4.1 Evolution of bacterial strains toward methionine analog usage
The aim of the projects presented here was to deepen our knowledge of the genetic code, including its evolution and flexibility, by using ncAAs and their incorporation on a proteome-wide scale as a tool.
The unambiguous proteome-wide replacement of the latest addition to the genetic code, Trp, with a number of different analogs was already shown231,491. The second part of this thesis discusses attempts at further alienating one of these strains from life as we know it. The first part of this thesis aims at adapting E. coli to replace another late addition to the genetic code, Met, which is the only other amino acid encoded by a single codon. As Met is not only used in protein biosynthesis but is also a precursor for the major cellular methyl donor, such a strain would not only increase our understanding of the genetic code but also our knowledge of transmethylation reactions and pose a potential platform for intriguing trans-alkylation reactions. Furthermore, the more strains with replaced cAA there are and the better the underlying mechanisms are understood, the more options there are for combining them and creating organisms with multiple amino acid replacements, thus alienating them farther and farther (and making them safer).
Such adaptation experiments require strains that are auxotrophic for the amino acid to be replaced.
Therefore, an MG1655 derivative auxotrophic under all cultivation conditions (cobalamin present/not present, aerobic/anaerobic, Hcy formation via SAH recycling) was created by knocking out the last two genes in the methionine biosynthesis pathway, metE, and metH. The resulting strain, denoted as
∆metEH::FRT, was cultivated in the presence of Eth/TfM for approximately 4 months with decreasing Met concentrations. Unfortunately, even after more than 50 passages no increase in optical densities, indicating adaptation to the Met analogs, could be observed. In comparison, during the adaptation experiments with [3,2]Tp, 4-F-indole, and 5-F-indole the Trp precursor indole was completely removed from the cultivation media within around 25 to 30 passages491,513. The lack of adaptation observed here was attributed to methionine’s role as a precursor for S-adenosyl methionine and the fact that the bacterial enzyme catalyzing this reaction (MAT) is not very permissible towards Met analogs.
Thus, two promiscuous archaeal MAT enzymes were chosen to replace the endogenous variant.
However, even after three different approaches, the endogenous metK gene could not be replaced with any of the two archaeal orthologs, suggesting that these orthologs do not support E. coli growth.
Due to the complexities of methionine’s role as SAM precursor and concomitant difficulties with the experiments, the aim of the project was amended to adaptation towards utilization of the close structural analog ethionine.
A metK point mutation known to increase Eth to SAE conversion in B. subtilis was established in
∆metEH::FRT, yielding the strain metK(I302V). Comparison of this new strain's cultivation characteristics with its ancestor revealed, that optical densities of the ancestral strain depend much stronger on the Eth concentration than those of metK(I302V). While as little as 15µM Eth suffice for metK(302V) to grow to optical densities of approximately 1.0, higher Eth concentrations do not increase OD600 values by much. In the case of ∆metEH::FRT, on the other hand, low Eth concentrations result in lower optical densities, while higher Eth concentrations result in higher optical densities than those of metk(I302V).
A second adaptive laboratory evolution was conducted with the new strain, which was restricted to 31 passages due to time constraints. While one of the six populations exhibited a striking increase in OD600
in passage 21, indicating a possible adaptation event, this effect could no longer be observed in the following passages. As discussed in chapter 3.1.4 (p. 37), the passage size chosen for a given ALE experiment is crucial to the outcome. The observations made in passage 21 and the following passages suggest, that a beneficial mutation might have arisen in this population, but was lost again during
passaging. It is possible that the passage size of 0.02 OD600, which was chosen because it produced satisfying results during the ALE experiments with Trp, might be too small to allow accumulation of beneficial mutations for the adaptation towards Met analog usage in a reasonable time frame.
Comparison of optical densities and CFU assessed after a month of cultivation to those taken before the adaptation experiment, revealed a slight increase in general fitness in the presence of Eth. The plots of the 31Eth populations resemble those of metK(I302V) cultivated in minimal media lacking Eth prior to the ALE experiment, while the plots of the 31Met control populations cultivated in the presence of the analog resemble those of the unadapted strain. These observations could be in line with the cells entering a state of stress upon the first confrontation with the synthetic amino acid, resulting in a noticeable drop of CFU after only 8 h of cultivation. After continuously being confronted with Eth for 31 passages, the number of CFU remains stable in the presence of Eth for at least 24 h.
The control populations, on the other hand, exhibit the same drop of CFU after approximately 8 h of cultivation in the presence of Eth, indicating a response to Eth in 31Eth populations rather than simply adaptation to prolonged cultivation in minimal media.
