title: Production of copolyesters of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates by E. coli containing an optimized PHA synthase gene.
abstract: BACKGROUND
  Microbial polyhydroxyalkanoates (PHA) are biopolyesters consisting of diverse monomers. PHA synthase PhaC2Ps cloned from Pseudomonas stutzeri 1317 is able to polymerize short-chain-length (scl) 3-hydroxybutyrate (3HB) monomers and medium-chain-length (mcl) 3-hydroxyalkanoates (3HA) with carbon chain lengths ranging from C6 to C12. However, the scl and mcl PHA production in Escherichia coli expressing PhaC2Ps is limited with very low PHA yield.
  
  
  RESULTS
  To improve the production of PHA with a wide range of monomer compositions in E. coli, a series of optimization strategies were applied on the PHA synthase PhaC2Ps. Codon optimization of the gene and mRNA stabilization with a hairpin structure were conducted and the function of the optimized PHA synthase was tested in E. coli. The transcript was more stable after the hairpin structure was introduced, and western blot analysis showed that both codon optimization and hairpin introduction increased the protein expression level. Compared with the wild type PhaC2Ps, the optimized PhaC2Ps increased poly-3-hydroxybutyrate (PHB) production by approximately 16-fold to 30% of the cell dry weight. When grown on dodecanoate, the recombinant E. coli harboring the optimized gene phaC2PsO with a hairpin structure in the 5' untranslated region was able to synthesize 4-fold more PHA consisting of 3HB and medium-chain-length 3HA compared to the recombinant harboring the wild type phaC2Ps.
  
  
  CONCLUSIONS
  The levels of both PHB and scl-mcl PHA in E. coli were significantly increased by series of optimization strategies applied on PHA synthase PhaC2Ps. These results indicate that strategies including codon optimization and mRNA stabilization are useful for heterologous PHA synthase expression and therefore enhance PHA production.
authors.affiliation: MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Gao
authors.forename: Xiao-Xi
authors.initials: XX
authors.lastname: Yuan
authors.forename: Zhen-Yu
authors.initials: ZY
authors.lastname: Shi
authors.forename: Ying-Ying
authors.initials: YY
authors.lastname: Guo
authors.forename: Xiao-Wen
authors.initials: XW
authors.lastname: Shen
authors.forename: Jin-Chun
authors.initials: JC
authors.lastname: Chen
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wu
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Chen
mesh_terms: D020155:3-Hydroxybutyric Acid / Q000378:metabolism; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000378:metabolism; D009154:Mutation; D011091:Polyesters / Q000378:metabolism; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis; D044223:Pseudomonas stutzeri / Q000201:enzymology; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D000094122:Polyhydroxybutyrates
chemical_list: D020155:3-Hydroxybutyric Acid; D000217:Acyltransferases; D001426:Bacterial Proteins; D006885:Hydroxybutyrates; D011091:Polyesters; D054813:Polyhydroxyalkanoates; D012333:RNA, Messenger; D011994:Recombinant Proteins; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C077225:poly(3-hydroxyalkanoic acid) synthase