Production of lycopene and β-Carotene from metabolically engineered escherichia coli

Abstract

ABSTRACT We generated recombinant lycopene and β-carotene utilizing metabolically engineered Escherichia coli DH5α. For this purpose, we constructed synthetic operons regulated by the araBAD promoter. Vectors harboring these operons exhibited diverse expression patterns dependant upon plasmid copy number. Carotenoid production from these recombinant E. coli was either constitutive or inducible depending upon plasmid copy number. Furthermore, the yields of both lycopene and β-carotene in E. coli cultures harboring high-copy vectors (pTB-EIB and pTB-EIBY) varied depending upon inocula state. This inocula dependence, solely associated with use of high copy vectors, was overcome through using low-copy vectors (pTB-EIBrop and pTB-EIBYrop) for recombinant carotenoid production. We performed fermentation process from laboratory-scale to pilot-scale. Various cultivation conditions, including temperature, medium composition, and induction time, were investigated and determined for high-level production of lycopene and β-carotene. Interestingly, lycopene and β-carotene were greatly increased at low-temperature condition (25 ℃). Through fed-batch fermentation in 300 L-bioreactor, maximal lycopene production reached up to 99 $mg/l^{-1}$ with 2.3 $mg/g^{-1}$ DCW. In addition, using this same technique, β-carotene production reached up to 110 $mg/ l^{-1}$ with 2.2 $mg/g^{-1}$ DCW. Although carotenoid content per cell is relatively low, our results showed that the high-level production of carotenoids could be achieved by optimizing culture conditions at various fermentation scales. Previous sequence analyses of the lycopene cyclase gene (crtY) revealed the protein product was translated from the ATG start codon in Escherichia coli. We found, however, this enzyme could be also obtained without ATG start codon. The research using crtY mutants revealed the GTG (Val) sequence located in-frame 24 bp downstream of the ATG was a potential start codon. This result suggests t...