FEMS Yeast Res. 2017 Jun 1;17(4). doi: 10.1093/femsyr/fox040.
The expression of codon optimised genes is a popular genetic engineering approach for the production of industrially relevant proteins. This study investigates and compares the expression of codon optimised and codon adapted amylase variants. The Aspergillus tubingensis raw starch hydrolysing α-amylase (amyA) and glucoamylase (glaA) encoding genes were redesigned using synonymous codons and expressed in Saccharomyces cerevisiae Y294. Codon optimisation to favour S. cerevisiae codon bias resulted in a decrease in extracellular enzyme activity of 72% (30.28 nkat ml-1) and 68% (4.08 nkat ml-1) compared to the expression of the native amyA and glaA genes, respectively, after 96 h of growth. However, a lower cultivation temperature and co-expression with the PDI1 gene increased extracellular activity levels of the codon optimised α-amylase and glucoamylase, respectively. Despite the identical amino acid sequence of GlaA, GlaA_Opt and GlaA_CBI, differential scanning fluorimetry revealed changes in the glucoamylase proteins' melting temperatures (>3°C). Shifts in the fluorescence curves suggest changes in glucoamylase tertiary structure. Results indicate that synonymous codon changes resulting from codon optimisation of amyA and glaA genes does not guarantee increased recombinant protein production and that there is crucial translational information present within the coding sequence that controls protein folding and secretion.
密码子优化基因的表达是一种常用于生产工业相关蛋白质的基因工程方法。本研究调查并比较了密码子优化和密码子适应的淀粉酶变体的表达。使用同义密码子重新设计了 Aspergillus tubingensis 生淀粉水解 α-淀粉酶（amyA）和葡糖淀粉酶（glaA）编码基因，并在酿酒酵母 Y294 中表达。为了有利于酿酒酵母密码子偏好性而进行的密码子优化导致胞外酶活性分别下降了 72%（30.28 nkat ml-1）和 68%（4.08 nkat ml-1），与表达天然 amyA 和 glaA 基因相比，分别在 96 小时的生长后。然而，较低的培养温度和与 PDI1 基因的共表达分别提高了密码子优化的α-淀粉酶和葡糖淀粉酶的胞外活性水平。尽管 GlaA、GlaA_Opt 和 GlaA_CBI 的氨基酸序列相同，但差示扫描荧光法显示了葡糖淀粉酶蛋白的熔点变化（>3°C）。荧光曲线的偏移表明了葡糖淀粉酶三级结构的变化。结果表明，amyA 和 glaA 基因的密码子优化导致的同义密码子变化并不能保证增加重组蛋白的产量，并且编码序列中存在控制蛋白质折叠和分泌的关键翻译信息。
