Submitted to: Molecular Genetics and Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/27/2005
Publication Date: 11/1/2005
Citation: Skory, C.D. 2005. Inhibition of non-homologous end joining and integration of DNA with transformation of Rhizopus oryzae. Molecular Genetics and Genomics. 274(4):373-83. Interpretive Summary: The filamentous fungus Rhizopus has long been recognized as an important microorganism that can be both beneficial and harmful. This fungus is well known for the ability to produce industrial enzymes (e.g. glucoamylase, lipase), organic acids (e.g. lactate, fumarate), corticosteroids, and even fermented foods. However, it is also a plant pathogen, that results in significant loss of agricultural commodities. Unfortunately, research with Rhizopus has been hampered by a lack of molecular techniques for the genetic manipulation of this fungus. This void of understanding impedes the ability to study the organism and to exploit its valuable traits. A cornerstone of genetic engineering involves being able to put modified genes back into the organism of interest. In order to be useful, the fungus must be able to replicate this DNA and repair any damage that might occur during this manipulation process. This work describes novel methods of introducing genes into Rhizopus in a controlled and targeted manner. The results of this study will allow new strategies to be developed that can exploit this discovery and allow more rapid progression of the industrial utilization of this valuable organism.
Technical Abstract: The study of the filamentous fungus Rhizopus is of significant value because of the organism's industrial importance, clinical detriment, and agricultural problems. Yet, research has yielded very few advances that allow site directed integration of DNA used for transformation. This is because plasmids tend to replicate in high molecular weight concatenated structures and rarely integrate into the chromosome of Mucorales fungi. Even if plasmids are linearized with restriction endonucleases prior to transformation, they mostly recircularize and replicate autonomously. This work examined methods that might interfere with this religation process, select against plasmids that had recircularized, and encourage strand invasion that would hopefully lead to the integration of the plasmid. In vitro methods were used to determine if the structure of the double strand break had any effect on the ability to rejoin plasmid ends. There was very little difference with cell free end-joining between linearized plasmids with 5' overhangs, 3' overhangs, or blunt ends. Additionally, dephosphorylation seemed to make little difference. Transformation of plasmids prepared in the same manner confirmed that they were easily religated, with almost all prototrophic isolates having autonomous plasmid replication. It was possible to block this religation by modifying the free DNA ends of the linearized plasmid with oligonucleotide adapters which were blocked in the 3'-hydroxyl position with a amino modifier C7 and which contained phosphorothioate nucleotides for nuclease resistance. However, gene replacement and repair of the genomic auxotrophic mutation was the predominant event with these plasmids. The most successful approach for integration was to design a plasmid, containing a truncated non-functional pyrG gene for selection. Autonomous replication of this plasmid would not support prototrophic growth, but site directed integration of the plasmid could be directed such that recombination in the chromosome would restore functionality of the pyrG gene. Transformants isolated with this selective plasmid were found to have 100% integration, with multi-copy insertion being common.