In silico analysis of phag-like protein in ralstonia eutropha H16, potentially involved in Polyhydroxyalkanoates synthesis

Melissa Uribe Acosta; Andrés Felipe Villa Restrepo

doi:10.33571/rpolitec.v15n29a5

Autores/as

Melissa Uribe Acosta University of Antioquia https://orcid.org/0000-0003-3755-1802
Andrés Felipe Villa Restrepo University of Antioquia

DOI:

https://doi.org/10.33571/rpolitec.v15n29a5

Palabras clave:

polyhydroxyalkanoates, ralstonia eutropha H16, non-related carbon sources, protein function prediction

Resumen

Los polihidroxialcanoatos (PHA) son sintetizados por las bacterias como material de reserva de carbono. La proteína PhaG dirige el carbono proveniente de fuentes de carbono no relacionadas como el glicerol, que son metabolizados a través de la síntesis de ácidos grasos de novo (FAS), hacia la síntesis de PHA. El gen que codifica esta proteína no ha sido aún encontrado en el genoma de Ralstonia eutropha H16, un organismo modelo. A través de la comparación con proteínas PhaG ya conocidas, una proteína similar a PhaG, fue encontrada siendo codificada por el gen H16_A0147 y la presencia del gen confirmada preliminarmente utilizando PCR. Este es el primer estudio que muestra la presencia y características de una proteína similar a PhaG en R. eutropha H16 y representa el primer paso en la identificación de una conexión entre las rutas metabólicas FAS y de PHA en esta bacteria modelo. Estudios de bloqueo de genes y actividad enzimática son necesarios para confirmar esta relación potencial que podría mejorar la producción industrial de PHA y la utilización de residuos agroindustriales como el glicerol.

Polyhydroxyalkanoates (PHA) are synthesised by bacteria as carbon storage material. The protein PhaG directs carbon from non-related carbon sources such as glycerol, metabolised through fatty acid de novo synthesis (FAS) pathway, with PHA synthesis. The gene that codifies for this protein has not yet been found in the genome of Ralstonia eutropha H16, a model organism. By bioinformatic comparison to already known PhaG proteins, a PhaG-like protein was found codified by gene H16_A0147 and presence of the gene was preliminary confirmed by PCR. This is the first study that shows the presence and characteristics of a PhaG-like protein in R. eutropha H16 and represents the first step for the identification of a connection between FAS and PHA pathways in this model bacterium. Further gene deletion and enzymatic activity studies are necessary to confirm this potential relationship, which could improve industrial PHA production and utilisation of agro-industrial residues such as glycerol.

Métricas de artículo

Resumen: 631 HTML (English): 425 PDF (English): 330 XML (English): 73

Métricas PlumX

Biografía del autor/a

Melissa Uribe Acosta, University of Antioquia

Microbióloga industrial y ambiental, estudiante de Maestría en Biología y docente de laboratorio de ecología microbiana de y en la Universidad de Antioquia.

Andrés Felipe Villa Restrepo, University of Antioquia

PhD Microbiología, docente de la Universidad de Antioquia

Citas

Aldor I.S. and Keasling, J.D. (2003). Process Design for Microbial Plastic Factories: Metabolic Engineering of Polyhydroxyalkanoates. Curr. Opin. Biotechnol. 14 475–483. DOI: 10.1016/j.copbio.2003.09.002

Reddy, C.S.K. Ghai, R., Rashmi. and Kalia, V.C. (2003). Polyhydroxyalkanoates: An Overview. Biores Tech 87, 137–146. DOI: 10.1016/S0960-8524(02)00212-2

Urtivia, V., Villegas, P., González, M. and Seeger, M. (2014). Bacterial Production of the Biodegradable Plastics Polyhydroxyalkanoates. Intern J Biol Macromol. 70: 208–213. DOI: 10.1016/j.ijbiomac.2014.06.001

Lau, N., Foong, C.P., Kurihara, Y., Sudesh, K. and Matsui, M. (2014). RNA-Seq Analysis Provides Insights for Understanding Photoautotrophic Polyhydroxyalkanoate Production in Recombinant Synechocystis Sp. PLOS one. Vol 9(1). DOI: 10.1371/journal.pone.0086368

Rehm, B.H.A., Kruger, N. and Steinbuchel, A. (1998). A New Metabolic Link between Fatty Acid de novo Synthesis and Polyhydroxyalkanoic Acid Synthesis: The phaG Gene from Pseudomonas putida KT2440 Encodes a 3-hydroxyacyl- acyl carrier Protein-coenzyme a Transferase. J. Biol.

