Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China.
Appl Biochem Biotechnol. 2012 Sep;168(1):1-9. doi: 10.1007/s12010-011-9268-1. Epub 2011 May 3.
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 P(43) 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.
异丁醇可以通过α-酮异戊酸经异源酮酸脱羧酶 (KDC) 和醇脱氢酶 (ADH) 催化生物合成。在这项工作中，设计了枯草芽孢杆菌中的异丁醇生物合成途径，枯草芽孢杆菌是一种显著的耐溶剂宿主。为此，构建了一个表达 KDC 和 ADH 的质粒 pPKA，其表达受枯草芽孢杆菌强启动子 P(43)的控制。在含有 pPKA 质粒的重组枯草芽孢杆菌的产物中检测到了异丁醇，而野生型菌株则没有检测到。还研究了葡萄糖浓度和缬氨酸添加、初始 pH 值、接种量和培养基工作体积等培养基成分以及操作参数对异丁醇生产的影响。在葡萄糖浓度为 3%、缬氨酸添加量为 2%、初始 pH 值为 7.0、接种量为 1%、工作体积为 50 mL/250 mL 的条件下，经过 35 小时培养，异丁醇产量达到 0.607 g/L 的最大值。尽管重组菌的异丁醇产量较低，但这是首次在工程枯草芽孢杆菌中成功尝试生产异丁醇，结果表明其作为异丁醇生产细胞工厂具有巨大的潜力。
