Design of a single-chain multi-enzyme fusion protein establishing the polyhydroxybutyrate biosynthesis pathway

Mullaney, Jane A. and Rehm, Bernd H. A. (2010) Design of a single-chain multi-enzyme fusion protein establishing the polyhydroxybutyrate biosynthesis pathway. Journal of Biotechnology, 147 1: 31-36. doi:10.1016/j.jbiotec.2010.02.021


Author Mullaney, Jane A.
Rehm, Bernd H. A.
Title Design of a single-chain multi-enzyme fusion protein establishing the polyhydroxybutyrate biosynthesis pathway
Journal name Journal of Biotechnology   Check publisher's open access policy
ISSN 0168-1656
1873-4863
Publication date 2010-01-01
Year available 2010
Sub-type Article (original research)
DOI 10.1016/j.jbiotec.2010.02.021
Open Access Status Not Open Access
Volume 147
Issue 1
Start page 31
End page 36
Total pages 6
Place of publication Amsterdam, The Netherlands
Publisher Elsevier BV
Language eng
Subject 1305 Biotechnology
2402 Applied Microbiology and Biotechnology
Abstract Polyhydroxyalkanoates are biodegradable biocompatible polymers naturally produced by various bacteria and archaea. Biotechnological production in transgenic plants has already been demonstrated with efficient polyhydroxybutyrate production requiring targeting of the enzymes to the chloroplasts. Three enzymes are required to establish the polyhydroxybutyrate biosynthesis pathway in non-naturally producing microorganisms or plants. To facilitate production of biopolyesters in plants, a gene encoding a translational fusion of the polyhydroxybutyrate biosynthesis enzymes PhaA (β-ketothiolase), PhaB (acetoacetyl-CoA reductase) and PhaC (PHA synthase) was constructed. Escherichia coli harboring a plasmid encoding this fusion protein (PhaA-PhaB-PhaC) under control of the lac promoter accumulated polyhydroxybutyrate contributing to 0.4% (w/w) of cellular dry weight. Insertion of an extended linker between PhaA and PhaB increased polyhydroxybutyrate accumulation to 3.9% (w/w) of cellular dry weight. Introduction of a second plasmid encoding PhaA and PhaB restored polyhydroxybutyrate accumulation to wildtype levels of about 35% (w/w) of cellular dry weight suggesting that the functions of PhaA and/or PhaB were limiting factors. Deletion of PhaA in trans led to significantly reduced polyhydroxybutyrate production suggesting that the PhaA activity in the fusion protein is reduced. This study showed that a single-chain translational fusion protein comprising the three enzymes essential for polyhydroxybutyrate synthesis can be engineered which will strongly facilitate the establishment of recombinant polyhydroxybutyrate production organisms particularly requiring targeting to sub-cellular compartments such as the chloroplasts in plants.
Keyword Biopolyester
Biopolymer
Fusion protein
Polyhydroxyalkanoate
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: UQ Diamantina Institute Publications
 
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Created: Mon, 19 May 2014, 21:27:59 EST by Jane Mullaney on behalf of UQ Diamantina Institute