| P-carotene is a kind of liposoluble carotenoid widely found in nature and is commonly used in food,cosmetics,animal husbandry and other industries.Y.lipolytica is a lipid-producing microorganism with a well-researched genetic background and can synthesize acetyl CoA and FPP.Its intracellularly independent liposome provides storage space for ?-carotene,which makes it a promissing strain for producing ?-carotene.As the fermenting carbon source of the strain,the amount of glucose entering the cell is controlled by hexokinase.The utilization rate of glucose not only affects the growth of the strain,but also affects the accumulation of?-carotene and the length of fermentation time.However,the relative research can not be found so far.In Y.lipolytica,HMGS provides a catalytic substrate for the rate-limiting enzyme HMGR of the MVA pathway.There are many research works on HMGR,but there is no research on HMGS at present,and the relationship between HMGS and ?-carotene synthesis is not clear.In view of the above problems,in this paper,Y-L,which could synthesis ?-carotene as the expression of gene of tHMG,GGS1,carRA and carB,is used as the parent strain.The main research results are as follows:(1)Increasing the copy number of Hxk and erg13 to promote the utilization of glucose and increase the amount of downstream HMG-CoA,thereby increasing the production of ?-carotene.The results showed that overexpression of Hxk and erg13 genes did not affect the growth of the strain.In Y.L-Hxk,?-carotene production increased by 1.17 times to 4.86 mg/g DCW;hexokinase activity increased by 3.24 times;strain utilization of glucose increased;intracellular ATP levels decreased.Among the strains with increased ergl3 copy number,the ?-carotene production was highest in Y.L-ergl3-ergl3,reaching 19.9 mg/g DCW,which was 7.9 times higher than Y.L.With the increase of copy number,the mRNA level of erg13 increased gradually.However,the content of HMG-CoA was inconsistent with the transcription level,and reached the highest level in Y.L-ergl3-ergl3,in which was 1.33 times higher than that of Y.L.(2)Knocking out gut2 to push glycerol to the ?-carotene biosynthesis pathway and increasing the content of intracellular liposomes to provide more storage space for?-carotene.The results showed that the biomass of YL-?gut2 was decreased,and the yield of ?-carotene was increased by 0.4 times to 3.13 mg/g DCW.The production reached the highest after 144 h of fermentation while YL is 24 hours late.The lipid droplets in the cells were significantly increased under the observation of the Confocal microscope.(3)Constructing strain Y.L-gut2::erg13-erg13 to combine the two chapters of this article.Two copies of erg13 were expressed and expressed Hxk gene to construct a stable genetic strain.The results showed that YL gut2::erg13-erg13 could grow normally and biomass during fermentation;the yield of ?-carotene increased by 3.27 times to 9.56 mg/g DCW;the content of HMG-CoA increased by 3.12 times;intracellular ATP level improve.In summary,this study used Y.L as the parent strain.Overexpression of hexokinase promoted the utilization of glucose and shortened the fermentation cycle of the strain.Overexpression of HMG-CoA synthetase provided more catalysis substrate for the rate-limiting enzyme HMGR.The increasing of the supply of precursor substances promoted the production of ?-carotene.Finally,The increasing of the amount of intracellular liposomes expanded the storage space of ?-carotene,and further increase the production of ?-carotene.Shortening the fermentation time can greatly reduce the industrial production cost.It lays a theoretical foundation for the industrial production of ?-carotene using Yarrowia lipolytica.It provides a new idea for the regulation of ?-carotene metabolic balance by increasing the catalytic substrate,which affects the rate-limiting step. |
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