Journal of Bioscience and Bioengineering

VOL. 121 No. 4, 355e364, 2016

www.elsevier.com/locate/jbiosc

Discovery of a new polyhydroxyalkanoate synthase from limestone soil through

metagenomic approach

Yen Teng Tai,1 Choon Pin Foong,1 Nazalan Najimudin,1 and Kumar Sudesh1, 2, *

School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia1 and Centre for Chemical Biology, Universiti Sains Malaysia, 11800 Penang, Malaysia2

Received 20 May 2015; accepted 11 August 2015

Available online 20 October 2015
PHA synthase (PhaC) is the key enzyme in the production of biodegradable plastics known as polyhydroxyalkanoate
(PHA). Nevertheless, most of these enzymes are isolated from cultivable bacteria using traditional isolation method.
Most of the microorganisms found in nature could not be successfully cultivated due to the lack of knowledge on their
growth conditions. In this study, a culture-independent approach was applied. The presence of phaC genes in limestone
soil was screened using primers targeting the class I and II PHA synthases. Based on the partial gene sequences, a total of
19 gene clusters have been identified and 7 clones were selected for full length amplification through genome walking.
The complete phaC gene sequence of one of the clones (SC8) was obtained and it revealed 81% nucleotide identity to the
PHA synthase gene of Chromobacterium violaceum ATCC 12472. This gene obtained from uncultured bacterium was
successfully cloned and expressed in a Cupriavidus necator PHBL4 PHA-negative mutant resulting in the accumulation
of significant amount of PHA. The PHA synthase activity of this transformant was 64 ± 12 U/g proteins. This paper
presents a pioneering study on the discovery of phaC in a limestone area using metagenomic approach. Through this
study, a new functional phaC was discovered from uncultured bacterium. Phylogenetic classification for all the phaCs
isolated from this study has revealed that limestone hill harbors a great diversity of PhaCs with activities that have not
yet been investigated.
Ó 2015, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Polyhydroxyalkanoate; PHA synthase gene; Metagenomic; Genome walking; Limestone; Uncultured bacterium]

In this modern era, plastics play a vital role in many aspects of and vegetable oils. It can be synthesized by a wide range of gram-
our lives. Plastics have been widely used in daily products ranging positive and gram-negative bacteria, for instance Cupriavidus
from food packages, containers, furniture, toys, shoes, clothes, necator (previously known as Ralstonia eutropha), Pseudomonas
electronic devices and even medical devices. However, they are oleovorans, Bacillus sp., Chromobacterium sp. and Burkholderia sp.
produced from petroleum, an extract of non-renewable fossil fuels. (3e7). PHA is stored as intracellular storage materials in the cyto-
They pose several setbacks such as waste accumulation due to their plasm of bacteria under stress conditions such as in the event of
non-biodegradability. As such, the widespread use of plastics has limitation of nitrogen, phosphorus or magnesium although carbon
caused global environmental issues due to its non-renewability and is in excess (8).
long-term non-sustainability. An increasing cost of production due In order to synthesize and accumulate PHA in the bacterial cell
to depletion of oil reserves is another discouraging factor on the cytoplasm, several enzymes are involved. One of the key enzymes is
petrochemical-based plastics. the PHA synthase (PhaC), which catalyzes the stereo-selective
In the USA, Europe and Japan, about 55 million tons of post- conversion of (R)-3-hydroxyacyl-CoA substrates to PHA with the
consumer plastic wastes were generated and ended up in landfill concomitant release of CoA. There are four classes of PHA synthase
sites (1). In Malaysia, plastics also contribute to a large amount of enzymes based on their subunit composition, representative spe-
the solid wastes in Kuala Lumpur and an increase in the volume of cies and substrate specificity. The type of PHA produced is strongly
such waste has been observed year on year (2). In order to over- dependent on the classes of PHA synthase in the bacteria. However,
come problems of plastics waste management, bioplastics offer a most of these enzymes have been isolated from cultivable bacteria
good alternative to replace some of these petrochemical-based because the classical method of enzyme discovery involves
plastics. Hence, polyhydroxyalkanoate (PHA) is a promising sub- isolating, culturing and growing of microorganisms under different
stitute due to its biodegradability in the environment. conditions. Unfortunately, 99% of the microorganisms found in
PHA is a type of naturally occurring polyester that can be pro- nature are still uncultivable (9).
duced from renewable resources such as agricultural wastes, sugar Metagenomics, sometimes referred to as community genomics
or environmental genomics, is the sequencing and analysis of DNA
of microorganisms recovered from an environment, without the
* Corresponding author at: School of Biological Sciences, Universiti Sains Malaysia,
need of culturing. By generating metagenomic libraries from
11800 Penang, Malaysia. Tel.: þ60 46534367; fax: þ60 46534060. different environmental niches, different types of novel genes or
E-mail address: [email protected] (K. Sudesh). enzymes have been successfully isolated. Previous study showed

