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.