CN116121091A - 一种高产熊果酸或齐墩果酸的酿酒酵母工程菌株及其应用 - Google Patents
一种高产熊果酸或齐墩果酸的酿酒酵母工程菌株及其应用 Download PDFInfo
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- CN116121091A CN116121091A CN202211281300.2A CN202211281300A CN116121091A CN 116121091 A CN116121091 A CN 116121091A CN 202211281300 A CN202211281300 A CN 202211281300A CN 116121091 A CN116121091 A CN 116121091A
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Abstract
本发明公开了一种高产熊果酸或齐墩果酸的酿酒酵母工程菌株,表达长春花来源的香树脂醇合酶CrMAS和香树脂醇C‑28位氧化酶CrAO以及拟南芥来源的细胞色素‑NADPH‑还原酶AtCPR1,使得重组酿酒酵母能够合成熊果酸和齐墩果酸。进一步通过增加CrMAS的拷贝数,敲除苹果酸合酶MLS1以抑制前体乙酰辅酶A的竞争途径,使得重组酿酒酵母能够高效合成熊果酸和齐墩果酸。
Description
技术领域
本发明涉及一种高产熊果酸或齐墩果酸的酿酒酵母工程菌株及其应用,属于生物技术领域。
背景技术
熊果酸(Ursolic acid,UA)及其同分异构体齐墩果酸(Oleanolic acid,OA)属于五环三萜类化合物。熊果酸既可以游离酸形式又可以皂苷形式存在,普遍存在植物中,比如枇杷、迷迭香、白花蛇舌草、女贞、欧活血丹、蔓越等,以及苹果、梨和李子等一些水果的蜡涂层。由于具有镇静、降低血糖、抗炎、抗菌、抗溃疡、抗氧化、抗糖尿病等多种生物学效应,熊果酸被广泛用于医药和化妆品。除此之外,有广泛的研究阐释熊果酸的药用价值,研究表明熊果酸能够调控与癌症、心血管和神经损伤有关的信号通路,近期还有研究发现熊果酸在治疗COVID-19方面显示出了良好的潜力。齐墩果酸已经从多达1620种植物中分离检测到,其也时常被发现存在于植物表皮蜡层中,防止水分流失并且作为对病原虫的第一道防线。有证据表明,齐墩果酸具有抗癌、抗氧化、抗炎或抗菌作用,在保护肝脏、保护胃、降血脂和抗动脉粥样硬化方面也具有良好的活性。
目前,熊果酸和齐墩果酸主要通过植物提取获得。但效率低、成本高限制了该技术的应用。利用微生物细胞工厂生产熊果酸和齐墩果酸等高附加值的天然化合物可以解决与植物提取相关的问题。陆春哲(酿酒酵母生物合成熊果酸和齐墩果酸的研究)构建了一种合成熊果酸和齐墩果酸的酿酒酵母菌株,具体地,以酿酒酵母WTE菌株作为出发菌株,导入多功能香树脂醇合酶编码基因CrAS、鲨烯环氧酶编码基因ERG1、截短的3-羟基-3甲基戊二酰CoA还原酶编码基因tHMG1和法尼基焦磷酸合成酶编码基因ERG20,再导入香树脂醇C-28位氧化酶编码基因CYP716A12/CYP716AL1和拟南芥细胞色素-NADPH-还原酶1编码基因AtCPR1,将该工程菌株进行发酵生产,最高得到熊果酸和齐墩果酸的产量也仅为37.9mg/L和45.56mg/L。因此,仍需寻找一种更优的熊果酸和齐墩果酸合成方式。
发明内容
为解决上述问题,本发明提供了一种高产熊果酸或齐墩果酸的酿酒酵母工程菌株菌株,表达长春花来源的香树脂醇合酶CrMAS和香树脂醇C-28位氧化酶CrAO以及拟南芥来源的细胞色素-NADPH-还原酶AtCPR1,使得重组酿酒酵母能够合成熊果酸和齐墩果酸。进一步通过增加CrMAS的拷贝数,敲除苹果酸合酶MLS1以抑制前体乙酰辅酶A的竞争途径,使得重组酿酒酵母能够高效合成熊果酸和齐墩果酸。
本发明的第一个目的是提供一种高产熊果酸或齐墩果酸的酿酒酵母工程菌株,所述酿酒酵母工程菌株强化表达了香树脂醇合酶CrMAS、香树脂醇C-28位氧化酶CrAO、细胞色素-NADPH-还原酶AtCPR1和鲨烯环氧酶ERG1,敲除了苹果酸合酶MLS1。
