doi.org/10.26434/chemrxiv.12063243.v1

Molecular Packaging of Biocatalysts Using a Robust Protein CageNikola Loncar, Henriette J. Rozeboom, Linda E. Franken, Marc C. A. Stuart, Marco Fraaije

Submitted date: 02/04/2020 • Posted date: 03/04/2020Licence: CC BY-NC-ND 4.0Citation information: Loncar, Nikola; Rozeboom, Henriette J.; Franken, Linda E.; C. A. Stuart, Marc; Fraaije,Marco (2020): Molecular Packaging of Biocatalysts Using a Robust Protein Cage. ChemRxiv. Preprint.https://doi.org/10.26434/chemrxiv.12063243.v1

In this paper, we report on the discovery of a novel, robust protein cage (encapsulin) that we could use forpackaging various biocatalysts. We have elucidated the structure of the stable encapsulin by electronmicroscopy and X-ray diffraction. Furthermore, we developed an effective expression system for theencapsulin and a facile protocol for preparing encapsulated enzymes. By packaging and testing variousenzymes (varying in size, oligomeric structure, and cofactor type) we demonstrate that, throughencapsulation, the enzymes become significantly more stable. We also provide evidence that the pores of theencapsulin, through a size-exclusion effect, can modulate the substrate acceptance profile of an encapsulatedenzyme.

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Molecular Packaging of Biocatalysts Using a Robust Protein CageNikola Lončar†,‡, Henriette J. Rozeboom†, Linda E. Franken#,§, Marc C. A. Stuart#, and Marco W. Fraaije†,*

† Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands‡ GECCO Biotech, Nijenborgh 4, 9747AG, Groningen, The Netherlands

# Department of Electron Microscopy, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands

KEYWORDS: encapsulin, biocatalysis, oxidations, substrate specificity, crystal structure

ABSTRACT: Using a newly discovered encapsulin from Mycolicibacterium hassiacum, severalbiocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produceas recombinant protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of60 protomers (diameter of 23 nm) with narrow pores that connect the interior with the outside. Bydeveloping an effective coexpression and isolation procedure, the effect of packaging a variety ofbiocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stabilityof the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in theencapsulin. Due to the restricted diameters of the encapsulin pores (5 - 9 Å), the protein cage protectsthe encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuabletools to tune the properties of biocatalysts such as stability and substrate specificity.

INTRODUCTIONBacteria differ from eukaryotes by amongst

others being devoid of organelles.Compartmentalization provides eukaryotes with away to separate and optimize metabolicprocesses. Although typical membrane-encompassed organelles are lacking, bacteriahave found other ways to compartmentalizeenzymes by using proteinaceous structures.While the reason for bringing together a selectedset of enzymes within a protein shell is not alwaysclear, bacterial microcompartments areconserved and widespread in the bacterialkingdom.1,2 This strongly suggests that they areessential for proper functioning of theencapsulated enzymes. This type of subcellularco-localization may be to facilitate substratetransfer between connected reactions and toshield the separated metabolism frominterference by other cellular components.Moreover, encapsulation of enzymes within aprotein cage can promote the reaction rate as aconsequence of the high effective concentrationof enzyme, which leads to rapid formation of theenzyme–substrate complex.3,4 Experimental workdone on the different bacterial protein-based

compartments has revealed that the selectiveincorporation of target proteins depends on N- orC-terminal recognition peptides.5,6 This is similarto protein targeting in eukaryotic organelles, suchas the N- or C-terminal peroxisomal targetingsequences.7 Bacterial microcompartments arenormally larger (50-200 nm) and composed ofseveral building proteins while encapsulins aremuch smaller and composed of only one protein.

Only about a decade ago a newly recognizedclass of bacterial protein-basednanocompartments was described.8 These so-called encapsulins are formed by self-assembly of60 or 180 identical encapsulin protomers to formcages with a diameter of roughly 24 or 32 nm,which are divided into four families.2 In nature,these bacterial protein cages typically containone or a few different proteins, often enzymes.The encapsulated proteins are targeted to theinterior of encapsulins by virtue of a N- or C-terminal targeting peptide. The rationale for thetranslocation of specific proteins into encapsulinsis still enigmatic. In most cases, encapsulinscontain a ferritin-like protein or a dye-decolorizingperoxidase (DyP), while in some other cases othermetal-containing proteins have been identified.9 A

general feature of the cargo proteins seems tobe an involvement in redox processes, andtherefore it has been hypothesized thatencapsulins play a role in detoxificationprocesses.

Several viral nanoparticles and bacterialmicrocompartments have been produced asrecombinant proteins and have been studiedwith various cargo proteins in the context offundamental science, nanotechnology or as atool in cell biology.9-17 Examples of benefits ofencapsulation are the encapsulation of ahydrogenase and multiple enzymes inbacteriophage capsids which improved thestability of the respective enzymes.18,19 It hasalso been shown that foreign proteins can betargeted to encapsulins.20 This served asstarting point to explore the value of theseprotein cages for biocatalysis. In this work, wedescribe the structural features of anencapsulin originating from themesothermophile Mycolicibacterium hassiacum(EncMh) and its use as protein cage for severalbiocatalysts. Except for establishing a highlyeffective production procedure for this robustencapsulin, we elucidated its structure by cryo-electron microscopy and X-ray crystallography.Furthermore, we demonstrate that variousenzymes can be packaged in a functional formin EncMh and be used as highly stableencapsulated biocatalysts.

RESULTS AND DISCUSSIONM. hassiacum is an actinobacterium which

tolerates temperatures up to 65 °C.21 Byanalyzing its genome for encapsulin homologswe have identified a small operon consisting oftwo genes encoding a putative encapsulin(EncM, WP_005630281.1) and a putative DyP-type peroxidase (EKF22245.1). The putativeencapsulin has a predicted molecular mass of29 kDa and shares 34% sequence identity withthe encapsulin from Thermotoga maritima.8 Theputative DyP carries a C-terminal sequence thatresembles the targeting sequence used forincorporation into encapsulins. Based on theseobservations we decided to clone the gene

encoding for EncMh for expression inEscherichia coli. Upon screening expressionconditions we achieved a remarkably highexpression level. SDS-PAGE analysis revealedthat most of the intracellular protein of theexpression host was represented by EncMh.While EncMh could be purified to homogeneityby column chromatography (anion exchangefollowed by size exclusion) (Figure S1), we alsodeveloped a simple purification protocol thatmerely depends on the selective precipitationof EncMh by 5% PEG8000. Both purificationprocedures yield an impressive amount of 1 gpurified EncMh from 1 L growth medium,making EncMh an interesting candidate forfurther studies. EncMh was found to beresistant to proteolytic degradation by bovinetrypsin and proteinase K (Figure S2), asreported for other encapsulins.20 Additionally,EncMh displays a high thermostability as theoligomeric state was found to be stable up to50 °C (Figure S3). As expected for encapsulins,gelpermeation chromatography alreadysuggested that EncMh forms large oligomers.Cryo-electron microscopy analysis revealedthat EncMh forms large spherical structureswith a diameter of about 22 nm, which is in linewith other reported data on encapsulins with aT=1 symmetry.9 By class averaging anicosahedral architecture became apparent(Figure 1).

Figure 1. (Left) A typical micrograph of EncMh (scale bar: 100 nm).(Right) Four representative class averages of particles: images are theaverage sum of 1299, 602, 559 and 218 particles (top to bottom).

For a more detailed structural analysis, we set out tocrystallize EncMh. This resulted in well-diffracting crystalsand allowed us to determine a high-resolution structure ofEncMh. The 2.5 Å electron density map showed continuousdensity for all residues (1–265) in all fifteen chains (A-O) inthe asymmetric unit and has an R/R free of 17.2/21.1%. Datacollection and phasing details are summarized in Table S1.Five protein chains form a pentamer and the T=1icosahedral capsid is composed of 12 pentameric EncMholigomers (Figure 2). The unit cell contains twoicosahedrons with a diameter of 23 nm and a thickness ofthe protein layer of 2.0 - 2.5 nm. These data are perfectly inline with the CryoEM data (Figure 1). A comparison ofEncMh with other capsid proteins shows that it is mostsimilar to the encapsulin from T. maritima (PDB 3DKT)8

with a rmsd value of 1.8 Å on 251 Cα atoms. The EncMhmonomer shows the HK97 fold, the principal fold of manycapsids, and consists of a P-domain (peripheral domain),A-domain (axial domain) and E-loop (extension loop)(Figure S4).22 Its N-terminus is located on the inside of thecapsule while the C-terminus is on the outside. The P-domain (residues 1-43, 77-135 and 220-254) is composedof a three-stranded antiparallel β-sheet (β4-β11-β12) withon one side two small and two long α-helices (α1-α4). Asmall F-loop (44-46) is inserted after α2 and subsequently

the E-loop (47-76) is inserted which consists of a kinkedtwo-stranded β-sheet (β2-β3) and is involved in the two-foldinteraction in EncMh (Figure S4). Helix α4 contains a 9residue excursion; Gly122-Gly130, called the G-loop.22 TheA-domain (residues 136-219 and 254-265) is composed of amixed five-stranded β-sheet (β5-β6-β9-β10-β13), called β-hinge, that links the A- and P- domains of HK97-likeproteins and is flanked by three α-helices (α5-α7).22

Interestingly, Cys134, the ultimate residue of α4, is involvedin a disulfide bridge with Cys254, the ultimate residue ofβ12 from the P-domain. This disulfide is not observed instructures of known homologous encapsulins and maycontribute to the stability of EncMh.

Figure 2. Crystal structure of EncMh (PDB:6I9G). Left: the 60-mericEncMh with three pentameric units in yellow. Right: a pentamer withprotomers in different colors.

The tip of the A-domain is involved in the icosahedral five-fold axis (Figure S5). At this axis an opening in the shell ofEncMh is present with a diameter of 8 Å diameter which isshaped by residues His187-Gly-Tyr189 together withArg197. One of the bound sulfate ions occupies theentrance to the capsid at the five-fold axis while 5 sulfateions are bound close to the entrance. The three-fold axis islocated near the N-terminal methionine where no clear porecan be identified. However two additional pores are presentat the interface of two monomers, one with a diameter of 9Å and one with a diameter of 5 Å (Figure S6). The porediameters are similar to the pores of the carboxysomes andmetabolosomes that contain pores with a diameter of 4-10Å.23

Having a robust encapsulin at hand which can be easilyexpressed and isolated, we embarked on a study to exploreEncMh as packaging protein for biocatalysts. Thegenerated encapsulated biocatalysts were tested forbeneficial effects exerted by being packaged in EncMh. Wehave explored the encapsulation of biocatalysts fromdifferent enzyme classes (catalases, monooxygenases,oxidases, and peroxidases), containing different types ofcofactors (heme, flavin and copper), and of different sizes(up to 270 kDa) (Figures S7-S11). For targeting theenzymes to the interior of EncMh, a C-terminal 30 residuespeptide (PPPLPDSEPDREIPADDGSLGIGSLKGTRS) wasadded to each enzyme. This targeting peptide is used by thenative DyP of M. hassiacum. Co-expression of EncMh andeach target enzyme was accomplished by using a two-plasmid system, where the pBAD-EncMh and a pET-enzymevector were cotransformed (for details on vector design seeSI). As expression strain, E. coli BL21-AI was used becauseit allows the use of both arabinose and IPTG as inducers.Co-precipitation of EncMh with cargo protein during PEGprecipitation and co-elution in gelpermeationchromatography was taken as a proof of successful loadingof EncMh. CryoEM imaging was used to verify that cargoprotein had not aggregated on the outside of EncMh(Figure S12).

