Using Cupriavidus necator H16 to provide a roadmap for increasing electroporation efficiency in non-model bacteria

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

ABSTRACT Bacteria are a treasure trove of metabolic reactions, but most industrial biotechnology applications rely on a limited set of established host organisms. In contrast, adopting non-model bacteria for the production of various chemicals of interest is often hampered by their limited genetic amenability coupled with their low transformation efficiency. In this study, we propose a series of steps that can be taken to increase electroporation efficiency in non-model bacteria. As a test strain, we use Cupriavidus necator H16, a lithoautotrophic bacterium that has been engineered to produce a wide range of products from CO 2 and hydrogen. However, its low electroporation efficiency hinders the high-throughput genetic modifications required to develop C. necator into an industrially relevant host organism. First, we propose a species-independent technique based on natively methylated DNA and Golden Gate assembly to increase one-pot cloning and electroporation efficiency by 70-fold. Second, bioinformatic tools were used to predict defense systems and develop a restriction avoidance strategy that was used to introduce suicide plasmids by electroporation to obtain a domesticated strain. The results are discussed in the context of metabolic engineering of non-model bacteria. TABLE OF CONTENT

Authors 5

1. Matteo Vajente

   University of Groningen

   Affiliation as printed

   Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713AV, The Netherlands

   Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan , Groningen 973AV, The Netherlands

2. Riccardo Clerici Aachen Institute of Applied Microbiology

   RWTH Aachen University

   Affiliation as printed

   Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany

   Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Worringerweg 1, 5074 Aachen, Germany

3. Hendrik Ballerstedt Aachen Institute of Applied Microbiology

   RWTH Aachen University

   Affiliation as printed

   Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany

   Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Worringerweg 1, 5074 Aachen, Germany

4. Lars M. Blank Aachen Institute of Applied Microbiology

   RWTH Aachen University

   Affiliation as printed

   Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany

   Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Worringerweg 1, 5074 Aachen, Germany

5. Sandy Schmidt corresponding

   University of Groningen

   Affiliation as printed

   Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713AV, The Netherlands

   Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan , Groningen 973AV, The Netherlands