Ascomycin Production Research Articles

Boosting ascomycin production by n-butanol addition in fermentation of Streptomyces hygroscopicus var. ascomyceticus ATCC 14891

Boosting ascomycin production by n-butanol addition in fermentation of Streptomyces hygroscopicus var. ascomyceticus ATCC 14891

Enhanced ascomycin production in Streptomyces hygroscopicus var. ascomyceticus by employing polyhydroxybutyrate as an intracellular carbon reservoir and optimizing carbon addition

BackgroundAscomycin 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 prokaryotic microorganisms. Developing polyhydroxybutyrate as an intracellular carbon reservoir for precursor synthesis is of great significance to improve the yield of ascomycin.ResultsThe fermentation characteristics of the parent strain S. hygroscopicus var. ascomyceticus FS35 showed that the accumulation and decomposition of polyhydroxybutyrate was respectively correlated with cell growth and ascomycin production. The co-overexpression of the exogenous polyhydroxybutyrate synthesis gene phaC and native polyhydroxybutyrate decomposition gene fkbU increased both the biomass and ascomycin yield. Comparative transcriptional analysis showed that the storage of polyhydroxybutyrate during the exponential phase accelerated biosynthesis processes by stimulating the utilization of carbon sources, while the decomposition of polyhydroxybutyrate during the stationary phase increased the biosynthesis of ascomycin precursors by enhancing the metabolic flux through primary pathways. The comparative analysis of cofactor concentrations confirmed that the biosynthesis of polyhydroxybutyrate depended on the supply of NADH. At low sugar concentrations found in the late exponential phase, the optimization of carbon source addition further strengthened the polyhydroxybutyrate metabolism by increasing the total concentration of cofactors. Finally, in the fermentation medium with 22 g/L starch and 52 g/L dextrin, the ascomycin yield of the co-overexpression strain was increased to 626.30 mg/L, which was 2.11-fold higher than that of the parent strain in the initial medium (296.29 mg/L).ConclusionsHere we report for the first time that polyhydroxybutyrate metabolism is beneficial for cell growth and ascomycin production by acting as an intracellular carbon reservoir, stored as polymers when carbon sources are abundant and depolymerized into monomers for the biosynthesis of precursors when carbon sources are insufficient. The successful application of polyhydroxybutyrate in increasing the output of ascomycin provides a new strategy for improving the yields of other secondary metabolites.

Generation of Streptomyces hygroscopicus cell factories with enhanced ascomycin production by combined elicitation and pathway‐engineering strategies

Ascomycin (FK520) is a macrocyclic antibiotic that also exhibits antifungal and immunosuppressive activity. However, its relatively low titer and yield have hampered commercial application. Here, we have successfully constructed an efficient ascomycin-producing strain of Streptomyces hygroscopicus with high titer and yield, using a novel combinatorial engineering approach based on the identification of targets involved in both metabolic and transcriptional regulation. First, we investigated the effects of different chemicals on ascomycin accumulation and found that dimethyl sulfoxide best stimulated ascomycin overproduction. We next compared intracellular metabolic and transcriptional profiles after dimethyl sulfoxide and control treatments and identified potential target genes (zwf and aroA, involved in metabolic precursor pathways; and luxR, iclR, fadR, and fkbN, involved in transcriptional regulation). These candidate genes were then engineered to produce strains with individual and combinatorial overexpression. Combined overexpression of aroA, fkbN, and luxR resulted in the highest yield of ascomycin (1258.30 ± 33.49 mg/L), 4.12-fold higher than the control yield (305.60 ± 16.90 mg/L). This integrative multilevel approach identified novel determinants involved in both metabolic and transcriptional regulation, resulting in the diversion of carbon flux towards ascomycin accumulation. This approach could be applied to boost the production of a variety of useful bacterial metabolites.

Identification of the regulon FkbN for ascomycin biosynthesis and its interspecies conservation analysis as LAL family regulator

Ascomycin is a 23-membered polyketide macrolide with high immunosuppressant activity, produced by Streptomyces hygroscopicus var. ascomyceticus. However, the specific regulatory mechanism of the ascomycin biosynthesis pathway remains poorly understood. Here, we show that FkbN is a pathway-specific positive regulator for the ascomycin biosynthesis. Inactivation of fkbN led to a complete loss of ascomycin production ability. Compared with the parental strain, the ascomycin yield of a constructed fkbN overexpression strain increased by 400%–1800 mg/L. Transcriptional levels of the genes of encoding precursor, PKS (polyketide synthase) and NRPS (nonribosomal peptide synthetases) were blocked in the fkbN in-frame deletion strain and greatly upregulated in the fkbN overexpression strain. Electrophoretic mobility shift assays (EMSAs) revealed that FkbN-DBD protein specifically bound to the intergenic regions of fkbR1-fkbE and fkbB-fkbO. To explore the high conservation among regulators homologous to FkbN, EMSAs showed that the FkbN-DBD protein directly bound to the putative promoters of different macrolide biosynthetic gene clusters. We did further study in the fkbN deletion strain by heterologous genetic complementation with single copy of the homologous LAL family regulators. Results showed that all the heterologous complementation strains restored the production of ascomycin. Introduction of a single copy of fkbN into the tacrolimus producing strain S. tsukubaensis or rapamycin producing strain S. hygroscopicus, respectively, both enhanced the yields of the corresponding antibiotics.

