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This study describes a comparison of the polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 of Pseudomonas mendocina. The P. mendocina pha gene locus, encoding two PHA synthase genes [phaC1Pm and phaC2Pm flanking a PHA depolymerase gene (phaZ)], was cloned, and the nucleotide sequences of phaC1Pm (1,677 bp), phaZ (1,034 bp), and phaC2Pm (1,680 bp) were determined. The amino acid sequences deduced from phaC1Pm and phaC2Pm showed highest similarities to the corresponding PHA synthases from other pseudomonads sensu stricto. The two PHA synthase genes conferred PHA synthesis to the PHA-negative mutants P. putida GPp104 and Ralstonia eutropha PHB–4. In P. putida GPp104, phaC1Pm and phaC2Pm mediated PHA synthesis of medium-chain-length hydroxyalkanoates (C6–C12) as often reported for other pseudomonads. In contrast, in R. eutropha PHB–4, either PHA synthase gene also led to the incorporation of 3-hydroxybutyrate (3HB) into PHA. Recombinant strains of R. eutropha PHB–4 harboring either P. mendocina phaC gene even accumulated a homopolyester of 3HB during cultivation with gluconate, with poly(3HB) amounting to more than 80% of the cell dry matter if phaC2 was expressed. Interestingly, recombinant cells harboring the phaC1 synthase gene accumulated higher amounts of PHA when cultivated with fatty acids as sole carbon source, whereas recombinant cells harboring PhaC2 synthase accumulated higher amounts when gluconate was used as carbon source in storage experiments in either host. Furthermore, isogenic phaC1 and phaC2 knock-out mutants of P. mendocina provided evidence that PhaC1 is the major enzyme for PHA synthesis in P. mendocina, whereas PhaC2 contributes to the accumulation of PHA in this bacterium to only a minor extent, and then only when cultivated on gluconate.

Eleven different cyanobacteria were investigated with respect to their capabilities to synthesize poly-3-hydroxybutyrate [poly(3HB)] and the type of poly-beta-hydroxyalkanoic acid (PHA) synthase accounting for the synthesis of this polyester. Several methods, including (i) Southern blot analysis using a phaC-specific DNA probe, (ii) Western blot analysis using specific polyclonal anti-PhaE antibodies raised in this study against PhaE of Synechocystis sp. strain PCC 6803, (iii) generation and sequence analysis of PCR products using phaC-specific oligonucleotides as primers, and/or (iv) cloning and sequence analysis of PHA synthase structural genes, were used to provide evidence for the presence of a type-III PHA synthase in the following cyanobacteria: Synechococcus sp. strains MA19 and PCC 6715, Chlorogloeopsis fritschii PCC 6912, Anabaena cylindrica SAG 1403-2, Cyanothece sp. strains PCC 7424, PCC 8303 and PCC 8801, and Gloeocapsa sp. strain PCC 7428. The screening was compared with corresponding studies using crude protein extracts and genomic DNA of Synechocystis sp. strain PCC 6803, as a positive control, which is so far the only cyanobacterium for which molecular data of the PHA synthase genes are available. No evidence for the presence of a type-III PHA synthase could be obtained for only three of the eleven investigated cyanobacteria (Stanieria sp. strain PCC 7437, Cyanothece sp. strain PCC 8955 and Gloeothece sp. strain PCC 6501). The entire PHA synthase structural genes of the two thermophilic cyanobacteria Synechococcus sp. strain MA19 and Chlorogloeopsis fritschii PCC 6912, and in addition a central region of the phaC gene of Cyanothece sp. strain PCC 8303, were cloned, sequenced and also heterologously expressed in Escherichia coli.

Many bacteria are able to synthesize polyesters of hydroxyalkanoic acids (PHA), which occur as insoluble cytoplasmic inclusions in the cell and can contribute significantly to the cellular dry matter. More than 100 different hydroxyalkanoic acids have been detected as constituents in PHA. This article summarizes strategies and possibilities to obtain these biodegradable and thermoplastic/elastomeric polymers by fermentation of wild-type bacteria as well as of mutants and recombinant strains from renewable resources, waste substrates or special precursor substrates. In addition, in vitro biosynthesis of PHA employing the purified polymerizing enzyme (PHA synthase) is described. Throughout analysis and cloning of the bacterial PHA biosynthesis genes enabled scientists to also establish this pathway in non-PHA producing eukaryotic organisms such as yeast, plants and animals. This will allow new processes for cheap and economic production of PHA.

