Publication highlights

  Ulrich Markel Copyright: BioVI Ulrich Markel

Ulrich Markel, Khalil D. Essani, Volkan Besirlioglu, Johannes Schiffels, Wolfgang R. Streit and Ulrich Schwaneberg*, Chem. Soc. Rev., 2020, 49, 233-262. DOI:10.1039/C8CS00981C

Here, we review state-of-the-art and up-and-coming ultrahigh-throughput methods for the screening of large libraries in directed enzyme evolution.

 
  Advances in ultrahigh-throughput screening for directed enzyme evolution Copyright: Chemical Society Reviews State-of-the-art ultrahigh-throughput screening methods use cells and biomimetic compartments for the directed evolution of enzymes and are promising for the field of functional metagenomics.

Enzymes often need to be re-engineered or optimized in order to exploit their full potential. (Semi-) rational design requires detailed knowledge about structure function relationships. In turn, directed evolution methodologies can improve an enzyme’s properties without structural knowledge by iterative rounds of diversity generation and screening. Current diversity generation methods allow us to generate huge libraries but conventional screening on agar plates or in microtiter plates fails to interrogate the full generated diversity in reasonable time. Ultrahigh-throughput screening methods drastically increase the number of enzyme variants we can screen and speed up biocatalyst design ultimately widening our knowledge about sequence function relationships. In the present review, we summarize recent advances in ultrahigh-throughput screening for the directed evolution of enzymes. We discuss the importance of compartmentalization to link genotype and phenotype and illustrate how cells and biomimetic compartments can serve this function. Finally, we discuss how new functional metagenomics approaches can profit from ultrahigh-throughput screening to identify natural biocatalysts for novel chemical transformations.

 

Chemoenzymatic cascade for stilbene production from cinnamic acid catalyzed by ferulic acid decarboxylase and an artificial metathease

Daniel Sauer, Stephanie Mertens Copyright: BIO VI Daniel Sauer and Stephanie Mertens

M. A. Stephanie Mertens,‡ Daniel F. Sauer,‡ Ulrich Markel, Johannes Schiffels, Jun Okuda,* Ulrich Schwaneberg,* Catalysis Science & Technology, 2019, DOI: 10.1039/C9CY01412H

The combination of a decarboxylase and an artificial metathease in a chemoenzymatic cascade reaction for stilbene production with efficient removal of metal contamination is reported.

 
  Chemoenzymatic cascade for stilbene production from cinnamic acid catalyzed by ferulic acid decarboxylase and an artificial metathease Copyright: Catalysis Science & Technology Combination of a decarboxylase and an artificial metathease in a chemoenzymatic cascade reaction for stilbene production

A chemoenzymatic cascade reaction involving a biocatalyst and a biohybrid catalyst for the production of stilbene derivatives was designed. Stepwise conversion of cinnamic acid as a renewable resource to valuable compounds was achieved in one pot in aqueous solution and under mild reaction conditions. The ferulic acid decarboxylase FDC1 from Saccharomyces cerevisiae was used for the conversion of cinnamic acid. In a following reaction, cross-metathesis of the styrene intermediate was performed with an artificial metathease, FhuA-GH, based on a Grubbs-Hoveyda catalyst incorporated into an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A, FhuA. Intermediate workup steps and isolation of the styrene intermediates was not required, as both reaction steps proceeded in aqueous solution. In comparison to the protein-free catalyst, cascade reactions with the artificial metathease revealed a significant lower metal content after a simple extraction step. The cascade reaction is the first example of the combination of biocatalysts and biohybrid catalysts for efficient removal of metal impurities in the product fraction.

 

Tailor-made membrane channel enables chiral separation of racemates

Copyright: BioVI Deepak Anand

Deepak Anand, Gaurao V. Dhoke, Julia Gehrmann, Tayebeh M. Garakani, Mehdi D. Davari, Marco Bocola, Leilei Zhu, and Ulrich Schwaneberg, Chemical Communications, 2019. DOI: 10.1039/c9cc00154a.

Chiral resolution of arginine enantiomers was achieved through an engineered Escherichia coli outer membrane protein FhuA.

 
  Schematic representation of chiral resolution of arginine racemate through engineered FhuA channel. Copyright: Chem. Commun. Schematic representation of chiral resolution of arginine racemate through engineered FhuA channel.

