Establishment of a metabolic pathway to introduce the 3-hydroxyhexanoate unit into LA-based polyesters via a reverse reaction of β-oxidation in Escherichia coli LS5218

New lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate (3HB)-co-3-hydroxyhexananoate (3HHx)]s, were produced in recombinant Escherichia coli LS5218 harboring three genes encoding LA-polymerizing enzyme (LPE), propionyl-coenzyme A (CoA) transferase (PCT) and (R)-specific enoyl-CoA hydratase (PhaJ4). When the recombinant LS5218 was grown on glucose with the feeding of butyrate, 3HB-CoA and 3HHx-CoA were supplied, probably via reverse reactions of the β-oxidation pathway and PhaJ4. LPE copolymerized the two monomers with LA-CoA, which was generated by PCT, to yield the terpolymers. Gas chromatography analysis revealed that the terpolymers consisted of 2.7 to 34 mol% LA, 38 to 81 mol% 3HB and 17 to 33 mol% 3HHx units, which can be varied depending on the butyrate concentration fed in the medium. In addition, 1H-13C COSY NMR analysis provided evidence for a linkage between LA and 3HHx units in the polymer

Engineering of polyhydroxyalkanoate synthase by Ser477X/Gln481X saturation mutagenesis for efficient production of 3-hydroxybutyrate-based copolyesters

Class II polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 (PhaC1_[Ps]) synthesizes 3-hydroxybutyrate (3HB)-based copolyesters, P[3HB-co-3-hydroxyalkanoate (3HA)]. Four sites (130, 325, 477, and 481) in PhaC1_[Ps] that affect the cellular content and 3HB fraction of P(3HB-co-3HA) produced have been identified. Simple combination of beneficial mutations at the sites successfully increased 3HB fraction in the copolymers (62 mol%). However, polymer content was often largely decreased (0.2 wt%) regardless of an enhancement in 3HB fraction, compared to the wild-type enzyme (14 mol% 3HB and 12 wt%) [Matsumoto et al. (2006) Biomacromolecules, 7:2436-2442]. In the present study, we attempted to explore residues combination at the four sites to overcome the problem. Here, pairwise saturation mutagenesis at the neighboring sites 477 and 481 of PhaC1_[Ps] was performed using single and double mutations at sites 130 and 325 as templates, to increase 3HB fraction in the copolymer without reducing the polymer content in recombinant Escherichia coli. These useful PhaC1_[Ps] mutants were screened based on enhanced P(3HB) content, and were subsequently applied to P(3HB-co-3HA) production. Among the mutants tested, the Ser325Cys/Ser477Lys/Gln481Leu mutant exhibited increased 3HB fraction in copolymer (63 mol%) and also polymer content (18 wt%), indicating that mutation scrambling was effective for obtaining the desired mutants

Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium Sp. Strain Usm2

The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC[subscript Cs]). PhaC[subscript Cs] showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC[subscript Cs] expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC[subscript Cs] was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC[subscript Cs] of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC[subscript Cs] is a naturally occurring, highly active PHA synthase with superior polymerizing ability.Ministry of Science, Technology and Innovation, Malaysi