Team:NJTech China/Perspective

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Perspective

In this special year, we were quarantined for a long time at home where we discussed and designed the project by searching literature. In summer, the team members returned to the lab under the regional and school regulations, conducted a series of wet-lab experiments, and have finally made encouraging progress. The impact of the epidemic was the main obstacle of fully realizing the concept of this project. We will continue the progress and plan future work in detail.

1. Promoter Engineering and Cell Factory

Cell metabolism in organisms is largely regulated at the transcriptional level, and the 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 model eukaryotic yeast Saccharomyces cerevisiae^[1]. This year, our hybrid promoter engineering approach entails combining core promoters pTEF with upstream activation sequences (UAS elements) to enable fine-tuned control and amplification of gene expression. The expression of phenylacetaldehyde synthase (PAAS) is increased by high activity engineering promoter. The phenylacetaldehyde synthase pathway is artificially delivered to S. cerevisiae for 2-phenylethanol (2-PE) biosynthesis.

In the future, we will try more diverse promoter engineering strategies, including site-directed mutagenesis, error-prone PCR (Ep-PCR), sequence randomization of non-conserved region (NCR), hybrid-promoter design, and transcription factor-binding sites (TFBSs) modification, etc^[2]. These diversified modification methods will eventually form a complex and rich library of engineered promoters. After that, neural network models can screen engineered promoters that meet actual needs from the library, and molecular dynamics models make it easier to accurately characterize promoters. Moreover, we will try to transfer the complex enzyme system under the control of Synthetic Hybrid Promoters into a simpler system for metabolic engineering research. The regulation strategy can also be further optimized. Other regulate methods such as light regulate can be tested later. However, the Ehrlich pathway naturally present in the S. cerevisiae to produce 2-PE may compete with the imported PAAS pathway from plants, which may aggravate cell metabolic burden^[3]. More refined modification plans will be implemented to reduce damage to the normal metabolic process of cells.

2. Artificial Microbial Consortia System

Mixed cultures of microorganisms are common in natural ecological systems, which are often used for the treatment of waste materials discharged from industries, as well as for the production of bio-based products and bioactive compounds. The mixed cultures of yeast and microalgae would benefit in terms of the economical improvement of biosynthesis production. It has the potential to address three important societal needs: (i) the development of new energy sources; (ii) the protection of aquatic environments; and (iii) the reduction of the global anthropogenic greenhouse effect^[4]. 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^[5]. At present, the research of yeast-microalgae microbial consortia system focuses on aquaculture^[6], fine chemical production^[7] and biodiesel production^[8].

This year, we design and construct a yeast-microalgae microbial consortia system to promote green and low-carbon transformation, facilitating the achievement of carbon neutrality goal and high-quality economic development by reducing carbon dioxide emissions. The autotroph to heterotroph culture volume ratio seems to play a key role in the control of CO₂ evolution. The specific growth rate of the autotroph and the heterotroph are also affecting the CO₂ production and uptake, respectively, as these processes are directly related to microbial growth rates. The culture conditions including the ratio of yeast to microalga, initial pH, the addition of molasses, agitation speed, and light intensity will be optimized in the future^[9]. Our project will further prove the feasibility that study the yeast-microalgae microbial consortia system through omics technology^[10].

3. 3D Printing Technology

Based on digital model files, 3D printing is an emerging technology that uses powdery metal or plastic and other adhesive materials to construct objects by means of extrusion, sintering, melting, light curing, injection and layer-by-layer printing by 3D printer. The process is basically to first model by computer modeling software, three-dimensional design model, and then build three-dimensional model "partition" into layer by layer section, so as to guide the printer to print layer by layer, and finally establish the printing object^[11].

In recent years, relevant researchers have been constantly innovating on this basis, making its contributions in various fields more prominent. For example, researchers have found that extruded 3D printing in suspension can mask the collapse occurring when printed in air^[12], a major breakthrough for biology. Based on the advantages of 3D printing technology such as high efficiency, precision and convenience, we plan to use 3D printing technology to print bioactive materials and finally assemble them into bioreactor to improve the production efficiency of 2-PE. We believe that the application prospects of 3D printing are unlimited.

References

1. Blazeck, J.; Garg, R.; Reed, B.; Alper, H. S., Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters. Biotechnol Bioeng 2012, 109 (11), 2884-95.

2. Xu, N.; Wei, L.; Liu, J., Recent advances in the applications of promoter engineering for the optimization of metabolite biosynthesis. World J Microbiol Biotechnol 2019, 35 (2), 33.

3. Wang, Z.; Jiang, M.; Guo, X.; Liu, Z.; He, X., Reconstruction of metabolic module with improved promoter strength increases the productivity of 2-phenylethanol in Saccharomyces cerevisiae. Microb Cell Fact 2018, 17 (1), 60.

4. 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.

5. Santos, C. A.; Reis, A., Microalgal symbiosis in biotechnology. Appl Microbiol Biotechnol 2014, 98 (13), 5839-46.

6. Cai, S.; Hu, C.; Du, S., Comparisons of growth and biochemical composition between mixed culture of alga and yeast and monocultures. J Biosci Bioeng 2007, 104 (5), 391-7.

7. Dong, Q. L.; Zhao, X. M., In situ carbon dioxide fixation in the process of natural astaxanthin production by a mixed culture of Haematococcus pluvialis and Phaffia rhodozyma. Catal Today 2004, 98 (4), 537-544.

8. Cheirsilp, B.; Kitcha, S.; Torpee, S., Co-culture of an oleaginous yeast Rhodotorula glutinis and a microalga Chlorella vulgaris for biomass and lipid production using pure and crude glycerol as a sole carbon source. Ann Microbiol 2012, 62 (3), 987-993.

9. Shokrkar, H.; Ebrahimi, S.; Zamani, M., Bioethanol production from acidic and enzymatic hydrolysates of mixed microalgae culture. Fuel 2017, 200, 380-386.

10. Fazzini, R. A. B.; Preto, M. J.; Quintas, A. C. P.; Bielecka, A.; dos Santos, V. A. P. M., Consortia modulation of the stress response: proteomic analysis of single strain versus mixed culture. Environ Microbiol 2010, 12 (9), 2436-2449.

11. Richard, C.; Neild, A.; Cadarso, V. J., The emerging role of microfluidics in multi-material 3D bioprinting. Lab Chip 2020, 20 (12), 2044-2056.

12. McCormack, A.; Highley, C. B.; Leslie, N. R.; Melchels, F. P. W., 3D Printing in Suspension Baths: Keeping the Promises of Bioprinting Afloat. Trends Biotechnol 2020, 38 (6), 584-593.