title: High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources.
abstract: Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z-factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.
authors.affiliation: Department of Chemical and Environmental Engineering, UC Riverside, Riverside, California, USA.
authors.forename: Ann-Kathrin
authors.initials: AK
authors.lastname: Löbs
authors.affiliation: Department of Chemical and Environmental Engineering, UC Riverside, Riverside, California, USA.
authors.forename: Jyun-Liang
authors.initials: JL
authors.lastname: Lin
authors.affiliation: Department of Chemical and Environmental Engineering, UC Riverside, Riverside, California, USA.
authors.forename: Megan
authors.initials: M
authors.lastname: Cook
authors.affiliation: Department of Chemical and Environmental Engineering, UC Riverside, Riverside, California, USA. iwheeldon@engr.ucr.edu.
authors.forename: Ian
authors.initials: I
authors.lastname: Wheeldon
mesh_terms: D000085:Acetates / Q000032:analysis / Q000302:isolation & purification*; D002151:Calorimetry; D002244:Carbon / Q000378:metabolism*; D020650:Combinatorial Chemistry Techniques; D004952:Esters / Q000032:analysis / Q000302:isolation & purification; D005285:Fermentation; D005290:Ferric Compounds / Q000737:chemistry; D005818:Genetic Engineering; D057166:High-Throughput Screening Assays / Q000379:methods*; D006877:Hydroxamic Acids / Q000737:chemistry; D007716:Kluyveromyces / Q000235:genetics / Q000254:growth & development*
chemical_list: D000085:Acetates; D004952:Esters; D005290:Ferric Compounds; D006877:Hydroxamic Acids; C074579:iron (III) hydroxamate; D002244:Carbon; C007650:ethyl acetate
keywords: Ethyl acetate; Ferric hydroxamate; High throughput screening; Kluyveromyces marxianus