Comparative Genomic and Genetic Functional Analysis of Industrial L-Leucine– and L-Valine–Producing Corynebacterium glutamicum Strains

Abstract

Corynebacterium glutamicum is an excellent platform for the production of amino acids, and is widely used in the fermentation industry. Most industrial strains are traditionally obtained by repeated processes of random mutation and selection, but the genotype of these strains is often unclear owing to the absence of genomic information. As such, it is difficult to improve the growth and amino acid production of these strains via metabolic engineering. In this study, we generated a complete genome map of an industrial L-valine-producing strain, C. glutamicum XV. In order to establish the relationship between genotypes and physiological characteristics, a comparative genomic analysis was performed to explore the core genome, structural variations, and gene mutations referring to an industrial L-leucine-producing strain, C. glutamicum CP, and the widely used C. glutamicum ATCC 13032. The results indicate that a 36,349 bp repeat sequence in the CP genome contained an additional copy each of lrp and brnFE genes, which benefited the export of L-leucine. However, in XV, the kgd and panB genes were disrupted by nucleotide insertion, which increase the availability of precursors to synthesize L-valine. Moreover, the specific amino acid substitutions in key enzymes increased their activities. Additionally, a novel strategy is proposed to remodel central carbon metabolism and reduce pyruvate consumption without having a negative impact on cell growth by introducing the CP-derived mutant H + /citrate symporter. These results further our understanding regarding the metabolic networks in these strains and help to elucidate the influence of different genotypes on these processes.

Effect of low-level ultrasound treatment on the production of L-leucine by Corynebacterium glutamicum in fed-batch culture

“…The leucine-producing strain Corynebacterium glutamicum CP was engineered through multiple rounds of random mutagenesis and screening [ 25 ]. The complete genome sequence of C. glutamicum CP was reported by Gui et al [ 26 ].…”

“…The L-leucine concentration was measured by high-performance liquid chromatography (HPLC) using an LC20AT system (Shimadzu, Kyoto, Japan) equipped with an Agilent ZORBAX Eclipse AA column (4.6 × 150 mm, 5 µm; Agilent Technologies, Palo Alto, CA, USA) and a UV detector (360 nm). 50% acetonitrile and 50 mM sodium acetate were used as the mobile phase at a flow rate of 1 mL/min [ 25 ]. All samples were measured in triplicate.…”

Effect of Ultrasound on L-leucine Production by Corynebacterium Glutamicum in Fed-batch Culture

“…Amino acid concentrations were measured by high-performance liquid chromatography (HPLC) using an LC20AT system (Shimadzu, Kyoto, Japan) equipped with an Agilent ZORBAX Eclipse AA column (4.6 × 150 mm, 5 µm; Agilent Technologies, Palo Alto, CA, USA) with UV detection (360 nm). Acetate-buffered acetonitrile was used as the mobile phase at a flow rate of 1 mL/min [54]. All data were measured in triplicates.…”

Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids

“…The subsequent overexpression of R529H/G532D/L535V IPMS in the knockout strain resulted in 38.1 g/L l -leucine accumulation in a 50-L automated fermenter [ 64 ]. Moreover, C. glutamicum AN02 with G127D/I197V/R529H/G561D IPMS, IPMI, and IPMD overexpression resulted in more leucine (13.1 g/L) than a strain overexpressing only G127D/I197V/R529H/G561D IPMS [ 65 ]. Inserting the terminator with the strength of 40.4 a.u.…”

“…A comparison of intracellular metabolites and BCAAs production by wild-type C. glutamicum , C. glutamicum XV (producing l -valine), and C. glutamicum CP (producing l -leucine) [ 89 ] revealed a more active pentose phosphate pathway in the XV or CP strains than the wild type, indicating that these strains could produce more NADPH than the wild type. C. glutamicum XV and CP were obtained via multiple rounds of random mutagenesis [ 65 ], and these two strains are accessible at the China General Microbiological Culture Collection Center with the identifiers of CGMCC11425 (CP) and CGMCC 1.15672 (XV), respectively [ 89 ]. The transcription of most genes in l -valine and l -leucine biosynthetic pathways is up-regulated in the XV and CP strains [ 89 ].…”