Self-Assembling Protein Scaffold System for Easy in Vitro

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A self-assembling protein scaffold system for easy in vitro co-immobilization of biocatalytic cascade enzymes Guoqiang Zhang, Maureen Quin, and Claudia Schmidt-Dannert ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b00986 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 13, 2018

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ACS Catalysis

A self-assembling protein scaffold system for easy in vitro co-immobilization of biocatalytic cascade enzymes

2 3 4 5 Guoqiang Zhang, Maureen B. Quin* and Claudia Schmidt-Dannert* 6 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Ave, St. Paul, MN 55108, USA. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

ABSTRACT: Biocatalytic cascades represent an attractive approach for the synthesis of valuable chemicals. To be competitive with chemical synthesis, cascade reactions need to be efficient, robust, self-sufficient and ideally performed as one-pot in vitro reactions. Immobilization of enzymes has the potential to improve enzyme stability and increase reaction efficiency. However, co-immobilization of multiple different enzymes on the same solid surface is difficult, requiring optimized chemistry for each catalyst. To address this challenge, we set out to develop an easy-to-adapt, genetically programmable and self-assembling protein scaffolding system for the simple immobilization of biocatalytic cascades. We adopted the self-assembling properties of the bacterial microcompartment protein EutM from Salmonella enterica to engineer scaffolds for covalent linkage with biocatalysts using SpyTag-SpyCatcher covalent bond formation. Our results show that our scaffolding system can be readily isolated from E. coli, self-assembles, and remains stable in vitro under a range of conditions relevant for biocatalysis. Furthermore, cargo proteins spontaneously covalently attach to the protein scaffolds in vitro. As initial proof-of-concept, we co-immobilize a dual enzyme cascade for chiral amine synthesis and show that scaffolding of the cascade reduces the time required to reach final conversions, compared to the free enzyme system. Detailed analyses suggest that this may result from stabilization of the enzymes upon immobilization on the protein scaffolds. Together these results establish the groundwork for future protein-based scaffolding of enzyme cascades for in vitro or in vivo biocatalysis.

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In vitro biocatalytic systems are increasingly being developed as an alternative to chemical synthetic routes. Enzymes can serve as economically viable catalysts in the production of valuable compounds1-4. Advances in systems and synthetic biology have provided easy access to biocatalysts, inspiring the design of new enzymatic cascades. Enzymes from multiple different sources can be combined in one pot reactions to achieve diverse and complex multi-step chemical transformations5-8. There are several advantages associated with in vitro, one pot biocatalytic cascades, including the fact that there is no need to isolate reaction intermediates as they are directly transferred to the next step of the reaction sequence, and isolation of the product is relatively straightforward. Overall, this can reduce cost, time, and effort, and can increase final yield of product9. Yet, developing an efficient, one-pot biocatalytic cascade is not without its challenges. Designing an effective enzyme cascade requires careful consideration of several factors that can affect catalyst and cascade performance, including optimizing reaction conditions for each biocatalyst, stoichiometric balancing of enzyme activities to prevent substrate or product inhibition, and maintaining enzyme stability to achieve adequate turn over numbers. In nature, cells perform numerous enzyme cascade reactions simultaneously, and with high efficiency. Spatial organization of the enzymes within these cellular metabolic networks plays a key role in ensuring optimal substrate concentrations and microenvironments for enzyme catalysis and stability1, 10-16. Similarly, spatial organization of enzymes could be employed to create robust and highly efficient cell-free, in vitro biocatalytic cascade reactions17-23. One approach to achieve this would be to co-immobilize biocatalytic enzyme cascades on an inert, solid support. Enzymes immobilized on different support materials are routinely used in biotransformations24-25 enabling biocatalyst

KEYWORDS: Self-assembling, protein scaffolds, SpyTag/SpyCatcher, enzyme cascade, chiral amine, biocatalysis

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recycling and stabilization26. However, co-immobilizing multiple different enzymes on the same support can be challenging, either requiring that immobilization chemistries are carefully chosen for each catalyst27, or that the enzymes are each engineered with a different immobilization tag28-29. Our goal in this work was to develop an easy-to-produce and easy-to-use, self-assembling scaffolding system for the straightforward co-immobilization of enzymes for in vitro biocatalysis. Protein-based scaffolds have the advantage of being genetically programmable i.e. they can be easily engineered for enzyme-friendly and versatile attachment via genetically fused peptide tags. They can also be produced recombinantly at a relatively low cost using a microbial host, and unlike e.g. nucleic acid scaffolds, protein scaffolds are sufficiently robust for industrial processes30-33. Additionally, both microenvironment and proximity of catalysts determine cascade reaction efficiency11, and protein scaffold surfaces could provide optimal microenvironments for catalysis34. As such, different protein- or peptide-based scaffolding systems have been explored for the spatial organization of single and multi-enzyme systems in vivo (e.g. metabolic engineering) and in vitro (e.g. synthetic biology) 18, 30, 33, 35-40 . In this work, we sought to explore whether selfassembling protein arrays could be used to develop a scaffolding system for facile co-immobilization of biocatalysts to its surface and subsequent in vitro operation of an industrially relevant biocatalytic cascade reaction. Based on our previous work on engineering protein nanocompartments in E. coli using shell proteins from the ethanolamine utilization (Eut) bacterial microcompartment from Salmonella enterica41-43, we chose the 9.8 kDa shell protein EutM to create self-assembling protein scaffolds. Structural studies of

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EutM homologs have shown that it assembles into hexamers which self-organize into the arrays that form the facets of microcompartment shells44-46. We serendipitously observed that overexpression of the S. enterica shell protein EutM in E. coli resulted in the formation of a thick axial protein filament that in some cases prevented correct cell division41. Intrigued by these self-assembly properties, we reasoned that EutM could serve as a building block for the design of an in vitro protein-based scaffolding system for biocatalytic cascades. Here, we show that heterologously expressed EutM protein scaffolds can be readily isolated in vitro and remain stable under a range of biocatalysis relevant process conditions. We demonstrate that cargo proteins can be rapidly covalently attached to the scaffolds via a short, genetically fused peptide tag. Finally, as a proof-of-concept, we characterize and optimize the co-immobilization of a dual enzyme cascade for chiral amine synthesis. Our results indicate that improvements in rate of reaction obtained by the scaffolded enzyme system versus the free system are caused by stabilization of the enzyme(s) upon immobilization on the protein scaffold. This work will serve as a framework for future design and optimization of other one-pot cascade reactions with multiple enzymes co-immobilized onto our developed scaffolding system.

