Enhanced lycopene production in Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate.
To increase expression of lycopene synthetic genes crtE, crtB, crtI, and ipiHP1, the four exogenous genes were cloned into a high copy pTrc99A vector with a strong trc promoter. Recombinant Escherichia coli harboring pT-LYCm4 produced 17 mg/L of lycopene. The mevalonate lower pathway, composed of mvaK1, mvaK2, mvaD, and idi, was engineered to produce pSSN12Didi for an efficient supply of the lycopene building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Mevalonate was supplied as a substrate for the mevalonate lower pathway. Lycopene production in E. coli harboring pT-LYCm4 and pSSN12Didi with supplementation of 3.3 mM mevalonate was more than threefold greater than bacteria with pT-LYCm4 only. Lycopene production was dependent on mevalonate concentration supplied in the culture. Clump formation was observed as cells accumulated more lycopene. Further clumping was prevented by adding the surfactant Tween 80 0.5% (w/v), which also increased lycopene production and cell growth. When recombinant E. coli harboring pT-LYCm4 and pSSN12Didi was cultivated in 2YT medium containing 2% (w/v) glycerol as a carbon source, 6.6 mM mevalonate for the mevalonate lower pathway, and 0.5% (w/v) Tween 80 to prevent clump formation, lycopene production was 102 mg/L and 22 mg/g dry cell weight, and cell growth had an OD(600) value of 15 for 72 h.
Yoon
Sang-Hwal
SH
Department of Food Science & Nutrition, Gyeongsang National University, Jinju, Korea.
Lee
Young-Mi
YM
Kim
Ju-Eun
JE
Lee
Sook-Hee
SH
Lee
Joo-Hee
JH
Kim
Jae-Yean
JY
Jung
Kyung-Hwa
KH
Shin
Yong-Chul
YC
Keasling
Jay D
JD
Kim
Seon-Won
SW
D002338:Carotenoids / Q000096:biosynthesis*; D018929:Cell Culture Techniques / Q000379:methods*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D045782:Hemiterpenes / Q000096:biosynthesis*; D000077276:Lycopene; D008798:Mevalonic Acid / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism
D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C004809:isopentenyl pyrophosphate; D002338:Carotenoids; D008798:Mevalonic Acid; D000077276:Lycopene