Affibody scaffolds improve sesquiterpene production in Saccharomyces cerevisiae

Stefan Tippmann, Josefine Anfelt, Florian David, Jacqueline M. Rand, Verena Siewers, Mathias Uhlén, Jens Nielsen, Elton Paul Hudson

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Abstract

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 co-localized through attachment to a synthetic scaffold via non-covalent 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 co-localization 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.
Original languageEnglish
JournalA C S Synthetic Biology
Volume6
Issue number1
Pages (from-to)19-28
Number of pages10
ISSN2161-5063
DOIs
Publication statusPublished - 2017

Keywords

  • Affibodies
  • Biofuels
  • Isoprenoids
  • Metabolic engineering
  • PHB
  • Yeast

Cite this

Tippmann, S., Anfelt, J., David, F., Rand, J. M., Siewers, V., Uhlén, M., Nielsen, J., & Hudson, E. P. (2017). Affibody scaffolds improve sesquiterpene production in Saccharomyces cerevisiae. A C S Synthetic Biology, 6(1), 19-28. https://doi.org/10.1021/acssynbio.6b00109