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Improvement of l-arginine production by in silico genome-scale metabolic network model guided genetic engineering.

ABSTRACT: Genome-scale metabolic network model (GSMM) is an important in silico tool that can efficiently predict the target genes to be modulated. A Corynebacterium crenatum argB-M4 Cc_iKK446_arginine model was constructed on the basis of the GSMM of Corynebacterium glutamicum ATCC 13032 Cg_iKK446. Sixty-four gene deletion sites, twenty-four gene enhancement sites, and seven gene attenuation sites were determined for the improvement of l-arginine production in engineered C. crenatum. Among these sites, the effects of disrupting putP, cgl2310, pta, and Ncgl1221 and overexpressing lysE on l-arginine production were investigated. Moreover, the strain CCM007 with deleted putP, cgl2310, pta, and Ncgl1221 and overexpressed lysE produced 24.85 g/L l-arginine. This finding indicated a 106.8% improvement in l-arginine production compared with that in CCM01. GSMM is an excellent tool for identifying target genes to promote l-arginine accumulation in engineered C. crenatum.

SUBMITTER: Huang M

PROVIDER: S-EPMC7031459 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Improvement of l-arginine production by in silico genome-scale metabolic network model guided genetic engineering.

Huang Mingzhu M Zhao Yue Y Li Rong R Huang Weihua W Chen Xuelan X

3 Biotech 20200219 3

Genome-scale metabolic network model (GSMM) is an important in silico tool that can efficiently predict the target genes to be modulated. A Corynebacterium crenatum argB-M4 Cc_iKK446_arginine model was constructed on the basis of the GSMM of Corynebacterium glutamicum ATCC 13032 Cg_iKK446. Sixty-four gene deletion sites, twenty-four gene enhancement sites, and seven gene attenuation sites were determined for the improvement of l-arginine production in engineered C. crenatum. ...[more]

PMID: 32140378

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Project description:BackgroundAscomycin is a 23-membered polyketide macrolide with high immunosuppressant and antifungal activity. As the lower production in bio-fermentation, global metabolic analysis is required to further explore its biosynthetic network and determine the key limiting steps for rationally engineering. To achieve this goal, an engineering approach guided by a metabolic network model was implemented to better understand ascomycin biosynthesis and improve its production.ResultsThe metabolic conservation of Streptomyces species was first investigated by comparing the metabolic enzymes of Streptomyces coelicolor A3(2) with those of 31 Streptomyces strains, the results showed that more than 72% of the examined proteins had high sequence similarity with counterparts in every surveyed strain. And it was found that metabolic reactions are more highly conserved than the enzymes themselves because of its lower diversity of metabolic functions than that of genes. The main source of the observed metabolic differences was from the diversity of secondary metabolism. According to the high conservation of primary metabolic reactions in Streptomyces species, the metabolic network model of Streptomyces hygroscopicus var. ascomyceticus was constructed based on the latest reported metabolic model of S. coelicolor A3(2) and validated experimentally. By coupling with flux balance analysis and using minimization of metabolic adjustment algorithm, potential targets for ascomycin overproduction were predicted. Since several of the preferred targets were highly associated with ethylmalonyl-CoA biosynthesis, two target genes hcd (encoding 3-hydroxybutyryl-CoA dehydrogenase) and ccr (encoding crotonyl-CoA carboxylase/reductase) were selected for overexpression in S. hygroscopicus var. ascomyceticus FS35. Both the mutants HA-Hcd and HA-Ccr showed higher ascomycin titer, which was consistent with the model predictions. Furthermore, the combined effects of the two genes were evaluated and the strain HA-Hcd-Ccr with hcd and ccr overexpression exhibited the highest ascomycin production (up to 438.95 mg/L), 1.43-folds improvement than that of the parent strain FS35 (305.56 mg/L).ConclusionsThe successful constructing and experimental validation of the metabolic model of S. hygroscopicus var. ascomyceticus showed that the general metabolic network model of Streptomyces species could be used to analyze the intracellular metabolism and predict the potential key limiting steps for target metabolites overproduction. The corresponding overexpression strains of the two identified genes (hcd and ccr) using the constructed model all displayed higher ascomycin titer. The strategy for yield improvement developed here could also be extended to the improvement of other secondary metabolites in Streptomyces species.

Dataset Information

Improvement of l-arginine production by in silico genome-scale metabolic network model guided genetic engineering.

Publications

Improvement of l-arginine production by in silico genome-scale metabolic network model guided genetic engineering.

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