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RESULTS AND DISCUSSION Design and in vivo testing of protein scaffolding system We first established that S. enterica EutM, which forms protein filaments when recombinantly overexpressed in E. coli41, could also self-assemble as structural scaffolds in vitro. When we overexpressed and purified EutM, the isolated protein readily precipitated out of solution as large crystalline arrays, with obvious hexameric organization and symmetry (Figure 1a). These results indicated that EutM could serve as a building block for the design of an in vitro protein-based scaffolding system. We hypothesized that attachment of cargo enzymes to the protein scaffold could be achieved by genetically fusing cognate interacting protein domains or peptides to the N- or C-terminus of cargo enzymes and EutM scaffold building blocks. To create the attachment points, we chose the well-established

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Figure 1. Protein-based scaffolding system. (a) Purified S. enterica EutM readily forms higher order structures in vitro. Negative stain TEM of the white precipitate formed in the tube shows crystalline EutM scaffolds (left) composed of hexameric tiles (middle, highlighted with a red dashed line) that are assembled from structurally ordered arrays of EutM hexamers (right, red dashed line indicates ordered crystalline lattice). Insert highlights individual EutM hexamers. (b) Enzymes are covalently attached to scaffolds by fusing a SpyCatcher domain (green) to the C-terminus of EutM monomers and a SpyTag peptide sequence (blue) to the N- or C-terminus of cargo-enzymes. SpyCatcher and SpyTag form a covalent isopeptide bond (yellow), attaching enzymes to scaffolds.

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SpyTag/SpyCatcher system as a convenient protein-protein ligation tool47-49. Fusion of a short SpyTag peptide (~1.5 kDa) to the N- or C-terminus of different cargo proteins, and the SpyCatcher domain (~9.5 kDa) to the C-terminus of EutM (creating EutM-SpyCatcher, ~21 kDa) should then facilitate rapid autocatalytic isopeptide bond formation between cargo and scaffolds as illustrated in Figure 1b. We first confirmed in vivo that the designed fusion protein EutM-SpyCatcher formed scaffolds, and that the SpyCatcher domain was accessible for cargo attachment. We co-expressed EutM-SpyCatcher as scaffolds, and SpyTag fused fluorescent proteins GFP and mCherry as cargo. Fluorescence microscopy imaging of the E. coli cells showed that both N- and C-terminal configurations of SpyTag on fluorescent proteins resulted in the formation of fluorescent foci when co-expressed with EutMSpyCatcher. Contrastingly, fluorescent proteins with no SpyTag fusion, or SpyTag fused cargo expressed in the absence of EutMSpyCatcher, resulted in diffuse fluorescence (Figure S1 and Figure S2). From these results we concluded that the SpyTag was directing the fluorescent protein cargo to EutM-SpyCatcher in vivo, and that EutM-SpyCatcher may have been assembling into higher order structures within the cytoplasm. To confirm whether EutM-SpyCatcher was in fact assembling into large structures, we visualized the ultrastructure of the E. coli cells by thin cell section transmission electron microscopy (TEM) (Figure S3). As we had previously observed41, EutM formed a thick protein filament along the axis of the cells, that extended the length of cells. It appeared that the protein filament was striated i.e. there were clearly defined lines along the length of the protein filament, suggesting that the axial protein filament could be composed of thinner protein fibers that are aligned in an ordered fashion. We noted that EutM-SpyCatcher also formed a higher order protein assembly within the cells, however, these structures appeared thinner and shorter than EutM, with fibril-like arrays that were aligned together in a less regular fashion throughout the cytoplasm, and not extending the full length of the cell. We concluded that the C-terminal fusion of the SpyCatcher domain to EutM slightly altered but did not abolish


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ACS Catalysis

Figure 2. EutM-SpyCatcher scaffolds and SpyTag-cargo loading in vitro. (a) His-tagged EutM-SpyCatcher (2 mg mL-1, 1x PBS pH 7.4) purified from E. coli forms arrays of protein fibrils visualized by negative stain TEM. (b) Purified SpyTag-GFP and EutM-SpyCatcher were mixed at 1:1 molar ratio (10 µM each) in 1 x PBS pH 7.4 at RT for 30 min and isopeptide bond formation analyzed by SDS-PAGE, alongside unreactive controls of EutM and GFP. The final SpyTag-SpyCatcher complexes are labelled with asterisks. (c) Fluorescence microscopy confirmed the efficient loading of SpyTag-GFP/GFP-SpyTag on EutM-SpyCatcher scaffolds by mixing equal volumes of 1 mg mL-1 GFP cargo and 2 mg mL-1 EutM-SpyCatcher in 1 x PBS pH 7.4 prior to incubation at RT for 30 min. its self-assembly properties in vivo. In vitro characterization of the scaffolding system Having confirmed our initial design of EutM-SpyCatcher scaffolds in vivo, we characterized the formation of scaffolds in vitro using purified protein. EutM-SpyCatcher (with an Nterminal His-tag) was overexpressed and isolated by one-step nickel affinity chromatography, to a high degree of purity. Negative stain TEM of the purified protein confirmed that it selfassembled into scaffolds at room temperature and pH 7.4 (Figure 2a). Compared to our previous observation that EutM formed rigid, hexameric arrays (Figure 1a), we noted that EutMSpyCatcher instead formed clusters or “nests” (µms in size) that were composed of long, flexible protein fibrils (Figure 2a, left TEM image). Individual fibrils were ~40 nm in width and several hundred nm in length (Figure 2a, middle and right TEM images). These structures were reminiscent of the fibril-like structures that we had observed by thin cell sectioning of cells (Figure S3). Because the EutM-SpyCatcher fibrils were long and thin, we were concerned that they may not be stable enough for typical in vitro biocatalytic reaction conditions50. We therefore tested scaffold assembly at a range of pH values, in a protein concentration dependent fashion. While self-assembly occurred most readily at pH 7 at a concentration of ~1 mg mL-1, we found that the fibril-like scaffolds also formed under a wide range of pH conditions (pH 5-9), including in 2 M ammonia buffer (pH 8.7) required for the enzyme cascade tested below (described in more detail in the next section) (Figure S4). Furthermore, when the protein scaffolds were incubated for 12 hrs at elevated temperatures (40 oC and 50 °C versus 30 °C in Tris-HCl buffer pH 8.0), we detected a slight improvement in scaffold structure, 3

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with fibrils appearing longer (Figure S5). It could be that increasing temperature may improve EutM-SpyCatcher selfassembly by enhancing macromolecular crowding51, however, more detailed structural studies beyond the scope of this work would be required to confirm this hypothesis. When the scaffolds were incubated under the same conditions for 12 hrs at 60 oC and 70 oC, scaffolds were not formed any longer. We concluded that EutM-SpyCatcher scaffolds were stable during prolonged incubation and were not disrupted by elevated temperatures up to 50 oC. Having established that robust scaffolds assemble in vitro, we tested SpyTag:SpyCatcher mediated cargo immobilization on scaffolds. We mixed different molar ratios of purified SpyTag fused fluorescent protein GFP with different molar ratios of purified EutM-SpyCatcher (Figure 2b and Figure S6). SDSPAGE analysis of the mixtures showed that isopeptide bond formation occurred spontaneously, and within minutes, with a covalent complex of EutM-SpyCatcher:SpyTag-GFP detectable as a ~52 kDa band. We did note that despite varying reaction time, and molar ratio of the two proteins, a small amount of EutMSpyCatcher and/or SpyTag-GFP always remained unattached, a phenomenon that has been previously observed (~80 % reaction completion has previously been reported for the SpyTag/SpyCatcher system)48. Additionally, the reaction between GFP-SpyTag and EutM-SpyCatcher was slightly less efficient compared to SpyTag-GFP, potentially due to steric hindrances in the isopeptide bond formation process. As a final validation of our protein scaffolding system, cargo loading onto preassembled EutM-SpyCatcher scaffolds was confirmed by light microscopy. At magnifications afforded by the


