Team:NJTech China/Engineering

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Engineering Success

In short, the purpose of our project this year is to establish a robust, stable, and controllable artificial microbial consortia system for the green and efficiency phenylethanol biosynthetic. We have designed and characterized plant-derived phenylacetaldehyde synthase (PAAS) according to the phenethylamine pathway of plants. Phenylethylamine pathway is artificially delivered to S. cerevisiae for phenylethanol biosynthesis. Well-characterized promoters are essential for metabolic engineering and synthetic biology efforts in the model eukaryotic yeast Saccharomyces cerevisiae. However, the expression level of allogenic genes is not satisfactory. To enable fine-tuned control and amplification of gene expression, we designed the hybrid promoters pTEF, trying to entails combining core promoters pTEF with upstream activation sequences (UAS elements). By reading literature, we learned that upstream activating sequences (UAS elements) can serve as synthetic transcriptional amplifiers and can modulate the expression of any promoter element. We constructed the UAS_CLB-pTEF-GFP strain and UAS_CIT-pTEF-GFP strain to increase the expression of phenylacetaldehyde synthase by high activity engineering promoter. As a result, the hybrid promoters can be used for improving the expression of PAAS. In addition, to reduce carbon dioxide, we proposed to establish a yeast-microalgae microbial consortia system based on nutritional complementation and metabolites exchange. We tried to use 3D printing technology to combine microalgae on bioactive materials and mixed cultured with yeast. The "living material", PVA-SA hydrogel, we used, offer significant advantages as a scaffold for living materials because it provides a cellular environment that is similar to natural biofilms.

Characterization and optimization of the PAAS in recombinant yeast strains

1.1 Inspiration and design

After reviewing the literature, we found that there is an Ehrlich pathway in S. cerevisiae, which can produce 2-phenylethanol (2-PE) from glucose. In addition, there is phenylethylamine pathway in petunia and vanda for de novo efficient synthesis of 2-PE, and it is simpler and more convenient than the Ehrlich pathway. More importantly, it can be uncoupled with cell growth for fractional optimization.

In our project, the key gene paas is heterologously expressed in yeasts to produce 2-PE from L-phenylalanine. Considering the compatibility between different plant genes and yeast genes, three different paas genes were obtained from rose, petunia and vanda, respectively and integrated into S. cerevisiae after codon optimization^[1]. This constructed pathway significantly simplified the route from phenylalanine to 2-PE, which will be more convenient in terms of scaling up and with fewer variables (gene expression, exterior co-factors, etc.). Moreover, this enzyme can be further subjected to protein engineering for improvement of its activity.

1.2 Construction of the recombinant yeast strains: pRS426-Petunia, pRS426-Vanda, pRS426-Rosa

We first constructed the recombinant plasmids carrying the exogenous gene paas from petunia, vanda and rose, respectively. The plasmid skeletons were the integrated plasmid pRS406 and the high copy plasmid pRS426. The recombinant plasmid was introduced into Escherichia coli DH5α by transformation. Figure 1 is the nucleic acid electrophoresis verification of the plasmids PCR, from which it can be seen that all the recombinant plasmids have been successfully introduced into E.coli DH5α.

Fig. 1 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

Then, we transformed the recombinant plasmids into Saccharomyces cerevisiae BY4741. Figure 2 shows the colony PCR verification diagram of recombinant yeast strains. After that, through gene sequencing, we confirmed that we successfully constructed three recombinant yeast strains: pRS426-Petunia, pRS426-Vanda and pRS426-Rose.

Fig. 2 Nucleic acid electrophoresis verification of the plasmids PCR

M: Trans2K Plus DNA Marker

1-8: pRS406-Petunia

9-16: pRS406-Vanda

17-24: pRS406-Rose

25-32: pRS426-Petunia

33-40: pRS426-Vanda

41-48: pRS426-Rose

1.3 Characterization of the PAAS by high performance liquid chromatography (HPLC)

Fig. 3 The growth curve of the wild type, pRS426-Petunia, pRS426-Vanda, pRS426-Rosa

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. 4 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 remarkably 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).

Research on UAS

2.1 Inspiration and design

Promoters are considered as basic regulatory elements responsible for transcription initiation. Well-characterized promoters are essential for metabolic engineering and synthetic biology efforts in the Saccharomyces cerevisiae. Promoter engineering provides sophisticated and diverse expression strategies for metabolic engineering. Upstream activating sequences (UAS elements) can serve as synthetic transcriptional amplifiers and can modulate the expression of any promoter element. Several upstream promoter sequences have previously been identified as transcriptional enhancing elements in S. cerevisiae, including a 240 bp sequence 5' of the mitotic cyclin clb2 gene , a 203 bp sequence 5' of the tef1 gene and a 275 bp sequence 5' of the mitochondrial citrate synthase cit1 gene referred to henceforth as UAS_CLB, UAS_TEF and UAS_CIT, respectively. So, overall this year our team makes efforts to combine core promoter with upstream activating sequences (UAS elements) to enhance transcription efficiency and tune the expression of the target gene^[2].