Study on growth and ascomycin production by Streptomyces hygroscopicus subsp. Ascomyceticus

Abstract The objective of this study undertaken was to acquire maximum information by the shake flask studies for Ascomycin production with Streptomyces hygroscopicus subsp. ascomyceticus ATCC 14891. The morphological observation of culture on plate media was carried out visually and under stereo microscope. The colonies obtained were granular and attained grayish black color on maturity. The microscopic examination of mycelia was performed at 20× and 100× (oil immersion) magnification. To improve the Ascomycin production in shake flask studies, two different approaches were evaluated. In the first set of experiments, “Two stage Fermentation” approach was adopted wherein; matured lab inoculum was transferred directly in different production media. The second approach consisted of set of experiments with “Three stage Fermentation” which involved transfer of grown lab inoculum into seed media and grown seed culture into production media. The age of seed culture was investigated for the betterment of the yield. The maximum concentration of ascomycin obtained was 0.5 mg/gm in the second approach on selected P8 media. Media selection was done on the basis of carbon source, nitrogen source, trace salts, buffer to maintain pH throughout the process and precursor as supplement. Simultaneously, microscopic observation was captured in the present study. Thus it could be concluded that “Three stage Fermentation” on selected media was the best option for scale up of ascomycin.

Enhancement of ascomycin production via a combination of atmospheric and room temperature plasma mutagenesis in Streptomyces hygroscopicus and medium optimization

Ascomycin, a key intermediate for chemical synthesis of immunosuppressive drug pimecrolimus, is produced by Streptomyces hygroscopicus var. ascomyceticus. In order to improve the strain production, the original S. hygroscopicus ATCC 14891 strain was treated here with atmospheric and room temperature plasma to obtain a stable high-producing S. hygroscopicus SFK-36 strain which produced 495.3 mg/L ascomycin, a 32.5% increase in ascomycin compared to the ATCC 14891. Then, fermentation medium was optimized using response surface methodology to further enhance ascomycin production. In the optimized medium containing 81.0 g/L soluble starch, 57.4 g/L peanut meal, and 15.8 g/L soybean oil, the ascomycin yield reached 1466.3 mg/L in flask culture. Furthermore, the fermentation process was carried out in a 5 L fermenter, and the ascomycin yield reached 1476.9 mg/L, which is the highest ascomycin yield reported so far. Therefore, traditional mutagenesis breeding combined with medium optimization is an effective approach for the enhancement of ascomycin production.

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

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.

Engineering of the LysR family transcriptional regulator FkbR1 and its target gene to improve ascomycin production.

Ascomycin (FK520), a macrocyclic polyketide natural antibiotic, displays high anti-fungal and immunosuppressive activity. In this study, the LysR family transcriptional regulator FkbR1 was characterized, and its role in ascomycin biosynthesis was explored by gene deletion, complementation, and overexpression. Inactivation of fkbR1 led to 67.5% reduction of ascomycin production, which was restored by complementation of fkbR1. Overexpression of fkbR1 resulted in a 33.5% increase in ascomycin production compared with the parent strain FS35. These findings indicated that FkbR1 was a positive regulator for ascomycin production. Quantitative RT-PCR analysis revealed that the expressions of fkbE, fkbF, fkbS, and fkbU were downregulated in the fkbR1 deletion strain and upregulated in the fkbR1 overexpression strain. Electrophoretic mobility shift assays (EMSAs) in vitro and chromatin immunoprecipitation (ChIP)-qPCR assays in vivo indicated that FkbR1 bound to the intergenic region of fkbR1-fkbE. To investigate the roles of the target genes fkbE and fkbF in ascomycin production, the deletion and overexpressions of fkbE and fkbF were implemented, respectively. Overexpression of fkbE resulted in a 45.6% increase in ascomycin production, but little change was observed in fkbF overexpression strain. To further enhance ascomycin production, the fkbR1 and fkbE combinatorial overexpression strain OfkbRE was constructed with the ascomycin yield increased by 69.9% to 536.7mg/L compared with that of the parent strain. Our research provided a helpful strategy to increase ascomycin production via engineering FkbR1 and its target gene.

Integration of parallel 13 C-labeling experiments and in silico pathway analysis for enhanced production of ascomycin.