进一步地,所述酿酒酵母工程菌株还敲除了柠檬酸合酶CIT2。
进一步地,所述酿酒酵母工程菌株通过Pgal1启动子强化表达香树脂醇合酶CrMAS和香树脂醇C-28位氧化酶CrAO,通过Pgal10启动子强化表达细胞色素-NADPH-还原酶AtCPR1,通过PTEF1启动子强化表达鲨烯环氧酶ERG1。
进一步地,所述香树脂醇合酶CrMAS的Gene ID为JN991165.1,所述香树脂醇C-28位氧化酶CrAO的Gene ID为AEX07772.1,所述细胞色素-NADPH-还原酶AtCPR1的核苷酸序列如SEQ ID NO.1所示,所述鲨烯环氧酶ERG1的Gene ID为853086,所述苹果酸合酶MLS1的Gene ID为855606。本文中所述的Gene ID均指NCBI平台。
进一步地,所述柠檬酸合酶CIT2的Gene ID为850361。
进一步地,所述的强化表达为在出发菌株基因组上增加至少一个拷贝数的香树脂醇合酶CrMAS、香树脂醇C-28位氧化酶CrAO、细胞色素-NADPH-还原酶AtCPR1和鲨烯环氧酶ERG1。
本发明在酿酒酵母基因组上强化表达了3个拷贝数的长春花来源的香树脂醇合酶CrMAS、1个拷贝数的长春花来源的香树脂醇C-28位氧化酶CrAO、1个拷贝数的拟南芥来源的细胞色素-NADPH-还原酶AtCPR1和1个拷贝数的鲨烯环氧酶ERG1,以及敲除苹果酸合酶MLS1和柠檬酸合酶CIT2抑制乙酰辅酶A的竞争途径,提高熊果酸和齐墩果酸的产量分别至约289.6mg/L和107.0mg/L。
进一步地,以强化表达了3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1、异戊烯基焦磷酸异构酶IDI、法尼基焦磷酸合酶ERG20和内质网大小调节因子的相关基因INO2、SCS22、TCB2和IST2,且敲除了ROX1基因的酿酒酵母为出发菌株。具体地,以专利CN113502235A中所述重组酿酒酵母Y6为出发菌株。
进一步地,所述重组酿酒酵母Y6以酿酒酵母BY4741为出发菌株。
进一步地,通过PGPD启动子强化表达3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1,通过PPGK1启动子强化表达内质网大小调节因子的相关基因INO2和IST2,通过PTEF1启动子强化表达异戊烯基焦磷酸异构酶IDI、法尼基焦磷酸合酶ERG20和内质网大小调节因子的相关基因TCB2,通过PGAP启动子强化表达内质网大小调节因子的相关基因SCS22。
进一步地,所述3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1的Gene ID为42650,异戊烯基焦磷酸异构酶IDI的Gene ID为855986,法尼基焦磷酸合酶ERG20的Gene ID为853272,内质网大小调节因子的相关基因INO2的Gene ID为851701,SCS22的Gene ID为852186,TCB2的Gene ID为855637,IST2的Gene ID为852382,ROX1的Gene ID为856178。
进一步地,PGPD启动子的核苷酸序列如SEQ ID NO.6所示,所述PPGK1启动子的核苷酸序列SEQ ID NO.7所示,所述PGAP启动子的核苷酸序列SEQ IDNO.8所示。
本发明的第二个目的是提供一种制备熊果酸的方法,包括采用上述酿酒酵母工程菌株发酵生产的步骤。
本发明的第三个目的是提供一种制备齐墩果酸的方法,包括采用上述酿酒酵母工程菌株发酵生产的步骤。
进一步地,所述的应用为,以大豆蛋白胨、葡萄糖、甘油、蔗糖为底物,发酵生产熊果酸和/或齐墩果酸。
进一步地,将上述酿酒酵母工程菌株活化后在种子培养基中培养得到种子液,再将种子液接种至发酵培养基中进行发酵培养制备熊果酸或齐墩果酸。
进一步地,所述种子培养基为YPD培养基。
进一步地,所述发酵培养基包含以下组分:30~60g/L大豆蛋白胨、15~35g/L蔗糖、15~35g/L葡萄糖和10-25g/L半乳糖。
