Background
Herein, to improve the production of AMPs, the RBS library of T7 RNA polymerase based on Escherichia coli BL21 (DE3) was constructed, and then the most suitable expression host could be screened by high throughput screening. Besides, promoter engineering is also an effective strategy to improve the expression of genes. Promoter plays a significant role in gene transcription because it controls the binding of RNA polymerase to DNA.
Therefore, we are trying to use promoter engineering to improve BioBrick BBa_K3578010 from the 2020 iGEM worldshaper-Nanjing Team, and improve the ability of Yarrowia lipolytica to utilize the starch as an improvement.
We hypothesized that the expression level of glucoamylase could be increased through using the more potent promoters. For this, we scanned the genome of Y. lipolytica and characterized 20 endogenous promoters in Y. lipolytica.
Design
Given Y. lipolytica's metabolism with a high propensity for flux to tricarboxylic acid (TCA) cycle intermediates, the promoters chosen are primarily centering around the carbon metabolism and tricarboxylic acid cycle. We selected 20 endogenous promoters (Table1) in Y. lipolytica for genetic parts characterization. Specifically, these genes include:
YALI0E00638p, YALI0F07711p, YALI0A15972p, YALI0E26004p, YALI0D02277p, YALI0F16819p, YALI0D06325p, YALI0D23683p, YALI0F02607p, YALI0D06215p, YALI0C19965p, YALI0E02090p, YALI0D06930p, YALI0A15147p, YALI0A16379g, YALI0F0960p, YALI0C19624p, YALI0B10406p, YALI0E18590p, YALI0D17864p.
Next, to test the strength of these promoters, we amplified the 1500 bp promoter and 5′ UTR (5′-untranslated region) sequences of the target genes, and further constructed 20 recombinant plasmids by Gibson Assembly method. Recombinant plasmids were constructed based on the original plasmid pYLXP'-PTEF-Nluc harboring with the nanoluc luciferase gene (Nluc) as the reporter by Gibson Assembly method. For example, the recombinant plasmid of pYLXP’-P1-Nluc was constructed using linearized pYLXP'-PTEF-Nluc (digested by AvrII and NheI) and the PCR-amplified promoter P1 sequences by Gibson Assembly.
Then, 20 recombinant plasmids were transformed into Y. lipolytica po1fk. In brief, one milliliter cells were harvested during the exponential growth phase (16-24 h) from 2 mL YPD medium (yeast extract 10 g/L, peptone 20 g/L, and glucose 20 g/L) in the 14-mL shake tube, and washed twice with 100 mM phosphate buffer (pH 7.0). Then, cells were resuspended in 105 µL transformation solution, containing 90 µL 50% PEG4000, 5 µL lithium acetate (2M), 5 µL boiled single stand DNA (salmon sperm, denatured) and 5 µL DNA products (including 200-500 ng of plasmids, lined plasmids or DNA fragments), and incubated at 39 ℃ for 1 h, then spread on selected plates. The transformants were selected by using the leucine-defective solid medium.
Subsequently, shaking flasks were performed to characterize the strength of promoters by the Nluc analysis, which has been reported in the previous study (Liu et al., 2019).
Furthermore, the identified more potent promoter was used to express glucoamylase.
Results
After amplifying the 1500 bp promoter and 5′ UTR (5′-untranslated region) sequences of the target genes, we assembled these sequences with linearized pYLXP'-PTEF-Nluc (digested by AvrII and NheI) to construct the recombinant plasmids (Fig. 1 and Fig. 2), including pYLXP’-P1-Nluc, pYLXP’-P2-Nluc, pYLXP’-P3-Nluc, pYLXP’-P4-Nluc, pYLXP’-P5-Nluc, pYLXP’-P6-Nluc, pYLXP’-P7-Nluc, pYLXP’-P8-Nluc, pYLXP’-P9-Nluc, pYLXP’-P10-Nluc, pYLXP’-P11-Nluc, pYLXP’-P12-Nluc, pYLXP’-P13-Nluc, pYLXP’-P14-Nluc, pYLXP’-P15-Nluc, pYLXP’-P16-Nluc, pYLXP’-P17-Nluc, pYLXP’-P18-Nluc, pYLXP’-P19-Nluc, and pYLXP’-P20-Nluc.
These recombinant plasmids were further transformed into Y. lipolytica po1f (Fig. 2), and obtained strains po1f-P1-Nluc, po1f-P2-Nluc, po1f-P3-Nluc, po1f-P4-Nluc, po1f-P5-Nluc, po1f-P6-Nluc, po1f-P7-Nluc, po1f-P8-Nluc, po1f-P9-Nluc, po1f-P10-Nluc, po1f-P11-Nluc, po1f-P12-Nluc, po1f-P13-Nluc, po1f-P14-Nluc, po1f-P15-Nluc, po1f-P16-Nluc, po1f-P17-Nluc, po1f-P18-Nluc, po1f-P19-Nluc, and po1f-P20-Nluc. Next, we performed the shaking flask of these engineering strains and tested the Nluc analysis.
The results of the Nluc analysis showed that strains po1f-P2-Nluc and po1f-P17-Nluc generated the highest Nluc activities compared with other strains (Fig. 3), indicating promoter P2 and P17 are two strong promoters.
Then, we replaced the promoter of plasmid pUC-Pexp-AnGlu (BBa_K3578010) with identified promoters P2 and P17, and obtained the recombinant plasmids pUC-P2-AnGlu and pUC-P17-AnGlu (Fig. 4), respectively. Further, we transformed plasmids pUC-P2-AnGlu and pUC-P17-AnGlu into po1f, and obtained strains po1f-P2-AnGlu and po1f-P17-AnGlu. Subsequently, we performed the shaking flask with using starch as the sole carbon source.
As shown in Fig. 5, we can see that po1f-P2-AnGlu and po1f-P17-AnGlu both showed better cell growth than that of the control strain po1f-Pexp-AnGlu (BBa_K3578010), indicating that glycolase has been overexpressed by promoters P2 and P17. Moreover, among these strains, the cell growth of po1f-P2-AnGlu was the highest. That is to say that the use of strong promoter expression can improve glycolase activity, indicating that our strategy is indeed effective.
[1] Liu, H., Marsafari, M., Deng, L., Xu, P. 2019. Understanding lipogenesis by dynamically profiling transcriptional activity of lipogenic promoters in Yarrowia lipolytica. Appl Microbiol Biotechnol, 103(7), 3167-3179.