As initial test we examined whether a small heme-containing protein, bacterial hemoglobin VsHb(WP_019959060.1), could be packaged in EncMh bycoexpressing both proteins. VsHb was cloned with andwithout C-terminal targeting peptide resulting in proteins of21 kDa and 18 kDa, respectively. These two proteins wereindividually coexpressed with EncMh and purified usingPEG precipitation. VsHb with the encapsulation tag couldbe clearly observed in the isolated red-colored EncMh,

while the VsHb without encapsulation tag did notcoprecipitate with EncMh but ended up in the solublefraction. This demonstrates that the employed C-terminaltag is efficient in loading EncMh with foreign cargoproteins (Figure S4). Next, we decided to test loading ofEncMh with various enzymes.

First, a bacterial peroxidase, DyP fromSaccharomonospora viridis DSM43017 (SviDyP), wassuccessfully packaged in EncMh. This peroxidase wasrecently shown to be active on various dyes and showedpotential in modifying lignin-containing biomass.24

Purification included, except for PEG precipitation, anaffinity chromatography step to remove any traces of non-encapsulated SviDyP (containing a His-tag). Both EncMHand SviDyP were well expressed as evidenced from SDS-PAGE analysis (Figure S7). The obtained protein samplecontained only the two overexpressed proteins anddisplayed a clear red color due to the presence of the hemecofactor in SviDyP. Gel permeation confirmed that theperoxidase had been incorporated in the encapsulin. Thepackaged peroxidase was found to be active on ABTS, acommonly used peroxidase substrate.25 Using ABTS as testsubstrate, we examined the effect of encapsulation on thekinetic parameters of SviDyP (Figure 3). Non-encapsulatedenzyme displayed a kcat of 24.5 ± 0.9 s-1 and a Km of 0.15 ±0.02 mM while encapsulated SviDyP displayed a slightlylower kcat (17.9 ± 1.9 s-1) and higher Km (0.55 ± 0.1 mM).Whether the packaging in EncMh has a stabilizing effect onthe stability of the peroxidase was tested by incubating theencapsulated enzyme and the free enzyme at 40°C andmonitoring peroxidase activity in time. This revealed thatthe packaging in EncMh has a strongly stabilizing effect(Figure S13). While the unpackaged peroxidase lost itsactivity within 30 min, the packaged peroxidase showedeven an increase in activity in the first few hours and onlyafter 25 h the activity decreased significantly (Figure S13).The increase in activity upon incubation at higher

temperature of enzymes from (semi)thermophiles has beenobserved before26, 27 and may suggest that an optimalcatalytically competent conformation is attained at a highertemperature. The stabilizing effect of encapsulation mayalso be due to a molecular crowding effect: the intenseprotein-protein interactions in the capsule preventsirreversible unfolding and aggregation of the cargo protein.

Figure 3. The effect of encapsulation on activity of SviDyP.

To challenge the capacity of EncMh, we also coexpressed atetrameric heme-containing catalase of 270 kDa fromThermo-bifida fusca (TfuCat).28 Encapsulation of TfuCatwas successful as evidenced by the isolation of intensely

brown-colored encapsulin. The encapsulation of TfuCatshows that EncMh can accommodate cargo proteinsranging from 17 kDa (VsHb) to relatively large cargoproteins such as catalase. The packaged catalase retainedactivity on hydrogen peroxide and was also able to oxidizecatechol, as was reported for the isolated enzyme.28

Encapsulated TfuCat showed similar behavior as SviDyP.Upon encapsulation, TfuCat displayed a slightly lowercatalytic efficiency (kcat/Km) when compared with the non-encapsulated catalase (0.7 vs. 1.6 106 s-1M-1).

Encapsulation of flavoenzymes was tested as well. Itwas found to be possible to package a prototypical Baeyer-Villiger monooxygenase, cyclohexanone monooxygenase(CHMO).29 Encapsulated CHMO did not show activitytowards cyclohexanone, although CHMO has beenpackaged in a correctly folded state (FAD-bound) asevidenced by the yellow appearance. In this case it is likelythat NADPH, the coenzyme required for activity, is not ableto enter the interior of the encapsulin. Pores of ~10 Åshould allow passage of substrates of up to 800 g/mol.30

Since the measured size of the pore is 9 Å and the molecularmass of NADPH is 744 g/mol it is likely that NADPHcannot pass through the pores. This is not a problem withABTS used as substrate for SviDyP, with a molecular massof 515 g/mol. These observations may also provide anadditional raison d’être for encapsulins: they function asmolecular sieves for the enzymes that are in theencapsulins, thereby preventing undesired reactions. Incontrast to CHMO, an encapsulated FAD-containingcarbohydrate oxidase, mChitO,31 was found to be activewhen packaged in EncMh (Figure S14). This oxidaseaccepts various oligosaccharides. All testedoligosaccharides (cellobiose, maltotriose and cellotetraose)were found to be oxidized, indicating that EncMh allowspassage of such hydrophilic molecules, where the highestmolar weight is the one of cellotetraose (667 g/mol), whichfits within the expected molecular weight cut-off.31

Furthermore, as was observed for the peroxidase, mChitOshowed improved thermostability at 50°C by retaining halfof its initial activity after 4h, while soluble, non-encapsulated mChitO loses almost all the activity within 1h(Figure 4).

Figure 4. The effect of encapsulation on stability of ChitO. The X-axiscorresponds to the time of incubation at 50°C. Activity of 100% wastaken for the activity of the starting enzyme preparation (0.1 U/ml).

Finally, also a bacterial copper-containing oxidase, alaccase, was coexpressed with EncMh. This resulted in an

encapsulated laccase as evidenced from the greenappearance of the isolated encapsulin preparation. Activityof the laccase in EncMh was verified using ABTS andsyringaldazine (MW 360 g/mol). As bacterial laccase isalready highly thermostable,32 we were not able to assessthe effect of encapsulation on thermal stability. Thecompartmentalization of enzymes within encapsulin offersthe possibility of tuning the substrate scope of the enzyme.We decided to explore the effects of the added selectivityupon packaging biocatalysts in EncMh by preforming acascade reaction involving SviDyP. Recently, we reportedon the use of eugenol as starting material and eugenoloxidase and peroxidase as biocatalysts for the production oflignin-like material.33-35 Herein, we tested the effect of usingencapsulated SviDyP on the characteristics of the formedlignin oligomers. It was found that the packaged peroxidasecan still support formation of lignin-like material asevidenced by formation of insoluble lignin-like oligomers.Interestingly, the end-product contains a slighlty differentrelative abundance of the linkages as compared to thesystem where peroxidase is not encapsulated. Thisapproach to perform a cascade reaction results in a lowerabundance of dibenzodioxin, b-O-4, b-b and b-5 linkages,while promoting formation of a-O-4 linkages (Table S1).

CONCLUSIONSThe results above demonstrate that EncMh can be used topackage all kinds of biocatalysts, including heme-, flavin-and metallo-enzymes of different sizes and oligomerizationstates. Successful incorporation of cofactor containingenzymes shows that the enzymes are incorporated in theencapsulin nanocage after folding and cofactorincorporation. This is in line with the proposed mechanismof cargo loading which relies on association of the C-terminal cargo loading peptide to encapsulin protomersduring the self-assembly process. This work also shows thatthe approach of coexpressing EncMh with a target proteinprovides a generic method for producing encapsulinsloaded with any desired protein. While this concept hasbeen shown in other studies, for example for creatingartificial organelles in yeast36 or size-selective RNApackaging and creation of artificial proteasomes,37,38 wenow demonstrate that encapsulin-packaged enzymes can beused as biocatalysts. Through engineering of theencapsulin, it may also be possible to tune the catalyticproperties of the enzyme. Recently, the groups of Lutz andHilvert have shown that through modifying the pores ofencapsulins or other protein-based cages, such cages attainspecific permeation properties.31, 37, 39 This concept of addingan additional filtering layer around an enzyme may alsohave been the evolutionary trigger for bacteria to evolvesuch protein-based nanocages.

ASSOCIATED CONTENT Supporting Information. Details on thematerials and methods used, additional data andsequence information can be found in theSupporting Information. This material is availablefree of charge via the Internet athttp://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author* m.w.fraaije@rug.nl

Present Addresses§ Department of Structural Cell Biology of Viruses, Heinrich-Pette-Institut, Leibniz-Institut für Experimentelle Virologie - Centre for Structural Systems Biology, Notkestraße 85, 22607, Hamburg, Germany

Author ContributionsThe manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Author ContributionsThe authors declare no competing financial interest.

ACKNOWLEDGMENT The authors are thankful to Dr. M. Trajković forhelp with interpretation of 2D NMR spectra oflignin oligomers and Dr. M.J.L.J. Fürst for help withcreating the pENC vector.

REFERENCES (1) Kerfeld, C. A.; Aussignargues, C.; Zarzycki, J.; Cai,F.; Sutter, M. Bacterial Microcompartments. Nat. Rev.Microbiol. 2018, 16, 277-290. (2) Giessen, T. W.; Silver, P. A. Widespread Distributionof Encapsulin Nanocompartments Reveals FunctionalDiversity. Nat. Microbiology 2017, 2, 17029. (3) Comellas-Aragones, M.; Engelkamp, H.; Claessen,V. I.; Sommerdijk, N. A.; Rowan, A. E.; Christianen, P. C.;Maan, J.C.; Verduin, B. J.; Cornelissen, J. J.; Nolte, R. J. AVirus-based Single-enzyme Nanoreactor. Nat.Nanotechnol 2007, 2, 635-639. (4) Minten, I.J.; Claessen, V. I.; Blank, K.; Rowan, A. E.;Nolte, R. J. M.; Cornelissen, J. J. L. M. Catalytic Capsids:the Art of Confinement. Chem. Sci. 2011, 2, 358-362. (5) Cassidy-Amstutz, C.; Oltrogge, L.; Going, C. C.; Lee,A.; Teng, P.; Quintanilla, D.; East-Seletsky, A.; Williams,E.R; Savage, D.F. Identification of a Minimal Peptide Tagfor in Vivo and in Vitro Loading of Encapsulin.Biochemistry 2016, 55, 3461-3468. (6) Aussignargues, C.; Paasch, B. C.; Gonzalez-Esquer,R.; Erbilgin, O.; Kerfeld, O. A. BacterialMicrocompartment Assembly: The Key Role ofEncapsulation Peptides. Commun. Integr. Biol. 2015, 8,e1039755. (7) Kalel, V.C.; Erdmann, R. Unraveling of the Structureand Function of Peroxisomal Protein Import Machineries.Subcell Biochem. 2018, 89, 299-321. (8) Sutter, M. ;Boehringer, D.; Gutmann, S.; Gunther,S.; Prangishvili, D.; Loessner, M. J.; Stetter, K. O.; Weber-Ban, E.; Ban, N. Structural Basis of EnzymeEncapsulation into a Bacterial Nanocompartment. Nat.Struct. Mol. Biol. 2008, 15, 939-947. (9) Giessen, T. W.; Silver, P. A. Converting a NaturalProtein Compartment into a Nanofactory for the Size-Constrained Synthesis of Antimicrobial SilverNanoparticles. ACS Synth. Biol. 2016, 5, 1497-1504. (10) McHugh, C. A.; Fontana, J.; Nemecek, D; Cheng, N,;Aksyuk, A. A.; Heymann, J. B.; Winkler, D. C.; Lam, A. S.;Wall, J. S.; Steven, A. C.; Hoiczyk, E. A Virus Capsid-likeNanocompartment that Stores Iron and Protects Bacteriafrom Oxidative Stress. EMBO J. 2014, 33, 1896-1911. (11) Frey, R.; Hayashi, T.; Hilvert, D. Enzyme-mediatedPolymerization inside Engineered Protein Cages. Chem.Commun. (Camb) 2016, 52, 10423-10426. (12) Beck, T.; Tetter, S.; Kunzle, M.; Hilvert, D.Construction of Matryoshka-type Structures fromSupercharged Protein Nanocages. Angew. Chem. Int. EdEngl. 2015, 54, 937-940.

