BioMatNet Item: FAIR-CT96-1780 - PHAstics: Sustainable Production in Biodegradable Polyesters in Starch-Storing Crop Plants

FAIR-CT96-1780
PHAstics: Sustainable Production in Biodegradable Polyesters in Starch-Storing Crop Plants

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Biotechnology : FAIR Area 1.2 - Green Chemicals and Polymers Chain : Paints/Coatings/Plastics : Plant Genetics : Separation/Fractionation

	Proposal No:	FAIR-CT96-1780
	Date Prepared:	January 2000, May 1999, April 1998
	Source:	Final Report Abstract Second Annual Progress Report First Annual Progress Report

Final Report Abstract

Objectives

The main objective of the current project was the sustainable production and application of biodegradable polyesters, poly (3-hydroxyalkanoates), PHAS, by starch-storing crop plants, supported by knowledge generated by bacterial PHA pathway engineering and fermentation. It is anticipated that the broad applicability of these biodegradable polyesters will contribute to a considerable decrease in the current accumulation of non-degradable plastics and a large number of other commodities produced from mineral oils. These objectives were approached as outlined below.

Activities

Bacterial genes and pathways that specify the production of a number of different fatty acids and PHA precursors were isolated and characterised. Their corresponding gene products were expressed in recombinant bacteria and yeast cells, and analysed. One important aspect of the current project was also to identify novel pathways in order to generate new co-polyesters for an increased flexibility in PHA species that can be used for a broad field of applications. Within this objective of special importance was the notion that in starch-storing crop plants the ultimate precursor for de novo fatty acid biosynthesis would be sucrose (LJDP-glucose) which would normally be converted to starch. Therefore, bacterial species employed for PHA synthesis were genetically modified in such a way that they were able to use carbohydrates such as gluconate as substrate for de novo PHA synthesis.

Based on these results minimum gene sets were identified that theoretically would be needed to produce PHA in yeasts and starch-storing crop plants. These genes included the R. eutropha phbB (ketothiolase) and phbC (PBB polymerase) genes for PBB biosynthesis, the P. oleovorans phaC1 and phaC2 PHA polymerase genes and the P. putida phaG (acyl-CoA transferase) and E coli 'tesA (truncated thioesterase) precursor supply genes.

In the oleaginous yeast Cryptococcus curvatus PHB has been produced using the R. eutropha phbB and phbC genes. Suitable PHA biosynthetic genes and appropriate plant signal sequences for tissue (pea seed, potato tuber), compartment (plastid) and stage-specific expression were introduced into normal starch-storing crops (pea and potato). Standard transformation procedures were employed using Agrobacterium tumefaciens and newly developed methods for multiple gene transfer into potato using particle bombardment. The target crops included normal and aberrant representatives, in which the accumulation of starch had been reduced or abolished in favour of the formation of hexes and/or lipids.

For pea rug- (rugosus, wrinkled) mutants were employed, whereas for potato ADP-glucose phosphorylase (AGP-ase) antisense transformants became available (containing the nptII resistance marker). For the latter transformation vectors were constructed based on pGREEN that would allow re-transformation and selection for Basta-resistance. A pGREEN-based set of vectors was also used for primary selection of pea transgenics.

Results

A large number of bacterial mutants and transgenics have been produced, which allow high-yield production of conventional and new (co-)polyesters. A method was developed using Nile Red fluorescence for semi on-line measurements of the PHA production during bacterial fermentation, which indicates the optimal time of harvesting. PHB production in C curvatus shows for the first time that this organisms can be genetically modified with a bacterial gene that influences its fatty acid profile.

Hundreds of transgenic potato and peas were produced, potentially containing all essential combinations of genes mentioned above. A large number of these still await genomic and mRNA expression analyses. The first generation of potato transformants contains the phaC polymerase gene and shows a mRNA of expected size. However, the accompanying PhaC polymerase enzyme activity could not be detected, possibly due to the low levels of expression in plastids.

Methods were developed for the purification of 'green'mcl-PHAs and for the assessment of their physico-chemical characteristics in order to ascertain their practical applicability. A clear example of a totally new application was found by using mcl-PHA latex as an environmentally safe paint binder (patent pending).

