Engineering a Metabolic Pathway for Isobutanol Biosynthesis in Bacillus subtilis
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Isobutanol can be biosynthesized via α-ketoisovalerate catalyzed by heterologous keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH). In this work, isobutanol biosynthesis pathway was designed in Bacillus subtilis, a notable solvent-tolerant host. In order to do that, a plasmid pPKA expressing KDC and ADH under the control of a B. subtilis strong promoter P43 was constructed. Isobutanol was detected in the products of the recombinant B. subtilis harboring pPKA plasmid, whereas none was detected by the wild-type strain. Effects of the medium ingredients such as glucose concentration and valine addition, and operating parameters such as initial pH, inoculation volume, and medium work volume on isobutanol production were also investigated. Isobutanol production reached to the maximum of 0.607 g/L after 35-h cultivation under the conditions: glucose concentration of 3%, valine addition of 2%, initial pH of 7.0, inoculum of 1%, and work volume of 50 mL/250 mL. Though the isobutanol production by the recombinant was low, it was the first successful attempt to produce isobutanol in engineered B. subtilis, and the results showed its great potential as an isobutanol-producing cell factory.
KeywordsIsobutanol Bacillus subtilis Genetic engineering Biosynthesis
The authors appreciate the kind donation of the strain B. subtilis 168 and the plasmid pHP13 from Dr. Danier R. Zeigler and BGSC, the Ohio State University, OH, USA. The authors are also thankful to Prof. Pingsheng Ma, School of Chemical Engineering and Technology, Tianjin University, China, for the donation of the strain S. cerevisiae W303-1A. This research was financially supported by the National 973 Project of China (No. 2007CB714302), the National Natural Science Foundation of China (No. 20976124 and No. 20906070), the Innovation Foundation of Tianjin University, and the Program of Introducing Talents of Discipline to Universities (No. B06006).
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