Research Article: Revelation of the ability of Burkholderia sp. USM (JCM 15050) PHA synthase to polymerize 4-hydroxybutyrate monomer

Date Published: August 9, 2012

Publisher: Springer

Author(s): Nyok-Sean Lau, Kumar Sudesh.

http://doi.org/10.1186/2191-0855-2-41

Abstract

The nutrition-versatility of Burkholderia sp. strain USM (JCM 15050) has initiated the studies on the use of this bacterium for polyhydroxyalkanoate (PHA) production. To date, the Burkholderia sp. has been reported to synthesize 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxy-4-methylvalerate monomers. In this study, the PHA biosynthetic genes of this strain were successfully cloned and characterized. The PHA biosynthetic cluster of this strain consisted of a PHA synthase (phaC), β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthesis regulator (phaR). The translated products of these genes revealed identities to corresponding proteins of Burkholderia vietnamiensis (99–100 %) and Cupriavidus necator H16 (63–89%). Heterologous expression of phaCBs conferred PHA synthesis to the PHA-negative Cupriavidus necator PHB¯4, confirming that phaCBs encoded functionally active protein. PHA synthase activity measurements revealed that the crude extracts of C. necator PHB¯4 transformant showed higher synthase activity (243 U/g) compared to that of wild-types Burkholderia sp. (151 U/g) and C. necator H16 (180 U/g). Interestingly, the transformant C. necator PHB¯4 harbouring Burkholderia sp. PHA synthase gene accumulated poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with 4-hydroxybutyrate monomer as high as up to 87 mol% from sodium 4-hydroxybutyrate. The wild type Burkholderia sp. did not have the ability to produce this copolymer.

Partial Text

Since the introduction of phenoformaldehyde plastic in 1909 by Leo Hendrik Baekeland, petrochemical plastics have developed into a major industry and an indispensable commodity for modern life (Meikle, [1995]). It is estimated that more than 100 million tonnes of plastics are produced yearly. Most of these plastics end up after their useful life as discarded waste and some are disposed into the marine environment which pose a threat to the aquatic wildlife. In recent years, the widespread and increasing use of petrochemical plastics has raised concerns about the adverse impact of these recalcitrant plastics on the environment. Hence, biobased and biodegradable polymers are gaining widespread interest and acceptance as an alternative to some synthetic plastics. Polyhydroxyalkanoate (PHA), a storage and reserve compound accumulated naturally in the cytoplasm of numerous bacteria, is being considered as one of the most attractive and promising biodegradable thermoplastics for various industrial and biomedical applications (Sudesh and Iwata, [2008]).

Bacteria belonging to the genus Burkholderia was first used in 1989 for the production of P(3HB) homopolymer from fructose (Ramsay et al., [1989]). They are one of the most nutritionally versatile microorganisms that are capable of utilizing a wide range of carbon sources. In this study, the PHA biosynthetic genes from a locally isolated Burkholderia sp. strain USM (JCM 15050) were successfully amplified. The PHA biosynthetic genes of Burkholderia sp. consisted of a PHA synthase (phaC), β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthesis regulator (phaR). The phaCphaA and phaB seems to be organized in an operon and the structural organization of these genes is closely related to other bacteria harbouring type I PHA synthase e.g. C. necator and Alcaligenes latus (Choi et al., [1998]; Rehm and Steinbüchel, [2002]) (Figure 2). Although the PHA synthase and other PHA biosynthetic genes are often found clustered in the bacterial genomes, there are some exceptions to these observations. In the genomes of Caulobacter crescentusParacoccus denitrificansMethylobacterium extorquens and Aeromonas caviae, the genes related to PHA biosynthesis are not directly linked to the PHA synthase (Rehm and Steinbüchel, [2002]). Some bacteria e.g. P. denitrificans contains other genes related to PHA synthesis (phasin, phaP and phaR) map close to the PHA locus. Both C. necator and Burkholderia sp. PHA locus also possess a putative phaR immediately downstream of phaB. The phaR encodes a protein with putative regulatory function in PHA metabolism (York et al. [2002]; Stubbe and Tian, [2003]; Pötter and Steinbüchel, [2006]).

The authors declare that they have no competing interests.

 

Source:

http://doi.org/10.1186/2191-0855-2-41

 

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