ABSTRACT
The two-component system "AfsQ1/Q2" plays a crucial role to activate the production of antibiotics ACT, RED, and CDA through directly binding the promoters of pathway-specific activator genes actII-ORF4, redZ, and cdaR respectively when grown under glutamate-supplemented minimal medium in Streptomyces coelicolor. In this report, we demonstrated that the RspA1/A2 (a homologous protein of two-component system AfsQ1/Q2) plays a regulatory role in salinomycin biosynthesis in Streptomyces albus. Gene deletion and complementation experiments showed that the RspA1/A2 promoted salinomycin production but inhibited cell growth when cultured in YMG medium supplemented with 3% soybean oil. More importantly, RspA1/A2 strengthens salinomycin biosynthesis by directly affecting the transcription of the pathway-specific activator gene slnR. Meanwhile, RspA1/A2 plays a negative role in the regulation of nitrogen assimilation and urea decarboxylation by interacting with the promoters of genes gdhA, glnA, amtB, and SLNWT_1828/1829. Gene sigW is located downstream of rspA1/A2 and encodes an extracytoplasmic function sigma factor. Moreover, it negatively regulates the salinomycin biosynthesis and promotes cell growth, which antagonizes the function of RspA1/A2. In short, these useful findings are proved helpful to enrich the understanding of the regulatory pathways of antibiotic biosynthesis by an ECF σ factor-TCS signal transduction system in Streptomyces.
Subject(s)
Bacterial Proteins/metabolism , Pyrans/metabolism , Streptomyces/metabolism , Gene Expression Regulation, Bacterial/genetics , Promoter Regions, Genetic/genetics , Urea/metabolismABSTRACT
In our previous study, a two-component-system (TCS) RspA1/A2 was identified and proven to play a positive role in the regulation of salinomycin (antibiotic) biosynthesis in Streptomyces albus. However, the regulatory mechanism of RspA1/A2 using a carbon source (glucose or acetate) for the cell growth of S. albus is still unclear till present research work. Therefore, in this work, the mechanistic pathway of RspA1/A2 on carbon source metabolism is unveiled. Firstly, this work reports that the response regulator RspA1 gene rspA1 knocked-out mutant ΔrspA1 exhibits lower biomass accumulation and lower glucose consumption rates as compared to the parental strain A30 when cultivated in a defined minimal medium (MM) complemented with 75 mM glutamate. Further, it is demonstrated that the regulation of TCS RspA1/A2 on the phosphoenolpyruvate-pyruvate-oxaloacetate node results in decreasing the intracellular acetyl-CoA pool in mutant ΔrspA1. Subsequently, it was verified that the RspA1 could not only directly interact with the promoter regions of key genes encoding AMP-forming acetyl-CoA synthase (ACS), citrate synthase (CS), and pyruvate dehydrogenase complex (PDH) but also bind promoter regions of the genes pyc, pck, and glpX in gluconeogenesis. In addition, the transcriptomic data analysis showed that pyruvate and glutamate transformations supported robust TCS RspA1/A2-dependent regulation of glucose metabolism, which led to a decreased flux of pyruvate into the TCA cycle and an increased flux of gluconeogenesis pathway in mutant ΔrspA1. Finally, a new transcriptional regulatory network of TCS RspA1/A2 on primary metabolism across central carbon metabolic pathways including the glycolysis pathway, TCA cycle, and gluconeogenesis pathway is proposed.
ABSTRACT
Salinomycin, an important polyketide, has been widely utilized in agriculture to inhibit growth of pathogenic bacteria. In addition, salinomycin has great potential in treatment of cancer cells. Due to inherited characteristics and beneficial potential, its demand is also inclining. Therefore, there is an urgent need to increase the current high demand of salinomycin. In order to obtain a high-yield mutant strain of salinomycin, the present work has developed an efficient breeding process of Streptomyces albus by using atmospheric and room temperature plasma (ARTP) combined with ribosome engineering. In this study, we investigate the presented method as it has the advantage of significantly shortening mutant screening duration by using an agar block diffusion method, as compared to other traditional strain breeding methods. As a result, the obtained mutant Tet30Chl25 with tetracycline and chloramphenicol resistance provided a salinomycin yield of 34,712 mg/L in shake flask culture, which was over 2.0-fold the parental strain S12. In addition, comparative transcriptome analysis of low and high yield mutants, and a parental strain revealed the mechanistic insight of biosynthesis pathways, in which metabolic pathways including butanoate metabolism, starch and sucrose metabolism and glyoxylate metabolism were closely associated with salinomycin biosynthesis. Moreover, we also confirmed that enhanced flux of glyoxylate metabolism via overexpression gene of isocitrate lyase (icl) promoted salinomycin biosynthesis. Based on these results, it has been successfully verified that the overexpression of crotonyl-CoA reductase gene (crr) and transcriptional regulator genes (orf 3 and orf 15), located in salinomycin synthesis gene cluster, is possibly responsible for the increase in salinomycin production in a typical strain Streptomyces albus DSM41398. Conclusively, a tentative regulatory model of ribosome engineering combined with ARTP in S. ablus is proposed to explore the roles of transcriptional regulators and stringent responses in the biosynthesis regulation of salinomycin.
ABSTRACT
Herein, an enhanced green production of xanthan gum has been achieved by utilizing orange peels. Response surface methodology and kinetic modeling were adapted for the process optimization and its influence on scale up production respectively. Optimal conditions for the maximum xanthan production were 1.62% acid hydrolysis, 85% carbon source of orange peel hydrolysate and 30.4°C temperature. Furthermore, the optimized treatment was conducted in the batch culture fermentor to observe the associated variations during scale up process. In bio-fermentor, to the first time ever, xanthan production along with reducing sugar conversion and utilization rates reached 30.19g/L, 69.29% and 99.99%, respectively. Employed characterization techniques of FTIR, XRD and HPLC confirmed the fermented product as xanthan gum and obtained an average molecular weight of 1.01×106g/mol. This work on account of optimized process parameters presented maximum xanthan production from a waste material.
