Polyhydroxyalkanoates (PHA) are polyesters of various hydroxyalkanoates accumulated in numerous bacteria. All of the monomeric units of PHA are enantiomerically pure and in R-configuration. R-Hydroxyalkanoic acids can be widely used as chiral starting materials in fine chemical, pharmaceutical and medical industries. In this study, we established an efficient method for the production of chiral hydroxyalkanoic acid monomers from PHA. Pseudomonas putida cells containing PHA were resuspended in phosphate buffer at different pH. We observed that the optimal initial pH for intracellular PHA degradation and monomer release was at pH 8-11 with pH 11 as the best. At initial pH 11, PHA containing 3-hydroxyoctanoic acid and 3-hydroxyhexanoic acid was degraded with an efficiency of over 90% (w/w) in 9 h, and the yield of the corresponding monomers was also over 90%. Under the same conditions, unsaturated monomers were also effectively produced from PHA containing 3-hydroxy-6-heptenoic acid, 3-hydroxy-8-nonenoic acid, and 3-hydroxy-10-undecenoic acid. The monomers (e.g., 3-hydroxyoctanoic acid) were further isolated using solid phase extraction and purified on reversed phase semipreparative liquid chromatography. We confirmed that the purified 3-hydroxyoctanoic acid monomer has exclusively the R-configuration.

The regulation of medium-chain-length polyhydroxyalkanoates (mcl-PHA) metabolism in Pseudomonas putida GPo1 was studied by analysis of enzymes bound to PHA granules and enzymes involved in fatty acid oxidation. N-terminal sequencing of granule-bound enzymes revealed the presence of PHA polymerase (PhaC) and PHA depolymerase (PhaZ) and an acyl-CoA synthetase (ACS1), which recently was found to be associated with PHA granules by in vivo study. The acs1 knockout mutant accumulated 30-50% less PHA than its parental strain, confirming the involvement of ACS1 in PHA metabolism. Isolated PHA granules showed both PhaC and PhaZ activities. PhaC activity was found to be sensitive to the ratio of [R-3-hydroxyacyl-CoA]/[CoA] in which free CoA was a mild competitive inhibitor. Fatty acid oxidation was regulated by the [acetyl-CoA]/[CoA] and [NADH]/[NAD] ratios, with high ratios resulting in accumulation and low ratios leading to rapid oxidation of 3-hydroxyacyl-CoA. These results suggest that PHA metabolism is likely to be controlled by the [acetyl-CoA]/[CoA] and [NADH]/[NAD] ratios. The physiological roles of simultaneous PHA accumulation and degradation are also discussed.

The growing awareness of the importance of chirality in conjunction with biological activity has led to an increasing demand for efficient methods for the industrial synthesis of enantiomerically pure compounds. Polyhydroxyalkanotes (PHAs) are a family of polyesters consisting of over 140 chiral R-hydroxycarboxylic acids (R-HAs), representing a promising source for obtaining chiral chemicals from renewable carbon sources. Although some R-HAs have been produced for some time and certain knowledge of the production processes has been gained, large-scale production has not yet been possible. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in biotechnological processes for R-HA production.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-010-2530-6) contains supplementary material, which is available to authorized users.

An efficient method to prepare enantiomerically pure (R)-3-hydroxycarboxylic acids from bacterial polyhydroxyalkanoates (PHAs) accumulated by Pseudomonas putida GPo1 is reported in this study. (R)-3-Hydroxycarboxylic acids from whole cells were obtained when conditions were provided to promote in vivo depolymerization of intracellular PHA. The monomers were secreted into the extracellular environment. They were separated and purified by acidic precipitation, preparative reversed-phase column chromatography, and subsequent solvent extraction. Eight (R)-3-hydroxycarboxylic acids were isolated: (R)-3-hydroxyoctanoic acid, (R)-3-hydroxyhexanoic acid, (R)-3-hydroxy-10-undecenoic acid, (R)-3-hydroxy-8-nonenoic acid, (R)-3-hydroxy-6-heptenoic acid, (R)-3-hydroxyundecanoic acid, (R)-3-hydroxynonanoic acid, and (R)-3-hydroxyheptanoic acid. The overall yield based on released monomers was around 78 wt % for (R)-3-hydroxyoctanoic acid. All obtained monomers had a purity of over 95 wt %. The physical properties of the purified monomers and their antimicrobial activities were also investigated.