Dataset Information

Metabolic network model guided engineering ethylmalonyl-CoA pathway to improve ascomycin production in Streptomyces hygroscopicus var. ascomyceticus.

ABSTRACT:

Background

Ascomycin 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.

Results

The 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).

Conclusions

The 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.

SUBMITTER: Wang J

PROVIDER: S-EPMC5627430 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Publications

Metabolic network model guided engineering ethylmalonyl-CoA pathway to improve ascomycin production in Streptomyces hygroscopicus var. ascomyceticus.

Wang Junhua J Wang Cheng C Song Kejing K Wen Jianping J

Microbial cell factories 20171003 1

<h4>Background</h4>Ascomycin 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.<h4>Results</h4>The ...[more]

PMID: 28974216

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Project description:Malaria is one of the life-threatening diseases in the world. The spread of resistance to antimalarial drugs is a major challenge, and resistance to artemisinin has been reported in the Southeast Asian region. In the previous study, the active compound of Streptomyces hygroscopicus subsp. Hygroscopicus (S. hygroscopicus), eponemycin, has been shown to have antimalarial effects. To further analyze the effects of other active compounds on the Plasmodium parasite, identifying and analyzing the effectiveness of compounds contained in S. hygroscopicus through instrumentation of liquid chromatography/mass spectrometry (LC/MS) and in silico studies were very useful. This study aimed at identifying other derivative compounds from S. hygroscopicus and screening the antimalarial activity of the compound by assessing the binding affinity, pharmacokinetic profile, and bond interaction. The derivative compounds were identified using LC/MS. Protein targets for derivative compounds were found through literature studies, and the results of identification of compounds and protein targets were reconstructed into three-dimensional models. Prediction of pharmacokinetic profiles was carried out using Swiss ADME. Screening of protein targets for the derivative compound was carried out using the reverse molecular docking method. Analyzing bond interaction was done by LigPlot. One compound from S. hygroscopicus, i.e., 6,7-dinitro-2-[1, 2, 4]triazole-4-yl-benzo[de]isoquinoline-1,3-dione, was successfully identified using LC/MS. This compound was an isoquinoline derivative compound. Through literature studies with inclusion criteria, thirteen protein targets were obtained for reverse molecular docking. This isoquinoline derivative had the potential to bind to each protein target. The pharmacokinetic profile showed that this compound had the drug-likeness criteria. Conclusion. 6,7-Dinitro-2-[1, 2, 4]triazole-4-yl-benzo[de]isoquinoline-1,3-dione has antimalarial activity as shown by reverse molecular docking studies and pharmacokinetic profiles. The best inhibitory ability of compounds based on bond affinity is with adenylosuccinate synthetase.

Project description:The cyclodipeptide pulcherriminic acid, produced by Bacillus licheniformis, is derived from cyclo(l-Leu-l-Leu) and possesses excellent antibacterial activities. In this study, we achieved the high-level production of pulcherriminic acid via multistep metabolic engineering of B. licheniformis DWc9n*. First, we increased leucine (Leu) supply by overexpressing the ilvBHC-leuABCD operon and ilvD, involved in Leu biosynthesis, to obtain strain W1, and the engineered strain W2 was further attained by the deletion of gene bkdAB, encoding a branched-chain ?-keto acid dehydrogenase in W1. As a result, the intracellular Leu content and pulcherriminic acid yield of W2 reached 147.4?mg/g DCW (dry cell weight) and 189.9?mg/liter, which were 227.6% and 48.9% higher than those of DWc9n*, respectively. Second, strain W3 was constructed through overexpressing the leucyl-tRNA synthase gene leuS in W2, and it produced 367.7?mg/liter pulcherriminic acid. Third, the original promoter of the pulcherriminic acid synthetase cluster yvmC-cypX in W3 was replaced with a proven strong promoter, PbacA, to produce the strain W4, and its pulcherriminic acid yield was increased to 507.4?mg/liter. Finally, pulcherriminic acid secretion was strengthened via overexpressing the transporter gene yvmA in W4, resulting in the W4/pHY-yvmA strain, which yielded 556.1?mg/liter pulcherriminic acid, increased by 337.8% compared to DWc9n*, which is currently the highest pulcherriminic acid yield to the best of our knowledge. Taken together, we provided an efficient strategy for enhancing pulcherriminic acid production, which could apply to the high-level production of other cyclodipeptides.IMPORTANCE Pulcherriminic acid is a cyclodipeptide derived from cyclo(l-Leu-l-Leu), which shares the same iron chelation group with hydroxamate sidephores. Generally, pulcherriminic acid-producing strains could be the perfect candidates for antibacterial and anti-plant-pathogenic fungal agents. In this study, we obtained the promising W4/pHY-yvmA pulcherriminic acid-producing strain via a multistep metabolic modification. The engineered W4/pHY-yvmA strain is able to achieve 556.1?mg/liter pulcherriminic acid production, which is the highest yield so far to the best of our knowledge.

Dataset Information

Metabolic network model guided engineering ethylmalonyl-CoA pathway to improve ascomycin production in Streptomyces hygroscopicus var. ascomyceticus.

Background

Results

Conclusions

Publications

Metabolic network model guided engineering ethylmalonyl-CoA pathway to improve ascomycin production in Streptomyces hygroscopicus var. ascomyceticus.

Similar Datasets

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