3 Results and Discussion
3.1.5 Plug and play with metK
Two promising metK candidates were chosen: the gene from S. solfataricus, because of its ability to convert branched Met analogs, and the gene from M. jannaschii, as it is one of the most permissible variants towards a range of Met analogs. As both enzymes have already been shown to convert Eth efficiently to S-adenosyl ethionine (SAE)469,470, it was attempted to replace the endogenous metK gene in the strain ∆metEH::FRT with one of these genes. In another study, Parungao and coworkers have successfully substituted the E. coli metK gene with bacterial and eukaryotic orthologs supplied on plasmids, demonstrating the ability of E. coli to survive with foreign MAT variants481. These promising results were encouraging for the approach described here. Simultaneously, both enzymes were overexpressed heterogeneously in E. coli to test in an in vitro assay, if one of these variants is also capable of TfMet turnover.
3.1.5.1 1
stapproach: Rescue plasmid
Therefore, the metK genes from S. solfataricus and M. jannaschii were codon-optimized for expression in E. coli and ordered from GeneArt. To knock out the endogenous gene, the λ red system, which assists in recombination (for more details see chapter 5.2.17.2, p. 114), was employed in an approach analogous to the one described in chapter 3.2.2.1 (p. 66) for the knockout of the trpS gene. As metK is also an essential gene, a copy needs to be supplied on a rescue plasmid to be able to replace the chromosomal gene with a selection marker. To test whether the enzymes from S. solfataricus and/or M. jannaschii are capable of supporting E. coli growth, they were cloned on the rescue plasmid and it was attempted to replace the endogenous metK with a kanamycin resistance cassette (Figure 20, step 1). In the event of a successful knockout, the resistance cassette in the chromosome would then be replaced with the S. solfataricus or M. jannaschii gene (Figure 20, step 2). The rescue plasmid would be removed (Figure 20, step 3) in order to start another adaptation experiment with a plasmid-free setup. Analogously to the trpS rescue plasmid, these plasmids harbored 3 I-SceI restriction sites to allow for their removal.
The successful assembly of all constructs was verified via sequencing analysis.
Figure 20 I Overview of the 1st approach for the replacement of the E. coli metK with either the M. jannaschii or S. solfataricus metK. Mj: M. jannaschii, Ss: S. solfataricus. The plasmid harboring the λ red system and the helper plasmid have temperature-sensitive origins of replication and can be removed via incubation at 42°C. The linearized pieces of the rescue plasmid are digested by endogenous restriction enzymes.
ALE 2.0
(and elimination of red system)
However, even after several KO attempts, no colonies where the chromosomal metK gene was replaced with the kanamycin resistance cassette could be obtained. Figure 21 shows the agarose gel of the colony PCR of a representative colony (one of many picked colonies). Only bands corresponding to the wt (metK in chromosome still present) can be seen and the fragment corresponding to the kanamycin resistance (C1+C4) is not formed, indicating that the KO attempt was unsuccessful.
Figure 21 I Colony PCR of a representative clone from the first metK KO approach. Left: Agarose gel of the colony PCR. Bands are shown for a representative clone from the KO attempt (∆metK) as well as ∆metEH as wt ctrl. Right: Schematic overview showing where the primers bind and the fragment lengths of their PCR products.
This could either mean, that both foreign MAT copies do not support the growth of E. coli, or that the expression of these genes from a plasmid is insufficient, due to plasmid copy number and/or expression from the lac promotor employed on the plasmid. In their study, Parungao and coworkers observed starvation for Met in their engineered strains. There is a very sensitive intracellular equilibrium for Met and SAM, as both products regulate their own biosynthesis via feedback inhibition.
Met starvation was likely caused by the overproduction of SAM from the metK genes on high copy plasmids481.
