A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms.
To examine as randomly as possible the role of the beta-ketoacyl and acyl carrier protein (ACP) components of bacterial type II polyketide synthases (PKSs), homologs of the chain-length-factor (CLF) genes were cloned from the environmental community of microorganisms. With PCR primers derived from conserved regions of known ketosynthase (KSalpha) and ACP genes specifying the formation of 16- to 24-carbon polyketides, two CLF (KSbeta) genes were cloned from unclassified streptomycetes isolated from the soil, and two were cloned from soil DNA without the prior isolation of the parent microorganism. The sequence and deduced product of each gene were distinct from those of known KSbeta genes and, by phylogenetic analysis, belonged to antibiotic-producing PKS gene clusters. Hybrid PKS gene cassettes were constructed with each novel KSbeta gene substituted for the actI-ORF2 or tcmL KSbeta subunit genes, along with the respective actI-ORF1 or tcmK KSalpha, tcmM ACP, and tcmN cyclase genes, and were found to produce an octaketide or decaketide product characteristic of the ones known to be made by the heterologous KSalpha gene partner. Since substantially less than 1% of the microorganisms present in soil are thought to be cultivatable by standard methods, this work demonstrates a potential way to gain access to a more extensive range of microbial molecular diversity and to biosynthetic pathways whose products can be tested for biological applications.
Seow
K T
KT
Terragen Diversity Inc., Vancouver, British Columbia, Canada.
Meurer
G
G
Gerlitz
M
M
Wendt-Pienkowski
E
E
Hutchinson
C R
CR
Davies
J
J
D000213:Acyl Carrier Protein / Q000235:genetics; D000595:Amino Acid Sequence; D000873:Anthracenes / Q000378:metabolism; D001431:Bacteriological Techniques; D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000145:classification / Q000235:genetics*; D005810:Multigene Family; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013302:Streptomyces / Q000235:genetics*
D000213:Acyl Carrier Protein; D000873:Anthracenes; D004269:DNA, Bacterial; D009097:Multienzyme Complexes; C083905:tetracenomycin F2