Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli

Ayaka Hiroe, Kenji Tsuge, Christopher T. Nomura, Mitsuhiro Itaya, Takeharu Tsuge

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

Ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineered Escherichia coli is an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized as phaCAB) encodes the essential proteins for the production of P(3HB) in the native producer, Ralstonia eutropha. The three genes of the phaCAB operon are phaC, which encodes the polyhydroxyalkanoate (PHA) synthase, phaA, which encodes a 3-ketothiolase, and phaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of the phaCAB operon (phaABC, phaACB, phaBAC, phaBCA, phaCAB, and phaCBA) on an expression plasmid in genetically engineered E. coli was examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of the pha genes relative to the promoter. The most balanced production result was achieved in the strain harboring the phaBCA expression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production.

Original languageEnglish
Pages (from-to)3177-3184
Number of pages8
JournalApplied and Environmental Microbiology
Volume78
Issue number9
DOIs
Publication statusPublished - 2012 May

Fingerprint

3-hydroxybutyric acid
Gene Order
operon
Operon
Molecular Weight
polyhydroxyalkanoates
molecular weight
Escherichia coli
gene
plasmid
Cupriavidus necator
plasmids
Plasmids
genes
Acetyl-CoA C-Acyltransferase
coenzyme A
Population Groups
films (materials)
limiting factor
Genes

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Food Science
  • Biotechnology
  • Ecology

Cite this

Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli. / Hiroe, Ayaka; Tsuge, Kenji; Nomura, Christopher T.; Itaya, Mitsuhiro; Tsuge, Takeharu.

In: Applied and Environmental Microbiology, Vol. 78, No. 9, 05.2012, p. 3177-3184.

Research output: Contribution to journalArticle

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