3.1.5.2 2
ndapproach: CRISPR/Cas9
Thus, to rule out effects caused by the expression vector, another KO approach was tested, where the E. coli gene was attempted to be replaced by the S. solfataricus/M. jannaschii gene directly on the chromosome while retaining the original metK promoter. To this end, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) based approach was tested. The CRISPR/Cas system is an ancient immune system in bacteria as a protection against foreign DNA (e.g. from viruses). After the survival of an infection, bits of the foreign DNA are stored in a CRISPR array, which serves as a library and helps the cell “remember” foreign DNA for a quick response upon a recurring infection with the same pathogen. Together with a trans-activating CRISPR RNA (tracrRNA) and a Cas protein, the foreign DNA is precisely targeted and cleaved (for more details on CRISPR/Cas see section 5.2.17.2, p. 114). Emmanuelle Charpentier, who came across the system by
C1+C5 C1+C4 C1+C2
C1
C2 C5
C1 C4
M
KanR Ec metK
FRT FRT
1209 bp
843 bp
1 2 3 4 5 6 ΔmetK ΔmetEH C1+C5
C1+C4 C1+C2
1544 bp
530 bp C2
500 bp 1000 bp 1200 bp 1500 bp
studying pathogenic bacteria in the hope of discovering a new antibiotic, collaborated with Jennifer Doudna, who came across CRISPR as an expert for small regulatory RNA, to develop a genetic tool which they published in their seminal paper in 2012482. Since then, CRISPR/Cas has revolutionized genetic engineering, and Charpentier together with Doudna were awarded the Nobel Prize in Chemistry 2020 “for the development of a method for genome editing”.
By now, numerous CRISPR/Cas systems for a variety of applications and several Cas proteins for the targeting of a wide range of DNA sequences have been published483. Most systems, however, require the design of a new guide RNA (gRNA, a tracrRNA/CRISPR RNA chimera) for each target, which, despite the availability of several online tools, can be cumbersome and Cas9 off-target effects limit the targetable sequence space484,485. Therefore, a more general system that does not require the design of specific gRNAs for each target was chosen here. Zhao and coworkers developed a technique they coined “the CRISPR/Cas9-assisted gRNA-free one-step (CAGO) genome editing technique”486. Critical for their approach was the design of a universal CRISPR/Cas9 recognition sequence (N20PAM) with minimal sequence homology to the E. coli genome to minimize Cas9 off-target effects. This universal N20PAM can be incorporated into any desired editing cassette and is targeted by its homologous gRNA, which is supplied on the pCAGO plasmid. Additionally, the cas9 gene, as well as the λ red recombination system, are supplied on the pCAGO plasmid.
Figure 22 I Schematic overview of processes involved in the CAGO technique. Left homo: left homology region, R short: first 40-50 bp of the right homology region, CmR: chloramphenicol resistance cassette, Right homo: right homology region. The scissors represent Cas9-mediated DNA cleavage of the universal N20PAM sequence, DSB: double-strand break.
Step one of the CAGO editing technique is the design and assembly of a suitable editing cassette, which should be comprised of three homology regions targeting the desired gene locus, a selection marker, and the universal N20PAM for later removal of the selection marker (Figure 22). In the case of a simple gene KO, no additional insert is needed. However, if editing of a gene locus or insertions of any kind are desired, an appropriate insert bearing the desired modification needs to be incorporated into the editing cassette. In this study, the goal was to replace the E. coli metK gene with a chloramphenicol resistance cassette followed by a copy of either the S. solfataricus or M. jannaschii metK gene. After λ red-mediated recombination of the editing cassette at the desired locus (Figure 22, step two), the success of the recombination is verified via colony-PCR and/or sequencing analysis. Positive clones are then subjected to step three, where the selection marker is removed via Cas9-mediated DNA cleavage and λ red-assisted recombination. Finally, the pCAGO plasmid bearing a temperature-sensitive ori is cured by incubation at 42°C (step four, not shown in the figure).