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Figure 3. Co-immobilization of a dual enzyme cascade for chiral amine synthesis. (a) Schematic of dual enzyme cascade coimmobilized on EutM-SpyCatcher protein scaffolds. An alcohol dehydrogenase (ADH) oxidizes an alcohol substrate into the corresponding ketone intermediate that is subsequently reduced by an amine dehydrogenase (AmDH) into a chiral amine. In this study, a Prelog AA-ADH with broad substrate specificity was combined with an engineered, stable chimeric Chl1-AmDH for the conversion (S)-2hexanol to (R)-2-aminohexane. (b) SDS-PAGE analysis confirms enzyme cargo loading to EutM-SpyCatcher scaffolds under amination reaction conditions (2 M ammonium chloride buffer, pH 8.7) prior to co-factor and substrate additions. Enzyme cascades (SpyTag fused ADH (6 µM) and AmDH (150 µM)) were mixed at different molar ratios with EutM-SpyCatcher (see Table S3 for concentrations). Corresponding control reactions were performed with untagged enzymes. “=” represents the isopeptide formed between SpyTag and SpyCatcher. (c) Visualization of the enzyme-loaded scaffolds under amination reaction conditions by negative stain TEM. Top: free enzyme cascade of SpyTag-ADH (6 µM) and SpyTag-AmDH (150 µM); middle: EutM-SpyCatcher (780 µM) forms fibril-like scaffolds; bottom: large structures are formed by (SpyTag-ADH+SpyTag-AmDH):EutM-SpyCatcher scaffolds at 1:5 ratio. All images were taken at a magnification of x 53, 000. The scale bars represent 100 nm. light microscope, EutM-SpyCatcher scaffolds appeared as 34 amines in a highly enantioselective manner. Because the two thinfilms (>100 µm in size) that were folded over, indicating more 35 enzymes catalyze redox opposite reactions, this cascade is selfflexible structures presumably due to arrays formed from dense 36 sufficient, using ammonium ion/ammonia in the buffer to clusters of EutM-SpyCatcher fibrils (Figure 2a and Figure 2c). 37 regenerate the cofactor. Owing to these properties, we considered Addition of SpyTag-GFP or GFP-SpyTag to EutM-SpyCatcher 38 this dual enzyme cascade as an ideal model with which to test films rendered the films fluorescent, confirming that cargo protein 39 whether our genetically programmable scaffolding system could can be efficiently immobilized on pre-fabricated scaffolds. In 40 be used to co-immobilize an industrially relevant biocatalytic contrast, untagged GFP mixed with EutM-SpyCatcher films 41 cascade. From the enzymes tested in52, we chose a Prelog AAresulted in diffuse fluorescence, indicating that the cargo only 42 ADH53 (referred to as ADH) with broad substrate specificity54, interacted specifically with scaffolds when fused to the SpyTag 43 and a stability engineered chimeric Ch1-AmDH55 (referred to as (Figure 2c). Negative stain TEM showed that GFP cargo 44 AmDH) for co-immobilization on EutM-SpyCatcher scaffolds. As attachment did not affect the morphology of the EutM- 45 our model reaction, we chose the conversion of (S)-2-hexanol to 46 (R)-2-aminohexane because substrate and reaction products are SpyCatcher scaffolds (Figure S7). 47 commercially available, and the conversion was shown to be Prototyping scaffolding system for a dual enzyme cascade To demonstrate the utility of our protein scaffolding system for 48 catalyzed by the two enzymes in 48 hrs with 9552% efficiency and in vitro biocatalysis, we chose to co-immobilize an elegant dual 49 > 99 % enantiomeric excess (ee) to the R-amine (Figure 3a). enzyme cascade for chiral amine synthesis developed by the 50 To achieve optimal cascade efficiency, biocatalysts that are coTurner group52. In this system, an NAD+-dependent alcohol 51 immobilized should theoretically be loaded on scaffolds at a ratio dehydrogenase (ADH) and an NADH-dependent-amine- 52 that is dependent upon enzyme activities. Because kinetics dehydrogenase (AmDH) are combined to convert alcohols to 4


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ACS Catalysis Table 1. Kinetic Properties of ADH and AmDH Compared to SpyTag Fused Enzymes under Amination Reaction Conditions Parameter

ADHa

SpyTag-ADHa

AmDHa

SpyTag-AmDHa

Specific activity (mU mg-1)

3451±210

4392±273

457±17

480±18

Km (mM)b

0.09±0.01

0.08±0.01

4.3±0.6

4.9±0.5

kcat (s-1)

1.6±0.1

1.9±0.1

0.35±0.01

0.39±0.01

kcat /Km (M-1 s-1)

1.8×104

2.4×104

81

80

a Specific activities were measured with 20 mM (S)-2-hexanol (ADH) or 20 mM 2-hexanone (AmDH) in 2 M ammonium chloride (NH4Cl/NH3) buffer (pH 8.7) at 30 °C by monitoring the NADH concentration. One unit (U) was defined as the amount of enzyme that produces/consumes 1 µmol of NADH per min. Activities were measured with 0.02 mg mL-1 of purified enzymes with and without an Nterminal SpyTag downstream of a His-Tag and a thrombin cleavage site. b Km values were measured for (S)-2-hexanol (ADH/SpyTagADH) and 2-hexanone (AmDH/SpyTag-AmDH), respectively. Activities were measured in triplicate and error bars indicate standard deviation from the mean.

