Why Phosphate?

We need phosphate (+5 valence)

     Phosphate is the very form in which phosphorus could be utilized by earth creatures [1]. What makes it widely known is that it is one of the fundamental components for innumerable substances designed by the nature, such as cell membranes, nucleic acids, phosphorylated proteins and even human bones. Besides, phosphate is actually an indispensable substance to almost everything in people's daily life as well as agricultural and industrial production of human society [2].

Differences in biogeochemical cycle

     Other than the gaseous cycle of carbon, nitrogen and oxygen, the biogeochemical cycle of phosphorus is in the form of sedimentary, that is, soluble phosphate is released in the leaching process, then flows into rivers through surface runoff and eventually deposits in the sea. Therefore, approximately 85% of phosphorus on the earth today derives from the mining of marine sedimentary phosphorus deposits. More importantly, in the earth's phosphorus minerals, including marine sedimentary phosphate mine, almost all forms of phosphorus are at +5 valence [3].

Phosphorus on terrestrial planets

     Although the composition varies, the "bio-planetary chemical cycle" of the above elements on terrestrial planets will not change, and neither will the way people obtain phosphorus. The meteorites left on the earth have brought us its message from terrestrial planets. What encourages us is the considerable amount of phosphorus there. However, it is mainly in the form of phosphite, hypophosphite, or even phosphosiderite, which exists in a lower oxidation state [4, 5]. Under natural conditions, it is such a difficult task for phosphorus to be further oxidized to a form assimilated by life, namely phosphate. Even worse, phosphite is toxic to earth creatures.

Synthetic Biology: Opportunity, Challenge and Strategy

Opportunity: phosphite dehydrogenase in bacteria

     Thanks to the diversity of microorganisms on the earth, phosphite dehydrogenases present in a few bacteria, capable of oxidizing phosphite to phosphate [6], provide an amazing clue to solving this problem.

Challenge: spurring bacteria to overcome laziness

     The depressing fact is that bacteria are "passive", or even "lazy", in the matter of phosphite oxidation. Though having the ability to oxidize phosphite, they do so only to meet their own growth needs [7]. Once there is enough phosphate, they are unwilling to spend even one more second for this process, bringing us a challenging situation to get through. Therefore, only by spurring bacteria to overcome this "inherent laziness" can they continue to manufacture phosphate from phosphite coming from the environment.

Strategy: coupling polyphosphate (polyP) synthesis to create phosphorus starvation

     From the perspective of recycling, the assimilated phosphate (Pi) in bacterial cells can be bifurcated:

     (1) Fixed Pi, such as those in cell membranes, nucleic acids and phosphorylated proteins, exist in the cell's own components, which are difficult to be recycled in the short term.



     (2) Free Pi, such as Pi used in synthesis of intracellular ATP, are recycled in the ATP regeneration process (ATP ↔ ADP + Pi).
    Our strategy is based on the common fact that ADP + Pi ↔ ATP is essential for the survival of bacteria. Due to the existence of intracellular free Pi, bacterial cells are essentially not deficient in phosphorus. However, if we use synthetic biology methods to trap the mobile Pi inside the cell, such as "pouring" it into the synthesis of polyphosphate (polyP) [8], then we can force the bacteria to constantly remain "phosphorus starvation" state, so as to reach the goal of spurring them to continuously transport phosphite from the environment into the cell and oxidize it to Pi.

What is and why is polyP?

     PolyP is a linear polymer consisting of 3-1000 phosphate residues, linked by high-energy phosphoanhydride bonds similar to those possessed by ATP [9]. In bacterial cells, its synthesis is catalyzed by polyphosphate kinase using ATP as a substrate. It stores phosphate residues in a high-density form inside bacterial cells [10]. More importantly, when the external environment becomes severe, polyP will be hydrolyzed by bacterial endogenous exopolyphosphatase (PPX) and released outside the cell in the form of phosphate [11]. By operating in this way, we can easily recover phosphate directly from the growth matrix of the bacteria. It follows that the accumulation of intracellular polyP will be equivalent to the final production of Pi. It is a turbine that drives the bacteria to oxidize more phosphite, in addition to an intermediate product of our project.

More Challenges Before Landing the Project - Chassis Supporting System and Adaptive Hardware Design

Chassis Supporting system

    Given that the synthetic biology process described above is an aerobic and energy-consuming procedure, we need to provide oxygen and assimilable carbon sources to our engineered chassis. Based on the knowledge that the environment of terrestrial planets is different from that on the earth, we chose blue-green algae, known as "miniature oxygen and organic substance factories" [12], to build a life supporting system for the engineered chassis. The system produces oxygen and organics for the engineered chassis inside through photosynthesis, a negative carbon emission process.

Adaptive hardware design

     Throughout the field of manufacturing, the value of technology is reflected in whether it can be applied for reality and yield products. Using phosphite as a substrate to produce phosphate, truly finishing an implemented and close-loop project so as to serve all aspects of social production and life, is the ultimate goal of our team. Therefore, based on the above synthetic biology strategies, it is particularly necessary to develop adaptive hardware suitable for environments on terrestrial planets. To this end, we developed a solar-energy-driven sequence batch bioreactor and performed a bench-scale test of phosphate production.



     Of course, apart from what have mentioned above, there are still quantities of problems waiting to be solved. Our project this year is merely a beginning!

Reference

[1] R. Bieleski, Phosphate pools, phosphate transport, and phosphate availability, Annual Review of Plant Physiology 24(1) (1973) 225-252.
[2] S.C. Kamerlin, P.K. Sharma, R.B. Prasad, A. Warshel, Why nature really chose phosphate, Quarterly Reviews of Biophysics 46(1) (2013) 1-132.
[3] W.L. Lindsay, P.L. Vlek, S.H. Chien, Phosphate minerals, Minerals in Soil Environments 1 (1989) 1089-1130.
[4] M. Pasek, D. Lauretta, Extraterrestrial flux of potentially prebiotic C, N, and P to the early Earth, Origins of Life Evolution of Biospheres 38(1) (2008) 5-21.
[5] M.A. Pasek, J.P. Harnmeijer, R. Buick, M. Gull, Z. Atlas, Evidence for reactive reduced phosphorus species in the early Archean ocean, Proceedings of the National Academy of Sciences 110(25) (2013) 10089-10094.
[6] H.A. Relyea, W.A.J.B.c. Van Der Donk, Mechanism and applications of phosphite dehydrogenase, 33(3) (2005) 171-189.
[7] L. Casida Jr, Microbial oxidation and utilization of orthophosphite during growth, Journal of Bacteriology 80(2) (1960) 237-241.
[8] M.R. Brown, A. Kornberg, The long and short of it–polyphosphate, PPK and bacterial survival, Trends in Biochemical Sciences 33(6) (2008) 284-290.
[9] M.R. Brown, A. Kornberg, Inorganic polyphosphate in the origin and survival of species, Proceedings of the National Academy of Sciences 101(46) (2004) 16085-16087.
[10] L. Xie, U. Jakob, Inorganic polyphosphate, a multifunctional polyanionic protein scaffold, Journal of Biological Chemistry 294(6) (2019) 2180-2190.
[11] S.J. Van Dien, S. Keyhani, C. Yang, J. Keasling, Manipulation of independent synthesis and degradation of polyphosphate in Escherichia coli for investigation of phosphate secretion from the cell, Applied Environmental Microbiology 63(5) (1997) 1689-1695.
[12] 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.