(13) Snijder, J.; van de Waterbeemd, M.; Damoc, E.;Denisov, E.; Grinfeld, D.; Bennett, A.; Agbandje-McKenna, M.; Makarov, A.; Heck, A. J. Defining theStoichiometry and Cargo Load of Viral and BacterialNanoparticles by Orbitrap Mass Spectrometry. J. Am.Chem. Soc. 2014, 136, 7295-7299. (14) Kwak, M.; Minten, I. J.; Anaya, D. M.; Musser, A. J.;Brasch, M.; Nolte, R. J.; Mullen, K.; Cornelissen, J. J.;Herrmann, A. Virus-like Particles Templated by DNAMicelles: A General Method for Loading VirusNanocarriers. J. Am. Chem. Soc. 2010, 132, 7834-7835. (15) Minten, I. J.; Nolte, R. J.; Cornelissen, J. J. ComplexAssembly Behavior During the Encapsulation of GreenFluorescent Protein Analogs in Virus Derived ProteinCapsules. Macromol. Biosci. 2010, 10, 539-545. (16) Moon, H.; Lee, J.; Min, J.; Kang, S. DevelopingGenetically Engineered Encapsulin Protein CageNanoparticles as a Targeted Delivery Nanoplatform.Biomacromolecules 2014, 15, 3794-3801. (17) Moon, H.; Lee, J.; Kim, H.; Heo, S.; Min, J.; Kang, S.Genetically Engineering Encapsulin Protein CageNanoparticle as a SCC-7 Cell Targeting OpticalNanoprobe. Biomater. Res. 2014, 18, 21. (18) Jordan, P. C.; Patterson, D. P.; Saboda, K. N.;Edwards, E. J.; Miettinen, H. M.; Basu, G.; Thielges, M. C.;Douglas, T. Self-assembling Biomolecular Catalysts forHydrogen Production. Nat. Chemistry 2016, 8, 179-185. (19) Giessen, T. W.; Silver, P. A. A Catalytic NanoreactorBased on in Vivo Encapsulation of Multiple Enzymes inan Engineered Protein Nanocompartment.Chembiochem 2016, 17, 1931-1935. (20) Tamura, A.; Fukutani, Y.; Takami, T.; Fujii, M.;Nakaguchi, Y.; Murakami, Y.; Noguchi, K.; Yohda, M.;Odaka, M. Packaging Guest Proteins into the EncapsulinNanocompartment from Rhodococcus erythropolis N771.Biotechnol. Bioeng. 2015, 112, 13-20. (21) Tiago, I.; Maranha, A.; Mendes, V.; Alarico, S.;Moynihan, P. J.; Clarke, A. J.; Macedo-Ribeiro, S.; Pereira,P. J.; Empadinhas, N. Genome Sequence ofMycobacterium hassiacum DSM 44199, a Rare Source ofHeat-Stable Mycobacterial Proteins. J. Bacteriol. 2012,194, 7010-7011. (22) Suhanovsky, M. M.; Teschke, C. M. Nature׳sFavorite Building Block: Deciphering Folding and CapsidAssembly of Proteins with the HK97-fold. Virology 2015,479, 487-497. (23) Plegaria, J. S.; Kerfeld, C. A. EngineeringNanoreactors using Bacterial MicrocompartmentArchitectures. Curr. Opin. Biotechnol. 2018, 51, 1-7. (24) Yu, W.; Liu, W.; Huang, H.; Zheng, F.; Wang, X.;Wu, Y.; Li, K.; Xie, X.; Jin, Y. Application of a Novel Alkali-Tolerant Thermostable DyP-Type Peroxidase fromSaccharomonospora viridis DSM 43017 in Biobleachingof Eucalyptus Kraft Pulp. PLoS One 2014, 9, e110319. (25) Contreras, H.; Joens, M. S.; McMath, L. M.; Le, V. P.;Tullius, M. V.; Kimmey, J. M.; Bionghi, N.; Horwitz, M. A.;Fitzpatrick, J. A.; Goulding, C. W. Characterization of aMycobacterium tuberculosis Nanocompartment and ItsPotential Cargo Proteins. J. Biol. Chem. 2014, 289,18279-18289. (26) Fraaije, M. W.; Wu, J.; Heuts, D. P. H. M.; vanHellemond, E. W.; Spelberg, J. H. L.; Janssen, D. B.Discovery of a Thermostable Baeyer-VilligerMonooxygenase by Genome Mining. Appl. Microbiol.Biotechnol. 2005, 66, 393-400. (27) van Bloois, E.; Torres Pazmino, D. E.; Winter, R. T.;Fraaije, M. W. A Robust and Extracellular Heme-Containing Peroxidase from Thermobifida fusca asPrototype of a Bacterial Peroxidase Superfamily. Appl.Microbiol. Biotechnol. 2010, 86, 1419-1430. (28) Lončar, N.; Fraaije, M. W. Not so Monofunctional -a Case of Thermostable Thermobifida fusca catalasewith Peroxidase Activity. Appl. Microbiol. Biotechnol.2015, 99, 2225-2232. (29) Torres Pazmiño, D. E.; Dudek, H. M.; Fraaije, M. W.Baeyer-Villiger monooxygenases: recent advances and

future challenges. Curr. Opin. Chem. Biol. 2010, 14, 138-144. (30) Williams, E. M.; Jung, S. M.; Coffman, J. L.; Lutz, S.Pore Engineering for Enhanced Mass Transport inEncapsulin Nanocompartments. ACS Synth. Biol. 2018,7, 2514-2517. (31) Ferrari, A. R.; Lee, M.; Fraaije, M. W. Expanding theSubstrate Scope of Chitooligosaccharide Oxidase fromFusarium graminearum by Structure‐inspiredMutagenesis. Biotechnol. Bioeng. 2015, 112, 1074-1080. (32) Lončar, N. Božić, Z. Vujčić, Expression andCharacterization of a Thermostable Organic Solvent-tolerant Laccase from Bacillus licheniformis ATCC 9945a.J. Mol. Catal. B: Enzym. 2016, 134, 390 - 395. (33) Habib, M.; Trajkovic, M.; Fraaije, M. W. TheBiocatalytic Synthesis of Syringaresinol from 2,6-Dimethoxy-4-allylphenol in One-Pot Using a TailoredOxidase/Peroxidase System. ACS Catal. 2018, 8, 5549-5552. (34) Habib, M.; Deuss, P. J.; Lončar, N.; Trajkovic, M.;Fraaije, M. W. A Biocatalytic One-Pot Approach for the

Preparation of Lignin Oligomers Using anOxidase/Peroxidase Cascade Enzyme System. Adv.Synth. Catal. 2017, 359, 3354-3361. (35) Colpa, D. I.; Lončar, N.; Schmidt, M.; Fraaije, M. W.Creating Oxidase-Peroxidase Fusion Enzymes as aToolbox for Cascade Reactions. Chembiochem 2017, 18,2226-2230. (36) Lau, Y. H.; Giessen, T. W.; Altenburg, W. J.; Silver, P.A. Prokaryotic Nanocompartments Form SyntheticOrganelles in a Eukaryote. Nat. Communications 2018,9, 1311. (37) Azuma, Y.; Bader, D. L. V.; Hilvert, D. SubstrateSorting by a Supercharged Nanoreactor. J. Am. Chem.Soc. 2018, 140, 860-863. (38) Azuma, Y.; Edwardson, T. G. V.; Terasaka, N.;Hilvert, D. Modular Protein Cages for Size-Selective RNAPackaging in Vivo. J. Am. Chem. Soc. 2018, 140, 566-569. (39) Azuma, Y.; Herger, M.; Hilvert, D. Diversification ofProtein Cage Structure Using Circularly PermutedSubunits. J. Am. Chem. Soc. 2018, 140, 558-561.

Table of Contents artwork

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download fileview on ChemRxivEncMh.docx (3.64 MiB)

Supporting Information

Molecular packaging of biocatalysts using a robust protein cageNikola Lončar†,‡, Henriette J. Rozeboom†, Linda E. Franken#,§, Marc C. A. Stuart#, and Marco W. Fraaije†,*

† Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG Groningen,The Netherlands‡ GECCO Biotech, Nijenborgh 4, 9747AG, Groningen, The Netherlands

# Department of Electron Microscopy, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands

1

Table of ContentsTable of Contents 2Material and Methods 3Reagents and enzymes 3Strains and plasmids 3Expression and purification 3Proteolysis resistance of encapsulin 4Co-expression of encapsulin and cargo enzymes 4Cryo- Transmission Electron Microscopy 5Crystallization and structure determination 5Steady-state kinetic analyses 6Thermal stability assays 7Conversions of eugenol with EugO-SviDyP and EugO-EncMh/SviDyP 7Results and Discussion 8SDS-PAGE analysis of purified EncMh 8Proteolysis resistance of encapsulin 9Thermal stability of encapsulin 9Data collection and refinement statistics 10Structure of EncMh 11Co-expression of EncMh with VsHb and VsHb (without targeting peptide) 13Co-expression of EncMh with BliLacc 13Co-expression of EncMh with SviDyP and TfuCat 14Co-expression of EncMh with CHMO and ChitO3x 14Co-expression of EncMh with CHMO 15Cryo-EM verification of enzyme incorporation 15Enzyme activity 16The effect of encapsulation on stability of SviDyP 16Testing activity of EncMh / BliLacc 17Lignin oligomers analysis 17Multiple sequence alignment 18Sequences of the genes and proteins used in this study 19References 23

2

Material and Methods

Reagents and enzymes

Restriction enzymes were purchased from New England Biolabs, Phusion polymerase and prestained

protein ladders from Thermo Scientific and Ni2+-Sepharose HP from GE Lifescience. PfuUltra Hot Start

II Master Mix was purchased from Agilent. All other chemicals were bought from Sigma-Aldrich and

were of analytical grade.

Strains and plasmids

Escherichia coli NEB10β was used for routine cloning and maintenance of all plasmid constructs. This

strain was also used for overexpression of EncMh. The gene encoding EncMh (RefSeq code:

YP_289707.1) was synthesized as codon optimized for E. coli by GeneScript. The EncMh-encoding

gene was recloned form pUC into the pBadNdeIHis vector using NdeI and HindIII restriction sites to

give a pBAD-EncMh construct for expression of encapsulin without tag. pBadNdeIHis is a pBAD/Myc-

HisA-derived expression vector (Invitrogen) in which the NdeI site is removed and the NcoI site is

replaced by NdeI. For verification, all created constructs have been sequenced (GATC Biotech).

Construction of cargo co-expression vectors

Genes of the respective cargo proteins were amplified from already published vectors or synthesized

as double stranded DNA fragments (gblocks) by IDT DNA or GeneScript. The following cargo proteins

have been used in this study: Vitreoscilla hemoglobin VsHb (WP_019959060.1), Saccharomonospora

viridis DSM 43017 (SviDyP) [1, 2], Thermobifida fusca catalase (TfuCat) [3], Bacillus licheniformis

laccase (BliLacc) [4], chitooligosaccharide oxidase Q268R G270E S410R (ChitO3x) [5], and cyclo-

hexanone monooxygenase (AcCHMO). These genes were cloned into the pENC vector based on

pET28 with a changed origin of replication to p15A in order to be compatible with pBAD vector, and

with an introduced BsaI restriction sites (to facilitate Golden Gate cloning). These changes have been

introduced using Gibson assembly. Briefly, the p15A ori was amplified from pEVOL-pAzF (Addgene

plasmid #31186) and the pET-28a(+) vector was amplified excluding pBR322 ori. These two fragments

were assembled using NEB’s Gibson assembly mix, with all the primers designed using Geneious

software (Gibson cloning option; vector map available on request).

The gene encoding cargo protein was cloned into pENC in such way that, when expressed, it contains

a N-terminal 6xHis-tag and also C-terminal fusion tag of 30 amino acids from DyP-peroxidase from the

same operon as the original encapsulin gene. After sequencing, both vectors (pBAD-EncMh and

pENC-cargo) were co-transformed into E. coli BL21-AI strain (ThermoFisher) and selected on LB plates

containing ampicillin, kanamycin and tetracyclin.

