Meet the Proteins
We have selected ten genes from Microlunatus phosphovorus that encode for proteins relevant to our project goals: phosphate remediation and recovery. Only one of these proteins has already been characterized prior to our project.
These ten proteins can be broken up into four different categories:
Inorganic phosphate transporters (Pit) transport metal-chelated phosphate across the cell membrane using the proton motive force . We have selected three Pit gene homologs from M. phosphovorus for expression in E. coli.
Phosphate transfer (kinase)
Polyphosphate kinases (PPK) catalyze the reversible transfer of phosphate between nucleoside phosphates and polyphosphates.  There are three PPK subtypes: PPK1 favors polyphosphate synthesis, PAP favors polyphosphate hydrolysis, and PPK2’s affinity for hydrolysis or synthesis varies by homolog.  We have selected four PPK homologs from M. phosphovorus to express in E. coli: one PPK1, two PPK2s, and one similar to both a PPK2 and PAP.
Phosphate transfer (glucokinase)
Polyphosphate-dependent glucokinases (PPGK) catalyze the first step of glycolysis, the phosphorylation of glucose using a phosphate group from polyphosphate or ATP. We have selected two PPGK homologs from M. phosphovorus to express in E. coli, one of which is the only protein of the ten chosen that have been characterized. The characterized PPGK cannot use ATP, and is referred to as PPGK ATP-independent (ATPI) .
Preparation for Export
Exopolyphosphatase (PPX) hydrolyzes of the terminal phosphate of a polyphosphate chain  The presence of PPX decreases polyphosphate accumulation, which allows PPX to maintain polyphosphate balance.  There are two variants of PPX proteins, PPX1 and PPX2, and we have selected one PPX2 homolog from M. phosphovorus to express in E. coli.
A Closer Look
Inorganic Phosphate Transporters
The primary structure of E. coli’s PitA was found on Uniprot . Primary structures (amino acid sequence) were compared using BLAST . Since the amino acid sequence of all M. phosphovorus Pits have relatively low identity similarities to one another and the Pit native to E. coli, they likely function in different capacities. Because of this low similarity, we believe that expressing and characterizing all three Pit genes would be worthwhile and that expressing M. phosphovorus Pits could be beneficial in increasing E. coli’s ability to uptake phosphate.
Important note: The NCBI Reference Sequence, and not the GenBank sequence, was used for PPK2 homolog C. This is the only case in which the two sequences vary from one another.
While none of the four PPKs in M. phosphovorus have been previously characterized, the affinity for polyphosphate hydrolysis or synthesis could be predicted by phylogenetic analysis for PPK1, PPK2 homolog A, and PPK2 homolog B. **PPK2 homolog C is phylogenetically similar to other uncharacterized PPKs .
The primary structure of E. coli’s PitA was found on Uniprot. . Primary structures (amino acid sequence) were compared using BLAST .
* No significant similarity was found for PPK1 and PPK2 homolog B.
Since the amino acid sequence of all M. phosphovorus PPKs have relatively low identity similarities to one another and the PPK native to E. coli, they likely function in different capacities. Because of this low similarity, we believe that expressing and characterizing all four PPK genes would be worthwhile and that expressing M. phosphovorus PPKs could be beneficial in modifying E. coli’s ability to store polyphosphate.
PPGK ATP-independent cannot use ATP to phosphorylate glucose, and it is also the only protein of the ten used that has been previously characterized . The second PPGK homolog’s ability to utilize ATP is unknown.
Primary structures were compared using BLAST . Because PPGKs are not native to E. coli, we believe that expressing PPGK could be beneficial in allowing E. coli to utilize its polyphosphate reserve. As M. phosphovorus’s PPGKs have only a 50% similarity to one another, we believe that characterizing both would be useful, as their functioning capacity may differ.
Identity similarity of PPX2 from M. phosphovorus and PPX2 from E. coli: 31%
Primary structures were compared using BLAST . As the amino acid sequences have a relatively low level of similarity, it is probable that they function at different capacities, which is why we believe expressing M. phosphovorus’s PPX2 could be beneficial in increasing E. coli’s ability to export phosphate.
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