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Engineering Bacillus subtilis for isobutanol production by heterologous Ehrlich pathway construction and the biosynthetic 2-ketoisovalerate precursor pathway overexpression

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Abstract

In the present work, Bacillus subtilis was engineered as the cell factory for isobutanol production due to its high tolerance to isobutanol. Initially, an efficient heterologous Ehrlich pathway controlled by the promoter P43 was introduced into B. subtilis for the isobutanol biosynthesis. Further, investigation of acetolactate synthase of B. subtilis, ketol-acid reductoisomerase, and dihydroxy-acid dehydratase of Corynebacterium glutamicum responsible for 2-ketoisovalerate precursor biosynthesis showed that acetolactate synthase played an important role in isobutanol biosynthesis. The overexpression of acetolactate synthase led to a 2.8-fold isobutanol production compared with the control. Apart from isobutanol, alcoholic profile analysis also confirmed the existence of 1.21 g/L ethanol, 1.06 g/L 2-phenylethanol, as well as traces of 2-methyl-1-butanol and 3-methyl-1-butanol in the fermentation broth. Under microaerobic condition, the engineered B. subtilis produced up to 2.62 g/L isobutanol in shake-flask fed-batch fermentation, which was 21.3% higher than that in batch fermentation.

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Acknowledgments

The authors appreciate the kind donation of strain B. subtilis 168 and plasmid pHP13, pDG364 and pDG1730 from Dr. Danier R. Zeigler and the Bacillus Genetic Stock Center, The Ohio State University. This research was financially supported by the National 973 Project of China (No. 2007CB714302), the Key Program of National Natural Science Foundation of China (Grant No. 20936002), and National Nature Science Foundation of China (No. 20976124 and No. 20906070).

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  1. Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China

    Shanshan Li, Jianping Wen & Xiaoqiang Jia

  2. Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, 300072, China

    Jianping Wen & Xiaoqiang Jia

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  1. Shanshan Li
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Correspondence to Jianping Wen.

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Li, S., Wen, J. & Jia, X. Engineering Bacillus subtilis for isobutanol production by heterologous Ehrlich pathway construction and the biosynthetic 2-ketoisovalerate precursor pathway overexpression. Appl Microbiol Biotechnol 91, 577–589 (2011). https://doi.org/10.1007/s00253-011-3280-9

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