AGRE-0006 Novel Biosynthetic Routes for and Biodegradation of Polyhydroxy Alkanoates Made by Genetically Engineered Strains of Bacteria and Plants

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Biopolymers/Gums : Biotechnology : ECLAIR Cluster III - Carbohydrates : Paints/Coatings/Plastics

PHB expression in tobacco plant (control is on the right). The gas chromatograph shows peaks corresponding to PHB after extraction from plant.

SUMMARY

A number of species of bacteria produce granules of material within their cells which have the properties of thermoformable plastics and which are both biodegradable and compatible with living systems. Biodegradability is of value in many applications where the object concerned is likely to be placed into the open environment (eg horticultural mulches) but only expected to last a finite time, where it may be discarded as litter or disposed of through biological treatment systems (composting, anaerobic digestion). The properties of biological compatibility is of value in medical applications. A main constraint in the use of such microbial products, including polyhydroxybutyrate and its copolymer with hydroxyvaleric acid, is economic. This constraint can be overcome by increasing productivity, simplifying the process and extending the range of materials. These were some of the objectives of this project, supported under the ECLAIR programme, which extended to examination of biodegradation in a number of environmental situations. The main objectives focused on both the various microorganisms which produce such materials and the biochemistry and genetics of the synthetic pathways. This knowledge was then used to facilitate an alternative to fermentation as the manufacturing process - that is to grow the material in plants. To do this the individual genes have to be identified, cloned and monitored in the recipient host plant using specific antibody assays. Some of the genes involved have been expressed in tobacco and rape plants. Extensive information has been collected on the microbiology and biochemistry of synthesis of a number of materials in various types of bacteria. The rates of degradation of various materials have also been compared under different environmental conditions, and have been shown to depend on environment, temperature and polymer composition.

INTRODUCTION

PHB (poly-ß-hydroxybutyrate) is a thermoplastic polyester that is accumulated as an energy reserve material by various microorganisms including species of bacteria such as Alcaligenes, Bacillus, Pseudomonas and Rhizobium. Such bacteria can be cultivated in large scale fermenters and the product recovered by breaking the harvested cells followed by solvent extraction. Both PHB and its copolymer with hydroxyvaleric acid (pHB/v), which may also be produced in the same way, belong to a class of compounds known as polyhydroxy alkanoates (PHA). These materials behave as thermoformable plastics and are biodegradable and compatible with living organisms (biocompatible). Hence, they have potential applications in agricultural, industrial and medical fields since the polymers can be made into films, bottles, sheets, fibres etc. 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 was developed by ICI. However, in order to maximise the opportunities for use of these interesting materials, it was necessary to improve the economics of the process by increasing productivity and simplifying the process, as well as extending the range of materials available. More information was also required concerning biodegradation of the materials under varying conditions in natural environments.

APPROACH AND RESULTS

The pathways through which other PHBs are produced were studied in various organisms. This included investigations of PHA synthesis (consisting of medium-chain length 3-hydroxyalkanoic acids) in Pseudonomas aeruginosa PAOI and in another Pseudomonas species capable of synthesising a polymer blend. Elucidation of the biosynthesis of a polymer produced by Rhodococcus sp PP2 was also investigated. Two possible biochemical sources of propionyl-CoA have now been identified in this organism but further work has also shown that these may not be the sole routes leading to HV (hydroxy valerate). However, properties of the purified enzymes immediately responsible for PHA biosynthesis in Rhodococcus sp PP2 have been established. It has been found that although the synthase enzyme is capable of utilising both isomers of
3-hydroxybutyrate, the polymer produced by this organism is optically pure. The physical/mechanical properties of high HV polymer from Rhodococcus sp. PP2 have been investigated as have the changes in physical properties of various PHB/HV (poly-B-hydroxybutyrate hydroxy-valerate) copolymers after biodegradation under various conditions (see below). It was found that the presence of different levels of pigment produced by Rhodococcus sp. PP2 altered the physical properties of the isolated polymer, although the monomer composition remained unchanged. These studies included species which are capable of synthesising either long chain PHAs or PHB/HV copolymer solely from glucose. The biochemical pathways including key metabolic control points for synthesis of these polymers have been investigated in various organisms and the genetics of PHAMCL biosynthesis investigated in Pseudomonas aeruginosa PA01. The pha locus is not transcribed as a single operon and, as in Pseudomonas putida, unsaturated monomers have been detected in PHA synthesised by this organism. However, it has not been possible to construct an artificial PHA biosynthetic system using a mammalian thioesterase enzyme in any of the bacteria tried. Genetic analysis of PHA biosynthetic genes from other Pseudomonads has been completed and has shown that one strain possesses three functionally active PHA synthases. The PHA synthase gene from Rhodococcus sp PP2 has been sequenced and an open reading frame (ORF) has been found to encode a small protein associated with polymer granules. This protein has been isolated and characterised.

Batch fermentation studies have been completed to study the kinetics of PHB production using the self cloned strains of Alcalignes and alternative substrates. Continuous fermentation has been used to study the kinetics of PHB production with three mutants of Alcalignes. The kinetics of glucose transport in Alicagenes have been studied and the effect of different substrates on the rate of polymer production investigated as a way of understanding and improving productivity. Mutants with improved rates of polymer production have been isolated. Laboratory scale fermentations to make HO/HD (hydroxyoctanoate/hydroxydecanoate) copolymers from gluconate using Pseudomonas have been carried out and the physical/mechanical properties of HO/HD have been investigated. Large numbers of transformant rape and tobacco plants have now been generated for each of the three PHB genes. Biochemical and genetic analysis has shown expression of each of the three genes in rape plants and increased level of PHB over control plants correlated with expression of the synthase gene. Plants have also been generated by cotransformation and cross-pollination with more than one gene and resulting seeds collected for analysis of the progeny. Methods for plant plastid expression of each of the genes are complete or nearing completion. Although PHB and related polymers are classified as biodegradable, prior to this project little work had been done on the actual mechanism and organisms involved. These aspects were investigated over a range of conditions in various forms, including powder, film and fabricated articles, reflecting locations in which the products might be used, discarded or disposed of (soil, landfills, sewage works, etc).

PARTICIPANTS

ICI (now Zeneca) Seeds Bracknell (UK), Institut für Mikrobiologie der Georg-August Universitat, Gottingen (Germany), University of Hull, Department. of Applied Biology (UK), Rijksuniversiteit Gent (Belgium).