title: Hyperproduction of poly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli.
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 (1)H and (13)C 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.
authors.affiliation: Department of Biological Science and Biotechnology, MOE Key Lab of Bioinformatics and Systems Biology, School of Life Sciences, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, 100084, China.
authors.forename: Xiao-Yun
authors.initials: XY
authors.lastname: Zhou
authors.forename: Xiao-Xi
authors.initials: XX
authors.lastname: Yuan
authors.forename: Zhen-Yu
authors.initials: ZY
authors.lastname: Shi
authors.forename: De-Chuang
authors.initials: DC
authors.lastname: Meng
authors.forename: Wen-Jun
authors.initials: WJ
authors.lastname: Jiang
authors.forename: Lin-Ping
authors.initials: LP
authors.lastname: Wu
authors.forename: Jin-Chun
authors.initials: JC
authors.lastname: Chen
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Chen
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D047010:Clostridium kluyveri / Q000378:metabolism; D020118:Cupriavidus necator / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005947:Glucose / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D050644:Succinate-Semialdehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011091:Polyesters; D011994:Recombinant Proteins; C107955:poly(4-hydroxybutanoate); D019802:Succinic Acid; D050644:Succinate-Semialdehyde Dehydrogenase; C547505:gabD protein, E coli; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; C060970:poly-beta-hydroxybutyrate polymerase; D005947:Glucose