Abstract
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 ΔilvBN ΔilvIH ΔilvGME::PL-ilvBNN17KDA) 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.
Highlights
L-Valine is a branched-chain amino acid (BCAA) that is widely used in dietary products, pharmaceuticals, and cosmetics, as an animal feed additive and as a precursor in the chemical synthesis of antibiotics and herbicides (Park and Lee, 2010)
We showed an increase in valine production in E. coli under microaerobic conditions due to the improvement of the redox cofactor balance (NADH synthesis/NADPH consumption) by the introduction of NAD-specific LeuDH from B. subtilis encoded by the bcd gene instead of AT encoded by ilvE
LeuDH from different species is widely used for the synthesis of a range of compounds by biotransformation, such as L-ABA from threonine (Tap et al, 2014) and L-tert-leucine from trimethylpyruvate (TMP) (Zhu et al, 2016)
Summary
L-Valine (hereinafter, valine) is a branched-chain amino acid (BCAA) that is widely used in dietary products, pharmaceuticals, and cosmetics, as an animal feed additive and as a precursor in the chemical synthesis of antibiotics and herbicides (Park and Lee, 2010). In some cases, anaerobic cultivation may increase the product yield, as E. coli is a metabolically versatile bacterium able to respond to changes in oxygen availability. This approach exploits a flexible biochemistry in which aerobic respiration is preferred to anaerobic respiration, which in turn is preferred to fermentation (Partridge et al, 2007). E. coli cells can be intentionally adapted to microaerobic conditions, e.g., by laboratory adaptive evolution (Partridge et al, 2007)
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