BioChem - DOS

Affibody Scaffolds Improve Sesquiterpene Production in Saccharomyces cerevisiae

ACS Synthetic biology

Tippmann, Stefan; Anfelt, Josefine; David, Florian; Rand, Jacqueline M.; Siewers, Verena; Uhlé n, Mathias; Nielsen, Jens; Hudson, Elton P.

<P>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 <I>via</I> 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 <I>in vivo</I>, and was first employed for colocalization of farnesyl diphosphate synthase and farnesene synthase in <I>S.？cerevisiae</I>. 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 <I>Y</I><SUB><I>SFar</I></SUB> 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 <I>E.？coli</I>. 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.</P><P><B>Graphic Abstract</B><BR><IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/asbcd6/2017/asbcd6.2017.6.issue-1/acssynbio.6b00109/production/images/medium/sb-2016-00109t_0006.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/sb6b00109'>ACS Electronic Supporting Info</A></P>

2017

American Chemical Society

2161-5063

6

1

pp.19-28

10.1021/acssynbio.6b00109.s001

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART77246973

https://doi.org/10.1021/acssynbio.6b00109.s001

affibodies; isoprenoids; biofuels; PHB; yeast; metabolic engineering