Bioelectrochemical detection of L-lactate respiration using genetically modified Hansenula polymorpha yeast cells overexpressing flavocytochrome b2.
In general, L-lactate respiration is difficult to detect in living yeast cells due to the small activity of L-lactate oxidizing enzymes within the mitochondria. Genetically modified cells of methylotrophic yeast Hansenula polymorpha overproducing L-lactate:cytochrome c-oxidoreductase (EC 1.1.2.3, also known as flavocytochrome b(2), FC b(2)) were physically immobilized by means of a dialysis membrane onto various types of electrode materials in order to investigate the possibility of electrochemically detecting L-lactate respiration. It could be shown that in the case of genetically modified Hansenula polymorpha cells in contrast to cells from the parental strain, enhanced L-lactate-dependent respiration could be detected. Due to overproduction of FC b(2) the O(2) reduction current is decreased upon addition of L-lactate to the electrolyte solution. The electron transfer pathway in the L-lactate-dependent respiration process involves a cascade over three redox proteins, FC b(2), cytochrome c and Complex-IV, starting with L-lactate oxidation and ending with oxygen reduction. By means of selective inhibition of Complex IV with CN(-), lactate respiration could be proven for causing the decrease in the O(2) reduction.
Shkil
Halyna
H
Analytische Chemie-Elektroanalytik & Sensorik, Ruhr-Universität Bochum, Universitätsstr. 150, D-44780, Bochum, Germany.
Stoica
Leonard
L
Dmytruk
Kostyantyn
K
Smutok
Oleh
O
Gonchar
Mykhailo
M
Sibirny
Andriy
A
Schuhmann
Wolfgang
W
D015374:Biosensing Techniques; D004563:Electrochemistry; D004566:Electrodes; D004579:Electron Transport; D015870:Gene Expression; D005818:Genetic Engineering; D006108:Graphite / Q000737:chemistry; D042844:L-Lactate Dehydrogenase (Cytochrome) / Q000235:genetics*; D019344:Lactic Acid / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism
D019344:Lactic Acid; D006108:Graphite; D042844:L-Lactate Dehydrogenase (Cytochrome); D010100:Oxygen