3.2 Optimization of IUP expression cassette based on promotor engineering and IDI selection
Studies have shown that promoters are valuable tool for controlling and regulating gene transcriptional level . Synthetic constitutive promoters like J231 family have been widely applied in gene controlling, however, exhibiting different activities according to genetic host background, and thus strength-varied promoters needed to be tested . Hence, promoter engineering strategy was conducted on two genes involved in IUP,ThiMEc and IPKMTH . To be specific, constitutive promotors and inducible promotor library was built respectively. Firstly, the constitutive promoter ofIPKMTH was replaced with PJ23106(BBa_J23106, medium strength) and PJ23109 (BBa_J23109, low strength). Based on these, we then inserted PJ23119, PJ23106 and PJ23109 to controlThiM , obtaining a promoter library (3*3=9 types) (Figure 2A). Strains harboring these plasmids were fermented and strain YZ3-1 had the highest amount of lycopene titer (0.59 mg/OD600) (Figure 2B). L-rhamnose inducible promoter (PRha), arabinose inducible (PBAD), tac promoter (Ptac) and anhydrotetracycline inducible promoter (Ptet) are commonly used inducible promoters for tunable heterologous gene expression in E.coli . Thus, the constitutive promoter PJ23119 in pC19IUP was replaced with these 4 kinds of inducible promotors, meanwhile, controlling bothThiMEc and IPKMTH (Figure 2C) and thus four inducible IUP-harbored plasmids, pTacIUPidi, pTetIUPidi, pBADIUPidi, pRhaIUPidi were assembled, followed by being transformed with pEBI to BL21(DE3), creating strains YZ4, YZ5, YZ6 and YZ7, accordingly. After the same fermentation condition, 4 strains produced 1.1, 0.56, 0.58 and 1.96 mg/OD600 lycopene (Figure 2D). Comparing strain YZ3-1 and YZ7, it was the PRha that was the most effective in controlling IUP for lycopene titer, reaching almost 22.8-fold that of the control strain without IUP. This may be due to the fact that the PRha has tight expression with almost none leakage enabling better cell growth in uninduced stage and increased IUP expression level in induced stage.
To further increase the lycopene titer, the ratio of prenol and isoprene was changed. This is because the block may occur during the C5 to C40 conversion process. It is worth noting that IPP and DMAPP are two key precursors of terpenoids and the former one is especially critical in generating farnesyl diphosphate (FPP), geranylpyrophosphate (GPP) and geranylgeranyl diphosphate (GGPP) . Besides, two isopentenol isomers (isoprenol/prenol) can turn into IPP and DMAPP accordingly through IUP pathway, so changing the ratio of these two substrates is a direct method of C5 supplement balance . Therefore, 1 g/L prenol was replaced with 1 g/L 1:1 prenol/isoprenol. Surprisingly, the accumulation of lycopene reached 2.3 mg/OD600 (Figure 2E), demonstrating this limitation step and the poor performance of IDIEc we employed.
It’s reported type II IDI behaves better than that of type I , thus we then replaced IDIEc with IDIBc and IDISc of type II (Figure 2C). It came out that the lycopene production was respectively increased by 2.06 and 2.66-fold compared with the strain harboring original IDIEc, the later reaching 4.42 mg/OD600, 51.5-fold that of the control strain (Figure 2F). Base on this result, we decide to employ IDISc in the following experiment.
Optimization of IUP expression cassette based on construction of RBS library of ThiMEc
ThiMEc was reported being capable of catalyzing phosphorylation reaction, the overexpression of which combined with other optimizations brought about 200 mg/L geranoids . It is also the first enzyme in IUP utilizing substrates, determining the influx of IUP . According to the first part, it could be concluded that the lower transcriptional level of IPKMTH and higher transcriptional level of ThiMEc benefited the lycopene respectively. Therefore, the online ‘UTR designer’ (https://sbi.postech.ac.kr/utr_designer/#) was turned to predict the expression level of two genes , it turned out that compared to IPKMTH (value=1283574.22),ThiMEc , in contrast, was in relatively low expression level (RBS0(GCAGGAGCA)value =40772.93). Thus, the RBS library of ThiMEc (pRhaIUP-nRBSThiM-idisc) was constructed through circular PCR to screen the most suitable RBS for expression of ThiMEc so as to increase lycopene titer (Figure 3B). In our study, 135 strains were randomly picked and fermented in 24-well plates, of which 11 candidates with higher lycopene accumulation were screened by the intense of red color compared with the control strain. The results demonstrated that strain YZ72-5 harboring the RBS5 (AAGGGGGGA) is the optimal, accumulating 6.5 mg/OD600lycopene titer, reaching almost 1.5-fold of the control strain YZ72 (Figure 3C). To further estimate the expression strength ofThiMEc , we recorded its fluorescence value per unit cell with GFP using a reporter. It demonstrated that RBS5 contributes a higher translational level(3-fold) than RBS0 (Figure 3D). Meanwhile, the expression level of ThiMEcwith RBS5 was also predicted using online ‘UTR designer’ mentioned above, the value increased to 861937.03, which showed the identical trend of out verification.