Affibody Scaffolds Improve Sesquiterpene Production in Saccharomyces cerevisiae.
Journal article, 2017

Enzyme fusions have been widely used as a tool in metabolic engineering to increase pathway efficiency by reducing substrate loss and accumulation of toxic intermediates. Alternatively, enzymes can be colocalized through attachment to a synthetic scaffold via noncovalent interactions. Here we describe the use of affibodies for enzyme tagging and scaffolding. The scaffolding is based on the recognition of affibodies to their anti-idiotypic partners in vivo, and was first employed for colocalization of farnesyl diphosphate synthase and farnesene synthase in S. cerevisiae. Different parameters were modulated to improve the system, and the enzyme:scaffold ratio was most critical for its functionality. Ultimately, the yield of farnesene on glucose YSFar could be improved by 135% in fed-batch cultivations using a 2-site affibody scaffold. The scaffolding strategy was then extended to a three-enzyme polyhydroxybutyrate (PHB) pathway, heterologously expressed in E. coli. Within a narrow range of enzyme and scaffold induction, the affibody tagging and scaffolding increased PHB production 7-fold. This work demonstrates how the versatile affibody can be used for metabolic engineering purposes.

Author

Stefan Tippmann

Novo Nordisk Foundation Center for Biosustainability

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

J. Anfelt

Royal Institute of Technology (KTH)

Florian David

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Novo Nordisk Foundation Center for Biosustainability

Jacqueline M Rand

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

University of Wisconsin Madison

Verena Siewers

Novo Nordisk Foundation Center for Biosustainability

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Mathias Uhlen

Technical University of Denmark (DTU)

Novo Nordisk Foundation Center for Biosustainability

Royal Institute of Technology (KTH)

Jens B Nielsen

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Novo Nordisk Foundation Center for Biosustainability

E. P. Hudson

Royal Institute of Technology (KTH)

ACS Synthetic Biology

2161-5063 (eISSN)

Vol. 6 1 19-28

Subject Categories

Biological Sciences

Areas of Advance

Energy

Life Science Engineering (2010-2018)

DOI

10.1021/acssynbio.6b00109

PubMed

27560952

More information

Latest update

1/22/2021