【作者】 李敏；

【导师】 于洪巍；

【作者基本信息】 浙江大学 ， 生物化工， 2021， 硕士

【摘要】 虾青素是一种抗氧化能力较强的红色类胡萝卜素,在营养保健品、饲料以及化妆品等产业具有较高的应用价值。为了提高虾青素的生产效率以实现虾青素的工业化生产,针对实验室现有虾青素生产菌株代谢压力大、虾青素比例较低等关键问题,本文从脂滴调控和代谢途径优化两个方面,进一步提高了酿酒酵母工程菌株的虾青素产量。首先,结合多功能CRISPR(可同时进行基因表达水平上调、下调和基因敲除)基因编辑系统和虾青素的颜色效应,以实验室现有低产虾青素酿酒酵母菌株YPP9为报告菌株,构建了脂滴调控基因组合文库及相应的高通量筛选方法,并通过筛选获得了一些能提高虾青素产量的基因组合,包括参与磷脂酰胆碱合成的OPI3,与内质网压力有关的HRD1,seipin类似物FLD1,参与甾醇酯合成的ARE1,参与中性脂合成的DGA1和LRO1,其中抑制OPI3或敲除HRD1使YPP9的虾青素产量提高到原本的2.25倍和2.45倍。随后通过组合调控对这些基因之间的相互作用进行了研究,发现只有HRD1和OP13的表达调控之间存在协同关系。为了提高虾青素产量并阐明脂滴调控在虾青素生产菌株中的作用,进一步在高产虾青素的温度调控菌株Yast-TS14中对上述脂滴调控基因及其组合进行了反向代谢工程验证。在YastTS-14的基础上,单独敲除OPI3后的Yast-TS15菌株虾青素产量提高了 43%,达到了 8.99±0.62 mg/g DCW,并且虾青素的比例从27%上升到36%,单独敲除HRD1的Yast-TS16菌株虾青素产量提高了 15%,达到7.21±0.20 mg/g DCW,单独敲除FLD1的Yast-TS18菌株虾青素产量提高了 10%,达到6.89±0.29mg/g DCW,而上调ARE1的Yast-TS19菌株虾青素产量没有显著变化。在构建的双敲除菌株中,双敲除HRD1和OPI3的Yast-TS17的虾青素产量最高,在摇瓶培养中干渣含量达到8.66±0.26 mg/g DCW,略低于Yast-TS15,但是产量最高,为 48.68± 1.15 mg/L。对 Yast-TS15、Yast-TS16、Yast-TS17、Yast-TS18 菌株的激光共聚焦显微镜观察显示,敲除HRD1使得酿酒酵母形成更多的脂滴,敲除FLD1使得脂滴形态变得不稳定,敲除OPI3的菌株在SD培养基中形成巨型脂滴,而同时敲除HRD1和OPI3则使酿酒酵母中脂滴的数量和大小都有所提升。虽然脂滴调控能够有效提高虾青素的产量,但是在产量最高的Yast-TS17中虾青素的比例仍然较低(约32%),且β-胡萝卜素酮基化产物大量积累,这说明β-胡萝卜素羟化酶与β-胡萝卜素酮化酶的活性不匹配。事实上,在课题组前期构建的一系列产虾青素酿酒酵母菌株中,都存在虾青素比例低且其酮基化前体显著积累的问题。为了解决这个问题,对β-胡萝卜素羟化酶OCrtZM1和β-胡萝卜素酮化酶OBKTM29的相对表达强度进行了优化。首先对PGAL1和PGAL10启动子的强度进行了表征,然后将活性偏低的OCrtZM1置于较强的PGAL1启动子下表达,而将OBKTM29置于较弱的PGAL10启动子下表达,得到的菌株YM-27与以PGAL1控制OBKTM29而以PGAL10控制OCrtZM1的YPP-27相比虾青素干渣含量和虾青素占总类胡萝卜素的比例均有显著的提升(7.42±0.30 mg/g DCW vs.4.66±0.07 mg/g DCW,34%vs24%)。这个结果表明通过调整β-胡萝卜素酮化酶和羟化酶的表达强度实现两者之间的平衡从而促进虾青素合成的思路是有效的。进一步在β-胡萝卜素高度积累的碳源调控菌株Ycarot-02(-)中,对PGAL1-OCrtZM1和PGAL10-OBKTM29的拷贝数进行优化,构建了 OCrtZM1和OBKTM29的拷贝数比分别为1:1、2:2和2:1的菌株YM-1、YM-2和YM-4。其中,YM-4的虾青素比例达到了95%,但是由于生物量严重下降,虾青素产量仅为24.68±2.24mg/L,干渣含量也低于YM-1,只有6.7±0.23mg/g DCW。这说明高纯度虾青素的积累对底盘细胞造成了巨大的压力。因此,转而在能够更严谨地分离细胞生长和产物合成阶段的温度调控体系中进行途径优化,构建了OCrtZM1和OBKTM29拷贝数比分别为1:1、2:1、2:2 和 3:2 的菌株 YM5-TS、YM6-TS、YM7-TS、YM8-TS。与碳源调控菌株相似,当OCrtZM1和OBKTM29比例为2:1时虾青素比例最高,达到32%,主要是番茄红素积累过多,酮基化产物不多,且温度调控避免了虾青素积累对生物量的不良影响,在摇瓶发酵中虾青素产量达到了 40.77±1.13 mg/L。在此菌株基础上,引入脂滴调控策略,即敲除HRD1和OPI3,并增加一个基因拷贝的β-胡萝卜素合成的限速酶CrtYB,构建菌株YM12-TS,其虾青素产量达到了 10.02±0.65mg/g DCW和52.92±2.81 mg/L。最终在温度调控的两阶段发酵中,通过对变温时间点进行优化,最终OD600达到254.2,虾青素产量达到446.4 mg/L,虾青素比例为55%。本研究结合脂滴调控和代谢优化,构建了高产虾青素的酿酒酵母工程菌株,并通过高密度发酵获得了当前报道的酿酒酵母合成虾青素的最高产量。这项工作将为虾青素的发酵法合成提供技术支持,其中的脂滴调控策略还将为其他疏水性产物的生物合成提供借鉴。更多还原

