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Phasins are proteins located on the surface of poly-3-hydroxybutyrate (P3HB) granules that affect the metabolism of the polymer, the size and number of the granules, and some also have stress-protecting and growth-promoting effects. This study evaluated the effect of inactivating two new phasins (PhbP2 or PhbP3) on the cellular growth, production, and molecular mass of P3HB in cultures under low or high oxygen transfer rates (OTR). The results revealed that under high OTRₘₐₓ conditions (between 8.1 and 8.9 mmol L−1 h−1), the absence of phasins PhbP2 and PhbP3 resulted in a strong negative effect on the growth rate; in contrast, the rates of specific oxygen consumption increased in both cases. This behavior was not observed under a low oxygen transfer rate (3.9 ± 0.71 mol L−1 h−1), where cellular growth and oxygen consumption were the same for the different strains evaluated. It was observed that at high OTR, the absence of PhbP3 affected the production of P3HB, decreasing it by 30% at the end of cultivation. In contrast, the molecular weight remained constant over time. In summary, the absence of phasin PhbP3 significantly impacted the growth rate and polymer synthesis, particularly at high maximum oxygen transfer rates (OTRₘₐₓ). [ABSTRACT FROM AUTHOR]

A novel poly-3-hydroxybutyrate depolymerase was identified in Azotobacter vinelandii. This enzyme, now designated PhbZ1, is associated to the poly-3-hydroxybutyrate (PHB) granules and when expressed in Escherichia coli, it showed in vitro PHB depolymerizing activity on native or artificial PHB granules, but not on crystalline PHB. Native PHB (nPHB) granules isolated from a PhbZ1 mutant had a diminished endogenous in vitro hydrolysis of the polyester, when compared to the granules of the wild-type strain. This in vitro degradation was also tested in the presence of free coenzyme A. Thiolytic degradation of the polymer was observed in the nPHB granules of the wild type, resulting in the formation of 3-hydroxybutyryl-CoA, but was absent in the granules of the mutant. It was previously reported that cultures of A. vinelandii OP grown in a bioreactor showed a decrease in the weight average molecular weight (Mw) of the PHB after 20 h of culture, with an increase in the fraction of polymers of lower molecular weight. This decrease was correlated with an increase in the PHB depolymerase activity during the culture. Here, we show that in the phbZ1 mutant, neither the decrease in the Mw nor the appearance of a low molecular weight polymers occurred. In addition, a higher PHB accumulation was observed in the cultures of the phbZ1 mutant. These results suggest that PhbZ1 has a role in the degradation of PHB in cultures in bioreactors and its inactivation allows the production of a polymer of a uniform high molecular weight. [ABSTRACT FROM AUTHOR]

Poly-3-hydroxybutyrate (P3HB) is an intracellular polyester produced by numerous bacteria, including Azotobacter vinelandii . Thermo-mechanical properties and biomedical applications of P3HB depend on its molecular mass (MM), which in turn is controlled by the balance between synthesis and degradation of the polymer during its biosynthesis. The aim of this study was to determine the activity levels of enzymes involved in the synthesis and degradation of P3HB and their effect on the molecular mass of the polymer produced by A. vinelandii strain OP under the conditions of dissolved oxygen tension of 1 and 15%. The results show that the MM of P3HB changed between the exponential and stationary growth phases, under both oxygen conditions. During the exponential growth phase, the mean molecular mass (MMM) of P3HB was high (4800 kDa), coincident with a high activity P3HB synthase and with a low activity of P3HB depolymerase. In contrast, during the stationary phase, the P3HB MM decreased to 3600 kDa, because of the increased activity of the P3HB depolymerase. [ABSTRACT FROM AUTHOR]

Poly-3-hydroxybutyrate (P3HB) is a biopolymer produced by Azotobacter vinelandii. The physicochemical properties and applications of P3HB are strongly influenced by its weight-average molecular mass ( Mw ), and in A. vinelandii, it could be influenced by the culture conditions. The aim of this study was to evaluate the effect of the P3HB content of the inoculum on the Mw of the polymer produced by A. vinelandii OP in bioreactor cultures. A. vinelandii cells containing 20, 50 and 70% of P3HB were used as inoculum. The P3HB content in the inoculum affected the volumetric P3HB productivity ( q P3HB ) and the Mw of P3HB. Those cultures inoculated with cells containing 20% of P3HB, achieved the highest q P3HB (0.17 ± 0.018 g P3HB L −1 h −1 ); whereas a P3HB content of 70% was reflected as a low q P3HB (0.021 ± 0.002 g P3HB L −1 h −1 ). On the other hand, using an inoculum with 70% of polymer content, the Mw of the biopolymer remained stable at values close to 3200 kDa; whereas, when an inoculum with 20% of P3HB was used, the Mw decreased drastically during early stages of cultivation. These results show that manipulating the P3HB content of the inoculum is possible to produce biopolymers with a suitable Mw . [ABSTRACT FROM AUTHOR]