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Poly(3-hydroxybutyrate) [P(3HB)] is the most representative polyhydroxyalkanoate (PHA), which is a storage polyester for prokaryotic cells. P(3HB)-producing recombinant Escherichia coli secretes diethylene glycol (DEG)-terminated 3HB oligomers (3HBO-DEG) through a PHA synthase-mediated chain transfer and alcoholysis reactions with externally added DEG. The purpose of this study was to optimize the culture conditions for the secretory production of 3HBO-DEG with jar fermenters. First, the effects of culture conditions, such as agitation speed, culture temperature, culture pH, and medium composition on 3HBO-DEG production, were investigated in a batch culture using 250-ml mini jar fermenters. Based on the best culture conditions, a fed-batch culture was conducted by feeding glucose to further increase the 3HBO-DEG titer. Consequently, the optimized culture conditions were reproduced using a 2-L jar fermenter. This study successfully demonstrates a high titer of 3HBO-DEG, up to 34.8 g/L, by optimizing the culture conditions, showing the feasibility of a new synthetic strategy for PHA-based materials by combining secretory oligomer production and subsequent chemical reaction.

Polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC) catalyzes not only PHA polymerization but also alcoholytic cleavage of PHA chains. The alcoholysis activity of PhaRC is expressed when a hydroxyacyl-CoA monomer is absent but an alcohol compound is present. In this study, we performed alanine mutagenesis of the putative catalytic triad (Cys, Asp, and His) in the PhaC subunit to identify the active site residues for polymerization and alcoholysis activities. Individual substitution of each triad residue with alanine resulted in loss of both polymerization and alcoholysis activities, suggesting that these residues are commonly shared between polymerization and alcoholysis reactions. The loss of activity was also observed following mutagenesis of the triad to other amino acids, except for one PhaRC mutant with a C151S substitution, which lost polymerization activity but still possessed cleavage activity towards PHA chains. The low-molecular-weight PHA isolated from the PhaRC(C151S)-expressing strain showed a lower ratio of alcohol capping at the P(3HB) carboxy terminus than did that from the wild-type-expressing strain. This observation implies that hydrolysis activity of PhaRC might be elicited by the C151S mutation. [ABSTRACT FROM AUTHOR]

Many microorganisms harbor genes necessary to synthesize biodegradable plastics known as polyhydroxyalkanoates (PHAs). We surveyed a genomic database and discovered a new cluster of class IV PHA synthase genes (phaRC). These genes are different in sequence and operon structure from any previously reported PHA synthase. The newly discovered PhaRC synthase was demonstrated to produce PHAs in recombinantEscherichia coli. [ABSTRACT FROM PUBLISHER]

Poly(3-hydroxybutyrate) [P(3HB)] is the most representative polyhydroxyalkanoate (PHA), which is a storage polyester for prokaryotic cells. P(3HB)-producing recombinant Escherichia coli secretes diethylene glycol (DEG)-terminated 3HB oligomers (3HBO-DEG) through a PHA synthase-mediated chain transfer and alcoholysis reactions with externally added DEG. The purpose of this study was to optimize the culture conditions for the secretory production of 3HBO-DEG with jar fermenters. First, the effects of culture conditions, such as agitation speed, culture temperature, culture pH, and medium composition on 3HBO-DEG production, were investigated in a batch culture using 250-ml mini jar fermenters. Based on the best culture conditions, a fed-batch culture was conducted by feeding glucose to further increase the 3HBO-DEG titer. Consequently, the optimized culture conditions were reproduced using a 2-L jar fermenter. This study successfully demonstrates a high titer of 3HBO-DEG, up to 34.8 g/L, by optimizing the culture conditions, showing the feasibility of a new synthetic strategy for PHA-based materials by combining secretory oligomer production and subsequent chemical reaction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sakurai, Mizuno, Miyahara, Hiroe, Taguchi and Tsuge.)