Cloning, characterization and expression of a gene encoding dihydroxyacetone synthase in Mycobacterium sp. strain JC1 DSM 3803

Jae Gu Seo, Sae W. Park, Hyuk Park, Seo Y. Kim, Young T. Ro, Eungbin Kim, Jin W. Cho, Young M. Kim

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Dihydroxyacetone synthase (DHAS) is a key enzyme involved in the assimilation of methanol in Mycobacterium sp. strain JC1 DSM 3803. The structural gene encoding DHAS in Mycobacterium sp. strain JC1 was cloned using random-primed probes synthesized after PCR with synthetic primers based on the amino acid sequences conserved in two yeast DHASs and several transketolases. The cloned gene, dasS, had an ORF of 2193 nt, encoding a protein with a calculated molecular mass of 78 197 Da. The deduced amino acid sequence of dasS contained an internal sequence of Mycobacterium sp. strain JC1 DHAS and exhibited 29.2 and 27.3 % identity with those of Candida boidinii and Hansenula polymorpha enzymes, respectively. Escherichia coli transformed with the cloned gene produced a novel protein with a molecular mass of ∼78 kDa, which cross-reacted with anti-DHAS antiserum and exhibited DHAS activity. Primer-extension analysis revealed that the transcriptional start site of the gene was the nucleotide A located 31 bp upstream from the dasS start codon. RT-PCR showed that dasS was transcribed as a monocistronic message. Northern hybridization and β-galactosidase assay with the putative promoter region of dasS revealed that the gene was transcribed only in cells growing on methanol. The expression of dasS in Mycobacterium sp. strain JC1 was free from catabolite repression.

Original languageEnglish
Pages (from-to)4174-4182
Number of pages9
JournalMicrobiology
Volume153
Issue number12
DOIs
Publication statusPublished - 2007 Dec 1

Fingerprint

formaldehyde transketolase
Mycobacterium
Organism Cloning
Gene Expression
Genes
Methanol
Amino Acid Sequence
Galactosidases
Catabolite Repression
Polymerase Chain Reaction
Initiator Codon
Pichia
Enzymes
Candida
Genetic Promoter Regions
Open Reading Frames
Immune Sera
Proteins
Nucleotides
Yeasts

All Science Journal Classification (ASJC) codes

  • Microbiology

Cite this

Seo, Jae Gu ; Park, Sae W. ; Park, Hyuk ; Kim, Seo Y. ; Ro, Young T. ; Kim, Eungbin ; Cho, Jin W. ; Kim, Young M. / Cloning, characterization and expression of a gene encoding dihydroxyacetone synthase in Mycobacterium sp. strain JC1 DSM 3803. In: Microbiology. 2007 ; Vol. 153, No. 12. pp. 4174-4182.
@article{102edc5962cc496ba2e82c1ae574f07a,
title = "Cloning, characterization and expression of a gene encoding dihydroxyacetone synthase in Mycobacterium sp. strain JC1 DSM 3803",
abstract = "Dihydroxyacetone synthase (DHAS) is a key enzyme involved in the assimilation of methanol in Mycobacterium sp. strain JC1 DSM 3803. The structural gene encoding DHAS in Mycobacterium sp. strain JC1 was cloned using random-primed probes synthesized after PCR with synthetic primers based on the amino acid sequences conserved in two yeast DHASs and several transketolases. The cloned gene, dasS, had an ORF of 2193 nt, encoding a protein with a calculated molecular mass of 78 197 Da. The deduced amino acid sequence of dasS contained an internal sequence of Mycobacterium sp. strain JC1 DHAS and exhibited 29.2 and 27.3 {\%} identity with those of Candida boidinii and Hansenula polymorpha enzymes, respectively. Escherichia coli transformed with the cloned gene produced a novel protein with a molecular mass of ∼78 kDa, which cross-reacted with anti-DHAS antiserum and exhibited DHAS activity. Primer-extension analysis revealed that the transcriptional start site of the gene was the nucleotide A located 31 bp upstream from the dasS start codon. RT-PCR showed that dasS was transcribed as a monocistronic message. Northern hybridization and β-galactosidase assay with the putative promoter region of dasS revealed that the gene was transcribed only in cells growing on methanol. The expression of dasS in Mycobacterium sp. strain JC1 was free from catabolite repression.",
author = "Seo, {Jae Gu} and Park, {Sae W.} and Hyuk Park and Kim, {Seo Y.} and Ro, {Young T.} and Eungbin Kim and Cho, {Jin W.} and Kim, {Young M.}",
year = "2007",
month = "12",
day = "1",
doi = "10.1099/mic.0.2007/011965-0",
language = "English",
volume = "153",
pages = "4174--4182",
journal = "Microbiology",
issn = "1350-0872",
publisher = "Society for General Microbiology",
number = "12",

