Metabolic Engineering of Escherichia coli for Production of Enantiomerically Pure ( R )-(−)-Hydroxycarboxylic Acids

Abstract

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid 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 produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By 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 produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.Polyhydroxyalkanoates (PHAs) are a group of completely biodegradable polyesters that are synthesized and accumulated by various bacteria (1,5,9,27). More than 140 kinds of carboxylic acids hydroxylated at the 3-, 4-, 5-, or 6-position, all in the (R)-configuration if they possess a chiral center on the position of hydroxyl group, can be incorporated into PHAs by employing different bacteria under various culture conditions (5, 9, 28). Therefore, it was reasoned that various enantiomerically pure (R)-(Ϫ)-hydroxycarboxylic acids (RHAs) might be conveniently prepared by depolymerizing biosynthesized PHAs. These RHAs contain two functional groups that are convenient to modify for the synthesis of various chiral compounds, especially fine chemicals such as antibiotics, vitamins, perfumes, and pheromones (3, 9, 21, 23). For example, (R)-(Ϫ)-3-hydroxybutyric acid (R3HB) is an important precursor of 4-acetoxyazetidinone, which in turn is used to make carbapenem antibiotics, which have close to a billion-dollar market. Poly-(R)-(Ϫ)-3-hydroxybutyrate (PHB) is the most ubiquitous member of the PHAs. Methods for producing R3HB by chemical digestion of PHB have been reported (13,23,24). In these methods, however, organic solvents were used in large amounts, and the production efficiency was rather low due to complicated processes.The metabolism for the synthesis and degradation of PHB plays an important role in many bacteria for the reservation and reutilization of excess carbon and energy sources and reducing power (1). The metabolic pathway for the synthesis and degradation of PHB in Ralstonia eutropha was already proposed (1,5,11,18,22,25). PHB is synthesized from acetylcoenzyme A (CoA) by three sequential enzymatic reactions catalyzed by ␤-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase (18,22,25). When the conditions are met, PHB is depolymerized to R3HB by intracellular PHA depolymerase and oligomer hydrolase (1,5,11). Then the R3HB dehydrogenase converts R3HB to acetoacetate, which is further metabolized in th...

Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase

“…coli, Lee et al reported the production of 9.6 g L -1 3HB (Lee and Lee 2003). This is of great interest, but also requires careful confirmation, because there is a counter-claim that the high (R)-3HB production was not reproducible, based on both theoretical grounds and experimental evidence (Uchino et al 2008).…”

Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

“…The quantification of 3HB by NAD-dependent 3HB dehydrogenase is possible but is also time-consuming, allows the detection of monomers only, and gives no online information for the hydrolysis reaction. 3HB oligomers that are major products of most PHB depolymerases can only be indirectly taken into account either by chemical cleavage of 3HB oligomers to monomers by alkali treatment (29) or by enzymatic hydrolysis by 3HB oligomer hydrolase (40). Some exotic methods for recording the degradation of polyesters include electron microscopy (35,38), 1 H-nuclear magnetic resonance (47), or tracking of radiolabeled polymers (18,50), but apparently, they are not suitable for routine assays.…”

Metabolic Engineering of Escherichia coli for Enhanced Production of ( R )- and ( S )-3-Hydroxybutyrate

“…The biosynthesis of 3HB has typically been achieved by two different mechanisms: depolymerization (in vitro or in vivo) of microbially synthesized poly-(R)-3-hydroxybutyric acid (PHB) (8,13) or direct synthesis of 3HB without a PHB intermediate (9,12,15). However, due to the stereospecific constraints of PHB synthesis, in which polymers are composed exclusively of (R)-3HB monomer units, the synthesis of (S)-3HB from PHB is effectively impossible.…”