Expression and purification

E. coli NEB10β cells expressing genes cloned in pBAD-EncMh were grown in LB medium at 37⁰C to

saturation overnight. The following day, cultures were diluted 1:100 into fresh TBamp media and

grown until OD600=1.25, when 0.02% L-arabinose (final concentration) was added to induce the

expression of EncMh. This expression was carried out at 30⁰C and 180 rpm for 24h. Cells were

harvested by centrifugation at 6000 rpm using JLA 10.500 rotor and washed once with 50 mM TrisHCl

at pH 7.5 with 150 mM NaCl. Pelleted cells were resuspended in the same buffer and disrupted by

sonication using Vibra cell sonicator. Cell-free extract was obtained after centrifugation at 17000 rpm

using JLA 17 rotor at 4⁰C for 1 h.

For the purification of EncMh two strategies were developed:

3

1) For cryoEM and crystallography: ion exchange chromatography using 60 mL Q-Sepharose FF

column was used as first step, aiming at binding of the majority of host cell proteins to the resin,

while EncMh does not bind due to its large size. The column was equilibrated in 20 mM TrisHCl buffer

pH 8.0 and, after applying the sample, the column was washed with the starting buffer. Elution was

done with a gradient from 0 - 0.5 M NaCl in 10 column volumes. Fractions were assayed for purity

and pooled and concentrated as described above. Preparative gel filtration on a Superdex 200

XK26/60 column was used as a polishing step. The column was equilibrated and eluted using 50 mM

TrisHCl pH 7.5 containing 150 mM NaCl and 10% glycerol.

2) A fast purification approach was developed, that uses a precipitation strategy with PEG-8000

solutions. Series of PEG-8000 solutions (2, 4, 6, 8, 10, 12, 14, 16, 18 and 20%) in 50 mM TrisHCl pH 7.5

with 2M NaCl were mixed with equal volumes of cell-free extract containing encapsulin. Solutions

were incubated on a nutating shaker at 4° for 1h. After 1h the solutions were centrifuged at 4000 x g

for 10 min. Supernatant was removed and kept for analysis. A control sample was mixed with the

same buffer without PEG-8000. Resulting pellets were dissolved by overnight incubation with 1 mL of

50 mM TrisHCl pH 7.5 150 mM NaCl with 5% glycerol at 4°C. Final solutions were clear, without visible

precipitates. Purity of encapsulin was analyzed by SDS PAGE.

Proteolysis resistance of encapsulin

Purified encapsulin (10 mg/mL) was incubated for 16h with bovine trypsin and Proteinase K to study

resistance to proteolytic degradation. Trypsin from bovine pancreas (1 mg/mL) was dissolved in 50

mM acetate buffer, while Proteinase K from T. album (20U/mg, 1 mg/mL) was dissolved in 50 mM

TrisHCl pH 8.0 with 10 mM CaCl2. Reactions contained 50 µL of 10 mg/mL ENC, 400 µL TrisHCl pH 8 10

mM CaCl2 with 2 mM DTT and 50 µL of protease solution. The control samples contained 50 µL of

encapsulin solution and 450 µL TrisHCl pH 8, 10 mM CaCl2 with 2 mM DTT. Samples were incubated at

37°C at 500 rpm for 16h in an Eppendorf thermomixer. Samples were then analyzed by SDS PAGE.

Co-expression of encapsulin and cargo enzymes

For co-expression, the One Shot® BL21-AI™ E. coli strain from ThermoFisher Scientific was used. This

strain contains a chromosomal insertion of the gene encoding T7 RNA polymerase into the araB locus

of the araBAD operon, placing regulation of T7 RNAP under the control of the arabinose-inducible

araBAD promoter, while at the same time it enables the use of pBAD vectors.

1 µL of each of the constructs pBAD-ENC and pENC-cargo (~50 ng/µL) was used for co-transformation

of 50 µL of chemically competent BL21-AI cells. Cells were incubated on ice for 30 min followed by 30

s heat-shock at 42°C. 250 µL of SOC medium was added to the cells and recovery was done at 37°C

for 1 h and 650 rpm in an Eppendorf thermomixer. Cells were plated on freshly made LB plates

containing ampicillin, kanamycin and tetracycline. Two single colonies were used for inoculation of

LBamp/kan media as starting cultures. On the following day overnight cultures were diluted in

TBamp/kan 1:100 and incubated at 37°C until OD600 = 0.6. Expression of both encapsulin and cargo

enzyme was initiated by addition of 1 mM IPTG and 0.2 % arabinose (final concentrations). Cultures

were then incubated for 16-20 h at 30°C and at 135 rpm (5 cm orbital shaker).

Cultures reached OD600=15-18 at the end of growth. Cells were then harvested using centrifugation

at 4000 rpm for 30 min at 4°C, resuspended in 50 mM TrisHCl at pH 7.5 with 150 mM NaCl and

sonicated on ice using a Vibra cell sonicator (for 50 mL cultures 3 min time, 3s on / 6s off, 70%

amplitude). Samples were clarified using centrifugation at 12000 rpm for 1h at 4°C. Clear cell-free

extract (CFE) was loaded on a pre-equilibrated Ni-Sepharose column to remove the excess of cargo-

enzyme. In some cases, the flow-through fraction from the Ni-Sepharose column was ran over 5 mL

4

Q-Sepharose column to remove excess of the E.coli proteins in order to achieve a higher purity

preparation for further steps. Encapsulin does not bind or binds only weakly to Q-sepharose.

Collected flow-through fractions from Ni-Sepharose (or Ni-Sepharose and Q-Sepharose) were mixed

on ice with equal volumes of 10% PEG-8000 solution in 50 mM TrisHCl at pH 7.5 with 2 M NaCl and

further treated as described above. A polishing step, if needed, was done using the preparative

Superdex 200 column XK26/60 with 50 mM TrisHCl at pH 7.5 and 150 mM NaCl. Flow rates were 2

ml/min and sample volumes were 5-15 mL. Encapsulin and encapsulin loaded with cargo enzyme

eluted in void volume of the column. The purity was checked using SDS PAGE.

Cryo-Transmission Electron Microscopy

On a glow-discharged copper grid with holey carbon film (quantifoil 3.5/1) we applied three

microliters of 10 mg/mL EncMh solution. This was blotted for 5 seconds and plunge-frozen with a

Vitrobot (FEI, Eindhoven, The Netherlands) in liquid ethane. The grid was transferred to a cryo-

transfer holder (Gatan model 626) and visualized in a Tecnai G2 20 Twin electron microscope (FEI,

Eindhoven, the Netherlands), that was equipped with an LaB6 cathode and operated at 200 kV.

Images were recorded with an UltraScan 4000 UHS CCD camera (Gatan, Pleasanton, CA, USA) using

low-dose mode. GRACE software [6] was used for semi-automated data acquisition to record 246

images with predetermined defocusses of 1000, 1600, 2000 and 2500 nm and with 80,000-fold

nominal magnification leading to a pixel size of 0.3 nm at the specimen level.

These micrographs were further processed using the software package Xmipp 3.0 [7]. Automated

particle picking [8] yielded 5169 ctf-corrected particles, which were sorted into 10 classes using the

maximum-likelyhood method of the Relion software that was implemented into the Xmipp

interphase [9, 10]. The best 4 classes are visualised in Figure 1 along with a representative micrograph

at 2500 nm defocus. This micrograph was cropped and had adjustments of levels, brightness and

contrast in Adobe Photoshop CS6.

Crystallization and structure determination

Preliminary screening of crystallization conditions was performed with Wizard, Cryo (Emerald

Biosystems, Bainbridge Island, USA), PACT and JCSG+ (Qiagen Systems, Maryland, USA) by the sitting

drop vapor-diffusion method in 96-well MRC2 plates (Hampton research, Aliso Viejo, USA) using a

Mosquito crystallization robot (Molecular Dimensions Ltd, Newmarket, England). Encapsulin (EncMh)

crystals were obtained at room temperature from ammonium sulfate. Optimization of the

crystallization condition using hanging drop vapor-diffusion yielded crystals that grew within several

days when 1 µL protein solution (6.0 mg mL-1 in 20 mM TrisHCl pH 7.0) was mixed with 1 µL reservoir

solution containing 1.3-1.6 M ammonium sulfate with 0.1 M MES buffer, pH 5.5.

Prior to data collection, crystals were briefly soaked in a cryoprotectant solution, consisting of 30%

glycerol, 2.0 M ammonium sulfate and 0.1 M acetate pH 5.5. Initial X-ray diffraction data to 3.4 Å

were collected on an in-house MarDTB Goniostat System using Cu-Kα radiation from a Bruker

MicrostarH rotating-anode generator equipped with HeliosMX mirrors at 100K. Intensity data were

processed using iMOSFLM [11] and the CCP4 package [12]. The space group was I222, with unit cell

dimensions of a = 232.2, b = 242.8, c = 272.4 Å. With fifteen monomers of 29 kDa in the asymmetric

unit, the VM is 4.4 Å3/Da [13] with a calculated solvent content of 72%. A high resolution native

dataset to 2.5 Å was collected on beamline P13 at the EMBL (Hamburg, Germany). The mutant crystal

was briefly soaked in a cryoprotectant solution, consisting of 30% glycerol, 2.0 M ammonium sulfate

and 0.1 M MES buffer, pH 5.5. A dataset to 2.6 Å was also collected on beamline P13. Intensity data

were processed as above. A summary of data collection statistics is given in Table S1.

5

Using the FFAS03 server [14] and SCWRL [15], a homology model for EncMh was generated.

Thermotoga maritima Encapsulin (Maritimacin) (PDBcode 3DKT [16] was used as a template, having a

sequence identity of 32%. Molecular replacement was performed with PHASER [17]. PHENIX Phase

and Build [18] was used for automatic building and the model was refined with REFMAC5 using 15-

fold local NCS restraints [19]. COOT [20] was used for manual rebuilding and map inspection. The

quality of the model was analyzed with MolProbity [21]; secondary structure elements were assigned

with DSSP [22]. Figures were prepared with PyMOL [23] and ESPript [24]. Atomic coordinates and

experimental structure factor amplitudes for Encapsulin have been deposited in the RCSB Protein

Data Bank and are accessible under entry code 6I9G.

Steady-state kinetic analyses

Purified enzymes containing encapsulation targeting peptide (TB) and EncMh loaded with cargo

enzymes have been tested for activity using JASCO V-660 spectrophotometer. Control reactions were

included empty encapsulin solution of the same concentration (encapsulin expressed without specific

cargo enzyme).

Concentration of SviDyP in non-encapsulated and encapsulated form was estimated using the

absorbance of Soret band and the extinction coefficient used for heme-containing proteins [25].

Based on these values dilutions were made to achieve equal concentration of SviDyP in non-

encapsulated and encapsulated form. The same approach was used for the activity assay of TfuCat.

For the SviDyP activity assay, reaction mixture of a total of 1 mL contained 0.05-1.0 mM ABTS in 50

mM acetate pH 5 and 50 µL or either SviDyP or EncMh/SviDyP (1.4 µM stock solution). The reaction

was started by adding 50 µL 20 mM H2O2. Change in absorbance was measured at 414 nm. For data

analysis the following equation was used: Y=kobs*X/(Km + X*(1+X/Ki)) to account for the observed

substrate inhibition (Ki for SviDyP = 9.7 +/- 1.7 mM and for EncMh/SviDyP = 4.0 +/- 0.9 mM). Fitting

was done using GraphPad Prism version 6.07.

For assaying catalase activity, a previously published procedure was followed [4]. In brief, the reaction

mixtures contained 10-50 mM H2O2 in 50 mM TrisHCl pH 8.5 solution. Reaction was started by adding

either TfuCat or EncMh/TfuCat (final concentration 5 nM). Measurements were done in duplicates

using quartz cuvettes at 240nm.