Chiral molecules are of large economic value in chemical, pharmaceutical, and food industries. Separation of enantiomers can be achieved by various methods but it still remains a challenging task. Chiral protein-polymer membranes would be an attractive, cost-effective, and scalable alternative for chiral separation. The main challenges lie in the design of filter regions within the channel proteins and the development of screening systems to identify chiral channel protein variants. In the present study, we report for the first time a chiral β-barrel channel based on ferric hydroxamate uptake component A – FhuA, an outer membrane protein of E. coli. Two filter regions were identified and redesigned through screening of multi-site saturation mutagenesis libraries, in order to achieve the chiral separation of a D-/L-arginine racemate. Screening resulted in identification of FhuAF4 variant showing an improved enantiomeric excess of 24% at 52% conversion compared to the parent FhuA variant. Interestingly, even a subtle change of just two amino acids considerably influenced the selectivity of the FhuA channel. Steered molecular dynamic simulations indicated that the separation is based on diffusivity differences of two enantiomers through FhuAF4. It is likely that with the identified filter region and multi-site saturation libraries further improvements are achievable for separation of other amino acids and a broader range of enantiomers. The chiral FhuA channel proteins would be an excellent scaffold for generation of chiral membranes based on protein-polymer conjugates with a high potential for novel and scalable downstream processes.

We kindly acknowledge the German Federal Ministry of Education and Research (BMBF) for providing financial support to the Chiral Membranes I project. Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under the project RWTH0116.

 

Towards evolution of artificial metalloenzymes – A protein engineer’s perspective

Photos of Ulrich Markel and Daniel Sauer Copyright: BioVI

Ulrich Markel,# Daniel F. Sauer,# Johannes Schiffels, Jun Okuda, and Ulrich Schwaneberg, Ang. Chem. Int. Ed., 2018 , DOI: 10.1002/ange.201811042

In the present publication, an overview of the early approaches of directed evolution of biohybrid catalyst is given.

 
  Scheme of directed evolution of biohybrid catalysts Copyright: Ang. Chem. Directed evolution of artificial metalloenzymes/biohybrid catalysts. In contrast to the directed evolution of natural enzymes, the directed evolution of artificial metalloenzymes/biohybrid catalysts requires two additional steps (orange).

The incorporation of artificial metal-cofactors into protein scaffolds yields a new class of catalysts termed biohybrid catalysts or artificial metalloenzymes. In addition to modification of the artificial cofactor, these biohybrid catalysts can be modified at the second coordination sphere provided by the protein scaffold. Protein engineering provides tremendous potential to tailor such biohybrid catalysts but requires a robust screening system and sophisticated metal cofactor conjugation. In this minireview, we give an overview of the recent efforts in this field. We describe high-throughput screening methods applied for the directed evolution of biohybrid catalysts and we illustrate how non-chiral catalysts catalyze reactions enantioselectively by highlighting the first successes in this emerging field. Furthermore, we summarize the potential and limitations that need to be overcome to advance from biohybrid catalysts to true artificial metalloenzymes.

We gratefully acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG) through the International Research Training Group ‘Selectivity in Chemo- and Biocatalysis’ (SeleCa) and the Bundesministerium für Bildung und Forschung (BMBF) (FKZ: 031B0297). #: These authors contributed equally

 

Directed OmniChange evolution converts P450 BM3 into an alkyltrimethylammonium hydroxylase

Photo of Yu Ji Copyright: Bio VI Yu Ji, first author of the study on Omnichange evolution of P450BM3 into a alkyltrimethylammonium hydroxylase

Yu Ji, Alan Maurice Mertens, Christoph Gertler, Sallama Fekiri, Merve Keser, Daniel F. Sauer, Kilian E. C. Smith, and Ulrich Schwaneberg*, Chemistry - A European Journal, 2018, doi:10.1002/chem. 201803806

 
 

Reengineering of the P450 BM3 substrate specificity towards the hydroxylation of CTAB by OmniChange multi-site mutagenesis method

  Image of reaction educts and products Copyright: Chemistry – A European Journal Directed OmniChange evolution converts P450 BM3 into an alkyltrimethylammonium hydroxylase.

