Inactivation of an intracellular poly-3-hydroxybutyrate depolymerase of Azotobacter vinelandii allows to obtain a polymer of uniform high molecular mass
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A novel poly-3-hydroxybutyrate depolymerase was identified in Azotobacter vinelandii. This enzyme, now designated PhbZ1, is associated to the poly-3-hydroxybutyrate (PHB) granules and when expressed in Escherichia coli, it showed in vitro PHB depolymerizing activity on native or artificial PHB granules, but not on crystalline PHB. Native PHB (nPHB) granules isolated from a PhbZ1 mutant had a diminished endogenous in vitro hydrolysis of the polyester, when compared to the granules of the wild-type strain. This in vitro degradation was also tested in the presence of free coenzyme A. Thiolytic degradation of the polymer was observed in the nPHB granules of the wild type, resulting in the formation of 3-hydroxybutyryl-CoA, but was absent in the granules of the mutant. It was previously reported that cultures of A. vinelandii OP grown in a bioreactor showed a decrease in the weight average molecular weight (Mw) of the PHB after 20 h of culture, with an increase in the fraction of polymers of lower molecular weight. This decrease was correlated with an increase in the PHB depolymerase activity during the culture. Here, we show that in the phbZ1 mutant, neither the decrease in the Mw nor the appearance of a low molecular weight polymers occurred. In addition, a higher PHB accumulation was observed in the cultures of the phbZ1 mutant. These results suggest that PhbZ1 has a role in the degradation of PHB in cultures in bioreactors and its inactivation allows the production of a polymer of a uniform high molecular weight.
KeywordsPolyhydroxybutyrate Depolymerase Bioplastic Molecular mass
The authors are grateful to Soledad Moreno and Ramón de Anda for the technical assistance and Déborah Yanajara-Parra for her assistance in the purification of the PHB granules. L. Adaya is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México, and was supported by a scholarship from Consejo Nacional de Ciencia y Tecnología (México). This work was supported by grants 255158 and 238535 from Consejo Nacional de Ciencia y Tecnología and IT200415 form Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT) DGAPA-UNAM, as well as by a grant of the Deutsche Forschungsgemeinschaft to D. J.
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Agus J, Kahar P, Abe H, Doi Y, Tsuge T (2006) Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli. Polym Degrad Stab 91(8):1645–1650. https://doi.org/10.1016/j.polymdegradstab.2005.12.011 CrossRefGoogle Scholar
- Agus J, Kahar P, Hyakutake M, Tomizawa S, Abe H, Tsuge T, Satoh Y, Tajima K (2010) Unusual change in molecular weight of polyhydroxyalkanoate (PHA) during cultivation of PHA-accumulating Escherichia coli. Polym Degrad Stab 95(12):2250–2254. https://doi.org/10.1016/j.polymdegradstab.2010.09.009 CrossRefGoogle Scholar
- Arias S, Bassas-Galia M, Molinari G, Timmis KN (2013) Tight coupling of polymerization and depolymerization of polyhydroxyalkanoates ensures efficient management of carbon resources in Pseudomonas putida. Microb Biotechnol 6(5):551–563. https://doi.org/10.1111/1751-7915.12040 CrossRefPubMedPubMedCentralGoogle Scholar
- Arikawa H, Sato S, Fujiki T, Matsumoto K (2016) A study on the relation between poly(3-hydroxybutyrate) depolymerases or oligomer hydrolases and molecular weight of polyhydroxyalkanoates accumulating in Cupriavidus necator H16. J Biotechnol 227:94–102. https://doi.org/10.1016/j.jbiotec.2016.04.004 CrossRefPubMedGoogle Scholar
- Bocanegra JK, Da Cruz Pradella JG, Da Silva LF, Taciro MK, Gomez JGC (2013) Influence of pH on the molecular weight of poly-3-hydroxybutyric acid (P3HB) produced by recombinant Escherichia coli. Appl Biochem Biotechnol 170(6):1336–1347. https://doi.org/10.1007/s12010-013-0257-4 CrossRefPubMedGoogle Scholar
- Centeno-Leija S, Huerta-Beristain G, Giles-Gómez M, Bolivar F, Gosset G, Martínez A (2014) Improving poly-3-hydroxybutyrate production in Escherichia coli by combining the increase in the NADPH pool and acetyl-CoA availability. Antonie Van Leeuwenhoek 105(4):687–696. https://doi.org/10.1007/s10482-014-0124-5 CrossRefPubMedGoogle Scholar
- Doi Y, Segawa A, Kawaguchi Y, Kunioka M (1990) Cyclic nature of poly(3-hydroxyalkanoate) metabolism in Alcaligenes eutrophus. FEMS Microbiol Lett 67(1-2):165–170. https://doi.org/10.1111/j.1574-6968.1990.tb13856.x CrossRefGoogle Scholar
- Hernández-Eligio A, Moreno S, Castellanos M, Castañeda M, Nuñez C, Muriel-Millán LF, Espín G (2012) RsmA post-transcriptionally controls PhbR expression and polyhydroxybutyrate biosynthesis in Azotobacter vinelandii. Microbiol (United Kingdom) 158:1953–1963Google Scholar