parameters have not been published for the dual enzyme cascade with our chosen model substrate (S)-2-hexanol52, we therefore initially measured the activities of the nickel-affinity chromatography purified enzymes ADH and AmDH (with an Nterminal His-tag) using previously published substrates and reaction conditions, to establish kinetic parameters for our own comparison (Table S1 and S2). Preliminary analyses of ADH with (S)-1-phenylethanol53 and AmDH with acetophenone55 as test substrates provided kinetic parameters that were in the same order of magnitude as previously published results. Our measured specific activity for AmDH with acetophenone was slightly lower than previously published data55 (191 mU mg-1 versus 301 mU mg-1), which may be due to the fact that, in our hands, AmDH precipitated at 60 oC, which is in contrast to published results (however, the enzyme did not precipitate at 30 oC, which is the temperature used for the animation cascade reaction). We did note that ADH activity was inhibited at substrate concentrations >30 µM (Figure S8), corroborating published work that ADH is substrate inhibited53. We also found that AmDH has a much lower affinity for its substrate (Km) than ADH (Table S1 and S2), resulting in a 1000-fold lower kcat/Km. Removing the His tag from the enzymes did not have a significant effect on the Km or kcat of the enzymes, although it increased the specific activity of ADH (by ~50%) and slightly decreased the specific activity of AmDH (by ~6%). Having established reliable enzyme activities, we then measured the kinetics of ADH with (S)-2-hexanol, and AmDH with 2-hexanone under amination conditions (2 M ammonium chloride buffer, pH 8.7)52 (Table S1 and S2). Again, we observed that AmDH had a lower affinity for its substrate than ADH (Km values were 4.3 mM and 0.09 mM, respectively), which resulted in a 3 orders of magnitude difference in kcat/Km (81 M-1 s-1 for AmDH and 1.8 x 104 M-1 s-1 for ADH). Specific activities were 457 mU mg-1 for AmDH and 3451 mU mg-1 for ADH. Again, we noted that ADH activity appears to be substrate inhibited, in particular when (S)-2-hexanol is present at concentrations > 5 mM (Figure S8). Removal of the His-tag had no significant impact on the kcat/Km and only slightly changed the specific activities of the enzymes (~8% increase for ADH and (~13% decrease for AmDH). For the purpose of immobilizing the dual enzyme cascade on our protein scaffold, a SpyTag was added both in a C- and Nterminal configuration to ADH and AmDH. To ensure that the addition of the SpyTag had not adversely affected the enzymes, activities of SpyTag fused enzymes were compared to our previously established specific activities for ADH and AmDH with (S)-2-hexanol and 2-hexanone, respectively. ADH activity was significantly disrupted by a C-terminal fusion of SpyTag (Figure S9), which may be due to the tag causing steric hinderances that could prevent correct positioning of the substrate 5

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binding loop in all four subunits of the tetrameric enzyme53. On the other hand, an N-terminal fusion of SpyTag slightly improved the activity of AmDH and ADH (AmDH specific activity increased from 457 mU mg-1 to 480 mU mg-1, and ADH specific activity increased from 3451 mU mg-1 to 4392 mU mg-1) (Table 1). Removing the N-terminal His tag from the SpyTag fused enzymes had no significant effect on the specific activity of AmDH and the specific activity of ADH was only slightly diminished (Figure S9). It was reported that removal the His-Tag from AmDH was necessary to prevent enzyme precipitation in solutions containing AmDH and ADH52. Chelation of Mg2+ ions by the His-Tag on AmDH was speculated to destabilize ADH homotetramer formation. In our hands, preliminary studies with His-tagged AmDH and ADH mixtures did not cause protein precipitation as observed in52. We therefore concluded that an additional thrombin cleavage step to remove the N-terminal HisTag from SpyTag fused enzymes would not be necessary prior to immobilization on protein scaffolds, and all subsequent experiments were carried out with His-tagged enzymes. The measured kinetic parameters (Table 1) indicated that AmDH would be the rate limiting enzyme in the two-step amination cascade. Balancing the lower activity and higher Km of AmDH with the significantly more active ADH in a cascade reaction would therefore require a higher protein concentration of AmDH compared to ADH. To identify optimal amounts of enzyme for cascade reactions, we measured the volumetric and specific activities of AmDH and ADH in amination buffer at different protein concentrations (Figure S10). The specific activity of AmDH was strongly dependent on protein concentration (potentially due to the formation of soluble aggregates), decreasing from e.g. 457 mU mg-1 at a protein concentration of 0.02 mg mL-1, to 246 mU mg -1 at a protein concentration of 0.2 mg mL-1, and decreasing further to 37 mU mg-1 at a protein concentration of 5 mg mL-1. A concentration of ~5-7 mg mL-1 of AmDH therefore afforded the best volumetric activity, as such 150 µM (185 mU mL-1, 7.4 mg mL-1) AmDH was subsequently used in all cascade reactions (Table S3). To balance the cascade, ADH was added at a concentration of 6 µM (0.2 mg mL-1) to all cascade reactions. This resulted in a 3.5-fold higher total activity (650 mU mL-1) of ADH compared to AmDH (185 mU mL-1) (Table S3). Finally, isopeptide bond formation under amination reaction conditions was confirmed by mixing EutM-SpyCatcher with SpyTag fused enzymes (and untagged enzymes as a control) at the identified concentrations (6 µM (0.2 mg mL-1) for SpyTag-ADH and 150 µM (7.4 mg mL-1) for SpyTag-AmDH, Table S3). Assuming that enzyme distribution on scaffolds would influence cascade efficiency56, different molar ratios of enzyme mixture to


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Figure 4. One-pot amination reaction with free and EutM-SpyCatcher scaffolded dual-enzyme cascade. (a) Characterization of conversion rates of (S)-2-hexanol to (R)-2-aminohexane by free and scaffolded SpyTag-ADH/AmDH dual enzyme cascades (controls contain untagged ADH/AmDH) containing increasing molar ratios of EutM-SpyCatcher. Conversion rates are shown after 12 and 24 hrs (see Dataset S1 for complete conversion time courses for all reactions). (b) Time course of amination reaction by free SpyTag- enzyme cascade. (c) Time course of amination reaction by scaffolded SpyTag- enzyme cascade with 1:5 molar ratio of SpyTag-enzymes and EutM-SpyCatcher. Data are the average of three replicate experiments and error bars are the standard error of the mean. All cascade reactions (a-c) were performed in a 3 mL reaction volume with ammonium chloride buffer (2 M, pH 8.7) at 30 °C and 190 rpm containing 20 mM (S)-2-hexanol, 1 mM NAD+, 6 µM ADH, 150 µM AmDH and EutM-SpyCatcher (scaffold) added to obtain differing molar ratios of enzymes to scaffold (see Table S3 for protein concentrations). First time point for reaction was analyzed after 0.5 hrs. Conversion rates are shown as percentage (%) of alcohol converted to ketone intermediate and final amine product. scaffold were tested (Figure 3b). As seen with GFP (Figure 2b), isopeptide bond formation proceeded rapidly; at a 1:1 molar ratio (calculated based on measured protein concentrations) all EutM-SpyCatcher was converted into higher molecular weight complexes as detectable by SDS-PAGE analysis. With increasing molar ratios of EutM-SpyCatcher in the mixtures, proportional amounts of EutM-SpyCatcher remained unmodified as expected. A small amount of SpyTag-AmDH remained unbound, even when EutM-SpyCatcher was present in excess, suggesting that a small portion of SpyTag-AmDH does not display a tag that is conformationally accessible for interaction with the SpyCatcher domain. Negative stain TEM of the enzymes immobilized on the scaffolds showed that the attachment of SpyTag fused enzymes to EutM-SpyCatcher resulted in a dense film-like material covering the fibril-like EutM-SpyCatcher scaffolds (Figure 3c, bottom panel: note that enzyme-immobilized scaffolds appear thicker and more darkly stained than scaffolds lacking enzymes, middle 6

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panel). No scaffold-like structures or amorphous aggregates were observed in the enzyme only control. This change in scaffold morphology when loaded with enzymes ADH and AmDH is in contrast to GFP loading on scaffolds, which did not change the appearance of EutM-SpyCatcher (Figure S7) and must relate to the properties of the enzymes. ADH is a homotetramer (PDB: 2EW8, 2EWM)53, while AmDH might associate as a homodimer based on its sequence similarity to PheDH from Rhodococcus sp. M4 (PDB: 1BW9, 1C1D)57-58. The quaternary structures of the enzymes and potential multipoint attachment of these multimeric complexes may be responsible for the observed altered scaffold morphology.

