产肌醇酿酒酵母基因工程菌的构建

Construction of genetically engineered Saccharomyces cerevisiae for inositol production

摘要:

【目的】过表达酿酒酵母肌醇合成关键酶基因 INO1，促进肌醇合成，构建能够分泌肌醇的基因工程菌株。【方法】构建 rDNA介导的 INO1基因多拷贝整合表达载体 pURIH，电转化酿酒酵母 Y01菌株，构建工程菌株 YI2-1和 YI2-2，荧光定量 PCR方法分析 INO1基因表达量。敲除 KanMX抗性基因，HPLC检测重组菌发酵液中肌醇含量。【结果】获得 INO1基因过表达菌株 YI2-1和 YI2-2，YI2-1的 INO1基因表达量是出发菌 Y01的 16.235倍。敲除 KanMX抗性基因的菌株命名为 YI2-1△KP，初步检测 YI2-1△KP产肌醇量为 627 mg/L。【结论】 rDNA介导的 INO1基因多拷贝整合表达载体 pURIH能够有效地过表达目的基因；过表达菌株合成的肌醇不仅能满足自身的需要，而且能够向胞外分泌，具有潜在的工业应用价值。

Abstract:

[Objective] This study aimed to construct a genetically engineered Saccharomyces cerevisiae strain for inositol production by overexpressing its INO1 gene that encodes inositol-1-phosphate synthase. [Methods] The integrated multi-copy expression vector pURIH was constructed via an rDNA-mediated method and transformed into S. cerevisiae Y01 strain. The expression levels of INO1 in genetically engineered strains were analyzed through quantitative RT-PCR. was detected through HPLC. [Results] Two genetically engineered strains, namely, YI2-1 and YI2-2, were constructed. These strains could highly express INO1. YI2-1 produced 16.235 times more inositol than Y01 did. The KanMX resistance gene was knocked out in the YI2-1ΔKP strain, which efficiently produced up to 627 mg/L inositol. [Conclusion] pURIH could be applied to overexpress homologous INO1 in S. cerevisiae. The amount of inositol produced by the engineered strain was sufficient and thus could be employed in engineering applications.