Publication highlights

 

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.

 

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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.