Team:NJTech China/Results

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Results

1. Construction of engineered E. coli DH5α and Saccharomyces cerevisiae BY4741

The designed plasmids (pRS406-Petunia, pRS406-Vanda, pRS406-Rosa, pRS426-Petunia, pRS426-Vanda, pRS426-Rosa) were synthesized by GenScript.

Fig. 1 Plasmid maps of the recombinant plasmids

A: pRS406-Petunia; B: pRS406-Vanda; C: pRS406-Rosa

D: pRS426-Petunia; E: pRS426-Vanda; F: pRS426-Rosa

The transformation of the recombinant plasmids into the E. coli DH5α.

The plasmids were extracted using plasmids extraction kit and verified by plasmids PCR. The target fragment was about 2300bp.

Fig. 2 Nucleic acid electrophoresis verification of the plasmids PCR

M:Trans2K Plus DNA Marker

1:pRS426-Petunia

2: pRS426-Vanda

3: pRS426-Rosa

4:pRS406-Petunia

5: pRS406-Vanda

6: pRS406-Rosa

Fig. 3 Results of plasmid sequencing

A: pRS406-Petunia; B: pRS426-Petunia; C: pRS406-Vanda

D: pRS426-Vanda; E: pRS406-Rosa; F: pRS426-Rosa

As can be seen from the figure 3, we have successfully constructed the recombinant strains of E. coli.

The transformation of the recombinant plasmids into the S. cerevisiae BY4741. We performed the electrotransformation, followed by the validation of colony PCR. The target fragment was about 2300bp.

Fig. 4 Nucleic acid electrophoresis verification of the colony PCR

M:Trans2K Plus DNA Marker

1-8: pRS406-P;

9-16: pRS406-V;

17-24: pRS406-R;

25-32: pRS426-P;

33-40: pRS426-V;

41-48: pRS426-R.

Figure 4 showed that the recombinant plasmids may be successfully introduced into yeasts, and the genome of the recombinant strains needed to be extracted for further verification.

Fig. 5 Nucleic acid electrophoresis verification of the genome PCR

M:Trans2K Plus DNA Marker

1: BY4741-pRS426-P2 genome;

2: BY4741-pRS426-P8 genome;

3: BY4741-pRS426-V1 genome;

4: BY4741-pRS426-V3 genome;

5: BY4741-pRS426-R1 genome;

6: BY4741-pRS426-R4 genome.

The yeast genome was extracted by the yeast genome extraction kit and verified by genome PCR. The target fragment was about 2300bp. As can be seen in figure above, the recombinant plasmids of pRS426 were successfully introduced into the S. cerevisiae BY4741. Due to time constraints, we were unable to complete the construction of the recombinant yeast strains with recombinant plasmids of pRS406. In the future, we will continue to complete the construction of recombinant strains. To ensure that the recombinant plasmids of pRS426 were introduced into yeasts, we sent the extracted genome to the company for sequencing.

Fig. 6 Results of genome sequencing

A: BY4741-pRS426-Petunia; B: BY4741-pRS426-Vanda; C: BY4741-pRS426-Rosa

The results of sequencing were right. The recombinant yeast strains with the recombinant plasmids of pRS426 have been successfully constructed.

2. Construction of the phenylacetaldehyde synthase (PAAS) metabolic pathway in yeast to produce 2-phenylethanol (2-PE)

Fig. 7 The growth curve of recombinant strains

The figure above shows the changes of biomass concentration in WT, BY4741-pRS426-Petunia, BY4741-pRS426-Vanda, BY4741-pRS426-Rosa yeast over time. The OD₆₀₀ of different strain cultures is measured at the designated time points (0h, 24h,48h,72h). The result shows that the growth trend of the recombinant strain is basically the same as the wild-type strain, indicating that the introduction of heterogeneous gene has no significant effect on the growth of yeast.

Fig. 8 The 2-PE production of the recombinant strains

After 72 hours of fermentation, 2-PE production of the wild-type of BY4741, pRS426-Petunia, pRS426-Vanda and pRS426-Rosa was tested by HPLC. Data represent means of triplicate culture ± standard error.

Saccharomyces cerevisiae BY4741 contains Ehrlich pathway and other metabolic pathways to operate simultaneously to produce 2-PE, so the wild-type of BY4741 has a certain amount of 2-PE production (1.205g/L). After the introduction of heterogeneous paas gene, the 2-PE production has remarkablely increased. Among them, the production of 2-PE produced by the yeast strain which was introduced petunia-paas increased the most(1.570g/L), followed by the strain which was introduced vanda-paas (1.514g/L) and rosa-paas (1.341g/L).

