AM Azevedo

AS Vangnai

B Han

C Verduyn

CV Agu

FM Dickinson

J Fibla

JE Dueber

M Scheel

MAJ Siemerink

TJ Mangos

V Ujor

X Ying

Y Zhou

Scientific Reports

AbstractWe 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.</jats:p

Chidozie Victor Agu

Stella M. Lai

Victor Ujor

Pradip K. Biswas

Andy Jones

Venkat Gopalan

Thaddeus Chukwuemeka Ezeji

Crossref

Development of a high-throughput assay for rapid screening of butanologenic strains

Development of a high-throughput assay for rapid screening of butanologenic strains

Abstract

Similar works

Full text

Available Versions

Crossref