Douglas E. Dennis

Polyhydroxybutyrate, a Biodegradable Thermoplastic, Produced in Transgenic Plants

Polyhydroxybutyrate (PHB), a high molecular weight polyester, is accumulated as a storage carbon in many species of bacteria and is a biodegradable thermoplastic. To produce PHB by genetic engineering in plants, genes from the bacterium Alcaligenes eutrophus that encoded the two enzymes required to convert acetoacetyl—coenzyme A to PHB were placed under transcriptional control of the cauliflower mosaic virus 35S promoter and introduced into Arabidopsis thaliana. Transgenic plant lines that contained both genes accumulated PHB as electron-lucent granules in the cytoplasm, nucleus, and vacuole; the size and appearance of these granules were similar to the PHB granules that accumulate in bacteria.

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinant Escherichia coli grown on glucose

A recombinant Escherichia coli strain has been developed that produces poly(3-hydroxybutyrate-co-4-hydroxybutyrate) when grown in complex medium containing glucose. This has been accomplished by introducing into E. coli DH5 alpha separate plasmids harboring the polyhydroxyalkanoate (PHA) biosynthesis genes from Ralstonia eutropha (formerly named Alcaligenes eutrophus) and the succinate degradation genes from Clostridium kluyveri, respectively. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) levels reached 50% of the cell dry weight and contained up to 2.8 mol.% 4-hydroxybutyrate. The molecular weight of the polymer was 1.8 x 10(6).

Polyhydroxyalkanoate production in recombinant Escherichia coli

The bacterial species Escherichia coli has proven to be a powerful tool in the molecular analysis of polyhydroxyalkanoate (PHA) biosynthesis. In addition, E. coli holds promise as a source for economical PHA production. Using this microorganism, clones have been developed in our laboratory which direct the synthesis of poly-beta-hydroxybutyrate (PHB) to levels as high as 95% of the cell dry weight. These clones have been further enhanced by the addition of a genetically mediated lysis system that allows the PHB granules to be released gently and efficiently. This paper describes these developments, as well as the use of an E. coli strain to produce the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-3HV).

Formation of poly(3-hydroxybutyrate-co -3-hydroxyhexanoate) by PHA synthase from Ralstonia eutropha

The acetoacetyl-CoA reductase and the polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (formerly Alcaligenes eutrophus) were expressed in Escherichia coli, Klebsiella aerogenes, and PHA-negative mutants of R. eutropha and Pseudomonas putida. While expression in E. coli strains resulted in the accumulation of poly(3-hydroxybutyrate) [PHB], strains of R. eutropha, P. putida and K. aerogenes accumulated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [poly(3HB-co-3HHx)] when even chain fatty acids were provided as carbon source, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)] when odd chain fatty acids were provided as carbon source. This suggests that fatty acid degradation can be directly accessed employing only the acetoacetyl-CoA reductase and the PHA synthase. This is also the first proof that the PHA synthase from R. eutropha can incorporate 3-hydroxyhexanoate (3HHx) into PHA and has, therefore, a broader substrate specificity than previously described.

Perspectives on the production of polyhydroxyalkanoates in plants

Poly-β-hydroxybutyrate (PBH) was recently shown to be produced in genetically engineered plants which expressed the genes from Alcaligenes eutrophus responsible for the formation of PHB from acetoacetyl-CoA. The transgenic plants accumulated PHB as granules which were similar in size and appearance to the bacterial PHB granules. These observations suggest that large scale production of PHB and other polyhydroxyalkanoates in genetically altered crop plants may be feasible.

Protein organization on the PHA inclusion cytoplasmic boundary

Polyhydroxyalkanoate (PHA) cellular inclusions consist of polyesters, phospholipids, and proteins. Both the polymerase and the depolymerase enzymes are active components of the structure. Recently, proteins associated with these inclusions have been described in a number of bacterial species. In order to further clarify the structure and function of these proteins in relation to polymer inclusions, ultrastructural studies of isolated polymer inclusions were initiated. The surface boundary characteristics of polymer inclusions, produced by several genera of bacteria, two different Pseudomonas putida deletion mutants and by Escherichia coli recombinants, were examined. The recombinant E. coli carried either the PHB biosynthesis operon (phaCAB) from Ralstonia eutropha alone, or both this operon and a gene encoding an inclusion surface protein of R. eutropha (phaP). The results support two suggestions: (i) specific genes in the PHA gene cluster code for the proteins forming the surface boundary arrays which characterize the polymer inclusion; and (ii) transfer of such a gene would result in subcellular compartmentalization of accumulating polymer. Although the proteins appear to serve a similar function among different genera, nevertheless, the different surface proteins are encoded by a variety of non-homologous genetic sequences.