3. Establishment of the yeast-microalgae interaction system

To further increase production of 2-PE, the improvements we made is to design a yeast-microalgae system. The mixed cultures performed better due to the higher carbon dioxide available for microalgae use in photosynthesis and higher oxygen availability for heterotrophy of yeast, leading to reduced microalgae production costs while maintaining alga production reliability.

During yeast cultivation, organic acids were synthesized and the pH of the culture dropped slightly. In addition, the media acidified with organic acids (e.g. acetic, lactic acids) are more inhibitor to yeast growth compared with those acidified with mineral acids. When CO₂ dissolves in water at neutral pH, bicarbonate (HCO₃^-) is formed. During microalgal photosynthesis activity HCO₃^- is converted to CO₂ and hydroxide ion (OH^-). Hence, when CO₂ is consumed by microalgae, the OH^- is formed, and the pH becomes more alkaline. So microalgae can improve the stability of the mixed culture and promote the production of 2-PE.

Fig. 9 The 2-PE production in the yeast-microalgae microbial consortia system

However, the results do not meet our expectations when our recombinant yeast was directly co-cultured with microalgae. The decline in the production of 2-PE may be due to the insufficient nutrients resulted by substrate competition and different nutritional conditions of yeasts and microalgae, posing great challenges to the stability of the artificial microbial consortia system.

To solve the problems above, we have tried immobilized cell techique to realize the spatial compartmentalization of microbial coculture, using polyvinyl alcohol-sodium alginate (PVA-SA) as supporter material. The composite supporter material has the benefits of superior biocompatibility, better processibility, stronger mechanic stiffness, and chemical inertia. In our project, immobilized cell technique is combined with 3D printing technology. Thus, mass exchange at the medium (PVA-SA) interface can be improved by optimizing 3D geometries, resulting in high catalytic efficiency. Finally, the production of 2-PE is increased from 1.585 g/L to 1.755 g/L (Fig. 9).

Due to limited time, we are unable to make further improvements to our artificial microbial consortia system, including adding time of microalgae and materials used in 3D printed. We believe that the yeast-microalgae interaction system combined with immobilized cell technique and 3D printing technology has a promising future.

4. Transformation of yeast

The plasmid pRS426 was extracted using AXYGEN AxyPrep Plasmid Miniprep Kit, and the extracted plasmid was digested by BamH Ι and EcoR Ι single digestion. The enzyme digestion result was verified by agarose gel electrophoresis. Using BY4741 genome as a template, pTEF -Primer-F, pTEF-Primer-R as primers to amplify the TEF promoter; using BY4741 genome as a template, UAS_TEF-Primer-F, USA_TEF-Primer-R/ UA_SCLB-Primer-F, USA_CLB-Primer-R/ UAS_CIT-Primer-F, USA_CIT-Primer-R as primers to amplify the USA_TEF/ USA_CLB/ UAS_CIT fragments. Using pRS415 genome as a template, GFP-Primer-F and CYC1-Primer-R are primers to amplify GFP-CYC1 fragments. After one-step cloning, the yeast is transformed by the recombinant plasmid. The yeast genome was extracted using the Zoman Yeast Genomic DNA Extraction Kit. Using the proposed genome as a template and M13-F and M13-R as primers, genomic PCR was performed to verify whether our target gene was successfully transformed the strains. As shown in figure 1, it has been verified that the recombinant yeast containing the plasmid pRS426-pTEF-GFP-CYC1, pRS426-UAS_TEF-pTEF-GFP-CYC1, pRS426-UAS_CLB-pTEF-GFP-CYC1, pRS426-UAS_CIT-pTEF-GFP-CYC1 have been obtained.

Fig. 10 Electrophoresis verification of PCR products of recombinant yeast

5. Characterization of the natural and hybrid promoters with flow cytometry

The yeast culture with recombinant plasmid was normalized to OD₆₀₀=0.01 after 48h culture and shaking for 15h shaking. The results measured by flow cytometry are shown in figure 11. It's shown that the function of promoter can be improved by adding tandem UAS elements to the upsteam of pTEF. The UAS_CIT -pTEFpromoter strength was expanded beyond the pTEF by 1.44-fold in terms of mean fluorescence intensity. UAS_CLB-pTEF and UAS_TEF-pTEF promoter resulted 1.2-fold improvment.

Fig. 11 The fluorescence intensity of GFP expressed by different promoters