Two gene loci, which hybridized with pdhA, the structural gene of the E1 component of the Alcaligenes eutrophus pyruvate dehydrogenase complex [Hein, S. & Steinbuchel, A. (1994) J. Bacteriol. 176, 4394–4408], were identified on two nonrelated A. eutrophus chromosomal Bam HI fragments by using a pdhA -specific DNA probe. These data indicated that A. eutrophus possesses, beside PdhA, two additional distinct pyruvate dehydrogenases (E1). A 6.8-kbp genomic Bam HI fragment of A. eutrophus was cloned, and sequence analysis of a 3.896-kbp region revealed the structural gene pdhE (2.694 kbp) for a second pyruvate dehydrogenase (E1), which was not clustered with structural genes for other components of 2-oxo acid dehydrogenase complexes. The A. eutrophus pdhE gene product (898 amino acid residues) exhibited significant similarities to the E1 components of the pyruvate dehydrogenase complexes of A. eutrophus, Neisseria meningitidis, Escherichia coli and Azotobacter vinelandii, which are also composed of only one type of subunit. Heterologous expression of pdhE in the aceEF deletion mutant E. coli YYC202 was demonstrated by spectrometric detection of enzyme activities and by phenotypic complementation to acetate prototrophy. These complementation studies indicated that the E1 component of the A. eutrophus pyruvate dehydrogenase complex can be replaced by a functionally active pdhE gene product.

Mutants with defects in components of the glutathione-glutaredoxin (GSH/Grx) system of Rhodobacter capsulatus were constructed to study its role in defense against oxidative stress and the redox-dependent formation of photosynthetic complexes. The lack of the glutaredoxin 3 gene ( grxC ) or the glutathione synthetase B gene ( gshB ) resulted in lower growth rates under aerobic conditions and higher sensitivity to oxidative stress, confirming the role of the GSH/Grx system in oxidative stress defense. Both mutants are highly sensitive to disulfide stress, indicating a major contribution of the GSH/Grx system to the thiol-disulfide redox buffer in the cytoplasm. Like mutations in the thioredoxin system, mutations in the GSH/Grx system affected the formation of photosynthetic complexes, which is redox dependent in R. capsulatus . Expression of the genes grxC , gshB , grxA for glutaredoxin 1, and gorA for glutathione reductase, all encoding components of the GSH/Grx system, was not induced by oxidative stress. Other genes, for which a role in oxidative stress was established in Escherichia coli , acnA , fpr , fur , and katG , were strongly induced by oxidative stress in R. capsulatus . Mutations in the grxC , and/or gshB , and/or trxC (thioredoxin 2) genes affected expression of these genes, indicating an interplay of the different defense systems against oxidative stress. The OxyR and the SoxRS regulons control the expression of many genes involved in oxidative stress defense in E. coli in response to H 2 O 2 and superoxide, respectively. Our data and the available genome sequence of R. capsulatus suggest that a SoxRS system is lacking but an alternative superoxide specific regulator exists in R. capsulatus. While the expression of gorA and grxA is regulated by H 2 O 2 in E. coli this is not the case in R. capsulatus , indicating that the OxyR regulons of these two species are significantly different.

Intensive research on the physiology, biochemistry, and molecular genetics of the metabolism of polyhydroxyalkanoates (PHA) during the last 15 years has revealed a dramatic increase of our knowledge on the biosynthesis of these polyesters in bacteria. This mainly very basic research has revealed several new, hitherto not described enzymes and pathways. In addition, many genes encoding the enzymes of these pathways and in particular the key enzyme of PHA biosynthesis, PHA synthase, were cloned and characterized at a molecular level. This knowledge was utilized to establish PHA biosynthesis in many prokaryotic and eukaryotic organisms, which were unable to synthesize PHAs, and to apply the methodology of metabolic engineering, thus opening new perspectives for the production of various PHAs by fermentation biotechnology or agriculture in economically feasible processes. This contribution summarizes the properties of PHA synthases and gives an overview on the genes for these enzymes and other enzymes of PHA biosynthesis that have been cloned and are available. It also summarizes our current knowledge on the regulation at the enzyme and gene level of PHA biosynthesis in bacteria.

During cultivation under storage conditions with BG11 medium containing acetate as a carbon source, Synechocystis sp. PCC6803 accumulated poly(3-hydroxybutyrate) up to 10% (w/w) of the cell dry weight. Our analysis of the complete Synechocystis sp. PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open reading frame slr1829, which is located colinear and upstream of phaC, most probably represent a polyhydroxyalkanoic acid (PHA) synthase gene. The open reading frame slr1829 was therefore designated as phaE. The phaE and phaC gene products exhibited striking sequence similarities to the corresponding PHA synthase subunits PhaE and PhaC of Thiocystis violacea, Chromatium vinosum, and Thiocapsa pfennigii. The Synechocystis sp. PCC6803 genes were cloned using PCR and were heterologously expressed in Escherichia coli and in Alcaligenes eutrophus. Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3-hydroxybutyrate) in the PHA-negative mutant A. eutrophus PHB–4. These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp. PCC6803 PHA synthase. PHA granules were detected by electron microscopy in these cells, and the PHA-granule-associated proteins were studied. Western blot analysis of Synechocystis sp. PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raised against the PHA synthases of A. eutrophus (PhaC) and of C. vinosum (PhaE and PhaC) revealed no immunoreaction.