【Abstract】 As a strong antioxidant,astaxanthin is widely applied in nutritional and healthy products,aquaculture,pharmaceutical,food and cosmetic industries.To improve the efficiency of astaxanthin biosynthesis in S.cerevisiae,a combinatorial engineering strategy composed of lipid droplet engineering and pathway optimization was applied to relieve the metabolic burden resulted from astaxanthin accumulation and meanwhile enhance the proportion of astaxanthin in total carotenoids produced.Firstly,a library of lipid droplet engineering genes was constructed based on Trifunctional CRISPR system.Meanwhile,a color-based high-throughput screening method was established using the previously constructed YPP9 strain with moderate astaxanthin yield as the reporter to avoid color saturation.As a result,several targets were screened out that could improve the astaxanthin yield of YPP9,including OPI3,HRD1,FLD1,ARE,DGA1 and LRO1.In particular,the down-regulation of OPI3 and deletion of HRD1 led to 125%and 145%increase of astaxanthin yield,respectively.Based on the screening results,combinations of every two targets with different manipulations were tested,with only the combination of HRD1 deletion and OPI3 down-regulation exhibited synergistic effect.The effectiveness and possible mechanism of lipid droplet engineering were further examined in Yast-TS14 which produced more astaxanthin than YPP9.Yast-TS15(ΔOPI3),Yast-TS16(ΔHRD1),Yast-TS18(ΔFLD1)and Yast-TS19(ARE1 overexpression)were constructed based on Yast-TS 14.The astaxanthin yield of Yast-TS 15 reached 8.99±0.62 mg/g DCW and the astaxanthin proportion in carotenoids was 36%,both were higher than those of Yast-TS 14(6.27±0.09 mg/g DCW,27%astaxanthin).The astaxanthin yields of Yast-TS 16 and Yast-TS 18 were also improved by 15%and 10%,reaching 7.21±0.20 mg/g DCW and 6.89±0.29 mg/g DCW,respectively.However,the upregulation of ARE1 did not show any difference in Yast-TS 19.Among all the double-knockout strains constructed,YastTS20(ΔHRD1ΔOP13)exhibited highest astaxanthin titer,which reached 48.68±1.15 mg/L.Subsequent NileRed staining and Laser Scanning Confocal Microscopy of YastTS15,Yast-TS16 and Yast-TS 18 showed the formation of more lipid droplets after HRD1 knockout,unstable morphology of lipid droplets after FLD1 knockout and formation of giant lipid droplet in OPI3 knockout strain when cultured in SD medium.In the double-knockout strain Yast-TS20,both the size and number of lipid droplets were increased.Although the astaxanthin yield was enhanced by lipid droplet engineering,the proportion of astaxanthin in carotenoids was still low.In Yast-TS17,the percentage of astaxanthin was only 32%,and there was significant accumulation of the ketolated products of β-carotene,namely,canthaxanthin and echinenone.These results implied imbalance of β-carotene hydroxylation and ketolation activities in biosynthesis of astaxanthin,which was actually a common