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Genome-scale metabolic network guided engineering of Streptomyces tsukubaensis for FK506 production improvement.

ABSTRACT: BACKGROUND: FK506 is an important immunosuppressant, which can be produced by Streptomyces tsukubaensis. However, the production capacity of the strain is very low. Hereby, a computational guided engineering approach was proposed in order to improve the intracellular precursor and cofactor availability of FK506 in S. tsukubaensis. RESULTS: First, a genome-scale metabolic model of S. tsukubaensis was constructed based on its annotated genome and biochemical information. Subsequently, several potential genetic targets (knockout or overexpression) that guaranteed an improved yield of FK506 were identified by the recently developed methodology. To validate the model predictions, each target gene was manipulated in the parent strain D852, respectively. All the engineered strains showed a higher FK506 production, compared with D852. Furthermore, the combined effect of the genetic modifications was evaluated. Results showed that the strain HT-?GDH-DAZ with gdhA-deletion and dahp-, accA2-, zwf2-overexpression enhanced FK506 concentration up to 398.9 mg/L, compared with 143.5 mg/L of the parent strain D852. Finally, fed-batch fermentations of HT-?GDH-DAZ were carried out, which led to the FK506 production of 435.9 mg/L, 1.47-fold higher than the parent strain D852 (158.7 mg/L). CONCLUSIONS: Results confirmed that the promising targets led to an increase in FK506 titer. The present work is the first attempt to engineer the primary precursor pathways to improve FK506 production in S. tsukubaensis with genome-scale metabolic network guided metabolic engineering. The relationship between model prediction and experimental results demonstrates the rationality and validity of this approach for target identification. This strategy can also be applied to the improvement of other important secondary metabolites.

SUBMITTER: Huang D

PROVIDER: S-EPMC3680238 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Genome-scale metabolic network guided engineering of Streptomyces tsukubaensis for FK506 production improvement.

Huang Di D Li Shanshan S Xia Menglei M Wen Jianping J Jia Xiaoqiang X

Microbial cell factories 20130524

<h4>Background</h4>FK506 is an important immunosuppressant, which can be produced by Streptomyces tsukubaensis. However, the production capacity of the strain is very low. Hereby, a computational guided engineering approach was proposed in order to improve the intracellular precursor and cofactor availability of FK506 in S. tsukubaensis.<h4>Results</h4>First, a genome-scale metabolic model of S. tsukubaensis was constructed based on its annotated genome and biochemical information. Subsequently, ...[more]

PMID: 23705993

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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.

Project description:BACKGROUND: FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influence on the final yield of target product and can play an important role in development of industrial bioprocesses. RESULTS: Three putative regulatory elements, namely fkbR, belonging to the LysR-type family, fkbN, a large ATP-binding regulator of the LuxR family (LAL-type) and allN, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of fkbN caused a complete disruption of FK506 biosynthesis, while inactivation of fkbR resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the allN gene. Using RT-PCR and a reporter system based on a chalcone synthase rppA, we demonstrated, that in the wild type as well as in fkbN- and fkbR-inactivated strains, fkbR is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas fkbN expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of fkbN (or fkbR) does not abolish the transcription of the genes in the FK506 gene cluster in general, but may reduce expression of some of the tested biosynthetic genes. Finally, introduction of a second copy of the fkbR or fkbN genes under the control of the strong ermE* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively. CONCLUSIONS: Our results clearly demonstrate the positive regulatory role of fkbR and fkbN genes in FK506 biosynthesis in S. tsukubaensis NRRL 18488. We have shown that regulatory mechanisms can differ substantially from other, even apparently closely similar FK506-producing strains, reported in literature. Finally, we have demonstrated the potential of these genetically modified strains of S. tsukubaensis for improving the yield of fermentative processes for production of FK506.

Dataset Information

Genome-scale metabolic network guided engineering of Streptomyces tsukubaensis for FK506 production improvement.

Publications

Genome-scale metabolic network guided engineering of Streptomyces tsukubaensis for FK506 production improvement.

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