For the editing cassette, four PCR fragments with BsaI recognition sites were generated and the cassette was assembled in a one-pot golden gate reaction. Hereby, alternating cycles of digestion with the BsaI restriction enzyme and ligation with the T4 DNA ligase yielded the desired cassette, which was further amplified in a final PCR reaction due to the high DNA requirements during electroporation and recombination. Three different editing cassettes were generated: one bearing the S. solfataricus metK
Left homo Mj / Ss metK (ctrl: Ec metK) CmR Right homo
R short R short
N20PAM
Left Right
Chromosome
Left homo Mj / Ss metK (ctrl: Ec metK) CmR Right homo
R short
R short N20PAM
Chromosome
Step 1
Assembly of editing cassette
Step 2
Recombination into target locus
Left homo Mj / Ss metK (ctrl: Ec metK) CmR
R short N20
Right homo PAM
DSB
Left homo Mj / Ss metK (ctrl: Ec metK)
R short
Right homo
Step 3
Cas9 - mediated DNA cleavage and
red - mediated recombination λ
Chromosome with desired insert / mutation
gene, one harboring the M. jannaschii metK gene, and a control harboring the E. coli gene to monitor any potential problems with this new editing technique. The successful assembly was verified via sequencing analysis.
For the introduction of the editing cassette into the target organism, the λ red system was induced from pCAGO in ∆metEH::FRT, the cells were made electrocompetent, and electroporated with about 400 ng of the editing cassette. After approximately 2 h of recovery in rich media lacking glucose (to avoid suppression of the lac promoter and concomitant λ red expression), the cells were plated on Agar-plates with Cm and incubated overnight at 30 °C. Single colonies were picked and the success of the recombination was monitored via sequencing analysis.
Figure 23 I Sequencing chromatogram of a representative colony from the CAGO attempt with the M. jannaschii and S. solfataricus editing cassettes. The sequencing chromatograms are aligned to the E. coli control cassette with its left homology arm (dark blue), the E. coli metK gene (green), R short fragment (light blue), Cm resistance cassette (grey), N20PAM (purple), and right homology arm (dark blue).
However, all sequenced colonies exhibited the control cassette with the E. coli metK gene, even those transformed with the M. jannaschii and S. solfataricus cassettes. To rule out any cross-contamination with the control cassette, the M. jannaschii and S. solfataricus cassettes were reassembled with fresh stocks and fresh water without handling the control cassette in parallel. Additionally, the freshly assembled editing cassettes were once again sequenced immediately before the transformation.
Nevertheless, sequencing analyses after another recombination attempt revealed the same outcome as before: the E. coli metK gene followed by the homology regions, Cm resistance cassette, and N20PAM (Figure 23). As the correctness of the editing cassettes was confirmed and contamination with the control was ruled out, it seems likely that these archaeal genes are not able to support E. coli growth and cannot substitute for the endogenous metK. This is further supported by the lack of success of the first KO approach. Furthermore, bacteria and archaea are two entirely different kingdoms and therefore not closely related. It is perhaps not too surprising, that substituting such an essential gene with an ortholog from another kingdom does not support growth. In fact, it is a common approach to obtain orthogonal aaRS/tRNA pairs for stop-codon suppression, that do not interact with any endogenous aaRSs, tRNAs, or amino acids, from another kingdom308. The presence of the Cm resistance, however, is curious. The sequencing results look like the cells retained their endogenous metK gene, while only the part of the editing cassette bearing the Cm resistance and right homology regions were recombined into the chromosome. Such a recombination event may have been driven by the presence of Cm in the agar plates, but partial recombination of editing cassettes is highly unusual and a mechanism is not known. The recombination events leading to these results were not studied further. Instead, it was decided to focus on a third approach.
MG1655 metK regi...