phenomenon observed in all previously constructed astaxanthin-producing strains.To solve this problem,the expression levels of OCrtZMl and OBKTM29 was optimized through promoter exchange and copy number adjustment.First,the promoter strength of PGAL1 and PGAL10 was compared,showing that PGAL1 was stronger than PGAL10.Strain YM-27 was then constructed by placing OCrtZM1 under PGAL1 and OBKTM29 under PGAL10.As compared to YPP-27 which expressed OCrtZM1 under PGAL10 and OBKTM29 under PGAL1,both the astaxanthin yield and proportion were increased in YM-27(7.42±10.30 mg/g DCW vs.4.66±0.07 mg/g DCW,34%vs.24%).This result suggested reconstruction of metabolic balance by expression modulation of β-carotene hydroxylase and ketolase as an effective strategy.Subsequently,the copy numbers of PGAL1-OCrtZM1 and PGAL10-OBKTM29 were adjusted in the glucose-responsive β-carotene-accumulating strain Ycarot-02(-),generating strains YM-1,YM-2 and YM-4 with an OCrtZM1/OBKTM29 ratio of 1:1,2:2,2:1,respectively.The astaxanthin proportion of YM-4 reached as high as 95%,however,the biomass decreased significantly,and the astaxanthin titer was only 24.68±2.24 mg/L.This result implied that accumulation of high-purity astaxanthin might cause huge metabolic burden.Therefore,we turned to the temperature-responsive strains which could separate cell growth from product accumulation with more stringent control.Strains YM5-TS,YM6-TS,YM7-TS and YM8-TS were constructed by introducing 1:1,2:1,2:2,3:2 ratios of PGAL1-OCrtZMl and PGAL10-OBKTM29.Similarly,strain YM6-TS with an OCrtZM1/OBKTM29 ratio of 2:1 showed the highest astaxanthin proportion,and the adoption of temperature control avoided the damage of astaxanthin production on cell growth,leading to an astaxanthin titer of 40.77±1.13 mg/L in shakeflask cultures.By introducing the lipid droplet engineering strain into YM6-TS and overexpressing an additional copy of CrtYB which encodes one of the rate-limiting enzymes in β-carotene biosynthesis,the resulting strain YM12-TS produced astaxanthin at 10.02±0.65 mg/g DCW and 52.92±2.81 mg/L.Finally,temperature-regulated highdensity fermentation of YM13-TS was conducted,with a final biomass of OD600=254.2 and astaxanthin titer of 446.4 mg/L.In summary,by combining lipid droplet engineering and pathway balancing,an efficient astaxanthin-producing S.cerevisiae strain was constructed,leading to the highest ever reported astaxanthin production in engineered yeast.This study lays a foundation for fermentative production of astaxanthin and provides insights in solving the storage problems of hydrophobic products in cell factory design.更多还原