进一步地,将重组酿酒酵母于28~32℃下在种子培养基中活化,得到种子液,将种子液以5-10%的接种量接入发酵培养基中,在28~32℃下发酵培养。
进一步地,将所述的酵母菌种子液在YPD培养基中,于28~32℃,220~280rpm条件下培养24-30h,得到种子液。
进一步地,将制备得到的种子液以5-10%(v/v)的接种量接入发酵培养基中,在28~32℃下发酵培养96-120h。
本发明的有益效果:
本发明提供的高产熊果酸或齐墩果酸的酿酒酵母工程菌株,通过表达长春花来源的香树脂醇合酶CrMAS和香树脂醇C-28位氧化酶CrAO以及拟南芥来源的细胞色素-NADPH-还原酶AtCPR1,使得重组酿酒酵母能够合成熊果酸和齐墩果酸。进一步通过增加CrMAS的拷贝数,敲除苹果酸合酶MLS1以抑制前体乙酰辅酶A的竞争途径,使得重组酿酒酵母能够高效合成熊果酸和齐墩果酸,产量分别达到222.4mg/L、83.2mg/L。最后,对柠檬酸合酶CIT2进行敲除,产量进一步提升,熊果酸发酵产量达到289.6mg/L,齐墩果酸发酵产量达到107.0mg/L。
具体实施方式
下面结合具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
下述实施例中所涉及的材料如下:
重组酿酒酵母Y6为出发菌株,出自专利CN113502235A。
下述实施例中所涉及的培养基如下:
SD LEU平板:YNB培养基1.7g/L,葡萄糖20g/L,L-组氨酸100mg/L,L-色氨酸100g/L,尿嘧啶100mg/L,琼脂粉15g/L。
SD Ura平板:YNB培养基1.7g/L,葡萄糖20g/L,L-亮氨酸100mg/L,L-色氨酸100g/L,L-组氨酸100g/L,琼脂粉15g/L。
YPD固体平板:10g/L酵母粉,20g/L蛋白胨,20g/L葡萄糖,15g/L琼脂粉。
下述实施例中所涉及的检测方法如下:
熊果酸和齐墩果酸产量的测定:
使用Agilent 1260进行高效液相色谱检测,色谱柱为C18 ODS(5μm,250×4.6mm,Thermo Fisher Scientific,Waltham,MA,USA)。流动相:甲醇:纯乙腈=6:4,流速0.8mL/min,柱温25℃,波长为210nm,进样体积为20μL。
实施例1:构建酿酒酵母菌株UO1
具体步骤如下:
(1)人工合成基因片段Pgal1-CrMAS-TCYC1(核苷酸序列如SEQ ID NO.2所示);
以酿酒酵母Y6基因组为模板,采用表1所述的引物序列,用引物416d-UP-F、416d-UP-R扩增得到基因片段416d-UP;
用引物416d-DOWN-F、416d-DOWN-R扩增得到基因片段416d-DOWN;
以质粒pMHyLp-LEU(核苷酸序列如SEQ ID NO.3所示)为模板,采用引物416d-loxLEU-F、416d-loxLEU-R扩增得到416d-loxLEU片段。
(2)将步骤(1)中的四个片段Pgal1-CrMAS-TCYC1、416d-UP、416d-DOWN、416d-loxLEU采用PCR进行融合PCR,跑胶得到的正确条带进行柱回收后得到融合基因片段416d-Pgal1-CrMAS-TCYC1。
(3)将步骤(2)中的融合基因片段转化入酿酒酵母Y6菌株感受态中,在SD LEU平板上于28-30℃培养2-3天,使用表1所述的引物YZ-416d-F、YZ-416d-R进行单菌落PCR验证。挑选条带正确的单菌落。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒(专利CN113502235A中核苷酸序列SEQ ID NO.3)质粒,在SD Ura平板上于28-30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-氟乳清酸的YPD平板上,于30℃培养2-3天。将长出的单菌落分别在SD Ura、SD LEU、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的重组酿酒酵母菌株,命名为酿酒酵母UO1。
表1:引物序列
