Comparative Characterization and Identification of Poly-3-Hydroxybutyrate Producing Bacteria with Subsequent Optimization of Polymer Yield

In this work, the strains Bacillus megaterium BM 1, Azotobacter chrocococcumAz 3, Bacillus araybhattay RA 5 were used as an effective producer of poly-3-hydroxybutyrate P(3HB). The purpose of the study was to isolate and obtain an effective producer of P(3HB) isolated from regional chestnut soils of northern Kazakhstan. This study demonstrates the possibility of combining the protective system of cells to physical stress as a way to optimize the synthesis of PHA by strains. Molecular identification of strains and amplification of the phbC gene, transmission electron microscope (TEM), extracted and dried PHB were subjected to Fourier infrared transmission spectroscopy (FTIR). The melting point of the isolated P(3HB) was determined. The optimal concentration of bean broth for the synthesis of P(3HB) for the modified type of Bacillus megaterium RAZ 3 was 20 g/L, at which the dry weight of cells was 25.7 g/L−1 and P(3HB) yield of 13.83 g/L−1, while the percentage yield of P(3HB) was 53.75%. The FTIR spectra of the extracted polymer showed noticeable peaks at long wavelengths. Based on a proof of concept, this study demonstrates encouraging results.

ThephbC(poly-β-hydroxybutyrate synthase) gene ofRhizobium(Sinorhizobium)melilotiand characterization ofphbCmutants

Defined insertion mutations have been constructed in theRhizobium (Sinorhizobium) meliloti phbC gene, which encodes poly-β-hydroxybutyrate (PHB) synthase. The locus was isolated and subcloned from a genomic library of R. meliloti Rm1021 by complementation of a phbC mutation of Alcaligenes eutrophus. PHB production was detected in wild-type R. meliloti under nutrient-limited conditions but not in rich medium. No PHB production was detected in the R. meliloti phbC mutants. The DNA sequence of the R. meliloti phbC gene was determined. The deduced polypeptide sequence is homologous to previously identified PhbCs from other bacteria. The R. meliloti phbC locus maps to pRmeSU47a, the smaller of the two megaplasmids in this strain.Key words: Rhizobium meliloti, PHB, PHA, poly-β-hydroxybutyrate, phbC.

High-level poly(β-hydroxybutyrate) production in recombinantEscherichia coliin sugar-free, complex medium

The poly(β-hydroxybutyrate) (PHB) biosynthetic genes of Alcaligenes eutrophus that are organized in a single operon (phbCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type phb operon from plasmid p4A leads to the formation of 10 or 50–80% PHB/cell dry mass when the cells are grown in Luria–Bertani medium alone or supplemented with 1% glucose (w/v), respectively. To further stimulate PHB formation independent of additional carbon source in Luria–Bertani medium, molecular methods have been applied to provide efficient E. coli transcription and translation signals for the PHB synthase gene (phbC). The lac promoter present upstream of the phbC sequence allows its expression to be controlled depending on the LacI status of the chosen host strain. The T7 gene 10 ribosome binding site is utilized for translational initiation. PHB production in E. coli was compared in strains either harboring plasmid p4A containing the intact phbCAB operon or harboring two compatible plasmids carrying the β-ketothiolase (phbA) and acetoacetyl-CoA-reductase (phbB) genes under transcriptional control of the lac promoter–operator region and also carrying separately the phbC gene with its natural promoter sequence. In addition, plasmid pSYN allowing the phbC gene to be expressed under new transcription and translation conditions combined with plasmid pUMS gave rise to the same amount of PHB formation (70% PHB cell dry mass) in E. coli when grown in Luria–Bertani medium without glucose supplement.Key words: PHB production, engineered phbCAB operon.