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Applied and Environmental Microbiology, October 2007, p. 6277-6283, Vol. 73, No. 19
0099-2240/07/$08.00+0     doi:10.1128/AEM.00861-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Isopentenol Biosynthetic Genes from Bacillus subtilis by a Screening Method Based on Isoprenoid Precursor Toxicity{triangledown}

Sydnor T. Withers,1,2 Shayin S. Gottlieb,3 Bonny Lieu,3 Jack D. Newman,3 and Jay D. Keasling1,2,4,5*

Department of Chemical Engineering, University of California, Berkeley, California,1 Department of Bioengineering, University of California, Berkeley, California,2 Amyris Biotechnologies, Emeryville, California,3 Synthetic Biology Department, Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California,4 California Institute for Quantitative Biomedical Research (QB3), University of California, Berkeley, California5

Received 17 April 2007/ Accepted 8 August 2007

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 6051 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.

* Corresponding author. Mailing address: Berkeley Center for Synthetic Biology, 717 Potter Street, Building 977, Mail Code 3224, University of California, Berkeley, CA 94720-3224. Phone: (510) 495-2620. Fax: (510) 495-2630. E-mail: keasling{at}berkeley.edu

{triangledown} Published ahead of print on 10 August 2007.

Applied and Environmental Microbiology, October 2007, p. 6277-6283, Vol. 73, No. 19
0099-2240/07/$08.00+0     doi:10.1128/AEM.00861-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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