Team:SCUT-China/Improve
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Overview
We wanted to find efficient promoter elements in yeast to help us achieve yield increase. So we tested the strength of 14 promoters from different metabolic pathways. And the experiments showed that the intensity of PDC1(BBa_K2365033) was the highest among them. Although the intensity of PDC1 is high in the early stage of fermentation compared to other promoters, it decreases significantly in the middle and late stages. (Fig.1) Therefore, we hope to construct a more stable promoter with high activity.
Fig.1 a) Fermentation results-genome level LEU2::P-VS-T; b) Time curve of Valencene; c) Glucose, Ethanol concentration and Valencene production.
Design
We determine that the large decrease in intensity before and after PDC1 may be due to the low activation of PDC1 in response to the carbon source at the late stage of fermentation. Different UASs on each promoter give it the ability to respond to different carbon sources [1]. Therefore, we can recombine the UASs from different promoters to change promoters, making them adapt for different carbon sources and activate production in the diauxic shift automatically.
After design and experimental validation, we designed three more ideal hybrid UAS promoters, M2 (BBa_K3772039), M5 (BBa_K3772042), and M8 (BBa_K3772045), using PDC1p as a prototype.
Hybrid UAS promoters M2, M5 and M8 were constructed by Over-lap PCR as shown in the Fig.2.
Fig.2 Multi copy addition of UAS
Results
We next constructed hybrid UAS Promoter-VS-SAG1t expression cassettes (BBa_K3772060, BBa_K3772063 and BBa_K3772066) to compare the expression intensity of the hybrid promoters by the yield of valenece.
According to the experimental results (Fig.3), the production of Valencene of PDC1p was 4.31mg/L and that of M2 was increased by 18.9% (5.13 mg/L) while M5 was being increased by 29.5% (5.58mg/L). Compared with M5, the Valencene yield of M8 increased by 27.0% (6.75 mg/L). Finally, the yield of Valencene controlled by the modified M8 mutant strain was increased by 56.6% (6.75 mg/L) compared with PDC1p.
Fig.3 The production of Valencene. a) Comparison of products between M1p, M2p and PDC1p; b) Comparison of products between M1-7p and PDC1p; c) Comparison of products between M5p and M8-10p; d) Comparison of products of all hybrid UAS Promoters in PDC1p.
And to illustrate in more detail the characteristics of our highest transcriptional intensity hybrid UAS promoter M8, we compared the time curve and specific rate curve of Valencene production between the original PDC1p and the M8 promoter.
The results showed that the overall level of Valencene fermentation in M8 was higher than that in PDC1, and the yield of Valencene in M8 was significantly higher than that in PDC1 at the late stage of fermentation
(Fig.4), indicating that the transformation was effective.Fig.4 Time curve of Valencene between M8 and PDC1p.
By analyzing Glucose, Ethanol concentration and Valencene production between M8 and PDC1 (Fig.5), we could find the difference in the activity of M8 and PDC1 during the consumption of different carbon sources.
Fig.5 Glucose, Ethanol concentration and Valencene production between M8p and PDC1p.
During glucose consumption (0-15 h), there was no significant difference in Valencene Valencene production between M8p and PDC1p, while during the ethanol consumption phase (15-70 h), the Valencene production corresponding to M8p continued to rise with ethanol consumption, with significant growth, while PDC1p grew slowly.
The Fig.6 comparing the Valencene ratio production rate of M8 with that of PDC1 more visually reflects that the Valencene production rate of M8 is much higher than that of PDC1p at the late stage of fermentation (48h-72h).
Fig.6 Specific production rate of product.
In order to verify that the increase of Valencene production is the results of the increase of mRNA at the transcription level, we conducted real-time quantitative PCR between M8 & PDC1. To be specific, we sampled the 9h and 26h fermentation broth to extract RNA from cells and reverse transcribed cDNA for qPCR verification, and the results (Fig.7) showed that the transcription level of M8 at the late fermentation (26h) was significantly higher than that of PDC1. These results indicated that the increase of Valencene production was due to the increased transcription level of Valencene synthase gene.
Fig.7 The differences of M8 & PDC1 in transcription level
Finally, by transcribing Valencene synthase using the M8 promoter, the yield of Nootkatone synthesis with other enzymes showed a significant increase in the yield of Valencene, Nootkatol and total terpene compared to PDC1. (Fig.8)
Fig.8 the yield of Valencene, Nootkatol and total terpene between M8p and PDC1p
In summary, our improvement of BBa_K2365033 resulted in a significant increase in promoter activity of the improved parts M2 (BBa_K3772039), M5 (BBa_K3772042), and M8 (BBa_K3772045). And the M8 promoter was shown to have high activity even in the late stage of fermentation.
References
[1] Cao, L., et al., Two-stage transcriptional reprogramming in Saccharomyces cerevisiae for optimizing ethanol production from xylose. Metabolic Engineering, 2014. 24: p. 150-159.