Production of Medium-Chain-Length Poly(3-Hydroxyalkanoates) from Gluconate by RecombinantEscherichia coli

Abstract

It was shown recently that recombinant Escherichia coli, defective in the β-oxidation cycle and harboring a medium-chain-length (MCL) poly(3-hydroxyalkanoate) (PHA) polymerase-encoding gene of Pseudomonas, is able to produce MCL PHA from fatty acids but not from sugars or gluconate (S. Langenbach, B. H. A. Rehm, and A. Steinbüchel, FEMS Microbiol. Lett. 150:303–309, 1997; Q. Ren, Ph.D. thesis, ETH Zürich, Zürich, Switzerland, 1997). In this study, we report the formation of MCL PHA from gluconate by recombinant E. coli. By introduction of genes coding for an MCL PHA polymerase and the cytosolic thioesterase I (′thioesterase I) into E. coli JMU193, we were able to engineer a pathway for the synthesis of MCL PHA from gluconate. We used two expression systems, i.e., thebad promoter and alk promoter, for the ′thioesterase I- and PHA polymerase-encoding genes, respectively, which enabled us to modulate their expression independently over a range of inducer concentrations, which resulted in a maximum MCL PHA accumulation of 2.3% of cell dry weight from gluconate. We found that the amount of PHA and the ′thioesterase I activity are directly correlated. Moreover, the polymer accumulated in the recombinantE. coli consisted mainly of 3-hydroxyoctanoate monomers. On the basis of our data, we propose an MCL PHA biosynthesis pathway scheme for recombinant E. coli JMU193, harboring PHA polymerase and ′thioesterase I, when grown on gluconate, which involves both de novo fatty acid synthesis and β-oxidation.

The isolation of poly(3‐hydroxybutyrate) from recombinant Escherichia coli XL1‐blue using the digestion method

“…For example, short-chain-length (scl) PHAs (for example, PHB, poly(ˇ-hydroxybutyrate-co--hydroxyvalerate) (P(3HB-co-3HV))) were synthesised, and the content of PHA compared to the dry cell weight was 20-80% (Hein et al, 1997;Valentin and Dennis, 1997;Slater et al, 1998;Liu and Steinbuchel, 2000;Kahar et al, 2005;Nikel et al, 2006;Gong et al, 2008). Medium-chain-length (mcl) PHAs (for example, poly(3-hydroxyhexanoate), poly(3hydroxydodecanoate)) and poly(3-hydroxyoctanoate) copolymer) can also be produced (Kim et al, 1997;Klinke et al, 1999;Chen et al, 2001;Jung et al, 2001).…”

Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum

“…Thioesterase I encoded by the tesA gene shows broad specificity for C12 to C18 acyl-CoA esters [10]. Thioesterase I was reported to mediate acyl-ACP intermediates from the fatty acid de novo biosynthesis pathway to fatty acid β-oxidation in E. coli [11]. On the other hand, thioesterase II encoded by the tesB gene has broader substrate specificity, and is capable of hydrolysis of C6 to C18 acyl-CoA esters [9].…”

Characterization of temperature-sensitive and lipopolysaccharide overproducing transposon mutants ofPseudomonas putidaCA-3 affected in PHA accumulation

“…While the study of genes and their products directly related to mclPHA biosynthesis (e.g. PHA polymerase, PHA depolymerase) has been reported (Klinke et al, 1999;Jendrossek & Handrick, 2002;Luengo et al, 2003), there is a dearth of information on the role of other genes and gene products involved in secondary pathways such as nitrogen metabolism and stress responses.…”