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Operation of scaffold immobilized dual cascade reaction Having established the basic parameters for co-immobilization of the dual enzyme cascade on our protein scaffold, we began testing the effect of immobilizing different molar ratios of enzyme (SpyTag-ADH + SpyTag-AmDH) to EutM-SpyCatcher scaffold (molar ratios 1:0 to 1:6, see Table S3 for protein concentrations),


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Figure 5. Effect of EutM-SpyCatcher protein scaffolding on enzyme stabilities. (a) Relative activity of free (1:0) and immobilized on scaffolds (1:6, 1:18) SpyTag-ADH. (b) Relative activity of free (1:0) and immobilized on scaffolds (1:6, 1:18) SpyTag-AmDH. 30 µM purified enzyme was mixed with different molar ratios of EutM-SpyCatcher and incubated under amination reaction conditions at 30 °C and activities were measured every 12 hrs for 48 hrs. Relative activity assumes 100 % activity (set as 1.0) of the enzyme at the beginning of the experiment. Figure S13 shows complete relative activity time courses (including additional molar ratios) of SpyTagged enzymes (and as controls, untagged enzymes) in the presence or absence of EutM-SpyCatcher. following cascade reaction conditions that have been previously published52. For each of the reactions, substrate conversion and product formation were quantified by GC-FID over time, up to 48 hrs (Figure S11 and Dataset S1 for complete conversion data). A final conversion of 85 % from (S)-2-hexanol to 2-aminohexane was achieved after 48 hrs in control reactions with (6 µM ADH + 150 µM AmDH (each without a SpyTag fusion) and without EutM-SpyCatcher in the reaction) (Dataset S1). This is lower than the 95 % conversion in 48 hrs reported by Mutti et al.52, however, it is likely that we used significantly lower catalyst concentrations i.e. potentially ~ 9-fold less ADH (compare our 0.2 mg mL-1, 650 mU mL-1 to the previously reported 1.8 mg mL-1, and an estimated ~ 5,600 mU mL-1) and potentially ~ 200-fold less AmDH (compare our 7.4 mg mL-1, 185 mU mL-1 to an estimated ~ 36,000-40,000 mU mL-1 (note that not all protein concentrations, and no specific activities were previously reported52, which makes direct comparisons between cascade systems difficult)). Interestingly, we found that the final conversion after 48 hrs with 6 µM SpyTag-ADH + 150 µM SpyTag-AmDH in the absence of EutM-SpyCatcher was 93 %. Therefore, addition of the SpyTag to the dual enzyme cascade improved the overall conversion by 8 %, without addition of protein scaffold, corroborating our finding that addition of an Nterminal SpyTag improves enzyme specific activity (Table 1, Figure S9). Final conversions of the scaffolded SpyTag-enzymes with differing molar ratios of EutM-SpyCatcher were all in the range 92 %-94 % after 48 hrs, similar to the unscaffolded SpyTag-enzyme cascade (Dataset S1). While final conversions (i.e. after 48 hrs) by the SpyTagenzyme cascade were unchanged upon immobilization on the scaffold, we did observe a significant improvement in conversions at earlier time points (i.e. after 12 hrs and 24 hrs) (Figure 4a, Dataset S1). After 12 hrs reaction time, conversion by the control reaction cascade ADH + AmDH was 36 %, while SpyTag-ADH + SpyTag-AmDH cascade conversion was 49 %. This again indicates that fusion of SpyTag to the dual enzyme cascade improved activity. When we co-immobilized SpyTag-ADH + SpyTag-AmDH on EutM-SpyCatcher scaffolds, at a 1:1, 1:3, 1:5 and 1:6 molar ratio, conversions after 12 hrs further increased 4 %, 8 %, 10 % and 16 %, respectively. Likewise, conversions after 24 hrs were increased further by 6 %, 15 %, 18 % and 20 % when SpyTag-ADH + SpyTag-AmDH were co-immobilized on EutMSpyCatcher scaffolds at a 1:1, 1:3, 1:5 and 1:6 molar ratio. In the 7

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case of 1:5 and 1:6 molar ratios, almost complete conversion was reached after 24 hrs (89 % and 91 %, respectively). This indicates that the rate of reaction is significantly improved when the SpyTag-enzyme cascade is immobilized on EutM-SpyCatcher scaffolds at a 1:5 or 1:6 ratio (Dataset S1) i.e. reaching almost final conversion in 24 hrs as opposed to 36 or 48 hrs as shown in Figure 4b and 4c for the free vs. the scaffolded (at a 1:5 molar ratio) cascades. Finally, we determined the enantioselectivity of the reaction by chiral GC-FID, to confirm whether or not immobilization of the enzyme AmDH altered its selectivity in the reduction of 2hexanone. (R)-2-aminohexane was produced with an enantiomeric excess of > 99 % (Figure S12), which is in agreement with previously published data for the free enzyme under these reaction conditions52. Covalent attachment of AmDH to the protein scaffold therefore did not change its (R)-selectivity.

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Immobilization on EutM-SpyCatcher scaffolds stabilizes enzymes Immobilization of enzymes on different support materials is a widely used technique to stabilize catalysts26, 59-60. We hypothesized that cascade attachment to our protein scaffolds may stabilize the enzymes, resulting in the shorter reaction times required to reach final conversions of ~ 90 % (Figure 4). We tested this by monitoring the relative activities of ADH and AmDH, as well as SpyTag-ADH and SpyTag-AmDH, over time in the presence and absence of different molar ratios of EutMSpyCatcher (Figure 5, see Figure S13 and S14 for complete dataset). We found that ADH was significantly less stable than AmDH. After 48 hrs incubation time, only ~40 % relative activity of ADH remained, compared to ~65 % remaining AmDH relative activity. Adding EutM-SpyCatcher scaffolds (without affording scaffold immobilization) to ADH and AmDH had no significant stabilizing effect (Figure S14). Additionally, control reactions of SpyTag-ADH and SpyTag-AmDH in the absence of EutMSpyCatcher showed a similar decrease in relative activity over time (Figure S14). We found that SpyTag-AmDH and SpyTag-ADH stabilities were improved only when the enzymes were immobilized on EutM-SpyCatcher scaffolds. In the case of SpyTag-AmDH, stabilization after 48 hrs was apparent at all ratios of enzyme:scaffold tested (Figure 5, Figure S14), with 1:6 SpyTagAmDH:EutM-SpyCatcher scaffolded enzyme retaining ~79 %