Contrary to EncMh/SviDyP and EncMh/TfuCat, for encapsulated forms of AcCHMO, ChitO and BliLacc

it was not possible to use characteristic wavelength maxima to estimate the concentration of cargo

enzymes. Therefore, for these cases, only qualitative assay was used to check whether enzyme

retained the activity. The activity assay for EncMh/AcCHMO was performed by spectrophotometric

measurement of NADPH consumption at 340 nm in presence of 1 mM cyclohexanone. NADPH

concentration used in the assay was 0.1 mM. The assay was done for 10 min in 200 µL 50 mM TrisHCl

pH 7.5. Three different concentrations of EncMh/AcCHMO were tested. As positive control non-

encapsulated CHMOenc was taken and as negative control encapsulin without cargo enzyme.

For the mChitO (ChitO Q268R G270E S410R) activity assay, an HRP-based assay was performed in

MTP format in 200 µL by mixing 5 µL HRP (0.4 U/µL), 20 µL DCHBS (10 mM), 20 µL AAP (1 mM), 100

µL enzyme diluted in 50 mM TrisHCl at pH 7.5, and 50 µL of substrate solution (25-100 mM stock).

Measurements were done at 25°C at 515 nm using ε515= 27 mM-1 cm-1. Non-encapsulated mChitO was

used as a positive control and reaction mixtures without mChitO (but with all other components)

were used as negative control. The substrates used and their final concentration were as follows:

cellobiose (25 mM), maltotriose (25 mM) and cellotetraose (6 mM).

6

The activity of BliLacc and EncMh/BliLacc was assayed using the canonical laccase substrate

syrgingaldazine (0.1 mM) in 50 mM TrisHCl pH 7.0, following the absorbance at 525 nm.

Thermal stability assays

The thermal stability of encapsulin was assessed using the ThermoDLS method: DLS (dynamic light

scattering) measurements were performed in a 4 μL cuvette using a DynaPro NanoStar instrument

(Wyatt Technology, Santa Barbara, CA, USA) with a temperature ramp from 298 K to 333 K with an

acquisition every 1°C. Results were analyzed with dynamics software, version 7 (Wyatt Technology).

The thermal stability of encapsulated SviDyP was determined by measuring residual activity upon

incubating aliquots of SviDyP and EncMh/SviDyP at 40⁰C in a water bath. Samples were withdrawn at

specific time points and the activity was determined spectrophotometrically using a BioTek SMX plate

reader. The assay was performed at room temperature by mixing 120 µL of 50 mM Na-acetate buffer

at pH 5.0, 20 µL of 10 mM ABTS solution (final concentration 1 mM), 50 µL of 4 mM H2O2 (final

concentration 1 mM) and 10 µL of the SviDyP solution or ENC/SviDyP solution diluted to the same

starting amount of units as measured against 1 mM ABTS. All measurements were done in triplicate.

The thermal stability of encapsulated mChitO was determined in similar manner, by incubation of

mChitO and EncMh/mChitO at 50⁰C in a water bath. Samples were withdrawn at specific time points

and the activity was determined spectrophotometrically using a BioTek SMX plate reader using

cellobiose as a substrate and the HRP-coupled assay as described above.

Conversions of eugenol with EugO-SviDyP and EugO-EncMh/SviDyP

Production of lignin-like material was tested using EugO and EncMh/SviDyP. The reaction was carried

out in 500 mL glass flasks with 50 mL reaction medium containing 10 mM eugenol in 50 mM K-

phosphate at pH 6, 100 nM EugO and either SviDyP or 3 ml of EncMh/SviDyP preparation (10

mg/mL). Reaction was incubated at 24°C 50 rpm using orbital shaker with 2.5 cm orbital for 48h. The

insoluble fraction was removed by centrifugation at 12000 rpm, washed twice with water, freeze-

dried and 10 mg of powder was taken to be analyzed by 2D NMR as described before [26].

 

7

Results and Discussion

SDS-PAGE analysis of purified EncMh

Figure S1 Purification of native (empty) encapsulin produced in 200 mL TB culture.

A. SDS PAGE analysis of 60 nL flow-through fractions of cell-free extract containing encapsulin that

were first applied to Q-Sepharose in 20 mM TrisHCl pH 8.

B. Pooled flow-through fractions from A. were applied on a Superdex 200 column 26/60 (320 mL).

Fractions containing encapsulin elute immediately after the void volume of the column (Ve ~100 mL).

The calculated yield according to the concentration by Bradford assay was 150 mg and by extinction

coefficient it is 188 mg, which roughly corresponds to the purified yield of 940 mg/L TB.

An alternative purification protocol was tested as well, employing the molecular crowding effect of

PEG. Buffer containing different percentages (w/v) of PEG-8000 was mixed with cell-free extract

containing EncMh as indicated in C and D. After the precipitation, the remaining supernatants (C) and

redissolved pellets (D) were analyzed by SDS PAGE to determine the minimal concentratrion of PEG-

8000 to achieve quantitative precipitation of EncMh.

 

8

Proteolysis resistance of encapsulin

Incubation of encapsulin with proteases and subsequent SDS PAGE analysis shows that encapsulin is

resistant to proteolysis (Figure S2). The lower molecular weight bands correspond to the respective

proteases.

Figure S2 SDS PAGE analysis of samples of encapsulin treated with bovine trypsin (lane 2) and

Proteinase K (lane 3). Control sample was incubated without proteases (lane 1).

Thermal stability of encapsulin

According to the ThermoDLS measurement, the hydrodynamic radius of encapsulin starts increasing

at temperatures higher than 50°C Figure S3). The increase of the hydrodynamic radius reflects the

aggregation of the particles and it is taken as a measure of the thermal stability.

Figure S3 DLS thermal ramping of Encapsulin (5 mg/mL pH 7.5), hydrodynamic radius is plotted as

function of temperature.

9

Data collection and refinement statistics

Table S1 Data collection and refinement statistics. Values in parentheses are for the highest resolution

shell.

10

Structure of EncMh

Figure S4 (left) Close up of the monomer of encapsulin EncMh. P-domain (peripheral domain), A-

domain (axial domain) and E-loop (extension loop) (Figure 2B) [29]. Its N-terminus is located on the

inside of the capsule while the C-terminus is on the outside. The P-domain is colored in pink, the F-

loop in dark blue sticks, the E-loop in cyan, the G-loop in orange sticks and the A-domain in light blue.

The disulfide Cys134-Cys254 is shown in sticks. The elongated E-loops (cyan and grey from another

monomer) are involved in 2-fold interaction. (right) structure of pentamer.

Figure S5 Close up of the icosahedral five-fold axis (A) view from the inside, (B) view from the outside.

The A-domain is colored in light blue, structural elements from one monomer are colored in darker

blue. Residues forming the pore and sulfate ions are shown in sticks.

11

Figure S6 Pores in the shell of the encapsulin at interface of two monomers in surface represent-

tation. One pore has a diameter of 9.0 Å and the other one has a diameter of 5.0 Å. Coloring as in

Figure S4.

12

Co-expression of EncMh with VsHb and VsHb (without targeting peptide)

Figure S7 A. SDS PAGE analysis of co-expression of encapsulin with VsHb with C-terminal targeting

peptide and B. VsHb without targeting peptide. Legend: CFE – cell-free extract, NiS – flowthrough

fraction collected after running the cfe over Ni-Sepharose column, PEG SN – 10x diluted SN obtained

after PEG-precipitation, PEG pellet – pellet obtained by PEG precipitation redissolved in buffer,

contains encapsulin and corresponding cargo protein, el – cargo protein eluted from Ni-Sepharose

column, this fraction corresponds to the excess of cargo protein which was not encapsulated.

Co-expression of EncMh with BliLacc

Figure S8 SDS PAGE analysis of co-expression of encapsulin with BliLacc. A. PEG-purified(enriched)

EncMh/BliLacc (lanes 2 and 3). B. Proteins removed by PEG-prescipitation (remaining supernatant)

can be observed in lanes 7-8.

13

Co-expression of EncMh with SviDyP and TfuCat

Figure S9 A. SDS PAGE analysis of full extract (lanes, 1 and 2) and cell-free extracts (lanes 6 and 7) of

culture expressing EncMh/SviDyP and EncMh/TfuCat (lanes 3 and 4 for full extract and lanes 8 and 9

for cell-free extract). B. Samples purified with PEG precipitation, lanes 2 and 3 EncMh/SviDyP and

lanes 4 and 5 EncMh/TfuCat.

Co-expression of EncMh with CHMO

Figure S10 SDS PAGE analysis of the expression and purification of the ENC/CHMO, performed in

biological duplicate. A. full extract (lanes 2 and 7), cell-free extract (lanes 3 and 8), flow-through

fraction from Ni-Sepharose (lanes 4 and 9), flow-through fraction from Ni-Sepharose ran over Q-

Sepharose (lanes 5 and 10). Lanes 11 and 12 – CHMO containing enc-tag which bound to Ni-

Sepharose and was eluted with imidazole. B. Final preparation of ENC/CHMO which was obtained by

running samples from A (lanes 5 and 10) on the Superdex 200 column.

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Co-expression of EncMh with mChitO

Figure S11 SDS PAGE analysis of the expression and purification of the ENC/ChitO3x, performed in

biological duplicate. A. full extract (lanes 2 and 7), cell-free extract (lanes 3 and 8), flow-through

fraction from Ni-Sepharose (lanes 4 and 9), flow-through fraction from Ni-Sepharose ran over Q-

Sepharose (lanes 5 and 10). B. Final preparation of ENC/ChitO3x which was obtained by running

samples from A (lanes 5 and 10) on the Superdex 200 column.

Cryo-EM verification of enzyme incorporation

Selective precipitation by PEG-8000 of encapsulin and various cargo enzymes and their co-elution

during gel-filtration chromatography is a clear indication that cargo enzyme is located in the lumen of

encapsulin. An additional step of IMAC using Ni-Sepharose serves as a measure to prevent any non-

incorporated enzyme to be present in the preparation. Furthermore, CryoEM was used in this study

to confirm the formation of encapsulin oligomers while it also revealed that no significant amounts of

protein aggregates.

Figure S12 Cryo-TEM image of encapsulin with cargo enzyme (SviDyP) inside. Though individual

enzymes cannot be distinguished, the micrograph confirms that the encapsulin cages from discrete

structures. Scale bar represents 50 nm.

15

Enzyme activity

Figure S13 The effect of encapsulation on stability of SviDyP. X-axis indicates time of incubation of

enzyme solution at 40⁰C. Activity assay was performed at 25⁰C.

EncMh / mChitO (ChitO Q268R G270E S410R) activity assay was performed using a coupled HRP-

based assay in MTP format using non-encapsulated mChitO as positive control and a reaction mixture

without mChitO (but with all other components) was used as negative control. Substrates used and

their final concentration were as follows: cellobiose (25 mM), maltotriose (25 mM) and cellotetraose

(6 mM). Encapsulated mChitO showed the same substrate scope as non-encapsulated enzyme, being

active on cellobiose, maltotriose and cellotetraose.

Figure S14 Example of activity assays for non-encapsulated mChitO and for duplicates of

EncMh/mChitO (ENC-ChtI3x-1 and ENC-ChtI3x-2) using three different substrates (cellobiose,

maltotriose and cellotetraose). C- control, 1 – cellobiose, 2-maltotriose, 3- cellotetraose. ChitO3x

denotes mChitO.

16

Testing activity of EncMh / BliLacc

EncMh loaded with BliLacc shows activity with canonical substrate for laccases, syringaldazine (SGZ),

with observed rates of ~1.2 min-1 as calculated per mg of total protein (including EncMh and

encapsulated BliLacc). The activity assay (Fig S11) shows two phases, where first phase is faster and

second phase, which is slower possibly due to the diffusion limitation for the oxidized product. The

rate was calculated from the first phase.

