Publication highlightsCopyright: © BioVI
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.
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 metatheaseCopyright: © BIO VI
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.
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 racematesCopyright: © BioVI
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.
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 perspectiveCopyright: © 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.
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 hydroxylaseCopyright: © Bio VI
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 methodCopyright: © Chemistry – A European Journal
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.
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.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.
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.
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.