Second Annual Progress Report

Objectives
The main objective of the current research project is the production of biodegradable plastics poly(3-hydroxy-alkanoates) by starch-storing crop plants via biotechnological approaches. For this purpose more knowledge is needed on genes involved in the synthesis of PHAs and their precursors, both in homologous and heterologous microbial systems. In addition, pathways leading to novel types of PHAs are being investigated.

Description of work
The project involves the identification, isolation and characterisation of bacterial genes involved in the biosynthesis of different mcl-PHAs and PHB. In addition, genes that mediate the supply of sufficient and adequate precursors both from bacterial and plant origin will similarly be isolated and characterised. In different combinations these genes will be introduced, firstly into bacteria and yeasts in order to identify the minimum gene set required for plants, and then introduced into low-starch potato and pea. PHAs to be produced will be extracted, processed, characterised and applications will sought in the food and packaging industry.

A number of these bacterial genes involved in mcl-PHA and PHB have been made available to Partners 1 and 4 for plant transformations already.

Low-starch potato varieties and pea mutants will be and are available, respectively. For the introduction of several essential genes into potato and pea multiple transformations and crossing of single transgenics is envisaged. Potato transformants containing and expressing the P. oleovorans phaC2 gene (in pET100) in microtubers have been generated, but no PHA has yet been detected, suggesting that the supply of precursors is insufficient or inadequate (or plant transformation vectors need optimisation). A microtuber induction system has been optimised for potato cultivars used. Also the P. oleovorans phaCl and Alcaligenes eutrophus phb-genes have been cloned into plant transformation vectors and used for transformation. Alcaligenes eutrophus has now been renamed to Ralstonia eutropha. A second marker (bar) system is currently under construction, which will allow re-transformation of available and coming primary potato transformants for Bialaphos-resistance and the introduction of genes for enhanced precursor supply. Within the framework of a Madam Curie Fellowship (FAIR-CT98-5036) a promising approach of simultaneous introduction of a number of different genes via Particle Bombardment is addressed. For pea the work has been initiated with vector constructions also based on bar selection, using the R. eutropha phbA, phbB and phbC genes and plastidial targeting sequences. Initial transformation experiments have yielded PPT resistant plant material that is currently being characterised. Similarly, phaCl, phaC2 and phaG genes will be inserted into vectors for pea transformation.

Achievements
A number of bacterial genes involved in the biosynthesis of mcl-PHA and PHB have been isolated, characterised and expressed in heterologous bacterial hosts. Also different copolymers have been produced. Further insight in the pathways involved is gained by precursor feeding experiments and by the production of specific mutants.

A number of PHA and PHB synthetic genes have become available for plant work.

A new pea plastidial targeting sequence has been isolated. The first putative pea transgenics have been isolated and multiplied for analysis. The first set of transgenic potato plants containing and expressing the P. oleovorans phaC2 gene has been produced and is currently further analysed. The same set of PHA synthetic genes has also been inserted into yeast (Cryptococcus curvatus) transformation vectors and initial transformations were carried out.

Future actions
A number of approaches for work on bacteria concerns: (i) heterologous expression of mcl-PHA synthase genes in the already available ß-oxidation mutants from R. eutropha, (ii) the production of 4HV-containing polyesters, (iii) the catabolism of levulinic acid in R. eutropha, (iv) theapplication of different E. coli fad-mutants and Pseudomonas transposon mutants, (v) identification and analysis of required gene(s) for the production of mcl-PHAs from non-related carbon sources, such as glucose, and (vi) the production, extraction and characterisation of PHAs (e.g. molecular weight determination, glass transition temperature, melting temperature) from recombinant E. coli strains. Further research, also based on literature, will focus on the identification and isolation of bacterial and plant genes involved in the supply of PHA precursors and de novo PH A synthesis. These genes will be introduced into the oleaginous yeast Cryptococcus curvatus, which already contains considerable amounts of fatty acids as storage compounds and also into plants by re-transformation and/or crossing.

Once transgenic potato and pea phaC1 transformants will be available, proper targeting into plastids will be monitored using available antibodies (ETHz).

The detection of PHAs in plants will firstly be performed following routine methods involving Nile Blue and Nile Red staining and later by more sophisticated techniques like in situ natural abundance ¹³C NMR. Once PHAs will have been detected methods for their extraction, processing, characterisation and their applications will be addressed.

