Affibody Scaffolds Improve Sesquiterpene Production in Saccharomyces cerevisiae.
Artikel i vetenskaplig tidskrift, 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.
Författare
Stefan Tippmann
Novo Nordisk Fonden
Chalmers, Biologi och bioteknik, Systembiologi
J. Anfelt
Kungliga Tekniska Högskolan (KTH)
Florian David
Chalmers, Biologi och bioteknik, Systembiologi
Novo Nordisk Fonden
Jacqueline M Rand
Chalmers, Biologi och bioteknik, Systembiologi
University of Wisconsin Madison
Verena Siewers
Chalmers, Biologi och bioteknik, Systembiologi
Novo Nordisk Fonden
Mathias Uhlen
Novo Nordisk Fonden
Danmarks Tekniske Universitet (DTU)
Kungliga Tekniska Högskolan (KTH)
Jens B Nielsen
Novo Nordisk Fonden
Chalmers, Biologi och bioteknik, Systembiologi
E. P. Hudson
Kungliga Tekniska Högskolan (KTH)
ACS Synthetic Biology
2161-5063 (eISSN)
Vol. 6 1 19-28Ämneskategorier (SSIF 2011)
Biologiska vetenskaper
Styrkeområden
Energi
Livsvetenskaper och teknik (2010-2018)
DOI
10.1021/acssynbio.6b00109
PubMed
27560952