Level 1

Promoter

Design of Promoter Screening and Characterization of Their Strengths

First of all, we downloaded the sequences of several promoters in the yeast genome from the EPD (Eucaryotic promoter database). After excluding the promoters with the cutting sites of the restriction enzyme BsmBI and BsaI, we identified 29 promoters. In order to clone each promoter into the target vector, we used a pair of primers with specific prefix sequences to individually amplify the promoters. After obtaining the promoter fragments, the sequences of the ccdB in the vector of TU11-ccdB-mcherry-T4 were replaced by the sequences of target promoters, and thus the TU11-promoter-mcherry-T4 plasmids were constructed through the Goldengate method, followed by transformation into S. cerevisiae (Figure 1). The strength of each promotor was characterized by their ability in enhancing the fluorescence intensity of mCherry (Figure 2).

Figure 1. Yeast characterization plates. Images of Petri dishes with yeast colonies: the difference of mCherry fluorescence intensity can be clearly visualized when exposed to blue light.

Figure 2. Strengths of promoters. The data of mCherry fluorescence intensity were obtained from the reporters using the different promoters.

Construction of Standardized Promoters

After finishing strength characterization of 29 promoters, we selected 10 of them with relatively high strengths, and integrated them into the pccdK2-P vector by the Goldengate method to construct the pccdk2-promoter. For each primer, the nucleotides (nts) in green text indicate the BsaI recognition sequences, while the nts in red text represent the sticky ends after the digestion of BsaI. The bases with fluorescent yellow are extra sequences to increase the efficiency of digestion, and “nnnn…nnn” represents specific sequences to amply your target. For different targets, only the "nnnn...nnn" region needs to be changed. We used the following primers to amplify the promoters.

5':agcgtg GGTCTCgGGCTnnnnnnnnnnnnnnnnnnnn

3':gtgctg GGTCTCaCATCnnnnnnnnnnnnnnnnnnnnn

Figure 3. Agarose gel electrophoresis images for the PCR products of the standardized parts. From The constructions are for the pccdK2-P1 (left) and pccdK2-P5 (right). The predicted size of the PCR band is 536 bps. The average positive rate was over 90%. NC: negative control without any template.

Optimization of the PGK1 Promoter Truncation

We noticed that the PGK1 promoter in the part library of the iGEM official website had a length of 1,500 bps, which could be relatively too long when used as a transcription unit. Therefore, we conducted experiments of PGK1 promoter truncation. We amplified the PGK1 promoter sequence from the yeast genome with different lengths of 200, 400, 600 and 800 bps, and determined their relative strengths (Figure 4).

Figure 4. Characterization of the strengths of the truncated PGK promoters.

ORF

Construction of Standardized ORF

In order to prove the efficiency of the YeastFab assembly strategy and integrate the lycopene production system into the genome of S. cerevisiae, we first identified the key enzyme genes crtB, crtI and crtE in the lycopene synthesis pathway, and then added the genes of terpenoid synthesis precursors in the MVA pathway to increase the production. We also used the Goldengate approach with the restriction enzyme BsaI to insert the target gene into pccdK2-O. The primers used to amplify the target gene is shown as follows. We have built a total of 10 standard plasmids of ORFs. The glue diagrams of colony PCR are correct (Figure 5）.

5':agcgtg GGTCTCaGATGnnnnnnnnnnnnnnnnnnnn

3':gtgctg GGTCTCgGCTAnnnnnnnnnnnnnnnnnnnnn

Figure 5. Colony PCR to screen for the positive clones in the construction of standard parts of the lycopene synthesis pathway. The predicted PCR band sizes are: (a) pccdK2-tHMG: 1,715 bps; (b) pccdK2-crtE: 1,310 bps; (c) pccdK2-ERG10: 1,328 bps; (d) pccdK2-ERG19: 1,319 bps. NC: negative control without any template.

Terminator

Construction of Standard Parts of Terminators

In this part, we used the same method as that in the promoter construction. We selected 10 terminators, and used the following primers to amplify the terminators.

5':agcgtg GGTCTCtTAGCnnnnnnnnnnnnnnnnnnnn

3':gtgctg GGTCTCgGAGGnnnnnnnnnnnnnnnnnnnn

Figure 6. The result of colony PCR in constructing the standard plasmids of terminators. 1-5 are for the TADH1 colony PCR with a predicted band of 356 bps; 6-10 are for the TENO1 colony PCR with a predicted band of 356 bps. NC: negative control without any template.

