Affibody Scaffolds Improve Sesquiterpene Production in Saccharomyces cerevisiae.
Artikel i vetenskaplig tidskrift, 2016

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.

Författare

Stefan Tippmann

Chalmers, Biologi och bioteknik, Systembiologi

J. Anfelt

Kungliga Tekniska Högskolan (KTH)

Florian David

Chalmers, Biologi och bioteknik, Systembiologi

Jacqueline M Rand

Chalmers University of Technology

University of Wisconsin Madison

Verena Siewers

Chalmers, Biologi och bioteknik, Systembiologi

Mathias Uhlen

Danmarks Tekniske Universitet (DTU)

Kungliga Tekniska Högskolan (KTH)

Jens B Nielsen

Chalmers, Biologi och bioteknik, Systembiologi

E. P. Hudson

Kungliga Tekniska Högskolan (KTH)

ACS Synthetic Biology

2161-5063 (eISSN)

19-28

Ämneskategorier

Biologiska vetenskaper

Styrkeområden

Energi

Livsvetenskaper och teknik

DOI

10.1021/acssynbio.6b00109

PubMed

27560952