Biosynthesis of poly(3-hydroxypropionate-co-3-hydroxybutyrate) with fully controllable structures from glycerol.

As the most representative biodegradable thermoplastic, poly(3-hydroxybutyrate) (P3HB) has a limited range of applications because of its poor thermal and physical properties. To improve its properties, a novel biosynthetic system was designed to produce poly(3-hydroxypropionate-co-3-hydroxybutyrate) (P(3HP-co-3HB)) with fully controllable structures from inexpensive carbon source. In this system, two parallel synthetic pathways controlled by independent regulatory systems were used to produce the 3HP and 3HB monomers, respectively. Through tuning the expression level of appropriate genes, P(3HP-co-3HB) copolyesters were synthesized with a wide range of 3HP fraction from 11.5 mol% to 94.6 mol%. Differential scanning calorimetry analysis demonstrated that the thermal properties of P(3HP-co-3HB) copolymer were totally dependent on its composition. The bioreactor cultivation was also performed and accumulated 9.8 g/L P(48.2 mol% 3HP-co-3HB) using glycerol as sole carbon source, which represented the highest production so far.

Biosynthesis of poly(3-hydroxypropionate) from glycerol by recombinant Escherichia coli.

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In this study, a P3HP biosynthetic pathway from glycerol was constructed in recombinant Escherichia coli. The genes for glycerol dehydratase and its reactivating factor (dhaB123 and gdrAB, from Klebsiella pneumoniae), propionaldehyde dehydrogenase (pduP, from Salmonella typhimurium), and polyhydroxyalkanoate synthase (phaC1, from Cupriavidus necator) were cloned and expressed in E. coli. After culture condition optimization, the final engineered strain accumulated 10.1 g/L P3HP (46.4% of the cell dry weight) using glycerol and glucose as cosubstrates in an aerobic fed-batch fermentation. To date, this is the highest P3HP production without addition of any expensive precursor.

Prokaryotic expression and characterization of human AC3-33 protein.

The transcription factor, AP-1, plays an important role in cellular proliferation, transformation and death. We previously showed that AC3-33 (GenBank name: c3orf33, FLJ31139), significantly inhibited transcriptional activity of AP-1. In this study, we report a method to express and purify AC3-33 in E. coli using glutathione-S-transferase (GST) fusion system. A GST-fusion protein was created by insertion of AC3-33 gene into a pGEX-4T-1 vector. The fusion protein, GST-AC3-33, was expressed in BL21 strain, and purified by GSH-affinity chromatography followed by thrombin cleavage. The digested product was further purified in a GSH-affinity column. After cleavage and purification, the recombinant AC3-33 protein exhibited the expected size of 29 kDa by SDS-PAGE and Western blotting and inhibited transcriptional activity of AP-1 in a dual-luciferase reporter assay. The bioactive recombinant GST-AC3-33, can be used to decipher the physiological and biochemical role of this protein.