1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2015.08.008
356 TAI ET AL. J. BIOSCI. BIOENG.,

that less than 1% of all bacterial species have been cultured due to CTAB) were added together with 100 mL of proteinase K (10 mgmL1). The soil
the lack of knowledge of their growth conditions. For example, the samples were incubated at 37 C for 30 min by horizontal shaking at 225 rpm.
After that, 3 mL of 10% SDS solution was added to each sample and incubated at
commonly used culture medium may be biased towards certain 65 C for 2 h with gentle end-over-end inversions every 15e20 min.
types of bacteria (9,10). Therefore, metagenomics enables the study Following incubation, each suspension of soil samples was centrifuged at
of various communities of microorganisms by deciphering their 6000 g for 10 min at room temperature and was transferred into a new sterile
genetic information from DNA that is extracted directly and thus 50 mL centrifuge tube. The soil pellets were extracted again by adding 4.5 mL of the
DNA extraction buffer and 1 mL of 10% SDS, vortexing for 10 s and incubating at 65 C
sidestepping the need for culturing or isolation. Due to its ability to
for 10 min. The soil samples were once again centrifuged and the supernatants from
uncover hidden diversity of microbial communities, metagenomics the two cycles of extractions were combined. Subsequently, an equal volume of
offers a powerful approach. chloroform isoamyl alcohol (24:1, vol/vol) was added in. The aqueous phase was
Many studies have been performed on metagenomic samples recovered by centrifugation and precipitated with 0.6 volume of isopropanol
from different niches such as canine oral cavity, sewage sludge and at 20 C overnight. On the following day, the soil samples were centrifuged at
16,000 g for 20 min at room temperature. Subsequently, the pellet was washed
bovine rumen (11e13). Besides that, there are also reports on soil with cold 70% ethanol and resuspended in sterile deionized water to give a final
metagenomes yielding new knowledge of the uncultured world volume of 200 mL. Gel electrophoresis was carried out on 0.8% agarose gel using
(14e17). Since Malaysia is well known as one of the twelve mega Mupid-exU System (Takara, Japan) with 50 V for 1 h to observe the quality of the
biodiversity countries in the world, its natural diversity serves as a extracted DNA. The agarose gel containing DNA band of approximately 20 kb in size
was excised and subjected to gel purification with Wizard SV Gel and PCR Clean-Up
natural gene bank of biodiversity. There are an enormous number
System (Promega, USA) according to the manufacturer’s protocol.
of unknown PHA-producing microorganisms that are yet to be
Screening of PHA synthase genes from limestone soil PCR was performed
explored in these natural environments. using class I and II degenerate primers (G-D and G-1R) to amplify the partial PHA
The presence of PHA producing bacteria in different environ- synthase (phaC) gene from the soil genomic DNA (22) (Table S2). The optimized PCR
ments including a limestone area was previously reported (18). reaction mixture contained 1 Green GoTaq Flexi Buffer, 2.5 mmol l1 MgCl2
Limestone hills and outcrops are common in Malaysia especially in Solution, 0.2 mmol l1 dNTP Mix, 1 mmol l1 of upstream and downstream
primers, 1.25 U of GoTaq DNA Polymerase (Promega, USA) and 100 ng of purified
the northern region. Ipoh, also known as Hill City is surrounded by soil DNA. The PCR run was performed in an MJ Mini Gradient Thermal Cycler (Bio-
limestone outcrops and caves making it a top tourist attraction site Rad, USA) and consisted of one cycle of 95 C for 2 min and 39 cycles of 95 C for
in Malaysia. As these caves are abundant in calcium carbonate, 30 s, 55.5 C for 1 min, 72 C for 2 min; one cycle at 72 C for 5 min. The PCR
organisms in these niches are able to endure highly alkaline envi- product was purified with Wizard SV Gel and PCR Clean-Up System (Promega, USA).
The PCR product was ligated into pGEM-T Easy Vector (Promega, USA) and
ronment. Besides that, these organisms are able to withstand the
transformed into E. coli JM109 High Efficiency Competent Cells (Promega, USA) ac-
severity of exceedingly arid conditions during the dry season. Thus, cording to the manufacturer’s protocols. The transformation culture was plated onto
limestone was chosen in this study because it provides a unique LB/ampicillin/IPTG/X-Gal plates and the plates were incubated at 37 C for 16 h.
periodically stressful habitat to bacteria. It is believed that there White colonies were sub-cultured onto LB/ampicillin plates and incubated overnight
will be an enormous amount of PHA producing bacteria in this area at 37 C.

