Tailoring the properties of (catalytically)-active inclusion bodies.

Jager VD, Kloss R, Grünberger A, Seide S, Hahn D, Karmainski T, Piqueray M, Embruch J, Longerich S, Mackfeld U, Jaeger K-E, et al. (2019)
Microbial cell factories 18(1): 33.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
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Jager, V D; Kloss, R; Grünberger, AlexanderUniBi; Seide, S; Hahn, D; Karmainski, T; Piqueray, M; Embruch, J; Longerich, S; Mackfeld, U; Jaeger, K-E; Wiechert, W
Abstract / Bemerkung
BACKGROUND: Immobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically. RESULTS: We here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein. CONCLUSION: We here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.
Microbial cell factories
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Jager VD, Kloss R, Grünberger A, et al. Tailoring the properties of (catalytically)-active inclusion bodies. Microbial cell factories. 2019;18(1): 33.
Jager, V. D., Kloss, R., Grünberger, A., Seide, S., Hahn, D., Karmainski, T., Piqueray, M., et al. (2019). Tailoring the properties of (catalytically)-active inclusion bodies. Microbial cell factories, 18(1), 33. doi:10.1186/s12934-019-1081-5
Jager, V D, Kloss, R, Grünberger, Alexander, Seide, S, Hahn, D, Karmainski, T, Piqueray, M, et al. 2019. “Tailoring the properties of (catalytically)-active inclusion bodies.”. Microbial cell factories 18 (1): 33.
Jager, V. D., Kloss, R., Grünberger, A., Seide, S., Hahn, D., Karmainski, T., Piqueray, M., Embruch, J., Longerich, S., Mackfeld, U., et al. (2019). Tailoring the properties of (catalytically)-active inclusion bodies. Microbial cell factories 18:33.
Jager, V.D., et al., 2019. Tailoring the properties of (catalytically)-active inclusion bodies. Microbial cell factories, 18(1): 33.
V.D. Jager, et al., “Tailoring the properties of (catalytically)-active inclusion bodies.”, Microbial cell factories, vol. 18, 2019, : 33.
Jager, V.D., Kloss, R., Grünberger, A., Seide, S., Hahn, D., Karmainski, T., Piqueray, M., Embruch, J., Longerich, S., Mackfeld, U., Jaeger, K.-E., Wiechert, W., Pohl, M., Krauss, U.: Tailoring the properties of (catalytically)-active inclusion bodies. Microbial cell factories. 18, : 33 (2019).
Jager, V D, Kloss, R, Grünberger, Alexander, Seide, S, Hahn, D, Karmainski, T, Piqueray, M, Embruch, J, Longerich, S, Mackfeld, U, Jaeger, K-E, Wiechert, W, Pohl, M, and Krauss, U. “Tailoring the properties of (catalytically)-active inclusion bodies.”. Microbial cell factories 18.1 (2019): 33.

94 References

Daten bereitgestellt von Europe PubMed Central.

