Team:UFRGS Brazil/Design
OVERVIEW
The Phn Operon is one of the nine transcriptional unities that are present in the Phosphate Regulon (Pho), a global regulatory mechanism involved in bacterial Pi management. It is responsible for the catabolism of phosphates and it has 14 cistrons (phnCDEFGHIJKLMNOP) that, together, are responsible for controlling the Carbon-Phosphorus (C-P) lyase activity[1].
The Pho regulon is one of the most efficient and sensible mechanism in bacteria and works activating Pi transporters, extracellular enzymes for Pi obtention from organic phosphonates, and other enzymes inolved in storage and saving of Pi.
The C-P lyases
The C-P lyases pathway is activated upon conditions of phosphate starvation and it is responsible for converting phosphonates into 5-phosphoribosyl-α-1-diphosphate (PRPP), utilizing ATP[2].
The PnhCDEFGHIJKLMNOP operon components
PhCDE
It encodes an ATP-binding cassette, where phnC has the ATP-binding site, phnD is a periplasmic binding protein for phosphonates and phnE is a membrane spanning transport protein. The complex formed is composed of two proteins derived from PhnC, two from phnE and one solute-binding protein from phnD. altogether these proteins are responsible for the transport of an alkylphosphonates and an aminoalkylphosphonates, thourgh the reactions:
The PhnF
The cistron phnf is responsible for the production of a transcriptional regulator with two domains, an amino-terminal domain that contains a potential helix-turn-helix DNA-binding motif and a carboxy-terminal domain involved in effector recognition. Its exact function is not known, but it was inferred by sequence similarity to Mycobacterium smegmatis. The phnF represses the phnCDE, in phophorus replete conditions.
The PhnGHIJKLM
It forms the C-P lyase core complex and is responsible for the coupling of phosphonate to ATP and C-P bond cleavage. In this complex, the PhnI, a nucleosidase capable of deglycosylating ATP and GTP to ribose 5-triphosphate, is supported by PhnG, PhnH, and PhnL in the catalysis of the transfer of phosphonate moiety to a 5′-triphosphate alkyl phosphonate intermediate , according to the following reaction:
Source:EcoCyc:Escherichia coli K-12 substr. MG1655 Reaction: 2.7.8.37
Access: Oct, 2019
After that, PhnM catalyses the pyrophosphate release through the reaction α-D-ribose-1-(methyl)phosphonate-5-triphosphate + H2O → α-D-ribose-1-methylphosphonate 5-phosphate + diphosphate + H+ allowing phnJ to cleave de C-P lyase bond via an S-adenosyl methionine (SAM)-dependent radical mechanism, thus converting the resulting ribose cyclic phosphate into PRPP. This reaction is possible because of the interaction of phnK trough the via its α helices 3 and 4 with PhnJ, exposing its active site.
The PhnN
It encodes a ribosyl 1,5-bisphosphate phosphokinase and catalyses the following reaction:
Source:EcoCyc:Escherichia coli K-12 substr. MG1655 Reaction: 2.7.4.23
Access: Oct, 2019
The PhnO
The PhnO cistron encodes aminophosphonate N-acetyltransferase that is involved in the utilization of aminomethylphosphonate as a source of phosphate, according to the reaction below:
Source:EcoCyc:Escherichia coli K-12 substr. MG1655 Reaction: 2.3.1.280
Access: Oct, 2019
The PhnP
The last cistron of the phnCDEFGHIJKLMNOP, the phnP, encodes phosphoribosyl cyclic phosphodiesterase. You can see the reaction below:
Source:EcoCyc:Escherichia coli K-12 substr. MG1655 Reaction: 3.1.4.55
Access: Oct, 2019
CHASSIS
We chose Escherichia coli subtr. MG1655 to be our chassis for engineering. Firstly due to E. coli being one of the most used and studied microorganisms to date and our labs already had standardized protocols for transformation and manipulation of this strain. And most importantly, E. coli MG1655 have an endogenous phnCDEFGHIJKLMNOP (C-P lyase) operon, but with a frame-shift at phnE, that impossibilitates phosponate utilization. Correcting this frame-shift potentially enable E. coli K12 to use glyphosate as a phosphorous source without generating AMPA.
Experimental design
How we pretend to develop Glyphosate-eating bacteria?
To overcome this issue, we developed a cassette to correct phnE frame-shift and exclude phnF (repressor) from the operon in one transformation step. Unfortunately, it was not possible to include a antibiotic selection in this construction due to the operon disposition in tandem.
As previously described, this operon is under control of a phosphate-dependent promoter, active only in lack of phosphate in the medium. We also developed three different promoter cassettes to increase expression of this operon, with Chloranfenicol resistance for selection.
How did we construct the promoter cassettes ?
The chloramphenicol resistance gene and the individual promoters were synthetized separately, and fused together through single-joint PCR.
Transformation
We transformed all constructions in the chassis using Lambda-red recombinase mediated transformation.
How to prove our design?
We used M9 and MSM2 minimal medium supplemented with different concentrations of glyphosate as sole phosphate source for selection and for testing. Glyphosate was measured in liquid media in partnership with a federal laboratory (LFDA) using UPLC. E. coli K12 and mutants harboring the constitutive promoters and corrected operon had their growth compared in the same minimal medias (MSM2). Growth was measured in 96-well plates at SpectraMAX i3. Growth curves were made to evaluate growth with glyphosate as the only phosphorus source.
Reaction Overview:
Pathways for the conversion of phosphorus of glyphosate and AMPA to Pi. A) Conversion of glyphosate. B) Conversion of AMPA.
Source:Hove-Jensen et. al,2014
References:
HOVE-JENSEN, B.; ZECHEL, D. L.; JOCHIMSEN, B. Utilization of Glyphosate as Phosphate Source: Biochemistry and Genetics of Bacterial Carbon-Phosphorus Lyase.Microbiology and Molecular Biology Reviews, v. 78, n. 1, p. 176–197, 1 mar. 2014.
JOCHIMSEN, B. et al. Five phosphonate operon gene products as components of a multi-subunit complex of the carbon-phosphorus lyase pathway. [s.d.].
KAMAT, S. S.; WILLIAMS, H. J.; RAUSHEL, F. M.Phosphonates to Phosphate: A Functional Annotation of the Essential Genes of the Phn Operon in Escherichia coli. [s.l: s.n.]. Available at:
KESELER, I. M. et al. The EcoCyc database: Reflecting new knowledge about Escherichia coli K-12. Nucleic Acids Research, v. 45, n. D1, p. D543–D550, 1 jan. 2017.
SEWERYN, P. et al. Structural insights into the bacterial carbon-phosphorus lyase machinery.Nature, v. 525, n. 7567, p. 68–72, 3 set. 2015.
THE UNIPROT CONSORTIUM. UniProt: a worldwide hub of protein knowledge.Nucleic Acids Res. 47: D506-515, 2019.