Bolaform surfactants are a novel class of compounds with a wide range of industrial and technical applications. Bolaform surfactants are capable of forming of very small micelles and therefore are more effective than contemporary surfactants.

However, their production is expensive and involves the use of strong acids and large amounts of solvents. An alternative green synthesis route is the direct hydroxylation through monooxygenases as performed in nature.

 
 

In this study, the OmniChange multi-site mutagenesis method was applied for reengineering of the P450 BM3 substrate specificity towards the hydroxylation of CTAB by simultaneous mutagenesis of four relevant positions. Improved variants were identified in a two-step screening system. Then 10 promising P450 BM3 variants were analyzed by HPLC-MS/MS. Four P450 BM3 variants had significantly improved productivities and were kinetically characterized after purification. Interestingly all four variants were capable to di-hydroxylate CTAB and coupling efficiency up to 92.5% were obtained.

Notably, di-hydroxylation products of CTAB with bolaform surfactant properties have for the first time been produced. Bolaform surfactants are biodegradable and sustainable surfactants that offer excellent solubility properties and novel possibilities for drug delivery and/or compound formulations. Additionally, the two-step screening system proved to be efficient to boost coupling efficiency and can likely be used in many other P450 evolution campaigns to generate robust P450 catalysts. This work was funded by the China Scholarship Council, No. 201608080082.

Please find the link to this publication here.

 

A loop engineering strategy improves laccase lcc2 activity in ionic liquid and aqueous solution

Photo of Anne Wallraf Copyright: Bio VI

A. M. Wallraf, H. Liu, L. Zhu, G. Khalfallaha, C. Simons, H. Alibiglou, M. D. Davari and U. Schwaneberg, Green Chemistry, 2018, DOI: 10.1039/C7GC03776G

 

Importance of a domain-connecting loop in laccase for increased activity in ionic liquid (IL) was identified.

Laccases are involved in lignin degradation. EMIM- and BMIM-based ionic liquids show excellent solubilization of wooden biomass but impede laccase activity. Protein engineering to improve the activity and resistance of laccases in ILs is promising for lignin valorization for the sustainable production of fuels and bulk high-value chemicals . We report for the first time an efficient semi-rational design with focus on a domain-connecting loop of a laccase lcc2 loop L1 from Trametes versicolor.

The loop engineering strategy is based on a KnowVolution campaign and can be divided into three steps. Prediction of seven resistance increasing positions out of 37 amino acids in L1 -residues 284-320- was performed by in silico SSM analysis with FoldX. These seven positions were saturated by SSM and four beneficial positions were subjected to simultaneous SSM using OmniChange. The OmniChange variants OM1 -A285P/A310R/A312E/A318G- and OM3 (A310D/A312P/A318R) showed a 3.9-fold -535.8 ± 36.9 U/mg- and 1.6-fold -216.8 ± 5.3 U/mg- increased specific activity in aqueous solution - lcc2 WT, 138.9 ± 6.5 U/mg, respectively, and up to 8.4-fold increased activity in 35% EMIM EtSO4 and aqueous solution when compared to lcc2 WT. Hydrogen bond pattern analysis revealed that both variants harbor an increased number of hydrogen bonds within the loop and between domains two and three which resulted in increased IL resistance.

 
  Flow scheme of the loop engineering experiment Copyright: Royal Society of Chemistry
 
 

Entropy analysis indicated that the substitution of alanine at each selected amino acid position A285, A310, A312, and A318 reduced the flexibility of the loop L1. Conservational analysis with ConSurf server showed that the long domain-connecting loop L1 is a conserved feature in fungal laccases and suggests loop engineering as a useful strategy for increasing laccase activity in ILs and aqueous solutions. This work was funded by Deutsche Forschungsgemeinschaft DFG in the frame of the research cluster “ Tailor-Made Fuels from Biomass ” TMFB.

