Application of leucine dehydrogenase Bcd from Bacillus subtilis for l-valine synthesis in Escherichia coli under microaerobic conditions

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 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.

Metabolic Engineering of Bacillus licheniformis for Sustainable Production of Isobutanol

“…subtilis; the l -valine titer in recombinant E. coli strain was significantly improved under microaerobic conditions, while we made a conflicting result that knockdown of bcd increased l -valine production via inhibiting l -valine degradation . The contradictory results may be due to different enzymes, strains, media compositions, and fermentation conditions.…”

“…29,42 It was reported that the overexpression of bcd significantly increased L-valine production. 42,43 For example, the aminotransferase was replaced by NAD + -dependent leucine dehydrogenase BCD from B. subtilis; the L-valine titer in recombinant E. coli strain was significantly improved under microaerobic conditions, 43 while we made a conflicting result that knockdown of bcd increased L-valine production via inhibiting L-valine degradation. 29 The contradictory results may be due to different enzymes, strains, media compositions, and fermentation conditions.…”

“…Leucine dehydrogenase BCD contributes to the conversion between 2-keto acid and branched chain amino acids (BCAAs). The role of bcd on l -valine production ha been investigated. , It was reported that the overexpression of bcd significantly increased l -valine production. , For example, the aminotransferase was replaced by NAD + -dependent leucine dehydrogenase BCD from B. subtilis; the l -valine titer in recombinant E.…”

Rational metabolic engineering of <i>Corynebacterium glutamicum</i> to create a producer of L-valine

“…Replacement of enzyme specificity from NADPH to NADH to adapt the amino acid production process to microaerobic conditions has also been done in the development of E. colibased valine producer (Savrasova, Stoynova, 2019) and C. glutamicum-based leucine and L-ornithine producers (Jiang et al, 2013;Wang et al, 2019b). In all cases, this resulted in an increased yield of the target product.…”

Engineering of microbial cells for L-valine production: challenges and opportunities

“…Besides, microaerobic environments have been shown to improve the productivity of organisms. Ekaterina A. Savrasova et al used E. coli for L-valine production under microaerobic conditions, this study introduced leucine dehydrogenase Bcd in B. subtilis , which altered the NADPH-dependent metabolic pathway, resulting in a final L-valine yield of 9.1 g L −1 [ 58 ].…”