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relative activity, compared to ~67 % for unscaffolded SpyTagAmDH. Likewise, at 24 hrs, ~88 % relative activity was retained by 1:6 SpyTag-AmDH:EutM-SpyCatcher scaffolded enzyme, compared to ~74 % retained by unscaffolded SpyTag-AmDH. This 14 % increase in activity remaining in immobilized SpyTagAmDH, compared to non-immobilized SpyTag-AmDH, could be partially responsible for the 20 % increase in substrate conversion obtained with the immobilized cascade at 24 hrs (Figure 4). Interestingly, while the scaffold stabilizing effect on SpyTagAmDH was apparent with all molar ratios of EutM-SpyCatcher, the stability of SpyTag-ADH only appeared to be improved at molar ratios of 1:6 and greater. Contrastingly, at molar ratios of 1:1 and 1:3 SpyTag-ADH:EutM-SpyCatcher, the relative SpyTagADH activity was decreased at all time points (Figure S14). We suggest that this may be due to improper attachment of the homotetrameric enzyme to the scaffold at ratios where available SpyCatcher domains are lower in number, which may cause disruption of the quaternary structure of the enzyme, and therefore loss of activity. Previous studies have shown that the ADH tetramer can dissociate as a dimer when in contact with solid surfaces, which would prevent correct positioning of the substrate binding loop53. On the other hand, the 1:6 SpyTag-ADH:EutMSpyCatcher scaffolded enzyme retained ~60 % relative activity after 24 hrs, and ~44 % relative activity after 48 hrs, compared to unscaffolded SpyTag-ADH retaining ~50 % relative activity after 24 hrs, and ~34 % relative activity after 48 hrs (Figure 5, Figure S14). Again, this 10 % increase in activity remaining in immobilized SpyTag-ADH, compared to non-immobilized SpyTag-ADH, may play a role in the increase in substrate conversion by the immobilized cascade at 24 hrs (Figure 4). Together, the stabilization of both enzymes in the cascade upon immobilization on EutM-SpyCatcher scaffolds may therefore have increased the rate of reaction compared to the nonimmobilized system. We speculate that the surface of the EutMSpyCatcher scaffold may provide a favorable microenvironment for enzyme stability26. The cascade reaction has to be performed at a high pH of 8.7, while the calculated pI values of EutM and EutM-SpyCatcher are 6.7 and 5.7, respectively.

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CONCLUSIONS In summary, our goal in this work was to create an easy-to-use, scalable and stable system for the co-immobilization of cascade enzymes on protein-based scaffolds for in vitro biocatalysis. We have designed and created a genetically encoded protein scaffolding system that can be easily manufactured by E. coli and used for the colocalization of enzymes, using a single short peptide fusion. The scaffolds self-organize in vivo and in vitro and remain stable under a variety of pH and temperature conditions. We have demonstrated that the scaffolding system spontaneously and rapidly immobilizes different types of cargo via covalent bond formation. We showed initial proof-of-concept of the utility of this system for dual-enzyme biocatalysis using an amination cascade as a model system. Following careful and detailed analyses of the kinetic parameters of the cascade enzymes to try and balance activities and loading of enzymes on scaffolds, our results indicate that immobilization of the enzyme cascade can reduce the amount of time required to reach final conversion yields (achieving ~ 90 % conversion in 24 hrs, compared to 48 hrs for the free system). We confirm that immobilization of the biocatalysts on our protein scaffold improves stability of the enzymes (residual enzyme activity after 24 hrs reaction time increased by up to 20 %) at certain loading ratios. Whether or not the observed increase in cascade throughput with the scaffolded system is solely the result of increased enzyme stability, or whether proximity induced substrate channeling plays a role18, 22, 32-34, 61 , remains to be investigated. Future work will include scaleup and optimization of this cascade reaction, as well as co8

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immobilization of more complex multi-enzyme cascades, to explore the use of this system in diverse in vitro biocatalytic cascade reactions. Finally, the ease with which this protein scaffolding system can be engineered makes it an optimal platform for future work implementing synthetic biology approaches to create a toolbox of scaffold building blocks for the rapid production and prototyping of protein immobilization systems. Work is underway to engineer protein scaffolds that incorporate different building blocks variants to control the spacing and co-localization of multiple catalysts, scaffold assembly properties, and the surface properties of protein scaffolds to provide tailored microenvironments for specific reactions34. Furthermore, this scaffolding system may also be adapted for future in vivo colocalization of enzymes, depending on the cascade reaction requirements such as substrate access in cells and ease with which products can be separated from cells37. This work therefore represents a step on the path towards creating optimized protein scaffolding systems for the easy co-immobilization of multi-enzyme biocatalytic cascades.

86 METHODS 87 Experimental procedures are provided as Supporting Information. 88 ASSOCIATED CONTENT 89 AUTHOR INFORMATION 90 Corresponding Authors 91 *Email: [email protected] (C.S.-D.) 92 *Email: [email protected] (M.B.Q.) 93 94 95 96

ORCID Claudia Schmidt-Dannert: 0000-0002-0559-3656 Maureen Quin: 0000-0001-7193-639X Guoqiang Zhang: 0000-0003-0999-9394

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Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Complete experimental procedures (PDF) Supporting Figures, Tables and Data Set (PDF)

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ACKNOWLEDGMENTS We would like to thank Dr. Kelly Aukema and Dr. James Christenson (University of Minnesota) for assistance in developing the GC procedures and Dr. Gail Celio at the University of Minnesota Imaging Center for preparing thin cell sections. This research was supported by grants from the National Science Foundation (MCB-12644429 to C.S.-D.) and Defense Threat Reduction Agency (HDTRA-15-0004 to C.S.-D.), and by funding from the Biotechnology Institute at the University of Minnesota through the Biocatalysis Initiative.

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REFERENCES 1. Agapakis, C. M.; Boyle, P. M.; Silver, P. A., Natural Strategies for the Spatial Optimization of Metabolism in Synthetic Biology, Nat Chem Biol 2012, 8, 527-535. 2. Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.; Moore, J. C.; Robins, K., Engineering the Third Wave of Biocatalysis, Nature 2012, 485, 185-194. 3. France, S. P.; Hepworth, L. J.; Turner, N. J.; Flitsch, S. L., Constructing Biocatalytic Cascades: In Vitro and in Vivo Approaches to de Novo Multi-Enzyme Pathways, ACS Catalysis 2017, 7, 710-724.