Homogeneous biocatalysis in organic solvents and water-organic mixtures.
Castro GR, Knubovets T., Crit. Rev. Biotechnol. 23(3), 2003
PMID: 14743990
Enzyme Engineering for In Situ Immobilization.
Rehm FB, Chen S, Rehm BH., Molecules 21(10), 2016
PMID: 27754434
Understanding enzyme immobilisation
Hanefeld U, Gardossi L, Magner E., 2009
Enzyme immobilisation in biocatalysis: why, what and how.
Sheldon RA, van Pelt S., Chem Soc Rev 42(15), 2013
PMID: 23532151
Principles, techniques, and applications of biocatalyst immobilization for industrial application.
Es I, Vieira JD, Amaral AC., Appl. Microbiol. Biotechnol. 99(5), 2015
PMID: 25616529
The sol-gel encapsulation of enzymes
Pierre AC., 2004
Guidelines and cost analysis for catalyst production in biocatalytic processes
Tufvesson P, Lima-Ramos J, Nordblad M, Woodley JM., 2011
Fusion of a coiled-coil domain facilitates the high-level production of catalytically active enzyme inclusion bodies
Diener M, Kopka B, Pohl M, Jaeger KE, Krauss U., 2016
Catalytically active inclusion bodies of L-lysine decarboxylase from E. coli for 1,5-diaminopentane production.
Kloss R, Limberg MH, Mackfeld U, Hahn D, Grunberger A, Jager VD, Krauss U, Oldiges M, Pohl M., Sci Rep 8(1), 2018
PMID: 29643457
Small surfactant-like peptides can drive soluble proteins into active aggregates.
Zhou B, Xing L, Wu W, Zhang XE, Lin Z., Microb. Cell Fact. 11(), 2012
PMID: 22251949
Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8.
Wang X, Zhou B, Hu W, Zhao Q, Lin Z., Microb. Cell Fact. 14(), 2015
PMID: 26077447
Generation of catalytic protein particles in Escherichia coli cells using the cellulose-binding domain from Cellulomonas fimi as a fusion partner
Choi S-L, Lee SJ, Ha J-S, Song JJ, Rhee YH, Lee S-G., 2011
Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins.
Garcia-Fruitos E, Gonzalez-Montalban N, Morell M, Vera A, Ferraz RM, Aris A, Ventura S, Villaverde A., Microb. Cell Fact. 4(), 2005
PMID: 16156893
Insoluble protein applications: the use of bacterial inclusion bodies as biocatalysts.
Hrabarova E, Achbergerova L, Nahalka J., Methods Mol. Biol. 1258(), 2015
PMID: 25447879
Targeting lectin activity into inclusion bodies for the characterisation of glycoproteins.
Nahalka J, Mislovicova D, Kavcova H., Mol Biosyst 5(8), 2009
PMID: 19603115
Aggresomes, inclusion bodies and protein aggregation.
Kopito RR., Trends Cell Biol. 10(12), 2000
PMID: 11121744
Recombinant protein folding and misfolding in Escherichia coli.
Baneyx F, Mujacic M., Nat. Biotechnol. 22(11), 2004
PMID: 15529165
A Synthetic Reaction Cascade Implemented by Colocalization of Two Proteins within Catalytically Active Inclusion Bodies.
Jager VD, Lamm R, Kloß R, Kaganovitch E, Grunberger A, Pohl M, Buchs J, Jaeger KE, Krauss U., ACS Synth Biol 7(9), 2018
PMID: 30053372
In vivo enzyme immobilization by inclusion body display.
Steinmann B, Christmann A, Heiseler T, Fritz J, Kolmar H., Appl. Environ. Microbiol. 76(16), 2010
PMID: 20581198

Functional inclusion bodies produced in the yeast Pichia pastoris.
Rueda F, Gasser B, Sanchez-Chardi A, Roldan M, Villegas S, Puxbaum V, Ferrer-Miralles N, Unzueta U, Vazquez E, Garcia-Fruitos E, Mattanovich D, Villaverde A., Microb. Cell Fact. 15(1), 2016
PMID: 27716225
Functional inclusion bodies produced in bacteria as naturally occurring nanopills for advanced cell therapies.
Vazquez E, Corchero JL, Burgueno JF, Seras-Franzoso J, Kosoy A, Bosser R, Mendoza R, Martinez-Lainez JM, Rinas U, Fernandez E, Ruiz-Avila L, Garcia-Fruitos E, Villaverde A., Adv. Mater. Weinheim 24(13), 2012
PMID: 22410789
Crystal structure of a naturally occurring parallel right-handed coiled coil tetramer.
Stetefeld J, Jenny M, Schulthess T, Landwehr R, Engel J, Kammerer RA., Nat. Struct. Biol. 7(9), 2000
PMID: 10966648
Tailor-made catalytically active inclusion bodies for different applications in biocatalysis
Kloss R, Karmainski T, Jäger VD, Hahn D, Grünberger A, Baumgart M, Krauss U, Jaeger K-E, Wiechert W, Pohl M., 2018
The structure of alpha-helical coiled coils.
Lupas AN, Gruber M., Adv. Protein Chem. 70(), 2005
PMID: 15837513
New currency for old rope: from coiled-coil assemblies to α-helical barrels.
Woolfson DN, Bartlett GJ, Bruning M, Thomson AR., Curr. Opin. Struct. Biol. 22(4), 2012
PMID: 22445228
Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein.
Wachter RM, Elsliger MA, Kallio K, Hanson GT, Remington SJ., Structure 6(10), 1998
PMID: 9782051
Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.
Zacharias DA, Violin JD, Newton AC, Tsien RY., Science 296(5569), 2002
PMID: 11988576
Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.
Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY., Nat. Biotechnol. 22(12), 2004
PMID: 15558047
Stereoselective bioreduction of bulky-bulky ketones by a novel ADH from Ralstonia sp.
Lavandera I, Kern A, Ferreira-Silva B, Glieder A, de Wildeman S, Kroutil W., J. Org. Chem. 73(15), 2008
PMID: 18597534
Biochemical characterization of an alcohol dehydrogenase from Ralstonia sp.
Kulig J, Frese A, Kroutil W, Pohl M, Rother D., Biotechnol. Bioeng. 110(7), 2013
PMID: 23381774
Benzoylformate decarboxylase from Pseudomonas putida as stable catalyst for the synthesis of chiral 2-hydroxy ketones.
Iding H, Dunnwald T, Greiner L, Liese A, Muller M, Siegert P, Grotzinger J, Demir AS, Pohl M., Chemistry 6(8), 2000
PMID: 10840971
Rational protein design of ThDP-dependent enzymes-engineering stereoselectivity.
Gocke D, Walter L, Gauchenova E, Kolter G, Knoll M, Berthold CL, Schneider G, Pleiss J, Muller M, Pohl M., Chembiochem 9(3), 2008
PMID: 18224647