416d-DOWN-F | gctcgaaggctttaatttgcggcctgctccttcactattttaacatgtggaattcttg |
416d-DOWN-R | atatccacatcaatggctaatggcaaaac |
416d-loxLEU-F | ctatatgttgataattagcgttgcctcatcaggaaacagctatgaccatgattacg |
416d-loxLEU-R | tggatatgtatatggtggtaatgccatgttaaaacgacggccagtgccaa |
416d-UP-F | ggccttttgaaaagcaagcataaaagatctaaac |
416d-UP-R | gtaatcatggtcatagctgtttcctgatgaggcaacgctaattatcaacatatagattg |
YZ-416d-F | ttgctatttgggaaccacctgttc |
YZ-416d-R | caataatctatatgctcaccaatcagatttactctgc |
实施例2:构建酿酒酵母菌株UO2
(1)人工合成基因片段Pgal1-CrAO-TTDH3-Pgal10-AtCPR1-TCYC1(核苷酸序列如SEQ IDNO.4所示);
以酿酒酵母Y6基因组为模板,用引物208a-UP-F、208a-UP-R扩增得到基因片段208a-UP;
用引物208a-DOWN-F、208a-DOWN-R扩增得到基因片段208a-DOWN;
以质粒pMHyLp-LEU为模板,采用引物208a-loxLEU-F、208a-loxLEU-R扩增得到208a-LEU片段。
(2)将步骤(1)中的四个片段Pgal1-CrAO-TTDH3-Pgal10-AtCPR1-TCYC1、208a-UP、208a-DOWN、208a-LEU采用PCR进行融合PCR,跑胶得到的正确条带进行柱回收后得到融合基因片段208a-Pgal1-CrAO-TTDH3-Pgal10-AtCPR1-TCYC1。
(3)将步骤(2)中的基因片段转化入实施例1制备得到的UO1菌株感受态中,在SDLEU平板上于28-30℃培养2-3天,使用引物YZ-208a-F、YZ-208a-R进行单菌落PCR验证。挑选条带正确的单菌落。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于28-30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-氟乳清酸的YPD平板上,于28-30℃培养2-3天。将长出的单菌落分别在SD Ura、SD LEU、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的酿酒酵母菌株UO2。
表2引物序列
208a-DOWN-F | gacgctcgaaggctttaatttgcggccgatcacgacggcaatgacaaaaact |
208a-DOWN-R | gaggcctgcacagacacttg |
208a-UP-F | gctaaacatgccgtctccgaag |
208a-UP-R | catggtcatagctgtttcctggagcactttacacagtgcaggaac |
208a-loxLEU-F | gttcctgcactgtgtaaagtgctccaggaaacagctatgaccatgattacg |
208a-loxLEU-R | gctgtatagctcatatctttccctttaaaacgacggccagtgcca |
YZ-208a-F | tggaaattgctaattctaaagctcctggtg |
YZ-208a-R | gaagatggttttcagacaaactcctacaca |
实施例3:构建酿酒酵母菌株UO3
具体步骤如下:
(1)人工合成基因片段PTEF1-ERG1-TTDH3(核苷酸序列如SEQ ID NO.5所示);
以酿酒酵母Y6基因组为模板,采用表3所述的引物序列,用引物1021b-UP-F、1021b-UP-R扩增得到基因片段1021b-UP;
