. Furthermore, ethanol fermentation was carried out with the recombinants and medium containing 200 g/L raw Jerusalem artichoke meal, and ethanol concentrations of 55 g/L and 52 g/L were obtained, with ethanol yields of 0.495 and 0.453, respectively, equivalent to 96.9% and 88.6% of the theoretical value.