The applicability of the statistical tools coupled with artificial intelligence techniques was tested to optimize the critical medium components for the production of extracellular cholesterol ...oxidase (COD; an enzyme of commercial interest) from Streptomyces rimosus MTCC 10792. The initial medium component screening was performed using Placket-Burman design with yeast extract, dextrose, starch and ammonium carbonate as significant factors. Response surface methodology (RSM) was attempted to develop a statistical model with a significant coefficient of determination (R
= 0.89847), followed by model optimization using Genetic Algorithm (GA). RSM-GA based optimization approach predicted that the combination of yeast extract, dextrose, starch and ammonium carbonate at concentrations 0.99, 0.8, 0.1, and 0.05 g/100 ml respectively, has resulted in 3.6 folds increase in COD production (5.41 U/ml) in comparison with the un-optimized medium (1.5 U/ml). COD was purified 10.34 folds having specific activity of 12.37 U/mg with molecular mass of 54 kDa. The enzyme was stable at pH 7.0 and 40 °C temperature. The apparent Michaelis constant (K
) and V
values of COD were 0.043 mM and 2.21 μmol/min/mg, respectively. This is the first communication reporting RSM-GA based medium optimization, purification and characterization of COD by S. rimosus isolated from the forest soil of eastern India.
Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by ...expanding its capability with the introduction of its own in vitro transcription‐translation (TX‐TL) system. TX‐TL is a fast and expanding technology for bottom‐up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX‐TL platform by reporting a streamlined protocol for cell‐extract preparation, demonstrating high‐yield synthesis of a codon‐optimized sfGFP reporter and the prototyping of a synthetic tetracycline‐inducible promoter in S. venezuelae TX‐TL based on the tetO‐TetR repressor system. The aim of this system is to provide a host for the homologous production of exotic enzymes from Actinobacteria secondary metabolism in vitro. As an example, the authors demonstrate the soluble synthesis of a selection of enzymes (12–70 kDa) from the Streptomyces rimosus oxytetracycline pathway.
Streptomyces venezuelae is a developing host for synthetic biology and metabolic engineering. To accelerate the understanding of this host and access secondary metabolism enzymes, the authors have characterized a high‐yield S. venezuelae in vitro transcription‐translation (TX‐TL) system for synthetic biology. The system utilizes 3‐phosphoglyceric acid (3‐PGA) to regenerate energy and has been shown to synthesize up to 1.3 µM of sfGFP (under batch synthesis) and a range of enzymes from the oxytetracycline biosynthesis pathway.
Oxytetracycline which is derived from Streptomyces rimosus, inhibits a wide range of bacteria and is industrially important. The underlying biosynthetic processes are complex and hinder rational ...engineering, so industrial manufacturing currently relies on classical mutants for production. While the biochemistry underlying oxytetracycline synthesis is known to involve polyketide synthase, hyperproducing strains of S. rimosus have not been extensively studied, limiting our knowledge on fundamental mechanisms that drive production. In this study, a multiomics analysis of S. rimosus is performed and wild-type and hyperproducing strains are compared. Insights into the metabolic and regulatory networks driving oxytetracycline formation were obtained. The overproducer exhibited increased acetyl-CoA and malonyl CoA supply, upregulated oxytetracycline biosynthesis, reduced competing byproduct formation, and streamlined morphology. These features were used to synthesize bhimamycin, an antibiotic, and a novel microbial chassis strain was created. A cluster deletion derivative showed enhanced bhimamycin production. This study suggests that the precursor supply should be globally increased to further increase the expression of the oxytetracycline cluster while maintaining the natural cluster sequence. The mutagenized hyperproducer S. rimosus HP126 exhibited numerous mutations, including large genomic rearrangements, due to natural genetic instability, and single nucleotide changes. More complex mutations were found than those typically observed in mutagenized bacteria, impacting gene expression, and complicating rational engineering. Overall, the approach revealed key traits influencing oxytetracycline production in S. rimosus, suggesting that similar studies for other antibiotics could uncover general mechanisms to improve production.