Aqueous release and purification of poly(β-hydroxybutyrate) from Escherichia coli

Abstract The poly(β-hydroxybutyrate) (PHB) biosynthetic genes of Ralstonia eutropha that are organized in a single operon ( phaCAB ) have been cloned in Escherichia coli, where the expression of the genes in the wild-type pha operon from plasmid pTZ18U-PHB leads to the formation of 50–80% PHB/celldry mass when the cells are grown in Luria–Bertani medium supplemented with 1% glucose (w/v). In combination with the phaCAB genes, expression of cloned lysis gene E of bacteriophage PhiX174 from plasmid pSH2 has been used to release PHB granules produced in E. coli . It was shown that small PHB granules in a semiliquid stage are squeezed out of the cells through the E-lysis tunnel structure which is characterized by a small opening in the envelope with borders of fused inner and outer membranes. All envelope components remain intact after E-lysis and can be removed from the mixture of released PHB granules by density gradient centrifugation. In addition, a modified E-lysis procedure is described which enables the release of PHB from cell pellets in pure water or low ionic strength buffer. PHB granules in aqueous solution can be aggregated by divalent cations. Addition of glassmilk speeds up the agglomeration of PHB granules and binding to glass beads can either be used for collection or further purification of PHB in aqueous solutions.

Preliminary analysis of polyhydroxyalkanoate inclusions using atomic force microscopy

Atomic force microscopy analysis of polyhydroxyalkanoate (PHA) inclusions isolated from sonicated Ralstonia eutropha cells revealed that they exhibit two types of surface structure and shape; rough and ovoid, or smooth and spherical. Smooth inclusions possessed linear surface structures that were in parallel arrays with 7-nm spacing. Occasionally, cracks or fissures could be seen on the surface of the rough inclusions, which allowed a measurement of approximately 4 nm for the thickness of the boundary layer. When the rough inclusions were imaged at higher resolution, globular structures, 35 nm in diameter, having a central pore could be seen. These globular structures were connected by a network of 4-nm-wide linear structures. When the inclusions were treated with sodium lauryl sulfate, the boundary layer of the inclusion deteriorated in a manner that would be consistent with a lipid envelope. When the boundary layer was largely gone, 35-nm globular disks could be imaged laying on the surface of the filter beside the inclusions. These data have facilitated the development of a preliminary model for PHA inclusion structure that is more advanced than previous models.

Investigation of the function of proteins associated to polyhydroxyalkanoate inclusions in Pseudomonas putida BMO1

Polyhydroxyalkanoate (PHA) granule associated proteins from Pseudomonas oleovorans were purified and the N-terminal sequences of two major proteins migrating in sodium dodecyl sulfate polyacrylamide gels with a relative molecular mass of 18 and 43 kDa (GA1 and GA2, respectively) were analyzed. Radiolabeled degenerate probes deduced from these amino acid sequences were used to identify genomic DNA fragments from P. oleovorans and Pseudomonas putida encoding GA1 and GA2. DNA sequence analysis of the fragments obtained from P. putida revealed that the genes encoding these proteins were adjacent to phaC2 and ORF3, the PHA synthase II gene and an open reading frame of unknown function, respectively, found at the P. oleovorans and P. aeruginosa PHA synthase gene locus. The open reading frames encoding GA1, GA2 and ORF3 or smaller fragments beginning at GA1 were inactivated by chromosomal insertion of the Tn5 kanamycin resistance gene block (neo). When these mutants were grown on mineral salts agar media under nitrogen limitation, containing gluconate or decanoate as carbon sources, they appeared more translucent than the wild-type grown under similar conditions. Gas-chromatographic analysis of the cellular dry mass revealed that the mutant strains accumulated 30-50% less PHA than the P. putida wild type.

Molecular Characterization of the Poly-ß-Hydroxybutyrate Biosynthetic Pathway of Alcaligenes Eutrophus H16

The poly-s-hydroxybutyrate biosynthetic pathway of Alcaligenes eutrophus H16 has been cloned into Escherichia coli, where it has been the subject of a series of molecular analyses. The DNA sequence of the pathway has been determined and analyzed for open reading frames and secondary structure. In vivo transcription/translation studies have identified the size of the gene products and these have been aligned with specific open reading frames. A putative promoter has been located approximately 300 bases upstream from the start of translation on the basis of deletion mutants and consensus sequence comparisons. PHB is made to levels approaching 90% of the cell weight, in large part due to the gene dosage effect of the multicopy plasmid on which the pathway resides. PHB production varies considerably in response to media alterations.