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Letter
A Whole Cell E. coli Display Platform for Artificial Metalloenzymes: Polyphenylacetylene Production with a Rhodium-Nitrobindin Metalloprotein Alexander R. Grimm, Daniel F. Sauer, Tino Polen, Leilei Zhu, Takashi Hayashi, Jun Okuda, and Ulrich Schwaneberg ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b04369 • Publication Date (Web): 16 Feb 2018 Downloaded from http://pubs.acs.org on February 17, 2018
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ACS Catalysis
A Whole Cell E. coli Display Platform for Artificial Metalloenzymes: Polyphenylacetylene Production with a Rhodium-Nitrobindin Metalloprotein Alexander R. Grimm,† Daniel F. Sauer,‡ Tino Polen,§ Leilei Zhu,†,┴ Takashi Hayashi,║ Jun Okuda,‡ and Ulrich Schwaneberg*,†,# †
Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074 Aachen, Germany Institute of Inorganic Chemistry, RWTH Aachen University, Landoltweg 1, D-52056 Aachen, Germany § IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany ║ Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan # DWI - Leibniz-Institute for Interactive Materials, Forckenbeckstrasse 50, D-52074 Aachen, Germany ‡
ABSTRACT: Whole cell catalysis is in many cases a prerequisite for the cost-effective production of chemicals by biotechnological means. Synthetic metal catalysts for bioorthogonal reactions can be inactivated within cells due to abundant thiol derivatives. Here a cell surface display-based whole cell biohybrid catalyst system (termed ArMt bugs) is reported as a generally applicable platform to unify cost-effective whole cell catalysis with biohybrid catalysis. An inactivated esterase autotransporter is employed to display the nitrobindin protein scaffold with a Rh catalyst on the E. coli surface. Stereoselective polymerization of phenylacetylene yielded a high TON (39*106 cell-1) for the ArMt bugs conversion platform.
Keywords: biohybrid catalysis, whole cell catalysis, cell surface display, protein engineering, metalloenzymes Biohybrid catalysts catalyze a broad range of industrially and synthetically important reactions including transfer hydrogenation, Diels-Alder reactions and olefin metathesis which have been summarized in comprehensive reviews1-8. Protein scaffolds used to generate biohybrid catalysts have been known to alter activity and selectivity of the incorporated catalysts and enable catalysis in aqueous systems. Employed scaffolds range from α-helical globular proteins (e.g. myoglobin9) to β-barrel proteins (e.g. streptavidin10-11 and nitrobindin12). Especially β-barrel proteins have proven to be efficient and robust scaffolds by providing a rigid β-stranded barrel (e.g. 10 β-sheets in nitrobindin). Nitrobindin has a hydrophilic exterior and a hydrophobic cavity which is wellsuited for the anchoring of metal catalysts12-13. For instance, anchoring of a Rh(cp)(cod) catalyst within the well-defined protein cavity of nitrobindin variant NB4 inverted the selectivity in the polymerization of phenylacetylene from 91% cis (free Rh(cp)(cod) catalyst) to 82% trans14. Today around 60% of industrial biocatalytic reactions are based on whole cell catalytic systems15 which are expected to reach 515 billion Euro in sales by 202016. Whole cell processes usually employ simple purification procedures17 and are as a rule of thumb 10 times more cost-effective than enzymes employed in cell-free systems18. Interestingly, industrial whole cell processes have until now mainly been limited to the chemical repertoire of natural enzymes17. Wilson et al. attempted to expand the catalytic range of whole cell systems by employing transition metal catalysis in vivo. The obtained results revealed that many metal catalysts are poisoned within cells or from cell debris; thiols such as glutathione were responsible for the
