Abstract:
Genes encoding polyhydroxyalkanoate synthase, β-ketothiolase and acetoacetyl CoA reductase are isolated from the publicly available bacterium  Chromatium vinosum . Recombinant genomes of plants or other species of bacteria which contain these genes are capable of producing polyalkanoate polymers. The nucleotide sequences of the said three genees have been determined.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to the production of polyhydroxyalkanoate by the culture of microorganisms which produce same. 
     Poly-3-hydroxybutyrate is a linear polyester of D(−)-3-hydroxybutyrate. It was first discovered in  Bacillus megaterium  in 1925. Polyhydroxy-butyrate accumulates in intracellular granules of a wide variety of bacteria. The granules appear to be membrane bound and can be stained with Sudan Black dye. The polymer is produced under conditions of nutrient limitation and acts as a reserve of carbon and energy. The molecular weight of the polyhydroxybutyrate varies from around 50,000 to greater than 1,000,000, depending on the microorganisms involved, the conditions of growth, and the method employed for extraction of the polyhydroxybutyrate. Polyhydroxybutyrate is an ideal carbon reserve as it exists in the cell in a highly reduced state, (it is virtually insoluble), and exerts negligible osmotic pressure. 
     Polyhydroxybutyrate and related poly-hydroxy-alkanoates, such as poly-3-hydroxyvalerate and poly-3-hydroxyoctanoate, are biodegradable thermo-plastics of considerable commercial importance. 
     The term “polyhydroxyalkanoate” as used hereinafter includes copolymers of polyhydroxy-butyrate with other polyhydroxyalkanoates such as poly-3-hydroxyvalerate. 
     2. Background Information 
     Polyhydroxyalkanoate is biodegradable and is broken down rapidly by soil microorganisms. It is thermoplastic (it melts at 180° C.) and can readily be moulded into diverse forms using technology well-established for the other thermoplastics materials such as high-density polyethylene which melts at around the same temperature (190° C.). The material is ideal for the production of biodegradable packaging which will degrade in landfill sites and sewage farms. The polymer is biocompatible, as well as biodegradable, and is well tolerated by the mammalian, including human, body, its degradation product, 3-hydroxybutyrate, is a normal mammalian metabolite. However, polyhydroxyalkanoate degrades only slowly in the body and its medical uses are limited to those applications where long term degradation is required. 
     Polyhydroxyalkanoate, produced by the microorganism  Alcaligenes eutrophus , is manufactured, as a copolymer with of polyhydroxy-butyrate and polhydroxyvalerate, by Imperial Chemical Industries PLC and sold under the Trade Mark BIOPOL. It is normally supplied in the form of pellets for thermoprocessing. However, polyhydroxyalkanoate is more expensive to manufacture by existing methods than, say, polyethylene. It is, therefore, desirable that new, more economic production of polyhydroxy-alkanoate be provided. 
     SUMMARY OF THE INVENTION 
     An object of the present invention is to provide materials and a method for the efficient production of polyhydroxyalkanoate. 
     According to the present invention there are provided gene fragments isolated from the bacterium  Chromatium vinosum  and encoding PHA polymerase, acetoacetyl CoA reductase and β-ketothiolase. 
     Preferably the  C. vinosum  is of the strain designated D, available to the public from the Deutsche Sammlung fur Mikroorganismen under the Accession Number 180. 
     The invention also provides a 16.5 kb EcoR1 fragment of  C. vinosum  DNA, designated PP10, hybridizable to a 5.2 kb SmaI/EcoR1 fragment, designated SE52 isolated from  Alcaligenes eutrophus  and known to contain all three of said genes responsible for expression of PHAS. 
     The invention further provides a fragment of the said PP10 fragment, designated SE45, encoding the PHA-synthase and β-ketothiolase genes and a region, designated SB24, encoding the acetoacetyl CoA reductase gene. 
     The invention also provides a recombinant genome of a microorganism, preferably a bacterium or a plant, which contains one or more of said fragments designated PP10, SE45 and region SB24. 
     Finally, the invention provides a method for the manufacture of PHAs, comprising culturing the microorganism  Chromatium vinosum , or a bacterium of a different species having stably incorporated within its genome by transformation one or more PHA synthesising genes from  Chromatium vinosum.    
