Study On Production Of ?-carotene By Engineering Of Yarrowia Lipolytica

?-Carotene is a carotenoid with anti-oxidation,anti-cancer and immune function,which is widely used in food additives,nutritional supplements,cosmetics,and other industries.Combined with the construction of biological components,foreign gene expression,and metabolic pathway regulation of metabolic engineering,the ?-carotene biosynthetic pathway was expressed while the isoprene pathway was optimized in Y.lipolytica.In addition,astaxanthin biosynthetic pathway was also introduced into Y.lipolytica for preliminary reasearch.?-Carotene biosynthesis genes from Blakeslea trispora or Mucor circinelloides were expressed in Y.lipolytica respectively.The ?-carotene production of engineering strains(YH202)haboring carRA and carB from B.trispora was 4.35 mg/L while that of the strains(YM202)having carRP and carB(M)from M.circinelloides was 3.90 mg/L,indicating that expression level of ?-carotene biosynthetic genes from B.trispora was better than that from M.circinelloides in Y.lipolytica.To increase the production of ?-carotene,it is necessary to enhance the supply of precursor.HMG-CoA reductase can catalyze HMG-CoA to mevalonate,which is an important rate-limiting step for mevalonate pathway.Overexpression of the catalytic domain of HMG-CoA reductase gene(HMGcata)enhanced the catalytic activity by approximately 65% in Y.lipolytica,simultaneously resulted in ?-carotene yield from 4.35 mg/L to 6.95 mg/L.Farnesyl diphosphate(FPP)branch is regulatory site in the ergosterol pathway where most FPP were synthesized to squalene catalyzed by aqualene synthase(encoded by ERG9).The MET3 promoter(sulphate adenyltransferase sulphurlyase gene promoter,ATP sulphurlyase gene promoter)or CTR3 promoter(copper-regulated promoter)from Saccharomyces cerevisiae was used to substitute with ERG9 endogenous promoter to reduce FPP towards to sterols,however the results were unsatisfactory,indicating that the MET3 or CTR3 promoter derived from S.cerevisiae does not play a role in Y.lipolytica.Carotenoids are mainly stored in lipid body of Y.lipolytica,so improvement of lipid body could increase the accumulation of ?-carotene.The ?-carotene production were investigated by regulation of synthesis and degradation of lipid body in Y.lipolytica H222 strain.The strain H306 harboring one copy of ?-carotene biosyethetic genes,?-carotene-producing strain H405 deleting GUT2 gene(encoding glycerol-3-phosphate dehydrogenase isomer),?-carotene-producing strain H512 deleting POX1-6 gene(encoding acyl-CoA oxidases),and ?-carotene-producing strain H611 deleting GUT2 and POX1-6 gene were constructed to analysis their growth status and ?-carotene production.Knockout of GUT2 resulted in decrease of 9.1% in biomass and 4.6-fold increase in ?-carotene yield,deletion of POX1-6 gene reduced 15.0% in biomass and increased 5.4 times in ?-carotene yield,while deletion of GUT2 and POX1-6 led to decrease of 30.2% in biomass and 11.5-fold increase in ?-carotene yield.The results suggested that improving lipid body could increase ?-carotene production,but inhibit the growth.The URA3/5-FOA screening system was applied to repeatably integrate multiple metabolic pathway genes into different parts of the genome of Y.lipolytica.Based on this system,three copies of ?-carotene synthetic genes and two copies of HMGcata genes were successfully introduced into the ATCC20362 strain to construct the high ?-carotene-producing strain YC607 where GUT2 and POX2 were deleted.With the continuous integration of genes,?-carotene yeild of modified strains were increased gradually.The modified strain YC607 could accumulate 72.89 mg/L of ?-carotene during shake flask culture,leading to approximately 16.7-fold increase than strain YH202(4.35 mg/L)which harbors one copy of GGS,carRA and carB.The effects of different concentration of glucose and glycerol on the synthesis of ?-carotene were investigated.It was found that during shake flask fermentation using 4% glycerol as a carbon source,the ?-carotene production was up to 96.88 mg/L.Furthermore,fed-batch cultivation was conducted in a 5-L bioreactor for YC607.During fed-batch cultivation feeding with glucose,the biomass and ?-carotene yield were 97.6 g/L and 443.02 mg/L.When fed-batch cultivation feeding with glycerol,the biomass was up to 130.0 g/L,while the yield of ?-carotene reached 613.63 mg/L.The crtZ(encoding ?-Carotene hydroxylase)and crt W(encoding ?-carotene ketone)from marine bacteria Agrobacterium aurantiacum were introduced into the genome of the strain YC607 for heterologous expression.Almost all of ?-carotene was converted,while 28.48 mg/L of canthaxanthin,3.64 mg/L of zeaxanthin and 1.42 mg/L of astaxanthin were produced,indicating that expressional capacity of crtZ was weak in Y.lipolytica.It is the first time to study the synthesis of canthaxanthin,zeaxanthin,and astaxanthin in Y.lipolytica.