CnRed: Efficient, Marker-free Genome Engineering of Cupriavidus necator H16 by Adapted Lambda Red Recombineering.

Due to its ability to utilize carbon dioxide, native intracellular accumulation of bioplastic precursors, and a high protein content, the bacterium Cupriavidus necator offers potential solutions for social problems tackled by modern biotechnology. Yet, engineering of high-performing chemolithotrophic production strains has so far been hindered by the lack of adequate genome editing methods. In this work we present the establishment of a lambda Red recombineering system for use in Cupriavidus necator H16. In combination with electroporation as DNA delivery system, it enables an efficient and fast gene deletion methodology utilizing either suicide plasmids or, for the first time, linear PCR product. The novel lambda Red system was validated for the modification of three different genomic loci and, as a proof-of-concept, ultimately utilized for stable genomic integration of Escherichia coli phytase gene appA into the phaC1 locus. A Cre/loxP system further enabled efficient marker recycling. The combination of a minimal transformation protocol with lambda Red recombineering and a Cre/loxP system offers a robust, freedom-to-operate synthetic biology tool in an increasingly important bacterial production host. This approach simplifies and accelerates genome engineering in C. necator and is expected to significantly enhance future strain development efforts.