Enhancement of ascomycin production in <i>Streptomyces hygroscopicus</i> var. <i>ascomyceticus</i> by combining resin HP20 addition and metabolic profiling analysis

Song

et al. 2017

Microb Cell Fact

Self Cite

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0787-5) contains supplementary material, which is available to authorized users.

Section: Introductionmentioning

confidence: 99%

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

Song

et al. 2017

Microb Cell Fact

Self Cite

“…In our previous study, metabolic pro ling analysis showed that lysine (Lys), used for the synthesis of precursors pipecolic, and valine (Val), used for the synthesis of precursors methylmalonyl-CoA, were the key primary metabolites in the production of FK520 under the condition of adding resin HP20. Adding 1.0 g/L Val to the fermentation broth at 48 h increased the yield of FK520 by 9.2 %, and adding 1.0 g/L Lys at 72 h increased the FK520 yield by 13.2 % [2]. Similarly, another previous research showed that for the shikimic acid-resistant strain SA68, adding 3 g/L shikimic acid, an important metabolite used for the synthesis of precursor DHCHC, to the fermentation broth at 48 h increased the yield of FK520 by 36 % [45].…”

Section: Discussionmentioning

confidence: 99%

“…ascomyceticus FS35 selected from S. hygroscopicus var. ascomyceticus ATCC 14891 after femtosecond laser irradiation was used as the parent strain [2]. All the strains and plasmids used in this work are listed in Additional le 1: Table S1.…”

Section: Methodsmentioning

confidence: 99%

“…ascomyceticus ATCC 14891 [1]. Because of its speci c structure, FK520 exhibits diverse biological and pharmacological activities, for example antifungal [2], antimalarial [3], immunosuppressive [4] and anticonvulsive effects [5]. Therefore, FK520 has shown broad application prospect in the eld of medicine and attracted extensive attention from researchers.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Increasing Ascomycin Yield in Streptomyces Hygroscopicus var. Ascomyceticus by Using Polyhydroxybutyrate as an Intracellular Carbon Supply Station

Yin

et al. 2020

Preprint

Self Cite

Background: Ascomycin is a multifunctional antibiotic produced by Streptomyces hygroscopicus var. ascomyceticus. As a secondary metabolite, the production of ascomycin is often limited by the shortage of precursors during the late fermentation phase. Polyhydroxybutyrate is an intracellular polymer accumulated by various microorganisms. The development of polyhydroxybutyrate as a useful carbon reservoir for precursor synthesis is of great significance for improving the yield of ascomycin.Results: The fermentation characteristics of the parent strain S. hygroscopicus var. ascomyceticus FS35 showed that the accumulation and decomposition of polyhydroxybutyrate were respectively correlated with the growth of strain and the production of ascomycin. The co-overexpression of exogenous polyhydroxybutyrate synthesis gene phaC and native polyhydroxybutyrate decomposition gene fkbU increased both strain biomass and ascomycin yield. Comparative transcription analysis showed that the storage of polyhydroxybutyrate during the exponential phase accelerated biosynthesis processes by stimulating the utilization of carbon sources, and the decomposition of polyhydroxybutyrate during the stationary phase increased the biosynthesis of ascomycin precursors by enhancing metabolic flux of primary pathways. The comparative analysis of cofactor concentration reflected that the biosynthesis of polyhydroxybutyrate depended on the supply of NADH pool. Under the condition of low sugar content in the later exponential phase, the optimization of carbon source addition further strengthened the polyhydroxybutyrate metabolism by increasing total concentration of cofactors. Finally, in the fermentation medium with 22 g/L starch and 52 g/L dextrin, the ascomycin yield of co-overexpression strain was increased to 626.30 mg/L, 2.11-fold higher than that of parent strain in the initial medium (296.29 mg/L). Conclusions: This paper reported for the first time that the polyhydroxybutyrate was beneficial to the growth of strain and the production of ascomycin by working as an intracellular carbon reservoir, storing as polymers when carbon sources are abundant and depolymerizing to monomers for the biosynthesis of precursors when carbon sources are insufficient. The successful application of polyhydroxybutyrate in increasing the output of ascomycin provided a new strategy for the high yield of other secondary metabolites.

“…[12] To date, only few studies exist in the literature that demonstrate the application of targeted GC-MS analysis of metabolites for the optimization of fermentation medium and parameters in industrial scale biomass productions. [13,14] The aim of the present study was to evaluate dynamics of the broth metabolome of a large industrial production scale fermentation with S. thermophiles using untargeted GC-MS metabolomics. Seven different batch fermentations were screened for the change of metabolites during the fermentation time.…”

Section: Introductionmentioning

confidence: 99%

Untargeted GC‐MS Metabolomics Reveals Changes in the Metabolite Dynamics of Industrial Scale Batch Fermentations of Streptoccoccus thermophilus Broth

Khakimov

Christiansen

Heins

et al. 2017

Biotechnology Journal

An industrial scale biomass production using batch or fed-batch fermentations usually optimized by selection of bacterial strains, tuning fermentation media, feeding strategy, and temperature. However, in-depth investigation of the biomass metabolome during the production may reveal new knowledge for better optimization. In this study, for the first time, the authors investigated seven fermentation batches performed on five Streptoccoccus thermophilus strains during the biomass production at Chr. Hansen (Denmark) in a real life large scale fermentation process. The study is designed to investigate effects of batch fermentation, fermentation time, production line, and yeast extract brands on the biomass metabolome using untargeted GC-MS metabolomics. Processing of the raw GC-MS data using PARAFAC2 revealed a total of 90 metabolites out of which 64 are identified. Partitioning of the data variance according to the experimental design was performed using ASCA and revealed that batch and fermentation time effects and their interaction term were the most significant effects. The yeast extract brand had a smaller impact on the biomass metabolome, while the production line showed no effect. This study shows that in-depth metabolic analysis of fermentation broth provides a new tool for advanced optimization of high-volume-low-cost biomass production by lowering the cost, increase the yield, and augment the product quality.