Overexpression of succinyl-CoA synthase for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) production in engineered Escherichia coli BL21(DE3).

AIM: This study aims to increase the 3-hydroxyvalerate (3HV) fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)] using succinyl-CoA synthase. METHODS AND RESULTS: Escherichia coli YH090, a polyhydroxyalkonate (PHA)-producing strain, was further engineered for overexpression of succinyl-CoA synthase genes (sucCD), and examined for P(HB-co-HV) copolymer production in the presence of various precursor molecules using mixture analysis. Glycerol, succinate and propionate were screened as important factors for controlling intracellular PHA accumulation and monomer composition. Glycerol concentrations exerted the greatest influence on the overall biomass concentration and the intracellular PHA content, while propionate concentrations in the presence of succinate influenced the 3HV content of the copolymer. Mixture analysis also demonstrated that the engineered strain has the capacity to accumulate up to 80% of its cell dry weight (CDW) as PHA with a variable fraction of 3HV monomer (maximum of 72 wt %) depending on the controlled conditions. CONCLUSIONS: Propionate is the principal precursor for 3HV monomer in P(HB-co-HV) biopolymer and its utilization requires conversion to propionyl-CoA. Engineered E. coli YHY99, overexpressing sucCD genes, leads to an increase of the succinyl-CoA pool, which enhances the conversion rate of propionate by providing a CoA supply to other acyltransferase enzymes that have a role in propionate utilization. SIGNIFICANCE AND IMPACT OF THE STUDY: Engineered E. coli YHY99 was able to utilize propionate with a 4·5-fold increase in rate, as compared to the control strain, and resulted in the synthesis of a copolymer with high 3HV monomer content.

Optimization and production of pyrrolidone antimicrobial agent from marine sponge-associated Streptomyces sp. MAPS15.

Twenty-nine actinobacterial strains were isolated from marine sponge Spongia officinalis and screened for antagonistic activity against various bacterial and fungal pathogens. The active antibiotic producer MAPS15 was identified as Streptomyces sp. using 16S rRNA phylogenetic analysis. The critical control factors were selected from Plackett-Burman (PB) factorial design and the bioprocess medium was optimized by central composite design (CCD) for the production of bioactive metabolite from Streptomyces sp. MAPS15. The maximum biomass and active compound production obtained with optimized medium was 6.13 g/L and 62.41 mg/L, respectively. The economical carbon source, paddy straw was applied for the enhanced production of bioactive compound. The purified active fraction was characterized and predicted as pyrrolidone derivative which showed broad spectrum of bioactivity towards indicator organisms. The predicted antimicrobial spectra suggested that the Streptomyces sp. MAPS15 can produce a suite of novel antimicrobial drugs.

Optimization of polyhydroxybutyrate production by marine Bacillus megaterium MSBN04 under solid state culture.

A marine sponge-associated bacterium Bacillus megaterium MSBN04 was used for the production of polyhydroxybutyrate (PHB) under solid state culture (SSC). A central composite design (CCD) was employed to optimize the production medium and to find out the interactive effects of four independent variables, viz. tapioca industry waste, palm jaggery, horse gram flour and trace element solution on PHB production. The maximum yield of PHB 8.637 mg g(-1) of substrate (tapioca industry waste) was achieved from biomass 15.203 mg g(-1) of substrate, using statistically optimized medium. The horse gram flour (nitrogen source) and trace element solution were found to be critical control factors for PHB synthesis. The (1)H NMR analysis revealed that the polymer was a PHB monomer. PHB obtained from this study having high molecular weight (6.7×10(5) Da) with low polydispersity index (PDI) value (1.71) and produced PHB was used to synthesize PHB polymeric nanoparticles using solvent displacement approach. Therefore, B. megaterium MSBN04 is an ideal candidate that can be exploited biotechnologically for the commercial production of PHB under solid state culture.

Process optimization and production of polyhydroxybutyrate using palm jaggery as economical carbon source by marine sponge-associated Bacillus licheniformis MSBN12.

The Polyhydroxybutyrate (PHB) producer, Bacillus licheniformis MSBN12 was isolated from the marine sponge Callyspongia diffusa. The PHB production of B. licheniformis MSBN12 was optimized using a four-factor Box-Behnken design to find the interactive effects of variables such as palm jaggery, wheat bran, seawater, and incubation temperature. The maximum yield of PHB (6.38 g/L) was achieved through response surface methodology-based optimization and the optimized conditions were further used for the batch and fed-batch fermentation. Maximum biomass was reached at 48 and 36 h of incubation with PHB accumulation of 62.91 and 67.16 % (w/w of dry cells) for batch and fed-batch process. The production of PHB under fed-batch process with B. licheniformis MSBN12 was increased threefold over shake flask culture when palm jaggery as sole carbon source. The ¹H NMR data was extrapolated with peaks of the PHB reference standard and confirmed as PHB analog.

A statistical approach for optimization of polyhydroxybutyrate production by marine Bacillus subtilis MSBN17.

The important biological macromolecule polyhydroxybutyrate (PHB) producing Bacillus subtilis was isolated from the marine sponge Callyspongia diffusa and identified by means of 16S rRNA analysis. The central composite design (CCD) was used to optimize the PHB production using cheap raw materials such as pulp industry waste (PIW), tamarind kernel powder (TKP), palm jaggery (PJ) and green gram flour (GGF). The extracted polymer was characterized by (1)H NMR analysis. The PIW was fed at three different intervals and the maximum production of PHB (19.08g/L) was attained after a period of 40h of incubation of B. subtilis. Dissolved oxygen, sodium chloride and nitrogen source were found to be the critical control factors that affected the PHB polymer production. The present investigation demonstrates an inexpensive model of producing PHB green thermoplastics in vitro for biomedical applications.

Synthesis of silver nanoparticles by polysaccharide bioflocculant produced from marine Bacillus subtilis MSBN17.

The polysaccharides are emerging as stabilizing and reducing agents for nanoparticles synthesis, however the commercial polysaccharides are not economically viable. Therefore, the exopolysaccharide from microbial origin such as bioflocculants are promising alternate for the synthesis and stabilization of nanoparticles. In this report, a bioflocculant (MSBF17) was produced from marine sponge-associated Bacillus subtilis MSBN17 under submerged fermentation using the economical substrates. The production was statistically optimized with most significant factors such as palm jaggery, NH(4)NO(2), K(2)HPO(4) and NaCl. The maximum bioflocculant production obtained with statistically optimized medium was 13.42 g/l. Based on the biochemical composition and FT-IR analysis, the flocculant compound was predicted as a polysaccharide derivative. The flocculating activity of the MSBF17 was invariably considerable at high salinity and temperature. It was found that the nano-scale silver can be synthesized in reverse micelles using the bioflocculant as stabilizer. The silver nanoparticles (AgNPs) were characterized by UV-spectroscopy, FT-IR and TEM analysis. The AgNPs were spherical shaped (60 nm) and stable for 5 months. Therefore, the bioflocculant-mediated synthesis of nanomaterials can be considered as environmental benign greener approach.