Chem. 273:24044-24051. DOI: 10.1074/jbc.273.37.24044

Nomura, C.T., Tanaka, T., Eguen, T.E., Appah, A.S., Matsumoto, K., Taguchi, S., Ortiz, L. and Doi, Y. (2008). FabG Mediates Polyhydroxyalkanoate Production from Both Related and Nonrelated Carbon Sources in Recombinant Escherichia coli LS5218. Biotechnol. 24: 42-351. DOI: 10.1021/bp070303y

Wang, Q., Zhuang, Q., Liang, Q. and QI, Q. (2013). Polyhydroxyalkanoic Acids from Structurally-unrelated Carbon Sources in Escherichia coli. Appl Microbiol Biotechnol. Vol 97:3301–3307. DOI: 10.1007/s00253-013-4809-x

Leong, Y.K., Show P.L., Ooi, C.W., Ling, T.C. and Lan, J.C. (2014). Current Trends in Polyhydroxyalkanoates (PHAs) Biosynthesis: Insights from the Recombinant Escherichia coli. J Biotech 180, 52-65. DOI: 10.1016/j.jbiotec.2014.03.020

Fiedler, S., Steinbuchel, A. and Rehm, B.H. (2000). PhaG-Mediated Synthesis of Poly(3-Hydroxyalkanoates) Consisting of Medium-Chain- Length Constituents from Nonrelated Carbon Sources in Recombinant Pseudomonas fragi. Appl. Environ. Microbiol. 66(5):2117-2124. DOI: 10.1128/aem.66.5.2117-2124.2000

Röttig, A., and Steinbüchel, A. (2013). Acyltransferases in Bacteria. Microbiol Mol Bio Reviews : MMBR, 77(2), 277–321. DOI: 10.1128/MMBR.00010-13

Pohlmann, A., Fricke, W. F., Reinecke, F., Kusian, B., Liesegang, H., Cramm, R., Eitinger, T., Ewering, C., Potter, M., Schwartz, E., Strittmatter, A., Voss, I., Gottschalk, G., Steinbuchel, A., Friedrich, B. and Bowien, B. (2006). Hydrogen-based Biotechnology: Genome Sequence of the Bioplastic-producing ‘‘Knallgas’’ Bacterium Ralstonia eutropha H16. Nat Biotechnol 24, 1257–1262. DOI: 10.1038/nbt1244

Peplinski, K., Ehrenreich, A., Doring, C., Bomeke, M., Reinecke, F., Hutmacher, C. and Steinbuchel, A. (2010). Genome-wide Transcriptome Analyses of the ‘Knallgas’ Bacterium Ralstonia eutropha H16 with Regard to Polyhydroxyalkanoate Metabolism. Microbiology, 156: 2136–2152. DOI: 10.1099/mic.0.038380-0

Lindenkamp, N., Peplinski, K., Volodina, E., Ehrenreich, A. and Steinbuchel, A. (2010). Impact of Multiple Beta-Ketothiolase Deletion Mutations in Ralstonia eutropha H16 on the Composition of 3-Mercaptopropionic Acid-Containing Copolymers. Appl. Environ. Microbiol. Vol. 76(16): 5373–5382. DOI: 10.1128/AEM.01058-10

Lindenkamp, N., Volodina, E. and Steinbuchel, A. (2012). Genetically Modified Strains of Ralstonia eutropha H16 with Beta-Ketothiolase Gene Deletions for Production of Copolyesters with Defined 3-Hydroxyvaleric Acid Contents. App. Environ. Microbio. 78(15):5375-83. DOI: 10.1128/AEM.00824-12

Nevill-Manning, C., Wu, T. and Brutlag, D. (1998). Highly Specific Protein Sequence Motifs for Genome Analysis. Proc. Natl Acad. Sci. 95:5865–5871. DOI: 10.1073/pnas.95.11.5865

Arsad H. (2009). Cloning and Characterisation of (R)-3-hydroxyacyl-acyl Carrier Proteincoenzyme A Transferase Gene (phaG) from Pseudomonas sp. USM 4-55. Trop. Life. Sci. Res. 20(2):1-14.

