Team:SCUT-China/Engineering

1. Overview

As a team participating in the synthetic Biology Competition, we plan to follow the engineering philosophy of "design-build-test-learn" to carry out our promoter engineering projects. In this process, we apply the “learning by doing” approach. Based on the experience learned from each round of modification, then we continue to design the next round of modification, and finally get the ideal result. We are good at summarizing experience from each modification and some exploring principle from existing research, so as to provide more experience for rational design of promoters in the future.

2. UAS adding location

2.1 Design

The YeTFaSCo database(http://yetfasco.ccbr.utoronto.ca/scanSeqs.php) was used to predict the transcription factor binding sites (TFBs) of PPDC1, PSED1L, PALD4. Selected version: 1.02 and resulted in more than 100 putative TFBSs with a matching score higher than 0.75. In order to avoid the incompleteness of using a single database and improve the accuracy of prediction, the YEASTRACT database (http://www.yeastract.com/formtfsbindingsites.php) also used for prediction and comparison. These TFBSs were classified into five groups according to the function (Table 1)

Table 1. Predicted putative TFBs on P_PDC1, P_SED1L, P_ALD4 by YeTFaSCo database and YEASTRACT database

According to the characteristics of P_SED1L and P_ALD4 to control the synthesis of Valencene, which mainly expressed in the later stage of fermentation. We rationally selected the transcription factor binding sites of CAT8p and ADR1p for promoter modification.

CAT8p is a Zinc cluster transcriptional activator, which regulate the expression include of most genes in gluconeogenesis, ethanol utilization and glyoxylate cycle^[1]. CAT8p can bind to carbon source response elements and activate target genes after glucose consumption, the binding motif is 5′-YCCNYTNRKCCG-3′^[2].

ADR1p is a Carbon source-responsive zinc-finger transcription factor, which first identified as a transcription factor activate the transcription of the alcohol dehydrogenase gene ADH2. It also activates genes involved glucose fermentation, glycerol metabolism and fatty acid utilization. The binding motif is 5′-TTGGRG-3′. In addition^[3-4], ADR1p and CAT8p may interact when activating the transcription of certain genes^[5].

In cells, transcription is a process of three-dimensional regulation. The transcription factor binding promoter has a positional effect. When the transcription factor binding site is at a position with high nucleosome affinity, the transcription factor may not be able to bind to the site due to steric hindrance^[6]. In order to find out which position of PDC1 can make the added UAS (upstream activation sequences) avoid the effect of steric hindrance. The reported UAS1 and the database predicted 100% binding UAS2 were added to the two key positions of PDC1, which can normally bind to transcription factors and activate. UAS1 is the CAT8 binding site of the FBP1 promoter, and UAS2 is the ADR1p binding site predicted to be 100% bound in the ALD4 promoter. Studies have shown that there may be a positive interaction between CAT8p and ADR1p, so we added UAS1 and UAS2 closer to each other. (Figure 1)

Considering promoter architecture constraints, (a) avoiding a change on other Predicted putative TFBSs for the activation of PDC1 promoter, (b) determining positions for CAT8p and ADR1p binding sites that are close proximity to the core promoter, (c) existing binding sites were replaced with new ones, reduce the changes to the local sequence context.

Figure 1. The addition of different positions of CAT8 and ADR1

2.2 Build

Using Overlap PCR method, the sequence at the key position of the PDC1 promoter is replaced. Taking the construction of the M1 mutant strain as an example, the construction method of the M2 strain was the same. Find the CAT8 binding site of FBP1 promoter through the literature and the ADR1 binding site from ALD4 promoter predicted by the database (Table 2).

Design the binding site motifs on the homology arm, (1) Using primer M1-F/PDC1-R to amplify fragment one, primer M1-R/PDC1-F to amplify fragment two, (2) assemble fragment 1 and fragment 2 to obtain M1 promoter, (3) Using primer PDC1-F/PDC1-R amplifies M1 promoter. Using BamHI and XbaI restriction enzymes to digest the M1 promoter and YEp181-PDC1p-VS-SAG1t vector, and react at 37°C for 2h. After the reaction, the promoter and vector were purified and ligated at 16°C overnight. Transform into E. coli DH5α, pick the correct transformants, and extract the YEp181-M1-VS-SAG1t plasmid.

Then, use donor DNA primers (Table4) to amplify the M1-VS-SAG1t expression cassette with a homology arm next to the LEU2 site. Transform the constructed gRNA expression plasmid and M1-VS-SAG1t expression cassette into yeast cells which can express Cas9 protein, and use the method of auxotrophic medium selection and colony PCR verification to obtain the strains that successfully knocked into the M1-VS-SAG1t expression cassette.