Alteration of the substrate specificity of benzoylformate decarboxylase from Pseudomonas putida by directed evolution.
Lingen B, Kolter-Jung D, Dunkelmann P, Feldmann R, Grotzinger J, Pohl M, Muller M., Chembiochem 4(8), 2003
PMID: 12898622
Design of the linkers which effectively separate domains of a bifunctional fusion protein.
Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T., Protein Eng. 14(8), 2001
PMID: 11579220
Fusion protein linkers: property, design and functionality.
Chen X, Zaro JL, Shen WC., Adv. Drug Deliv. Rev. 65(10), 2012
PMID: 23026637
Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA.
Kandiah E, Carriel D, Perard J, Malet H, Bacia M, Liu K, Chan SW, Houry WA, Ollagnier de Choudens S, Elsen S, Gutsche I., Sci Rep 6(), 2016
PMID: 27080013
Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase.
Kanjee U, Gutsche I, Alexopoulos E, Zhao B, El Bakkouri M, Thibault G, Liu K, Ramachandran S, Snider J, Pai EF, Houry WA., EMBO J. 30(5), 2011
PMID: 21278708
Structure of concatenated HAMP domains provides a mechanism for signal transduction.
Airola MV, Watts KJ, Bilwes AM, Crane BR., Structure 18(4), 2010
PMID: 20399181
Cross-linked α-L-rhamnosidase aggregates with potential application in food industry
Alvarenga AE, Amoroso MJ, Illanes A, Castro GR., Eur. Food Res. Technol. 238(5), 2014
PMID: IND500894132
Cross-linked aggregates of the hydroxynitrile lyase from Manihot esculenta: highly active and robust biocatalysts
Chmura A, van GM, Kielar F, van LM, van F, Sheldon RA., 2006
Encapsulation of Spherical Cross-Linked Phenylalanine Ammonia Lyase Aggregates in Mesoporous Biosilica.
Cui J, Zhao Y, Feng Y, Lin T, Zhong C, Tan Z, Jia S., J. Agric. Food Chem. 65(3), 2017
PMID: 28054483
A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates.
Mateo C, Palomo JM, van Langen LM, van Rantwijk F, Sheldon RA., Biotechnol. Bioeng. 86(3), 2004
PMID: 15083507
Cadaverine Production by Using Cross-Linked Enzyme Aggregate of Escherichia coli Lysine Decarboxylase.
Park SH, Soetyono F, Kim HK., J. Microbiol. Biotechnol. 27(2), 2017
PMID: 27780956
Preparation and characterization of cross linked enzyme aggregates (CLEAs) of Bacillus amyloliquefaciens alpha amylase
Talekar S, Waingade S, Gaikwad V, Patil S, Nagavekar N., 2012
Preparation of cross-linked enzyme aggregates of nitrile hydratase ES-NHT-118 from E coli by macromolecular cross-linking agent
Zhou LY, Mou HX, Gao J, Ma L, He Y, Jiang YJ., 2017
E. coli transports aggregated proteins to the poles by a specific and energy-dependent process.
Rokney A, Shagan M, Kessel M, Smith Y, Rosenshine I, Oppenheim AB., J. Mol. Biol. 392(3), 2009
PMID: 19596340
Functional protein-based nanomaterial produced in microorganisms recognized as safe: A new platform for biotechnology.
Cano-Garrido O, Sanchez-Chardi A, Pares S, Giro I, Tatkiewicz WI, Ferrer-Miralles N, Ratera I, Natalello A, Cubarsi R, Veciana J, Bach A, Villaverde A, Aris A, Garcia-Fruitos E., Acta Biomater 43(), 2016
PMID: 27452157
Sequence determinants of amyloid fibril formation.
Lopez de la Paz M, Serrano L., Proc. Natl. Acad. Sci. U.S.A. 101(1), 2003
PMID: 14691246
The amyloid stretch hypothesis: recruiting proteins toward the dark side.
Esteras-Chopo A, Serrano L, Lopez de la Paz M., Proc. Natl. Acad. Sci. U.S.A. 102(46), 2005
PMID: 16263932
Short protein segments can drive a non-fibrillizing protein into the amyloid state.
Teng PK, Eisenberg D., Protein Eng. Des. Sel. 22(8), 2009
PMID: 19602569
Rationalization of the effects of mutations on peptide and protein aggregation rates.
Chiti F, Stefani M, Taddei N, Ramponi G, Dobson CM., Nature 424(6950), 2003
PMID: 12917692
AGGRESCAN: a server for the prediction and evaluation of "hot spots" of aggregation in polypeptides.
Conchillo-Sole O, de Groot NS, Aviles FX, Vendrell J, Daura X, Ventura S., BMC Bioinformatics 8(), 2007
PMID: 17324296
Intrinsically unstructured proteins. Trends Biochemical
Tompa P., 2002
A novel strategy for the purification of recombinantly expressed unstructured protein domains.
Kalthoff C., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 786(1-2), 2003
PMID: 12651021
Native protein sequences are close to optimal for their structures.
Kuhlman B, Baker D., Proc. Natl. Acad. Sci. U.S.A. 97(19), 2000
PMID: 10984534
Protein structure prediction using Rosetta.
Rohl CA, Strauss CE, Misura KM, Baker D., Meth. Enzymol. 383(), 2004
PMID: 15063647

