Abstract The fermentation process is dynamically changing, and the metabolic status can be grasped through real-time monitoring of environmental parameters. In this study, a real-time and on-line monitoring experiment platform for substrates and products detection was developed based on non-contact type near-infrared (NIR) spectroscopy technology. The prediction models for monitoring the fermentation process of lactic acid, sophorolipids and sodium gluconate were established based on partial least-squares regression and internal cross-validation methods. Through fermentation verification, the accuracy and precision of the NIR model for the complex fermentation environments, different rheological properties (uniform system and multi-phase inhomogeneous system) and different parameter types (substrate, product and nutrients) have good applicability, and R2 is greater than 0.90, exhibiting a good linear relationship. The root mean square error shows that the model has high credibility. This research provides a basis for the application of NIR spectroscopy in complex fermentation systems.

The fermentation process is dynamically changing, and the metabolic status can be grasped through real-time monitoring of environmental parameters. In this study, a real-time and on-line monitoring experiment platform for substrates and products detection was developed based on non-contact type near-infrared (NIR) spectroscopy technology. The prediction models for monitoring the fermentation process of lactic acid, sophorolipids (SLs) and sodium gluconate (SG) were established based on partial least-squares regression and internal cross-validation methods. Through fermentation verification, the accuracy and precision of the NIR model for the complex fermentation environments, different rheological properties (uniform system and multi-phase inhomogeneous system) and different parameter types (substrate, product and nutrients) have good applicability, and R 2 was greater than 0.98, exhibiting a good linear relationship. The root mean square error of prediction shows that the model has high credibility. Through the control of appropriate glucose concentration in SG fermentation as well as glucose and oil concentrations SLs fermentation by NIR model, the titers of SG and SLs were increased to 11.8% and 26.8%, respectively. Although high cost of NIR spectrometer is a key issue for its wide application in an industrial scale. This work provides a basis for the application of NIR spectroscopy in complex fermentation systems.

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 .

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 inStreptomyces albus. However, the regulatory mechanism of RspA1/A2 using a carbon source (glucose or acetate) for the cell growth ofS. albusis 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 generspA1knocked-out mutant Delta rspA1 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 Delta rspA1. 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 genespyc,pck, andglpXin 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 Delta rspA1. 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.

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 inStreptomyces albus. However, the regulatory mechanism of RspA1/A2 using a carbon source (glucose or acetate) for the cell growth ofS. albusis 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 generspA1knocked-out mutant Delta rspA1 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 Delta rspA1. 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 genespyc,pck, andglpXin 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 Delta rspA1. 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.

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.

The purpose of this paper is to develop a more convenient,rapid and unpretreated method to measure the oil content in the culture medium.A quantitative method for determining the oil content in the fermentation medium using low field NMR(LF-NMR) instruments has been developed.All components of the medium were detected using the LF-NMR technology and the relaxation time of oil is about 100ms using LF-NMR.Except for soybean flour and wheat germ flour,no ingredients had significant effects on the oil peak area in the single-factor experiments.The oil content determined by LF-NMR tests showed that it had a good stability (CV=4.4％) and accuracy of about 1.0％.In addition,the oil contents determined by the method of N-hexane extraction and LF-NMR technology presented a linear relationship with a correlation R2 of 0.9581.The LF-NMR method is more convenient and accurate than the traditional methods of oil detections such as n-hexane extraction,indicating that LF-NMR method could be applied in the industrial process to monitor the oil content in the broth.

目的 优化高通量筛选盐霉素高产菌株流程,高通量筛选出具有遗传稳定性的产盐霉素高产菌株.方法 以核糖体工程理论为基础,常压室温等离子体(atmospheric and room temperature plasma,ARTP)及高通量筛选技术(high-throughputscreening, HTS)对产盐霉素的白色链霉菌(Streptomyces albus)S12诱变筛选,并以琼脂块扩散法为基础建立一种快速检测盐霉素的方法应用于高通量筛选过程初筛.结果 获得具有氯霉素和四环素双重抗性的高产菌株Tet30Ch125.摇瓶发酵实验结果表明:突变株Tet30Ch125盐霉素效价由初始菌株的16306U/mL提高到34712U/mL,提高了112％,且遗传稳定性良好.同时建立一种快速检测盐霉素的方法——琼脂块扩散法并应用于高通量筛选过程初筛中,缩短筛选周期12d,优化了筛选流程.结论 首次综合运用核糖体工程推理选育技术、常压室温等离子体(atmospheric and room temperature plasma,ARTP)及高通量筛选技术(high-throughput screening,HTS)对产盐霉素的白色链霉菌(S.albus)诱变筛选,是很好的菌种选育策略,适合工业菌种的选育.