Figure 7. Positive rates of the Level 1 constructions. Different colors refer to the percentages of colony PCR positive rates achieved during the construction of the Level 1 standard components. For example, red refers to the percentage of the number of constructions with 100% bacterial P positive rate in the total number of constructions.

Level 2

Constructions of Transcription Unit

It was the time to assemble the transcription units when all the desired parts were successfully constructed. In this level, the promoter, ORF and terminator of interests were released from their vectors, and assembled together into an accepting vector in a one-pot reaction^[1]. In the first round of transcription unit construction, we selected 5 promoters of different strengths (pccdk2-P1, P5, P7, P8 and P20), and the same terminator pccdk2-T4 to combine with pccdk2-crtE, crtI and crtB for assembly using BsmBI digestion. The colony PCR agarose gel electrophoresis for the screening of the transcription unit is shown below.

Figure 8. Representative agarose gel electrophoresis images of the colony PCR products in the construction of TU vectors. The predicted sizes of the PCR products are: (a) TU2-P1-crtE-T4: 2,104 bps; (b)TU5-P1-crtB-T4: 1,813 bps; (c) TU2-P7-crtE-T4: 2,105 bps; (d)TU2-P5-crtE-T4: 2,105 bps. NC: negative control without any template.

Figure 9. The Positive rates of the Level 2 constructions. It can be seen that only a small number of constructions in the Level 2 had positive rates below 80%, and the overall constructions at this level were very efficient.

Figure 10. . Image of agarose gel electrophoresis of plasmid digestion to verify inserted DNA fragments. The blue lines denote the bands with predicted sizes of 4,964 bps and 2,010 bps(1-2 for TU2-P1-crtE-T4, 3-4 for TU2-P5-crtE-T4, 5-6 for TU2-P7-crtE-T4, 7-8 for TU2-P8-crtE-T4, 9-10 for TU2-P20-crtE-T4). The orange lines denote the bands with predicted sizes of 4,964 bps and 2,577 bps (11 for TU4-P1-crtI-T4, 12 for TU4-P5-crtI-T4, 13 for TU4-P7-crtI-T4, and 14 for TU4-P8-crtI-T4). The last pink lines denote the bands with predicted sizes of 4,964 bps and 1,719 bps. (15-16 for TU5-P1-crtB-T4, 17-18 for TU5-P5-crtB-T4, 19-20 for TU5-P7-crtB-T4, 21-22 for TU5-P8-crtB-T4, and 23 for TU5-P20-crtB-T4). NC: negative control without any template. 

Level 3

Integration of the Lycopene Synthesis Pathway

To integrate the lycopene synthesis pathway into the yeast genome, we chose to transform gene clusters carrying selection markers and homology arms into yeast. As the yeast used by us was a kind of four-deficient strain lacking the ability to synthesize three different amino acids and uracil, we cultivated the yeast in a nutrient-deficient medium, and used the synthetase genes corresponding to these four substances as selection markers to screen for yeast colonies with successful integration of the lycopene gene cluster (Figure 11).

Figure 11. Schematic representation of homology arm integration of the lycopene gene cluster into the genome of S. cerevisiae.

Figure 12. Images of Petri dishes of the yeast with the integration of the lycopene synthesis pathway using the homology arms. (a) The experiments using the OGG homology arm; (b) The experiments using the APL4 homology arm; (c) The experiments using the TY homology arm with multi-integration sites. The yeast colonies of both OGG and APL4 were white, only the TY homologous arm integrated appeared as red colonies.

Based on Figure 12, all yeast colonies generated by the homologous arms with a single integration site were white, while the experiments using the TY homologous arms with multi-integration sites showed red colonies. It was obvious that our integration did not succeed according to the experimental results. Around this time, we participated in the CCIC at that time. The judges of the conference suggested us to use distinct terminators in different transcription units, because the use of the same terminator in different units may cause gene loss due to the strong recombination ability of S. cerevisiae. We accepted this valuable suggestion, and planned to use different terminators. We hoped that the new strategy could solve the problems of yeast redness and transmission degradation.

Figure 13. Schematic representation of gene loss when an identical terminator is used in two transcription units after integrated into the yeast genome. Homologous recombination may occur when both transcription units use T4 as their terminators, leading to the loss of the crtI gene.

