title: Identification of isopentenol biosynthetic genes from Bacillus subtilis by a screening method based on isoprenoid precursor toxicity.
abstract: We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6,051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol.
authors: Department of Chemical Engineering, University of California, Berkeley, California, USA.
authors: Sydnor T
authors: ST
authors: Withers
authors: Shayin S
authors: SS
authors: Gottlieb
authors: Bonny
authors: B
authors: Lieu
authors: Jack D
authors: JD
authors: Newman
authors: Jay D
authors: JD
authors: Keasling
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D002070:Butadienes / Q000378:metabolism / Q000633:toxicity*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005818:Genetic Engineering; D045782:Hemiterpenes / Q000378:metabolism / Q000633:toxicity*; D008798:Mevalonic Acid / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010420:Pentanes / Q000378:metabolism / Q000633:toxicity*; D000439:Pentanols / Q000378:metabolism*; D013729:Terpenes / Q000378:metabolism*
chemical_list: D002070:Butadienes; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D010420:Pentanes; D000439:Pentanols; D013729:Terpenes; C005059:isoprene; C056744:isopentenol; C004809:isopentenyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; D008798:Mevalonic Acid
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