用引物1021b-DOWN-F、1021b-DOWN-R扩增得到基因片段1021b-DOWN;
以质粒pMHyLp-LEU为模板,采用引物1021b-loxLEU-F、1021b-loxLEU-R扩增得到1021b-LEU片段。
(2)将步骤(1)中的片段Pgal1-CrMAS-TCYC1、PTEF1-ERG1-TTDH3、1021b-UP、1021b-DOWN、1021b-LEU采用PCR进行融合PCR,跑胶得到的正确条带进行柱回收后得到融合基因片段1021b-Pgal1-CrMAS-TCYC1-PTEF1-ERG1-TTDH3;
(3)将步骤(2)中得到的融合基因片段转化入实施例2制备得到的UO2菌株的感受态中,在SD LEU平板上于28-30℃培养2-3天,使用引物YZ-1021b-F、YZ-1021b-R进行单菌落PCR验证;挑选条带正确的单菌落。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于28-30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-氟乳清酸的YPD平板上,于28-30℃培养2-3天。将长出的单菌落分别在SD Ura、SD LEU、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的酿酒酵母菌株UO3。
表3引物序列:
1021b-DOWN-F | tgtatagctcatatctttcccttggcattatgagttaagagataatacgc |
1021b-DOWN-R | gagaaaggacttaatccgtacacaatga |
1021b-loxLEU-F | gccagtaattcgtcgcatctcccaggaaacagctatgaccatgattacgc |
1021b-loxLEU-R | aaaaaaggagtagaaacattttgaagctattaaaacgacggccagtgcca |
1021b-UP-F | ttggtaacagaagatggcagtatttcca |
1021b-UP-R | gcgtaatcatggtcatagctgtttcctgggagatgcgacgaattactggc |
YZ-1021b-F | gggtttgatgtatggtggtcaagc |
YZ-1021b-R | agagtgggaggaacaagatgct |
实施例4:构建酿酒酵母菌株UO4
具体步骤如下:
(1)以酿酒酵母Y6基因组为模板,采用表4所述的引物序列,用引物MLS1-UP-F、MLS1-UP-R扩增得到基因片段MLS1-UP;
用引物MLS1-DOWN-F、MLS1-DOWN-R扩增得到基因片段MLS1-DOWN;
以质粒pMHyLp-LEU为模板,采用引物MLS1-loxLEU-F、MLS1-loxLEU-R扩增得到INO2-LEU片段。
(2)将步骤(1)中的片段Pgal1-CrMAS-TCYC1、MLS1-UP、MLS1-DOWN、MLS1-LEU采用PCR进行融合PCR,跑胶得到的正确条带进行柱回收后得到融合基因片段MLS1-Pgal1-CrMAS-TCYC1;
(3)将步骤(2)中得到的融合基因片段转化入实施例3制备得到的UO3菌株的感受态中,在SD LEU平板上于28-30℃培养2-3天,使用引物YZ-MLS1-F、YZ-MLS1-R进行单菌落PCR验证;挑选条带正确的单菌落。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于28-30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-氟乳清酸的YPD平板上,于28-30℃培养2-3天。将长出的单菌落分别在SD Ura、SD LEU、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确敲除了MLS1的酿酒酵母菌株UO4。
表4引物序列