inactivation of an Ir-based transfer hydrogenation catalyst; pretreatment with oxidizing agents such as diamide was required to observe product formation19. How could a generally applicable and compatible whole cell conversion platform be designed to unify cost-effective whole cell catalysis with biohybrid catalysis? Microbial cell surface display (CSD) systems20-23 are a promising platform to avoid biohybrid catalyst poisoning by separating the whole cell production strain from the biohybrid catalysts which are presented on the cell surface. Cell surface display systems do not require an uptake of the metal catalyst in an often “inactivating” cellular environment and no membrane diffusion barrier limits the diffusion of substrates to biohybrid catalysts on the cell surface. Among the CSD systems found in nature, the inactivated esterase autotransporter (EstA) reported by Becker et al. was reported to be a reliable system, displaying active lipolytic enzymes up to 62 kDa in size (S. marcescens lipase)24. In a patent application (WO 2008063310 A2) a yeast cell surface display method for catalyst support was described for the first time. However, a cell surface display-based system for biohybrid catalysis has not yet been reported for bacterial hosts such as E. coli. Jeschek et al. were the first to show that whole cell biohybrid catalysis is possible in E. coli with their periplasm-based system for Ru-streptavidin-based metathesis catalysts25. Herein we report the first example of a bacterial cell surface display-based whole cell biohybrid catalyst (ArMt bugs, Figure 1a). The EstA autotransporter is employed to display the nitrobindin variant NB4 which acts as a protein scaffold (Figure 1a, Figure S1) for the covalent anchoring of a synthetic
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Rh(cp)(cod) catalyst 1 (Figure 1b). Onoda et al. previously reported catalyst 1 to catalyze the polymerization of phenylacetylene 3 to polyphenylacetylene 4 in a protein and cell free environment12; catalyst 1 showed a TON of 5000 with a 95% preference for cis in THF (Table 1, entry 1). Furthermore, Fukumoto et al. anchored catalyst 1 to a myoglobin variant (1@Mb(A125C))14 without changing its cis : trans selectivity (Table 1, entry 2). In the case of the whole cell biohybrid catalyst (ArMt bugs), the employed conjugation chemistry and linkage of catalyst 1 was performed as reported14. Additionally, a TEV proteasespecific cleavage site (peptide sequence ENLYFQG) was introduced between EstA and NB4 to facilitate analysis of the 1@NB4 biohybrid catalyst (resulting variant: csdNB4 TEV, Figure 1b, Figure S2, Tables S1-S8).
Figure 1. Scheme of CSD system for efficient whole cell catalysis with the 1@NB4 biohybrid catalyst. a) Localization of cell surface displayed biohybrid catalyst scaffold (csdNB4 TEV) consisting of NB4 (red) and the EstA autotransporter (blue, based on PDB structure 3KVN) in the outer E. coli membrane (yellow); b) Coupling reaction via a maleimide linker of Rh(cp)(cod) 1 to csdNB4 TEV; again NB4 shown in red, TEV cleavage site as dashed line, E-epitope as solid line, EstA autotransporter in blue, and E. coli membranes in yellow.
The expected size of the csdNB4 TEV fusion protein was first confirmed via SDS-PAGE analysis (calculated: 90.52 kDa (ExPASy ProtParam tool), Figure S3). The location of the csdNB4 TEV protein on the cell surface was confirmed via outer membrane fractionation26. A band corresponding in size to csdNB4 TEV (ca. 90±1 kDa) was found in the outer membrane fraction (OMF) of E. coli (Figure S4). The cell surface exposition of csdNB4 TEV was further confirmed by an immunofluorescent staining of the E-epitope. The E-epitope in the csdNB4 TEV can specifically be targeted with a FITCconjugated E-epitope antibody (Novus Biologicals)24. E. coli cells with displayed csdNB4 TEV exhibited a significantly increased fluorescence (5.3 times higher) when compared to the negative control (stained E. coli BL21 (DE3) gold pET22b cells without the gene encoding for csdNB4 TEV). Intrinsic fluorescence of the csdNB4 TEV fusion protein also could be excluded (Figure S5). In summary, these results proved the successful expression and outer membrane localization of the csdNB4 TEV fusion protein. The amount of csdNB4 TEV in the outer membrane fraction (OMF) of E. coli was quantified using an Experion device (an automated, microfluidic electrophoresis system, Bio-Rad,
