Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic.
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Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs. (+info)
Pregnenolone esterification in Saccharomyces cerevisiae. A potential detoxification mechanism.
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While studying the effect of steroids on the growth of the yeast Saccharomyces cerevisiae, we found that pregnenolone was converted into the acetate ester. This reaction was identified as a transfer of the acetyl group from acetyl-CoA to the 3beta-hydroxyl group of pregnenolone. The corresponding enzyme, acetyl-CoA:pregnenolone acetyltransferase (APAT) is specific for Delta5- or Delta4-3beta-hydroxysteroids and short-chain acyl-CoAs. The apparent Km for pregnenolone is approximately 0.5 microm. The protein associated with APAT activity was partially purified and finally isolated from an SDS/polyacrylamide gel. Tryptic peptides were generated and N-terminally sequenced. Two peptide sequences allowed the identification of an open reading frame (YGR177c, in the S. cerevisiae genome database) translating into a 62-kDa protein of hitherto unknown function. This protein encoded by a gene known as ATF2 displays 37% identity with an alcohol acetyltransferase encoded by the yeast gene ATF1. Disruption of ATF2 led to the complete elimination of APAT activity and consequently abolished the esterification of pregnenolone. In addition, a toxic effect of pregnenolone linked to the disruption of ATF2 was observed. Pregnenolone toxicity is more pronounced when the atf2-Delta mutation is introduced in a yeast strain devoid of the ATP-binding cassette transporters, PDR5 and SNQ2. Our results suggest that Atf2p (APAT) plays an active role in the detoxification of 3beta-hydroxysteroids in association with the efflux pumps Pdr5p and Snq2p. (+info)
A functional 4-hydroxysalicylate/hydroxyquinol degradative pathway gene cluster is linked to the initial dibenzo-p-dioxin pathway genes in Sphingomonas sp. strain RW1.
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The bacterium Sphingomonas sp. strain RW1 is able to use dibenzo-p-dioxin, dibenzofuran, and several hydroxylated derivatives as sole sources of carbon and energy. We have determined and analyzed the nucleic acid sequence of a 9,997-bp HindIII fragment downstream of cistrons dxnA1A2, which encode the dioxygenase component of the initial dioxygenase system of the corresponding catabolic pathways. This fragment contains 10 colinear open reading frames (ORFs), apparently organized in one compact operon. The enzymatic activities of some proteins encoded by these genes were analyzed in the strain RW1 and, after hyperexpression, in Escherichia coli. The first three ORFs of the locus, designated dxnC, ORF2, and fdx3, specify a protein with a low homology to bacterial siderophore receptors, a polypeptide representing no significant homology to known proteins, and a putative ferredoxin, respectively. dxnD encodes a 69-kDa phenol monooxygenase-like protein with activity for the turnover of 4-hydroxysalicylate, and dxnE codes for a 37-kDa protein whose sequence and activity are similar to those of known maleylacetate reductases. The following gene, dxnF, encodes a 33-kDa intradiol dioxygenase which efficiently cleaves hydroxyquinol, yielding maleylacetate, the ketoform of 3-hydroxy-cis,cis-muconate. The heteromeric protein encoded by dxnGH is a 3-oxoadipate succinyl coenzyme A (succinyl-CoA) transferase, whereas dxnI specifies a protein exhibiting marked homology to acetyl-CoA acetyltransferases (thiolases). The last ORF of the sequenced fragment codes for a putative transposase. DxnD, DxnF, DxnE, DxnGH, and DxnI (the activities of most of them have also been detected in strain RW1) thus form a complete 4-hydroxysalicylate/hydroxyquinol degradative pathway. A route for the mineralization of the growth substrates 3-hydroxydibenzofuran and 2-hydroxydibenzo-p-dioxin in Sphingomonas sp. strain RW1 thus suggests itself. (+info)
Origin of gene overlap: the case of TCP1 and ACAT2.
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The human acetyl-CoA acetyltransferase 2 gene, ACAT2, codes for a thiolase, an enzyme involved in lipid metabolism. The human T-complex protein 1 gene, TCP1, encodes a molecular chaperone of the chaperonin family. The two genes overlap by their 3'-untranslated regions, their coding sequences being located on opposite DNA strands in a tail-to-tail orientation. To find out how the overlap might have arisen in evolution, the homologous genes of the zebrafish, the African toad, caiman, platypus, opossum, and wallaby were identified. In each species, standard or long polymerase chain reactions were used to determine whether the ACAT2 and TCP1 homologs are closely linked and, if so, whether they overlap. The results reveal that the overlap apparently arose during the transition from therapsid reptiles to mammals and has been retained for >200 million years. Part of the overlapping untranslated region shows remarkable sequence conservation. The overlap presumably arose during the chromosomal rearrangement that brought the two unrelated and previously separated genes together. One or both of the transposed genes found by chance signals that are necessary for the processing of their transcripts to be present on the noncoding strand of the partner gene. (+info)
Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824.
