Affibody Scaffolds Improve Sesquiterpene Production in Saccharomyces cerevisiae

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• 作者：Stefan Tippmann ; Josefine Anfelt ; Florian David ; Jacqueline M. Rand ; Verena Siewers ; Mathias Uhlén ; Jens Nielsen ; Elton P. Hudson
• 刊名：ACS Synthetic Biology
• 出版年：2017
• 出版时间：January 20, 2017
• 年：2017
• 卷：6
• 期：1
• 页码：19-28
• 全文大小：445K
• ISSN：2161-5063

文摘

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 Y_SFar 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.