Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in Bacillus aryabhattai and cytotoxicity evaluation of PHBV/poly(ethylene glycol) blends.

The study described poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) accumulation in Bacillus aryabhattai PHB10 for the first time and evaluated the polymer induced cytotoxicity in-vitro with PHBV/poly(ethylene glycol) (PEG) blends. The B. aryabhattai strain produced 2.8 g/L PHBV, equivalent to 71.15% of cell dry mass in a medium supplemented with propionic acid, after 48 h incubation. The optimum temperature and pH for the copolymer accumulation was 31 °C and 7, respectively. The gas chromatography-mass spectrometry and nuclear magnetic resonance analyses confirmed the polymer obtained as PHBV. The differential scanning calorimetry analysis revealed that the melting point of the material as 90 °C and its thermal stability up to 220 °C. The average molecular weight (Mn) and polydispersity index of the sample was estimated by gel permeation chromatography analysis and observed as 128.508 kDa and 2.82, respectively. The PHBV showed tensile strength of 10.3 MPa and elongation at break of 13.3%. The PHBV and their blends with PEG were tested for cytotoxicity on human keratinocytes (HaCaT cells) and the cells incubated with PHBV/PEG2kDa blends were 99% viable, whereas with the PHBV alone showed comparatively higher cytotoxicity. The significant improvement in the cell viability of PHBV/PEG2kDa blends indicates its potential as a candidate for skin graft applications.

Enhanced production of poly(3-hydroxybutyrate) in recombinant Escherichia coli and EDTA-microwave-assisted cell lysis for polymer recovery.

Poly(3-hydroxybutyrate) (PHB) is a bacterial polymer of great commercial importance due to its properties similar to polypropylene. With an aim to develop a recombinant system for economical polymer production, PHB biosynthesis genes from Bacillus aryabhattai PHB10 were cloned in E. coli. The recombinant cells accumulated a maximum level of 6.22 g/L biopolymer utilizing glycerol in shake flasks. The extracted polymer was confirmed as PHB by GC-MS and NMR analyses. The polymer showed melting point at 171 °C, thermal stability in a temperature range of 0-140 °C and no weight loss up to 200 °C. PHB extracted from sodium hypochlorite lysed cells had average molecular weight of 143.108 kDa, polydispersity index (PDI) 1.81, tensile strength of 14.2 MPa and an elongation at break of 7.65%. This is the first report on high level polymer accumulation in recombinant E. coli solely expressing PHB biosynthesis genes from a Bacillus sp. As an alternative to sodium hypochlorite cell lysis mediated polymer extraction, the effect of combined treatment with ethylenediaminetetraacetic acid and microwave was studied which attained 93.75% yield. The polymer recovered through this method was 97.21% pure, showed 2.9-fold improvement in molecular weight and better PDI. The procedure is simple, with minimum polymer damage and more eco-friendly than the sodium hypochlorite lysis method.

Efficient methanol-degrading aerobic bacteria isolated from a wetland ecosystem.

Methylotrophs present in the soil play an important role in the regulation of one carbon compounds in the environment, and thereby aid in mitigating global warming. The study envisages the isolation and characterization of methanol-degrading bacteria from Kuttanad wetland ecosystem, India. Three methylotrophs, viz. Achromobacter spanius KUT14, Acinetobacter sp. KUT26 and Methylobacterium radiotolerans KUT39 were isolated and their phylogenetic positions were determined by constructing a phylogenetic tree based on 16S rDNA sequences. In vitro activity of methanol dehydrogenase enzyme, responsible for methanol oxidation was evaluated and the genes involved in methanol metabolism, mxaF and xoxF were partially amplified and sequenced. The specific activity of methanol dehydrogenase (451.9 nmol min-1 mg-1) observed in KUT39 is the highest, reported ever to our knowledge from a soil bacterium. KUT14 recorded the least activity of 50.15 nmol min-1 mg-1 and is the first report on methylotrophy in A. spanius.

Draft Genome Sequence of Bacillus aryabhattai Strain PHB10, a Poly(3-Hydroxybutyrate)-Accumulating Bacterium Isolated from Domestic Sewerage.

