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Affibody Scaffolds Improve Sesquiterpene Production in Saccharomyces cerevisiae.

Stefan Tippmann (Institutionen för biologi och bioteknik, Systembiologi) ; Josefine Anfelt ; Florian David (Institutionen för biologi och bioteknik, Systembiologi) ; Jacqueline M Rand ; Verena Siewers (Institutionen för biologi och bioteknik, Systembiologi) ; Mathias Uhlén ; Jens B. Nielsen (Institutionen för biologi och bioteknik, Systembiologi) ; Elton P Hudson
ACS synthetic biology (2161-5063). (2016)
[Artikel, refereegranskad vetenskaplig]

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.



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Denna post skapades 2017-01-05. Senast ändrad 2017-03-09.
CPL Pubid: 246665

 

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Institutionen för biologi och bioteknik, Systembiologi

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