To investigate the conversion of lignin derivatives to biopolyesters, 11 polyhydroxyalkanoate (PHA)-accumulating strains were cultured on mineral salt media containing each of the 18 lignin derivatives and hydroxybenzoic acids, including intermediates derived from the metabolism of lignin derivatives in bacteria. Most of the strains grew poorly in media containing lignin derivatives such as p-coumaric acid, caffeic acid, ferulic acid, and sinapinic acid. One of the strains, Pseudomonas putida Gpo1, grew in the presence of p-coumaric acid, ferulic acid, vanillic acid, 4hydroxybenzoic acid (4-HBA), and 3,4-dihydroxybenzoic acid (3,4-DHBA). Pseudomonas putida JCM 13063 also grew in the presence of 4-HBA, 3,4-DHBA, and vanillic acid. Another strain, Ralstonia eutropha H16, synthesized PHA from 4-HBA, 2,5-DHBA, and 3,4-DHBA. On the basis of the data obtained from these experiments, we suggest that the conversion of lignin derivatives into intermediates such as 4-HBA, 2,5-DHBA, 3,4-DHBA, and vanillic acid represents the major bottleneck in the synthesis of PHA from lignin derivatives.

Polyhydroxyalkanoate (PHA)-producing Bacillus strains possess class IV PHA synthases composed of two subunit types, namely, PhaR and PhaC. In the present study, PHA synthases from Bacillus megaterium NBRC15308(T) (PhaRC(Bm)), B. cereus YB-4 (PhaRC(YB4)), and hybrids (PhaR(Bm)C(YB4) and PhaR(YB4)C(Bm)) were expressed in Escherichia coli JM109 to characterize the molecular weight of the synthesized poly(3-hydroxybutyrate) [P(3HB)]. PhaRC(Bm) synthesized P(3HB) with a relatively high molecular weight (M(n) = 890 × 10(3)) during 72 h of cultivation, whereas PhaRC(YB4) synthesized low-molecular-weight P(3HB) (M(n) = 20 × 10(3)). The molecular weight of P(3HB) synthesized by PhaRC(YB4) decreased with increasing culture time and temperature. This time-dependent behavior was observed for hybrid synthase PhaR(Bm)C(YB4), but not for PhaR(YB4)C(Bm). These results suggest that the molecular weight change is caused by the PhaC(YB4) subunit. The homology between PhaCs from B. megaterium and B. cereus YB-4 is 71% (amino acid identity); however, PhaC(YB4) was found to have a previously unknown effect on the molecular weight of the P(3HB) synthesized in E. coli.

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Polyhydroxyalkanoate (PHA) synthases catalyze chain transfer (CT) reaction after polymerization reaction of PHA by transferring PHA chain from PHA synthase to a CT agent, resulting in covalent bonding of CT agent to PHA chain at the carboxyl end. Previous studies have shown that poly(ethylene glycol) (PEG) is an effective exogenous CT agent. This study aimed to compare the effects of PEG on CT reaction during poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis by using six PHA synthases in Escherichia coli JM109. The synthesized P(3HB) polymers were characterized in terms of molecular weight and end-group structure. Supplementation of PEG to the culture medium reduced P(3HB) molecular weights by up to 96% due to PEG-induced CT reaction. The P(3HB) polymers were subjected to (1)H NMR analysis to confirm the formation of a covalent bond between PEG and P(3HB) chain at the carboxyl end. This study revealed the reactivity of PHA synthases to PEG with respect to CT reaction in E. coli.

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