Engineering microbial biofuel tolerance and export using efflux pumps.

Many compounds being considered as candidates for advanced biofuels are toxic to microorganisms. This introduces an undesirable trade-off when engineering metabolic pathways for biofuel production because the engineered microbes must balance production against survival. Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing biofuel toxicity. To identify novel biofuel pumps, we used bioinformatics to generate a list of all efflux pumps from sequenced bacterial genomes and prioritized a subset of targets for cloning. The resulting library of 43 pumps was heterologously expressed in Escherichia coli, where we tested it against seven representative biofuels. By using a competitive growth assay, we efficiently distinguished pumps that improved survival. For two of the fuels (n-butanol and isopentanol), none of the pumps improved tolerance. For all other fuels, we identified pumps that restored growth in the presence of biofuel. We then tested a beneficial pump directly in a production strain and demonstrated that it improved biofuel yields. Our findings introduce new tools for engineering production strains and utilize the increasingly large database of sequenced genomes.

Targeted proteomics for metabolic pathway optimization: application to terpene production.

Successful metabolic engineering relies on methodologies that aid assembly and optimization of novel pathways in microbes. Many different factors may contribute to pathway performance, and problems due to mRNA abundance, protein abundance, or enzymatic activity may not be evident by monitoring product titers. To this end, synthetic biologists and metabolic engineers utilize a variety of analytical methods to identify the parts of the pathway that limit production. In this study, targeted proteomics, via selected-reaction monitoring (SRM) mass spectrometry, was used to measure protein levels in Escherichia coli strains engineered to produce the sesquiterpene, amorpha-4,11-diene. From this analysis, two mevalonate pathway proteins, mevalonate kinase (MK) and phosphomevalonate kinase (PMK) from Saccharomyces cerevisiae, were identified as potential bottlenecks. Codon-optimization of the genes encoding MK and PMK and expression from a stronger promoter led to significantly improved MK and PMK protein levels and over three-fold improved final amorpha-4,11-diene titer (>500 mg/L).

Functional genomic study of exogenous n-butanol stress in Escherichia coli.

n-Butanol has been proposed as an alternative biofuel to ethanol, and several industrially used microbes, including Escherichia coli, have been engineered to produce it. Unfortunately, n-butanol is more toxic than ethanol to these organisms. To understand the basis for its toxicity, cell-wide studies were conducted at the transcript, protein, and metabolite levels to obtain a global view of the n-butanol stress response. Analysis of the data indicates that n-butanol stress has components common to other stress responses, including perturbation of respiratory functions (nuo and cyo operons), oxidative stress (sodA, sodC, and yqhD), heat shock and cell envelope stress (rpoE, clpB, htpG, cpxR, and cpxP), and metabolite transport and biosynthesis (malE and opp operon). Assays using fluorescent dyes indicated a large increase in reactive oxygen species during n-butanol stress, confirming observations from the microarray and proteomics measurements. Mutant strains with mutations in several genes whose products changed most dramatically during n-butanol stress were examined for increased sensitivity to n-butanol. Results from these analyses allowed identification of key genes that were recruited to alleviate oxidative stress, protein misfolding, and other causes of growth defects. Cellular engineering based on these cues may assist in developing a high-titer, n-butanol-producing host.