| CPU_Nanjing - iGEM 2022

Overview

The ultimate goal of our project this year is to manufacture phosphate on terrestrial planets by utilizing phosphite and means of synthetic biology.

In terms of the overall design, phosphite serves as the substrate, polyphosphate (polyP) as the intermediate, and phosphate as the final product. To verify this assumption, we will couple two metabolic pathways, phosphite oxidation mediated by phosphite dehydrogenase (PTDH) [1] and polyP synthesis mediated by polyphosphate kinase (PPK) [2], and introduce them into E. coli afterwards. Intracellular polyP, a high-density form of phosphate, will be hydrolyzed to phosphate by endogenous exopolyphosphatase of E. coli under anaerobic conditions [3, 4]. Phosphate produced from polyP hydrolysis will be released into the extracellular medium/environment, where we can recover and obtain the final product.

Meanwhile, given that there is a lack of oxygen and nutrients on terrestrial planets, we need to find a feasible way to provide the heterotrophic E. coli with these matters so as to support its growth, making the aforementioned biological process thereof feasible.

Moreover, as a manufacture project, a suitable device needs to be developed. By doing so, we can achieve bench-scale phosphate production and verify the technological feasibility of our integrated design.

Aim 1 - Enhance intracellular phosphite oxidation by selecting a phosphite dehydrogenase of high activity

Intracellular phosphite oxidation, the first and also the rate-limiting step of the engineered metabolic pathway, is in strong need of phosphite dehydrogenase with high intracellular activity. Based on the literature report and NCBI query [5, 6], 2 to 3 kinds of phosphite dehydrogenase genes from bacterial sources were chemically synthesized. Then, the highly active one was identified after comparison tests.

Aim 2 - Enhance intracellular polyP accumulation by adopting mutant E. coli polyphosphate kinase

The intermediate of the engineered metabolic pathway is polyP, a compact polymer of phosphate. It is without doubt that the enhancement of polyP biosynthesis will improve the yield of the final product phosphate. Therefore, in addition to phosphite dehydrogenase with high intracellular activity, we also tend to use highly active polyphosphate kinase, which is available according to the method described by Bassin et al [7].

Aim 3 - Explore oxygen and nutrients to provide life support for E. coli

When culturing E. coli, glucose is widely used on the earth to prepare synthetic media, in which it serves as the carbon source. However, all kinds of organic matters, including glucose, may be unavailable on terrestrial planets. In addition, intracellular polyP accumulates under aerobic conditions, meaning that this process consumes oxygen. Under such circumstances, exploring a practicable method to satisfy these two conditions simultaneously is a pressing challenge we face. After considering several possible options, we eventually decided to culture the blue-green algae, which can not only fix carbon and release oxygen through photosynthesis, but also fix atmospheric nitrogen [8]. Meanwhile, given that the algae cannot be “eaten” raw by E. coli, we need to figure out a feasible way on terrestrial planets to “cook” the algae for E. coli.

Aim 4 - Technological feasibility test upon bench-scale phosphate production

    Bench-scale phosphate production can meet our needs of verifying the technological feasibility of our integrated design. Therefore, designing a suitable bioreactor is necessary. Considering the environment of terrestrial planets, liquid water, atmospheric carbon dioxide and nitrogen might be available resources. In order to maintain the vitality of the engineered E. coli and wild-type algae under these preset conditions, the following four components were considered while designing our bioreactor:

    (1) an independent chamber for the engineered E. coli, which serves as the initial inocula,

    (2) an independent chamber for algae culture, where carbon dioxide will be fixed through photosynthesis and converted into organic matters that stored in the form of algae biomass (i.e., organic matters),

    (3) an algae lysate preparation chamber, where the algae will be lysed by using a solar-energy-driven electric heater,

    (4) a phosphate production chamber that run on an aerobic-anaerobic cycle regime, where the phosphate will be produced by culturing the engineered E. coli using oxygen, and algae lysate produced from chamber 2 and 3.

Detailed information on the configuration of this reactor is provided in the Hardware part.

Reference

[1] H.A. Relyea, W.A.J.B.c. Van Der Donk, Mechanism and applications of phosphite dehydrogenase, 33(3) (2005) 171-189.
[2] N.N. Rao, M.R. Gómez-García, A. Kornberg, Inorganic polyphosphate: essential for growth and survival, Annual Review of Biochemistry 78 (2009) 605-647.
[3] M. Akiyama, E. Crooke, A. Kornberg, An exopolyphosphatase of Escherichia coli. The enzyme and its ppx gene in a polyphosphate operon, Journal of Biological Chemistry 268(1) (1993) 633-639.
[4] D. Ault-Riché, C.D. Fraley, C.-M. Tzeng, A. Kornberg, Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in Escherichia coli, Journal of Bacteriology 180(7) (1998) 1841-1847.
[5] R. Hirota, S.-t. Yamane, T. Fujibuchi, K. Motomura, T. Ishida, T. Ikeda, A. Kuroda, Isolation and characterization of a soluble and thermostable phosphite dehydrogenase from Ralstonia sp. strain 4506, Journal of Bioscience Bioengineering 113(4) (2012) 445-450.
[6] W.W. Metcalf, R.S. Wolfe, Molecular genetic analysis of phosphite and hypophosphite oxidation by Pseudomonas stutzeri WM88, Journal of Bacteriology 180(21) (1998) 5547-5558.
[7] A.K. Rudat, A. Pokhrel, T.J. Green, M. Gray, Mutations in Escherichia coli polyphosphate kinase that lead to dramatically increased in vivo polyphosphate levels, Journal of Bacteriology 200(6) (2018) e00697-17.
[8] N.K. Sharma, S.P. Tiwari, K. Tripathi, A.K. Rai, Sustainability and cyanobacteria (blue-green algae): facts and challenges, Journal of Applied Phycology 23(6) (2011) 1059-1081.