Estimating P-coverage of biosynthetic pathways in DNA libraries and screening by genetic selection: biotin biosynthesis in the marine microorganism Chromohalobacter.
Traditional approaches to natural product discovery involve cell-based screening of natural product extracts followed by compound isolation and characterization. Their importance notwithstanding, continued mining leads to depletion of natural resources and the reisolation of previously identified metabolites. Metagenomic strategies aimed at localizing the biosynthetic cluster genes and expressing them in surrogate hosts offers one possible alternative. A fundamental question that naturally arises when pursuing such a strategy is, how large must the genomic library be to effectively represent the genome of an organism(s) and the biosynthetic gene clusters they harbor? Such an issue is certainly augmented in the absence of expensive robotics to expedite colony picking and/or screening of clones. We have developed an algorism, named BPC (biosynthetic pathway coverage), supported by molecular simulations to deduce the number of BAC clones required to achieve proper coverage of the genome and their respective biosynthetic pathways. The strategy has been applied to the construction of a large-insert BAC library from a marine microorganism, Hon6 (isolated from Honokohau, Maui) thought to represent a new species. The genomic library is constructed with a BAC yeast shuttle vector pClasper lacZ paving the way for the culturing of libraries in both prokaryotic and eukaryotic hosts. Flow cytometric methods are utilized to estimate the genome size of the organism and BPC implemented to assess P-coverage or percent coverage. A genetic selection strategy is illustrated, applications of which could expedite screening efforts in the identification and localization of biosynthetic pathways from marine microbial consortia, offering a powerful complement to genome sequencing and degenerate probe strategies. Implementing this approach, we report on the biotin biosynthetic pathway from the marine microorganism Hon6.
Kim
Eun Jin
EJ
Department of Chemistry, Texas A&M University, College Station, MS 3255, TX 77843, USA.
Angell
Scott
S
Janes
Jeff
J
Watanabe
Coran M H
CM
D001710:Biotin / Q000096:biosynthesis* / Q000235:genetics; D004247:DNA / Q000235:genetics; D005434:Flow Cytometry; D015698:Genomic Library; D044129:Halomonadaceae / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D013045:Species Specificity
D001710:Biotin; D004247:DNA