due to such stressful conditions that favor the accumulation of PHA. Sequence analyses All of the positive transformants were subjected to
plasmid DNA extraction using QIAprep Plasmid Miniprep Kit (Qiagen, German). The
This is the first attempt to explore PhaC diversity from limestone
plasmids were sent to Genomics Bioscience, Kuala Lumpur for DNA sequencing. The
using a culture-independent approach. In this study, screening of sequence analyses were carried out according to previous reports with modifica-
class I and II PHA synthase genes was carried out from limestone tions (23,24). Each of the obtained sequence was trimmed by removing the pGEM-T
soil metagenomic samples of Gunung Lang, Ipoh, Perak. The aim Easy Vector sequence using BioEdit (25). After that, the sequences were used to
check for open reading frame through Expasy (http://web.expasy.org/translate/).
was to identify new PHA synthase genes from uncultivable bacteria.
Sequences without proper open reading frame were removed from further analysis.
Subsequently, the substrate specificity and enzyme activity of the The identity of each clone was determined by using the Basic Local Alignment
PHA synthase was determined by heterologous expression of the Search Tool (BLAST) analysis of the translated amino acid sequence. Sequence
identified gene in a PHA negative mutant strain. similarities among the clones were identified using MegAlign (DNASTAR, USA) and
those with more than 90% sequence similarities were classified under the same gene
group. All the gene groups were then further analyzed by MEGA 5.2 (26) using
MATERIALS AND METHODS CLUSTAL W and a phylogenetic tree was drawn using neighbor-joining method with
bootstrap value of 1000.
Full length PHA synthase gene amplification through genome
Bacterial strains, plasmids and maintenance Bacterial strains and plas-
walking The full length phaC gene amplification was carried out using inverse
mids used and constructed in this study are shown in Table S1. C. necator was
affinity nested-PCR (IAN-PCR) as described by previous studies (24,27). Primers used
routinely cultivated at 30 C in a nutrient rich (NR) medium composed of 10 g L1
are listed in Table S2. The extracted genomic DNA (5 mg) was digested using ApaI,
of meat extract, 10 g L1 of peptone, and 2 g L1 of yeast extract (19). Cultures of
BamHI, HindIII and NotI restriction enzymes (REs) respectively (Thermo Scientific,
Escherichia coli JM109 and S17-1 were grown at 37 C in a Luria Bertani (LB)
USA) and then self-ligated using DNA Ligation Kit Ver 2.1 (TaKaRa, Japan) at 16 C
medium for general cloning and transconjugation experiments, respectively.
for 45 min. Since the size of complete PHA synthase gene is approximately 2 kb
Kanamycin (50 mg mL1) or ampicillin (100 mg mL1) was added to the media
and thus, the genomic DNA cannot be over-digested (size of fragments < 2 kb) in
when necessary. Plasmid pGEM-T Easy (Promega, USA) and pBBR1MCS-2 (20)
order to obtain the complete gene. The self-ligated DNA was used as a template
were used for cloning purpose.
for the first round inverse PCR. A PCR mixture consisted of 2.5 U LA Taq DNA
Sample collection and soil pretreatment Soil sampling was carried out at polymerase (TaKaRa, Japan), 0.4 mmol l1 each dNTP, 1 GC buffer I, and
Gunung Lang, Ipoh, Perak (GPS coordinate: 4.622N, 101.0888E) in May, 2013. The 0.4 mmol l1 each primer in a total volume of 50 mL. The PCR conditions were as
location of the soil was underneath a limestone hill. The soil sample was collected follows: 94 C for 1 min, followed by 40 cycles of 98 C for 20 s, 68 C for 20 s, and
from about 5 cm deep from the top layer. Rock sample was also collected from the a final step at 72 C for 10 min. After that, the PCR products were purified using
limestone hill. The materials were then transported to the laboratory under sterile the QIAquick PCR Purification kit (Qiagen, Germany) and subjected to affinity
conditions. In the laboratory, the plant roots and humus were removed from the soil purification using Dynal Dynabeads Kilobase Binder kit (Invitrogen, USA). Then,
samples in a sterile condition. The samples were then stored in a freezer at a tem- 2 mL of the product was used as a template for second round nested PCR. The PCR
perature of 20 C. mixture and conditions used were similar as described earlier (Ta ¼ 50 C).
Rock analysis The limestone sample was sent to the Archeology Centre, Purified PCR products were cloned into vector pGEM-T Easy and JM109 High
Universiti Sains Malaysia (USM) for X-Ray Fluorescence (XRF) analysis to confirm the Efficiency Competent Cells (Promega, USA). The plasmids were extracted and sent
identity of the rock and at the same time, determine the major elements present. for DNA sequencing. Each clone was identified with the BLAST using translated
DNA extraction and purification from limestone soil The DNA was amino acid sequence. Full length PHA synthase gene was determined by
extracted using a sodium dodecyl sulphate (SDS)-based method for DNA recovery assembling the sequenced DNA fragments using SeqMan (DNASTAR, USA).
from soil with some modifications (21). Hexadecylmethylammonium bromide Construction of C. necator PHBL4 transformant Forward and reverse
(CTAB) was used to reduce humic acids contamination. Approximately 5 g of soil primers (SC8F and SC8R) were designed based on the complete phaC gene sequence
samples were weighed in sterile 50 mL centrifuge tubes. Subsequently, 13.5 mL of with each primer containing additional restriction sites at the 50 -end (Table S2). PCR
DNA extraction buffer (100 mmol l1 TriseHCl [pH 8.0], 100 mmol l1 sodium was carried out using the enzyme LA Taq (TaKaRa, Japan) with the following
EDTA [pH 8.0], 100 mmol l1 sodium phosphate [pH 8.0], 1.5 mol l1 NaCl, 1% conditions: 94 C for 1 min followed by 35 cycles of 98 C for 20 s, 63 C for 20 s
VOL. 121, 2016 NEW PhaC FROM LIMESTONE METAGENOME 357