800 1,000 1,200 1,400 1,600 1,800 2,000 2,200 2,400 2,600 2,800 3,000 3,200 3,400 3,600 3,800 4,000 4,200 4,400 4,562
3,086,242 3,086,442 3,086,642 152 352 552 752 952 1,152 578 778 978 1,178 3,400 3,600 3,800 4,000 4,200 4,400 4,562
Consensus
3,086,242 3,086,442 3,086,642 152 352 552 752 952 1,152 578 778 978 1,178 3,400 3,600 3,800 4,000 4,200 4,400 4,562
galP gene
3.1.5.3 3
rdapproach: Point mutation
Due to the complex nature of methionine’s involvement in translation initiation, elongation, and the synthesis of the major methyl donor SAM, it was decided to simplify the goal of this project and focus solely on the adaptation to the closer structural analog ethionine. The purification optimizations (Appendix 9.1, p. 156) of the M. jannaschii and S. solfataricus MATs for the in vitro assay to test for TfMet turnover were thus discontinued.
In their study, Dippe and coworkers demonstrate that a single point mutation, in the metK gene from Bacillus subtilis vastly improves its ethionine turnover to S-adenosyl ethionine471. With a sequence homology of 65%, the MAT enzymes from B. subtilis and E. coli are closely related and the mutated residue (I302 in E. coli) is highly conserved throughout all tested MAT enzymes. Indeed, the crystal structure of the E. coli MAT reveals that the mutation of the isoleucine at position 302 to the shorter side chain of valine likely affords more space to accommodate the ethyl group of ethionine (Figure 24).
This mechanism has also been proposed by Dippe and coworkers for the success of their mutation in the B. subtilis enzyme471. Therefore, this point mutation was inserted in the metK gene in the strain
∆metEH::FRT to start a novel adaptation experiment.
Figure 24 I Crystal structure of the E. coli MAT with bound SAM (PDB: 1RG9). The isoleucine at position 302 is highlighted in red and bound SAM is shown as sticks colored by elements. The distance between the methyl group from SAM and the Ile side chain is shown in yellow (3.6 Å).
An editing cassette analogous to the control cassette described in the previous chapter was constructed, but bearing the desired I302V point mutation in the E. coli metK gene. The λ red system was induced and the editing cassette was transformed in ∆metEH::FRT. After incubation at 30°C overnight single colonies were picked and the recombination of the editing cassette at the metK locus was verified via sequencing analysis. After induction of both, the λ red system and Cas9, the cells were incubated at 30°C overnight in liquid culture and then spread on agar plates for the separation of single colonies. The removal of the resistance marker was verified via sequencing analysis (Figure 25 a). The pCAGO plasmid was cured via incubation at 42°C and removal of the plasmid was verified by streaking the cells on agar plates with and without the pCAGO selection marker ampicillin (Amp). This step was repeated until no cell growth on Amp could be observed (Figure 25 b). The resulting strain was termed
∆metEH::FRT metK(I302V) (abbreviated as metK(I302V)) and bears the isoleucine to valine point mutation at position 302 of the E. coli metK gene.
Figure 25 I Establishing the strain ∆metEH::FRT metK(I302V). a) Verification of the isoleucine to valine point mutation at position 302 (ATC -> GTC) of the E. coli metK. b) LB-agar plate verifying the removal of the pCAGO plasmid. Left: ampicillin supplementation, right: without ampicillin.
Next, the growth behavior of the new strain with the modified metK gene was compared to its ancestor
∆metEH::FRT, which was used in the first ALE experiment. Both strains were cultivated in NMM19 supplied with 15 µM Met and Eth concentrations ranging from 0-1000 µM Eth, OD600 measurements were taken over the time course of 48 h (Figure 26). The optical densities increase with the Eth concentration, whereby for ∆metEH::FRT there is a noticeable gap between cultures cultivated with up to 150 µM Eth and those cultivated with 500 µM Eth and 1000 µM Eth. In the case of metK(I302V) the optical densities do not vary as much, especially during exponential growth the OD600 values are very similar, regardless of the Eth concentration. While in the case of metK(I302V) as little as 15 µM Eth is enough for growth up to approximately OD600 = 1.1, the same amount of Eth only results in a maximal OD600 of around 0.9 for ∆metEH::FRT. At a concentration of 1 mM Eth, however, the ancestral strain reaches a higher optical density of approximately 1.7, while the new strain only reaches values of around 1.5. Thus, high ethionine concentrations seem to have less impact on the growth of the new strain than on its ancestor ∆metEH::FRT.
