Dataset Information

Characterization of the highly active polyhydroxyalkanoate synthase of Chromobacterium sp. strain USM2.

ABSTRACT: The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.

SUBMITTER: Bhubalan K

PROVIDER: S-EPMC3126384 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterization of the highly active polyhydroxyalkanoate synthase of Chromobacterium sp. strain USM2.

Bhubalan Kesaven K Chuah Jo-Ann JA Shozui Fumi F Brigham Christopher J CJ Taguchi Seiichi S Sinskey Anthony J AJ Rha Chokyun C Sudesh Kumar K

Applied and environmental microbiology 20110311 9

The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoat ...[more]

PMID: 21398494

Similar Datasets

Project description:Clostridium sp. strain TO-931 can rapidly convert the primary bile acid cholic acid to a potentially toxic compound, deoxycholic acid. Mixed oligonucleotide probes were used to isolate a gene fragment encoding a putative bile acid transporter from Clostridium sp. strain TO-931. This DNA fragment had 60% nucleotide sequence identity to a known bile acid transporter gene from Eubacterium sp. strain VPI 12708, another bile acid-7alpha-dehydroxylating intestinal bacterium. The DNA (9.15 kb) surrounding the transporter gene was cloned from Clostridium sp. strain TO-931 and sequenced. Within this larger DNA fragment was a 7.9-kb region, containing six successive open reading frames (ORFs), that was encoded by a single 8.1-kb transcript, as determined by Northern blot analysis. The gene arrangement and DNA sequence of the Clostridium sp. strain TO-931 operon are similar to those of a Eubacterium sp. strain VPI 12708 bile acid-inducible operon containing nine ORFs. Several genes in the Eubacterium sp. strain VPI 12708 operon have been shown to encode products required for bile acid 7alpha-dehydroxylation. In Clostridium sp. strain TO-931, genes potentially encoding bile acid-coenzyme A (CoA) ligase, 3alpha-hydroxysteroid dehydrogenase, bile acid 7alpha-dehydratase, bile acid-CoA hydrolase, and a bile acid transporter were similar in size and exhibited amino acid homology to similar gene products from Eubacterium sp. strain VPI 12708 (encoded by baiB, baiA, baiE, baiF, and baiG, respectively). However, no genes similar to Eubacterium sp. strain VPI 12708 biaH or baiI were found in the Clostridium sp. strain TO-931 bai operon, and the two putative Eubacterium sp. strain VPI 12708 genes, baiC and baiD, were arranged in one continuous ORF in Clostridium sp. strain TO-931. Intergene regions showed no significant DNA sequence similarity, but primer extension analysis identified a region 115 bp upstream from the first ORF that exhibited 58% identity to a bai operator/promoter region identified in Eubacterium sp. strain VPI 12708. These results indicate that the gene organization, gene product amino acid sequences, and promoters of the bile acid-inducible operons of Clostridium sp. strain TO-931 and Eubacterium sp. strain VPI 12708 are highly conserved.

Project description:Given the continued high prevalence of mosquito-transmitted diseases, there is a clear need to develop novel disease and vector control strategies. Biopesticides of microbial origin represent a promising source of new approaches to target disease-transmitting mosquito populations. Here, we describe the development and characterization of a novel mosquito biopesticide, derived from an air-dried, nonlive preparation of the bacterium Chromobacterium sp. Panama (family: Neisseriaceae). This preparation rapidly and effectively kills the larvae of prominent mosquito vectors, including the dengue and Zika vector Aedes aegypti and the human malaria vector Anopheles gambiae During semi-field trials in Puerto Rico, we observed high efficacy of the biopesticide against field-derived A. aegypti populations, and against A. aegypti and Culex species larvae in natural breeding water, indicating the suitability of the biopesticide for use under more natural conditions. In addition to high efficacy, the nonlive Csp_P biopesticide has a low effective dose, a long shelf life, and high heat stability and can be incorporated into attractive larval baits, all of which are desirable characteristics for a biopesticide.IMPORTANCE We have developed a novel preparation to kill mosquitoes from an abundant soil bacterium, Chromobacterium sp. Panama. This preparation is an air-dried powder containing no live bacteria, and it can be incorporated into an attractive bait and fed directly to mosquito larvae. We demonstrate that the preparation has broad spectrum activity against the larval form of the mosquitoes responsible for the transmission of malaria and the dengue, chikungunya, yellow fever, West Nile, and Zika viruses, as well as mosquito larvae that are already resistant to commonly used mosquitocidal chemicals. Our preparation possesses many favorable traits: it kills at a low dosage, and it does not lose activity when exposed to high temperatures, all of which suggest that this preparation could eventually become an effective new tool for controlling mosquitoes and the diseases they spread.

Dataset Information

Characterization of the highly active polyhydroxyalkanoate synthase of Chromobacterium sp. strain USM2.

Publications

Characterization of the highly active polyhydroxyalkanoate synthase of Chromobacterium sp. strain USM2.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets