A Markerless Gene Replacement Method For Pseudomonas Mendocina NK-01 And Its Application

Pseudomonas mendocina NK-01 is a gram negative bacteria, which can use glucose as the only carbon source to synthetize Medium-chain-length polyhydroxyalkanoates (PHAMCL) and alginate oligosaccharides (AO).At present, the synthesis of PHA and alginate have been elucidated, and the whole genome of this strain has been sequenced. Analysis the genomic information, we found that there are a large number of redundant and unessential genes or genomic island in the genome of P. mendocina NK-01. This study is intended to construct a markerless gene replacement method to knockout the function gene and to research how to streamline the genome.upp is uracil phosphoribosyltransferase encoding gene, which also is widely used as a counter-selection marker in markerless gene system. The strain knockded out this gene can be resistant to 5-fluorine uracil, so as to achieve the purpose of screening the deletion mutant.In this study, using this counter-selection marker combined with suicide plasmid, we construct a markerless gene replacement method in P. mendocina NK-01.Meanwhile, base on the method algA,involed alginate synthesis,and six part of large fragment respectively 8kb,9kb,10kb,32kb,41kb,51kb (total 151kb,3% of whole genomic sequence)have been continuous knocked out.Besides, we also studied and determined those deletion mutation about the strain growth, shake flask fermentation, batch fermentation and related products. Compared with the wild type, algA gene mutation strain, P. mendocina NK-01 ΔalgA growth was similar to wild type, but the yield of PHA,0.330g/L, is 1.4 times than the wild type. However, streamlining the 151kb mutation strain P. mendocina NK-01 Δ151, growth status and product yield were decreased, the alginate and PHA yield were 0.258g/L and 0.160g/L. But the structure and physical properties of PHA did not change between the mutation strain and the wild type.