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Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases

  • Applied Microbial and Cell Physiology
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Abstract

Various hydroxyacyl coenzyme A (CoA) thioesters were synthesized from the corresponding hydroxyalkanoic acid (such as e.g. [3-14C]d-(−)-hydroxybutyric acid, [1-14C]d-lactic acid, [1-14C]l-lactic acid, etc.) and from acetyl-CoA employing the propionate CoA transferase of Clostridium propionicum. Preparative isolation of the thioesters on hydrophobic matrices and analysis by HPLC are reported. These thioesters were subjected to a radiometric or a spectrometric assay of polyhydroxyalkanoic acid (PHA) synthase activity. The latter was based on the release of CoA from, for example, d-(−)-3-hydroxybutyryl-CoA, which was detected spectroscopically at 412 nm by reduction of 5,5′-dithiobis(2-nitrobenzoic acid) and provided a convenient assay of poly(3-hydroxybutyrate) synthase. When [1-14C]lactyl-CoA was used as substrate in a PHA synthase assay employing crude extracts obtained from various wild-type strains, [1-14C]lactyl-CoA was used as a substrate at a rate that was only less than 10−4 of the rate than with [3-14C]d-(−)-3-hydroxybutyryl-CoA or was negligible. One exception was a recombinant strain of Escherichia coli, which overexpressed the PHA synthase complex of Chromatium vinosum and which used [1-14C]d-lactyl-CoA as substrate at a relatively high rate.

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References

  • Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472

    CAS  PubMed  Google Scholar 

  • Anderson AJ, Haywood GW, Williams DR, Dawes EA (1990) The production of polyhydroxyalkanoates from unrelated carbon sources. In: Dawes EA (ed) Novel biodegradable microbial polymers. Kluwer, Dordrecht, pp 119–129

    Google Scholar 

  • Bullock WO, Fernandez JM, Stuart JM (1987) XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. BioTechniques 5:376–379

    Google Scholar 

  • Bousfield IJ, Green PN (1985) Reclassification of bacteria of the genus Protomonas Urakami and Komagata 1984 in the genus Methylobacterium (Patt, Cole and Hanson) emend. Green and Bousfield 1983. Int J Syst Bacteriol 32:209

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Davis DH, Doudoroff M, Stanier RY, Mandel M (1969) Proposal to reject the genus Hydrogenomonas: taxomomic implications. Int J Syst Bacteriol 19:375–390

    Google Scholar 

  • Decker K (1959) Die aktivierte Essigsäure. Ferdinand Enke, Stuttgart

    Google Scholar 

  • Decker K (1962) l-(+)-β-Hydroxybutyryl-Coenzym A. In: Bergmeyer HU (ed) Methoden der enzymatischen Analyse, 1st edn. Verlag Chemie GmbH, Weinheim, pp 441–444

    Google Scholar 

  • Decker K (1985a) Acetyl coenyzme A. In: Bergmeyer HU, Bergmeyer J, Grassl M (eds) Methods of enzymatic analysis, vol 7, 3rd edn. VCH, Weinheim, pp 186–193

    Google Scholar 

  • Decker K (1985b) Acetyl coenzyme A. In: Bergmeyer HU, Bergmeyer J, Grassl M (eds) Methods of enzymatic analysis, vol 7, 3rd edn. VCH, Weinheim, pp 201–206

    Google Scholar 

  • De Buysere MS, Olson MS (1983) The analysis of acyl-coenzyme A derivatives by reverse-phase high performance liquid chromatography. Anal Biochem 133:373–379

    Google Scholar 

  • De Smet MJ, Eggink G, Witholt B, Kingma J, Wynberg H (1983) Characterization of intracellular inclusions formed by Pseudomonas oleovorans during growth on octane. J Bacteriol 154:870–878

    Google Scholar 

  • Doi Y, Tamaki A, Kunioka M, Soga K (1987) Biosynthesis of terpolyesters of 3-hydroxybutyrate, 3-hydroxyvalerate, and 5-hydroxyvalerate in Alcaligenes eutrophus from 5-chloropentanoic and pentanoic acids. Makromol Chem Rapid Commun 8:631–635

    Google Scholar 

  • Ellmann GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82:70–77

    Google Scholar 

  • Fründ C, Priefert H, Steinbüchel A, Schlegel HG (1989) Biochemical and genetic analysis of acetoin catabolism in Alcaligenes eutrophus. J Bacteriol 171:6539–6548

    Google Scholar 

  • Fukui T, Yoshimoto A, Matsumoto M, Hosokawa S, Saito T, Nishikawa H, Tomita K (1976) Enzymatic synthesis of poly-β-hydroxybutyrate in Zoogloea ramigera. Arch Microbiol 110:149–156

