Abstract
Backgroundl-Glutamate is an important precursor in the l-arginine (l-Arg) biosynthetic pathway. Various methods, including polyoxyethylene sorbitan monopalmitate (Tween 40) addition and dtsR1 disruption, have been widely used to induce l-glutamate overproduction in Corynebacterium glutamicum. In this study, a novel strategy for l-Arg overproduction through Tween 40 trigger and ΔdtsR1 mutant were proposed in Corynebacterium crenatum.ResultsCorynebacterium crenatum mutant (CCM01) was selected as a host strain, whose argR was lethal via mutagenesis screening, the proB gene was knocked out, and argB was replaced by argB M4 (E19R, H26E, D311R, and D312R) to release l-Arg feedback resistance. After Tween 40 trigger in the logarithmic period, l-Arg production increased from 15.22 to 17.73 g/L in CCM01 strain. When NCgl1221 and dtsR1 disruption (CCM03), l-Arg production drastically increased to 27.45 g/L and then further to 29.97 g/L after Tween 40 trigger. Moreover, the specific activity of α-oxoglutarate dehydrogenase complex (ODHC) decreased, whereas the regeneration of NADP+/NADPH significantly increased after dtsR1 disruption and Tween 40 trigger. Results of real-time PCR showed that the transcriptional levels of odhA, sucB, and lpdA (encoding three subunits of the ODHC complex) were downregulated after Tween 40 trigger or dtsR1 disruption. By contrast, zwf transcription (encoding glucose-6-phosphate dehydrogenase) showed no significant difference among CCM01, CCM02 (ΔNCgl1221), and CCM03 (ΔNCgl1221ΔdtsR1) strains without Tween 40 trigger but evidently increased by 5.50 folds after Tween 40 trigger.ConclusionA novel strategy for l-Arg overproduction by dtsR1 disruption and Tween 40 trigger in C. crenatum was reported. Tween 40 addition exhibited a bifunctional mechanism for l-Arg overproduction, including reduced ODHC activity and enhanced NADPH pools accumulation by downregulated dtsR1 expression and upregulated zwf expression, respectively.
Highlights
Background lArginine (l-Arg) is a semi-essential amino acid that is widely used as an additive in food, cosmetic, and pharmaceutical industries because of its ability to promoteIn recent decades, various strategies based on genetic engineering technology have been designed to improve industrial levels of l-Arg
Extracellular l-glutamate in CCM02 strain decreased to an undetectable level (Fig. 3), whereas l-Arg production increased to 19.56 g/L after Tween 40 trigger
The results showed that extracellular l-Arg production in CCM03 strain drastically increased to 27.45 g/L, whereas l-lysine production declined by 39.56 % to 1.06 g/L compared with 1.75 g/L in CCM01 + Tween 40 condition (Fig. 3)
Summary
Background lArginine (l-Arg) is a semi-essential amino acid that is widely used as an additive in food, cosmetic, and pharmaceutical industries because of its ability to promoteIn recent decades, various strategies based on genetic engineering technology have been designed to improve industrial levels of l-Arg. Chen et al Microb Cell Fact (2015) 14:119 an engineered E. coli strain for l-Arg overproduction by deleting the speC, speF, argA, adiA, and argR genes, introducing feedback-resistant argA214, and overexpressing argO genes, whose l-Arg final production was achieved at 11.64 g/L in 1-L batch fermentation. Xu et al [11] performed site-directed mutagenesis of N-acetyl-l-glutamate kinase (including E19R, H26E, and H268D) to alleviate feedback inhibition by l-Arg; lArg production improved by about 41.7 % as compared with that of the initial strain. Lee et al [14, 15] reported a metabolically engineered C. glutamicum for production of l-Arg at the industrial-scale based on systems metabolic engineering, including random mutagenesis to release feedback inhibition, knocking out argR and farR genes to remove repressors, increasing NADPH and carbamoyl phosphate pools, and deleting NCgl1221 gene to avoid l-glutamate exporter. L-Arg production distinctly increased to 92.5 g/L by fed-batch fermentation in a 5-L bioreactor
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