     The biosynthesis of polyhydroxyalkanoate from the substrate, acetyl-CoA involves three enzyme-catalysed steps. 
     The three enzymes involved are β-ketot hiolase, acetoactyl-CoA-reductase and polyhydroxy-butyrate-synthase, the genes for which have been cloned from  Chromatium vinosum . The three genes are known to facilitate production of polyhydroxyalkanoates, the reactions involved being represented as follows:                           
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     The invention will now be described with reference to the accompanying drawings, of which; 
     FIG. 1 is the physical map of the 16.5 kb EcoR1 fragment of  Chromatium vinosum  DNA, designated PP10. The positions of the restriction sites and positions and names of the sub-fragments are shown. PHA-synthase and β-ketothiolase genes are located in fragment SE45 and acetoacetyl CoA reductase in region SB24; 
     FIG. 2 is the map of PP10 showing the positions of the β-ketothiolase and acetoacetyl CoA reductase genes and of the PHA-synthase gene open reading frames ORF2 and ORF3. 
     FIG. 3 is the complete nucleotide sequence of fragment SE45 (SEQ ID NO:1). The transcriptional start sites and terminators for the β-ketothiolase gene and for ORF3 and ORF2 are shown. The positions of the “−10” and “−35” sequences are also shown, as are the positions of the putative ribosome binding sites (“s/d”). Translational start and stop (*) codon are also marked and the amino acid sequences of the β-ketothiolase (SEQ ID NO:2), ORF2 (SEQ ID NO:3) and ORF3 (SEQ ID NO:4) are give. 
     FIG. 4 shows the alignment of the amino acid sequences of  Chromatium vinosum  ORF3 (SEQ ID NO:4) with PHA polymerase of  Alcaligenes eutrophus  (SEQ ID NO:5) and PHA polymerases 1 and 2 of  Pseudomonas oleovorans  (SEQ ID NO:6 and SEQ ID NO:7). 
     FIG. 5 shows the complete nucleotide sequence of the DNA encoding PHA synthesis genes from  Chromatium vinosum  (SEQ ID NO:8) The positions of PHA polymerase (phbC) (SEQ ID NO:4), acetoacetyl CoA reductase (phbB) (SEQ ID NO:9) and ketothiolase (phbA) (SEQ ID NO:2), genes are shown and also; the positions of ORF2 (SEQ ID NO:3), ORF4, (SEQ ID NO:10) ORF5 (SEQ ID NO:11) and ORF7 (SEQ ID NO:12). 
     FIG. 6 shows the alignment of the amino acid sequences of ketothiolases encoded by  C. vinosum  (C.v.) (SEQ ID NO:2),  A. eutrophus  (A.e.) (SEQ ID NO:13),  Zoogloea ramigera  (Z.r ) (SEQ ID NO:14),  Escherichia coli  (E.c) (SEQ ID NO:10),  Saccharomyces uvarum  (S.u) (SEQ ID NO:17) and  Rattus norvegicus  (R.n.) (SEQ ID NO:17). 
     FIG. 7 shows the alignment of the amino acid sequences of acetoacetyl CoA reductases encoded by  C. vinosum  (Cv) (SEQ ID NO:17),  A. eutrophus  (A.e. ) (SEQ ID NO:18) and  Z. ramigera  (Z.r.) (SEQ ID NO:18) 
     FIG. 8 is a Table (Table 1) showing the heterolocous expression in  Escherichia coli  of DNA fragments from  C. vinosum . Activities of PHA biosynthetic enzymes expressed by the different fragments are shown. The levels of PHA accumulated in  E. coli  transformed with the fragments are also given. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Example 
     The organism  C. vinosum  was a gift from Dr J. Imhoff of the University of Bonn, Germany. 
     1. Isolation of DNA Fragments from  C. vinosum  Encoding PHA Synthesis Genes 
     A 5.2 kb SmaI/EcoRI fragment (SE52), which codes for all three PHA biosynthetic genes has previously been isolated from  Alcaligenes eutrophus  [Schubert et al., J. Bacteriol. 170 (1988)]. This fragment was used to detect PHA biosynthetic genes of  C. vinosum . EcoRI restricted genomic DNA of  C. vinosum  was blotted on to a nylon membrane and hybridized with biotinylated SE52 DNA. One signal appeared, representing a DNA fragment of 16.5 kb. 
