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Team:Hong Kong HKU/project/cargo
From 2013.igem.org
Polyphosphate Kinase
The enhanced biological phosphorous removal process is necessarily dependent on the ability of sludge microorganism (PAOs) to take up phosphate and to store it intracellularly in the form of polyP. For polyP formation to occur, phosphate must first be transported into the microbial cell and subsequently converted into ATP before incorporation in the polyP polymer. To improve the efficiency of the whole process, there are two steps we can work on:
(1) Phosphate transport system: increase phosphate uptake by engineering biological phosphate transport system.
(2) PolyP synthesis: engineer microbes to favour the formation of PolyP over the hydrolysis of PolyP, so that higher cell PolyP concentration and longer PolyP chain can be achieved.
Phosphate transport system
There are two major phosphate transport system in bacterial cells:
A. The inorganic phosphate transport system (Pit) is constitutively expressed and has a relatively low specificity for phosphate. Pit transport neutral metal phosphates, each in symport with a proton.
B. The phosphate-specific transport system (Pst) transport both H2PO4- and HPO42-, but not neutral metal phosphates. Unlike constitutively expressed Pit system, Pst system is phosphate-starvation inducible.
More future research on microbial polyP metabolism in the EBPR process is needed to investigate how phosphate transport system activities affect polyP production. Clarification of the mechanism by which in-fluent phosphate is converted into ATP and used as substrate for polyP synthesis. Due to the much unknown details and limit of time, we choose to focus on the enhancement of PolyP synthesis efficiency inside bacteria.
PolyP synthesis
Polyphosphate kinase 1 (PPK1) is the most extensively studied polyp-synthesizing enzyme and has been detected in a wide range of prokaryotes. It catalyzes the transfer of the terminal phosphate of ATP to an active-site histidine residue, the initial step in the processive synthesis of a long PolyP chain. The reaction is reversible but favors synthesis.
Although PPK’s involvement in the EBPR process is still unclear, we hypothesize over-expressing PPK can favour the PolyP synthesis and further increase the phosphate uptake. Hence we clone the ppk1 gene and put it under a controllable expression system. We notice that metabolic reactions are dynamic, maintaining a homeostasis inside the bacterial cells. PolyP synthesis by PPK1 may be counteracted by numerous “enemies” inside the bacteria.
Enemies against PolyP synthesis
There are variety of hydrolases and phosphotransferases known to utilize polyP as a substrate in bacteria. Namely,
Exopolyphosphatase (PPX) (EC 3.6.1.11)
It is the major polyP-degrading enzyme in bacterial cells. This enzyme catalyzes the processive hydrolytic cleavage of Pi from the end of the polyP chain and the reaction may continue until only pyrophosphate (PPi) remains.
Polyphosphate Glucokinase (EC 2.7.1.63)
It catalyzes an attack by glucose at the end of the polyP chain
AMP Phosphotransferase
This enzyme catalyzes the attack at the end of the polyP chain by adenosine monophosphate (AMP) to produce ADP:
ADP Phosphotransferase
This enzyme is proposed to catalyse the polyphosphate-dependent phosphorylation of nucleoside diposphates such as ADP, the reverse activity of PPK:
Considering the vast number of polyP degradation pathways, we wonder, is there a way to isolate polyP synthesis from the surrounding polyP degrading enzymes? Just like the compartmentation in eukaryotes, can we also achieve compartmentation of metabolic reactions inside bacterial cells?
Hence, we come up with the idea of using Bacterial Micocompartment.
The enhanced biological phosphorous removal process is necessarily dependent on the ability of sludge microorganism (PAOs) to take up phosphate and to store it intracellularly in the form of polyP. For polyP formation to occur, phosphate must first be transported into the microbial cell and subsequently converted into ATP before incorporation in the polyP polymer. To improve the efficiency of the whole process, there are two steps we can work on:
(1) Phosphate transport system: increase phosphate uptake by engineering biological phosphate transport system.
(2) PolyP synthesis: engineer microbes to favour the formation of PolyP over the hydrolysis of PolyP, so that higher cell PolyP concentration and longer PolyP chain can be achieved.
Phosphate transport system
There are two major phosphate transport system in bacterial cells:
A. The inorganic phosphate transport system (Pit) is constitutively expressed and has a relatively low specificity for phosphate. Pit transport neutral metal phosphates, each in symport with a proton.
B. The phosphate-specific transport system (Pst) transport both H2PO4- and HPO42-, but not neutral metal phosphates. Unlike constitutively expressed Pit system, Pst system is phosphate-starvation inducible.
More future research on microbial polyP metabolism in the EBPR process is needed to investigate how phosphate transport system activities affect polyP production. Clarification of the mechanism by which in-fluent phosphate is converted into ATP and used as substrate for polyP synthesis. Due to the much unknown details and limit of time, we choose to focus on the enhancement of PolyP synthesis efficiency inside bacteria.
PolyP synthesis
Polyphosphate kinase 1 (PPK1) is the most extensively studied polyp-synthesizing enzyme and has been detected in a wide range of prokaryotes. It catalyzes the transfer of the terminal phosphate of ATP to an active-site histidine residue, the initial step in the processive synthesis of a long PolyP chain. The reaction is reversible but favors synthesis.
Although PPK’s involvement in the EBPR process is still unclear, we hypothesize over-expressing PPK can favour the PolyP synthesis and further increase the phosphate uptake. Hence we clone the ppk1 gene and put it under a controllable expression system. We notice that metabolic reactions are dynamic, maintaining a homeostasis inside the bacterial cells. PolyP synthesis by PPK1 may be counteracted by numerous “enemies” inside the bacteria.
Enemies against PolyP synthesis
There are variety of hydrolases and phosphotransferases known to utilize polyP as a substrate in bacteria. Namely,
Exopolyphosphatase (PPX) (EC 3.6.1.11)
It is the major polyP-degrading enzyme in bacterial cells. This enzyme catalyzes the processive hydrolytic cleavage of Pi from the end of the polyP chain and the reaction may continue until only pyrophosphate (PPi) remains.
Polyphosphate Glucokinase (EC 2.7.1.63)
It catalyzes an attack by glucose at the end of the polyP chain
AMP Phosphotransferase
This enzyme catalyzes the attack at the end of the polyP chain by adenosine monophosphate (AMP) to produce ADP:
ADP Phosphotransferase
This enzyme is proposed to catalyse the polyphosphate-dependent phosphorylation of nucleoside diposphates such as ADP, the reverse activity of PPK:
Considering the vast number of polyP degradation pathways, we wonder, is there a way to isolate polyP synthesis from the surrounding polyP degrading enzymes? Just like the compartmentation in eukaryotes, can we also achieve compartmentation of metabolic reactions inside bacterial cells?
Hence, we come up with the idea of using Bacterial Micocompartment.
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