Figure 26 I Comparison of optical densities of the new strain metK(I302V) and its ancestor ∆metEH::FRT in the presence of increasing Eth concentrations. a) OD600 values of ∆metEH::FRT cultivated in NMM19 supplied with 15 µM Met and Eth concentrations ranging from 0-1 mM measured over 48 h. b) OD600 values of metK(I302V) cultivated in NMM19 supplied with 15 µM Met and Eth concentrations ranging from 0-1 mM measured over 48 h.
a)
2,140 2,150 2,160 2,170 2,180 2,190 2,200 2,210 2,220 2,227
MG1655 metK...
2,140 2,150 2,160 2,170 2,180 2,190 2,200 2,210 2,220 2,227
C G T T G T G A A A T T C A G G T T T C C T A C G C AG T C G G C G T G G C T G A A C C G A C C T C C A T C A T G G T A G A A A C T T T C G G T A C T
Measuring optical densities at 600 nm is the most widely used and easiest way to monitor bacterial growth. However, these values make no statement about the viability of the measured cells, as dead cells are measured together with the live ones. Furthermore, changes in cell morphology may skewer the correlation between OD600 and cell number, and starvation for SAM is known to result in longer cells, as DNA replication and cell division are regulated by methylation385. As Eth is not a very good substrate for bacterial MATs and Met is only supplied in limiting concentrations, the cells in this study may also be somewhat starved for SAM. Therefore, in addition to measuring OD600 values, the viability of the cells was assessed by counting colony forming units (CFU).
As before, both strains were cultivated in NMM19 supplied with 15 µM Met as well as with 0 µM Eth, 15 µM Eth, 50 µM Eth, and 100 µM Eth. For the first 8 - 10 h samples were taken every 2 h with further samples taken after approximately 24 h, 30 h, and 48 h. OD600 values were measured and dilution series were spotted on agar plates with media compositions corresponding to those of the respective liquid culture. After incubation at 37°C, the CFU per mL of cell culture were counted and plotted with the corresponding OD600 values against the cultivation time (Figure 27).
The number of CFUs correlates with the corresponding OD600 values in cultures where no Eth is supplied. However, upon Eth supplementation, a clear discrepancy between OD600 values and CFU can be observed. The number of CFU starts to decrease after approximately 6 h, when the OD600 values are still increasing, indicating a diminished number of cells capable of reproduction while the cell mass is growing. This observation might be well in line with a functional protein biosynthesis, accounting for the increase in cell mass, and defective methylation, hampering DNA replication487,488, gene regulation489, and cell division385,490. In the time between around 8 h and 22 h of cultivation, the decrease in CFU is especially noticeable and this effect is even more pronounced for Eth concentrations of 50 µM and 100 µM.
Therefore, for the second ALE experiment, a distinctly lower Eth concentration of only 15 µM was chosen in contrast to 100 µM Eth during the first experiment. Furthermore, instead of employing the MOPS-buffered NMM used in the first ALE, it was decided to use regular, phosphate-buffered NMM, as it was successfully used in five different adaptation experiments in the Budisa lab (adaptation to [3,2]Tp231, 4-F-Trp, 5-F-Trp491, 6-F-Trp, 7-F-Trp [data not yet published]).
Figure 27 I Comparison of OD600 values and CFU between ∆metEH::FRT and metK(I302V) for increasing Eth concentrations.
a) Values for the strain ∆metEH::FRT with Eth concentrations increasing from 0-100 µM from top to bottom. b) Values for the strain metK(I302V) with Eth concentrations increasing from 0-100 µM from top to bottom. Values represent the mean of two (50 µM Eth, 100 µM Eth) and three (0 µM Eth, 15 µM Eth) experiments with the standard deviation as error bars.
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