     A λL47 gene bank from  C. vinosum  genomic DNA was prepared and plates with approximately 800 plaques were blotted on to nylon membranes and hybridized with biotinylated SE52 DNA. One positive recombinant phage was isolated, which harboured a 16.5 kb EcoRI fragment, which was designated PP10 (FIG.  1 ). With PP10 and a 9.4 kb EcoRI/PstI subfragient (EP94) of PP10, the phenotype of the wild type could be restored in PHA-negative mutants of  A. eutrophus.    
     Expression studies in  E. coli  (see below) showed that a 4.5 kb SmaI/EcoRI (SE45) subfragment of EP94 encodes for PHA synthase and β-ketothiolase. The nucleotide sequence of this fragment was determined by the dideoxy-chain termination method of Sanger et al. with alkaline denatured double stranded plasmid DNA. The T7-polymerase sequencing kit of Pharmacia, Uppsala, Sweden, was used with 7-deazaguanosine- 5 ′-tri-phosphate instead of dGTP. Most of the sequence was determined with a set of unidirectional overlapping deletion clones generated by exonuclease III digestion. For sequencing regions which were not covered by the deletion plasmids synthetic oligonucleotides were used. 
     It was not possible to clone the 4.9 kb SmaI/PstI fragment PS49 in a multi-copy vector. Therefore, fragment EP94 (FIG. 1) was treated with Exonuclease Bal31, ligated to Bluescript Sk and transferred to  E. coli  X1- 1 Blue. A clone was isolated which harboured Bluescript SK with a 5.5 kb fragment (B55) and which expressed β-ketothiolase and NADH-dependent reductase activity. 3146 base pairs of B55 were part of the SE45 fragment. The other part (approximately 2350 base pairs, SB24) has been sequenced applying the primer hopping strategy. The sequence and the position of the reductase gene on SB24 are known. The results of these studies, including the organisation of the PHA biosynthetic genes in  C. vinosum  and the sites of the ketothiolase, reductase and PHA synthase genes are shown in FIG.  2 . The determination of the full sequence of SB24 is in progress. 
     2. Sequence Analysis of the  C. vinosum  PHB Synthetic Genes 
     The nucleotide sequence of SE45 is shown in FIG. 3 (SEQ ID NO:1). The fragment size of SE45 is 4506 bp. 
     2.1 PHB Synthase 
     The fragment sequence corresponding to the PHB synthase gene is designated as ORF3. The determination of synthase activity of deleted plasmids containing SE45 (See below) gave evidence that expression of ORF2 is also required for expression of synthase activity. 
     ORF2 and ORF3 are transcribed as an operon. The determination of the transcription start site of ORF2 was conducted by S1 nuclease mapping. This site occurs at bp 3059 from the 3′ end of SE45. A putative “−10” site, given as 5′-ACAGAT-3′occurs at bp 3073-3078, and a “−35” site occurs at bp 3092-3099. A putative ribosome binding Site occurs at bp 3040-3045. The translation start codon commences at bp 3030. The translation stop codon occurs at bp 1958. 
     The putative ribosome binding site of ORF3 occurs at bp 1907-1911. The translation start ATG for ORF3 occurs at bp 1899, and the translation stop codon at bp 833. Putative transcriptional terminator sites occur at hairpin structures at bp 773-786 and 796-823. 
     ORF2 encodes, apolypypeptide of 358 amino acids with a MW of 40525 da (SEQ ID NO:5). ORF3 encodes a poly eptide of 356 amino acids with a MW of 39739 da (SEQ ID NO:4). The gene size of ORF3 is approximately 30% smaller as compared with the PHA polymerase genes of  A. eutrophus  and  P. oleovorans . The alignments of the primary structures of  C. vinosum  PHA polymerase,  A. eutrophus  PHA polymerase and  P. oleovorans  PHA polymerases 1 and 2 are shown in FIG.  4 . Thus the ORF3  C. vinosum  polymerase is shorter than the other polymerase enzymes, lacking the first 172 amino acids from the NH 2  terminus of the  A. eutrophus  PRA polymerase, and the first 148 amino acids of the  Pseudomonas polymerases . The amino acid sequence of ORF3 (SEQ ID NO:4) exhibited an overall homology of 25% to the polymerase of  A. eutrophus , with certain discrete regions of conserved sequence. 
