Optimizing the linker length for fusing an alcohol dehydrogenase with a cyclohexanone monooxygenase

A. Gran-Scheuch; F. Aalbers; Y. Woudstra; L. Parra; M.W. Fraaije

doi:10.1016/bs.mie.2020.09.008

Optimizing the linker length for fusing an alcohol dehydrogenase with a cyclohexanone monooxygenase

A. Gran-Scheuch, F. Aalbers, Y. Woudstra, L. Parra, M.W. Fraaije

Research output: Chapter in Book / Report / Conference proceeding › Chapter › Academic › peer-review

Abstract

© 2021 Elsevier Inc.The use of enzymes in organic synthesis is highly appealing due their remarkably high chemo-, regio- and enantioselectivity. Nevertheless, for biosynthetic routes to be industrially useful, the enzymes must fulfill several requirements. Particularly, in case of cofactor-dependent enzymes self-sufficient systems are highly valuable. This can be achieved by fusing enzymes with complementary cofactor dependency. Such bifunctional enzymes are also relatively easy to handle, may enhance stability, and promote product intermediate channeling. However, usually the characteristics of the linker, fusing the target enzymes, are not thoroughly evaluated. A poor linker design can lead to detrimental effects on expression levels, enzyme stability and/or enzyme performance. In this chapter, the effect of the length of a glycine-rich linker was explored for the case study of ɛ-caprolactone synthesis through an alcohol dehydrogenase-cyclohexanone monooxygenase fusion system. The procedure includes cloning of linker variants, expression analysis, determination of thermostability and effect on activity and conversion levels of 15 variants of different linker sizes. The protocols can also be used for the creation of other protein-protein fusions.

Original language	English
Title of host publication	Methods in Enzymology
Editors	M. Merkx
Publisher	Academic Press Inc.
Pages	107-143
ISBN (Print)	9780128208182
DOIs	https://doi.org/10.1016/bs.mie.2020.09.008
Publication status	Published - 1 Jan 2021
Externally published	Yes

Publication series

Name	Methods in Enzymology
ISSN (Print)	0076-6879
ISSN (Electronic)	1557-7988

Funding

This work received funding from the European Union (EU), project ROBOX (grant agreement no. 635734) under the EU's Horizon 2020 Program Research and Innovation actions H2020-LEIT BIO-2014-1, from the Institute for Biological and Medical Engineering from Pontificia Universidad Católica de Chile and CORFO's grant 14ENI2-26862. A PhD scholarship from CONICYT (Comisión Nacional de Investigación Científica y Tecnológica de Chile), from the graduated school of Pontificia Universidad Católica de Chile and from the Faculty of engineering of Pontificia Universidad Católica de Chile to AG-S are acknowledged. The views and opinions expressed in this article are only those of the authors and do not necessarily reflect those of the European Union Research Agency. The European Union is not liable for any use that may be made of the information contained herein. This work received funding from the European Union (EU), project ROBOX (grant agreement no. 635734) under the EU's Horizon 2020 Program Research and Innovation actions H2020-LEIT BIO-2014-1, from the Institute for Biological and Medical Engineering from Pontificia Universidad Cat?lica de Chile and CORFO's grant 14ENI2-26862. A PhD scholarship from CONICYT (Comisi?n Nacional de Investigaci?n Cient?fica y Tecnol?gica de Chile), from the graduated school of Pontificia Universidad Cat?lica de Chile and from the Faculty of engineering of Pontificia Universidad Cat?lica de Chile to AG-S are acknowledged. The views and opinions expressed in this article are only those of the authors and do not necessarily reflect those of the European Union Research Agency. The European Union is not liable for any use that may be made of the information contained herein.

Funders	Funder number
EU's Horizon 2020
European Union Research Agency
Institute for Biological and Medical Engineering
European Commission	635734
Comisión Nacional de Investigación Científica y Tecnológica
Horizon 2020	H2020-LEIT BIO-2014-1
Pontificia Universidad Católica de Chile	14ENI2-26862

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abstract = "{\textcopyright} 2021 Elsevier Inc.The use of enzymes in organic synthesis is highly appealing due their remarkably high chemo-, regio- and enantioselectivity. Nevertheless, for biosynthetic routes to be industrially useful, the enzymes must fulfill several requirements. Particularly, in case of cofactor-dependent enzymes self-sufficient systems are highly valuable. This can be achieved by fusing enzymes with complementary cofactor dependency. Such bifunctional enzymes are also relatively easy to handle, may enhance stability, and promote product intermediate channeling. However, usually the characteristics of the linker, fusing the target enzymes, are not thoroughly evaluated. A poor linker design can lead to detrimental effects on expression levels, enzyme stability and/or enzyme performance. In this chapter, the effect of the length of a glycine-rich linker was explored for the case study of ɛ-caprolactone synthesis through an alcohol dehydrogenase-cyclohexanone monooxygenase fusion system. The procedure includes cloning of linker variants, expression analysis, determination of thermostability and effect on activity and conversion levels of 15 variants of different linker sizes. The protocols can also be used for the creation of other protein-protein fusions.",

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AB - © 2021 Elsevier Inc.The use of enzymes in organic synthesis is highly appealing due their remarkably high chemo-, regio- and enantioselectivity. Nevertheless, for biosynthetic routes to be industrially useful, the enzymes must fulfill several requirements. Particularly, in case of cofactor-dependent enzymes self-sufficient systems are highly valuable. This can be achieved by fusing enzymes with complementary cofactor dependency. Such bifunctional enzymes are also relatively easy to handle, may enhance stability, and promote product intermediate channeling. However, usually the characteristics of the linker, fusing the target enzymes, are not thoroughly evaluated. A poor linker design can lead to detrimental effects on expression levels, enzyme stability and/or enzyme performance. In this chapter, the effect of the length of a glycine-rich linker was explored for the case study of ɛ-caprolactone synthesis through an alcohol dehydrogenase-cyclohexanone monooxygenase fusion system. The procedure includes cloning of linker variants, expression analysis, determination of thermostability and effect on activity and conversion levels of 15 variants of different linker sizes. The protocols can also be used for the creation of other protein-protein fusions.

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Optimizing the linker length for fusing an alcohol dehydrogenase with a cyclohexanone monooxygenase

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