Figure S15 Activity for EncMh/BliLacc (ENC-Lacc 1 and ENC-Lacc 2 stands for duplicates) using 0.1 mM

SGZ in TrisHCl pH 7.0 and measuring the absorbance at 525 nm.

Lignin oligomers analysis

The amount of linkages and end-groups was quantified by integration of the α-protons of the various

linkages. To express this in percentages, the total amount of visible linkages was taken into

consideration where the β-β linkage was divided by two as it involves two alkyl parts of a eugenol

substrate.

Table S2. The percentage of linkages forming the lignin oligomers in the insoluble fraction

17

Multiple sequence alignment

Figure S16 Multiple sequence alignment. Structure–based alignment of Mycobacterium hassiacum

Encapsulin (EncMh, shown as MhaEnc in the figure), Thermotoga maritima Encapsulin (TmaEnc, PDB

code 3DKT, Sutter, 2008), Myxococcus xanthus Encpasulin (MxaEnc, PDB code 4PT2, McHugh, 2014)

and Pyrococcus furiosus virus-like particle (PfuEnc, PDB code 2E0Z, Akita, 2007). The secondary

structure elements at the top of the sequence alignment are from the crystal structure of EncMh.

Helices are displayed as squiggles, β-strands are rendered as arrows and strict β-turns as TT letters.

The η symbol refers to 310-helices. The elements are colored as follows: the P-domain (peripheral

domain) in blue, the A-domain (axial domain) in pink and the E-loop (extension loop) in green.

Identical residues have a red background color, similar residues have a red color. Residues with a cyan

background have been mutated (HGY to AGA at the 5-fold pore) Residues in blue belong to the G-

loop (122-130). The disulfide linkage in EncMh is indicated in green italics below the sequences. The

figure was created with ESPript [24].

 

18

Sequences of the genes and proteins used in this study

EncMh synthetic gene sequence ATGGACAATCTGTATCGTGAACTGGCCCCGGTTACGGACGCTGCTTGGCATGAAATCGAAACGGAAGCTATTCGCACCTTTAAACGCCACATTGCCGGCCGTCGCGTGGTTGATGTTAGCGAACCGTCTGGTCCGGTTACCGCCGCAGTCAGTACCGGTCATCTGCGTGATATTTCCCCGCCGGGTGATGGTGTTGTGGCTCACCTGCGCGAATCAAAACCGCTGGTGCGTCTGCGCGTTCCGTTTACCGTCACGCGTTCGGCCATTGATGACGTGGAACGCGGTAGTCAGGATTCCGACTGGGATCCGGTTAAAGCAGCTGCGAAAAAACTGGCTTTTGTCGAAGACCGTGCGATCTTCGAAGGCTATCCGGCCGCACAGATTGATGGTATCCGCCAATGTACCAGCAATCCGGTCCTGCAGCTGCCGGATGATGCCCGTGACATTACGGATGTGATCGCTCAAGCGCTGTCTGAACTGCGTCTGGCCGGCGTCGATGGTCCGTATTCAGTGCTGCTGTCGGCAGAAGTGTACACCAAAGTTTCAGAAACCACGGAACATGGCTACCCGATTCGTGAACACCTGAATCGCCTGGTTGACGGCGATATTATCTGGGCCCCGGCAATCGATGGTGCATTTGTGCTGTCCACCCGTGGCGGTGACTTCGATCTGCGCCTGGGCACGGACGTGAGCATTGGTTATCTGTCTCATGATGCGGAAACCGTTCAACTGTACCTGGAAGAAACCCTGACGTTCCTGTGTTATACGAGCGAAGCATCGGTGGCACTGACGCCG

EncMh amino acid sequenceMDNLYRELAPVTDAAWHEIETEAIRTFKRHIAGRRVVDVSEPSGPVTAAVSTGHLRDISPPGDGVVAHLRESKPLVRLRVPFTVTRSAIDDVERGSQDSDWDPVKAAAKKLAFVEDRAIFEGYPAAQIDGIRQCTSNPVLQLPDDARDITDVIAQALSELRLAGVDGPYSVLLSAEVYTKVSETTEHGYPIREHLNRLVDGDIIWAPAIDGAFVLSTRGGDFDLRLGTDVSIGYLSHDAETVQLYLEETLTFLCYTSEASVALTP

Targeting peptide sequence (TB) CCGCCGCCGCTGCCGGATTCTGAACCGGACCGTGAAATCCCGGCGGACGATGGTTCCCTGGGCATTGGTTCGCTGAAAGGCACCCGTAGCTAA

Targeting peptide amino acid sequence PPPLPDSEPDREIPADDGSLGIGSLKGTRS

VsHb sequence (His-tag in blue and TB in red)ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGTTAGATCAACAAACAATCAACATCATTAAGGCCACAGTTCCGGTTTTAAAGGAACATGGAGTGACAATCACTACGACCTTCTACAAGAACTTATTCGCAAAGCACCCGGAGGTACGTCCTTTGTTCGATATGGGACGCCAAGAATCGCTTGAACAGCCAAAAGCATTAGCGATGACGGTCCTTGCCGCTGCTCAAAATATTGAGAACCTGCCAGCTATTCTGCCCGCCGTCAAGAAAATTGCCGTCAAGCACTGCCAGGCTGGCGTGGCCGCGGCTCATTATCCTATTGTAGGCCAGGAGCTTCTGGGAGCGATTAAGGAGGTCTTGGGTGACGCGGCTACCGATGATATCCTGGACGCCTGGGGCAAGGCGTATGGGGTTATTGCAGACGTTTTTATTCAGGTCGAAGCTGACTTGTACGCGCAGGCAGTCGAATCACCGCCGCCGCTGCCGGATTCTGAACCGGACCGTGAAATCCCGGCGGACGATGGTTCCCTGGGCATTGGTTCGCTGAAAGGCACCCGTAGCTAA

VsHb amino acid sequence (His-tag in blue and TB in red)MGSSHHHHHHSSGLVPRGSHMLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQESLEQPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVESPPPLPDSEPDREIPADDGSLGIGSLKGTRS

SviDyP sequence (His-tag in blue and TB in red)ATGGGCAGCAGCCATCATCATCATCATCACGGCAGCGGCGACGGCCGGGATCGTCCGGATCGGGGTGACATCGGGCGTGCCACCGTCGACTTCCACGGCGAACGGCAGGCCGGTGTCGCGACACCCGCGCAGGCCTTCGCGACGTTCGTGGCGTTCGACCTGCTCGACGGTGTGGACCGGGAGGCGTTGATCCGGTGGATGCGGGTGTGGACCGACGACATCGAGCGGCTCACCCGCGGTGCGCCCGCGTTGACCGACACCGAACCGGAACTGGCGTTGCTCCCGGCGCGGTTGACGGTGACGGTCGGCTTCGGTCCGGGTTTCCTGGCCGCGGCCGGGCGGGAGGAGCTACGGCCGAGCTGGCTGGCGCCGTTGCCCGAGTTCCCCATCGATCGGCTCCGGGAGGAGTTCAGCGGCGGTGATCTCGTGGCCCAGGTGTGCGCCGACGACGAGGTCACGGTCGCGCACGCGGTGCGGGTGCTGACCAAACAGGCCCGTTCGTTCGCGCGGCCGCGCTGGGTGCAGCGGGGTTTCCGGAACACGCCGGGAGCGGTGCCCGAAGGGGCCACCATGCGCAATCTCATGGGCCAGTTGGACGGTACGAGGAATCTGCGTCCCGGGCCCGACGATCGGCTGATCTGGATCTCGGACGGGCCCGAGTGGCTGCGCGGTGGCACCGGGATGGTGGTGCGGCGCATCGCCATGAACCTCGACACCTGGGACGAGCTGGACCGTCCCGCGCGGGAGTTGGTGATCGGCAGGCGGTTGGACAACGGTGCGCCGCTGACGGGTCGACACGAGCACGACGAGCCGGATCTGGAGGCGGTCGACGAGCGGGGTCTGTCGGTGATCCCGATGTTCGCGCACATCCGTCGGGCCCGTTCGGACAATCCCGACGAGCGGTTCCTGCGGCGTAGTTACAACTACGACGATCCGCCCGAACCGGGTGAGCTGTCCAACAGCGGCCTGGTGTTCGTCACCTTCCAGGCCGACATCGAGGCTCAGTTCACGCCGATCCAGCGGCGCCTCGCCGAATTGGACTCGCTGAACGACTGGACCACGCCGATCGGTTCGGCCGTGTTCGCCGTTCCGCGTGGCTGCCGACCGGGCGAGTACCTCGGCCAACCGCTGTTGGAAGCCCCGCCGCCGCTGCCGGATTCTGAACCGGACCGTGAAATCCCGGCGGACGATGGTTCCCTGGGCATTGGTTCGCTGAAAGGCACCCGTAGCTAA

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SviDyP amino acid sequence (His-tag in blue and TB in red)MGSSHHHHHHGSGDGRDRPDRGDIGRATVDFHGERQAGVATPAQAFATFVAFDLLDGVDREALIRWMRVWTDDIERLTRGAPALTDTEPELALLPARLTVTVGFGPGFLAAAGREELRPSWLAPLPEFPIDRLREEFSGGDLVAQVCADDEVTVAHAVRVLTKQARSFARPRWVQRGFRNTPGAVPEGATMRNLMGQLDGTRNLRPGPDDRLIWISDGPEWLRGGTGMVVRRIAMNLDTWDELDRPARELVIGRRLDNGAPLTGRHEHDEPDLEAVDERGLSVIPMFAHIRRARSDNPDERFLRRSYNYDDPPEPGELSNSGLVFVTFQADIEAQFTPIQRRLAELDSLNDWTTPIGSAVFAVPRGCRPGEYLGQPLLEAPPPLPDSEPDREIPADDGSLGIGSLKGTRS

