11/06/2012
Identifying the Best Biofuel-Producing Microbes
Strain improvement is a persistent challenge.
The Science
Researchers developed a generalized approach to screen or select for improved small-molecule biosynthesis using transcription factor-based biosensors. Using a tetracycline resistance gene 3′ of a small-molecule inducible promoter, host antibiotic resistance, and hence growth rate, was coupled to either small-molecule concentration in the growth medium or a small-molecule production phenotype. Biosensors were constructed for two important chemical classes, dicarboxylic acids and alcohols, using transcription factor-promoter pairs derived from Pseudomonas putida, Thauera butanivorans, or E. coli. Transcription factors were selected for specific activation by either succinate, adipate, or 1-butanol, and we demonstrate product-dependent growth in E. coli using all three compounds. The 1-butanol biosensor was applied in a proof-of-principle liquid culture screen to optimize 1-butanol biosynthesis in engineered E. coli, identifying a pathway variant yielding a 35% increase in 1-butanol specific productivity through optimization of enzyme expression levels. Lastly, to demonstrate the capacity to select for enzymatic activity, the 1-butanol biosensor was applied as synthetic selection, coupling in vivo 1-butanol biosynthesis to E. coli fitness, and an 120-fold enrichment for a 1-butanol production phenotype was observed following a single round of positive selection.
Summary
To use a microbe as a factory to make a desired product, a bacterial strain that already produces the compound is treated to generate many mutants, some of which may produce more of the product. From all these new variants, the challenge is to identify those microbes that make the largest amounts of the desired compound. This is particularly difficult when the target compound (e.g., a biofuel) does not confer any selective advantage to the microbe. To solve this problem, researchers at the U.S. Department of Energy’s (DOE) Lawrence Berkeley National Laboratory and DOE Joint BioEnergy Institute designed a “biosensor”—a genetic regulator that “senses” the presence of the desired product (e.g., butanol). The expression of a gene that confers an advantage to the microbe, such as resistance to the antibiotic tetracycline, is then induced by the presence of the biosensor. Butanol biosensor-containing Escherichia coli cells, for example, grow in the presence of the antibiotic only if the medium also contains butanol. Finally, plasmids capable of synthesizing various amounts of butanol were introduced into E. coli containing the butanol biosensor and growing in tetracycline-containing medium. High butanol-producing cells could readily be identified by their faster growth rates. This approach will facilitate the selection of microbial strains that produce large quantities of any small molecule, an important step toward the development of renewable biofuels.
Principal Investigator
Jay D. Keasling
Lawrence Berkeley National Laboratory
[email protected]
BER Program Manager
Pablo Rabinowicz
U.S. Department of Energy, Biological and Environmental Research (SC-33)
Biological Systems Science Division
[email protected]
References
Dietrich, J. A., D. L. Shis, A. Alikhani, and J. D. Keasling. 2012. “Transcription Factor-Based Screens and Synthetic Selections for Microbial Small-Molecule Biosynthesis,” ACS Synthetic Biology 2. DOI: 10.1021/sb300091d.