    Google Scholar 

  • Haywood GW, Anderson AJ, Dawes EA (1989a) The importance of PHB-synthase substrate speficity in polyhydroxyalkanoate synthesis by Alcaligenes eutrophus. FEMS Microbiol Lett 57:1–6

    Google Scholar 

  • Haywood GW, Anderson AJ, Dawes EA (1989b) A survey of the accumulation of novel polyhydroxyalkanoates by bacteria. Biotechnol Lett 11:471–476

    Google Scholar 

  • Haywood GW, Anderson AJ, Ewing DF, Dawes EA (1990) Accumulation of a polyhydroxyalkanoate containing primarily 3-hydroxydecanoate from simple carbohydrate substrates by Pseudomonas sp. NCIMB 40135. Appl Environ Microbiol 56:3354–3359

    Google Scholar 

  • Hilger U, Sattler K, Littkowsky U (1991) Untersuchungen zur Wachstums-assoziierten Akkumulation von Poly-β-hydroxybuttersäure bei Methylobacterium rhodesianum. Z Zentralbl Mikrobiol 146:83–88

    Google Scholar 

  • Holmes PA, Wright LF, Collins SH (1981) β-Hydroxybutyrate polymers. European patent application no. EP 052459

  • Hosokawa Y, Shinomura Y, Harris RA, Ozawa T (1986) Determination of short-chain acyl-coenzyme A esters by high-performance liquid chromatography. Anal Biochem 153:45–49

    Google Scholar 

  • Huismann GW, Wonink E, Meima R, Kazemier B, Terpstra P, Witholt B (1991) Metabolism of poly(3-hydroxyalkanoates) by Pseudomonas oleovorans: identification and sequences of genes and function of the encoded proteins in the synthesis and degradation of PHA. J Biol Chem 266:2191–2198

    Google Scholar 

  • Hustede E, Steinbüchel A, Schlegel HG (1993) Relationship between the photoproduction of hydrogen and the accumulation of PHB in non-sulphur purple bacteria. Appl Microbiol Biotechnol 39:87–93

    Google Scholar 

  • Kaudewitz F (1959) Inaktivierende und mutagene Wirkung salpetriger Säure auf Zellen von Escherichia coli. Z Naturforsch 14b:528–537

    Google Scholar 

  • King MT, Reiss PD (1985) Separation and measurement of short-chain coenzyme A compounds in rat liver by reversed-phase high performance liquid chromatography. Anal Biochem 146:173–179

    Google Scholar 

  • Kuchta RD, Abeles RH (1985) Lactate reduction in Clostridium propionicum. J Biol Chem 260:13181–13189

    Google Scholar 

  • Kunioka M, Nakamura Y, Doi Y (1988) New Bacterial copolyesters produced in Alcaligenes eutrophus from organic acids. Polymer Commun 29:174–176

    CAS  Google Scholar 

  • Lemoigne M (1926) Produits de deshydration et de polymerisation de lácide β-oxybutyric. Bull Soc Chim Biol (Paris) 8:770–782

    Google Scholar 

  • Liebergesell M, Steinbüchel A (1992) Cloning and nucleotide sequences of genes relevant for biosynthesis of polyhydroxyalkanoic acid in Chromatium vinosum. Eur J Biochem 209:135–150

    Google Scholar 

  • Liebergesell M, Steinbüchel A (1993) Cloning and molecular characterization of the poly(3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea. Appl Microbiol Biotechnol 38:493–501

    Google Scholar 

  • Liebergesell M, Mayer F, Steinbüchel A (1993) Analysis of polyhydroxyalkanoic acid-biosynthesis genes of anoxygenic phototrophic bacteria reveals synthesis of a polyester exhibiting an unusual composition. Appl Microbiol Biotechno, in press

  • Mieyal JJ, Webster LT, Siddiqui UA (1974) Benzoyl and hydroxybenzoyl esters of coenzyme A. J Biol Chem 249:2633–2640

    Google Scholar 

  • Pedrós-Alio C, Mas J, Guerrero R (1985) The influence of poly-β-hydroxybutyrate accumulation on cell volume and buoyant density in Alcaligenes eutrophus. Arch Microbiol 143:178–184

    Google Scholar 

  • Pieper U (1993) Biosynthese eines Copolymers aus 3-Hydroxybuttersäure und 3-Hydroxyvaleriansäure in Rhodococcus ruber NCIMB 40126: Physiologische, molekulargenetische und biochemische Untersuchungen. Dissertation, Georg-August-Universität Göttingen

