title: Electrochemical probing of in vivo 5-hydroxymethyl furfural reduction in Saccharomyces cerevisiae.
abstract: In this work, mediated amperometry was used to evaluate whether differences in intracellular nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) level could be observed between a genetically modified Saccharomyces cerevisiae strain, engineered for NADPH dependent 5-hydroxymethyl-2-furaldehyde (HMF) reduction, and its control strain. Cells overexpressing the alcohol dehydrogenase 6 gene (ADH6 strain) and cells carrying the corresponding control plasmid (control strain) were each immobilized on Au-microelectrodes. The real-time dynamics of NAD(P)H availability in the two strains, preincubated with HMF, was probed using the menadione-ferricyanide double mediator system. A lower intracellular NADPH level as the consequence of more effective HMF reduction was observed for the ADH6 strain both with and without added glucose, which increases the overall cellular NADPH level. The mediated amperometric signal during real-time monitoring of the concentration dependent HMF reduction in living cells could be translated into the cellular enzyme kinetic parameters: K(M,cell)(app), V(MAX), k(cat,cell), and k(cat,cell)/K)M,cell)(app). The results indicated that the overexpression of the ADH6 gene gave a 68% decrease in K(M,cell)(app) and 42% increase in V(MAX), resulting in a 4-fold increase in k(cat,cell)/K(M,cell)(app). These results demonstrate that the mediated amperometric method is useful for monitoring the short-term dynamics of NAD(P)H variations and determining cellular enzyme kinetic parameters in S. cerevisiae cells.
authors.affiliation: Department of Applied Microbiology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
authors.forename: Natalie V
authors.initials: NV
authors.lastname: Kostesha
authors.forename: João R M
authors.initials: JR
authors.lastname: Almeida
authors.forename: Arto R
authors.initials: AR
authors.lastname: Heiskanen
authors.forename: Marie F
authors.initials: MF
authors.lastname: Gorwa-Grauslund
authors.forename: Barbel
authors.initials: B
authors.lastname: Hahn-Hägerdal
authors.forename: Jenny
authors.initials: J
authors.lastname: Emnéus
mesh_terms: D004563:Electrochemistry; D005662:Furaldehyde / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006046:Gold / Q000737:chemistry*; D008839:Microelectrodes; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D013499:Surface Properties
chemical_list: D009249:NADP; C008046:5-hydroxymethylfurfural; D006046:Gold; D005662:Furaldehyde