Abstract
We report a Thermotoga hypogea (Th) alcohol dehydrogenase (ADH)-dependent spectrophotometric assay for quantifying the amount of butanol in growth media, an advance that will facilitate rapid high-throughput screening of hypo- and hyper-butanol-producing strains of solventogenic Clostridium species. While a colorimetric nitroblue tetrazolium chloride-based assay for quantitating butanol in acetone-butanol-ethanol (ABE) fermentation broth has been described previously, we determined that Saccharomyces cerevisiae (Sc) ADH used in this earlier study exhibits approximately 13-fold lower catalytic efficiency towards butanol than ethanol. Any Sc ADH-dependent assay for primary quantitation of butanol in an ethanol-butanol mixture is therefore subject to “ethanol interference”. To circumvent this limitation and better facilitate identification of hyper-butanol-producing Clostridia, we searched the literature for native ADHs that preferentially utilize butanol over ethanol and identified Th ADH as a candidate. Indeed, recombinant Th ADH exhibited a 6-fold higher catalytic efficiency with butanol than ethanol, as measured using the reduction of NADP+ to NADPH that accompanies alcohol oxidation. Moreover, the assay sensitivity was not affected by the presence of acetone, acetic acid or butyric acid (typical ABE fermentation products). We broadened the utility of our assay by adapting it to a high-throughput microtiter plate-based format, and piloted it successfully in an ongoing metabolic engineering initiative.
Highlights
Growing concerns over the environmental consequences of fossil fuel use have reinvigorated interest in renewable energy sources like biofuels
Because numerous challenges impeded attempts to develop an assay using Thauera butanivorans (Tb) butanol dehydrogenase (BOH), we focused our subsequent efforts on Thermotoga hypogea (Th) alcohol dehydrogenase (ADH)
This assay utilizes a recombinant Th ADH that can accurately measure the amount of butanol present in cell cultures because the activity of the enzyme is not affected by the presence of other major ABE fermentation products
Summary
Growing concerns over the environmental consequences of fossil fuel use have reinvigorated interest in renewable energy sources like biofuels (e.g., bio-butanol). Solventogenic Clostridium species like Clostridium beijerinckii (Cb) are the only known natural and reliable producers of butanol These microbes typically exhibit a biphasic metabolism wherein acetic and butyric acids produced during the acidogenic growth phase are re-assimilated to produce acetone, butanol, and ethanol (ABE) during the solventogenic phase. Current research on bio-butanol primarily focuses on maximizing production by butanologenic clostridia by enabling better utilization of commonly used biological feedstocks (e.g., lignocellulosic biomass). Such an approach relies heavily on the development of effective molecular tools for targeted metabolic engineering of desired phenotypes (e.g., high butanol production) in solventogenic Clostridium species[1]. As a robust alternative for the rapid and efficient screening of engineered strains of butanologenic clostridia, we developed a robust and high-throughput microtiter plate-based spectrophotometric assay that utilizes recombinant Thermotoga hypogea (Th) ADH, an enzyme that prefers butanol over ethanol[6] for rapid and efficient screening of engineered butanologenic clostridial strains
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