}

Cloning, characterization and expression of a gene encoding dihydroxyacetone synthase in Mycobacterium sp. strain JC1 DSM 3803. / Seo, Jae Gu; Park, Sae W.; Park, Hyuk; Kim, Seo Y.; Ro, Young T.; Kim, Eungbin; Cho, Jin W.; Kim, Young M.

In: Microbiology, Vol. 153, No. 12, 01.12.2007, p. 4174-4182.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Cloning, characterization and expression of a gene encoding dihydroxyacetone synthase in Mycobacterium sp. strain JC1 DSM 3803

AU - Seo, Jae Gu

AU - Park, Sae W.

AU - Park, Hyuk

AU - Kim, Seo Y.

AU - Ro, Young T.

AU - Kim, Eungbin

AU - Cho, Jin W.

AU - Kim, Young M.

PY - 2007/12/1

Y1 - 2007/12/1

N2 - Dihydroxyacetone synthase (DHAS) is a key enzyme involved in the assimilation of methanol in Mycobacterium sp. strain JC1 DSM 3803. The structural gene encoding DHAS in Mycobacterium sp. strain JC1 was cloned using random-primed probes synthesized after PCR with synthetic primers based on the amino acid sequences conserved in two yeast DHASs and several transketolases. The cloned gene, dasS, had an ORF of 2193 nt, encoding a protein with a calculated molecular mass of 78 197 Da. The deduced amino acid sequence of dasS contained an internal sequence of Mycobacterium sp. strain JC1 DHAS and exhibited 29.2 and 27.3 % identity with those of Candida boidinii and Hansenula polymorpha enzymes, respectively. Escherichia coli transformed with the cloned gene produced a novel protein with a molecular mass of ∼78 kDa, which cross-reacted with anti-DHAS antiserum and exhibited DHAS activity. Primer-extension analysis revealed that the transcriptional start site of the gene was the nucleotide A located 31 bp upstream from the dasS start codon. RT-PCR showed that dasS was transcribed as a monocistronic message. Northern hybridization and β-galactosidase assay with the putative promoter region of dasS revealed that the gene was transcribed only in cells growing on methanol. The expression of dasS in Mycobacterium sp. strain JC1 was free from catabolite repression.

AB - Dihydroxyacetone synthase (DHAS) is a key enzyme involved in the assimilation of methanol in Mycobacterium sp. strain JC1 DSM 3803. The structural gene encoding DHAS in Mycobacterium sp. strain JC1 was cloned using random-primed probes synthesized after PCR with synthetic primers based on the amino acid sequences conserved in two yeast DHASs and several transketolases. The cloned gene, dasS, had an ORF of 2193 nt, encoding a protein with a calculated molecular mass of 78 197 Da. The deduced amino acid sequence of dasS contained an internal sequence of Mycobacterium sp. strain JC1 DHAS and exhibited 29.2 and 27.3 % identity with those of Candida boidinii and Hansenula polymorpha enzymes, respectively. Escherichia coli transformed with the cloned gene produced a novel protein with a molecular mass of ∼78 kDa, which cross-reacted with anti-DHAS antiserum and exhibited DHAS activity. Primer-extension analysis revealed that the transcriptional start site of the gene was the nucleotide A located 31 bp upstream from the dasS start codon. RT-PCR showed that dasS was transcribed as a monocistronic message. Northern hybridization and β-galactosidase assay with the putative promoter region of dasS revealed that the gene was transcribed only in cells growing on methanol. The expression of dasS in Mycobacterium sp. strain JC1 was free from catabolite repression.

UR - http://www.scopus.com/inward/record.url?scp=37449002319&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=37449002319&partnerID=8YFLogxK

U2 - 10.1099/mic.0.2007/011965-0

DO - 10.1099/mic.0.2007/011965-0

M3 - Article

C2 - 18048931

AN - SCOPUS:37449002319

VL - 153

SP - 4174

EP - 4182

JO - Microbiology

JF - Microbiology

SN - 1350-0872

IS - 12

ER -