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NOVEL BIOSYNTHETIC ROUTES FOR AND BIODEGRADATION OF POLY- HYDROXY ALKANOATES MADE BY GENETICALLY ENGINEERED STRAINS OF BACTERIA AND PLANTS

Objective

Our strategic objective in this area is to produce industrially useful biodegradable polymers which are made from renewable agriculturally based feedstocks. At the end of this project we will have new and useful biodegradable materials, an improved manufacturing process, an increased understanding of the mechanism of biodegradability and, in the longer term, the prospect of a completely new route to the production of biodegradable materials via direct production in a crop plant.
Novel pathways of copolymer hydroxybutyrate/hydroxyvalerate (HB/HV) and polyhydroxydecanoate synthesis have been observed in Rhodococcus species and Pseudomonas polyhydroxyalkanoate respectively.

Physiological studies have provided clues as to the pathway of HB/HV synthesis in Rhodococcus. A number of genes of polyhydroxyalkanoate (PHA) synthesis have been cloned from both Rhodococcus and Pseudomonas. The properties of these novel polymers have been investigated. In addition, investigations of the effect of gene dosage on the physiology of PHB synthesis in recombinant organisms has been carried out. Expression vector systems have been constructed and individual genes of PHB synthesis have been sub cloned. Rates of biodegradation of both PHB and PHB/HV has been measured in a number of soils and aquatic environments. Microorganisms isolated from degrading polymer have been identified using chemotoxonomic techniques as well as conventional methodology.

Polyhydroxybutyrate (PHB) and its copolymer with hydroxyvaleric acid (PHB/HV) belong to a general class of compounds known as polyhydroxyalkanoates (PHA). These materials produced as granules within bacterial cells are thermoformable plastics which are biodegradable and biocompatible.
An extensive range of PHB negative mutants of Rhodococcus PP2 have been isolated and characterized with respect to the activity of the PHB biosynthetic enzymes.
Work is well advanced to clone PHA biosynthetic genes from Rhodococcus PP2 and Pseudomonas PP1. Putative PHB biosynthetic genes bave been detected in clones made from Rhodococcus deoxyribonucleic acid (DNA) by probing with labelled DNA from the Alcaligenes polymerase gene.
Clones of the polymerase gene have been isolated from Pseudomonas PP1 and other Pseudomonas species, and have been shown to be active when transferred to Alcaligenes.
Good progress has been made on a start to the sequencing of the polymerase genes from Pseudomonas PP1 and other Pseudomonas species.

Polyhydroxybutyrate (PHB) and its copolymer with hydroxyvaleric acid (PHB/HV) belong to a general class of compounds known as polyhydroxyalkanoates (PHA). These materials produced as granules within bacterial cells are thermoformable plastics which are biodegradable and biocompatible.
The enzymes immediately responsible for PHA synthesis have been isolated and some unusual features of the biosynthetic pathway identified. One enzyme has been purified and is to be used for N-terminal sequencing for probe design. Possible routes to the biochemical source of 3 hydroxyvalerate have been identified and are under active investigation.
A screen has been carried out to isolace further scrains of micro organisms capable of synthesizing PHB/HV from glucose alone. Several scrains of bacteria from coryneform genera have been shown to accumulate PHB/HV when grown on glucose.
The effect of growth conditions on yield and composition of polymer from PP2 has been studied. This physiological work has provided strong clues as to the biochemical route by which the organism is able to make PHB/HV.

Polyhydroxobutyrate (PHB) and its copolymer with hydroxyvaleric acid (PHB/HV) belong to a general class of compounds known as polyhydroxyalkanoates (PHA). These materials produced as granules within bacterial cells are thermoformable plastics which are biodegradable and biocompatible.
Molecular constructs for plant cell expression of cloned PHB biosynthetic genes have been designed. Good progress has been made in their construction and in subcloning of the individual genes of PHB synthesis. Experiments to assess the stability of PHB synthesizing enzymes derived from bacteria when in the environment of plant cell extracts have been carried out. PHB synthesising enzymes have been purified and will be used to make antibodies which will assist in the detection of clones expressing in plant cells.

Polyhydroxybutyrate (PHB) and its copolymer with hydroxyvaleric acid (PHB/HV) belong to a general class of compounds known as polyhydroxyalkanoates (PHA). These materials produced as granules within bacterial cells are thermoformable plastics which are biodegradable and biocompatible.
Laboratory fermentations to make PHB/HV copolymers from Rhodococcus have been carried out, although there was some difficulty due to extracellular polysaccharide synthesis. Physical properties of the material have been measured but the investigation of mechanical properties awaits larger quantities of polymer. The productivity of recombinant Alcaligenes strains has been investigated in laboratory fermenters with no significant effect on productivity being seen.
Poly beta-hydroxybutyrate (PHB) and its copolymer hydroxyvaleric acid (PHB/V) belong to a general class of compounds known as polyhydroxyalkanoates (PHA). These materials, produced as granules within bacterial cells, are thermoformable plastics which are both biodegradable and biocompatible.

A manufacturing process to produce PHB/V which involves the growth of a microorganism, Alcaligenes eutrophus, on an agricultural feedstock (sugar or glucose) supplemented by propionic acid, followed by a separation; a purification process is being developed by ICI. The purified material can be made into films, bottles, sheets, fibres, etc and has potential applications in agricultural, industrial, and medical fields. In order to maximize the opportunity for these interesting materials, it is necessary to improve the economics of the process (by increasing productivity and simplifying the process) and extend the range of materials available.

The scientific tasks of the project are as follows:
To study the enzymology of two novel routes to different PHA co-polymers which have recently been discovered by one of the project partners.
To clone genes for the key control steps of these novel pathways and transfer them to Alcaligenes eutrophus the organism used by ICI for PHB/V production. Further genetic work will compare the organization and structure of the PHA biosynthetic genes in the novel organisms.
The effect of cloned biosynthetic genes on the productivity of PHB/V manufacture will be studied.
The synthesis of PHA polymers is limited to prokaryotic organisms, but in recent years technology enabling transfer of prokaryotic genes into plants has been developed. Transfer of the PHA gene to plant cells and its subsequent expression and accumulation will be investigated.
In contrast to the fairly extensive literature on the biosynthesis of PHB little is known about the mechanism and kinetics of degradation. Some characteristics of intracellular depolymerization of PHB have been described but there are few reports of extracellular depolymerization. In order to determine the environmental fate of fabricated articles a much more extensive study is required and forms part of this programme.

Coordinator

ZENECA LTD.
Address
Stanhope Gate 15
W1Y 6LN London
United Kingdom

Participants (2)

GENT UNIVERSITY
Belgium
Address
Sint Pietersnieuwstraat 25
9000 Gent
GEORG-AUGUST-UNIVERSITY GOETTINGEN
Germany
Address
Grisebachstrasse 6
37077 Goettingen