Etablierung des stickstofffixierenden Alphaproteobakteriums Sinorhizobium meliloti als Chassis-Organismus in der Synthetischen Mikrobiologie

Synthetic biology requires strategies, which facilitate efficient genetic engineering. DNA editing in the range of single nucleotides up to whole genomes requires appropriate molecular methods. While powerful tools are available for prokaryotic model organisms, the repertoire for genetic engineering of rhizobia of the class alphaproteobacteria is still limited. The α-rhizobium Sinorhizobium meliloti is able to fix atmospheric nitrogen in symbiotic relationships with legumes. Therefore it is of high agricultural value. Due to its remarkable genome architecture, which besides a main chromosome exhibits a chromid (pSymB) and a megaplasmid (pSymA), S. meliloti emerged as model organism for studying multipartite genomes in bacteria. pSymA and pSymB carry ~45% of the genomic content and are based on repABC-type replication origins, which integrate these extra-chromosomal replicons into the cell cycle. The repABC region of pSymA was further identified to confer the chromosome-like features as single copy replication and stable inheritance to artificial DNA constructs. In this work, heterologous repABC cassettes derived from different rhizobial species were used to establish a novel shuttle-vector system for S. meliloti. This highly modular and standardized assembly system facilitates in-vitro construction of artificial mini replicons. Resulting pABC vectors serve as E. coli-compatible cloning vehicels and enable establishment of complex, single-copy expression systems. Furthermore, a cloning-free genome editing method and an optimized Cre/lox recombination system were developed and expand the genetic toolbox of S. meliloti. Respective engineering approaches were demonstrated by substantial genome rearrangements and large scale deletion and inversion systems. Cre/lox recombination strategies and an inducible repABC-type replication origin further facilitated in-vivo cloning, which enables new experimental approaches both in genome biology, as well as for synthetic applications. The robust function of Cre/lox and the wide distribution of repABC-based replicons among the alphaproteobacteria likely allow an application of the established methods in additional organisms to exploit their genetic resources for agricultural and biotechnological purposes.