Real-time detection of cofactor availability in genetically modified living Saccharomyces cerevisiae cells--simultaneous probing of different geno- and phenotypes.
This work describes a mediated amperometric method for simultaneous real-time probing of the NAD(P)H availability in two different phenotypes, fermentative and respiratory, of the phosphoglucose isomerase deletion mutant strain of S. cerevisiae, EBY44 [ENY.WA-1A pgi1-1D::URA3], and its parental strain, ENY.WA-1A. The developed method is based on multichannel detection using microelectrode arrays. Its versatility was demonstrated by using four microelectrode arrays for simultaneously monitoring the NAD(P)H availability of both geno- and phenotypes under the influence of two different carbon sources, glucose and fructose, as well as the cytosolic and mitochondrial inhibitor and uncoupler, dicoumarol. The obtained results indicate that the method is capable of accurately and reproducibly (overall relative standard error of mean 3.2%) mapping the real-time responses of the cells with different genotype-phenotype combinations. The ENY.WA cells showed the same response to glucose and fructose when dicoumarol was used; fermentative cells indicated the presence of cytosolic inhibition and respiratory cells a net effect of mitochondrial uncoupling. EBY44 cells showed cytosolic inhibition with the exception of respiratory cells when fructose was used as carbon source.
Kostesha
Natalie
N
Department of Applied Microbiology, Lund University, P.O. Box 124, S-221 00 Lund, Sweden.
Heiskanen
Arto
A
Spégel
Christer
C
Hahn-Hägerdal
Barbel
B
Gorwa-Grauslund
Marie-Francoise
MF
Emnéus
Jenny
J
D019069:Cell Respiration / Q000187:drug effects; D001728:Dicumarol / Q000494:pharmacology; D004563:Electrochemistry; D005285:Fermentation / Q000187:drug effects; D005818:Genetic Engineering; D005838:Genotype; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008839:Microelectrodes; D010084:Oxidation-Reduction; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D024483:Vitamin K 3 / Q000378:metabolism
D024483:Vitamin K 3; D001728:Dicumarol