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A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of beta-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional beta-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the beta-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the beta-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the beta-galactosidase produced by the lacZ gene from T. thermosulfurogenes EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of beta-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar. (+info)
A biosynthetic thiolase in complex with a reaction intermediate: the crystal structure provides new insights into the catalytic mechanism.
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BACKGROUND: Thiolases are ubiquitous and form a large family of dimeric or tetrameric enzymes with a conserved, five-layered alphabetaalphabetaalpha catalytic domain. Thiolases can function either degradatively, in the beta-oxidation pathway of fatty acids, or biosynthetically. Biosynthetic thiolases catalyze the biological Claisen condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in a wide range of biosynthetic pathways, including those that generate cholesterol, steroid hormones, and various energy-storage molecules. RESULTS: The crystal structure of the tetrameric biosynthetic thiolase from Zoogloea ramigera has been determined at 2.0 A resolution. The structure contains a striking and novel 'cage-like' tetramerization motif, which allows for some hinge motion of the two tight dimers with respect to each other. The protein crystals were flash-frozen after a short soak with the enzyme's substrate, acetoacetyl-CoA. A reaction intermediate was thus trapped: the enzyme tetramer is acetylated at Cys89 and has a CoA molecule bound in each of its active-site pockets. CONCLUSIONS: The shape of the substrate-binding pocket reveals the basis for the short-chain substrate specificity of the enzyme. The active-site architecture, and in particular the position of the covalently attached acetyl group, allow a more detailed reaction mechanism to be proposed in which Cys378 is involved in both steps of the reaction. The structure also suggests an important role for the thioester oxygen atom of the acetylated enzyme in catalysis. (+info)
Aberrant oxidation of the cholesterol side chain in bile acid synthesis of sterol carrier protein-2/sterol carrier protein-x knockout mice.
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Peroxisomal beta-oxidation plays an important role in the metabolism of a wide range of substrates, including various fatty acids and the steroid side chain in bile acid synthesis. Two distinct thiolases have been implicated to function in peroxisomal beta-oxidation: the long known 41-kDa beta-ketothiolase identified by Hashimoto and co-workers (Hijikata, M., Ishii, N., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8151-8158) and the recently discovered 60-kDa SCPx thiolase, that consists of an N-terminal domain with beta-ketothiolase activity and a C-terminal moiety of sterol carrier protein-2 (SCP2, a lipid carrier or transfer protein). Recently, gene targeting of the SCP2/SCPx gene has shown in mice that the SCPx beta-ketothiolase is involved in peroxisomal beta-oxidation of 2-methyl-branched chain fatty acids like pristanic acid. In our present work we have investigated bile acid synthesis in the SCP2/SCPx knockout mice. Specific inhibition of beta-oxidation at the thiolytic cleavage step in bile acid synthesis is supported by our finding of pronounced accumulation in bile and serum from the knockout mice of 3alpha,7alpha, 12alpha-trihydroxy-27-nor-5beta-cholestane-24-one (which is a known bile alcohol derivative of the cholic acid synthetic intermediate 3alpha,7alpha,12alpha-trihydroxy-24-keto-cholestano yl-coenzyme A). Moreover, these mice have elevated concentrations of bile acids with shortened side chains (i.e. 23-norcholic acid and 23-norchenodeoxycholic acid), which may be produced via alpha- rather than beta-oxidation. Our results demonstrate that the SCPx thiolase is critical for beta-oxidation of the steroid side chain in conversion of cholesterol into bile acids. (+info)
Peroxisomal fatty acid oxidation disorders and 58 kDa sterol carrier protein X (SCPx). Activity measurements in liver and fibroblasts using a newly developed method.
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Sterol carrier protein X (SCPx) plays a crucial role in the peroxisomal oxidation of branched-chain fatty acids. To investigate whether patients with an unresolved defect in peroxisomal beta-oxidation are deficient for SCPx, we developed a novel and specific assay to measure the activity of SCPx in both liver and fibroblast homogenates. The substrate used in the assay, 3alpha, 7alpha,12alpha-trihydroxy-24-keto-5beta-cholestanoy l-CoA (24-keto-THC-CoA), is produced by preincubating the enoyl-CoA of the bile acid intermediate THCA with a lysate from the yeast Saccharomyces cerevisiae expressing human D-bifunctional protein. After the preincubation period, liver or fibroblast homogenate is added plus CoASH, and the production of choloyl-CoA is determined by HPLC. The specificity of the assay was demonstrated by the finding of a full deficiency in fibroblasts from an SCPx knock-out mouse. In addition to SCPx activity measurements in fibroblasts from patients with a defect in peroxisomal beta-oxidation of unresolved etiology, we studied the stability and activity of SCPx in fibroblasts from patients with Zellweger syndrome, which lack functional peroxisomes. We found that SCPx is not only stable in the cytosol, but displays a higher activity in fibroblasts from patients with Zellweger syndrome than in control fibroblasts. Furthermore, in all patients studied with a defect in peroxisomal beta-oxidation of unknown origin, SCPx was found to be normally active, indicating that human SCPx deficiency remains to be identified. (+info)