Team:NJTech China/Design

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Design

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

The key enzyme phenylacetaldehyde synthase (PAAS) is heterologously expressed in the Saccharomyces cerevisiae to produce 2-PE, using L-phenylalanine as the substrate. We obtained the gene of the phenylacetaldehyde synthase from the Rose, Petunia and Vanda^[1], respectively. Because of the different codon preference of the species, these three genes were conducted codon optimization and introduced into the S. cerevisiae.

PAAS can convert L-phenylalanine to phenylacetaldehyde directly, which simplifies the original Erich pathway in the S. cerevisiae and reduces the metabolic burden of S. cerevisiae. The incorporation of PAAS instead of the three-enzyme Ehrlich pathway could lead to a simpler process that will be more convenient in terms of scaling up and with fewer variables (gene expression, exterior co-factors, etc.) that can influence the process in an unknown manner. Moreover, this enzyme can be further subjected to protein engineering for improvement of its activity^[2].

Fig. 1 The heterologous expression of PAAS from plants in Saccharomyces cerevisiae

2.Modification of natural TEF2 promoter——synthetic hybrid promoter approach improving transcription efficiency

Transcription control of specific genes is a direct and effective method to regulate metabolic flux. Promoters design with various strengths could realize the fine-tune of gene expression. So 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.

UAS is located upstream of the core promoter and serves as a binding site for specific transcription activators. The UAS is a crucial region of promoters which boosts gene expression^[3]. The tandem UAS elements of hybrid promoters could serve as synthetic transcriptional amplifiers to control expression levels^[4]. Several UAS elements have previously been identified as transcriptional enhancing elements in S. cerevisiae, including a 240 bp sequence 5’ of the mitotic cyclin coding gene clb2, a 275 bp sequence 5’ of the mitochondrial citrate synthase coding gene cit1 and a 203bp sequence 5’ of translational elongation factor coding gene tef1, termed UAS_CLB, UAS_CIT, UAS_TEF respectively^[5].

According to the literature, we found that even the strongest native promoters in S. cerevisiae are limited by a deficiency of UAS enhancer elements. Thus, we hold that the function of promoter can be improved by adding tandem UAS elements to the upstream of pTEF, thereby increasing the expression of PAAS. The modified promoters containing three copies from different origins are named UAS_CLB-pTEF, UAS_CIT-pTEF, UAS_TEF-pTEF, respectively.

Fig. 2 Improvement of pTEF

3. Establishment of the yeast-microalgae interaction system

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.

Fig. 3 Schematic diagram of carbon neutralisation

Fig. 4 Schematic of yeast-microalgae interaction system.

4. Utilization of 3D bioactive material printing technology to create a bioreactor

We used 3D printing technology to combine microalgae with bioactive materials and then performed a mixed culture of yeast Saccharomyces cerevisiae and microalgae. 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. Also, living material filaments were generated by electrospinning microbe-polymer solutions^[7]. This immobilized-cell bioreactor offers several advantages, such as reduced reactor down-time, ease of product removal, and minimized product inhibition and nutrient depletion. In addition, mass exchange at the medium (PVA-SA hydrogel) interface can be improved by optimizing 3D geometries, resulting in high catalytic efficiency. To improve the reactor performance, synergistic optimization of scaffold geometry, bioreactor design, and culture conditions are needed. 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^[8].

Fig. 5 Construction of the bioreactor using 3D printing.

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. Achmon, Y.; Ben-Barak Zelas, Z.; Fishman, A., Cloning Rosa hybrid phenylacetaldehyde synthase for the production of 2-phenylethanol in a whole cell Escherichia coli system. Appl Microbiol Biotechnol 2014, 98 (8), 3603-11.

3. Teixeira, M. C.; Monteiro, P. T.; Palma, M.; Costa, C.; Godinho, C. P.; Pais, P.; Cavalheiro, M.; Antunes, M.; Lemos, A.; Pedreira, T.; Sa-Correia, I., YEASTRACT: an upgraded database for the analysis of transcription regulatory networks in Saccharomyces cerevisiae. Nucleic Acids Res 2018, 46 (D1), D348-D353.

4. Da Silva, N. A.; Srikrishnan, S., Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae. FEMS Yeast Res 2012, 12 (2), 197-214.

5. 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.

6. 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.

7. 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.

8. 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.