and 72 C for 2 min with a final extension of 5 min at 72 C. The PCR products were TABLE 1. Major elements in limestone detected through XRF
digested using restriction enzymes ApaI and HindIII. The digested DNA was cloned analysis.
into a plasmid pBBR1MCS-2 with promoter from C. necator obtained from
Composition Weight percent (%)
previous study (24) and subsequently transformed into E. coli S17-1.
Transconjugation was carried out to transfer the plasmid from S17-1 to the PHA CaO 97.86
negative mutant PHB4 (28). SiO2 1.44
PHA production using C. necator PHBL4 transformant PHA biosynthesis MgO 0.56
of recombinant C. necator was carried out through one-stage batch cultivation in Fe2O3 0.04
 K2O 0.03
shake flasks. C. necator PHB 4 was used as a negative control while C. necator H16
was used as a positive control. Two loops of bacteria that were grown for 16e18 h P2O5 0.03
were inoculated into 50 mL of NR medium. The bacteria were cultured at 30 C MnO 0.02
and 200 rpm for 5 h to enrich the cell mass (OD600 nm ¼ 4.5e5.0). Approximately Na2O 0.02
3% (v/v) of the inoculum was then transferred into 50 mL of mineral medium Total 100
(MM) and incubated for 48 h under the same condition with the addition of
5 g L1 of crude palm kernel oil as carbon source for PHA accumulation. The MM
was prepared according to the following compositions (per liter): 3.32 g Na2HPO4,
2.80 g KH2PO4, 0.5 g NH4Cl, 0.25 g MgSO4$7H2O and 1 mL trace element solution (35). The calcium oxide content in this rock is 97.86% (Table 1)
which consisted of 0.22 g CoCl2$6H2O, 9.7 g FeCl3, 7.8 g CaCl2, 0.12 g NiCl2$6H2O, and thus fulfilling the criteria for carbonate rocks definition.
0.11 g CrCl3$6H2O and 0.16 g CuSO4$5H2O in 1 L of 0.1 N HCl (4). The cells were
harvested after a 48 h cultivation period. The cells were pelleted by centrifugation Screening of PHA synthase genes from limestone
at 5870 g for 5 min at 4 C. Residual oil was removed by adding approximately soil Using G-D and G-1R degenerate primers, phaC gene frag-
20 mL of hexane to the cell pellet followed by mixing using a vortex mixer and
centrifugation at 5870 g for 3 min at 4 C. A final centrifugation (5870 g for
ments of approximately 550 base pairs were obtained (22). A total of
5 min at 4 C) was performed with the addition of 50 mL distilled water to the 181 partial PHA synthase gene clones were generated. After DNA
pellet to remove the remaining hexane. The harvested cells were frozen at 20 C sequencing, the amino acid sequences of all the clones were
for 24 h prior to freeze drying for 48 h to remove water from the cells in the compared with each other and the amino acid sequences of known
frozen state. The PHA content and PHA composition of the freeze-dried cells were
proteins by using BLASTx analysis. Among these, 110 clones
determined using Shimadzu GC-2010 system equipped with column SPB-1
(Supelco, USA) and the internal standard used was caprylic acid methyl ester. contained phaC genes but only 87 clones (GenBank accession
Assessment of PHA synthase assay C. necator H16 and PHB4 transformant number: KP881518eKP881604) were considered as unique as
cells were subjected to PHA biosynthesis using CPKO as carbon source. After that, the some other clones have identical gene sequences. A phylogenetic
cells were harvested by centrifugation and resuspended in 20 mmol L1 TriseHCl tree was constructed based on their DNA sequences together with
(pH 8) at a ratio of 1 g of cells to 5 mL of TriseHCl buffer solution. Previous study the reference phaC gene using neighbor-joining method (Fig. 1).
showed that the highest synthase activity was achieved at 24 h of cultivation, thus
the cells used for this experiment were harvested at this time (29). The cells were
They can be grouped into 19 clusters and all of the identified phaC
disrupted through sonication at amplitude of 30 for 7 min with 10 s alternation genes belonged to the class I family although the degenerate
between on and off. This process was repeated three times and the cells primers used were targeted towards the genes for the class I and
suspension was kept in ice. Protein concentration was measured using the class II PhaC proteins. All of the partial phaC genes obtained were
Bradford assay with bovine serum albumin (BSA) as standard (30). A total of 20 mg
unique because they did not show close identity (<85%) to known
of the crude cell lysates was subjected to sodium dodecyl sulfate polyacrylamide
gel (SDS-PAGE) electrophoresis. On the other hand, the activity of PHA synthase PHA synthase genes (Table S3).
was determined from crude extracts of sonicated cells according to the
spectroscopic assay described previously with modifications (31,32). The total Full length PHA synthase gene amplification through
enzyme activity was determined by measuring the amount of CoA released from genome walking Within the 19 gene clusters, 7 clones (SC2
3HB-CoA. The crude protein extract of sonicated cell pellets containing 35e40 mg from gene cluster 1, SC8 from gene cluster 3, SC37 from gene cluster
of protein was added to PhaC assay buffer containing 0.6 mmol L1 3HB-CoA 6, SC61 from gene cluster 8, SC103 from gene cluster 15, SC137 from
lithium salt (SigmaeAldrich, USA), 150 mM potassium phosphate buffer pH 7.2,
gene cluster 16, and SC156 from gene cluster 19) were selected for
0.2% (w/v) glycerol and 0.05% (w/v) Hecameg. The mixture was incubated at 30 C
for 2 min after which 20 mL of the sample was removed at intermittent time full length amplification. These 7 clones were chosen because their
points of 20, 40, 60, 90 and 120 s to new 1.5 mL microcentrifuge tubes containing BLASTx results showed that they were similar to bacteria that have
50 mL chilled 0.6 N trichloroacetic acid (TCA) respectively. All tubes were not been studied for PHA production. The full length amplification
incubated at room temperature for 1 min prior to centrifugation at 9700 g for
involved three stages: inverse PCR, affinity purification and nested
1 min. Subsequently, 65 mL of the supernatants were transferred to new tubes and
385 mL of 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB) reaction solution was added.
PCR (27). The phaC gene of one of the clones (SC8) was successfully
Another tube was prepared for 0 s containing 50 mL of TCA, 20 of mL PhaC assay recovered (1662 bp) through this method. However, the open
buffer and 380 mL of DTNB reaction solution. The mixtures were mixed thoroughly reading frame (ORF) obtained was not complete and thus, an
and 450 mL was transferred to black-wall quartz cuvette. The concentration of CoA artificial stop codon was added at the end of the sequence. Based
was determined using Multiskan GO (Thermo Scientific, USA) with a molar
on the BLAST results, phaCSC8 (GenBank accession number:
absorption coefficient of 15.6  103 M1 cm1 at a wavelength of 412 nm (33) and
450 mL of 0.5 M potassium phosphate buffer pH 7.8 was used as the blank. One KP027476) which obtained from uncultured bacterium shows 81%
unit of enzyme activity (U) is defined as the amount of enzyme required to identity to the PHA synthase gene of Chromobacterium violaceum
catalyze the liberation of 1 mmol CoA in 1 min. ATCC 12472 (BLASTn) and 79% identity with PHA synthase gene
Accession number The sequences obtained from this study have been of beta proteobacterium L13 (BLASTx). The predicted amino acid
deposited into GenBank (NCBI). There were 87 partial phaC genes (GenBank
sequence was aligned with other commonly known PhaC
accession number: KP881518eKP881604) and 1 complete phaC gene (GenBank
accession number: KP027476) discovered in this study.
proteins and a putative lipase box (GXCXG) was detected (Fig. 2).
The residues with shaded regions were also found to be
conserved among all the sequences (36). The amino acid identity
of this PHA synthase was compared with the PHA synthase of
RESULTS
other well known PHA producing producers from four different
classes (Table 2). Among all these, PhaCSC8 shows the highest
Rock analysis The rock sample collected from the sampling amino acid identity to PhaC of Chromobacterium sp. USM2 which
site was confirmed to be of limestone origin using XRF. Limestone is 79%.
composes of seashells and is rich in calcium carbonate (CaCO3). The
calcium carbonate exists in two forms, namely aragonite and Construction of C. necator PHBL4 transformant Forward
calcite. Since aragonite is unstable, it undergoes transformation to and reverse primers were designed based on the complete gene
calcite, the main component of limestone (34). By definition, sequences with the addition of restriction sites (ApaI and HindIII)
carbonate rocks must contain more than 50% carbonate minerals (Table S2). The plasmid constructed (pBBR1MCS-2 ProCn PhaCSC8)
358 TAI ET AL. J. BIOSCI. BIOENG.,

