Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
DIVERSITY OF PHA DEPOLYMERASE ENCODING GENE Pattarawan Ruangsuj11,* , Wannaporn Muangsuwan21, Pichai Chaichanachaicharn31, Kosum Chansiri42 and Montri Yasawong51,# 1 Department of Biochemistry, Faculty of Pharmacy, Mahidol University 2 Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University *e-mail: [email protected], #e-mail: [email protected] Abstract Bacteria are able to use polyhydroxyalkanoates (PHAs) as carbon and energy sources. The PHAs were degraded by enzymes called PHA depolymerase. The enzymes are produced from environmental bacteria that contain phaZ gene. PHA depolymerase was encoded by phaZ gene. Thus, phaZ gene was a very important gene for degradation of bioplastics. In this study, phaZ gene sequences were collected from genome database of GenBank. The Bayesian trees were reconstructed for elucidation of the diversity of phaZ gene. There were 168 bacterial strains from the genome database of GenBank that contained at least one copy number of phaZ gene. Most of the phaZ gene was found in the bacterial strains in the phylum Proteobacteria (77.38%) and Actinobacteria (16.07%). These two phyla accounted 93.45% of the total phaZ gene sequences which were obtained from the genome database of GenBank. Phylogenetic analysis was represented two lineages of bacterial phaZ gene. There were phaZ group I (phaZ-I) and phaZ group II (phaZ-II). The phaZ gene of proteobacteria was distributed in both lineages (phaZ-I and phaZ-II). These meant that the proteobacteria may play a very important role for the degradation of bioplastic in environment. The phaZ group II was the most diverse group while the group consisted of six phyla of bacterial strains. The knowledge that gained from the study is essential for designing the experiments, which relates to bioplastic degradation such as primers and probes designed for detection of phaZ gene and etc. Keywords: phaZ gene, PHA depolymerase, phylogenetic Introduction Nowadays people use plastics that made from petroleum base, such as plastic bottles, plastic bags, or other materials that are made of plastic. Plastics are non-biodegradable products so they take several hundred years to degrade them. One way to destroy them is to burn, after that it generates the carbon dioxide which causes of global warming. Besides, chemical compounds in plastic can be absorbed by human bodies. These problems are very important; therefore petroleum plastics are replaced to be bioplastic or biodegradable plastics. In generally, plastic can be classified into 3 types; 1) lastics produced from petroleum such as polyethylene (PE) and polypropylene (PP). 2) Bioplastics produced from petroleum and Natural such as cellulose, collagen or starch and 3) Bioplastics produced from environmental bacteria by used glucose or starch in fermentation such as polyhydroxyalkanoates (PHAs) and poly-3-hydroxybutyrate (PHB). Advantages and disadvantages of plastic 3 types; 1) Plastics produced from petroleum it flexible and thermostable but non-biodegradable. 2) Bioplastics produced from petroleum and natural it easy degrades than petroleum plastics but partially non-biodegradable that is petroleum and few flexible, thermostable. 3) Bioplastics produced from environmental bacteria it completely biodegradable, flexible and thermostable than Type 2 but high cost due to spend culture medium and require a purification step. However, this plastic is friendly to environmental. Therefore, the mechanism degrades of plastics it interesting. Polyhydroxyalkanoates (PHAs) are linear polyesters. PHAs can be classified into two classes, owning to its carbon atom in chemical formula. The first is short-chain-length (SCL) that consists of four to five carbon atoms, and another is medium-chain-length (MCL) that contains six to fourteen carbon atoms. They were degraded by environmental bacteria and success to use as sources of plastic synthesis, called bioplastic. Currently, PHA was decomposed by enzymes called PHA depolymerase. They were encoded with phaZ gene [4]. That the enzyme mostly broken second and third ester linkages from the hydroxyl terminus to oligomers, dimers or monomer [5]. In generally, PHA depolymerases have 4 families: degrading intracellular granules have 2 type nPHAMCL depolymerases and nPHASCL depolymerases. The denatured extracellular PHA granules have 2 type dPHAMCL depolymerases and dPHASCL depolymerases. PHA depolymerase is important for degradation of bioplastic. This research will focus on phaZ gene diversity using phylogenetic technique. The preliminary study of phaZ gene diversity may helpful for designing futher study such as development of DNA sensor for detection of this gene. Methodology Construction of phaZ gene database The phaZ gene sequences were collected from the genome database of GenBank. Quality of the gene sequences was manually justified. Only high quality of the gene sequences was kept for further analysis. Phylogenetic analysis Multiple sequences alignment was performed based on iterative refinement method using MUSCLE version 3.8.31. Evolutionary model of phaZ gene was selected based on AIC and hLRTs criteria using MrModeltest version 2.3. Phylogenetic tree was analyzed based on Bayesian inference method using parallel version of MrBayes. The phylogenetic analysis was performed on computer cluster (KIRI cluster). The cluster was assembled from four IBM servers (x3250 M4), which were connected by a gigabit switch (HP ProCurve 1410-16G). The Bayesian posterior probabilities were obtained by performing two separate runs with twelve Markov chains. Each run was conducted with 1×107 generations and sampled every 100 generations. A consensus tree was calculated after discarding the first 25% of the iterations as burn-in. Phylogenetic tree drawing was performed using TreeView version 1.6.6. Results Construction of phaZ gene database There were 168 bacterial strains from the genome database of GenBank that contained at least one copy number of phaZ gene. The gene sequences had been collected and converted to the same orientation for futher analysis. The phaZ gene was distributed in ten phyla of bacteria (Figure 1.). There was no phaZ gene found in achaeal genome. Most of the phaZ gene was found in the bacterial strains in phylum Proteobacteria (77.38%) and Actinobacteria (16.07%) (Figure 1.). These two phyla accounted 93.45% of the total phaZ gene sequences which were obtained from the genome database of GenBank. 