Study On Gene Knockout Of Pox3in Yarrowia Lipolytica

The gamma-decalactone is a lactone which is used to formulate peach, coconuts, cream and orange flavors. For building a high-yield strain of gamma-decalactone, we try to knockout the pox3gene to enhance the gamma-decalactone production.Two chimeric primers and two verification primers was designed according to the sequence of pox3and pUG6which obtained from SGD and NCBI. Pox3gene cassette knockout was generated by using the chimeric primers to amplify the loxP-kanMX-loxP cassette from plasmid pUG6. After transformation into the yeast cells with lost pox3gene, the60bp nucleotides which homologous to the sequence upstream and downstream of pox3gene promote the recombination events that replaced the pox3with the loxP-kanMX-loxP cassette which carrying the heterologous kanMX marker and yield kan+transformants. The correct replacement of pox3with the loxP-kanMX-loxP cassette was verified by colony PCR. Then transform the plasmid pSH65with the bler marker into the positive colony, and incubate it in the galactose-containing medium to induce the expressing of Cre recombinase to excise the kanMX marker by recombination between the two loxP sites. Remove the plasmid pSH65from the cells; at last we get a pox3deletion yeast strain. Compared with the original strain, the mutants had no significant change in growth rate. Through the fermentation analysis, we see that DECA yield of mutant strain is1.13g/L, and the DECA yield of original strain is1.16g/L. This shows that the single gene knockout had no significant effect on improving the yield.Currently in the country, the method of gene knock-out which based with Cre/loxP system mainly used in the genetic transformation of Saccharomyces cerevisiae. Therefore, this study of the genetically engineered carried out a useful exploration of Yarrowia lipolytica.