Synthetic Biology of Polyhydroxyalkanoates (PHA).

Microbial polyhydroxyalkanoates (PHA) are a family of biodegradable and biocompatible polyesters which have been extensively studied using synthetic biology and metabolic engineering methods for improving production and for widening its diversity. Synthetic biology has allowed PHA to become composition controllable random copolymers, homopolymers, and block copolymers. Recent developments showed that it is possible to establish a microbial platform for producing not only random copolymers with controllable monomers and their ratios but also structurally defined homopolymers and block copolymers.

Production of poly(3-hydroxypropionate) and poly(3-hydroxybutyrate-co-3-hydroxypropionate) from glucose by engineering Escherichia coli.

Poly(3-hydroxypropionate) (P3HP) is the strongest family member of microbial polyhydroxyalkanoates (PHA) synthesized by bacteria grown on 1,3-propandiol or glycerol. In this study synthesis pathways of P3HP and its copolymer P3HB3HP of 3-hydroxybutyrate (3HB) and 3-hydroxypropionate (3HP) were assembled respectively to allow their synthesis from glucose, a more abundant carbon source. Recombinant Escherichia coli was constructed harboring the P3HP synthetic pathway consisting of heterologous genes encoding glycerol-3-phosphate dehydrogenase (gpd1), glycerol-3-P phosphatase (gpp2) from Saccharomyces cerevisiae that catalyzes formation of glycerol from glucose, and genes coding glycerol dehydratase (dhaB123) with its reactivating factors (gdrAB) from Klebsiella pneumoniae that transfer glycerol to 3-hydroxypropionaldehyde, as well as gene encoding propionaldehyde dehydrogenase (pdup) from Salmonella typhimurium which converts 3-hydroxypropionaldehyde to 3-hydroxypropionyl-CoA, together with the gene of PHA synthase (phaC) from Ralstonia eutropha which polymerizes 3-hydroxypropionyl-CoA into P3HP.

Engineering the diversity of polyesters.

Many bacteria have been found to produce various polyhydroxyalkanoates (PHA) biopolyesters. In many cases, it is not easy to control the structures of PHA including homopolymers, random copolymers and block copolymers as well as ratios of monomers in the copolymers. It has become possible to engineer bacteria for controllable synthesis of PHA with the desirable structures by creating new PHA synthesis pathways.

Production and characterization of poly(3-hydroxypropionate-co-4-hydroxybutyrate) with fully controllable structures by recombinant Escherichia coli containing an engineered pathway.

Copolyesters of 3-hydroxypropionate (3HP) and 4-hydroxybutyrate (4HB), abbreviated as P(3HP-co-4HB), was synthesized by Escherichia coli harboring a synthetic pathway consisting of five heterologous genes including orfZ encoding 4-hydroxybutyrate-coenzyme A transferase from Clostridium kluyveri, pcs' encoding the ACS domain of tri-functional propionyl-CoA ligase (PCS) from Chloroflexus aurantiacus, dhaT and aldD encoding dehydratase and aldehyde dehydrogenase from Pseudomonas putida KT2442, and phaC1 encoding PHA synthase from Ralstonia eutropha. When grown on mixtures of 1,3-propanediol (PDO) and 1,4-butanediol (BDO), compositions of 4HB in microbial P(3HP-co-4HB) were controllable ranging from 12 mol% to 82 mol% depending on PDO/BDO ratios. Nuclear magnetic resonance (NMR) spectra clearly indicated the polymers were random copolymers of 3HP and 4HB.

Production of 3-hydroxypropionate homopolymer and poly(3-hydroxypropionate-co-4-hydroxybutyrate) copolymer by recombinant Escherichia coli.

Conversion of 3-hydroxypropionate (3HP) from 1,3-propanediol (PDO) was improved by expressing dehydratase gene (dhaT) and aldehyde dehydrogenase gene (aldD) of Pseudomonas putida KT2442 under the promoter of phaCAB operon from Ralstonia eutropha H16. Expression of these genes in Aeromonas hydrophila 4AK4 produced up to 21 g/L 3HP in a fermentation process. To synthesize homopolymer poly(3-hydroxypropionate) (P3HP), and copolymer poly(3-hydroxypropionate-co-3-hydroxybutyrate) (P3HP4HB), dhaT and aldD were expressed in E.