Bacillus aryabhattai PHB10 is a poly(3-hydroxybutyrate) (PHB)-accumulating bacterium isolated from domestic sewerage. Here, we report the 4.19-Mb draft genome sequence, with 4,050 protein-coding genes and a G+C content of 37.5%. This sequence will be helpful in the study of the high-level PHB accumulation mechanism of the strain.

Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai

Abstract This study was focused on the polyhydroxybutyrate (PHB) accumulation property of Bacillus aryabhattai isolated from environment. Twenty-four polyhydroxyalkanoate (PHA) producers were screened out from sixty-two environmental bacterial isolates based on Sudan Black B colony staining. Based on their PHA accumulation property, six promising isolates were further screened out. The most productive isolate PHB10 was identified as B. aryabhattai PHB10. The polymer production maxima were 3.264 g/L, 2.181 g/L, 1.47 g/L, 1.742 g/L and 1.786 g/L in glucose, fructose, maltose, starch and glycerol respectively. The bacterial culture reached its stationary and declining phases at 18 h and 21 h respectively and indicated growth-associated PHB production. Nuclear Magnetic Resonance (NMR) spectra confirmed the material as PHB. The material has thermal stability between 30 and 140 °C, melting point at 170 °C and maximum thermal degradation at 287 °C. The molecular weight and poly dispersion index of the polymer were found as 199.7 kDa and 2.67 respectively. The bacterium B. aryabhattai accumulating PHB up to 75% of cell dry mass utilizing various carbon sources is a potential candidate for large scale production of bacterial polyhydroxybutyrate.

Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai.

This study was focused on the polyhydroxybutyrate (PHB) accumulation property of Bacillus aryabhattai isolated from environment. Twenty-four polyhydroxyalkanoate (PHA) producers were screened out from sixty-two environmental bacterial isolates based on Sudan Black B colony staining. Based on their PHA accumulation property, six promising isolates were further screened out. The most productive isolate PHB10 was identified as B. aryabhattai PHB10. The polymer production maxima were 3.264g/L, 2.181g/L, 1.47g/L, 1.742g/L and 1.786g/L in glucose, fructose, maltose, starch and glycerol respectively. The bacterial culture reached its stationary and declining phases at 18h and 21h respectively and indicated growth-associated PHB production. Nuclear Magnetic Resonance (NMR) spectra confirmed the material as PHB. The material has thermal stability between 30 and 140°C, melting point at 170°C and maximum thermal degradation at 287°C. The molecular weight and poly dispersion index of the polymer were found as 199.7kDa and 2.67 respectively. The bacterium B. aryabhattai accumulating PHB up to 75% of cell dry mass utilizing various carbon sources is a potential candidate for large scale production of bacterial polyhydroxybutyrate.

Stress effects caused by the expression of a mutant cellobiohydrolase I and proteasome inhibition in Trichoderma reesei Rut-C30.

Trichoderma reesei Rut-C30 is used widely as an expression host for various gene products. We have explored cellular effects caused by the expression of a mutant form of cellobiohydrolase I (CBHI), the major secreted protein of T. reesei using biochemical and transcriptomic analyses and confocal laser scanning microscopy. The mutated CBHI was tagged fluorescently with Venus to establish the subcellular location of the fusion protein and its potential association with the proteasome, an organelle assigned for the disposal of misfolded proteins. Expression of the mutant CBHI in the high protein-secreting host Rut-C30 caused physiological changes in the fungal hyphae, affected protein secretion and elicited ER stress. A massive upregulation of UPR- and ERAD-related genes sec61, der1, uba1, bip1, pdi1, prp1, cxl1 and lhs1 was observed by qRT-PCR in the CBHIΔ4-Venus strain with four mutations introduced in the DNA encoding the core domain of CBHI. Further stress was applied to this strain by inhibiting function of the proteasome with MG132 (N-benzoylcarbonyl(Cbz)-Leu-Leu-leucinal). The effect of MG132 was found to be specific to the proteasome-associated genes. There are no earlier reports on the effect of proteasome inhibition on protein quality control in filamentous fungi. Confocal fluorescence microscopy studies suggested that the mutant CBHI accumulated in the ER and colocalized with the fungal proteasome. These results provide an indication that there is a limit to how far T. reesei Rut-C30, already under secretion stress, can be pressed to produce higher protein yields.