In the present work, the approaches of submerged co-cultivation and microparticle-enhanced cultivation (MPEC) were combined and evaluated over the course of three case studies. The filamentous fungus
...Aspergillus terreus
was co-cultivated with
Penicillium rubens
,
Streptomyces rimosus
, or
Cerrena unicolor
in shake flasks with or without the addition of aluminum oxide microparticles. The influence of microparticles on the production of lovastatin, penicillin G, oxytetracycline, and laccase in co-cultures was compared with the effects recorded for the corresponding monocultures. In addition, the quantitative analyses of morphological parameters, sugars consumption, and by-products formation were performed. The study demonstrated that the influence of microparticles on the production of a given molecule in mono- and co-culture may differ considerably, e.g., the biosynthesis of oxytetracycline was shown to be inhibited due to the presence of aluminum oxide in “
A. terreus
vs.
S. rimosus
” co-cultivation variants but not in
S. rimosus
monocultures. The differences were also observed regarding the morphological characteristics, e.g., the microparticles-induced changes of projected area in the co-cultures and the corresponding monocultures were not always comparable. In addition, the study showed the importance of medium composition on the outcomes of MPEC, as exemplified by lovastatin production in
A. terreus
monocultures. Finally, the co-cultures of
A. terreus
with a white-rot fungus
C. unicolor
were described here for the first time.
Key points
• Aluminum oxide affects secondary metabolites production in submerged co-cultures.
• Mono- and co-cultures are differently impacted by the addition of aluminum oxide.
• Effect of aluminum oxide on metabolites production depends on medium composition.
The Cu super(2) super(+), Zn super(2) super(+) and Cr super(6) super(+) biosorption capacity of the Streptomyces rimosus biomass pretreated with NaOH was studied in the batch mode. Under optimal ...experimental conditions, a biosorption capacity of 30 mg Cu super(2) super(+) g super(-) super(1) biomass, 27.4 mg Zn super(2) super(+) g super(-) super(1) biomass and 26.7 mg Cr super(6) super(+) g super(-) super(1) biomass was obtained. The equilibrium data poorly fitted the Langmuir and Freundlich model isotherms over the whole range of initial Cu super(2) super(+), Zn super(2) super(+) and Cr super(6) super(+) concentrations tested (0-300 mg L super(-) super(1)).
The Cu super(2+), Zn super(2+) and Cr super(6+) biosorption capacity of the Streptomyces rimosus biomass pretreated with NaOH was studied in the batch mode. Under optimal experimental conditions, a ...biosorption capacity of 30 mg Cu super(2+) g super(-1) biomass, 27.4 mg Zn super(2+) g super(-1) biomass and 26.7 mg Cr super(6+) g super(-1) biomass was obtained. The equilibrium data poorly fitted the Langmuir and Freundlich model isotherms over the whole range of initial Cu super(2+), Zn super(2+) and Cr super(6+) concentrations tested (0-300 mg L super(-1)).
Paromomycin is a 2-deoxystreptamine aminocyclitol aminoglycoside antibiotic with broad spectrum activity against Gram-negative, Gram-positive bacteria and many protozoa. This study introduces a ...strategy for paromomycin production through solid-state fermentation using Streptomyces rimosus subsp. paromomycinus NRRL 2455. Solid state fermentation has gained enormous attention in the development of several products because of their numerous advantages over submerged liquid fermentation. After selecting the best solid substrate, a time course study of paromomycin production was carried out followed by optimization of environmental conditions using response surface methodology. Paromomycin yields obtained using this technique were also compared to those obtained using submerged liquid fermentation.
Upon screening of 6 different substrates, maximum paromomycin concentration (0.51 mg/g initial dry solids) was obtained with the cost-effective agro-industrial byproduct, corn bran, impregnated with aminoglycoside production media. Optimization of environmental conditions using D-optimal design yielded a 4.3-fold enhancement in paromomycin concentration reaching 2.21 mg/g initial dry solids at a pH of 8.5, inoculum size of 5% v/w and a temperature of 30 °C.
Compared to submerged liquid fermentation, solid state fermentation resulted in comparable paromomycin concentrations, cost reduction of raw materials, less energy consumption and waste water discharge, which have major implications in industrial fermentation. Therefore, solid state fermentation is a promising alternative to submerged liquid fermentation for paromomycin production. To the best of our knowledge, this is the first report on the optimized paromomycin production through solid state fermentation process.