  • Pries A, Priefert H, Krüger N, Steinbüchel A (1991) Identification and characterization of two Alcaligenes eutrophus gene loci relevant to the poly(β-hydroxybutyric acid)-leaky phenotype which exhibit homology to ptsH and ptsI of Escherichia coli. J Bacteriol 173:5843–5853

    Google Scholar 

  • Rogosa M (1969) Acidaminococcus gen. n., Acidaminococcus fermentans sp. n., anaerobic Gram-negative diplococci using amino acids as the sole energy source for growth. J Bacteriol 98:756–766

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Schlegel HG, Kaltwasser H, Gottschalk G (1961) Ein Submersverfahren zur Kultur wasserstoffoxidierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch Mikrobiol 38:209–222

    CAS  PubMed  Google Scholar 

  • Schlegel HB, Lafferty RM, Krauss I (1970) The isolation of mutants not accumulating poly-β-hydroxybutyric acid. Arch Microbiol 71:283–294

    Google Scholar 

  • Schubert P, Steinbüchel A, Schlegel HG (1988) Cloning of the Alcaligenes eutrophus genes for synthesis of poly-β-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. J Bacteriol 170:5837–5847

    Google Scholar 

  • Schubert P, Krüger N, Steinbüchel A (1991) Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N-terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. J Bacteriol 173:168–175

    Google Scholar 

  • Schweiger G (1986) Untersuchugunen zum Mechanismus der enzymatischen Dehydrierung von 2-Hydroxysäuren. Dissertation, Universität Regensburg

  • Schweiger G, Buckel W (1984) On the dehydration of (R)-lactate in the fermentation of alanine to propionate by Clostridium propionicum. FEBS Lett 171:79–84

    Google Scholar 

  • Simon EJ, Shemin D (1953) The preparation of S-succinyl-coenzyme A. J Am Chem Soc 75:2520

    Google Scholar 

  • Steinbüchel A (1991a) Polyhydroxyalkanoic acids. In: Byrom D (ed) Biomaterials. MacMillan, London, pp 123–213

    Google Scholar 

  • Steinbüchel A (1991b) Polyhydroxyfettsäuren — thermoplastisch verformbare und biologisch abbaubare Polyester aus Bakterien. Nachrichtenbl Chem Tech Lab 39:1112–1124

    Google Scholar 

  • Steinbüchel A, Pieper U (1992) Production of a copolyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid by a mutant of Alcaligenes eutrophus from single unrelated carbon sources. Appl Microbiol Biotechnol 37:1–6

    Google Scholar 

  • Steinbüchel A, Schlegel HG (1981) Die relative Respirationsrate (RRR), ein neuer Belüftungsparameter. In: Lafferty RM (ed) Fermentation. Springer, Vienna, pp 11–26

    Google Scholar 

  • Steinbüchel A, Schlegel HG (1991) Genetics of poly(β-hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus. Mol Microbiol 5:535–542

    Google Scholar 

  • Steinbüchel A, Hustede E, Liebergesell M, Timm A, Pieper U, Valentin H (1992) Molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria. FEMS Microbiol Rev 103:217–230

    Google Scholar 

  • Timm A, Steinbüchel A (1990) Formation of polyesters consisting of medium-chain-length 3-hydroxy alkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads. Appl Environ Microbiol 56:3360–3367

    Google Scholar 

  • Valentin HE, Schönebaum A, Steinbüchel A (1992) Identification of 4-hydroxyvaleric acids as a constituent in biosynthetic polyhydroxyalkanoic acids from bacteria. Appl Microbiol Biotechnol 36:507–514

    Google Scholar 

  • Vert M (1986) Biomedical polymers from chiral lactides and functional lactones. Properties and applications. Macromol Chem Macromol Symp 6:109–122

    Google Scholar 

  • Webster LT, Killenberg PG (1981) Coenzyme A thioesters of benzoic, hydroxybenzoic, phenylacetic, and bile acids. Methods Enzymol 77:430–436

    Google Scholar 

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  1. Institut für Mikrobiologie der Georg-August-Universität Göttingen, Grisebachstrasse 8, D-37077, Göttingen, Germany

    Henry E. Valentin & Alexander Steinbüchel

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  1. Henry E. Valentin
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  2. Alexander Steinbüchel
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Correspondence to: A. Steinbüchel

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Valentin, H.E., Steinbüchel, A. Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases. Appl Microbiol Biotechnol 40, 699–709 (1994). https://doi.org/10.1007/BF00173332

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  • DOI: https://doi.org/10.1007/BF00173332

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