Coenzyme Q10 production in recombinant Escherichia coli strains engineered with a heterologous decaprenyl diphosphate synthase gene and foreign mevalonate pathway.
In the present work, Escherichia coli DH5alpha was metabolically engineered for CoQ(10) production by the introduction of decaprenyl diphosphate synthase gene (ddsA) from Agrobacterium tumefaciens. Grown in 2YTG medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl, and 0.5% glycerol) with an initial pH of 7, the recombinant E. coli was capable of CoQ(10) production up to 470 microg/gDCW (dry cell weight). This value could be further elevated to 900 microg/gDCW simply by increasing the initial culture pH from 7 to 9. Supplementation of 4-hydroxy benzoate did not improve the productivity any further. However, engineering of a lower mevalonate semi-pathway so as to increase the isopentenyl diphosphate (IPP) supply of the recombinant strain using exogenous mevalonate efficiently increased the CoQ(10) production. Lower mevalonate semi-pathways of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, and Saccharomyces cerevisiae were tested. Among these, the pathway of Streptococcus pneumoniae proved to be superior, yielding CoQ(10) production of 2,700+/-115 microg/gDCW when supplemented with exogenous mevalonate of 3 mM. In order to construct a complete mevalonate pathway, the upper semi-pathway of the same bacterium, Streptococcus pneumoniae, was recruited. In a recombinant E. coli DH5alpha harboring three plasmids encoding for upper and lower mevalonate semi-pathways as well as DdsA enzyme, the heterologous mevalonate pathway could convert endogenous acetyl-CoA to IPP, resulting in CoQ(10) production of up to 2,428+/-75 microg/gDCW, without mevalonate supplementation. In contrast, a whole mevalonate pathway constructed in a single operon was found to be less efficient. However, it provided CoQ(10) production of up to 1,706+/-86 microg/gDCW, which was roughly 1.9 times higher than that obtained by ddsA alone.
Zahiri
Hossein Shahbani
HS
Department of Microbiology, Gyeongsang National University, Gazwa-dong 900, Jinju 660-701, Korea.
Yoon
Sang Hwal
SH
Keasling
Jay D
JD
Lee
Si Hyoung
SH
Won Kim
Seon
S
Yoon
Sung Chul
SC
Shin
Yong Chul
YC
D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D003067:Coenzymes; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D008798:Mevalonic Acid / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013045:Species Specificity; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism
D003067:Coenzymes; D011994:Recombinant Proteins; D014451:Ubiquinone; D019883:Alkyl and Aryl Transferases; C039821:decaprenyl pyrophosphate synthetase; C024989:coenzyme Q10; D008798:Mevalonic Acid