Fig. 5 Improvement of pTEF

2.2 Modification and Characterization of three hybrid promoters

The plasmid of pRS426 was extracted and single digested by BamHⅠ and EcoRⅠ. 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, USA_TEF-Primer-F, USA_TEF-Primer-R/ USA_CLB-Primer-F, USA_CLB-Primer-R/ UAS_CIT-Primer-F, UAS_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 6, it has been verified that the recombinant yeast containing the plasmid pRS426-pTEF-GFP-CYC1, pRS426-USA_TEF-pTEF-GFP-CYC1, pRS426-USA_CLB-pTEF-GFP-CYC1, pRS426-UAS_CIT-pTEF-GFP-CYC1 have been obtained.

Fig. 6 Electrophoresis verification of PCR products of recombinant yeast

A: M: DL5000 ( Vazyme ); 1-6: pRS425-pTEF-GFP-CYC1 ( 2140bp )

B: M: DL5000 ( Vazyme )

1-2: pRS426-USA_TEF-pTEF-GFP-CYC1 (3079bp)

3-4: pRS426-USA_CLB-pTEF-GFP-CYC1 (2981bp)

5-6: pRS426-UAS_CIT-pTEF-GFP-CYC1 (2863bp)

The yeast culture with recombinant plasmid was normalized to OD=0.01 after 48h culture and shaking for 15h shaking. The results measured by flow cytometry are shown in figure 7. It's shown that the function of promoter can be improved by adding tandem UAS elements to the upstream of pTEF. The UAS_CIT-pTEF promoter strength was expanded beyond the pTEF by 1.44-fold in terms of mean fluorescence intensity. USA_CLB-pTEF and USA_TEF-pTEF promoter resulted 1.2-fold improvement.

Fig. 7 The fluorescence intensity of GFP expressed by different promoters

The addition of a tandem UAS element upstream of the pTEF improves the promoter function. In contrast, the average fluorescence intensity of promoters of USA_CLB-pTEF, USA_TEF-pTEF and UAS_CIT-pTEF was higher than that of pTEF.

2.3 The application of Mathematical models in our project

Mathematical models are another important tool for characterizing promoters. In our project, neural network algorithms can predict the expression level of hybrid promoters.( https://2021.igem.org/Team:NJTech_China/Model )

Construction of the yeast-microalgae interaction system by 3D printing technology

3.1 Inspiration and design

In March 2021, China released the "Peak Carbon 2030 Study" and the "Carbon Neutral 2060 Study", explaining the significance of carbon peaking and carbon neutrality. Due to the higher carbon dioxide available for microalgae use in photosynthesis and higher oxygen availability for heterotrophy of yeast, we got the idea to design a yeast-microalgae system to reduce carbon dioxide while promoting yeast growth and 2-PE production as well. This yeast-microalgae interaction system is potential to address three important societal needs: (i) New mode production of 2-PE; (ii) The protection of aquatic environments; and (iii) The reduction of the global anthropogenic greenhouse effect^[6]. In the mixed culture, microalgae could generate oxygen for yeast while yeast provided CO₂ to microalgae and produce 2-PE. To achieve the stable co-cultivation, the growth conditions of the two organisms should be optimized to improve the stability of our system^[3].

We also used 3D printing technology to combine microalgae on bioactive materials and mixed cultured with yeast. The "living material", PVA-SA hydrogel, offer significant advantages as a scaffold for living materials because it provides a cellular environment that is similar to natural biofilms^[4]. Notably, the additive manufacturing of living whole cells can be used not only as a new bioreactor with high volumetric productivity and long lifetime, but also as a versatile platform for fundamental studies of microbial behaviors, communications, and interaction with the microenvironment^[5].

3.2 Mixed culture of the yeast-microalgae interaction system by 3D printing technology for production

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. 8 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 technique 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. 8).

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.

References

1. Kaminaga, Y.; Schnepp, J.; Peel, G.; Kish, C. M.; Ben-Nissan, G.; Weiss, D.; Orlova, I.; Lavie, O.; Rhodes, D.; Wood, K.; Porterfield, D. M.; Cooper, A. J.; Schloss, J. V.; Pichersky, E.; Vainstein, A.; Dudareva, N., Plant phenylacetaldehyde synthase is a bifunctional homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation. J Biol Chem 2006, 281 (33), 23357-66.

2. Keren, L.; Zackay, O.; Lotan-Pompan, M.; Barenholz, U.; Dekel, E.; Sasson, V.; Aidelberg, G.; Bren, A.; Zeevi, D.; Weinberger, A.; Alon, U.; Milo, R.; Segal, E., Promoters maintain their relative activity levels under different growth conditions. Mol Syst Biol 2013, 9, 701.

3. Cheirsilp, B.; Suwannarat, W.; Niyomdecha, R., Mixed culture of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris for lipid production from industrial wastes and its use as biodiesel feedstock. N Biotechnol 2011, 28 (4), 362-8.

4. Saha, A.; Johnston, T. G.; Shafranek, R. T.; Goodman, C. J.; Zalatan, J. G.; Storti, D. W.; Ganter, M. A.; Nelson, A., Additive Manufacturing of Catalytically Active Living Materials. ACS Appl Mater Interfaces 2018, 10 (16), 13373-13380.

5. Qian, F.; Zhu, C.; Knipe, J. M.; Ruelas, S.; Stolaroff, J. K.; DeOtte, J. R.; Duoss, E. B.; Spadaccini, C. M.; Henard, C. A.; Guarnieri, M. T.; Baker, S. E., Direct Writing of Tunable Living Inks for Bioprocess Intensification. Nano Lett 2019, 19 (9), 5829-5835.