【作者】 王静；

【导师】 孟永宏；

【作者基本信息】 陕西师范大学 ， 食品科学， 2019， 硕士

【摘要】 β-胡萝卜素是一种脂溶性类胡萝卜素,广泛存在于自然界中,可应用于食品、化妆品、畜牧业等行业。解脂亚罗酵母是遗传背景清晰,可合成乙酰辅酶A和法尼基焦磷酸的产油微生物,其胞内独立存在的脂质体有利于β-胡萝卜素储存。作为菌株的发酵碳源,葡萄糖进入细胞的量受到己糖激酶的控制,葡萄糖的利用速率不仅影响菌株的生长,对β-胡萝卜素的积累及发酵时长也会产生影响,然而目前这方面的研究还未开展。解脂亚罗酵母中的HMG-CoA合成酶(3-羟基-3-甲基-戊二酰辅酶A合成酶)为甲羟戊酸途径的限速酶HMG-CoA还原酶提供催化底物。关于HMG-CoA还原酶的相关研究工作有很多,但是目前还没有针对HMG-CoA合成酶的研究,其与β-胡萝卜素合成之间的关系亦不清晰。针对以上存在的问题,本文以整合tHMG、GGS1、carRA和carB的具有β-胡萝卜素合成能力的解脂亚罗酵母Y.L为出发菌株进行研究,取得的主要研究结果如下:(1)增加Hxk及erg13的拷贝数,促进葡萄糖的利用,提高下游HMG-CoA的量,从而提高β-胡萝卜素产量。结果显示,过表达Hxk及erg13基因不影响菌株的生长。在Y.L-Hxk中,β-胡萝卜素产量提高了 1.17倍,达到4.86mg/g DCW;己糖激酶的活性提高了 3.24倍;菌株对葡萄糖的利用速率提高;胞内的ATP水平降低。在erg13拷贝数增加的菌株中,β-胡萝卜素产量在Y.L-erg13-ergl3中最高,达到19.9 mg/g DCW,比Y.L提高了 7.9倍。随着拷贝数的增加erg13的mRNA水平逐步增加;HMG-CoA的含量与转录水平不一致,在Y.L-erg13-erg13达到最高,比YL提高了 1.33倍。(2)敲除gut2,推动甘油流向β-胡萝卜素生物合成途径,增加胞内脂质体的含量为β-胡萝卜素提供更大的储存空间。结果显示,缺失gut2的菌株即YL-Δ gut2中生物量降低,β-胡萝卜素产量提高至3.13 mg/g DCW,对菌体生长的影响使得在发酵144 h才达到最高产量,比Y.L晚了 24 h。在激光共聚焦显微镜下观察到脂质体明显增大。(3)构建稳定遗传菌株,整合表达两个拷贝数的erg13并表达Hxk基因得到最终菌株YL gut2::erg13-erg13/Hxk。结果表明,在发酵过程中Y.L gut2::erg13-erg13/Hxk可以正常生长;其中β-胡萝卜素产量提高3.27倍,达到9.56 mg/gDCW;HMG-CoA的含量增加3.12倍;胞内ATP水平提高。本研究的结果为利用解脂亚罗酵母进行工业生产β-胡萝卜素奠定了理论基础,通过增加催化底物进而影响限速步骤为β-胡萝卜素代谢平衡的调节提供了新思路。更多还原

【Abstract】 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 β-carotene,which makes it a promissing strain for producing β-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 ofβ-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 β-carotene synthesis is not clear.In view of the above problems,in this paper,Y-L,which could synthesis β-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 β-carotene.The results showed that overexpression of Hxk and erg13 genes did not affect the growth of the strain.In Y.L-Hxk,β-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 β-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 β-carotene biosynthesis pathway and increasing the content of intracellular liposomes to provide more storage space forβ-carotene.The results showed that the biomass of YL-Δgut2 was decreased,and the yield of β-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 β-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 β-carotene.Finally,The increasing of the amount of intracellular liposomes expanded the storage space of β-carotene,and further increase the production of β-carotene.Shortening the fermentation time can greatly reduce the industrial production cost.It lays a theoretical foundation for the industrial production of β-carotene using Yarrowia lipolytica.It provides a new idea for the regulation of β-carotene metabolic balance by increasing the catalytic substrate,which affects the rate-limiting step.更多还原