140 120 OTUS 100 80 60 40 20 0 AC AT BD CF CY DT FM PB SP TM Phylum Figure 1. Diversity of phaZ gene in bacterial genome database (GenBank). AC: Acidobacteria; AT: Actinobacteria; BD: Bacteroidetes; CF: Chloroflexi; CY: Cyanobacteria; DT: DeinococcusThermus; FM: Firmicutes; PB: Proteobacteria; SP: Spirochaetes; TM: Thermotogae. Phylogenetic analysis of bacterial phaZ gene Phylogenetic analysis was represented two lineages of bacterial phaZ gene that were phaZ group I (phaZ-I) and phaZ group II (phaZ-II) (Figure 2. and 3.). The first lineage was phaZ group I (Figure 2.). This group was able to divide into four subgroups, which were group A, B, C and D (Figure 2.). The member of phaZ group I was only the bacterial strains in phylum Proteobacteria. The second lineage was phaZ group II (Figure 3.). The group consisted of five subgroups that were subgroup A, B, C, D and E (Figure 3.). The members of phaZ-II subgroup A were bacterial strains in phylum Actinobacteria and Proteobacteria. PhaZ-II subgroup B contained only bacterial strains in the phylum Proteobacteria. PhaZ-II subgroup C consisted of bacterial strains in phylum Actinobacteria, Proteobacteria and Spirochaetes. PhaZ-II subgroup D contained bacterial strains in phylum Actinobacteria, Deinococcus-Thermus and Proteobacteria.The last subgroup of phaZ-II was subgroup D. This subgroup consisted of bacterial strains in phylum Bacteroidetes, Proteobacteria and Thermotogae. Figure 2. Bayesian inference tree based on complete phaZ gene sequences showing relationships of environmental bacteria and indicated to phaZ-I subgroup: A, B, C and D. Clade credibility values, expressed as percentages of 1×107 replications. Figure 3. Bayesian inference tree based on complete phaZ gene sequences showing relationships of environmental bacteria and indicated to phaZ-II subgroup: A, B, C, D and E. Clade credibility values, expressed as percentages of 1×107 replications. Discussion and Conclusion The phaZ gene was discovered from various kinds of environmental bacteria. Bacterial strains that belonged to the phylum Proteobacteria, accumulated 77.38% of phaZ gene. The phaZ gene of Proteobacteria was distributed in both lineages (phaZ-I and phaZ-II). These meant that the proteobacteria may play the very important role for the degradation of bioplastic in environment. However, the phaZ group II was the most diverse group while the group consisted of six phyla of bacterial strains that were phylum Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Proteobacteria, Spirochaetes and Thermotogae. Most of the bacterial strains that consisted of phaZ gene were isolated from soil samples and grown optimally 28-37°C with 7.2-8.0 of pH [6-9]. The knowledge that gained from the study is essential for designing the experiments, which relates to bioplastic degradation such as primers and probes designed for detection of phaZ gene and etc. References 1. 2. 3. 4. 5. 6. 7. 8. 9. Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev. 1990 Dec;54(4):450-72. Garcia B, Olivera ER, Minambres B, Fernandez-Valverde M, Canedo LM, Prieto MA, et al. Novel biodegradable aromatic plastics from a bacterial source. Genetic and biochemical studies on a route of the phenylacetyl-coa catabolon. J Biol Chem. 1999 Oct 8;274(41):29228-41. Prieto MA. From oil to bioplastics, a dream come true? J Bacteriol. 2007 Jan;189(2):289-90. de Eugenio LI, Garcia P, Luengo JM, Sanz JM, Roman JS, Garcia JL, et al. Biochemical evidence that phaZ gene encodes a specific intracellular medium chain length polyhydroxyalkanoate depolymerase in Pseudomonas putida KT2442: characterization of a paradigmatic enzyme. J Biol Chem. 2007 Feb 16;282(7):4951-62. Shirakura Y, Fukui T, Saito T, Okamoto Y, Narikawa T, Koide K, et al. Degradation of poly(3hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1. Biochimica et Biophysica Acta (BBA) - General Subjects. 1986;880(1):46-53. GILLIS M, VAN VAN T, BARDIN R, GOOR M, HEBBAR P, WILLEMS A, et al. Polyphasic Taxonomy in the Genus Burkholderia Leading to an Emended Description of the Genus and Proposition of Burkholderia vietnamiensis sp. nov. for N2-Fixing Isolates from Rice in Vietnam. International Journal of Systematic Bacteriology. 1995 April 1, 1995;45(2):274-89. Ghosh W, Bagchi A, Mandal S, Dam B, Roy P. Tetrathiobacter kashmirensis gen. nov., sp. nov., a novel mesophilic, neutrophilic, tetrathionate-oxidizing, facultatively chemolithotrophic betaproteobacterium isolated from soil from a temperate orchard in Jammu and Kashmir, India. Int J Syst Evol Microbiol. 2005 Sep;55(5):1779-87. Yang LL, Tang SK, Zhang YQ, Zhi XY, Wang D, Xu LH, et al. Thermobifida halotolerans sp. nov., isolated from a salt mine sample, and emended description of the genus Thermobifida. Int J Syst Evol Microbiol. 2008 Aug;58(8):1821-5. Ding X, Yin B, Qian L, Zeng Z, Yang Z, Li H, et al. Screening for novel quorum-sensing inhibitors to interfere with the formation of Pseudomonas aeruginosa biofilm. J Med Microbiol. 2011 Dec;60(12):1827-34. Acknowledgements: This work was financially supported by the National Research Council of Thailand (NRCT).