FIG. 1. Phylogenetic tree showing the known PHA producing bacteria and the 19 gene clusters (90% identity) identified from Gunung Lang, Ipoh. The PHA synthase genes can be
divided into four different classes (red, class I; blue, class II; green, class III and pink, class IV). Those marked with filled triangle were the gene clusters obtained in this study. The
clone SC8 is under gene cluster 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

was transconjugated from S17-1 to the PHA negative mutant PHB4 PAGE) electrophoresis to study the expression level of PhaC for both
for subsequent PHA production. strains. According to Rehm (36), the molecular weight of Class I PHA
synthase is around 60e73 kDa. Based on Fig. 3, it can be seen that
PHA production using C. necator PHBL4
PhaC presents in both strains. Clear bands at approximately 60 kDa
transformant PHA production was carried out to confirm that
confirmed that the phaC gene was expressed successfully. The PHA
the phaC gene was expressed and functional in the C. necator PHB4
synthesizing ability was studied further to investigate the
transformant. Table 3 shows the comparison of PHA content by
properties of this synthase and C. necator H16 was selected for
different bacterial strains when 5 g L1 of CPKO and 10 g L1 of
comparison. Previous reports have shown that the highest
fructose were used as the sole carbon source for cell growth and
synthase activity was achieved at 24 h of cultivation, thus the
PHA production respectively. The C. necator PHB4 transformant
cells used for this experiment were collected according to this
harboring the PHA synthase gene from SC8 was able to
time (29). Besides that, total protein extracts were used because
accumulate 55 wt. % of PHA of the cell dry weight when CPKO
the granule-bound form PHA synthase exhibits higher activity
was used as the carbon source. It was found to produce 14 wt. %
compared to the soluble form (37).
of PHA when 10 g L1 of fructose was used. On the other hand,
The 3HB-CoA was used as the substrate and the CoA release was
the C. necator H16 showed a relatively higher PHA production
measured to determine the total enzyme activity. Based on the
when these two carbon sources were used. For the negative
result, the enzymatic activity of PhaCSC8 was not as high as that of
mutant, only negligible amount of PHA was produced.
wild type C. necator H16 enzyme (Fig. 4). The specific activity of
Assessment of PHA synthase activity The crude cell lysates transformant PHB4 ProCn PhaCSC8 was 64  12 U/g protein, which
was subjected to sodium dodecyl sulfate polyacrylamide gel (SDS- is approximately one third of the specific activity for the C. necator
VOL. 121, 2016 NEW PhaC FROM LIMESTONE METAGENOME 359