- Hiroe A, Tsuge K, Nomura CT, Itaya M, Tsuge T (2012) Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli. Appl Environ Microbiol 78:3177–3184CrossRefPubMedPubMedCentralGoogle Scholar
- Jendrossek D, Handrick R (2002) Microbial degradation of polyhydroxyalkanoates. Annu Rev Microbiol 56(1):403–432. https://doi.org/10.1146/annurev.micro.56.012302.160838 CrossRefPubMedGoogle Scholar
- Jia Y, Kappock TJ, Frick T, Sinskey AJ, Stubbe J (2000) Lipases provide a new mechanistic model for polyhydroxybutyrate (PHB) synthases: characterization of the functional residues in Chromatium vinosum PHB synthase. Biochemistry 39(14):3927–3936. https://doi.org/10.1021/bi9928086 CrossRefPubMedGoogle Scholar
- Kobayashi T, Shiraki M, Abe T, Sugiyama A, Saito T (2003) Purification and properties of an intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZ2) in Ralstonia eutropha H16 and its identification as a novel intracellular poly(3-hydroxybutyrate) depolymerase. J Bacteriol 185:3485–3490CrossRefPubMedPubMedCentralGoogle Scholar
- Millán M, Segura D, Galindo E, Peña C (2016) Molecular mass of poly-3-hydroxybutyrate (P3HB) produced by Azotobacter vinelandii is determined by the ratio of synthesis and degradation under fixed dissolved oxygen tension. Process Biochem 51(8):950–958. https://doi.org/10.1016/j.procbio.2016.04.013 CrossRefGoogle Scholar
- Muriel-Millán LF, Castellanos M, Hernandez-Eligio JA, Moreno S, Espín G (2014) Posttranscriptional regulation of PhbR, the transcriptional activator of polyhydroxybutyrate synthesis, by iron and the sRNA ArrF in Azotobacter vinelandii. Appl Microbiol Biotechnol 98:2173–2182CrossRefPubMedGoogle Scholar
- Noguez R, Segura D, Moreno S, Hernandez A, Juarez K, Espín G (2008) Enzyme I NPr, NPr and IIA Ntr are involved in regulation of the poly-beta-hydroxybutyrate biosynthetic genes in Azotobacter vinelandii. J Mol Microbiol Biotechnol 15(4):244–254. https://doi.org/10.1159/000108658 CrossRefPubMedGoogle Scholar
- Peralta-Gil M, Segura D, Guzmán J, Servín-González L, Espín G (2002) Expression of the Azotobacter vinelandii poly-β-hydroxybutyrate biosynthetic phbBAC operon is driven by two overlapping promoters and is dependent on the transcriptional activator PhbR. J Bacteriol 184:5672–5677CrossRefPubMedPubMedCentralGoogle Scholar
- Saegusa H, Shiraki M, Kanai C, Saito T (2001) Cloning of an intracellular poly[D(−)-3-hydroxybutyrate] depolymerase gene from Ralstonia eutropha H16 and characterization of the gene product. J Bacteriol 183(1):94–100. https://doi.org/10.1128/JB.183.1.94-100.2001 CrossRefPubMedPubMedCentralGoogle Scholar
- Saegusa H, Shiraki M, Saito T (2002) Cloning of an intracellular D(−)-hydroxybutyrate-oligomer hydrolase gene from Ralstonia eutropha H16 and identification of the active site serine residue by site-directed mutagenesis. J Biosci Bioeng 94(2):106–112. https://doi.org/10.1016/S1389-1723(02)80128-1 CrossRefPubMedGoogle Scholar
- Sambrook JF, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York, pp 2100Google Scholar
- Shimizu H, Tamura S, Shioya S, Suga K-I (1993) Kinetic study of poly-D (−)-3-hydroxybutyric acid (PHB) production and its molecular weight distribution control in a fed-batch culture of Alcaligenes eutrophus. J Ferment Bioeng 76(6):465–469. https://doi.org/10.1016/0922-338X(93)90242-Z CrossRefGoogle Scholar
- Sznajder A, Jendrossek D (2014) To be or not to be a poly(3-Hydroxybutyrate) (PHB) depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, highly active PHB depolymerases with no detectable role in mobilization of accumulated PHB. Appl Environ Microbiol 80(16):4936–4946. https://doi.org/10.1128/AEM.01056-14 CrossRefPubMedPubMedCentralGoogle Scholar
- Tirapelle EF, Müller-Santos M, Tadra-Sfeir MZ, Kadowaki MAS, Steffens MBR, Monteiro RA, Souza EM, Pedrosa FO, Chubatsu LS (2013) Identification of proteins associated with polyhydroxybutyrate granules from Herbaspirillum seropedicae SmR1—old partners, new players. PLoS One 8(9):e75066. https://doi.org/10.1371/journal.pone.0075066 CrossRefPubMedPubMedCentralGoogle Scholar
- Uchino K, Saito T, Gebauer B, Jendrossek D (2007) Isolated poly(3-hydroxybutyrate) (PHB) granules are complex bacterial organelles catalyzing formation of PHB from acetyl coenzyme a (CoA) and degradation of PHB to acetyl-CoA. J Bacteriol 189(22):8250–8256. https://doi.org/10.1128/JB.00752-07 CrossRefPubMedPubMedCentralGoogle Scholar
- York GM, Lupberger J, Tian J, Lawrence AG, Stubbe J, Sinskey AJ (2003) Ralstonia eutropha H16 encodes two and possibly three intracellular poly[D-(−)-3-hydroxybutyrate] depolymerase genes. J Bacteriol 185(13):3788–3794. https://doi.org/10.1128/JB.185.13.3788-3794.2003 CrossRefPubMedPubMedCentralGoogle Scholar