Notes The authors declare no competing financial interests.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68

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4. Lopez-Gallego, F.; Schmidt-Dannert, C., Multienzymatic Synthesis, Curr Opin Chem Biol 2010, 14, 174-183. 5. Clomburg, J. M.; Crumbley, A. M.; Gonzalez, R., Industrial Biomanufacturing: The Future of Chemical Production, Science 2017, 355, 38. 6. Fessner, W.D., Systems Biocatalysis: Development and Engineering of Cell-free “artificial metabolisms” for Preparative Multi-enzymatic Synthesis, New Biotechnology 2015, 32, 658664. 7. Schrittwieser, J. H.; Velikogne, S.; Hall, M.; Kroutil, W., Artificial Biocatalytic Linear Cascades for Preparation of Organic Molecules, Chem Rev 2018, 118, 270-348. 8. Guterl, J. K.; Garbe, D.; Carsten, J.; Steffler, F.; Sommer, B.; Reisse, S.; Philipp, A.; Haack, M.; Ruhmann, B.; Koltermann, A.; Kettling, U.; Bruck, T.; Sieber, V., Cell-free Metabolic Engineering: Production of Chemicals by Minimized Reaction Cascades, ChemSusChem 2012, 5, 2165-2172. 9. Wang, Y.; Ren, H.; Zhao, H., Expanding the Boundary of Biocatalysis: Design and Optimization of In vitro Tandem Catalytic Reactions for Biochemical Production, Crit Rev Biochem Mol Biol 2018, 53, 115-129. 10. Giessen, T. W.; Silver, P. A., Encapsulation as a Strategy for the Design of Biological Compartmentalization, J Mol Biol 2016, 428, 916-927. 11. Kuchler, A.; Yoshimoto, M.; Luginbuhl, S.; Mavelli, F.; Walde, P., Enzymatic Reactions in Confined Environments, Nat Nanotechnol 2016, 11, 409-420. 12. Polka, J. K.; Hays, S. G.; Silver, P. A., Building Spatial Synthetic Biology with Compartments, Scaffolds, and Communities, Cold Spring Harb Perspect Biol 2016, 8, 1-16. 13. Proschel, M.; Detsch, R.; Boccaccini, A. R.; Sonnewald, U., Engineering of Metabolic Pathways by Artificial Enzyme Channels, Front Bioeng Biotechnol 2015, 3, 168. 14. Siu, K. H.; Chen, R. P.; Sun, Q.; Chen, L.; Tsai, S. L.; Chen, W., Synthetic Scaffolds for Pathway Enhancement, Curr Opin Biotechnol 2015, 36, 98-106. 15. Lim, F. Y.; Keller, N. P., Spatial and Temporal Control of Fungal Natural Product Synthesis, Natural Product Reports 2014, 31, 1277-1286. 16. Schmitt, D. L.; An, S., Spatial Organization of Metabolic Enzyme Complexes in Cells, Biochemistry 2017, 56, 3184-3196. 17. Chado, G. R.; Stoykovich, M. P.; Kaar, J. L., Role of Dimension and Spatial Arrangement on the Activity of Biocatalytic Cascade Reactions on Scaffolds, ACS Catalysis 2016, 6, 5161-5169. 18. Lin, J.-L.; Palomec, L.; Wheeldon, I., Design and Analysis of Enhanced Catalysis in Scaffolded Multienzyme Cascade Reactions, ACS Catalysis 2014, 4, 505-511. 19. Muschiol, J.; Peters, C.; Oberleitner, N.; Mihovilovic, M. D.; Bornscheuer, U. T.; Rudroff, F., Cascade CatalysisStrategies and Challenges En route to Preparative Synthetic Biology, Chem Commun (Camb) 2015, 51, 5798-5811. 20. Quin, M. B.; Wallin, K. K.; Zhang, G.; SchmidtDannert, C., Spatial Organization of Multi-enzyme Biocatalytic Cascades, Org Biomol Chem 2017, 15, 4260-4271. 21. Schmidt-Dannert, C.; Lopez-Gallego, F., A Roadmap for Biocatalysis - Functional and Spatial Orchestration of Enzyme Cascades, Microb Biotechnol 2016, 9, 601-609. 22. Wheeldon, I.; Minteer, S. D.; Banta, S.; Calabrese Barton, S.; Atanassov, P.; Sigma, M., Substrate Channelling as an Approach to Cascade Reactions, Nat Chem 2016, 8, 299-309. 23. Lopez-Gallego, F.; Jackson, E.; Betancor, L., Heterogeneous Systems Biocatalysis: The Path to the Fabrication of Self-Sufficient Artificial Metabolic Cells, Chemistry 2017, 23, 17841-17849. 24. Garcia-Galan, C.; Berenguer-Murcia, A.; FernandezLafuente, R.; Rodrigues, R. C., Potential of Different Enzyme 9

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Immobilization Strategies to Improve Enzyme Performance, Advanced Synthesis & Catalysis 2011, 353, 2885-2904. 25. Jia, F.; Narasimhan, B.; Mallapragada, S., Materialsbased Strategies for Multi-enzyme Immobilization and Colocalization: A review, Biotechnol Bioeng 2014, 111, 209-222. 26. Hoarau, M.; Badieyan, S.; Marsh, E. N. G., Immobilized Enzymes: Understanding Enzyme-Surface Interactions at the Molecular Level, Organic & Biomolecular Chemistry 2017, 15, 9539-9551. 27. Barbosa, O.; Ortiz, C.; Berenguer-Murcia, A.; Torres, R.; Rodrigues, R. C.; Fernandez-Lafuente, R., Strategies for the One-step Immobilization-purification of Enzymes as Industrial Biocatalysts, Biotechnol Adv 2015, 33, 435-456. 28. Benitez-Mateos, A. I.; Llarena, I.; Sanchez-Iglesias, A.; Lopez Gallego, F., Expanding One-pot Cell-free Protein Synthesis and Immobilization for On-demand Manufacturing of Biomaterials, ACS Synth Biol 2018, 7, 875-884. 29. Peschke, T.; Rabe, K. S.; Niemeyer, C. M., Orthogonal Surface Tags for Whole-Cell Biocatalysis, Angew Chem Int Ed Engl 2017, 56, 2183-2186. 30. Dueber, J. E.; Wu, G. C.; Malmirchegini, G. R.; Moon, T. S.; Petzold, C. J.; Ullal, A. V.; Prather, K. L.; Keasling, J. D., Synthetic Protein Scaffolds Provide Modular Control Over Metabolic Flux, Nat Biotechnol 2009, 27, 753-759. 31. Ferner-Ortner-Bleckmann, J.; Gelbmann, N.; Tesarz, M.; Egelseer, E. M.; Sleytr, U. B., Surface-layer Lattices as Patterning Element for Multimeric Extremozymes, Small 2013, 9, 3887-3894. 32. Wang, S. Z.; Zhang, Y. H.; Ren, H.; Wang, Y. L.; Jiang, W.; Fang, B. S., Strategies and Perspectives of Assembling Multienzyme Systems, Critical Reviews in Biotechnology 2017, 37, 1024-1037. 33. You, C.; Zhang, Y. H., Self-assembly of Synthetic Metabolons through Synthetic Protein Scaffolds: One-step Purification, Co-immobilization, and Substrate Channeling, ACS Synth Biol 2013, 2, 102-110. 34. Zhang, Y.; Tsitkov, S.; Hess, H., Proximity does not Contribute to Activity Enhancement in the Glucose Oxidasehorseradish Peroxidase Cascade, Nat Commun 2016, 7, 13982. 35. Hirakawa, H.; Nagamune, T., Molecular Assembly of P450 with Ferredoxin and Ferredoxin Reductase by Fusion to PCNA, Chembiochem 2010, 11, 1517-1520. 36. Horn, A. H.; Sticht, H., Synthetic Protein Scaffolds Based on Peptide Motifs and Cognate Adaptor Domains for Improving Metabolic Productivity, Front Bioeng Biotechnol 2015, 3, 191. 37. Lee, M. J.; Mantell, J.; Hodgson, L.; Alibhai, D.; Fletcher, J. M.; Brown, I. R.; Frank, S.; Xue, W. F.; Verkade, P.; Woolfson, D. N.; Warren, M. J., Engineered Synthetic Scaffolds for Organizing Proteins within the Bacterial Cytoplasm, Nature Chemical Biology 2018, 14, 142-147. 38. Liu, F.; Banta, S.; Chen, W., Functional Assembly of a Multi-enzyme Methanol Oxidation Cascade on a Surfacedisplayed Trifunctional Scaffold for Enhanced NADH Production, Chem Commun (Camb) 2013, 49, 3766-3768. 39. Tan, C. Y.; Hirakawa, H.; Nagamune, T., Supramolecular Protein Assembly Supports Immobilization of A Cytochrome P450 Monooxygenase System as Water-insoluble Gel, Sci Rep 2015, 5, 8648. 40. Visser, F.; Muller, B.; Rose, J.; Prufer, D.; Noll, G. A., Forizymes-Functionalised Artificial Forisomes as a Platform for the Production and Immobilisation of Single Enzymes and Multienzyme Complexes, Sci Rep 2016, 6, 30839. 41. Choudhary, S.; Quin, M. B.; Sanders, M. A.; Johnson, E. T.; Schmidt-Dannert, C., Engineered Protein Nanocompartments for Targeted Enzyme Localization, PLoS One 2012, 7, e33342.