Table 2. CAT8 and ADR1 binding sites

2.3 Test

The strain was inoculated into 10 ml of fermentation broth, the initial OD600 of the fermentation broth was controlled to be 0.05, and it was placed in a shaker at 30℃, 220 rpm for 64 hours. After 64 hours of shake flask fermentation, the concentration of valencene was detected by gas chromatography. (Figure 2)

Figure 2 Fermentation results of M1 and M2 mutant strains. (a) OD₆₀₀ of M1 and M2 mutant strain, (b) Valencene yield of M1 and M2 mutant strain.

The addition of CAT8 and ADR1 transcription factor binding sites at different positions shows different effects. No significant differences in Valencene production in M1, P_PDC1 were observed. The valencene production of the M2 mutant strain was increased by 18.9% compared to the original strain (P_PDC1).

2.4 Learn

In M2, UAS is added about 20 bp upstream of TATA-box located on P_PDC1, and there are continuous 17 bp A/T near the position of TATA-box, which may cause the structure of this position to be relatively loose and the nucleosome affinity rate Low ^[7], unable to form a nucleosome structure, transcription factors have a greater probability of binding to this site, and play a role in activating transcription ^[8]. In M1, UAS was added between -393 and -450 located in the upstream region of the PDC1 gene, and it did not work. The possible reason is that it is close to the original activation region, and there is dense transcription factor binding at this site, which hinders the combination of transcription factors to the added UAS. This suggests that we should transform on the basis of M2.

3. Add UAS

3.1 Design

The YeTFaSCo database and YEASTRACT database were used to predict the CAT8p binding site and ADR1p binding site of SED1L promoter, ALD4 promoter, and select data with a system score of 0.85 or more. According to the results of the first round of transformation, the CAT8p binding site was replaced with the predicted CAT8p binding site of P_SED1L and P_ALD4 on the basis of M2, but the original ADR1p binding sequence was not changed. (Figure 3)

Figure 3. the additional add of CAT8p-3 binding site

3.2 Build

Taking the construction of the M3 mutant strain as an example, the construction method of the M4-M7 strain was the same. Find the CAT8 binding site motifs and ADR1 binding site motif predicted by the database (Table 5).

Design the binding site motifs on the homology arm, (1) Using primer M3-F/PDC1-R to amplify fragment one, primer M3-R/PDC1-F to amplify fragment two, (2) assemble fragment 1 and fragment 2 to obtain M3 promoter, (3) Using primer PDC1-F/PDC1-R amplifies M3 promoter. Using BamHI and XbaI restriction enzymes to digest the M3 promoter and YEp181-PDC1p-VS-SAG1t vector, and react at 37°C for 2h. After the reaction, the promoter and vector were purified and ligated at 16°C overnight. Transform into E. coli DH5α, pick the correct transformants, and extract the YEp181-M3-VS-SAG1t plasmid.

Then, use donor DNA primers (Table 4) to amplify the M3-VS-SAG1t expression cassette with a homology arm next to the LEU2 site. Transform the constructed gRNA expression plasmid and M3-VS-SAG1t expression cassette into yeast cells which can express Cas9 protein, and use the method of auxotrophic medium selection and colony PCR verification to obtain the strains that successfully knocked into the M3-VS-SAG1t expression cassette.

Table 5. Predicted CAT8 and ADR1 binding site motif

3.3 Test

The strain was inoculated into 10 ml of fermentation broth, the initial OD600 of the fermentation broth was controlled to be 0.05, and it was placed in a shaker at 30℃, 220 rpm for 64 hours. After 64 hours of shake flask fermentation, the concentration of valencene was detected by gas chromatography. (Figure 4)

Figure 4. Fermentation results of M3-M7 mutant strains. (a) OD₆₀₀ of M3-M7 mutant strain, (b) Valencene yield of M3-M7 mutant strain.

The modified promoters showed different intensities. The Valencene yield of M3, M4, M6, and M7 mutant strains was not significantly different from that of the control strain. But the Valencene yield of the M5 mutant strain was increased by 29.5% (5.58 mg/L) compared to the control strain.

3.4 Learn

In M5, the CAT8 binding site-3 is derived from the SED1L promoter, which has superior performance compared to the CAT8 binding site from P_FBP1 reported in the article. And we observed that only CAT8 binding site-3 has such an effect, other CAT8 binding sites may be false positive data predicted by the database. This suggests that we can continue to add CAT8 binding site-3 in M5 to increase the expression strength of the promoter.

4. Multi copy of UAS

4.1 Design

Continue to add CAT8 binding site-3 from P_SED1L on the basis of M5, add in two locations, the first is added upstream of the original ADR1 binding site, and the second is added to the CAT8 binding site-3 between ADR1 binding site. (Figure 5)

Figure 5. The additional add of CAT8p-3 binding site

4.2 Build

Taking the construction of the M8 mutant strain as an example, the construction method of the M9 strain was the same. M10 is constructed on the basis of M8 or M9.