Pro260 chips; Figures S6-S7). Based on the determined concentration of csdNB4 TEV in the OMF, the number of csdNB4 TEV molecules per E. coli cell was calculated to be on average 12800 molecules27. MALDI-ToF-MS and ICPOES were subsequently performed to confirm coupling of the Rh(cp)(cod) catalyst 1 to the csdNB4 TEV cells (Figure 1b, Figures S8-S10). Coupling was performed by supplementing catalyst 1 to the E. coli cell suspension (slowly stirred for 30 minutes, centrifuged and washed to remove uncoupled catalyst; see “Rh(cp)(cod) coupling” in the experimental section; Figure S11). Structural integrity of NB4 under washing conditions (50% THF) was confirmed via CD spectroscopy (Figure S12). For MS analysis, the NB4 domain of csdNB4 TEV was cleaved (via the TEV cleavage site introduced between NB4 and EstA) and centrifuged (see Figure S8). MALDI-ToF-MS analysis yielded peaks corresponding to the expected m/z ratios which confirmed the covalent anchoring of catalyst 1 to the displayed csdNB4 TEV (NB4: 21581.7 m/z calculated, 21582.4 m/z found; 1@NB4: 22017.2 m/z calculated, 22017.7 m/z found, Figure S9). The cellular Rh content was determined via ICP-OES and as an ideal negative control a csdNB4 TEV variant was generated in which the cysteine used for covalent anchoring was “back”-substituted to glutamine (csdNB4 C96Q TEV; Q is the wild type-amino acid at position 96). The Rh content was approximately 13% higher in the ArMt bugs compared to the negative control (csdNB4 C96Q TEV-displaying cells; Figure S10). Despite the significant unspecific binding, no background activity was observed in control experiments; e.g. no product formation could be detected after supplementing substrate 3 to the “coupled” negative control variant (csdNB4 C96Q TEV) (Table 1, entry 6). The Rh(cp)(cod) catalyst 1 is inactive if not bound to the csdNB4 TEV. The latter is also in agreement with a report from Kourist et al., which described the deactivation of ruthenium metathesis catalysts in the presence of buffer in a biphasic system due to their lability towards water and air28. Furthermore, Jeschek et al. reported on the inhibition of their biotinylated Grubbs-Hoveyda type catalyst in E. coli in the absence of the corresponding streptavidin scaffold25. Polymerization of phenylacetylene 3 to polyphenylacetylene 4 was selected as a model reaction to investigate the catalytic performance of the generated ArMt bugs. Cis : trans ratios were determined via NMR analysis (see Figures S13-S15 for NMR results, see Figure S16 for GPC results). Without catalyst 1, the csdNB4 TEV whole cells did not exhibit any activity as expected (Table 1, entry 4). Upon coupling of catalyst 1 to csdNB4 TEV whole cells, polyphenylacetylene was produced with 80% trans (Table 1, entry 5, 12 h reaction time). This is in agreement with the published results for the soluble 1@NB4 (Table 1, entry 3)14. The coupled negative control csdNB4 C96Q TEV did not yield a detectable product formation; the latter proves that only catalyst 1, which is anchored within the hydrophobic NB4 cavity, exhibits activity. Additionally coupling and polyphenylacetylene formation were attempted with whole E. coli cells expressing NB4 in the cytosol. Indeed again, no polyphenylacetylene formation could be detected, indicating that the csdNB4 TEV displayed on the E. coli surface is exclusively responsible for the detected activity (Table 1, entry 7). The TON per cell was determined to be as high as TON = 39*106. The periplasmic whole cell biohybrid catalyst previously reported by Jeschek et al. achieved a TON of 0.5*106 per cell in the ring-closing metathesis (RCM) of diallyl-sulfonamide, with a background activity of
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ACS Catalysis approximately TON = 30*103 per cell (calculated from Figure herein presented ArMt bugs (Rh(cp)(cod) catalyst and phe4c in Jeschek et al. 2016)25. Since the catalyst and reaction nylacetylene polymerization, respectively), TONs of the two reported by Jeschek et al. (biotinylated Grubbs-Hoveyda type systems are difficult to compare. catalyst and olefin metathesis, respectively) differ from the Table 1. Cis : trans selectivity and TON of phenylacetylene 3 polymerization with catalyst 1 in a cell- and protein-free environment (Entry 1), at the surface of a myoglobin variant (Entry 2), anchored within the cavity of purified NB4 (Entry 3), coupled to csdNB4 TEV whole cells (Entry 5), coupled with various negative controls (Entries 6-8, see Table 1 footnote).
