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 .