Russell, B., Saqi, M.A., Sayle, R.A., Bates, P.A. and Sternberg, M.J.E. (1997). Recognition of Analogous and Homologous Protein Folds: Analysis of Sequence and Structure Conservation. J. Mol. Biol. 269:423-439. DOI: 10.1006/jmbi.1997.1019

Bedoya, O. and Tischer, I. (2015). Reducing Dimensionality in Remote Homology Detection Using Predicted Contact Maps. Comput. Biol. Med. 59:64-72. DOI: 10.1016/j.compbiomed.2015.01.020

Cao, B., Porollo, A., Adamczak, R., Jarrell, M. and Meller, J. (2006). Enhanced Recognition of Protein Transmembrane Domains with Prediction-based Structural Profiles. Bioinformatics. 22:303-9. DOI: 10.1093/bioinformatics/bti784

Cantu, D.C., Chen, Y. and Reilly, P.J. (2010). Thioesterases: A New Perspective Based on their Primary and Tertiary Structures. Protein Sci. 19:1281–1295. DOI: 10.1002/pro.417

Wang, Q., Tappel, R.C., Zhu, C. and Nomura, C.T. (2012). Development of a New Strategy for Production of Medium-chain-length Polyhydroxyalkanoates by Recombinant Escherichia coli via Inexpensive Non-fatty Acid Feedstocks. Appl Environ Microbiol. 78(2):519-27. DOI: 10.1128/AEM.07020-11

Bugg, T.D. (2004). Diverse Catalytic Activities in the Alphabeta-hydrolase Family of Enzymes: Activation of H2O, HCN, H2O2, and O2. Bioorg Chem. 32(5):367-375. DOI: 10.1016/j.bioorg.2004.05.005

Nardini, M. and Dijkstra, B.W. (1999). α/β Hydrolase Fold Enzymes: The Family Keeps Growing. Curr Opin Struct Biol. 9(6):732-737.

Stothard, P., Van Domselaar, G., Shrivastava, S., Guo, A., O'Neill, B., Cruz, J., Ellison, M. and Wishart, D.S. (2005). BacMap: An Interactive Picture Atlas of Annotated Bacterial Genomes. Nucleic Acids Res 33:D317-D320.

Brigham, C.J., Speth, D.R., Rha, C. and Sinskeya, A.J. (2012). Whole-Genome Microarray and Gene Deletion Studies Reveal Regulation of the Polyhydroxyalkanoate Production Cycle by the Stringent Response in Ralstonia eutropha H16. App Environ Microbiol. 78;8033–8044. DOI: 10.1128/AEM.01693-12

Shimizu, R., Chou, K., Orita, I., Suzuki, Y., Nakamura, S. and Fukui, T. (2013). Detection of Phase-dependent Transcriptomic Changes and Rubisco-mediated CO2 Fixation into Poly (3-hydroxybutyrate) under Heterotrophic Condition in Ralstonia eutropha H16 Based on RNA-seq and Gene Deletion Analyses. BioMed Cen Microbiol. 13:169. DOI: 10.1186/1471-2180-13-169

Tobin, K.M., O'Leary, N.D., Dobson, A.D. and O'Connor, K.E. (2007). Effect of Heterologous Expression of phaG [(R)-3-hydroxyacyl-ACP-CoA transferase] on Polyhydroxyalkanoate Accumulation from the Aromatic Hydrocarbon Phenylacetic Acid in Pseudomonas species. FEMS Microbiol Lett. 268(1):9-15. DOI: 10.1111/j.1574-6968.2006.00607.x