The nutritional requirements for antimicrobial activity of
Streptomyces rimosus
AG-P1441 were optimized using statistically-based experimental designs at a flask level. Based on a ...one-factor-at-a-time (OFAT) approach, glucose, corn starch and soybean meal were identified as the carbon and nitrogen sources having a significant effect on antimicrobial productivity. As a result of investigating the effect of glucose concentration, the highest antimicrobial activity was observed at 3% concentration. Response surface methodology (RSM) was then applied to optimize the growth medium components (corn starch, soybean meal, MgCl
2
and glutamate). Antimicrobial productivity increased sharply when the medium consisted of 3% glucose, 3.5% corn starch, 2.5% soybean meal, 1.2 mM MgCl
2
and 5.9 mM glutamate. The fermentation using optimized culture medium in a 5-L bioreactor allowed a significant increase in antimicrobial activity, evaluated by the paper disc assay, revealed a 29 mm inhibition zone diameter against
Phytophthora capsici
.
In this study, we employed a reporter-guided mutation selection (RGMS) strategy to improve the rimocidin production of Streptomyces rimosus M527, which is based on a single-reporter plasmid pAN and ...atmospheric and room temperature plasma (ARTP). In plasmid pAN, PrimA, a native promoter of the loading module of rimocidin biosynthesis (RimA) was chosen as a target, and the kanamycin resistance gene (neo) under the control of PrimA was chosen as the reporter gene. The integrative plasmid pAN was introduced into the chromosome of S. rimosus M527 by conjugation to yield the initial strain S. rimosus M527-pAN. Subsequently, mutants of M527-pAN were generated by ARTP. 79 mutants were obtained in total, of which 67 mutants showed a higher level of kanamycin resistance (Kanr) than that of the initial strain M527-pAN. The majority of mutants exhibited a slight increase in rimocidin production compared with M527-pAN. Notably, 3 mutants, M527-pAN-S34, S38, and S52, which exhibited highest kanamycin resistance among all Kanr mutants, showed 34%, 52%, and 45% increase in rimocidin production compared with M527-pAN, respectively. Quantitative RT-PCR analysis revealed that the transcriptional levels of neo and rim genes were increased in mutants M527-pAN-S34, S38, and S52 compared with M527-pAN. These results confirmed that the RGMS approach was successful in improving the rimocidin production in S. rimosus M527.
Aflatoxin B1 (AFB1) and zearalenone (ZON) are hazardous mycotoxins. AFB1 has cytotoxic, mutagenic and carcinogenic effects, whereas ZON can disrupt the endocrine system. Biodegradation by microbes is ...an effective method to eliminate these hazardous toxins. The aim of this work was to screen AFB1 and ZON biodegrading potential of one hundred and twenty-four Streptomyces strains deposited in the Actinomycetes strain collection of the Department of Environmental Safety and Ecotoxicology. Two different biotests were used for screening purposes: SOS-Chromotest was used to monitor genotoxicity and select microorganisms with the best AFB1 degrading potential. Estrogenic effect of ZON was measured with a yeast based bioluminescent test including human estrogen receptors Bioluminescent Yeast Estrogen System (BLYES). Biodegradation experiments were conducted with 1 mg l−1 AFB1 and 1 mg l−1 ZON concentration. On the base of the results, ten strains were selected for biodegradation experiments and Enzyme-linked Immunosorbent Assay tests (ELISA). The results of these tests Streptomyces cacaoi subsp. asoensis (K234) strain degraded AFB1 over 88 per cent and totally eliminated genotoxicity. Two strains of Streptomyces rimosus (K145, K189) degraded almost total amount of ZON and estrogenicity was not detected besides that.
•The biodagradation of AFB1 and ZON by several Streptomyces strains was tested.•Two different biotests were used for screening purposes, SOS-Chromotest and BLYES.•Analytical support was conducted by ELISA.•Streptomyces cacaoi subsp. asoensis strain degraded AFB1 over 88 per cent.•Two strains of Streptomyces rimosus degraded almost total amount of ZON.
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