Introduction
Welcome to our engineering page, where we present the engineering success of our case. The core theory of our case is derived from the YeastFab, a yeast DNA assembly strategy developed by Tsinghua University. Overall, based on the characteristics of our project, the engineering part is presented through multiple cycles. In each cycle, we will present our experimental design, construction process and validation assays according to the four basic processes of synthetic biology: Design-Build-Test-Learn.
In addition, the ultimate goal of our case is to significantly expand the technical concept of the YeastFab, achieve cross-species applications of the strategy, and significantly extend it to industrial production. To this end, we have made many attempts and efforts. We hope that any visitor on this page will be able to understand our engineering design, and every fan of synthetic biology will be inspired.
Cycle 1
Design
Up to now, we have found it very time-consuming and costly to construct a large number of DNA synthesis pathways simultaneously in real production. Although automated technology has given us a glimpse of the solution, machines are ultimately not humans and suffer from the lack of rational design thinking and the ability to handle emergencies. For this reason, we took note of the YeastFab technology developed by Tsinghua University, which can efficiently achieve large-scale DNA assembly. To accomplish efficient construction, we did the following design:
1. Based on the YeastFab technology, we replaced the mCherry red fluorescence gene by the ccdB toxin that can inhibit DNA unwinding activity as a new selection marker, which intuitively improved the efficiency of our screening and increased the rate to get positive clones.
2. We selected the integration site on the yeast genome, and homologous arms were designed at both ends of this site to guide targeted integration in the yeast genome.
3. To achieve high efficiency in DNA assembly using the YeastFab technology, we standardized our components by editing customized cleavage sites for BsaI and BsmBI to obtain customized sticky ends.
In our project, we demonstrated the process of our DNA assembly through the synthesis of lycopene.
Build
Based on the YeastFab technology principle, we designed the pccdK2 series of vectors (pccdK2-P, pccdK2-O and pccdK2-T) carrying the basic standard parts and the TU series of vectors (TU1-11) with integrated transcription units.
We used different promoters to drive the expression of the three genes TmcrtE, BtcrtI, PacrtB in the lycopene synthesis pathway. A combination of a moderately strong promoter to induce crtE, a strong promoter to drive crtI, and a weak promoter to induce crtB achieved a relatively decent lycopene yield [1]. Therefore, we constructed the three promoters of different strengths, crtE, crtI and crtB, and the tCPS1 terminator in the pccdK2 family of vectors by the Golden Gate assembly method according to this collocation, i.e., pccdK2-pTEF1, pccdK2-pACT1, pccdK2-pFBA1; pccdK2-crtE, pccdK2-crtB, pccdK2-crtI; pccdK2-tCPS1. We designated these constructions as the Level 1.
Next, we built the Level 2 constructs based on the foundation of the Level 1. We used the TU family of vectors to assemble the components of the Level 1 plasmids by the Golden Gate assembly method to form the transcription units TU2-pTEF1-crtE-tCPS1, TU4-pACT1-crtB-tCPS1 and TU5-pFBA1-crtB-tCPS1.
We then assembled the transcriptional unit fragments from recombinant plasmids in the Level 2 (i.e., the cascade of genes in TU-P-crt-T described above) to form a metabolic pathway carrying the deficiency screening marker Ura, and added homologous arms at both ends of the pathway, which would guide this pathway to the desired locus in the yeast genome for homologous recombination. Experimentally, we transformed this metabolic pathway plasmid carrying the screening marker and homologous arms into yeast to allow it integrated into the genome.
Figure 1. The process of standardized DNA assembly strategy
Test
We examined our integration results by three means.
1. We directly observed the integrated SD-Ura (Synthetic Dropout Media), and found a large amount of white colonies on the plates without any red one. The presence of colonies that grew normally in the deficient medium indicated that the screening marker gene was successfully integrated; however, lack of red colonies suggested that the synthetic pathway was either not integrated or not properly expressed.
2. We performed lycopene extraction from the colonies, and used HPLC to determine the presence of lycopene, which showed absence of detection.
3. We extracted the recombinant yeast genome and performed PCR to identify the integrated genes, and failed in identifying any inserted gene in the extracted yeast genomic DNA.
Learn
As we analyzed the results of this round of engineering, we made a lot of conjectures: we doubted the accuracy or efficiency of the genomic PCR, metabolic overload that could possibly lead to mutations or loss of genes, impurity of the chassis cells, etc., but we could neither find any essence of the problem nor make any meaningful progress. It was not until we attended the CCiC conference (Conference of China iGEMer Community) in August 2022 that we received valuable advice from the jury: due to the active homologous recombination in Saccharomyces cerevisiae, it is highly possible that multiple genes will be lost if the same combination of terminators is repetitively used in an integrated metabolic pathway. Therefore, we decided to increase the specificity of the terminators in homologous recombination in order to avoid gene loss mediated by potential recombination caused by identical terminator sequences. Inspired by the Cycle 1, we changed the engineering protocol and started a second cycle.
Cycle 2
Design
In the second round of construction, we designed to make crtE, crtI and crtB link to three different terminators tPRM9, tCPS1 and tSPG5, respectively. In this way, we could prevent the loss of any integrated region in the yeast genome caused by homologous recombination in the subsequent synthesis pathway. Following the same construction strategy as the Cycle1, the recombinant plasmid pccdK2-tPRM9/tCPS1/tSPG5 was created by combining pccdK2-T as a vector with different terminators as complementary parts of the Level 1.
