3.4 Optimization of fermentation condition for lycopene
production
It had been reported that the concentration of IPTG has a significant
effect on gene expression levels . Meanwhile, the excessive addition of
IPTG is toxic to cells, which illustrated the importance of optimization
. Therefore, the concentration of IPTG was tested, which controls the
expression of CrtE , CrtB and CrtI catalyzing for
synthesizing lycopene but is toxic to cells (Figure 4A). A concentration
gradient (0.5 mM/L, 0.1 mM/L, 0.05 mM/L, 0.01 mM/L and 0.005 mM/L) was
added respectively and it turned out that only strain adding 0.005 mM/L
IPTG behaves better than the original strain adding 0.1mM IPTG,
producing 6.86 mg/OD600 lycopene (Figure 4B).
As illustrated before, two kinds of isopentenol substrates contributes
to different C5 skeleton. Based on previous ratio 1p/1i (prenol:
isoprenol=1:1), the other different ratio: 100%p (p), prenol:
isoprenol=5:1, 3:1; 1:1; 1:3 and 1:5 and 100%i (i) were also tested. It
turned out that 1p/3i (prenol: isoprenol=1:3) is the most beneficial
proportion to balancing IPP/DMAPP in cellular, leading to about
7.94 mg/OD600lycopene, 1.4-fold of the original strain provided with 1p/1i (prenol:
isoprenol=1:1) (Figure 4C). Next, various substrates concentration
(1g/L, 2g/L and 3g/L) was added and it demonstrated that the best
concentration is 2g/L since it is not only the most favorable for
synthesizing lycopene (~8.67 mg/OD600)
but also leads to the moderate toxicity to cells compared with that of
1g/L and 3g/L (Figure 4D).
3.5 Rapid construction of the multicopy IUP
genome-harboring chassis through PtrCAST for lycopene production
Heterogeneous gene expressions usually relied on multiple compatible
plasmids, however, it brings about expression instability due to
unprecise copy number and higher fermentation cost because of antibiotic
addition, which is also unrealistic and reliable in industrial
production [31]. For instance, in the case of
lycopene production of E.coli , 3 plasmids were employed to
introduce MVA pathway and lycopene-related synthases, in which the
longer lag phase and lower mean productivity was observed . In another
case, only when adding more than 5-fold KmR than
normal level (50 mg/mL) can cells for astaxanthin production prevent
plasmids loss during fed-batch condition .
In our study, the IUP expression cassette was initially placed on
plasmid and might lead to similar problems mentioned above]. Therefore, the plasmid stability was first
measured by succession in CmR-free medium and it
showed that the plasmid stability decreased to lower 50% in the third
transfer (Figure S1). To address this problem, the genome multi-position
integration strategy was adopted. However, it was laborious and
time-consuming, and it’s really troublesome to construct a chassis in
need of serial manipulation ], though
highly-effective CRISPR/Cas system has been widely utilized in
engineering cell factories . Multicopy integration of chromosome based
on CASTs, more convenient and effective, was applied to construct strain
library and screening the most productive chassis . Importantly, the
CAST from Pseudoalteromonas translucida KMM520 was
employed to form a novel tool PtrCAST, performing multiple sites
integration of larger cargo (~15.4 kb) with higher
efficiency .
Thanks to this tool, a gift from Yang, several copies of IUP expression
cassette were successfully integrated into the genome of BL21(DE3). The
transposition targeting 8 loci was conducted through ptrTnsQCas-8array,
acquiring a mixed strain library with different IUP expression cassette
copies (Figure 5A-5B). It could be concluded that the lycopene
production performances of strains with different copy number varied
(Figure S2). Then, the pEBI was transferred into the strain library,
obtaining transformants which were selected for fermentation. Strains
with crimson were detected and sequenced, of which strain YZJ3 with 7
copies of IUP expression cassette performed best but failed to reach the
control strain YZ72-5 (Figure 5C and G). Therefore, the second
transposition was performed on YZJ3 targeting IS1 (Figure 5D). The same
procedure was followed mentioned above (Figure 5E-5F). Finally, strain
YZJ3-4 of 13 copies in total was obtained, resulting in 12.2
mg/OD600, 1.5-fold that of strain YZ72-5 using plasmids
(Figure 5G). In fed-batch fermentation, it produced 15.43
mg/OD600. Besides, we transferred this strain for ten
generations and examined each 13 loci and it showed that the IUP
expression cassettes were stably existed in each locus. Also, the
fermentation was conducted and it showed the similar level with the
first generation (Figure S3). It was reported that the genome position
had relationship with genome position, so that adoption of PtrCAST could
further screening for more suitable site-combination of IUP expression
cassettes, lowing the copy number and enhancing the effectiveness of
each cassette .