Deletion Of Pox4and Pox6Genes From Yarrowia Lipolytica And Its Effect On Yield Of γ-Decalactone

y-decalactone is widely used in various types of flavour, it has the good role of fixed flavour. Now yeast is the main strain employed for synthesis of y-decalactone. Yarrowia sp., Candida sp., Pichia sp.,and Sporidiobolus sp. are the main kinds of yeast employed for synthesis of y-decalactone. Yarrowia sp. has the most potential for synthesis of y-decalactone.The production process of y-decalactone employs ricinoleic acid, which is the main component of castor oil, as substrate. Through the P-oxidation ricinoleic acid carbon chain is shortened, and then generate the y-decalactone. This β-oxidation process involved pox1-pox6six genes which encoded acetyl-CoA oxidases. Acetyl-CoA oxidases are the enzymes for decomposition of y-decalactone. In this study, pox4and pox6genes were deleted from Yarrowia lipolytica to study the effect of y-decalactone production.The gene pox4and pox6disruption were mediated with a loxP-KanMX-loxP cassette centered in a PCR fragment employing80bp primer pairs in which60nucleotides fμLly match the upstream or downstream of the pox4and pox6open-reading frame. After transformed the linear disruption cassette with a Kan marker into Yarrowia lipolytica cells, selected transformants were checked by PCR for correct integration of the cassette and concurrent deletion of the genomic target sequence. After correctly integrated into the genome, the Kan marker was then efficiently removed by transforming pSH65plasmid and inducing the Cre expression. The expression of the Cre resμLted in the removal of the marker gene, leaving behind a single loxP site at the chromosomal locus. pSH65plasmid was then lost by continually incubated in YPD. At last, the deletion mutant of pox4and pox6gene were generated.It is shown from the GC resμLt that the yield of y-decalactone from the deletion mutant of pox4and pox6gene was not improved. However the byproduct of3-hydroxy-decalactone (3-OHDEC) and6-hydroxy-3-dodecenoic acid (6-OHAC) were improved.6-OHAC is the raw material for y-decalactone fermentation. By adding reductase into fermentation broth, the double bond is hydrogenated. Then two carbon atoms were removed by β-oxidation. At last y-decalactone was generated by the cyclization of carboxyl and hydroxyl.