MLS1-DOWN-F | tcgaaggctttaatttgcggcctctcccttgccccagtgtacac |
MLS1-DOWN-R | ttaaggatggctatcaacatcctttgaagtttccatta |
MLS1-loxLEU-F | aagtagtaaaagcacataaaagaattaagaaacaggaaacagctatgaccatgattacg |
MLS1-loxLEU-R | atggatatgtatatggtggtaatgccatgttaaaacgacggccagtgcca |
MLS1-UP-F | tgtctaatgcgaaggtacttttatttttttcagattca |
MLS1-UP-R | agctgtttcctgtttcttaattcttttatgtgcttttactactttgtttagttcaaaac |
YZ-MLS1-F | gggtttgatgtatggtggtcaagc |
YZ-MLS1-R | cgaggttgtggtattgttagtaccgt |
实施例5:构建酿酒酵母菌株UO5
具体步骤如下:
(1)以酿酒酵母Y6基因组为模板,采用表4所述的引物序列,用引物CIT2-UP-F、CIT2-UP-R扩增得到基因片段CIT2-UP;
用引物CIT2-DOWN-F、CIT2-DOWN-R扩增得到基因片段CIT2-DOWN;
以质粒pMHyLp-LEU为模板,采用引物CIT2-loxLEU-F、CIT2-loxLEU-R扩增得到CIT2-LEU片段。
(2)将步骤(1)中的片段CIT2-UP、CIT2-DOWN、CIT2-LEU采用PCR进行融合PCR,跑胶得到的正确条带进行柱回收后得到融合基因片段CIT2;
(3)将步骤(2)中得到的融合基因片段转化入实施例4制备得到的UO4菌株的感受态中,在SD LEU平板上于28-30℃培养2-3天,使用引物YZ-CIT2-F、YZ-CIT2-R进行单菌落PCR验证;挑选条带正确的单菌落。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于28-30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-氟乳清酸的YPD平板上,于28-30℃培养2-3天。将长出的单菌落分别在SD Ura、SD LEU、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的敲除了MLS1和CIT2的酿酒酵母菌株UO5。
表5引物序列
CIT2-DOWN-F | tcgaaggctttaatttgcggccttggattacatcctacttttacaccc |
CIT2-DOWN-R | ttacatgacagtacagcaggataataatc |
CIT2-loxLEU-F | aataatactagtaacaagaaaacaggaaacagctatgaccatgattacgc |
CIT2-loxLEU-R | atggatatgtatatggtggtaatgccatgttaaaacgacggccagtgccaa |
CIT2-UP-F | ctgcagcggactttacaga |
CIT2-UP-R | atggtcatagctgtttcctgttttcttgttactagtattattaaaacaaaaagttttga |
YZ-CIT2-F | aaacttcttgggctatgttgggtttga |
YZ-CIT2-R | gaagtactaacgaatatccccgcag |
实施例6:UO1~UO5酿酒酵母菌株发酵制备熊果酸和齐墩果酸
具体步骤如下:
(1)制备种子液
挑取重组酿酒酵母株菌UO1~UO5于3mL YPD培养基中,28~32℃,220~280rpm条件下培养24-32h。
(2)发酵培养
按5%~10%(v/v)的接种量转接至含有20~40mL大豆蛋白胨培养基的250mL平底烧瓶中,于28~32℃,220~280rpm条件下培养96-120h。