FIG. 2. Multiple sequence alignment of amino acid sequences of phaCSC8 and other commonly known phaC. The box indicates the position of the putative lipase box. Amino acid
residues with shaded regions were found to be conserved among all of the sequences.
360 TAI ET AL. J. BIOSCI. BIOENG.,

FIG. 2. (continued).
VOL. 121, 2016 NEW PhaC FROM LIMESTONE METAGENOME 361

FIG. 2. (continued).

H16 enzyme (173  8 U/g protein). Result of the in vivo experiment on this enzyme have to be carried out to comprehend its complete
correlated with the result of in vitro experiment where C. necator structure and properties. By far, the PhaC of C. necator is the most
H16 also exhibited higher PHA producing ability. extensively studied and it is commonly used to evaluate the per-
formance of other synthases (38). Different bacteria may have
DISCUSSION different PhaC. Therefore, it is worthwhile to identify more mi-
croorganisms with PhaC of different substrate specificity and ac-
The types of PhaC enzyme differ among different bacterial tivity. Although many PHA synthases have been identified, however
species and strains. PhaC is important in the determination of the most of these enzymes were from cultivable bacteria. Therefore, we
composition and properties of PHA (36). Since PhaC plays a domi- hope that with the help of metagenomic tools, more novel PHA
nant role in the PHA biosynthesis pathway, further investigations synthases can be found.
362 TAI ET AL. J. BIOSCI. BIOENG.,

TABLE 2. Amino acid identities of PhaCSC8 to PhaC of well known PHA producers
from different classes determined using BLASTp.

Organisms and gene Class of PhaC Accession number Identity (%)

Cupriavidus necator H16 PhaC I WP_011615085.1 47

Aeromonas cavie PhaC I D88825.1 40
Chromobacterium sp. USM2 PhaC I HM989943.1 79
Burkholderia sp. USM PhaC I JN022533.1 47
Pseudomonas oleovorans PhaC1 II M58445.1 36
Pseudomonas oleovorans PhaC2 II M58445.1 38
Bacillus megaterium PhaC III GU190757.1 24
Allochromatium vinosum PhaC IV EF067338.1 23

Little is known of the biodiversity in the limestone area

compared to the other habitats such as mangroves, forests and sea
water since not many studies have been carried out on this area.
FIG. 3. SDS-PAGE analysis of crude extracts of PhaCCn and PhaCSC8. SM, size marker; W,
The significance of limestone as an important habitat for rare
whole cell extract; S, supernatant; P, cell pellets; a, C. necator H16; b, C. necator
species has often been overlooked. No study has been carried out transformant PHBL4 ProCn PhaCSC8. Both strains were grown for 24 h using CPKO as
on the identification of PHA synthase gene from this area. This carbon sources in mineral medium (200 rpm, 30 C). The presence of bands at
makes limestone a suitable location for prospecting new PHA approximately 60 kDa indicates the PHA synthase and the expression level is about the
same based on the band intensity in the cell pellets (insoluble fraction).
synthase genes and culture independent method used in this study
can increase the chance of finding novel genes. In this study, se-
quences of 110 partial phaC genes were obtained and the BLASTx
In comparison to the PHA production of wild type H16, the
results are shown in Table S3. Among these 110 clones, only 87
performance of this transformant was poor. Since this mutant
clones (GenBank accession number: KP881518eKP881604) were
strain was derived from 1-nitroso-3-nitro-L-methylguanidine
different among each other as some clones contained identical gene
(NMG) treatment, thus, it is undefined. The mutations might occur
sequence. Based on the BLASTx results, the phaC genes identified
in the genome of the strain and have secondary effect on the cell
were mostly from microorganisms that are not well studied with
metabolism (41). Based on a recent study that investigated the
relatively low sequence identity (<85%) to known microorganisms
genotype of this negative mutant, it was found that several proteins
and therefore, we hypothesized that these partial phaC genes were
have been expressed increasingly compared to the wild type. Under
derived from uncultured bacteria. This showed that limestone soil
the growth conditions that favor the accumulation of PHA, this
provided a unique niche that harbored a great diversity of phaC
mutant strain was overwhelmed with acetyl-CoA and could not
genes that was worth investigating in the future.
direct these intermediates towards the synthesis of PHA. Therefore,
Based on the partial gene sequences obtained, a new functional
the mutant PHB-4 excreted large amounts of pyruvate and caused
PHA synthase was recovered from uncultured bacterium and
the pH of the medium to be lowered which impaired the growth of
expressed successfully in a PHA negative mutant (PHB4).
this bacterium (42). This can be seen from Table 3 where the
C. necator has long served as a model in the study of PHA produc-
negative mutant had lower cell dry weight. This happened to the
tion (39) and was thus used as a host in this study. The best pro-
transformant as well and it is believed that the slower growth rate
duction of PHA by this transformant was when CPKO was used as
of the host strain C. necator PHB4 had limited the overall pro-
carbon source where it can accumulate 55 wt. % of PHA. This
ductivity of PHA from CPKO.
experiment confirmed that the phaC gene was expressed and
Based on the SDS-PAGE analysis, the presence of bands at
functioned in the C. necator PHB4 transformant. Plant oil had al-
approximately 60 kDa confirmed that PhaC is present in both
ways resulted in better PHA yield compared to sugar due to the
strains. Although the intensity of bands for whole cell extracts for
higher number of carbon atoms in oil. This results in higher for-
both strains were same, the enzyme activity of PhaCSC8 appeared to
mation of 3HB monomer units (40). The substrate specificity of this
be lower than that of PhaC from C. necator H16. One of the plausible
transformant was evaluated using different structurally related
carbon sources but the PHA produced was only homopolymer of
poly-3-hydroxybutyrate [P(3HB)] (data not shown). 200
180
Activity (U/g protein)

