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Abstract
BackgroundThe microbial production of biofuels is complicated by a tradeoff between yield and toxicity of many fuels. Efflux pumps enable bacteria to tolerate toxic substances by their removal from the cells while bypassing the periplasm. Their use for the microbial production of biofuels can help to improve cell survival, product recovery, and productivity. However, no native efflux pump is known to act on the class of short-chain alcohols, important next-generation biofuels, and it was considered unlikely that such an efflux pump exists.ResultsWe report that controlled expression of the RND-type efflux pump TtgABC from Pseudomonas putida DOT-T1E strongly improved cell survival in highly toxic levels of the next-generation biofuels n-butanol, isobutanol, isoprenol, and isopentanol. GC-FID measurements indicated active efflux of n-butanol when the pump is expressed. Conversely, pump expression did not lead to faster growth in media supplemented with low concentrations of n-butanol and isopentanol.ConclusionsTtgABC is the first native efflux pump shown to act on multiple short-chain alcohols. Its controlled expression can be used to improve cell survival and increase production of biofuels as an orthogonal approach to metabolic engineering. Together with the increased interest in P. putida for metabolic engineering due to its flexible metabolism, high native tolerance to toxic substances, and various applications of engineering its metabolism, our findings endorse the strain as an excellent biocatalyst for the high-yield production of next-generation biofuels.
Highlights
The microbial production of biofuels is complicated by a tradeoff between yield and toxicity of many fuels
Since this phenomenon would have complicated observing an effect of the pump on toxic substances at intermediate expression levels, we first tested whether expression via PBAD is homogenous in the case of P. putida by placing GFP under control of PBAD and determining fluorescence after induction with 0, 1, 10, and 100 mM l-arabinose
We found that expression of TtgABC in E. coli inhibits growth (Fig. 1b), indicating that toxicity of the heterologous pump could have masked its effect on biofuel tolerance
Summary
The microbial production of biofuels is complicated by a tradeoff between yield and toxicity of many fuels. The high tolerance of several gram-negative bacteria to toxic substances compared to other organisms is attributed to a lower outer membrane permeability, periplasmic and cytosolic enzymatic degradation (e.g., β-lactamases), homeoviscous membrane adaptation, and multidrug efflux pumps [1,2,3]. The latter have the potential to improve cell survival in toxic environments, and to directly increase yield and productivity of production strains by removing the final product from the cells and facilitating extracellular product recovery [4]. It acts on specific aromatic hydrocarbons, such as toluene, m-xylene, and 1,2,4-trichlorobenzene, as well as antibiotics [15], in combination with the efflux systems
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