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Computational Molecular Biology
17
medium-chain-length (MCL-) PHAs are comprised of
6 to 14 carbon atoms (Gangoiti et al., 2012). Chiral
building blocks of polyhydroxyalkanoates monomers
may be utilized for synthesis of fine chemicals like
antibiotics, vitamins, aromatics and pheromones
(Guo-Qiang, 2009). It has been reported that some of
polyhydroxyalkanoic acids have antimicrobial activity
(Shuping, 2013; Ruth, 2007). PHA biosynthesis genes
vary in different PHA-producing organisms. PHA
biosynthesis loci have been categorized into three
major classes. In the type I system, PHA
synthase-encoding gene (
phbC
) is adjacent to
phbA
and
phbB
, as exemplified by the pha locus of
Ralstonia eutropha
(formerly
Alcaligenes eutrophus
).
These two genes, respectively code for
b-ketothiolase
and
acetoacetyl-CoA
reductase, two enzymes closely
related to the biosynthesis of scl-PHA. A locus in
which two synthase genes (
phaC1
and
phaC2
) are
separated by a PHA-depolymerization gene (
phaZ
) is
of type II PHA genetic organization system. The
mcl-PHA-producing Pseudomonads bears the type II
system. In type III PHA biosynthesis genetic
organization, the synthase enzyme is composed of two
polypeptide subunits encoded by
phbE
and
phbC
genes. The
phbA
and
phbB
genes are also located in
this locus, but are usually transcribed divergently from
the
phbE
and
phbC
genes. This type has been
observed in
Chromatium vinosum
,
Synechocystis
spp,
and
Thiocystis violacea
. Later, the pha locus of
Bacillus megaterium
was characterized as type IV
PHA biosynthesis genetic system (Hernandez-Eligio
et al., 2011; Poirier et al., 1995). In this paper, we
report a rapid and sensitive PCR procedure for
isolation and amplification of PHA synthase gene,
phaC1
, in a native
Pseudomonas
strain. The main aim
of our work was the cloning of
phaC1
gene from
native
Pseudomonad
strains to be expressed later in an
appropriate host for the intention of production of
biodegradable plastics, hence substitute the use of
petroleum-derived polymers as a non-degradable
plastic, Because of PHA synthase is a key enzyme
essential
for
bacterial
synthesis
of
polyhydroxyalkanoic
acid
biodegradable
polyesters.
1 Results
1.1 FT-IR analysis and confirmation of PHA
production
Purified PHB, mcl-PHA and P(HB + mcl-HA) showed
their strongest band at 1728 /cm, 1740 /cm and 1732
/cm respectively in FT-IR spectra (Shamala et al.,
2009). The strongest band in spectrum of mcl-PHA
was methylene C-H vibration near 2928 /cm. Other
characteristic band for mcl-PHA was visible near 1165
/cm in spectrum (Figure 1). It is demonstrated that the
band between 1728 /cm and 1744 /cm is characteristic
of PHA (Shamala et al., 2009).
Figure 1 FT-IR analysis of PHA produced by native
Pseudomonads
1.2 PCR amplification of
phaC1
gene
The PCR protocol was developed using genomic
DNAs purified from
P. putida
. This organism has been
reported to produce mcl-PHA. In this study, a distinct
540-bp PCR product was obtained (Figure 2). The size
of the PCR product agrees with the length of the
phaC1
and
phaC2
genes flanked by the I-179L/I-179R
primer-pair. When ELONGASE, formulated by its
manufacturer for long PCR amplification was used, an
additional ca. 3.4-kb amplicon was observed which
represents the DNA sequence flanked by the I-179L
and I-179R annealing sites in the
phaC1
and
phaC2
genes, respectively, and included the entire
pha Z
gene.
The PCR protocol developed using the purified
genomic-DNAs was then tested for the detection of
phaC
gene sequences in lysates prepared by heating
the suspension of bacterial colonies. The results
showed that the characteristic 540-bp PCR product
was produced even from the crude cell-lysates of
microorganisms shown to contain
phaC1
and
phaC2
genes (Steinbuchel and Schlegel, 1991).
Computational
Molecular Biology