This really cool ¹³C labeling experiment was done at the research center 'Forschunszentrum Jülich', where Prof. Dr. Wiechert offered all necessary equipment and components to us. Jannick Kappelmann instructed two iGEM Team Aachen members (Jonas R. & Tobias S.) during the whole experiment.

Summary

A batch fermentation with ¹³C labeled methanol as an additional carbon source was done to demonstrate the functionality of our methanol assimilation pathway. Afterwards ¹³C labeled carbon atoms were measured via mass spectrometry.

Two different constructs were tested in E. coli BL21 Gold (DE3).

Methanol Condensation Cycle scheme
Sugars are the first metabolites that will contain ¹³C if the pathway works successfully. The initial step is the assembly of formaldehyde.

One of them was the polycistronic expression cassette and the other one was the mdh behind a IPTG inducable T7 promoter in pSB1A30.

Different amounts of methanol were added to each reactor to make the polycistronic construct comparable with itself to generate more independent data.

Offgas analysis of one cultivation with the mdh construct was done constantly during the fermentation. Ideally, it detects labled carbondioxide which is produced via the detoxification pathway of E. coli as a response to the formaldehyde produced by the Mdh.
The samples for analysis of cytosolic metabolites were taken after one doubling time (ca. 2:25 h).
The analysis was done by mass spectrometry of fructose-bis-phosphate, glucose-6-phosphate and ribose-5-phosphate.

The MS-detection of cytosolic metabolites covered additionally DHAP, Xylulose-5-phosphate, Fructose-6-phosphate, E4P, 2Phosphoglycerat/3Phosphoglycerate, S7P, AMP & ADP. Nevertheless, these metabolites were not analysed because of natural isotope distributions, coelutions or too low concentrations.

Experimental Setup
Four 200 ml reactors for cultivation in the ¹³C experiment.

Achievements

We proved at methanol is not assimilated into the central metabolism of E. coli BL21 Gold (DE3) when the bacteria carries the polycistronic plasmid of the four missing enzymes to complete the MCC.
Despite no detectable production of ¹³C carbondioxide in the offgas we showed significant Mdh activity with samples from the respective reactor (see Mdh Characterization)

Expected Results

3 different sugars were analyzed: fructose-bis-phosphate, glucose-6-phosphate and ribose-5-phosphate.

Expected mass shift if ¹³C,d4 is assimilated: methanol - m+5, formaldehyde - m+3
- all other sugars in the subsequent pathway should have a shift of m+3 in the first cycle of the functional MCC.

Expected mass shift if ¹³C is assimilated: methanol - m+1, formaldehyde - m+1
- all other sugars in the subsequent pathway should have a shift of m+1 in the first cycle of the functional MCC.

Results

In no sample of the polycistronic clones was a mass shift of m+3 observable. The detected peak areas are similar to the analytic standard samples of ¹²C carbohydrates. The mass shifts m+1 & m+2 are as frequent as it is usual in the natural composition.

Plot of natural FBP, G6P & R5P as control
This figure shows the natural mass shift composition of the measured metabolites.

The measured data and average values of reactor 5 with #AW9K# and 0.75 M methanol do not differ from this mass composition.

The same is valid for reactor 6 with (#AW9K#) our polycistronic construct with 0.3 M methanol.

Our Mdh strain (#IGEM#) in reactor 7 does not differ at all.

Conclusion & Discussion

It was not possible to show a completely working "Methanol Condensation Cycle" ^[1] (MCC) in E. coli.

Although no labeled cytosolic metabolites could be detected we proved qualitatively the functionality of the Mdh. This leads to the assumption that the activity of the Mdh is too low to incorporate enough formaldehyde. Another possible explanation is that subsequent enzymes of the pathway do not function properly. The measured metabolites give no hint which of the implemented steps of the MCC does not work because the measured carbohydrates are only labeled if the assimilated molecule has passed the whole MCC.

The Xpk should have no influence on the assimilation of formaldehyde because without this enzyme the pathway equals the Ribulose-Monophosphate-Pathway (RuMP) which was already shown as a functional pathway if it is implemented in E. coli^[2].

Laboratory Notebook

References

↑ Bogorad IW, Chen CT, Theisen MK, Wu TY, Schlenz AR, Lam AT, Liao JC. Building carbon-carbon bonds using a biocatalytic methanol condensation cycle. Proc Natl Acad Sci U S A. 2014 Nov 11;111(45):15928-33. doi: 10.1073/pnas.1413470111. Epub 2014 Oct 29. PubMed PMID: 25355907; PubMed Central PMCID: PMC4234558.
↑ 1. Metab Eng. 2015 Mar;28:190-201. doi: 10.1016/j.ymben.2014.12.008. Epub 2015 Jan 14.Engineering Escherichia coli for methanol conversion. Müller JE(1), Meyer F(1), Litsanov B(1), Kiefer P(1), Potthoff E(1), Heux S(2), Quax WJ(3), Wendisch VF(4), Brautaset T(5), Portais JC(2), Vorholt JA(6).

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[1] Bogorad IW, Chen CT, Theisen MK, Wu TY, Schlenz AR, Lam AT, Liao JC. Building carbon-carbon bonds using a biocatalytic methanol condensation cycle. Proc Natl Acad Sci U S A. 2014 Nov 11;111(45):15928-33. doi: 10.1073/pnas.1413470111. Epub 2014 Oct 29. PubMed PMID: 25355907; PubMed Central PMCID: PMC4234558.

[2] 1. Metab Eng. 2015 Mar;28:190-201. doi: 10.1016/j.ymben.2014.12.008. Epub 2015 Jan 14.Engineering Escherichia coli for methanol conversion. Müller JE(1), Meyer F(1), Litsanov B(1), Kiefer P(1), Potthoff E(1), Heux S(2), Quax WJ(3), Wendisch VF(4), Brautaset T(5), Portais JC(2), Vorholt JA(6).

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