First Annual Progress Report

Description of work
The project involves the identification, isolation and characterisation of bacterial genes involved in the biosynthesis of different mcl-PHAs and PHB. In addition, genes that mediate the supply of sufficient and adequate precursors both from bacterial and plant origin will similarly be isolated and characterised. In different combinations these genes will be introduced, firstly into bacteria and yeasts in order to identify the minimum gene set required for plants, and then introduced into starch-less potato and pea. PHAs to be produced will be extracted, processed, characterised and applications will sought in the food and packaging industry.

State of progress
Partners 2 and 3 have made considerable progress in the isolation and characterisation of bacterial genes involved in the biosynthesis of different types of PHAs, including e.g 3HB, 3HV and 4HV-PHA-containing polyesters. Mutants have been generated and corresponding genes are being characterised to further elucidate pathways involved in the biosynthesis of PHAs. Also recombinant bacteria have been constructed able to produce a number of PHAs. A number of these bacterial genes involved in mcl-PHA and PHB have been made available to Partners 1 and 4 for plant transformations already. Further bacterial genes involved in precursor supply are now being identified and isolated. Starch-less potato varieties and pea mutants will be and are available, respectively. For the introduction of several essential genes into potato and pea multiple transformations and crossing of single transgenics is envisaged. Potato transformants containing the P. oleovorans phclC2 gene (in pET 100) have been generated, but no PHA has yet been detected in microtubers, suggesting that the supply of precursors is insufficient or inadequate (or plant transformation vectors need optimisation). A microtuber induction system has been optimised for potato cultivars used. Also the P. oleovorans phaC I and A. elutrophus phb-genes are now being cloned into plant transformation vectors. A second marker (bar) system is currently under construction, which will allow re-transformation of available and coming primary potato transformants for bialaphos-resistance and the introduction of genes for enhanced precursor supply. For pea the work has initiated with vector constructions also based on bar selection, using the A. elutrophus phbA, phbB and phbC genes and plastidial targeting sequences. Initial transformation experiments are underway. Similarly, the phb-genes are currently inserted into potato transformation vectors.

Achievements:
A number of bacterial genes involved in the biosynthesis of mcl-PHA and PHB have been isolated, characterised and expressed in heterologous bacterial hosts. Also different copolymers have been produced. Further insight in the pathways involved is gained by precursor feeding experiments and by the production of specific mutants. A number of PHA and PHB synthetic genes have become available for plant work. A new pea plastidial targeting sequence has been isolated. The first pea transformation vectors have been constructed. The first set of transgenic potato plants containing the P. oleovorans phaC2 gene has been produced and is currently further analysed.

Future activities
A number of approaches for work on bacteria concerns: (i) heterologous expression of mcl-PHA synthase genes in the already available (-oxidation mutants from A. elutrophus, (ii) the production of 4HV-containing polyesters, (iii) the catabolism of levulinic acid in A. elutrophus, (iv) the application of different E. coli fad-mutants and Pseudomonas transposon mutants, (v) identification and analysis of required gene(s) for the production of mcl-PHAs from non-related carbon sources, such as glucose, and (vi) the production, extraction and characterisation of PHAs (e.g. molecular weight determination, glass transition temperature, melting temperature) from recombinant E. coli strains. Further research, also based on literature, will focus on the identification and isolation of bacterial and plant genes involved in the supply of PHA precursors. These genes will be introduced into the oleaginous yeast Cryptococcus curvatus, which already contains considerable amounts of fatty acids as storage compounds and also into plants by re-transformation and/or crossing. A highly promising approach involving multiple, simultaneous transformation by particle bombardment has been validated for other plant species (JIC), but would need developing for potato and pea. However, this possibility will be considered since it would improve transformation efficiencies. Once transgenic potato and pea phaC1 transformants will be available, proper targeting into plastids will be monitored using available antibodies (ETHz). The detection of PHAs in plants will firstly be performed following routine methods and later by more sophisticated techniques like in situ ( natural abundance ¹³C NMR. Once PHAs have been detected methods for their extraction, processing, characterisation and applications will be addressed.