Ajinomoto-Genetika Research Institute, 1-St Dorozhny Proezd, b.1-1, Moscow, 117545, Russia.
J Ind Microbiol Biotechnol. 2011 Sep;38(9):1287-94. doi: 10.1007/s10295-010-0907-2. Epub 2010 Dec 15.
Microbiological synthesis of higher alcohols (1-butanol, isobutanol, 2-methyl-1-butanol, etc.) from plant biomass is critically important due to their advantages over ethanol as a motor fuel. In recent years, the use of branched-chain amino acid (BCAA) biosynthesis pathways together with heterologous Ehrlich pathway enzyme system (Hazelwood et al. in Appl Environ Microbiol 74:2259-2266, 2008) has been proposed by the Liao group as an alternative approach to aerobic production of higher alcohols as new-generation biofuels (Atsumi et al. in Nature 451:86-90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89-98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769-5775, 2008; Shen and Liao in Metab Eng 10:312-320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471-479, 2009). On the basis of these remarkable investigations, we re-engineered Escherichia coli valine-producing strain H-81, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar into isobutanol. To redirect valine biosynthesis to the production of alcohol, we also--as has been demonstrated previously (Atsumi et al. in Nature 451:86-90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89-98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769-5775, 2008; Shen and Liao in Metab Eng 10:312-320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471-479, 2009)--used enzymes of Ehrlich pathway. In particular, in our study, the following heterologous proteins were exploited: branched-chain 2-keto acid decarboxylase (BCKAD) encoded by the kdcA gene from Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by the ADH2 gene from Saccharomyces cerevisiae. We show that expression of both of these genes in the valine-producing strain H-81 results in accumulation of isobutanol instead of valine. Expression of BCKAD alone also resulted in isobutanol accumulation in the culture broth, supporting earlier obtained data (Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010) that native ADHs of E. coli are also capable of isobutanol production. Thus, in this work, isobutanol synthesis by E. coli was achieved using enzymes similar to but somewhat different from those previously used.
利用植物生物质微生物合成高级醇（1-丁醇、异丁醇、2-甲基-1-丁醇等）具有重要意义，因为它们作为燃料比乙醇具有优势。近年来，Liao 小组提出了一种替代方法，利用支链氨基酸（BCAA）生物合成途径和异源 Ehrlich 途径酶系统（Hazelwood 等人，Appl Environ Microbiol 74:2259-2266, 2008），以有氧生产作为新一代生物燃料的高级醇（Atsumi 等人，Nature 451:86-90, 2008; Atsumi 等人，Appl Microbiol Biotechnol 85:651-657, 2010; Cann 和 Liao，Appl Microbiol Biotechnol 81:89-98, 2008; Connor 和 Liao，Appl Environ Microbiol 74:5769-5775, 2008; Shen 和 Liao，Metab Eng 10:312-320, 2008; Yan 和 Liao，J Ind Microbiol Biotechnol 36:471-479, 2009）。在此基础上，我们对具有过表达 ilvGMED 操纵子的产缬氨酸大肠杆菌 H-81 进行了重新设计，以实现糖有氧转化为异丁醇。为了将缬氨酸生物合成定向转化为醇的生产，我们还--如前所述（Atsumi 等人，Nature 451:86-90, 2008; Atsumi 等人，Appl Microbiol Biotechnol 85:651-657, 2010; Cann 和 Liao，Appl Microbiol Biotechnol 81:89-98, 2008; Connor 和 Liao，Appl Environ Microbiol 74:5769-5775, 2008; Shen 和 Liao，Metab Eng 10:312-320, 2008; Yan 和 Liao，J Ind Microbiol Biotechnol 36:471-479, 2009）--使用 Ehrlich 途径的酶。特别是，在我们的研究中，利用了以下异源蛋白：来自乳球菌 lactis 的带有稀有密码子取代的分支链 2-酮酸脱羧酶（BCKAD），以及来自酿酒酵母的醇脱氢酶（ADH）。我们表明，在产缬氨酸的菌株 H-81 中表达这两个基因都会导致异丁醇的积累而不是缬氨酸。单独表达 BCKAD 也会导致发酵液中异丁醇的积累，这支持了之前获得的数据（Atsumi 等人，Appl Microbiol Biotechnol 85:651-657, 2010），即大肠杆菌的天然 ADH 也能够生产异丁醇。因此，在这项工作中，使用与之前使用的略有不同的类似酶实现了大肠杆菌的异丁醇合成。