TfuCat sequence (His-tag in blue and TB in red)ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCATGTTGGGAAGAGGTGGGCGCCTCCCTCCGGGGAAGTGTCGGGTTGGCCCCGCACATTGGCCCAGCCGACTCTCGTCCGCTGAGGTGAGCCCCACCACGCGGGCACTGTCGCGCCACACCGGAACTTCCGGGCCGGCGCCGTCCCGATCTCTACCAGCCATATCCACGATCAAGGAGACCGACTTGACCGAGACCAACGAGGCACTGTCCACTGCGAGCAACGCCACGGGCTCCACGCTCGACTCTGGTGCGCCTGCGGCTAGCGACCGCAACTCGCTGAGCGTGGGCAGCAACGGCCCGCTGCTGCTGCACGACGTACGGCTCGTTGAGACGCTCGCCCACTTCAACCGTGAGCGGGTCCCCGAGCGCAACCCGCACGCCAAGGGCGCCGGTGCCTTCGGTGTCTTCGAAACCACGGAGGACGTCTCCCAGTACACCAAGGCTGCCCTGTTCCAGAAGGGTGCCCGTACCGAGATGCTCGCCCGGTTCTCCACGGTCGCTGGTGAGCAGGGCTCCCCGGACACCTGGCGGGACGTGCGCGGTTTCGCTCTGAAGTTCTACACCTCTGAGGGCAACTACGACCTGGTCGGCAACAACACGCCGATCTTCTTCGTCCGCGACCCGATGAAGTTCCCGCACTTCATCCGCTCCCAGAAGCGGATGCCGGACACGGGTCTGCGCGACAACAACATGCAGTGGGACTTCTGGACCCTCAACCCGGAGACCGCCCACCAGGTCACCTACCTCATGGGTGACCGCGGCCTGCCGCGGACCTGGCGCCACATGAACGGCTACGGTTCGCACACCTACATGTGGATCAACGCCAAGGGCGAGAAGTTCTGGGTCAAGTACCACTTCAAAACCGACCAGGGCATCGAGAACATGACCAACGAGGAAGCCGAGCGTCTCGCCGGTGTGGACGCCGACTTCCACCGTCGTGACCTGGTCGAGGCCATCGAACGCGGGGACTACCCGAGCTGGACCCTCTACGTGCAGGTCATGCCGTACGAGGACGCCAAGACCTACCGGTTCAACCCGTTCGACCTGACCAAGGTCTGGCCGCACAGCGACTACCCGCTCATCAAGGTCGGCAAGATGACCTTGAACCGCAACCCGGAGAACTTCTTCGCCGAGATCGAGCAGGCCGCGTTCGCGCCGTCGAACCTGGTCCCGGGTATCGGCGTCTCCCCGGACAAGATGCTGCTGGGCCGCGTCTTCGCCTACGCGGACGCCCACCGGGCCCGGATCGGCACCAACTACTTCCAGCTGCCGGTGAACAAGCCGCGGGTGAAGGTCAACTCCTACACCTTCGACGGGCACATGACCTACGAGCACTCCGGCAAGGCCCCGGTGTACGCGCCCAACTCCTACGGCCGTCCCTGGTCTGACCAGACCGGCCCGGTCGAGGACAGCTGGGAGGCCGACGGCGAGCTGGTGCGCAGCGCCTACGAGCTGCACGCTGAGGACGACGACTTCTCGCAGGCCGGCACCCTGGTCCGCGAGGTCTTCGACGACGCCCAGCGGGACCGGCTCGTGGAGACGGTCGCCGACCACCTGTCCAAGGGTGTCGTCGAGCCGGTGCTCTCCCGGGCCTTCCAGTACTGGAAGAACATCGACGAGACCATCGGCGAGCGGATCGAGAAGCGGTACCACGAGATCGCTCGGCCGCCGCCGCTGCCGGATTCTGAACCGGACCGTGAAATCCCGGCGGACGATGGTTCCCTGGGCATTGGTTCGCTGAAAGGCACCCGTAGCTAATfuCat amino acid sequence (His-tag in blue and TB in red) MGSSHHHHHHSSGMLGRGGRLPPGKCRVGPAHWPSRLSSAEVSPTTRALSRHTGTSGPAPSRSLPAISTIKETDLTETNEALSTASNATGSTLDSGAPAASDRNSLSVGSNGPLLLHDVRLVETLAHFNRERVPERNPHAKGAGAFGVFETTEDVSQYTKAALFQKGARTEMLARFSTVAGEQGSPDTWRDVRGFALKFYTSEGNYDLVGNNTPIFFVRDPMKFPHFIRSQKRMPDTGLRDNNMQWDFWTLNPETAHQVTYLMGDRGLPRTWRHMNGYGSHTYMWINAKGEKFWVKYHFKTDQGIENMTNEEAERLAGVDADFHRRDLVEAIERGDYPSWTLYVQVMPYEDAKTYRFNPFDLTKVWPHSDYPLIKVGKMTLNRNPENFFAEIEQAAFAPSNLVPGIGVSPDKMLLGRVFAYADAHRARIGTNYFQLPVNKPRVKVNSYTFDGHMTYEHSGKAPVYAPNSYGRPWSDQTGPVEDSWEADGELVRSAYELHAEDDDFSQAGTLVREVFDDAQRDRLVETVADHLSKGVVEPVLSRAFQYWKNIDETIGERIEKRYHEIARPPPLPDSEPDREIPADDGSLGIGSLKGTRS

AcCHMO sequence (His-tag in blue and TB in red)ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGTCACAAAAAATGGATTTTGATGCTATCGTGATTGGTGGTGGTTTTGGCGGACTTTATGCAGTCAAAAAATTAAGAGACGAGCTCGAACTTAAGGTTCAGGCTTTTGATAAAGCCACGGATGTCGCAGGTACTTGGTACTGGAACCGTTACCCAGGTGCATTGACGGATACAGAAACCCACCTCTACTGCTATTCTTGGGATAAAGAATTACTACAATCGCTAGAAATCAAGAAAAAATATGTGCAAGGCCCTGATGTACGCAAGTATTTACAGCAAGTGGCTGAAAAGCATGATTTAAAGAAGAGCTATCAATTCAATACCGCGGTTCAATCGGCTCATTACAACGAAGCAGATGCCTTGTGGGAAGTCACCACTGAATATGGTGATAAGTACACGGCGCGTTTCCTCATCACTGCTTTAGGCTTATTGTCTGCGCCTAACTTGCCAAACATCAAAGGCATTAATCAGTTTAAAGGTGAGCTGCATCATACCAGCCGCTGGCCAGATGACGTAAGTTTTGAAGGTAAACGTGTCGGCGTGATTGGTACGGGTTCCACCGGTGTTCAGGTTATTACGGCTGTGGCACCTCTGGCTAAACACCTCACTGTCTTCCAGCGTTCTGCACAATACAGCGTTCCAATTGGCAATGATCCACTGTCTGAAGAAGATGTTAAAAAGATCAAAGACAATTATGACAAAATTTGGGATGGTGTATGGAATTCAGCCCTTGCCTTTGGCCTGAATGAAAGCACAGTGCCAGCAATGAGCGTATCAGCTGAAGAACGCAAGGCAGTTTTTGAAAAGGCATGGCAAACAGGTGGCGGTTTCCGTTTCATGTTTGAAACTTTCGGTGATATTGCCACCAATATGGAAGCCAATATCGAAGCGCAAAATTTCATTAAGGGTAAAATTGCTGAAATCGTCAAAGATCCAGCCATTGCACAGAAGCTCATGCCACAGGATTTGTATGCAAAACGTCCGTTGTGTGACAGTGGTTACTACAACACCTTTAACCGTGACAATGTCCGTTTAGAAGATGTGAAAGCCAATCCGATTGTTGAAATTACCGAAAACGGTGTGAAACTCGAAAATGGCGATTTCGTTGAATTAGACATGCTGATATGTGCCACAGGTTTT

20

GATGCCGTCGATGGCAACTATGTGCGCATGGACATTCAAGGTAAAAACGGCTTGGCCATGAAAGACTACTGGAAAGAAGGTCCGTCGAGCTATATGGGTGTCACCGTAAATAACTATCCAAACATGTTCATGGTGCTTGGACCGAATGGCCCGTTTACCAACCTGCCGCCATCAATTGAATCACAGGTGGAATGGATCAGTGATACCATTCAATACACGGTTGAAAACAATGTTGAATCCATTGAAGCGACAAAAGAAGCGGAAGAACAATGGACTCAAACTTGCGCCAATATTGCGGAAATGACCTTATTCCCTAAAGCGCAATCCTGGATTTTTGGTGCGAATATCCCGGGCAAGAAAAACACGGTTTACTTCTATCTCGGTGGTTTAAAAGAATATCGCAGTGCGCTAGCCAACTGCAAAAACCATGCCTATGAAGGTTTTGATATTCAATTACAACGTTCAGATATCAAGCAACCTGCCAATGCCCCGCCGCCGCTGCCGGATTCTGAACCGGACCGTGAAATCCCGGCGGACGATGGTTCCCTGGGCATTGGTTCGCTGAAAGGCACCCGTAGCTAA

AcCHMO amino acid sequence (His-tag in blue and TB in red)MGSSHHHHHHSSGLVPRGSHMSQKMDFDAIVIGGGFGGLYAVKKLRDELELKVQAFDKATDVAGTWYWNRYPGALTDTETHLYCYSWDKELLQSLEIKKKYVQGPDVRKYLQQVAEKHDLKKSYQFNTAVQSAHYNEADALWEVTTEYGDKYTARFLITALGLLSAPNLPNIKGINQFKGELHHTSRWPDDVSFEGKRVGVIGTGSTGVQVITAVAPLAKHLTVFQRSAQYSVPIGNDPLSEEDVKKIKDNYDKIWDGVWNSALAFGLNESTVPAMSVSAEERKAVFEKAWQTGGGFRFMFETFGDIATNMEANIEAQNFIKGKIAEIVKDPAIAQKLMPQDLYAKRPLCDSGYYNTFNRDNVRLEDVKANPIVEITENGVKLENGDFVELDMLICATGFDAVDGNYVRMDIQGKNGLAMKDYWKEGPSSYMGVTVNNYPNMFMVLGPNGPFTNLPPSIESQVEWISDTIQYTVENNVESIEATKEAEEQWTQTCANIAEMTLFPKAQSWIFGANIPGKKNTVYFYLGGLKEYRSALANCKNHAYEGFDIQLQRSDIKQPANAPPPLPDSEPDREIPADDGSLGIGSLKGTRS

ChitO Q268R G270E S410R sequence (His-tag in blue and TB in red)ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATGTCCCGACCAAGCGCGAAGCCGTTAACAGCTGTCTCACACAAGCCAAAGTGCCTACCGACGCACAGGGCTCTCAATCGTGGAAAGAAGACGGCACAGCTTACAATCTGAGACTTCCATTCGAACCAGCTGCCATCGCCGTTCCCACAACCGTCGCTCAAGTCTCCGCTGCCGTTGAATGTGGCGCCAAGCACGGCGTTGCAATTAGTGCCAAGAGTGGCGGCCACAGCTATACCTCTCTAGGCTTCGGTGGCGAAGATGGCCATCTCATGATTGAGCTCGACAGAATGTACAGCGTCAAGTTGGCCAAGGACGGCACCGCCAAAATCCAGCCTGGTGCTCGCCTTGGACATGTTGCTACTGAGCTCTGGAACCAGGGCAAGCGAGCTCTAGCTCATGGAACGTGCCCTGGAGTCGGCCTCGGTGGTCACGCCCTTCACGGAGGCTACGGAATGGTCGCCCGCAAGCACGGCCTCACTCTTGATCTTATGATCGGCGCCACAGTCGTTCTCCCCACCGGCAAAGTCGTCCACTGCTCCAAGACCGAGAACTCCGATCTCTTCTGGGGTATCCGTGGCGCCGGCGCAAACTTTGGTGTCGTTGTCGAGCTCGAGTTCCAGACATTTGCCGCACCTGAGAAGATCACCTACTTCGACATCGGCCTCAACTGGGATCAGAACACAGCTCCCCAGGGTCTTTATGACTTCCAGGAGTTTGGAAAGGGCATGCCTGCCGAGATCACCATGCGCATGGAGGTCTCTAAGAACGGATACAGCGTCGATGGTGCTTATATCGGTGATGAGGCCAGCTTGAGGAAGGCTCTGCAGCCTTTGGTCCAGAAGTTTGGCGGTGTTCAGGTCACTGCTACTACTGTTGACTGGATGGGTCTTGTTACTCACTTCGCCGGTGCTGGCGTCAACGTCAACCCTACCAGTGCCTCATACGACGCACATGACAACTTCTACGCCAGCAGTCTTGCAGCCCCCGCGTTGACCCTCGCCGAATTCAAGTCCTTCGTCAACTTCGTCTCCACCACCGGCAAGAGCAGCAGCCACTCTTGGTGGTTGCAAATGGACATCACCGGCGGCACATACTCCGCCGTCTCCAAGCCCAAGCCCAGCGACACCGCCTACGTCCACCGTGACACCCTCCTCCTCTTCCAATTCTACGACAGGGTTGCCGCCACTGCCCAGTACCCCTCTGATGGCTTCAACCTCATCAAGGGCTTGAGGCAGAGCATCTCCAGCTCTCTCAAGGCGGGAACTTGGGGTATGTACGCCAACTACCCTGACTCGCAGATCAAGAATGATCGCGCTACCGAGATGTACTGGGGAAGCAACGTTGCCAAGCTGGAGGCTGTCAAGGCCAAGTACGATCCTAAGAACTTGTTCCGCAACCCTCAGTCTATTAAGCCTAAGGCTCCGCCGCCGCTGCCGGATTCTGAACCGGACCGTGAAATCCCGGCGGACGATGGTTCCCTGGGCATTGGTTCGCTGAAAGGCACCCGTAGCTAA

