3.1 Assembling IUP pathway in E. coli enables lycopene production
pC19IUP was first designed for expression of IUP pathway to test its ability to produce lycopene inE. coli . In this study, IPK from Methanothermobacter thermautotrophicus (IPKMTH )] and ThiM from Escherichia coli(ThiMEc ) are employed, which was accordingly under the control of the constitutive promoter PJ23119(BBa_J23119, high strength) (Figure 1B). To better balance the ratio of IPP and DMAPP, idi from Escherichia coli(idiEc ) was also included. Downstream genes for lycopene production (CrtE , CrtB , CrtI ) were co-expressed in pEBI driven by inducible promoter Ptrc(a gift from Yang). Two strains were obtained, the control strain YZ0 with only MEP pathway and strain YZ1 with both MEP and IUP pathway. These two strains were cultivated in LB medium for about 3 h when we added a lower concentration of IPTG (0.01 mM) in combination with 1g/L prenol as substrate. When comparing the lycopene titer of two strains after 24 hours’ fermentation, YZ0 produced 0.5 mg/OD600 lycopene, which was nearly 6.2-fold of the control strain (0.086 mg/OD600), verifying the feasibility of IUP pathway.