160
TABLE 3. PHA biosynthesis of wild type, negative mutant and C. necator trans- 140
formant PHB4 ProCn PhaCSC8 using CPKO or fructose as the sole carbon source. 120
* **
Strain Carbon sources Cell dry weight (g/L) PHA content (wt. %) 100
H16 10 g/L fructose 4.5  0.1a
80  5a 80 173
H16 5 g/L CPKO 6.8  1.6b 94  3b
60
PHB4 10 g/L fructose 1.1  0.2c 2  1c
PHB4 5 g/L CPKO 0.1  0.0c 1  0c 40
64
SC8 10 g/L fructose 1.1  0.0c 14  1c 20
SC8 5 g/L CPKO 0.8  0.3c 55  2d
0
CPKO, crude palm kernel oil; H16, wild type C. necator; PHB4, PHA negative mutant H16 SC8
of C. necator; SC8, C. necator PHB4 transformant. Data shown are the mean of
triplicate tests. Mean data accompanied by different alphabet letters were signifi- Strains
cantly different (Tukey’s HSD test, p < 0.05). Cells were cultivated in 50 mL MM
supplemented with different concentrations of carbon sources for 48 h at 30 C, FIG. 4. PHA synthase activity of C. necator H16 and C. necator transformant PHBL4 ProCn
200 rpm in a 250 mL flask. PhaCSC8. Both strains were grown for 24 h using CPKO as carbon sources in mineral
* Cell dry weight shown is after freeze-drying. medium (200 rpm, 30 C). The PHA synthase activity of C. necator H16 was higher
** PHA content of the freeze-dried cells was determined by gas chromatography. compare to the transformant.
VOL. 121, 2016 NEW PhaC FROM LIMESTONE METAGENOME 363