ChitO Q268R G270E S410R amino acid sequence (His-tag in blue and TB in red)MGSSHHHHHHSSGLVPRGSHVPTKREAVNSCLTQAKVPTDAQGSQSWKEDGTAYNLRLPFEPAAIAVPTTVAQVSAAVECGAKHGVAISAKSGGHSYTSLGFGGEDGHLMIELDRMYSVKLAKDGTAKIQPGARLGHVATELWNQGKRALAHGTCPGVGLGGHALHGGYGMVARKHGLTLDLMIGATVVLPTGKVVHCSKTENSDLFWGIRGAGANFGVVVELEFQTFAAPEKITYFDIGLNWDQNTAPQGLYDFQEFGKGMPAEITMRMEVSKNGYSVDGAYIGDEASLRKALQPLVQKFGGVQVTATTVDWMGLVTHFAGAGVNVNPTSASYDAHDNFYASSLAAPALTLAEFKSFVNFVSTTGKSSSHSWWLQMDITGGTYSAVSKPKPSDTAYVHRDTLLLFQFYDRVAATAQYPSDGFNLIKGLRQSISSSLKAGTWGMYANYPDSQIKNDRATEMYWGSNVAKLEAVKAKYDPKNLFRNPQSIKPKAPPPLPDSEPDREIPADDGSLGIGSLKGTRS

BliLacc sequence (His-tag in blue and TB in red)ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGAAACTTGAAAAATTCGTTGACAAACTCCCCATTCCGAAAGTGCTTAAACCCCACAGCAAAAGCAAGGAAATGACCTATTATGAAGTCACGATGAAAGAATTTCAGCAGCAGCTCCACCGCGATCTGCCGCCGACCCGGCTGTTTGGATATAACGGGGTCTATCCCGGCCCCACCTTTGAAGTGCAAAAACACGAAAAAGTCGCGGTCAAATGGTTGAATAAGCTTCCGGATCATCATTTTCTCCCCGTCGACCATACAATTCATGACGACGGCCATCATGAACATGAAGTCAAAACGGTCGTTCATTTGCATGGAGGCCGGACACCGCCTGATAGCGACGGTTACCCGGAAGCCTGGTACACTAAAGATTTTCAAGTCAAAGGCCCTTTTTTTGAAAGGGAGGTGTATGAATATCCGAATGAGCAGGATGCTACGGCTCTCTGGTATCACGATCATGCAATGGCCATCACAAGGCTGAATGTATATGCGGGGCTTGTCGGTTTATATTTTATCCGCGACAGGGAAGAGCGGTCATTG

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AACTTGCCGAAGGGAGAATATGAAATTCCGCTCTTGATTCAGGATAAGTCCTTTCATGAAGATGGTTCATTGTTTTATCCGCGGCAGCCTGACAACCCTTCGCCGGATCTTCCCGACCCGTCGATTGTTCCGGCTTTTTGCGGTGATACCATTTTAGTCAACGGCAAGGTATGGCCTTATGATGAACTGGAACCTCGAAAATACCGTTTTCGGATACTGAACGCCTCCAATACGAGAATCTTTGAGCTGTATTTCGATCATGACATCACATTTCACCAAATCGGCACGGACGGTGGTCTTCTGCAGCATCCGGTTAAAGTCAATGAACTGGTGATCGCACCGGCTGAAAGATGCGATATCATCGTTGATTTTTCACGAGCTGAAGGAAAAACCGTGACATTGAAAAACCGGATCGGCTGCAGCGGACAAGACGCGGATCCCGATACAGATGCCAACATTATGCAATTCCGCATCTCAAAACCATTGAAGCAAAAAGATACAAGTTCATTGCCGAGAATATTGAGAAAACGCCCATTTTACCGGAGACACAAGATCAATACCCTTAGAAATCTGTCGTTGGGCGCGTCCCTTGACCAATATGGAAGACCTGTTCTGCTTTTAAACAACACAAAATGGCATGAACCGGTAACTGAAACTCCCGCCCTCGGCAGCACTGAGATCTGGTCGATCATCAATGCCGGAAGAGCGATCCATCCGATCCATTTACATCTTGTCCAATTTTTGATTCTCGACCACCGGCCGTTTGATATCGAACGGTATCAGGAAAACGGAGAACTAGTCTTTACAGGTCCGGCAGCTCCACCGGCACAGAATGAAAAGGGGCTGAAAGACACCGTCAAAGTACCTCCTGGCTCGGTGACGCGGATTATCGCAACCTTCGCGCCGTACAGCGGCAGATATGTGTGGCACTGCCACATTCTGGAGCACGAAGATTACGATATGATGCGCCCTCTTGAAGTAACGGATATTCGTCATCAACCGCCGCCGCTGCCGGATTCTGAACCGGACCGTGAAATCCCGGCGGACGATGGTTCCCTGGGCATTGGTTCGCTGAAAGGCACCCGTAGCTAA

BliLacc amino acid sequence (His-tag in blue and TB in red)MGSSHHHHHHSSGLVPRGSHMKLEKFVDKLPIPKVLKPHSKSKEMTYYEVTMKEFQQQLHRDLPPTRLFGYNGVYPGPTFEVQKHEKVAVKWLNKLPDHHFLPVDHTIHDDGHHEHEVKTVVHLHGGRTPPDSDGYPEAWYTKDFQVKGPFFEREVYEYPNEQDATALWYHDHAMAITRLNVYAGLVGLYFIRDREERSLNLPKGEYEIPLLIQDKSFHEDGSLFYPRQPDNPSPDLPDPSIVPAFCGDTILVNGKVWPYDELEPRKYRFRILNASNTRIFELYFDHDITFHQIGTDGGLLQHPVKVNELVIAPAERCDIIVDFSRAEGKTVTLKNRIGCSGQDADPDTDANIMQFRISKPLKQKDTSSLPRILRKRPFYRRHKINTLRNLSLGASLDQYGRPVLLLNNTKWHEPVTETPALGSTEIWSIINAGRAIHPIHLHLVQFLILDHRPFDIERYQENGELVFTGPAAPPAQNEKGLKDTVKVPPGSVTRIIATFAPYSGRYVWHCHILEHEDYDMMRPLEVTDIRHQPPPLPDSEPDREIPADDGSLGIGSLKGTRS 

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References[1] W. Yu, W. Liu, H. Huang, F. Zheng, X. Wang, Y. Wu, K. Li, X. Xie, Y. Jin, PLOS ONE 2014, 9, e110319.[2] D. I. Colpa, N. Lončar, M. Schmidt, M. W. Fraaije, Chembiochem : a European journal of chemical biology 2017, 18, 2226-2230.[3] N. Lončar, M. W. Fraaije, Appl. Microbiol. Biotechnol. 2015, 99, 2225-2232.[4] N. Lončar, N. Božić, Z. Vujčić, Journal of Molecular Catalysis B: Enzymatic, https://doi.org/10.1016/j.molcatb.2016.06.005.[5] A. R. Ferrari, M. Lee, M. W. Fraaije, Biotechnol. Bioeng. 2015, 112, 1074-1080.[6] G. T. Oostergetel, W. Keegstra, A. Brisson, Ultramicroscopy, https://doi.org/10.1016/S0304-3991(98)00022-9.[7] R. Marabini, I. M. Masegosa, M. C. San Mar n, S. Marco, J. J. Fernández, L. G. de la Fraga, C. tVaquerizo, J. M. Carazo, Journal of Structural Biology, https://doi.org/10.1006/jsbi.1996.0036.[8] A. Zaldívar-Peraza, C. O. S. Sorzano, J. Otón, J. Vargas, J. M. Carazo, J. M. de la Rosa-Trevín, R. Marabini, V. Abrishami, Y. Shkolnisky, Bioinformatics 2013, 29, 2460-2468.[9] S. H. W. Scheres, Journal of Molecular Biology, https://doi.org/10.1016/j.jmb.2011.11.010.[10] S. H. W. Scheres, Journal of Structural Biology, https://doi.org/10.1016/j.jsb.2012.09.006.[11] T. G. Battye, L. Kontogiannis, O. Johnson, H. R. Powell, A. G. Leslie, Acta Crystallogr. D 2011, 67, 271-281.[12] B. Reinhammar, Y. Oda, J. Inorg. Biochem., http://dx.doi.org/10.1016/S0162-0134(00)80177-4.[13] J. M. Harkin, M. J. Larsen, J. R. Obst, Mycologia 1974, 66, 469-476.[14] S. M. Cragg, G. T. Beckham, N. C. Bruce, T. D. Bugg, D. L. Distel, P. Dupree, A. G. Etxabe, B. S. Goodell, J. Jellison, J. E. McGeehan, S. J. McQueen-Mason, K. Schnorr, P. H. Walton, J. E. Watts, M. Zimmer, Curr. Opin. Chem. Biol. 2015, 29, 108-119.[15] D. Cannella, K. B. Mollers, N. U. Frigaard, P. E. Jensen, M. J. Bjerrum, K. S. Johansen, C. Felby, Nat. Commun. 2016, 7, 11134.[16] M. Sutter, D. Boehringer, S. Gutmann, S. Gunther, D. Prangishvili, M. J. Loessner, K. O. Stetter, E. Weber-Ban, N. Ban, Nat. Struct. Mol. Biol. 2008, 15, 939-947.[17] F. Sargent, F. A. Davidson, C. L. Kelly, R. Binny, N. Christodoulides, D. Gibson, E. Johansson, K. Kozyrska, L. L. Lado, J. Maccallum, R. Montague, B. Ortmann, R. Owen, S. J. Coulthurst, L. Dupuy, A. R. Prescott, T. Palmer, Microbiology 2013, 159, 2427-2436.[18] E. Strittmatter, S. Wachter, C. Liers, R. Ullrich, M. Hofrichter, D. A. Plattner, K. Piontek, Arch. Biochem. Biophys. 2013, 537, 161-167.[19] G. N. Murshudov, P. Skubak, A. A. Lebedev, N. S. Pannu, R. A. Steiner, R. A. Nicholls, M. D. Winn, F. Long, A. A. Vagin, Acta Crystallogr. D 2011, 67, 355-367.[20] P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Acta Crystallogr. D 2010, 66, 486-501.[21] V. B. Chen, W. B. 3. Arendall, J. J. Headd, D. A. Keedy, R. M. Immormino, G. J. Kapral, L. W. Murray, J. S. Richardson, D. C. Richardson, Acta Crystallogr. D 2010, 66, 12-21.[22] B. R. Scott, H. Z. Huang, J. Frickman, R. Halvorsen, K. S. Johansen, Biotechnol. Lett. 2016, 38, 425-434.[23] I. Patel, D. Kracher, S. Ma, S. Garajova, M. Haon, C. B. Faulds, J. G. Berrin, R. Ludwig, E. Record,Biotechnol. Biofuels 2016, 9, 108-016-0520-3. eCollection 2016.[24] X. Robert, P. Gouet, Nucleic Acids Res. 2014, 42, W320-W324.[25] C. Jakopitsch, J. Vlasits, B. Wiseman, P. C. Loewen, C. Obinger, Biochemistry 2007, 46, 1183-1193.[26] M. H. M. Habib, P. J. Deuss, N. Lončar, M. Trajkovic, M. W. Fraaije, Adv. Synth. Catal. 2017, 359,3354-3361.[27] R. Rahmanpour, T. D. Bugg, FEBS J. 2013, 280, 2097-2104.[28] C. Cassidy-Amstutz, L. Oltrogge, C. C. Going, A. Lee, P. Teng, D. Quintanilla, A. East-Seletsky, E. R. Williams, D. F. Savage, Biochemistry (N. Y. ) 2016, 55, 3461-3468.[29] M. M. Suhanovsky, C. M. Teschke, Virology 2015, 479, 487-497.

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