Microbial production of enantiomerically pure (R)-hydroxycarboxylic acids

Abstract

Enantiomerically pure (R)-hydroxycarboxylic acids (RHAs) that can be incorporated into bacterial polyhydroxyalkanoates (PHAs) as monomeric units, have great potential as a chiral building block to be widely used in the synthesis of various fine chemicals. For the efficient production of enantiomerically pure RHAs, several chemical and biological depolymerization of bacterial PHA was investigated. The sort of acid catalyst and the polyhydroxybutyrate (PHB) purification method shows some effects on the efficiency of acidic alcoholysis. When hydrochloric acid was used as an acid catalyst, the initial reactivity can be enhanced especially for the acidic methanolysis of PHB purified by simple digestion method with 0.2N NaOH than that observed when sulfuric acid was used. When 1.2 g of PHB was acidic acoholysed in 4.1 mL of reaction solution consisting 2 mL of 1,2-dichloroethane, 2 mL of methanol, and 0.1 mL of concentrated hydrochloric acid (38%), 84 % of PHB was converted to methyl R3HB in only 15 h of reaction. This rapid and high concentrated reaction will provide the economical profits by decreasing amount of expensive organic solvents and by providing higher productivity. A heterologous metabolism of PHA biosynthesis and degradation is established in Escherichia coli by introducing the Ralstonia eutropha or Alcaligenes latus PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By using this metabolically engineered E. coli, enantiomerically pure R3HB could be efficiently produced from glucose. By employing a two-plasmids system, developed as the PHA biosynthesis operon on a medium copy number plasmid and the PHA depolymerase gene on a high copy number plasmid, R3HB could be efficiently produced with a yield of 49.5% from glucose. By the integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be efficiently produced without plasmi...