Factors enhancing l-valine production by the growth-limited l-isoleucine auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB ilvNM13 (pECKAilvBNC)

Abstract

Cell growth limitation is known to be an important condition that enhances L: -valine synthesis in Corynebacterium glutamicum recombinant strains with L: -isoleucine auxotrophy. To identify whether it is the limited availability of L: -isoleucine itself or the L: -isoleucine limitation-induced rel-dependent ppGpp-mediated stringent response that is essential for the enhancement of L: -valine synthesis in growth-limited C. glutamicum cells, we deleted the rel gene, thereby constructing a relaxed (rel (-) ) C. glutamicum DeltailvA DeltapanB Deltarel ilvNM13 (pECKAilvBNC) strain. Variations in enzyme activity and L: -valine synthesis in rel (+) and rel (-) strains under conditions of L: -isoleucine excess and limitation were investigated. A sharp increase in acetohydroxy acid synthase (AHAS) activity, a slight increase in acetohydroxyacid isomeroreductase (AHAIR) activity, and a dramatic increase in L: -valine synthesis were observed in both rel (+) and rel (-) cells exposed to L: -isoleucine limitation. Although the positive effect of induction of the stringent response on AHAS and AHAIR upregulation in cells was not confirmed, we found the stringent response to be beneficial for maintaining increased AHAS, dihydroxyacid dehydratase, and transaminase B activity and L: -valine synthesis in cells during the stationary growth phase.

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

“…Decreasing 2-ketobutyrate availability was suggested also as a practical strategy for efficient pyruvate. Consequently, this can lead to the improvement of L-valine production and preventing the accumulation of L-isoleucine [ 47 ]. For instance, AceE is one of the key enzymes encoding pyruvate dehydrogenase complex.…”

“…When the L-valine synthesis pathway is enhanced, the energy molecule ATP is required. Recently, Denina and colleagues demonstrated that L-valine biosynthesis in C. glutamicum was related to relA gene that have a positive pact to increase ATP-coupled and decrease ATP-uncoupled respiration [ 47 ]. It has also demonstrated that ATP generated by the phosphotransacetylase- acetate kinase pathway plays an important role in L-valine production and cell growth [ 21 ].…”

Crystal Structure and Biochemical Characterization of Ketol-Acid Reductoisomerase from Corynebacterium glutamicum

“…l -Valine is well-known as a branched-chain amino acid (BCAA) such as leucine and isoleucine and is one of the essential amino acids . As l -valine can only be produced by microorganisms and plants, vertebrates are required to ingest l -valine from various additives or from food. − l -Valine is utilized in feed, food, dietary supplements, cosmetics, pharmaceuticals, and as a precursor of antibiotics or herbicides, as well as in many other commercial products. − Among them, feed additives for livestock account for the largest portion of industrial valine production, and in this form, l -valine is mainly consumed by poultry and pigs. Although the market size of l -valine is smaller than that of other amino acids, it is noted as an emerging industry in the essential amino acid market. , The global market of valine is growing at an average annual rate of more than 5%, and this trend is expected to continue for a while.…”

Understanding the high l-valine production in Corynebacterium glutamicum VWB-1 using transcriptomics and proteomics

“…Besides the production of L-glutamate and l -lysine based on a large scale genetically modified high-performance strains 3 , 4 , respectively, C. glutamicum is also predominantly used in microorganism for the microbial synthesis of l -valine and other branched-chain amino acids (BCAA) ( l -leucine and l -isoleucine) 5 – 7 . L-valine is an essential amino acid for vertebrates which is used as a feed additive and is a component of infusion solutions and cosmetics, it is also an important precursor in the chemical synthesis of herbicides 8 . In order to improve the efficiency of the l -valine production and other products of C. glutamicum , it is important to understand the composition and regulation of the metabolic pathways leading to these industrially important products.…”

“…The genome sequence knowledge also paved the way for the foundation of emerging genome-wide analysis techniques in fields of transcriptomics and proteomics 12 – 15 . While previous studies focused on specific genes or enzymes to enhance the l -valine biosynthesis ability of C. glutamicum from the viewpoint of carbon metabolic flux enhancement 8 , 16 , 17 , branched-chain amino acid transportation 18 and cofactors supply project 19 . All those methods have played a certain role in the process of improving the yield of l -valine, however, the regulation of l -valine metabolism in C. glutamicum is a much more complex process.…”