Download DIVERSITY OF PHA DEPOLYMERASE ENCODING GENE

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).