Cloning and characterization of an unusual polyhydroxyalkanoate acid synthase (PHAC) gene from a chromobacterium Sp. isolated locally
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Date
2009
Authors
Kam, Yew Chee
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Abstract
Chromobacterium sp. is a bacterium capable of producing polyhydroxyalkanoates
(PHA). The Chromobacterium sp. PHA synthase (PhaC) is able to polymerize shortchain-
length PHA monomers, such as 3-hydroxybutyrate and 3-hydroxyvalerate.
This bacterium is very interesting due to its ability to also produce a purple pigment,
violacein. Production of violacein makes its colony purple in color, a color very
unique among bacteria. Recently, this unique bacterium was successfully isolated
from the famous ‘Air Terjun Telaga Tujuh’ in Langkawi, Malaysia. 16S rRNA
analysis confirmed the identity of the isolate up to the genus level. In this study, the
PHA synthase (phaC) gene was successfully cloned by using the PCR amplication
technique. The Chromobacterium sp. phaC coding region consisted of 1704 bp,
which encodes for a putative 568 amino acid protein. Results show that the PhaC of
this bacterium has an unusual specificity towards 3-hydroxyvaleryl-CoA. This
enzyme specificity was further supported by the ability of this isolate to synthesize
poly(3-hydroxyvalerate) of 100 mol% purity from suitable substrates. When the
cloned phaC was expressed in the PHA-negative mutant of Cupriavidus necator
PHB–4, 64 wt% poly(3-hydroxybutyrate) of the dry cell weight was accumulated
with fructose as the carbon source. In addition, when suitable 3-hydroxyvalerate
(3HV) precursor carbon sources were used, PHA copolymers containing high molar
fractions of 3HV monomers were synthesized. Most striking is the ability of the
recombinant C. necator PHB–4 containing the Chromobacterium sp. phaC to
accumulate poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) when crude palm
kernel oil was used as the sole carbon source. This study demonstrates that the PhaC
of Chromobacterium sp. has a broad substrate specificity similar to that of
Aeromonas caviae.
Description
Master
Keywords
Biological Science , Cloning , Polyhydroxyalkanoate acid synthase , Chromobacterium sp.