     The amino acid sequence of ORF2 (SEQ ID NO:3) showed no significant homology to other enzymes in the NBRF gene bank. 
     2.2 βketothiolase 
     The β ketothiolase and acetoacetyl CoA reductase genes are transcribed in opposite direction to ORF2 and ORF3 (FIG.  2 ). A “−10” site in the identified ketothiolase promoter occurs at bp 3105-3111, and a “−35”site at bp 3082-3086. A putative ribosome binding site occurs at bp 3167-3171. The translation starts signal occurs at bp 3181. The translation stop codon occurs at bp 4361. 
     The aligments of the primary structures of β ketothiolases from  Chromatium vinosum  and other sources are shown in FIG.  5 . Considerable homology is apparent between the amino acid sequences of ketothiolases from  C. vinosum  and other bacterial and non-bacterial sources. 
     2.3 Acetoacetyl CoA Reductase 
     The alignments of the primary structures of acetoacetyl CoA reductases from  C. vinosum, A. eutrophus  and  z. ramigera  are shown in FIG.  6 . All three reductases are of similar chain length, while considerable homology is apparent between the sequences of reductases from these bacteria. 
     The  Chromatium vinosum  PHA synthetic genes therefore differ from the PHA synthetic genes of  A. eutrophus  and  P. oleovorans  in the following respects: 
     i) Whereas  A. eutrophus  PHB polymerase, acetoacetyl CoA reductase and β ketothiolase genes are all transcribed as an operon, in  C. vinosum  the ketothiolase and reductase genes are transcribed separately from the polymerase, and are transcribed in the opposite direction to the polymerase ORF3 and ORF2 genes. 
     ii) In contrast to  A. eutrophus , where one gene product is required for polymerase activity, in  C. vinosum  two gene products, represented by ORF2 and ORF3 are required for expression of polymerase activity. 
     iii) The  C. vinosum  ORF3 polymerase is 172 amino acids shorter, at the amino terminus, than the  A. eutrophus  polymerase, and 148 amino acids shorter than the  P. oleovorans  polymerases 1 and 2. The  C. vinosum  ORF3 shows only 25% homology with the primary sequence of the  A. eutrophus  polymerase. 
     iv) The  A. eutrophus  acetoacetyl CoA reductase enzyme involved in PHB synthesis is NADPH specific, while the  C. vinosum  enzyme exhibits a marked preference for NADH. 
     Between the structural genes for ketothiolase and acetoacetyl CoA reductase of  Chromatium vinosum , two open reading frames (ORF4 (SEQ ID NO:10), and ORF5 (SEQ ID NO:11) appear, and downstream from the reductase gene an ORF7 has been identified (FIG.  5 ). No additional ORFs were identified in the PHA coding region of  A. eutrophus.    
     3. Expression of  C. Vinosum  PHB Synthetic Genes in Other Bacteria. 
     With fragments PP10 and EP94 the ability to synthesise PHB could be restored to PHB negative mutants of  A. eutrophus . Recombinant strains of the FHB negative mutant  A. eutrophus  PHB-4, transformed with these fragments, were able to synthesise polymers containing 3-hydroxybutyrate and 3-hydroxyisovalerate at significant proportions, when supplied with appropriate substrates. 
     Studies on expression of  C. vinosum  DNA fragments in  E. coli  are presented in Table 1. Thus  E. coli  transformed with plasmids containing fragments PP10 and EP94 expressed PHB polymerase, acetoacetyl CoA reductase and β ketothiolase activities. They also synthesised PHB up to between 10 and 12% dry weight.  E. coli  transformed with plasmids containing fragment SE45 expressed PHB polymerase and β ketothiolase, but not acetoacetyl CoA reductase, and were unable to synthesise PHB. 
     4. Polymer Biochemistry 
     The specific optical rotations of methyl 3-hydroxybutyric acid liberated by methanolysis of PHB from  C. vinosum  (accumulated from acetate), from  A. eutrophus  PHB-4 pHP1014:PP10 (accumulated from fructose) and  E. coli  S17-1 pSUP202:PP10 (accumulated from glucose) were all negative. The determined values of the specific optical rotation were similar to those for PHB isolated from  A. eutrophus  (accumulated from fructose).