Department of Microbiology, Stellenbosch University, JC Smuts Building, De Beer Street, Stellenbosch, 7600, South Africa.
FEMS Yeast Res. 2019 Mar 1;19(2). doi: 10.1093/femsyr/foy127.
Amylases are used in a variety of industries that have a specific need for alternative enzymes capable of hydrolysing raw starch. Five α-amylase and five glucoamylase-encoding genes were expressed in the Saccharomyces cerevisiae Y294 laboratory strain to select for recombinant strains that best hydrolysed raw corn starch. Gene variants of four amylases were designed using codon optimisation and different secretion signals. The significant difference in activity levels among the gene variants confirms that codon optimisation of fungal genes for expression in S. cerevisiae does not guarantee improved recombinant protein production. The codon-optimised glucoamylase variant from Talaromyces emersonii (temG_Opt) yielded 3.3-fold higher extracellular activity relative to the native temG, whereas the codon-optimised T. emersonii α-amylase (temA_Opt) yielded 1.6-fold more extracellular activity than the native temA. The effect of four terminator sequences was also investigated using temG and temG_Opt as reporter genes, with the ALY2T terminator resulting in a 14% increase in glucoamylase activity relative to the gene cassettes containing the ENO1T terminator. This is the first report of engineered S. cerevisiae strains to express T. emersonii amylase variants, and these enzymes may have potential applications in the industrial conversion of raw starch under fermentation conditions.
淀粉酶被广泛应用于各种行业，这些行业对能够水解生淀粉的替代酶有特殊需求。为了筛选出最能水解生玉米淀粉的重组菌株，我们在酿酒酵母 Y294 实验室菌株中表达了 5 种α-淀粉酶和 5 种葡萄糖淀粉酶编码基因。使用密码子优化和不同的分泌信号设计了 4 种淀粉酶的基因变体。基因变体之间活性水平的显著差异证实，为在酿酒酵母中表达而对真菌基因进行密码子优化并不能保证提高重组蛋白的产量。与天然的 temG 相比，优化后的塔宾曲霉葡萄糖淀粉酶变体（temG_Opt）的胞外活性提高了 3.3 倍，而优化后的塔宾曲霉α-淀粉酶（temA_Opt）的胞外活性比天然的 temA 提高了 1.6 倍。我们还研究了 4 种终止子序列对 temG 和 temG_Opt 作为报告基因的影响，ALY2T 终止子使葡萄糖淀粉酶的活性相对于包含 ENO1T 终止子的基因盒提高了 14%。这是首次报道工程化酿酒酵母菌株表达塔宾曲霉淀粉酶变体，这些酶可能在发酵条件下对生淀粉的工业转化具有潜在应用。
