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

Abstract

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.

RSM–GA Based Optimization of Bacterial PHA Production and In Silico Modulation of Citrate Synthase for Enhancing PHA Production

“…The signals at 1.0 and 1.75 ppm were characterized by methyl group, whereas the signals at 2.507 and 4.490 ppm were characterized by methylene and methine groups, respectively (data/figure not shown). The results obtained were congruent with earlier reports of Pillai et al [36]. Both FTIR and NMR spectra of the extracted PHAs, along with crotonic acid assay, confirmed PHB form of the bioplastic production.…”

Analysis of Polyhydroxyalkanoates Granules in Haloferax mediterranei by Double-Fluorescence Staining with Nile Red and SYBR Green by Confocal Fluorescence Microscopy

“…Confocal fluorescence microscopy, at an excitation wavelength of 553 nm, revealed numerous brightly fluorescent red granules within the cells corresponding to PHA granules ( Figure 1 ). However, the identification of cells by phase contrast microscopy for the localization of PHA granules inside the cells [ 26 , 31 , 41 , 42 ] is not a suitable technique for haloarchaea due to the high concentration of salts in the culture medium as well as in the cytoplasm, making the image quality not optimal due to the presence of salt crystals ( Figure 1 ).…”

Process optimization, metabolic engineering interventions and commercialization of microbial polyhydroxyalkanoates production – A state‐of‐the art review

“…For example, about 93.75% and 51.12% of intracellular PHB could be recovered from recombinant E. coli and B. aryabhattai PHB10, respectively while using a combined EDTA (10 mM) and microwave (700 W) treatment for cell lysis. [ 160 ]…”