Entry 1
a
[Catalyst]
cis : trans
Turnover number (TON) 3
Mn
PDI
6400
3.0
1
95 : 5
5*10
2
1@Mb(A125C)
91 : 9
n.d.
46500
2.3
3
1@NB4
18 : 82
n.d.
38900
2.4
4
b
csdNB4 TEV
-
0
-
-
5b,c
ArMt bugs
20 : 80
39*106
7567
2.7
6b
csdNB4 C96Q TEV
-
0
-
-
7
b
8b
Intracellular NB4
-
0
-
-
csdNB4 TEV
-
0
-
-
Entry 1: Catalyst 1 in a cell- and protein-free environment (in THF). Entry 2 and 3: Fukumoto et al. (2014)14. Entry 4: Uncoupled csdNB4 TEV whole cells. Entry 5: ArMt bugs after coupling; TON per cell. Entry 6: Coupled csdNB4 C96Q TEV whole cells as negative control. Entry 7: Coupled whole cells carrying NB4 intracellularly. Entry 8: No rehydration (swelling) of lyophilized csdNB4 TEV whole cells before coupling. aTON per molecule. bTON per cell. c∆Mn = ±34%, ∆PDI = ±29%.
In summary, cell surface display proved to be an efficient system to generate a powerful whole cell biohybrid catalyst (TON = 39*106 per cell in the polymerization of phenylacetylene). The presented cell surface display-based system solves the challenge of separating biohybrid catalysts from inactivating compounds in the interior of E. coli cells, with the potential benefit that substrates do not have to translocate through membranes for conversion. From our point of view, the ArMt bugs demonstrate that compatible reaction conditions can be generated between whole cells systems and biohybrid catalysts to efficiently perform catalysis in whole cell systems. Furthermore, the employed EstA autotransporter was reported to successfully display enzymes up to 62 kDa in size24, indicating that a broad variety of scaffolds for biohybrid catalysts can likely be displayed on the E. coli surface and that ArMt bugs could be used as a generally applicable system for biohybrid catalysis. Additionally, the ArMt bugs display system will very likely be applicable to high-throughput screening and directed evolution campaigns to tune properties of biohybrid catalysts in the future.
Corresponding Author *E-mail:
[email protected].
Present Addresses Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin, China.
Funding Sources
Notes The authors declare no competing financial interest.
ABBREVIATIONS ArMt bugs, artificial metalloenzyme-displaying E. coli whole cells; CSD, cell surface display; csdNB4 TEV, fusion protein consisting of NB4-TEV protease cleavage site-EstA autotransporter; EstA, outer membrane-anchored esterase from Pseudomonas aeruginosa; FITC, fluorescein isothiocyanate; ICP-OES, inductively coupled plasma optical emission spectroscopy; MALDI-ToF-MS, matrix-assisted laser desorption/ionization mass spectrometry; Mb, myoglobin; NB, nitrobindin; NB4, nitrobindin variant 4; NMR, nuclear magnetic resonance; OMF, outer membrane fraction; RCM, ring-closing metathesis; Rh(cp)(cod), rhodium complex with cyclopentadiene and cyclooctadiene ligands; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TEV, tobacco etch virus; THF, tetrahydrofuran; TON, turnover number.
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AUTHOR INFORMATION
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This work was partially supported by JSPS KAKENHI Grant Number JP15H05804 to T.H.
Supporting Information. General comments, experimental section, generation, sequence, expression and characterization of cell surface display construct csdNB4 TEV, characterization of coupling with cell surface display construct csdNB4 TEV. This material is available free of charge via the Internet at http://pubs.acs.org.
ACKNOWLEDGMENTS We gratefully acknowledge the financial support by the Deutsche Forschungsgemeinschaft (DFG) through the International Re-
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search Training Group “Selectivity in Chemo- and Biocatalysis” (SeleCa), the Bundesministerium für Bildung und Forschung (BMBF) (FKZ: 031B0297), JSPS KAKENHI Grant Number JP15H05804 in Innovative Areas “Precisely Designed Catalysts with Customized Scaffolding” to T.H. Marcus Arlt is acknowledged for valuable discussions. Umicore, Frankfurt (Dr. A. Doppiu), is gratefully acknowledged for a generous gift of rhodium precursor.
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