Cofactor engineering of intracellular CoA/acetyl-CoA and its effect on metabolic flux redistribution in Escherichia coli.
Coenzyme A (CoA) and its thioester derivatives are important cofactors participating in over 100 different reactions in intermediary metabolism of microorganisms. The time profiles of intracellular CoA and acetyl-CoA levels were studied in an aerobic batch reactor. The CoA level starts at a high value and falls off gradually over the exponential and stationary growth phases, reaching negligible levels at the end of 24h. The acetyl-CoA level, on the other hand, increases initially reaching a maximum and decreases gradually reaching negligible levels after 24h. Overexpressing one of the upstream rate-controlling enzyme the pantothenate kinase with simultaneous supplementation of the precursor pantothenic acid to the culture medium increased the intracellular CoA/acetyl-CoA levels. It was found that supplementation of the precursor pantothenic acid is essential to increase CoA/acetyl-CoA levels. A 10-fold increase in CoA level was observed upon this overexpression in complex medium. Acetyl-CoA levels also increased (5-fold) but not as much as CoA, leaving much of the CoA in free unacetylated form. The increase in intracellular CoA/acetyl-CoA levels led to an increase in carbon flux to the acetate production pathway leading to formation of more acetate in complex medium, whereas no such change in metabolite redistribution was observed in minimal medium.
Vadali
Ravishankar V
RV
Department of Bioengineering, Rice University, Houston, TX 77005, USA.
Bennett
George N
GN
San
Ka-Yiu
KY
D000085:Acetates / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000235:genetics* / Q000378:metabolism*; D019149:Bioreactors / Q000382:microbiology*; D003065:Coenzyme A / Q000235:genetics* / Q000378:metabolism*; D003067:Coenzymes / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D042541:Intracellular Space / Q000201:enzymology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism
D000085:Acetates; D003067:Coenzymes; D011994:Recombinant Proteins; D000105:Acetyl Coenzyme A; D017853:Phosphotransferases (Alcohol Group Acceptor); C023088:pantothenate kinase; D003065:Coenzyme A