PHASTICS: Sustainable production of biodegradable polyesters in starch-storing crop plants

• Research unit: EU RFP
• Project number: 96.0348

Key words (English)

PHAs Poly-3-hydroxyalkanoates

Alternative project number (English)

EU project number: FAIR-CT96-1780

Research programs (English)

EU-programme: 4. Frame Research Programme - 4.3 Biomedical/Health research

Short description (English)

See abstract

Partners and International Organizations (English)

Coordinator: Instituut voor Agrotechn. Onderzoek ATO-DLO (NL)

Abstract (English)

Poiy-3-hydroxyalkanoates (PHAs), a large and versatile family of materials, currently receiving much attention because of their potential as renewable and biodegradable plastics. These biopolymers are stored by various bacteria as an energy reserve in much the same way as mammals accumulate fats. The overall objective of this contribution to produce medium-chain-length PHAs in recombinant Escherichia coli from different carbon sources was already achieved in the first two reporting periods. In addition, a new and alternative PHA synthetic pathway was engineered by introducing the acetoacetyl-CoA reductase encoding gene phbB of Ralstonia eutropha into E. coli equipped with the PHA polymerase of Pseudomonas oleovorans. The acetoacetyl-CoA reductase catalyses the reaction from ketoacyl-CoA to R-3-hydroxyalkanoyl-CoA, a reverse step of the b-oxidation, and therefore generates the precursor for the PHA polymerase. Introduction of the phbB gene into E. coli fadR/fadA strains caused significant changes in monomer composition and physical property of the polymer, such as increased molecular weight and loss of melting point. Furthermore, the 3 ketoacyl-ACP reductase encoding gene fabG of P. aeruginosa which is required for fatty acid synthesis was introduced into E. coli. It could be shown that the enzyme converts in addition to ACP coupled substrates the B-oxidation intermediate 3-ketoacyl-CoA to R-3-hydroxyalkanoyl-CoA, and therefore enhances polymer accumulation in PHA polymerase containing E. coli recombinants.
In order to redirect part of the carbon flow from the TCA cycle towards the fatty acid synthesis pathway and ultimately PHA biosynthesis, an isocitrate lyase knock out mutant of Pseudomonas putida KT2442 was generated by transposon mutagenesis. The mutant was not able to grow on acetate as sole carbon source due to disruption of the glyoxylate shunt. When bacteria were grown on gluconate or gluconate/fatty acid mixtures PHA accumulation was enhanced compared to the wild type strain.
Recently, more emphasis was put on the key enzyme, the PHA polymerase. An improved activity assay for PHA polymerases was developed. The activity assay is based on Coenzyme A release which can be detected using 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), a compound which specifically reacts with thiol groups. Using optimized conditions, activities up to 27 U/mg granule bound polymerase have been measured. The PHA polymerase was purified from overproducing recombinants by isolation of insoluble protein aggregates and refolding of those inclusion bodies in the presence of sepharose. Activities up to 4-5 U/mg purified PHA polymerase could be obtained and synthesized PHA product detected by gas chromatography. Further characterization of the enzyme is currently under way.

References in databases (English)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 96.0348