Dynamic and quantitative Ca2+ measurements using improved cameleons.
Miyawaki A, Griesbeck O, Heim R, Tsien RY., Proc. Natl. Acad. Sci. U.S.A. 96(5), 1999
PMID: 10051607
A guide to choosing fluorescent proteins.
Shaner NC, Steinbach PA, Tsien RY., Nat. Methods 2(12), 2005
PMID: 16299475
Protein production by auto-induction in high density shaking cultures.
Studier FW., Protein Expr. Purif. 41(1), 2005
PMID: 15915565
Stereoselective synthesis of bulky 1,2-diols with alcohol dehydrogenases
Kulig J, Simon RC, Rose CA, Husain SM, Hackh M, Ludeke S, Zeitler K, Kroutil W, Pohl M, Rother D., 2012
Factors influencing the operational stability of NADPH-dependent alcohol dehydrogenase and an NADH-dependent variant thereof in gas/solid reactors
Kulishova L, Dimoula K, Jordan M, Wirtz A, Hofmann D, Santiago-Schübel B, Fitter J, Pohl M, Spiess AC., 2010

Characterization of benzaldehyde lyase from Pseudomonas fluorescens: A versatile enzyme for asymmetric C-C bond formation.
Janzen E, Muller M, Kolter-Jung D, Kneen MM, McLeish MJ, Pohl M., Bioorg. Chem. 34(6), 2006
PMID: 17078994
Simple fed-batch technique for high cell density cultivation of Escherichia coli.
Korz DJ, Rinas U, Hellmuth K, Sanders EA, Deckwer WD., J. Biotechnol. 39(1), 1995
PMID: 7766011

Protein identification and analysis tools on the ExPASy server
Gasteiger E, Hoogland C, Gattiker A, Duvaud SE, Wilkins MR, Appel RD, Bairoch A., 2005
NIH Image to ImageJ: 25 years of image analysis.
Schneider CA, Rasband WS, Eliceiri KW., Nat. Methods 9(7), 2012
PMID: 22930834
Anatomy and ultrastructure of Wolffia columbiana and Wolffia borealis, two nonvascular aquatic angiosperms
White SL, Wise RR., 1998
The Protein Data Bank.
Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE., Nucleic Acids Res. 28(1), 2000
PMID: 10592235
Inference of macromolecular assemblies from crystalline state.
Krissinel E, Henrick K., J. Mol. Biol. 372(3), 2007
PMID: 17681537
Computational protein design with explicit consideration of surface hydrophobic patches.
Jacak R, Leaver-Fay A, Kuhlman B., Proteins 80(3), 2011
PMID: 22223219

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