Combinatorial library of synthetic inducible promoters responsive to higher alcohols

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Gaspar Morgado, Mario Leutert, Sven Panke

Bioprocess Laboratory, Switzerland

Higher alcohols ( C5) are promising for gasoline replacement as they have low hygroscopicity, high energy density and are compatible with transportation infrastructure. Linear and branched higher alcohols can be produced by engineered microorganisms expressing non-fermentative pathways [1,2]. Here, alcohols are biosynthesized in two steps: first, by decarboxylation of 2-ketoacids and second, reduction of the resulting aldehyde to alcohol. However, alcohols are toxic for microorganisms and improvement of cell tolerance do not increase production titers [3]. Therefore, other approaches, as feedback regulation systems and fine-tuning of pathway components are necessary for further optimization of alcohol production. To build such genetic constructs, regulatory elements orthogonally responsive to higher alcohols are mandatory. Previously, the alkB promoter (PalkB) was reported to be inducible by hexanol and heptanols but not butanol or propanol [4]. Thus, we investigated the suitability of PalkB as orthogonal responsive element using pentanol as proof of concept. We found that PalkB is not induced by metabolites like pyruvate, 2-ketoacids, aldehydes and amino acids, but a 40X fold induction was recorded for pentanol. Subsequently, we proceed with PalkB engineering in order to obtained variants with customized transfer functions. We constructed a synthetic combinatorial library of 78’125 promoter variants by variation of main promoter motifs. The library was cloned into a medium-copy plasmid and controlling GFP expression to allow readout. Then, a single-cell fluorescence sorting strategy was employed to specifically enrich and isolate hundreds of inducible variants. Finally, transfer functions were analyzed by flow cytometry and micro-fermentation. The resulting engineered promoters were able to span a wide range of strengths, inducibility ratios and more important, half maximal effective concentrations (EC50). [1] Atsumi et al. Nature. 2008. 451:86-9. [2] Zhang et al. PNAS. 2008. 105(52):20653-8. [3] Atsumi et al. Mol. Syst. Biol. 2010. 6:449. [4] Grund et al. J. Bacteriol. 1975. 123:546-56.