Engineering the leucine biosynthetic pathway for isoamyl alcohol overproduction in Saccharomyces cerevisiae

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• 作者：Jifeng Yuan ; Pranjul Mishra ; Chi Bun Ching
• 关键词：Ehrlich degradation pathway ; Isoamyl alcohol ; Isobutanol ; Artificial protein scaffold ; Enzyme co ; localization ; α ; IPM biosynthetic pathway
• 刊名：Journal of Industrial Microbiology & Biotechnology
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：44
• 期：1
• 页码：107-117
• 全文大小：
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Microbiology; Biochemistry, general; Inorganic Chemistry; Genetic Engineering; Biotechnology; Bioinformatics;
• 出版者：Springer Berlin Heidelberg
• ISSN：1476-5535
• 卷排序：44

文摘

Isoamyl alcohol can be used not only as a biofuel, but also as a precursor for various chemicals. Saccharomyces cerevisiae inherently produces a small amount of isoamyl alcohol via the leucine degradation pathway, but the yield is very low. In the current study, several strategies were devised to overproduce isoamyl alcohol in budding yeast. The engineered yeast cells with the cytosolic isoamyl alcohol biosynthetic pathway produced significantly higher amounts of isobutanol over isoamyl alcohol, suggesting that the majority of the metabolic flux was diverted to the isobutanol biosynthesis due to the broad substrate specificity of Ehrlich pathway enzymes. To channel the key intermediate 2-ketosiovalerate (KIV) towards α-IPM biosynthesis, we introduced an artificial protein scaffold to pull dihydroxyacid dehydratase and α-IPM synthase into the close proximity, and the resulting strain yielded more than twofold improvement of isoamyl alcohol. The best isoamyl alcohol producer yielded 522.76 ± 38.88 mg/L isoamyl alcohol, together with 540.30 ± 48.26 mg/L isobutanol and 82.56 ± 8.22 mg/L 2-methyl-1-butanol. To our best knowledge, our work represents the first study to bypass the native compartmentalized α-IPM biosynthesis pathway for the isoamyl alcohol overproduction in budding yeast. More importantly, artificial protein scaffold based on the feature of quaternary structure of enzymes would be useful in improving the catalytic efficiency and the product specificity of other enzymatic reactions.