Build
After completing the construction of the complementary parts as in Cycle 1 at the Level 1, we used this foundation to construct the following Level 2 in the Cycle 1, and generated TU2-pTEF1-crtE-tPRM9, TU4-pACT1-crtB- tCPS1 and TU5-pFBA1-crtB-tSPG5. Then, the target fragments in the Level 2 recombinant plasmids were assembled again to complete the Level 3 construction. The strategy was the same as that presented in Cycle 1.
Figure 2. Construction of transcription units after terminator replacements in level 2
Test
We used the same methods as above to test the parts of this cycle:
1. Direct observation. At this time, we found a large amount of red colonies and a few white colonies by direct observation of the SD-Ura, which supported the prediction that homologous recombination was responsible for the unsuccessful integration observed in the Cycle 1.
2. We extracted the lycopene from the red colonies and measured the yield using HPLC, which indeed displayed the presence of this product.
3. We extracted the genome of the yeast cultivated from the red colonies. Using it as a template in PCR amplification, we detected predicted bands for the gene fragments that we attempted to integrate.
These results demonstrated that our second round of integration was successful and the integrated pathway could be successfully expressed in yeast.
Learn
Although our integration was successful, we were not satisfied with the yield of lycopene, which did not meet our needs. After reflection and discussion, we realized that the cycle could be further optimized in two ways.
1. We could optimize the MVA synthesis pathway in the lycopene synthesis pathway.
2. We could explore the optimal incubation conditions of yeast.
These let us improve the protocol and start the third cycle (Cycle 3).
Cycle 3
Design
To further improve lycopene production, we interrogated the possibility of increasing its two precursors, IPP and DMAPP. For this purpose, we introduced the genes of tHMG and idi, two key enzymes in the MVA (mevalonic acid) pathway, into the original synthesis pathway. They could increase the consumption of Acetyal-CoA in the MVA pathway, and subsequently improve the productions of IPP and DMAPP, leading to enhanced lycopene production [2]. This strategy could enhance the synthesis of lycopene’s precursors, and theoretically increase the yield of lycopene.
Build
In the Cycle 3, we constructed pccdK2-tHMG and pccdK2-idi at the Level 1, TU2-pACT1-crtB-tCPS1, TU4-PA-tHMG-tTDH1 and TU5-PB-IDI-tADH1 at the Level 2, carrying a Leu screening marker using the same method as the two cycles above. We continued to ligate parts through the Golden Gate, using the transcription unit where crtB is located as the upstream homologous arm and the homologous arm of the original pathway as the downstream homologous arm for integration, allowing for repetitive recombinant integration at the same genomic locus. In this process, a selection marker was replaced, allowing us to make theoretically "countless" additions, knockouts or substitutions of transcription units with a limited number of selection markers.
Figure 3. Schematic representation of the yeast genome superposition principle.
Test
We extracted the genomic DNA of the yeast cultivated from the red colony for PCR. However, the PCR amplification always showed poor yields, which would need to be optimized in the following studies. On the other hand, the integrated fragments of crtE and idi genes from the first and second round of integrations were much easier to be amplified by PCR, indicating the success of our two rounds of integrations. We also extracted lycopene from the red colonies and measured the yield using HPLC. The yield of lycopene increased substantially from 0.56 mg/g DCW (dry cell weight) to 1.52 mg/g DCW, with an approximate 2.7-fold increase. These results demonstrated that the lycopene synthesis pathway was successfully optimized and the yield was improved through the two-round integrations.
Learn
After the two rounds of integrations described above, we found that the yield of lycopene was increased substantially. However, based on the experience of the second round of integration, we can continue the integration operation in the future and add other key enzyme genes in the synthesis pathway in addition to tHMG and IDI. This may help us to obtain optimal pairings to achieve the highest yield, with moderately increased number of integrated genes. It is also possible to optimize the lipid metabolism process by introducing the sterol ester synthesis genes ARE1 and ARE2, thus promoting lycopene production [3].
References
- Bahieldin, A., Gadalla, N. O., Al-Garni, S. M., Almehdar, H., Noor, S., Hassan, S. M., Shokry, A. M., Sabir, J. S., & Murata, N. (2014). Efficient production of lycopene in Saccharomyces cerevisiae by expression of synthetic crt genes from a plasmid harboring the ADH2 promoter. Plasmid, 72, 18–28. https://doi.org/10.1016/j.plasmid.2014.03.001
- Cheng, T., Wang, L., Sun, C., & Xie, C. (2022). Optimizing the downstream MVA pathway using a combination optimization strategy to increase lycopene yield in Escherichia coli. Microbial cell factories, 21(1), 121. https://doi.org/10.1186/s12934-022-01843-z
- Zhao, Y., Zhang, Y., Nielsen, J., & Liu, Z. (2021). Production of β-carotene in Saccharomyces cerevisiae through altering yeast lipid metabolism. Biotechnology and bioengineering, 118(5), 2043–2052. https://doi.org/10.1002/bit.27717