Herein, the hyper-producing strain for ascomycin was engineered based on 13 C-labeling experiments and elementary flux modes analysis (EFMA). First, the metabolism of non-model organism Streptomyces hygroscopicus var. ascomyceticus SA68 was investigated and an updated network model was reconstructed using 13 C- metabolic flux analysis. Based on the precise model, EFMA was further employed to predict genetic targets for higher ascomycin production. Chorismatase (FkbO) and pyruvate carboxylase (Pyc) were predicted as the promising overexpression and deletion targets, respectively. The corresponding mutant TD-FkbO and TD-ΔPyc exhibited the consistency effects between model prediction and experimental results. Finally, the combined genetic manipulations were performed, achieving a high-yield ascomycin engineering strain TD-ΔPyc-FkbO with production up to 610 mg/L, 84.8% improvement compared with the parent strain SA68. These results manifested that the integration of 13 C-labeling experiments and in silico pathway analysis could serve as a promising concept to enhance ascomycin production, as well as other valuable products. Biotechnol. Bioeng. 2017;114: 1036-1044. © 2016 Wiley Periodicals, Inc.

Reduction of foaming and enhancement of ascomycin production in rational Streptomyces hygroscopicus fermentation

Foaming reduces the working volume and limits the biosynthesis of macrolide immunosuppressant ascomycin (FK520) in the batch fermentation process of Streptomyces hygroscopicus FS-35 in a 7.5L bioreactor. To find the relation between FK520 production and foaming, effects of 10 fermentation parameters including organic acids and membrane permeability were investigated. The results suggest that acetate accumulation caused by short period oxygen deficiency and fast consumption of glucose is the reason for increased foaming and declined FK520 production. Therefore, a fed-batch fermentation strategy was developed to reduce the accumulation of acetate. After optimization, the maximum acetate concentration dropped from 320mg·L−1 to 157mg·L−1, decreased by 50.8%, and the maximum foam height reduced from 5.32cm to 3.74cm, decreased by 29.7%, while the maximum FK520 production increased from 375mg·L−1 to 421mg·L−1, improved by 12%.

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

Combinatorial approach of adsorbent resin HP20 addition and metabolic profiling analysis were carried out to enhance ascomycin production. Under the optimal condition of 5% m/v HP20 added at 24h, ascomycin production was increased to 380 from 300mg/L. To further rationally guide the improvement of ascomycin production, metabolic profiling analysis was employed to investigate the intracellular metabolite changes of Streptomyces hygroscopicus var. ascomyceticus FS35 in response to HP20 addition. A correlation between the metabolic profiles and ascomycin accumulation was revealed by partial least-squares to latent structures discriminant analysis, and 11 key metabolites that most contributed to metabolism differences and ascomycin biosynthesis were identified. Based on the analysis of metabolite changes together with their pathways, the potential key factors associated with ascomycin overproduction were determined. Finally, rationally designed fermentation strategies based on HP20 addition were performed as follows: 2% v/v n-hexadecane was added at 24h; 1.0g/L valine was supplemented at 48h; 1.0g/L lysine was added at 72h. The ascomycin production was ultimately improved to 460mg/L, a 53.3% enhancement compared with that obtained in initial condition. These results demonstrated that the combination of HP20 addition and metabolic profiling analysis could be successfully applied to the rational guidance of production improvement of ascomycin, as well as other clinically important compounds.

Analysis of ascomycin production enhanced by shikimic acid resistance and addition in Streptomyces hygroscopicus var. ascomyceticus

Shikimic acid resistance and addition to enhance immunosuppressant ascomycin production were investigated in this study. A shikimic acid resistant strain SA68 was obtained with ascomycin production of 330mg/L, displaying approximately 22% increment compared with the parental strain FS35 (270mg/L). Under the optimal condition of 3g/L shikimic acid added at 24h, ascomycin production was further increased by 36%, reaching 450mg/L. To get deep insights into the effects of shikimic acid resistance and addition on ascomycin biosynthesis, fermentation properties, enzymes activities, metabolites and genes transcriptional levels were analyzed and evaluated. The potential targets were revealed for higher ascomycin production, which were the amplification of carbon flux toward shikimic acid by enhancing 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) activities and eliminating the feedback inhibition of aromatic amino acids on DAHPS activities, and the elevation of FkbO activities by amplifying fkbO gene to increase the flux of shikimic acid to chorismic acid further to 4,5-dihydroxycyclohex-1-enecarboxylic acid in Streptomyces hygroscopicus var. ascomyceticus. The results indicated that the developed methods would be helpful for ascomycin large scale fermentation. Moreover, the potential targets revealed in this study are valuable to engineer the higher-yield ascomycin-producing strain, as well as other analogous macrolide compounds-producing strains.