Hyperproduction of poly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli
Open Access
- 2 May 2012
- journal article
- Published by Springer Science and Business Media LLC in Microbial Cell Factories
- Vol. 11 (1), 54
- https://doi.org/10.1186/1475-2859-11-54
Abstract
Background: Poly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as γ-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB). Results: Recombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (PhaP or phasins) including PhaP1, PhaP2, PhaP3 and PhaP4 from R. eutropha were heterologously expressed in the recombinant E. coli, respectively, leading to different levels of improvement in poly(4HB) production. Among them PhaP1 exhibited the highest capability for enhanced polymer synthesis. The recombinant E. coli produced 5.5 g L-1 cell dry weight containing 35.4% poly(4HB) using glucose as a sole carbon source in a 48 h shake flask growth. In a 6-L fermentor study, 11.5 g L-1 cell dry weight containing 68.2% poly(4HB) was obtained after 52 h of cultivation. This was the highest poly(4HB) yield using glucose as a sole carbon source reported so far. Poly(4HB) was structurally confirmed by gas chromatographic (GC) as well as 1H and 13C NMR studies. Conclusions: Significant level of poly(4HB) biosynthesis from glucose can be achieved in sad and gabD genes deficient strain of E. coli JM109 harboring an engineering pathway encoding succinate degradation genes and PHB synthase gene, together with expression of four PHA binding proteins PhaP or phasins, respectively. Over 68% poly(4HB) was produced in a fed-batch fermentation process, demonstrating the feasibility for enhanced poly(4HB) production using the recombinant strain for future cost effective commercial development.Keywords
This publication has 48 references indexed in Scilit:
- Synthesis of Diblock copolymer poly-3-hydroxybutyrate -block-poly-3-hydroxyhexanoate [PHB-b-PHHx] by a β-oxidation weakened Pseudomonas putida KT2442Microbial Cell Factories, 2012
- Comparative genomics study of polyhydroxyalkanoates (PHA) and ectoine relevant genes from Halomonas sp. TD01 revealed extensive horizontal gene transfer events and co-evolutionary relationshipsMicrobial Cell Factories, 2011
- Polyhydroxyalkanoates as a source of chemicals, polymers, and biofuelsCurrent Opinion in Biotechnology, 2011
- A transferable heterogeneous two-hybrid system in Escherichia coli based on polyhydroxyalkanoates synthesis regulatory protein PhaRMicrobial Cell Factories, 2011
- Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purificationMicrobial Cell Factories, 2010
- Bacillus subtilis as potential producer for polyhydroxyalkanoatesMicrobial Cell Factories, 2009
- Binding of the Major Phasin, PhaP1, fromRalstonia eutrophaH16 to Poly(3-Hydroxybutyrate) GranulesJournal of Bacteriology, 2008
- Increased diversification of polyhydroxyalkanoates by modification reactions for industrial and medical applicationsApplied Microbiology and Biotechnology, 2007
- Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16Nature Biotechnology, 2006
- The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutrophaMicrobiology, 2004