大豆蛋白胨培养基:30~60g/L大豆蛋白胨、15~35g/L蔗糖、15~35g/L葡萄糖,10-25g/L半乳糖。
(2)产物提取
取发酵液1mL,12000rpm离心5min,弃上清,加入1mL甲醇和0.5g玻璃珠进行研磨破碎,于12000rpm离心10min后,取上层甲醇进行熊果酸和齐墩果酸产量的测定。
结果如表6所示。
表6 UO1~UO5菌株产量
菌株 | 熊果酸产量(mg/L) | 齐墩果酸产量(mg/L) |
UO1 | 0 | 0 |
UO2 | 0 | 0 |
UO3 | 2.85±0.4 | 7.54±1.2 |
UO4 | 222.4±10.0 | 83.2±10.89 |
UO5 | 289.6±22.1 | 107.0±7.8 |
本发明以专利CN113502235A中所述重组酿酒酵母Y6为出发菌株,通过表达长春花来源的香树脂醇合酶CrMAS和香树脂醇C-28位氧化酶CrAO以及拟南芥来源的细胞色素-NADPH-还原酶AtCPR1,使得重组酿酒酵母能够合成熊果酸和齐墩果酸。进一步通过增加CrMAS以及鲨烯环氧酶ERG1的拷贝数,敲除苹果酸合酶MLS1和柠檬酸合酶CIT2以抑制前体乙酰辅酶A的竞争途径,使得重组酿酒酵母能够高效合成熊果酸和齐墩果酸。最后熊果酸发酵产量达到289.6mg/L,齐墩果酸发酵产量达到107.0mg/L。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种高产熊果酸或齐墩果酸的酿酒酵母工程菌株,其特征在于:所述酿酒酵母工程菌株强化表达了香树脂醇合酶CrMAS、香树脂醇C-28位氧化酶CrAO、细胞色素-NADPH-还原酶AtCPR1和鲨烯环氧酶ERG1,敲除了苹果酸合酶MLS1。
2.根据权利要求1所述的酿酒酵母工程菌株,其特征在于:所述酿酒酵母工程菌株还敲除了柠檬酸合酶CIT2。
3.根据权利要求1所述的酿酒酵母工程菌株,其特征在于:所述酿酒酵母工程菌株通过Pgal1启动子强化表达香树脂醇合酶CrMAS和香树脂醇C-28位氧化酶CrAO,通过Pgal10启动子强化表达细胞色素-NADPH-还原酶AtCPR1,通过PTEF1启动子强化表达鲨烯环氧酶ERG1。
4.根据权利要求1所述的酿酒酵母工程菌株,其特征在于:所述的强化表达为在出发菌株基因组上增加至少一个拷贝数的香树脂醇合酶CrMAS、香树脂醇C-28位氧化酶CrAO、细胞色素-NADPH-还原酶AtCPR1和鲨烯环氧酶ERG1。
5.根据权利要求1所述的酿酒酵母工程菌株,其特征在于:所述酿酒酵母工程菌株强化表达了3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1、异戊烯基焦磷酸异构酶IDI、法尼基焦磷酸合酶ERG20和内质网大小调节因子的相关基因INO2、SCS22、TCB2和IST2,敲除了ROX1基因。
6.根据权利要求5所述的酿酒酵母菌株,其特征在于:通过PGPD启动子强化表达3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1,通过PPGK1启动子强化表达内质网大小调节因子的相关基因INO2和IST2,通过PTEF1启动子强化表达异戊烯基焦磷酸异构酶IDI、法尼基焦磷酸合酶ERG20和内质网大小调节因子的相关基因TCB2,通过PGAP启动子强化表达内质网大小调节因子的相关基因SCS22。
7.一种制备熊果酸或齐墩果酸的方法,其特征在于:包括采用权利要求1-6任一项所述的酿酒酵母工程菌株发酵生产的步骤。
8.根据权利要求7所述的方法,其特征在于:将所述酿酒酵母工程菌株活化后在种子培养基中培养得到种子液,再将种子液接种至发酵培养基中进行发酵培养制备熊果酸或齐墩果酸。
9.根据权利要求8所述的方法,其特征在于:所述种子培养基为YPD培养基。
10.根据权利要求8所述的方法,其特征在于,所述发酵培养基包含以下组分:30~60g/L大豆蛋白胨、15~35g/L蔗糖、15~35g/L葡萄糖和10-25g/L半乳糖。
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