Design the CAT8 binding site motif on the homology arm, (1) Using primer M8-F/PDC1-R to amplify fragment one, primer M8-R/PDC1-F to amplify fragment two (Table 7), (2) assemble fragment 1 and fragment 2 to obtain M8 promoter, (3) Using primer PDC1-F/PDC1-R amplifies M8 promoter. Using BamHI and XbaI restriction enzymes to digest the M8 promoter and YEp181-PDC1p-VS-SAG1t vector, and react at 37°C for 2h. After the reaction, the promoter and vector were purified and ligated at 16°C overnight. Transform into E. coli DH5α, pick the correct transformants, and extract the YEp181-M8-VS-SAG1t plasmid.

Then, use donor DNA primers (Table 4) to amplify the M8-VS-SAG1t expression cassette with a homology arm next to the LEU2 site. Transform the constructed gRNA expression plasmid and M8-VS-SAG1t expression cassette into yeast cells which can express Cas9 protein, and use the method of auxotrophic medium selection and colony PCR verification to obtain the strains that successfully knocked into the M8-VS-SAG1t expression cassette.

4.3 Test

The strain was inoculated into 10 ml of fermentation broth, the initial OD600 of the fermentation broth was controlled to be 0.05, and it was placed in a shaker at 30℃, 220 rpm for 64 hours. After 64 hours of shake flask fermentation, the concentration of valencene was detected by gas chromatography. (Figure 6)

Figure 6. Figure 6 Fermentation results of M8-M10 mutant strains. (a) OD₆₀₀ of M8-M10 mutant strain, (b) Valencene yield of M8-M10 mutant strain.

The Valencene yield of M9 and M10 mutant strains was not significantly improved compared to control strain。However, the Valencene yield of the M8 mutant strain was increased by 27.0% (6.75 mg/L) compared to M5 strain, and 56.6% (6.75 mg/L) compared with the unmodified strain.

4.4 Learn

In M8, CAT8 binding sites-3 was added between the original CAT8 binding sites-3 and ADR1 binding site, which may increase the interaction between CAT8 transcription factor and ADR1 transcription factor. In M9, CAT8 binding site-3 was added to the upstream of the original CAT8 binding site-3, The transcription factor cannot effectively bind to this site, or the add changes the surrounding sequence and may have some negative effects, such as destroying the original binding site of transcription factor with activation. As a result, the Valencene yield of the M9 mutant strain could not be further increased. In M10, the two CAT8 binding sites-3 was added around the ADR1 binding site, which may prevent the binding of ADR1 transcription factors and affect the interaction between CAT8 transcription factors and ADR1 transcription factors.

5. Summary

The above is the content of the promoter engineering in the wet lab. Through three rounds of "design-build-test-learn", we not only obtained M1-M10 promoters with different strengths, but also summarized the engineering experience from each learning. Our work is summarized as follows. The work presented here, and the promoters designed and engineered through it, represent an attempt to identify yeast regulatory elements that respond to a condition of interest by designing regulatory parts responsive to such a signal––diauxic shift in this case.

6. References

1. Haurie V, Perrot M, Mini T, Jenö P, Sagliocco F, Boucherie H. The transcriptional activator Cat8p provides a major contribution to the reprogramming of carbon metabolism during the diauxic shift in Saccharomyces cerevisiae[J]. The Journal of Biological Chemistry, 2001, 276(1): 76-85.

2. Roth S, Kumme J, Schüller H-J. Transcriptional activators Cat8 and Sip4 discriminate between sequence variants of the carbon source-responsive promoter element in the yeast Saccharomyces cerevisiae[J]. Current Genetics, 2004, 45(3): 121-128.

3. Young ET, Dombek KM, Tachibana C, Ideker T. Multiple pathways are co-regulated by the protein kinase Snf1 and the transcription factors Adr1 and Cat8[J]. The Journal of Biological Chemistry, 2003, 278(28): 26146-26158.

4. Thukral SK, Eisen A, Young ET. Two monomers of yeast transcription factor ADR1 bind a palindromic sequence symmetrically to activate ADH2 expression[J]. Molecular and Cellular Biology, 1991, 11(3): 1566-1577.

5. Walther K, Schüller HJ. Adr1 and Cat8 synergistically activate the glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast Saccharomyces cerevisiae[J]. Microbiology (Reading, England), 2001, 147(Pt 8): 2037-2044.

6. Segal E, Widom J. Poly(dA:dT) tracts: major determinants of nucleosome organization[J]. Current Opinion in Structural Biology, 2009, 19(1): 65-71.

7. Anderson JD, Widom J. Poly(dA-dT) Promoter Elements Increase the Equilibrium Accessibility of Nucleosomal DNA Target Sites[J]. Molecular and Cellular Biology, 2001, 21(11): 3830.

8. Raveh-Sadka T, Levo M, Shabi U, Shany B, Keren L, Lotan-Pompan M, et al. Manipulating nucleosome disfavoring sequences allows fine-tune regulation of gene expression in yeast[J]. Nature Genetics, 2012, 44(7): 743-750.