Characterization of the [3,2]Tp-adapted strain TUB170 revealed a relaxation of the RpoS-mediated general stress response as the key mechanism for adaptation (chapter 1.3.1, p. 22, data not yet published). The general stress response is triggered upon entering stationary phase and provides protection against a number of different stresses514,515. It is also connected to the stringent response, which is triggered by amino acid starvation516,517. It would certainly be interesting to investigate whether an inactivation or attenuation of the general stress response would also be beneficial for the adaptation towards Eth utilization and if RpoS might already be involved in the slight relaxation of the 31Eth populations.
Furthermore, it would be interesting to investigate why there is such a large increase in OD600, upon Eth addition, and especially why this phenomenon can only be observed for the first few passages, and why there is such a large discrepancy in the number of corresponding CFU.
It might be worthwhile to continue the ALE experiments with Eth by reviving the “frozen fossils” from passage 31. Despite there being no general increase in optical densities after a month of cultivation, the stable number of CFU is promising and gives a tentative hope that a complete adaptation to Eth utilization might yet be achievable with the Met-auxotrophic strain established in this study.
Taken together, an E. coli strain auxotrophic for Met under all cultivation conditions was established and further improved for ethionine to S-adenosyl ethionine turnover. Finally, tolerance towards the ncAA ethionine could be improved within 31 passages of cultivation in the presence of this Met analog.
4.1 Biocontainment of TUB170
This project aimed to achieve biocontainment of the [3,2]Tp-adapted strain TUB170. While this Trp-auxotrophic strain survives on the non-canonical substrate [3,2]Tp, the complete substitution of the canonical amino acid with the synthetic substrate is only possible in the absence of Trp or its precursor indole. In this strain, incorporation of the ncAA relies on the endogenous TrpRS, which is unable to discriminate between its natural substrate Trp and the close structural analog [3,2]Tpa231. However, by replacing this promiscuous enzyme with one that exclusively recognizes the synthetic amino acid, biocontainment could be achieved, bringing us one step closer to creating safe synthetic organisms unable to survive in a natural environment.
Initial attempts to select an orthogonal translation system capable of distinguishing between Trp and [3,2]Tpa focused on the pyrrolysyl system due to its permissiveness towards anticodon mutations.
While selection systems for orthogonal translation systems require stop codons, the goal of this project was to incorporate the target ncAA at Trp codons. Thus, after selection of a suitable aaRS, the anticodon on its cognate tRNA would need to be mutated for suppression of Trp codons in the adapted strain TUB170. However, as extensive selection experiments with several different PylRS libraries and a variety of different selection conditions did not yield an enzyme capable of [3,2]Tpa incorporation, other systems more suitable for the incorporation of smaller, aromatic side chains were taken under consideration.
The first enzyme that came to mind was the TrpRS enzyme that already recognizes the target amino acid and even charges a tRNA with the correct anticodon. Changing the active site in a way that enables the enzyme to exclusively recognize [3,2]Tpa or at least prefer it over the natural substrate would result in a biocontained organism. An alternative selection system for selections with sense codons was established and suitable controls were implemented. However, a sub-optimally randomized TrpRS library, coupled with a propensity for false-positive results of the alternative selection system lead to reconsiderations of this approach. Furthermore, the goal for biocontained organisms is to keep escape frequencies below the NIH-defined threshold. Using the natural system as a starting point might encourage the reversal of introduced mutations to the wildtype and might thus not be the ideal strategy to obtain stable biocontainment.
Therefore, the spotlight fell on a related enzyme that also incorporates an aromatic amino acid and for which a very successful OTS already exists: the M. jannaschii TyrRS/tRNATyr pair. Selection experiments with a MjTyrRS-derived library yielded two promising candidates that showed potential in two different screening assays. Unfortunately, MS analysis revealed Gln incorporation as the main product, which is a common phenomenon during stop-codon suppression in the absence of an OTS503. Ambiguous interpretation of smaller peaks in the mass spectrum coupled with the promising results of the screening assays lead to the hypothesis, that these mutants do incorporate the target amino acid [3,2]Tpa, but not efficiently enough to suppress background suppression. However, efforts to improve variant 33 by creating an error-prone PCR-based library did not succeed.
Finally, the amino acid [3,2]Tpa was synthesized enzymatically from its precursor [3,2]Tp, as having to convert the precursor to the corresponding amino acid in addition to expressing the OTS and selection markers might have posed too much stress for the host cells. Furthermore, as the target ncAA had to be produced intracellularly from the precursor, there might not have been enough of the target ncAA present at the onset of the selections to drive selections towards [3,2]Tpa-incorporating variants. This might have been the reason for the lack of success of the previous selection experiments.
