White crystalline powder
Water; Aqueous acid
213.6±23.0 °C at 760 mmHg
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L-Valine is one of the three branched-chain amino acids (valine, leucine, and isoleucine) essential for animal health and important in metabolism; therefore, it is widely added in the products of food, medicine, and feed. L-Valine is predominantly produced through microbial fermentation, and the production efficiency largely depends on the quality of microorganisms. In recent years, continuing efforts have been made in revealing the mechanisms and regulation of L-valine biosynthesis in Corynebacterium glutamicum, the most utilitarian bacterium for amino acid production. Metabolic engineering based on the metabolic biosynthesis and regulation of L-valine provides an effective alternative to the traditional breeding for strain development. Industrially competitive L-valine-producing C. glutamicum strains have been constructed by genetically defined metabolic engineering. This article reviews the global metabolic and regulatory networks responsible for L-valine biosynthesis, the molecular mechanisms of regulation, and the strategies employed in C. glutamicum strain engineering.
Branched-chain amino acids; Corynebacterium glutamicum; Global metabolic and regulatory networks; L-Valine; L-Valine biosynthesis; Metabolic engineering; Metabolic regulation; Microbial fermentation; Strain development; Strain engineering
Production of L-valine from metabolically engineered Corynebacterium glutamicum.
Wang X1,2, Zhang H3, Quinn PJ4.
Evolutionary approaches are often undirected and mutagen-based yielding numerous mutations, which need elaborate screenings to identify relevant targets. We here apply Metabolic engineering to Guide Evolution (MGE), an evolutionary approach evolving and identifying new targets to improve microbial producer strains. MGE is based on the idea to impair the cell’s metabolism by metabolic engineering, thereby generating guided evolutionary pressure. It consists of three distinct phases: (i) metabolic engineering to create the evolutionary pressure on the applied strain followed by (ii) a cultivation phase with growth as straightforward screening indicator for the evolutionary event, and (iii) comparative whole genome sequencing (WGS), to identify mutations in the evolved strains, which are eventually re-engineered for verification. Applying MGE, we evolved the PEP and pyruvate carboxylase-deficient strain C. glutamicum Δppc Δpyc to grow on glucose as substrate with rates up to 0.31 ± 0.02 h-1 which corresponds to 80% of the growth rate of the wildtype strain. The intersection of the mutations identified by WGS revealed isocitrate dehydrogenase (ICD) as consistent target in three independently evolved mutants. Upon re-engineering in C. glutamicum Δppc Δpyc, the identified mutations led to diminished ICD activities and activated the glyoxylate shunt replenishing oxaloacetate required for growth. Intracellular relative quantitative metabolome analysis showed that the pools of citrate, isocitrate, cis-aconitate, and L-valine were significantly higher compared to the WT control. As an alternative to existing L-valine producer strains based on inactivated or attenuated pyruvate dehydrogenase complex, we finally engineered the PEP and pyruvate carboxylase-deficient C. glutamicum strains with identified ICD mutations for L-valine production by overexpression of the L-valine biosynthesis genes. Among them, C. glutamicum Δppc Δpyc ICDG407S (pJC4ilvBNCE) produced up to 8.9 ± 0.4 g L-valine L-1, with a product yield of 0.22 ± 0.01 g L-valine per g glucose.
Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
Corynebacterium glutamicum; Directed evolution; Glyoxylate shunt; Isocitrate dehydrogenase; L-valine production; Metabolic engineering
Metabolic engineering to guide evolution - Creating a novel mode for L-valine production with Corynebacterium glutamicum.
Schwentner A1, Feith A1, Munch E1, Busche T2, Ruckert C3, Kalinowski J3, Takors R1, Blombach B4.
In this study, an L-valine-producing strain was developed from Corynebacterium glutamicum ATCC13869 through deletion of the three genes aceE, alaT and ilvA combined with the overexpression of six genes ilvB, ilvN, ilvC, lrp1, brnF and brnE. Overexpression of lrp1 alone increased L-valine production by 16-fold. Deletion of the aceE, alaT and ilvA increased L-valine production by 44-fold. Overexpression of the six genes ilvB, ilvN, ilvC, lrp1, brnE and brnF in the triple deletion mutant WCC003 further increased L-valine production. The strain WCC003/pJYW-4-ilvBNC1-lrp1-brnFE produced 243mM L-valine in flask cultivation and 437mM (51g/L) L-valine in fed-batch fermentation and lacked detectable amino-acid byproduct such as l-alanine and l-isoleucine that are usually found in the fermentation of L-valine-producing C. glutamicum.
Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
Corynebacterium glutamicum; Ilva; Lrp; Metabolic engineering; alat; l-Valine production
Metabolic engineering of Corynebacterium glutamicum ATCC13869 for L-valine production.
Chen C1, Li Y2, Hu J2, Dong X2, Wang X3.
L-Valine is one of 20 proteinogenic amino acids. L-Valine is an essential amino acid.