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67

42. Held, M.; Kolb, A.; Perdue, S.; Hsu, S. Y.; Bloch, S. E.; Quin, M. B.; Schmidt-Dannert, C., Engineering Formation of Multiple Recombinant Eut Protein Nanocompartments in E. coli, Sci Rep 2016, 6, 24359. 43. Quin, M. B.; Perdue, S. A.; Hsu, S. Y.; SchmidtDannert, C., Encapsulation of Multiple Margo Proteins within Recombinant Eut Nanocompartments, Appl Microbiol Biotechnol 2016, 100, 9187-9200. 44. Pitts, A. C.; Tuck, L. R.; Faulds-Pain, A.; Lewis, R. J.; Marles-Wright, J., Structural Insight into the Clostridium difficile Ethanolamine Utilisation Microcompartment, PLoS One 2012, 7, e48360. 45. Takenoya, M.; Nikolakakis, K.; Sagermann, M., Crystallographic Insights into the Pore Structures and Mechanisms of the EutL and EutM Shell Proteins of the Ethanolamine-utilizing Microcompartment of Escherichia coli, J Bacteriol 2010, 192, 6056-6063. 46. Tanaka, S.; Sawaya, M. R.; Yeates, T. O., Structure and Mechanisms of a Protein-based Organelle in Escherichia coli, Science 2010, 327, 81-84. 47. Reddington, S. C.; Howarth, M., Secrets of a Covalent Interaction for Biomaterials and Biotechnology: SpyTag and SpyCatcher, Curr Opin Chem Biol 2015, 29, 94-99. 48. Zakeri, B.; Fierer, J. O.; Celik, E.; Chittock, E. C.; Schwarz-Linek, U.; Moy, V. T.; Howarth, M., Peptide Tag Forming a Rapid Covalent Bond to a Protein, through Engineering a Bacterial Adhesin, Proc Natl Acad Sci U S A 2012, 109, E690-E697. 49. Giessen, T. W.; Silver, P. A., A Catalytic Nanoreactor Based on in Vivo Encapsulation of Multiple Enzymes in an Engineered Protein Nanocompartment, Chembiochem 2016, 17, 1931-1935. 50. Bommarius, A. S.; Paye, M. F., Stabilizing Biocatalysts, Chem Soc Rev 2013, 42, 6534-6565. 51. Ellis, R. J., Macromolecular Crowding: Obvious but Underappreciated, Trends Biochem Sci 2001, 26, 597-604. 52. Mutti, F. G.; Knaus, T.; Scrutton, N. S.; Breuer, M.; Turner, N. J., Conversion of Alcohols to Enantiopure Amines through Dual-enzyme Hydrogen-borrowing Cascades, Science 2015, 349, 1525-1529. 53. Hoffken, H. W.; Duong, M.; Friedrich, T.; Breuer, M.; Hauer, B.; Reinhardt, R.; Rabus, R.; Heider, J., Crystal Structure and Enzyme Kinetics of the (S)-specific 1-phenylethanol Dehydrogenase of the Denitrifying Bacterium Strain EbN1, Biochemistry 2006, 45, 82-93. 54. Dudzik, A.; Snoch, W.; Borowiecki, P.; OpalinskaPiskorz, J.; Witko, M.; Heider, J.; Szaleniec, M., Asymmetric Reduction of Ketones and Beta-keto Esters by (S)-1phenylethanol Dehydrogenase from Denitrifying Bacterium Aromatoleum aromaticum, Applied Microbiology and Biotechnology 2015, 99, 5055-5069. 55. Bommarius, B. R.; Schurmann, M.; Bommarius, A. S., A Novel Chimeric Amine Dehydrogenase Shows Altered Substrate Specificity Compared to its Parent Enzymes, Chemical Communications 2014, 50, 14953-14955. 56. Fu, J.; Liu, M.; Liu, Y.; Woodbury, N. W.; Yan, H., Interenzyme Substrate Diffusion for an Enzyme Cascade Organized on Spatially Addressable DNA Nanostructures, J Am Chem Soc 2012, 134, 5516-5519. 57. Brunhuber, N. M.; Thoden, J. B.; Blanchard, J. S.; Vanhooke, J. L., Rhodococcus L-phenylalanine Dehydrogenase: Kinetics, Mechanism, and Structural Basis for Catalytic Specificity, Biochemistry 2000, 39, 9174-9187. 58. Vanhooke, J. L.; Thoden, J. B.; Brunhuber, N. M.; Blanchard, J. S.; Holden, H. M., Phenylalanine Dehydrogenase from Rhodococcus sp. M4: High-resolution X-ray Analyses of Inhibitory Ternary Complexes Reveal Key Features in the 10

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Oxidative Deamination Mechanism, Biochemistry 1999, 38, 23262339. 59. Luckarift, H. R.; Spain, J. C.; Naik, R. R.; Stone, M. O., Enzyme Immobilization in a Biomimetic Silica Support, Nature Biotechnology 2004, 22, 211-213. 60. Polizzi, K. M.; Bommarius, A. S.; Broering, J. M.; Chaparro-Riggers, J. F., Stability of Biocatalysts, Current Opinion in Chemical Biology 2007, 11, 220-225. 61. You, C.; Myung, S.; Zhang, Y. H. P., Facilitated Substrate Channeling in a Self-Assembled Trifunctional Enzyme Complex, Angewandte Chemie-International Edition 2012, 51, 8787-8790.


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Figure 2 138x85mm (300 x 300 DPI)


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Figure 4 218x191mm (150 x 150 DPI)


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Figure 5 338x113mm (150 x 150 DPI)


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ACS Catalysis

TOC 47x35mm (300 x 300 DPI)


Self-Assembling Protein Scaffold System for Easy in Vitro

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