Reassessment of the promising variants from previous selection experiments in the presence of the amino acid revealed that these mutants likely do not incorporate [3,2]Tpa. Last selection experiments,
this time in the presence of the amino acid rather than the precursor, failed to produce any promising candidates.
It is worth mentioning that [3,2]Tpa and its precursor [3,2]Tp are sensitive to light, temperature, and oxidation, resulting in the formation of polymers518,519. Furthermore, the low water solubility of the indole analog resulted in low yields of the enzymatic reaction. Therefore, only small amounts of the ncAA were available for selection experiments, and polymerization of the amino acid during cultivation might have further decreased the concentration available for stop-codon suppression. Thus, it is possible that extensive selection with larger amounts of [3,2]Tpa might yet yield the desired TpaRS, as a large excess of ncAAs is known to be conducive to ncAA incorporation308.
However, taken together, the results outlined above suggest that the creation of an enzyme capable of discriminating between Trp and [3,2]Tpa is unlikely to succeed. After all, this analog was chosen for the adaptation experiment precisely for its close structural resemblance to Trp and its good compatibility with the host protein expression system. This close resemblance and its concomitant minimal invasiveness in the proteome are likely the features that drove the success of the adaptation and at the same time represent the Achilles’ heel of the project described here.
Nevertheless, the importance of biocontainment has steadily increased over the years, which is perhaps more obvious now than ever. The need for effective containment strategies to prevent the spread of SARS-CoV-2 has gained wide-spread awareness throughout the general public. However, the health sector is only one of numerous areas where biocontainment is critical, especially with the increasing application of GMOs as sustainable industrial workhorses. For example, recently bio-solar cell factories for the fixation of CO2 to value-added chemicals were developed520,521. Such applications outside of a controlled laboratory environment increase the risk of GMOs escaping into nature, where the consequences cannot be foreseen. Therefore, Lee and coworkers have recently developed a biocontainment system for cyanobacteria producing α-farnesene, by knocking out the CO2
concentrating mechanism. These engineered microbes are no longer able to survive under ambient CO2 concentrations and depend on high CO2 concentrations for survival and α-farnesene production.
Farnesene is a renewable hydrocarbon building block and serves as a precursor for high-performance polymers and is a bio-jet fuel candidate522.
Another area where biocontainment plays a significant role is agriculture. Our growing world population is accompanied by an increasing demand for food without an expansion of arable land523,524. To avoid further loss of our natural ecosystems it is thus highly desirable to enhance food production without employing harmful chemicals. One promising approach is the utilization of plant growth-promoting rhizobacteria (PGPR)525, some of which are already commercial526,527. To avoid their spreading throughout the environment and colonizing off-target hosts, a few methods have been developed. A synthetic signaling circuit enables plant-host-specific communication and limits the expression of plant growth-promoting genes to the presence of the desired target528. That same circuit can also be wired to activate essential genes529,530 or control genetic “kill switches”531,532.
Genetic manipulation of plants relies on Agrobacterium tumefaciens as a transformation tool. Hence, prior to the release of transgenic plants, the bacterium must be completely eradicated from the plant, which is not always easy. Biocontainment strategies have the potential to facilitate Agrobacterium removal, but no truly satisfying systems have been generated to date533. Efforts towards Agrobacterium biocontainment include encoding the production of the sugar levan, which is toxic for Gram-negative bacteria, as a kill switch. However, a single mutation suffices to inactivate the toxic gene534. Another approach revolves around controlling the expression of the gene required for T-DNA transfer (virE2) by expressing it from an inducible promoter. However, this system is not completely tight and T-DNA transfer does not entirely depend on virE2 expression535.
More robust biocontainment could likely be achieved by combining multiple approaches, according to the principle “the farther the safer”. Thus, even though various biocontainment strategies for different fields and organisms have emerged in recent years, further improvement and development of new strategies, of which synthetic biology approaches show great promise, would be beneficial. For example, Rubini and Mayer recently reported a system that relies on the catalysis of abiotic reactions by biocompatible Pd and Ru catalysts. They employed an E. coli strain addicted to the ncAA 3-nitro-L-tyrosine (3nY) in the presence of ampicillin and managed to localize growth by spotting the catalyst on Agar plates containing Amp and allyloxycarbonyl-protected 3nY (alloc-3nY). The strain depends on alloc-3nY deprotection by the catalyst for growth. Further, they were able to reuse a second catalyst by entrapping Pd nanoparticles in polystyrene beads, thus creating a modular tool for biocontainment that can be transferred to other organisms and combined with other biocontainment strategies536.