Repair of plasmid DNA used for transformation of Rhizopus oryzae by gene conversion

Abstract

Techniques for genetic manipulation of the filamentous fungus Rhizopus have been hampered due to a lack of understanding regarding the recombination and replication mechanisms that affect the fate of introduced DNA. The ability to target chromosomal integration of a plasmid has been difficult because DNA transformed into Rhizopus rarely integrates and is autonomously replicated in a high molecular weight concatenated arrangement (i.e., series or chain). Linearization of the plasmid prior to transformation at a site having homology with the genomic DNA yields the highest frequency of integration, but repair of the double-strand break by end-joining is still the predominant event. We recently attempted to circumvent replication of the plasmid by introducing frameshift mutations in pyrG, the R. oryzae orotidine-5′-monophosphate decarboxylase gene used for selection of the vector. It was hypothesized that autonomous replication of the mutated plasmids would be incapable of restoring prototrophic growth, since the genomic pyrG also contained a mutation. However, homologous integration of the plasmid results in duplication of the pyrG gene, which can create a functional copy of pyrG if both the genomic and plasmid mutations are paired on the same duplicate copy. While this event was detected in one of the isolates, it represented less than 8% of the total transformants. The majority of transformants contained plasmid replicating autonomously in a concatenated arrangement. Sequence analysis showed that prototrophic growth was restored by repairing the non-functional pyrG sequence in the plasmid, while the genomic pyrG gene was unaltered. Frequent transfer of the genomic pyrG mutation to the plasmid suggests that gene conversion is likely occurring by recombination pathways involving break-induced replication or synthesis-dependent strand annealing.