After identifying the reason for the unsuccessful integration of the lycopene synthesis pathway into the yeast genome, we immediately reconstructed a series of transcription units with different terminators, and integrated them into the yeast genome through a single integration homologous arms. Ultimately, we obtained a petri dish with a large number of red colonies. We picked the red colonies and expanded them in YPD. We extracted the genomic DNA to verify the successful integration of the lycopene synthesis pathway. Meanwhile, we also determined the yield of lycopene by HPLC analysis.

Figure 14. Schematic diagram of the integration of the lycopene synthesis pathway in yeast. The genes marked with blue borders below are some of the genes that we designed primers to verify by PCR amplification.

Figure 15. URR-crtEIB. Image of a Petri dish containing red colonies after different terminators were used to integrate the lycopene synthesis pathway.

Figure 16. Agarose gel analysis of PCR amplification on extracted yeast genomic DNA to verify the integration of the lycopene synthesis pathway. Agarose gel electrophoresis of colony PCR extracted genome, 1-4 corresponding to the gene fragments marked in blue borders in Figure 14 respectively.

Figure 17. Standard curve of lycopene. Lycopene has a maximum absorption peak at the wavelength of 474 nm. The absorption peak area of different concentrations of lycopene standards at 475 nm was measured, and the standard curve of the proportional relationship between the peak area and the concentration was obtained.

Figure 18. Elution process of HPLC to detect the production of lycopene. The figure above shows the lycopene yield of the strain of URR-crtEIB.

Table 1. Lycopene production by the corresponding strains.

Response Surface Method

In the process of testing the integrated lycopene synthesis pathway, we found that the output did not meet up with our expectations. Therefore, we attempted to improve the yield from the perspective of fermentation. We modeled the fermentation process to find optimized conditions. We reviewed the literature and discovered that optimization through Response Surface Methods (RSM) was a common method.

Table 2. Experimental protocols for the three factors in the fermentation.

Y=5.5+0.2*A+0.15*B+0.25*C+0.01*A*B-0.14*A*C-0.01*B*C-0.05*A^2-0.0008*B^2+0.028*C^2-0.0008*A^2*B+0.0223*A^2*C-0.000042*A*B^2

Figure 19. (a) 3D plot of the effects of glucose concentration and dissolved oxygen on lycopene yield; (b) 3D plot of the effects of dissolved oxygen and inoculum on lycopene yield; (c) 3D plot of the effects of glucose concentration and inoculum on lycopene yield.

Superimposed Yeast Genome

The yield of lycopene by the engineered yeast was about 1.5 mg/g DCW as quantified by HPLC, which did not meet our satisfaction. Therefore, we planned to optimize the upstream of the MVA pathway. In addition to integrating the crtE, crtI and crtB into the yeast genome, we carried out the second round of integration of the genes of tHMG and IDI. For this purpose, we used the crtB as the upstream homologous arm, and a previous homologous arm as the downstream homologous arm. It was interesting to employ such a reorganization method, which allowed us to use limited filter tags to superimpose and integrate infinitely by different DNA fragments.

Figure 20. Schematic representation of the yeast genome superposition principle.

Figure 21. Images of cultured yeast cells derived from the red colonies of the yeast with the superposed genome.

Figure 22. Chromatographic elution of HPLC to determine the lycopene yield in the yeast cells derived from red colonies on transformed petri dishes of the yeast with superimposed genome. The figure above shows the lycopene yield of the strain of URR-crtEIB-tHMG-IDI.

Table 3. Production of lycopene by the URR-crtEIB-tHMG-IDI.

Figure 23. A dedication map of Northeast Forestry University (NEFU) to the 70^th anniversary using the lycopene product by engineered yeast strains. We would like to pay tribute to the forerunners who made outstanding contributions to the establishment and development of Northeast Forestry University, and also send a congratulation gift to our team of the NEFU_China for the ^th anniversary of NEFU.

Explore the Integration Efficiency of Plasmid and Homologous Arms：

In order to explore the advantages and disadvantages of homologous arm integration and plasmid integration, we used the URR^[2] homologous arms that are located in open chromatin area, or a series of TU plasmids to integrate mCherry into WAT21. As a result, we obtained correctly integrated strains by screening nutritionally deficiency markers and examining the fluorescence intensity of mCherry.

Figure 24. (a) Growth of URR1-P1-mCherry-T1-Ura3-URR2 integrated yeast after 96 h of cultivation. (b) Growth of TU-P1-mCherry-T1 integrated yeast after 96 h of cultivation. (c) Yellow：Characterization of URR1-P1-mCherry-T1-Ura3-URR2 mCherry. Green：Characterization of TU-P1-mCherry-T1 mCherry.