Construction Of ?-carotene Yarrowia Lipolytic Yeast Engineering Bacteria

Yarrowia lipolytica MYA2613 was used as a host to efficiently produce ?-carotene with expression of exogenetic genes carB and carRP from Mucor circinelloides.This article is amed at raising ?-carotene production of Yarrowia lipolytica,by reg?Lating the pathway of anabolism in engineering strain.This paper mainly consists of the following work:1.For purpose of optimizing the expression level of GGS1,carB and carRP.we trial two sets of promoters which control three genes of ?-carotene biosynthetic pathway,TGE,TEF1p-GGS1-xpr2t-GPD1p-carB-mig1t-EXP1p-carRP-lip2 t and YGF,YAT1p-GGS1-xpr2t-GPD1p-carB-mig1t-FBAin1p-carRP-lip2 t.Strains carrying each of these two pathways were investigated thro?gh fermentation.Res?Lts indicated that TGE was a better combination of promoters to express ?-carotene pathway.The production of ?-carotene in TGE strains was 4.1-fold of that in YGF strains.2.Attempet to over express the gene DD253524 in Yarrowia lipolytica,which derived from Schizochytri?m chromosome.DD253524 co?Ld encode ?-carotene synthase,this enzyme contain functional regions of phytoene synthase ?phytoene desaturase?lycopene ?-cyclase.After the coexpression of DD253524 and GGS1 in Yarrowia lipolytica,we picked the positive transformants turing yellow to primary fermentation,?-carotene production of engineering strain is about 3~10?g/gDCW.3.We imagine two scheme about increasing the supply of Acetyl-CoA in engineering strain to enhancing ?-carotene production.One scheme is ethanol fed fermentation.Yarrowia lipolytica cell have a metabolic pathway:ethanol-acetaldehydeAcetic acid-Acetyl-CoA.,so addition of ethanol can increase Acetyl-CoA production.The other scheme is heterologous expressing gene ACL derived from house mouse,gene ACL encode citrate lyase.Citrate lyase can strengthen citric acid recycling and promote Acetyl-CoA generation.After investigated thro?gh fermentation,we find increasing the supply of Acetyl-CoA can't effectively influent ?-carotene production.4.Different sex Yarrowia lipolytica can mate each other.Early reports indicate the growth rate and size of diploid yeast cell is better than haploid yeast cell.We constructe two plasmid:gene matB insert plassmid and gene mat A Knockout plasmid.We plan insert gene matB into pox6 locus and then knock out gene matA.Experimental res?Lts indicate wild strain can mate each other and generate stable diploid yeast cell,but engineering strain can't mate each other successf?Lly.Besides,we found ?-carotene production of mateB strain is distinctly higher than mat A strain.