Deletion Of Pox5Gene From Yarrowia Lipolytica And Impact On γ-decalactone Production

The γ-decalactone (GDL) is a kind of natural food flavor. The annual market for γ-decalactone has been grown in recent years. In order to construct a GDL-yield Yarrowia lipolytica (YL), we attempted to disrupt the POX5gene to block the catabolism of GDL in Yarrowia lipolytica. The haploid mutant YL-POX5was gained. It’s possible for increasing GDL in industry production and multi-gene knockout in Yarrowia lipolytica in future.In this paper, Cre/loxP system and KanMX resistance marker were used to construct the POX5gene knockout in Yarrowia lipolytica. Three pairs of amplimers and four verification primers were designed according to the sequences of POX5and plasmid pUG6. The5’end of amplimers included60nucleotides that were homologous to sites upstream or downstream of the genomic target sequence to be deleted,20nucleotides homologous to the loxP sit in the3’end. Verification primers A and D primers were located upstream and downstream of POX5respectively; primers B and C were within the KanMX. POX5gene knockout cassette, which included loxP-KanMX-loxP cassette from plasmid pUG6, was produced by PCR using the amplimers. After transformation of the linear disruption cassette with a Cre/loxP-mediated marker into the cells of Yarrowia lipolytica, disruption cassette loxP-KanMX-loxP replaced the POX5gene. Selected transformants were checked by PCR. Cre expression plasmid pSH65was then transformed into the positive cells. Plasmid pSH65carries the bler marker that confers resistance to antibiotic zeocin. Later, colony was incubated in galactose-containing medium to induce the expression of Cre recombinase that resulted in the removal of the marker gene.During PCR verification, some YL diploid mutants were found. The McClary medium was chosen to sporulate the cells. The concentration of helicase was2%to break ascus wall, desorbed haploid cells. After correct separation of YL haploid cell, plasmid pSH65was removed from the cell by streaking cells onto YPD plates. At last, the haploid mutant YL-POX5was gained.After ferment-cultivating, fermentation liquid was analyzed the production of GDL by gas chromatography. Compared with original YL, POX5knockout alone did not increase the production of GDL, but is ready to make combination knockouts with other.POX genes.