 
 

Cavity size engineering of a beta-barrel protein generates efficient biohybrid catalysts for olefin metathesis

Photo of Alexander Grimm and Daniel Sauer Copyright: Bio VI

Grimm, A.R.*, Sauer, D.F.*, Davari, M.D., Zhu, L., Bocola, M., Kato, S., Onoda, A., Hayashi, T., Okuda, J., Schwaneberg, U.; ACS Catalysis, 2018, 8, pp 3358–3364, DOI: 10.1021/acscatal.7b03652. (*contributed equally)

 
 

The successful application of the presented cavity size engineering strategy to biohybrid catalysis can potentially be transferred to other beta-barrel protein scaffolds to generate cavity sizes that match the sterical demands of synthetic catalysts.

 
  biohybrid catalyst Copyright: Bio VI

By incorporating a synthetic metal catalyst into a protein scaffold, a biohybrid catalyst is obtained. The protein scaffold can potentially alter the selectivity of the metal catalyst.and solubilizes it in water, while retaining their broad reaction scope. What’s new about this work is that until now, protein scaffolds for synthetic catalysts have mainly been engineered by exchanging individual amino acids. While a lot has been achieved using this conventional strategy, it is limited by the number of amino acids in the protein available for substitution. Here, cavity size engineering of a beta-barrel protein called nitrobindin was performed by duplicating multiple beta-strands to generate an expanded variant.

 
 

"Incorporation of a synthetic metal catalyst into a protein scaffold yielded a biohybrid catalyst that combines remarkable performance in aqueous media with the broad reaction scope of organometallic catalysts."

Alexander R. Grimm

It is the first time this has been done for biohybrid catalysis. By duplicating entire beta-strands, it was possible to covalently incorporate bulky catalysts and achieve excellent conversions in a whole series of olefin metathesis reactions – carbon-carbon double bond formation reactions. What’s exciting about this is that the design strategy that was used can potentially be transferred to other beta-barrel protein scaffolds to generate cavity sizes that match the sterical demands of synthetic catalysts. If high-throughput screening is applied to these new variants in the future, it should be possible to explore directed biohybrid catalyst evolution much more efficiently, which will likely further increase performance. This work was made possible through funding from the Deutsche Forschungsgemeinschaft and Bundesministerium für Bildung und Forschung.

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  Biohybrid Catalyst Copyright: ACS Catalysis Figure 1 Comparison of conversion of cavity size engineered biohybrid catalyst and protein-free catalyst in ring opening metathesis polymerization.
 
 

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A Whole Cell E. coli Display Platform for Artificial Metalloenzymes: Polyphenylacetylene Production with a Rhodium-Nitrobindin Metalloprotein

Photo of Alexander Grimm Copyright: Bio VI

Grimm, A.R., Sauer, D.F., Polen, T., Zhu, L., Hayashi T., Okuda J., Schwaneberg, U. (2018). A whole cell E. coli display platform for artificial metalloenzymes: polyphenylacetylene production with a rhodium-nitrobindin metalloprotein. ACS Catal., 8, 2611-2613.

 
 

A Whole Cell E. coli Display Platform for Artificial Metalloenzymes

 
  Function principle of the whole cell catalyst Copyright: Bio VI

In the latest publication of our colleageus from the Hybrid Catalysis & High Throughput Screening division, the first bacterial cell surface display-based whole cell biohybrid catalyst, termed ArMt bugs, was generated, characterized and applied to the stereoselective polymerization of phenylacetylene. Whole cell catalysis is very important for the cost-effective production of chemicals by biotechnological means. Despite the promising application of whole cells to biohybrid catalysis, researchers worldwide had to face the inactivation of their valuable synthetic metal catalysts within cells due to abundant thiols.

 
 

The authors therefore armed the surface of their Escherichia coli whole cells with rhodium-nitrobindin biohybrid catalysts via an esterase-based autotransporter to separate the catalysts from inhibiting cellular compounds. A high turnover number of 39,000,000 per cell in the polymerization of phenylacetylene and potential applications in directed evolution and high-throughput screening make the ArMt bugs an attractive platform for bioorthogonal reactions. This work was made possible through funding from the Deutsche Forschungsgemeinschaft and Bundesministerium für Bildung und Forschung.

