Forest soil metagenome gene cluster involved in antifungal activity expression in Escherichia coli.
Using two forest soils, we previously constructed two fosmid libraries containing 113,700 members in total. The libraries were screened to select active antifungal clones using Saccharomyces cerevisiae as a target fungus. One clone from the Yuseong pine tree rhizosphere soil library, pEAF66, showed S. cerevisiae growth inhibition. Despite an intensive effort, active chemicals were not isolated. DNA sequence analysis and transposon mutagenesis of pEAF66 revealed 39 open reading frames (ORFs) and indicated that eight ORFs, probably in one transcriptional unit, might be directly involved in the expression of antifungal activity in Escherichia coli. The deduced amino acid sequences of eight ORFs were similar to those of the core genes encoding type II family polyketide synthases, such as the acyl carrier protein (ACP), ACP synthases, aminotransferase, and ACP reductase. The gene cluster involved in antifungal activity was similar in organization to the putative antibiotic production locus of Pseudomonas putida KT2440, although we could not select a similar active clone from the KT2440 genomic DNA library in E. coli. ORFs encoding ATP binding cassette transporters and membrane proteins were located at both ends of the antifungal gene cluster. Upstream ORFs encoding an IclR family response regulator and a LysR family response regulator were involved in the positive regulation of antifungal gene expression. Our results suggested the metagenomic approach as an alternative to search for novel antifungal antibiotics from unculturable soil bacteria. This is the first report of an antifungal gene cluster obtained from a soil metagenome using S. cerevisiae as a target fungus.
Division of Applied Biology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea.
Eu Jin
EJ
Chung
He Kyoung
HK
Lim
Jin-Cheol
JC
Kim
Gyung Ja
GJ
Choi
Eun Jin
EJ
Park
Myung Hwan
MH
Lee
Young Ryun
YR
Chung
Seon-Woo
SW
Lee
D000935:Antifungal Agents / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015698:Genomic Library; D008969:Molecular Sequence Data; D005810:Multigene Family; D016254:Mutagenesis, Insertional / Q000379:methods; D028223:Pinus / Q000254:growth & development / Q000382:microbiology; D012441:Saccharomyces cerevisiae / Q000187:drug effects; D012988:Soil Microbiology; D014197:Trees / Q000254:growth & development / Q000382:microbiology*
D000935:Antifungal Agents; D001426:Bacterial Proteins; D004251:DNA Transposable Elements