4. Conclusion

In this study, 81 endogenous promoters in Y. lipolytica were systematically investigated and analyzed. For obtaining the endogenous promoters, we truncated the 1500 bp upstream sequences before the ATG site of the desired gene. Specifically, it has been reported that the core sequences of a promoter in fungi are about 200-300bp. As a result, our obtained promoters in this study should contain the binding sites of the transcription factors, thus can accurately reflect some properties of these endogenous promoters. However, Y. lipolytica could generate severe fluorescence background, which interferes with the reliabilities of conventional reporter systems. Specifically, a luciferase reporter system has been developed in Y. lipolytica , which is stable, efficient, and instant.[19]Herein, we chose this luciferase reporter system for the promoter analysis.
Next, to construct the reporter system plasmids pYLXP’-Pxx-Nluc , we inserted the promoter sequences into the chassis plasmid pYLXP’-Nluc , and obtained 82 recombinant promoter plasmids, including the PTEFpromoter. Further, constructed plasmids were transformed into po1g for the promoter analysis. Specifically, we obtained 15 strong promoters, 41 medium strength promoters and 25 weak promoters. Among them, the most potent promoter is PMnDH2 with a 1.60-fold strength of the PTEF promoter, reaching 6.87x107, which is responsible for transcribing sorbose reductase, catalyzing the reaction of sorbose to glucitol. Furthermore, the weakest promoter is PPHO89 (YALI0E23859g ), with a 0.06% strength of the PTEF promoter, which is responsible for transcribing sodium-dependent phosphate transporter. These results suggest that we obtained an endogenous promoter library with a strength spanning from 0.06% to 1.60-fold of the PTEF promoter. Although we only found several endogenous promoters with higher strength than PTEF in this study, gene expression levels do not blindly pursue high strength of the promoter.
We explored the relationship between the promoter activity and time (Fig. 5), and found that 88.9% promoters had the highest intensity in the logarithmic growth phase (before 36h). And, 9.9% promoters had the highest intensity in the stationary phase (48-96 h), while only one promoter (PPGM2, YALI0E02090g ) reached the highest transcriptional activity at 72h. In addition, we also found that the promoter strength is different in the specific metabolic pathways. For example, promoters in glycolysis are generally more robust. In conclusion, the promoter library we constructed is significant for enriching genetic expression elements for Y. lipolytica , and can be applied to construct microbial cell factories for the biomanufacturing applications.