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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - DrEAM (Directed Evolution of Artificial Metalloenzymes for In Vivo Applications)

Teaser

Summary

Living organisms rely on enzymes to sustain their vital function, including respiration, movement, digestion, reproduction, etc. These protein-based catalysts have evolved to display exquisite levels of activity and selectivity towards their natural substrates. Despite the attractive features of natural enzymes, the reaction repertoire that they catalyze remains somewhat limited. To produce high-added value molecules, chemists rely on both natural enzymes and small molecule catalysts (smolcats). These latter display features that are, in many ways, complementary to enzymes. Most importantly, smolcats give access to a very diverse range of reactions that have no equivalent in Nature but are very useful in chemical synthesis.
The DrEAM ERC project aims at combining attractive features of both enzymes and smolcats by designing and optimizing artificial metalloenzymes (ArMs). Building upon the vast reaction repertoire available to smolcats, the DrEAM creates ArMs that can catalyze new-to-nature reactions, thanks to the incorporation of smolcats within a protein scaffold.
We have demonstrated that ArMs can be optimized using directed evolution schemes, reminiscent of natural selection. Most importantly, these ArMs maintain their catalytic activity in a cellular environment. This feature allows the use of genetically engineered E. coli (resulting in a harmless microbe) as test-tube to produce high-added value chemicals by allowing natural enzymes and ArMs to work in concert. To achieve this ambitious goal, the following challenges are being addressed: i) optimize the incorporation of the smolcat within the host protein, overexpressed the E. coli; ii) improve the catalytic performance of the ArM in vivo to reach activities comparable to natural enzymes and iii) fine-tune the microbial factory to produce high-added value whereby waste and energy requirements are minimized.
The DrEAM team has developed three ArMs that catalyze new-to-nature reactions. These have been incorporated in E. coli, and directed evolution schemes have allowed improving their performance. We are in the process of combining these ArMs with natural enzymes to produce chemicals that are essential to E. coli\'s survival. This strategy allows us to improve further the ArM\'s performance relying on a selection scheme, reminiscent of Darwinian evolution: either evolve or die. Thus far, our efforts have been published in high impact open access publications and have received widespread appraisal (See, for example, Nat. Commun., DOI: 10.1038/s41467-018-04440-0, J. Am. Chem. Soc., DOI: 10.1021/jacs.8b07189 and Chem. Sci., DOI: 10.1039/c8sc00484f). The Principal Investigator was awarded the 2017 Royal Society of Chemistry award in recognition of his pioneering work on artificial metalloenzymes.
We anticipate that the engineered microbial factory developed within the DrEAM project will be widely applicable to produce high-added value chemicals by combining ArMs with natural enzymes.

Work performed

The work that forms the basis of the DrEAM proposal was published in Nature (2016, 537, 661) shortly prior to the initiation of the ERC project. Since October 2016, we have made significant progress towards the directed evolution of artificial metalloenzymes (ArM) for in vivo catalysis as summarized below.
In the DrEAM project, we proposed to assemble the ArM in the periplasm of E. coli. Although the periplasm offers several advantages to compartmentalize ArMs, it also presents the following challenges: i) it contains a peptidoglycan which hampers catalysis; ii) it contains proteins that may interfere with ArMs-catalyzed reactions; iii) it contains thiols and disulfides, albeit in lower concentration than in the cytoplasm; iv) it is separated from the medium by a semi-permeable outer-membrane, thus limiting cofactor and substrate uptake. These challenges were to be tackled in WP1 and WP2. As an alternative, we contemplated the possibility of displaying streptavidin (Sav) on the outer-surface of E. coli. This strategy offers the following attractive features: i) no physical barrier for cofactor and substrate uptake and ii) no detrimental thiols or proteins that may interfere with the ArM.
Accordingly, we set out to systematically compare the performance of ArMs either compartmentalized in the periplasm or displayed on E. coli\'s outer membrane. Two reactions were tested: transfer hydrogenation and allylic substitution. These efforts led to two publications (Chem. Sci., DOI: 10.1039/c8sc00484f and ACS Catal., DOI: 10.1021/acscatal.9b01006). The cofactor uptake was determined by ICP-MS and the turnover number of the ArMs was quantified by UPLC-MS. Gratifyingly, both systems performed equally well: up to 20\'000 biotinylated cofactor moieties are anchored within Sav both in the periplasm and on E. coli\'s outer membrane. Accordingly, the total turnover number/cofactor approaches 100 in vivo. In comparison, organometallic catalysts rarely display turnover numbers exceeding one in vivo. We compiled these data in a review (Curr. Op. Chem. Biol., DOI: 10.1016/j.copbio.2017.12.008). Two further publications investigated compartmentalization of ArMs within the periplasm of E. coli, testing various conditions to improve the cofactor uptake and the corresponding catalytic performance (transfer hydrogenation: J. Am. Chem. Soc., DOI: 10.1021/jacs.8b07189 and cyclopropanation Catal. Sci Technol., DOI: 10.1039/c8cy00646f).
Next, we co-expressed a pore protein (e.g. FhuA with its cork deleted inserted in the outer membrane and Sav secreted to the periplasm). Despite our efforts, the metabolic strain imposed to E. coli was high, leading to very limited Sav expression levels. Accordingly, the cofactor uptake, as determined by ICP-MS remained very low (< 5 % uptake, compared to > 15 % uptake for E. coli strains only secreting Sav to the periplasm. As alternative, we explored the possibility of using cell-penetrating polydisulfides (CPDs) to accumulate ArMs within mammalian cells. Relying on an engineered gene switch, the catalytic activity of the ArM led to the up-regulation of a luciferase gene. This work opens fascinating perspectives for the use of ArMs for medical applications. Accordingly, it received wide press-coverage (Nat. Commun., DOI: 10.1038/s41467-018-04440-0). The use of CPDs thus offers an attractive alternative to engineered pore proteins to accumulate ArMs in mammalian cells.
The DrEAM team has made significant progress in the design of a single chain dimer streptavidin (scdSav, WP3). We have engineered, structurally characterized and optimized the expression and catalytic performance of scdSav. For a challenging prochiral isoquinoline substrate, the catalytic performance of the artificial transfer-hydrogenase based on an evolved scdSav outperforms all other ArMs reported to date, both in terms of activity and selectivity. A publication is in preparation.
The main effort of the DrEAM team is currently focused on the implementation

Final results

As outlined in the DrEAM proposal, the ultimate aim of the research is to assemble and evolve an artificial metalloenzyme in an E. coli chassis to complement natural enzymes in vivo. With this goal in mind, we have demonstrated that both the periplasm and the outer membrane of E. coli are suitable to localize and evolve an ArM while maintaining the critical phenotype-genotype linkage.
Thus far, we have achieved over one hundred turnovers in vivo for an ArM\'s catalyzed reaction in vivo. To the best of our knowledge, this result far exceeds anything reported to date (typically single digit turnovers). We are confident that, relying either on selection schemes or high-throughput screening strategies, we can significantly further improve the in vivo catalytic performance of the ArM.