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  Functional principle whole cell catalyst Copyright: Bio VI
 
 

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In vitro flow cytometry-based screening

One of the main bottlenecks of directed evolution for successful tailoring of biocatalysts for industrial applications is ultrahigh throughput screening uHTS. uHTS enables analysis of up to 107 events per hour by which a coverage of the generated protein sequence space is ensured. Combination of flow cytometry based uHTS and cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. This is the first report where the flow cytometry screening system in combination with cell-free enzyme expression within emulsion compartments, termed InVitroFlow, was used for directed cellulase evolution. The celA2 mutant library containing high mutational load was generated and encapsulated in double emulsion compartments together with fluorogenic substrate and cell-free expression mixture, see Figure 4 Steps 1-3. The compartmentalization enables a genotype-phenotype linkage through an encapsulation of the gene, enzyme it encodes and generated fluorescent product within the same compartment. Compartment containing active enzyme variants can be sorted on flow cytometer based on the generation of fluorescent product, see Figure 4 Step 4. The genes contained in the sorted compartment can be isolated by PCR and can subsequently be used as template for further iterative rounds of directed evolution and/or cloned into a vector with a subsequent transformation into an expression host for MTP screening, see Figure 4 Steps 5-7.

  HTS platform Copyright: Bio VI  

Figure 4: InVitroFlow screening platform in 7 steps. Mutant library generation (1) and encapsulation into single (2) and double emulsion compartments (3) Analysis and sorting of the fluorescent compartments using flow cytometry (4). Isolation and amplification of DNA encoding for active enzyme variants using PCR (5) with subsequent cloning and transformation (6) for a fine characterization in MTP assay formats (7) or for a next iterative rounds of directed evolution.

The novel InVitroFlow screening platform was validated by screening a random mutant libraries and yielded improved cellulase variants, e.g. CelA2-H288F-M1, N273D/H288F/N468S, with 13.3-fold increased specific activity compared to CelA2 wildtype.

More detailed information on flow cytometry-based high-throughput screening can be found in the following publication:

Körfer, G., Pitzler, C., Vojcic, L., Martinez, R., and Schwaneberg, U. 2016. In vitro flow cytometry-based screening platform for cellulase engineering Sci Rep. 6:26128.

 

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High-troughput screening with "Fur Shell" hydrogels

High-throughput screening formats play a pivotal role in directed evolution experiments and enzyme discovery. A high-throughput screening system based on formation of fluorescent hydrogels around E. coli cells expressing active enzyme - "Fur Shell" - was established for phytase and later on the screening platform was advanced into a general Fur Shell based screening toolbox for directed evolution of hydrolases i.e. cellulase, esterase, and lipase. Cells expressing active hydrolase generate ß-D-glucose from glucose derived substrates as depicted in Fig. 3A which is subsequently converted by glucose oxidase under hydrogen peroxide production as depicted in Fig. 3B. Hydrogen peroxide serves as a source of hydroxyl radicals which initiates a fluorescent hydrogel formation around E. coli cells expressing active hydrolase variants as depicted in Fig. 1C.

 
  FurShell assay Copyright: Bio VI
 
 

Figure 3: Principle of Fur Shell technology using a coupled enzyme/GOx reaction leading to a formation of fluorescent hydrogel

The screening platform was validated by screening epPCR libraries for phytase, cellulase, esterase, and lipase in a single round of directed evolution and identification of improved variants 1.3 – 7-fold for four hydrolases. The presented Fur Shell screening platform is valuable prescreening system in order to isolate active enzyme variants and to minimize screening efforts in a cost effective manner.

The Fur Shell screening platform is a general platform for directed hydrolase evolution and it is easy to use and time efficient when compared to other reported flow cytometry screening systems in directed evolution. The principle of the Fur Shell technology can be adapted to other enzyme classes and has a potential to become a standard screening format in directed enzyme evolution. High throughput enabled by this technology will allow exploring a novel mutagenesis strategies and sampling through a protein sequence space even for a very short peptides. In addition, the presented platform is attractive for application in bio based interactive materials since it offers E. coli directed polymer capsule formation.

More detailed information on the topic of high-throughput screening can be found in these publications

Lülsdorf*, N., Pitzler*, C., Biggel, M., Martinez, R., Vojcic, L. and Schwaneberg, U. 2015. A flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogels. Chem Commun. 51, 41:8679-8682.

Pitzler, C., Wirtz, G., Vojcic, L., Hiltl S., Böker, A., Martinez, R., and Schwaneberg, U. 2014. A fluorescent hydrogel-based flow cytometry high-throughput screening platform for hydrolytic enzymes. Chem Biol. 21, 12:1733-1742.