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.

Microaerobic cultivation conditions have been shown experimentally and theoretically to improve the performance of a number of bioproduction systems. However, under these conditions, the production of l -valine by Escherichia coli is decreased mainly because of a redox cofactor imbalance and a decreased l -glutamate supply. The synthesis of one mole of l -valine from one mole of glucose generates two moles of NADH via glycolysis but consumes a total of two moles of NADPH, one in the ketol-acid reductoisomerase (KARI) reaction and the other in the regeneration of l -glutamate as an amino group donor for the branched-chain amino acid aminotransferase (BCAT) reaction. The improvement of l -valine synthesis under oxygen deprivation may be due to solving these problems. Increased l -valine synthesis under oxygen deprivation conditions was previously shown in Corynebacterium glutamicum (Hasegawa et al., 2012). In this study, we have proposed the use of NADH-dependent leucine dehydrogenase (LeuDH; EC 1.4.1.9) Bcd from B. subtilis instead of the native NADPH-dependent pathway including aminotransferase encoded by ilvE to improve l -valine production in E. coli under microaerobic conditions. We have created l -valine-producing strains on the base of the aminotransferase B-deficient strain V1 (B-7 Δ ilvBN Δ ilvIH Δ ilvGME: :P L -ilvBN N17K DA ) by introducing one chromosomal copy of the bcd gene or the ilvE gene. Evaluation of the l -valine production by the obtained strains under microaerobic and aerobic conditions revealed that leucine dehydrogenase Bcd had a higher potential for l -valine production under microaerobic conditions. The Bcd-possessing strain exhibited 2.2-fold higher l -valine accumulation (up to 9.1 g/L) and 2.0-fold higher yield (up to 35.3%) under microaerobic conditions than the IlvE - possessing strain. The obtained results could be interpreted as follows: an altering of redox cofactor balance in the l -valine biosynthesis pathway increased the production and yield by E. coli cells under microaerobic conditions. Thus, the effective synthesis of l -valine by means of “valine fermentation” was shown in E. coli . This methodology has the advantages of being an economical and environmentally friendly process.

No abstract