reasons was the loss of the C-terminal region where the more 4. Kahar, P., Tsuge, T., Taguchi, K., and Doi, Y.: High yield production of poly-
conserved C-terminal region is important for the enzyme activity of hydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant
strain, Polym. Degrad. Stab., 83, 79e86 (2004).
the PHA synthase (36). During the full length amplification, the stop 5. Valappil, S. P., Misra, S. K., Boccaccini, A. R., Keshavarz, T., Bucke, C., and
codon (TGA) was not found in the obtained sequence. Thus, an Roy, I.: Large-scale production and efficient recovery of PHB with desirable
artificial stop codon was added at the end of the sequence to material properties, from the newly characterised Bacillus cereus SPV,
generate a complete open reading frame and this synthase gene J. Biotechnol., 132, 251e258 (2007).
6. Lau, N. S., Chee, J. Y., Tsuge, T., and Sudesh, K.: Biosynthesis and mobilization
had a total of 554 amino acid residues. Based on Fig. 2, three amino
of a novel polyhydroxyalkanoate containing 3-hydroxy-4-methylvalerate
acids were missing in phaCSC8 after the valine residues in com- monomer produced by Burkholderia sp. USM (JCM15050), Bioresour. Tech-
parison with the phaCH16. Therefore, there is a chance that the nol., 101, 7916e7923 (2010).
functionally important C-terminal of PhaSC8 is truncated as C-ter- 7. Bhubalan, K., Kam, Y. C., Yong, K. H., and Sudesh, K.: Cloning and expression
minal region is located at the ending region of the PHA synthase of the PHA synthase gene from a locally isolated Chromobacterium sp. USM2,
Malays. J. Microbiol., 6, 81e90 (2010).
gene (43,44). Based on an earlier study, it is postulated that amino 8. Doi, Y.: Microbial polyesters. VCH Publishers, Inc., Tokyo Institute of Technol-
acids in this region are important for substrate specificity and ogy, Yokohama (1990).
enzyme activity (45). In addition, the suitability of host might affect 9. Amann, R. I., Ludwig, W., and Schleifer, K.-H.: Phylogenetic identification and
the performance of this synthase gene as well since the PhaCSC8 in situ detection of individual microbial cells without cultivation, Microbiol.
was cloned into C. necator PHB4. The characteristic of this syn- Rev., 59, 143e169 (1995).
10. Schloss, P. D. and Handelsman, J.: Biotechnological prospects from meta-
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be verified in future studies. 11. Sturgeon, A., Stull, J. W., Costa, M. C., and Weese, J. S.: Metagenomic analysis
Not many studies have been carried out on the identification of of the canine oral cavity as revealed by high-throughput pyrosequencing of the
PHA synthase gene through metagenomic approach. To the best of 16S rRNA gene, Vet. Microbiol., 162, 891e898 (2013).
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our knowledge, only three published studies have successfully sludge by metagenome analysis, Environ. Sci. Technol., 47, 1945e1951 (2013).
recovered the complete PHA synthase gene from metagenomic 13. Rashamuse, K. J., Visser, D. F., Hennessy, F., Kemp, J., Roux-van der
samples. Through phenotypic screening method, Schallmey et al. Merwe, M. P., Badenhorst, J., Ronneburg, T., Francis-Pope, R., and Brady, D.:
(14) have recovered three complete PHA synthase genes from soil Characterisation of two bifunctional cellulase-xylanase enzymes isolated from
a bovine rumen metagenome library, Curr. Microbiol., 66, 145e151 (2013).
metagenome. On the other hand, Cheema et al. (15) found nine
14. Schallmey, M., Ly, A., Wang, C., Meglei, G., Voget, S., Streit, W. R.,
partial phaC genes from oil contaminated soil but were only able to Driscoll, B. T., and Charles, T. C.: Harvesting of novel polyhydroxyalkanaote
recover one complete gene. In the latest publication by Foong et al. (PHA) synthase encoding genes froma soil metagenome library using pheno-
(24), three complete PHA synthase genes have been found from typic screening, FEMS Microbiol. Lett., 321, 150e156 (2011).
seawater metagenome but only one was functional. In this study, a 15. Cheema, S., Bassas-Galia, M., Sarma, P. M., Lal, B., and Arias, S.: Exploiting
metagenomic diversity for novel polyhydroxyalkanoate synthases: production
new PHA synthase gene has been isolated from limestone soil of a terpolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-
metagenome and it showed functional activity in a heterologous hydroxyoctanoate) with a recombinant Pseudomonas putida strain, Bioresour.
expression host. This newly discovered PHA synthase gene can be Technol., 103, 322e328 (2012).
added into the collection of known phaC gene and it is believed that 16. Frisli, T., Haverkamp, T. H. A., Jakobsen, K. S., Stenseth, N. C., and Rudi, K.:
Estimation of metagenome size and structure in an experimental soil micro-
more phaC genes will be discovered in the future.
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In conclusion, this paper presents a pioneering study on the 114, 141e151 (2012).
discovery of phaC in a limestone area using metagenomic approach. 17. Cheng, G., Hu, Y., Lu, N., Li, J., Wang, Z., Chen, Q., and Zhu, B.: Identification
Through this study, a new functional phaC was discovered. Phylo- of a novel fosfomycin-resistant UDP-N-acetylglucosamine enolpyruvyl
genetic classification for all the phaCs isolated from this study has transferase (MurA) from a soil metagenome, Biotechnol. Lett., 35, 273e278
(2013).
revealed that limestone hill harbors a great diversity of PhaCs with
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activities that have not yet been investigated. Dobrota , C.: Poly-b-hydroxybutyrate accumulation in bacterial consortia from
Supplementary data to this article can be found online at http:// different environments, Can. J. Microbiol., 58, 660e667 (2012).
dx.doi.org/10.1016/j.jbiosc.2015.08.008. 19. Doi, Y., Kitamura, S., and Abe, H.: Microbial synthesis and characterization of
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ACKNOWLEDGMENTS 20. Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A.,
Roop, R. M., II, and Peterson, K. M.: Four new derivatives of the broad-host-
This study was supported by the Long Term Research Grant range cloning vector pBBR1MCS, carrying different antibiotic-resistance cas-
Scheme (203/PKT/6725001) and Fundamental Research Grant settes, Gene, 166, 175e176 (1995).
21. Zhou, J., Bruns, M. A., and Tiedje, J. M.: DNA recovery from soils of diverse
Scheme (203/PBIOLOGI/6711361) (Universiti Sains Malaysia). We composition, Appl. Environ. Microbiol., 62, 316e322 (1996).
are grateful to Dr. Kovach ME for his kind gift of the pBBR1MCS 22. Romo, D. M. R., Grosso, M. V., Solano, N. C. M., and Castano, D. M.: A most
broad-host-range vector derivatives used in this study. YT Tai effective method for selecting a broad range of short and medium-chain-length
gratefully acknowledges the JPA scholarship from the Ministry of polyhidroxyalcanoate producing microorganisms, Electron. J. Biotechnol., 10,
348e357 (2007).
Higher Education Malaysia for financial support while CP Foong
23. Michinaka, A., Arou, J., Onuki, M., Satoh, H., and Mino, T.: Analysis of poly-
gratefully acknowledges the MyBrain15 from the Ministry of Higher hydroxyalkanoate (PHA) synthase gene in activated sludge that produces PHA
Education Malaysia for financial support. containing 3-hydroxy-2-methylvalerate, Biotechnol. Bioeng., 96, 871e880
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