Dataset Information

Identification and characterization of ?-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum.

ABSTRACT: Corynebacterium glutamicum is widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as ?-aminobutyric acid (GABA). In this study, a C. glutamicum GABA-specific transporter (GabP(Cg)) encoded by ncgl0464 was identified and characterized. GabP(Cg) plays a major role in GABA uptake and is essential to C. glutamicum growing on GABA. GABA uptake by GabP(Cg) was weakly competed by l-Asn and l-Gln and stimulated by sodium ion (Na(+)). The K(m) and V(max) values were determined to be 41.1 ± 4.5 ?M and 36.8 ± 2.6 nmol min(-1) (mg dry weight [DW])(-1), respectively, at pH 6.5 and 34.2 ± 1.1 ?M and 67.3 ± 1.0 nmol min(-1) (mg DW)(-1), respectively, at pH 7.5. GabP(Cg) has 29% amino acid sequence identity to a previously and functionally identified aromatic amino acid transporter (TyrP) of Escherichia coli but low identities to the currently known GABA transporters (17% and 15% to E. coli GabP and Bacillus subtilis GabP, respectively). The mutant RES167 ?ncgl0464/pGXKZ9 with the GabP(Cg) deletion showed 12.5% higher productivity of GABA than RES167/pGXKZ9. It is concluded that GabP(Cg) represents a new type of GABA transporter and is potentially important for engineering GABA-producing C. glutamicum strains.

SUBMITTER: Zhao Z

PROVIDER: S-EPMC3318837 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification and characterization of γ-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum.

Zhao Zhi Z Ding Jiu-Yuan JY Ma Wen-Hua WH Zhou Ning-Yi NY Liu Shuang-Jiang SJ

Applied and environmental microbiology 20120203 8

Corynebacterium glutamicum is widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as γ-aminobutyric acid (GABA). In this study, a C. glutamicum GABA-specific transporter (GabP(Cg)) encoded by ncgl0464 was identified and characterized. GabP(Cg) plays a major role in GABA uptake and is essential to C. glutamicum growing on GABA. GABA uptake by GabP(Cg) was weakly competed by l-As ...[more]

PMID: 22307305

Similar Datasets

Project description:The hyperphosphorylated guanosine derivatives ppGpp and pppGpp represent global regulators of the bacterial stress response, as they act as central elements of the stringent response system. Although it was assumed that both, (p)ppGpp synthesis and hydrolysis, are catalyzed by one bifunctional RSH-protein in the actinobacterial model organism Corynebacterium glutamicum ATCC 13032, two putative short alarmone synthetases (SASs) were identified by bioinformatic analyses. The predicted sequences of both enzymes, designated as RelP*Cg and RelSCg, exhibit high similarities to the conserved (p)ppGpp synthetase catalytic domain. In the context of sequence analysis, significant differences were found between the RelP variants of different C. glutamicum isolates. In contrast to the bifunctional RelA/SpoT homolog (RSH) protein RelCg, whose gene deletion results in a reduced growth rate, no change in growth characteristics were observed for deletion mutants of the putative SAS proteins under standard growth conditions. The growth deficit of the ?rel strain could be restored by the additional deletion of the gene encoding RelSCg, which clearly indicates a functional relationship between both enzymes. The predicted pyrophosphokinase activity of RelSCg was demonstrated by means of genetic complementation of an Escherichia coli ?relA?spoT strain. For the expression of RelP*Cg , as well as the slightly differing variant RelPCg from C. glutamicum AS1.542, no complementation was observed, concluding that both RelP versions possess no significant pyrophosphokinase activity in vivo. The results were confirmed by in vitro characterization of the corresponding proteins. In the course of this investigation, the additional conversion of GMP to pGpp was determined for the enzyme RelSCg. Since the SAS species analyzed extend both the network of stringent response related enzymes and the number of substances involved, the study of this class of enzymes is an important component in understanding the bacterial stress response. In addition to the comprehension of important biological processes, such as growth rate regulation and the survival of pathogenic species in the host organism, SAS enzymes can be used to produce novel hyperphosphorylated nucleotide species, such as pGpp.

Project description:To date, no information has been made available on the genetic traits that lead to increased carbon flow into the fatty acid biosynthetic pathway of Corynebacterium glutamicum. To develop basic technologies for engineering, we employed an approach that begins by isolating a fatty acid-secreting mutant without depending on mutagenic treatment. This was followed by genome analysis to characterize its genetic background. The selection of spontaneous mutants resistant to the palmitic acid ester surfactant Tween 40 resulted in the isolation of a desired mutant that produced oleic acid, suggesting that a single mutation would cause increased carbon flow down the pathway and subsequent excretion of the oversupplied fatty acid into the medium. Two additional rounds of selection of spontaneous cerulenin-resistant mutants led to increased production of the fatty acid in a stepwise manner. Whole-genome sequencing of the resulting best strain identified three specific mutations (fasR20, fasA63(up), and fasA2623). Allele-specific PCR analysis showed that the mutations arose in that order. Reconstitution experiments with these mutations revealed that only fasR20 gave rise to oleic acid production in the wild-type strain. The other two mutations contributed to an increase in oleic acid production. Deletion of fasR from the wild-type strain led to oleic acid production as well. Reverse transcription-quantitative PCR analysis revealed that the fasR20 mutation brought about upregulation of the fasA and fasB genes encoding fatty acid synthases IA and IB, respectively, by 1.31-fold ± 0.11-fold and 1.29-fold ± 0.12-fold, respectively, and of the accD1 gene encoding the β-subunit of acetyl-CoA carboxylase by 3.56-fold ± 0.97-fold. On the other hand, the fasA63(up) mutation upregulated the fasA gene by 2.67-fold ± 0.16-fold. In flask cultivation with 1% glucose, the fasR20 fasA63(up) fasA2623 triple mutant produced approximately 280 mg of fatty acids/liter, which consisted mainly of oleic acid (208 mg/liter) and palmitic acid (47 mg/liter).

Dataset Information

Identification and characterization of ?-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum.

Publications

Identification and characterization of γ-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets