AGOS: A Plug-and-Play Method for the Assembly of Artificial Gene Operons into Functional Biosynthetic Gene Clusters.
The generation of novel secondary metabolites by reengineering or refactoring biochemical pathways is a rewarding but also challenging goal of synthetic biology. For this, the development of tools for the reconstruction of secondary metabolite gene clusters as well as the challenge of understanding the obstacles in this process is of great interest. The artificial gene operon assembly system (AGOS) is a plug-and-play method developed as a tool to consecutively assemble artificial gene operons into a destination vector and subsequently express them under the control of a de-repressed promoter in a Streptomyces host strain. AGOS was designed as a set of entry plasmids for the construction of artificial gene operons and a SuperCos1 based destination vector, into which the constructed operons can be assembled by Red/ET-mediated recombination. To provide a proof-of-concept of this method, we disassembled the well-known novobiocin biosynthetic gene cluster into four gene operons, encoding for the different moieties of novobiocin. We then genetically reorganized these gene operons with the help of AGOS to finally obtain the complete novobiocin gene cluster again. The production of novobiocin precursors and of novobiocin could successfully be detected by LC-MS and LC-MS/MS. Furthermore, we demonstrated that the omission of terminator sequences only had a minor impact on product formation in our system.
Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen , Auf der Morgenstelle 8, Tübingen, 72076, Germany.
Patrick
P
Basitta
Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen , Auf der Morgenstelle 8, Tübingen, 72076, Germany.
Lucia
L
Westrich
Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen , Auf der Morgenstelle 8, Tübingen, 72076, Germany.
Manuela
M
Rösch
Andreas
A
Kulik
Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen , Auf der Morgenstelle 8, Tübingen, 72076, Germany.
Bertolt
B
Gust
Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen , Auf der Morgenstelle 8, Tübingen, 72076, Germany.
Alexander Kristian
0000-0002-4705-8539
AK
Apel
D002853:Chromatography, Liquid; D005813:Genes, Synthetic / Q000235:genetics*; D005810:Multigene Family / Q000235:genetics*; D009876:Operon / Q000235:genetics*; D013302:Streptomyces / Q000235:genetics; D053719:Tandem Mass Spectrometry
DNA assembly; Streptomyces; biosynthetic gene clusters; heterologous expression; refactoring