CN112920959B - 一种提高酵母菌中l-薄荷醇产量的方法 - Google Patents

一种提高酵母菌中l-薄荷醇产量的方法 Download PDF

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CN112920959B
CN112920959B CN202110163352.9A CN202110163352A CN112920959B CN 112920959 B CN112920959 B CN 112920959B CN 202110163352 A CN202110163352 A CN 202110163352A CN 112920959 B CN112920959 B CN 112920959B
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刘龙
陈坚
吕雪芹
堵国成
李江华
刘延峰
马骏
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Abstract

本发明公开了一种提高酵母菌中L‑薄荷醇产量的方法，本发明以酿酒酵母工程菌WMT1为出发菌株，采用组成型启动子表达人凝血酶基因F2，并采用在启动子前10BP处带有凝血酶适配体的诱导型启动子分别进行如下基因表达：共表达法尼基焦磷酸合酶突变体基因ERG20WW和橙花二磷酸合酶NPPS；以及，表达截短的柠檬烯合成酶基因LIS、柠檬烯羟基化酶基因L3H和细胞色素P450还原酶基因CPR；以及，表达反式异胡椒醇脱氢酶基因IPDH、异薄荷二烯酮还原酶基因IPR和类固醇异构酶基因KSI；以及，表达长叶薄荷酮还原酶基因PGR和薄荷醇还原酶基因MMR；以及，过表达融合蛋白tLIS‑L3H基因，最终L‑薄荷醇的发酵产量达到51.84mg/L。

Description

一种提高酵母菌中L-薄荷醇产量的方法
技术领域
本发明属于代谢工程技术领域，尤其涉及一种提高酵母菌中L-薄荷醇产量的方法。
背景技术
薄荷醇属于环状单萜类化合物，其有8种同分异构体。L-薄荷醇以其特有的薄荷香气以及冷却麻醉效果成为商业上最有应用价值的薄荷醇异构体。目前L-薄荷醇的主要应用有：作为食品添加剂，加入到饮料和糖果中增加薄荷风味；作为日化品赋香剂添加至牙膏和香水中，赋予产品清凉感；因其还具有抗菌、抗炎活性，可以作为局部抑菌剂和局部止痛药应用至医学上。尽管天然薄荷醇的价格不像天然香茅醇那样具有吸引力，但薄荷醇在全球的需求量可以达到4万吨/年，其市场规模与天然香茅醇相差无几。
核酸适配体是从随机序列的寡核苷酸库中筛选和分离的短核酸序列。到目前为止，已有数千种DNA或RNA适体被鉴定为多种靶标，包括蛋白质和小分子。近年来，核酸适体已被用于构建人工生物传感器，在转录和翻译水平上调控基因表达。其中，凝血酶及其适配体已经在枯草芽孢杆菌中被证明可以在转录水平上进行基因表达的上调，在翻译水平上进行基因表达的下调。但该系统还未在枯草芽孢杆菌以外的微生物中得到验证及应用，特别是作为模式真核微生物的酿酒酵母中，限制了该系统的应用范围。专利公开号为CN112175849A中构建的重组酿酒酵母工程菌WMT9仍然存在L-薄荷醇发酵产量低的缺点。因此，如何提高L-薄荷醇的合成效率，是微生物发酵法生产L-薄荷醇亟待解决问题。
鉴于上述原因，本发明人积极加以研究创新，以期创建一种新型的应用凝血酶结合适配体调控系统的产L-薄荷醇酿酒酵母工程菌。
发明内容
为解决上述技术问题，本发明通过代谢工程技术，提供了一种应用凝血酶结合适配体调控系统来提高L-薄荷醇发酵产量的方法。本发明采用凝血酶结合适配体系统调控MVA途径与L-薄荷醇合成途径，使得L-薄荷醇的发酵产量最终提高至51.84mg/L。
本发明的第一个目的是提供一种提高酵母菌中L-薄荷醇产量的方法，所述方法是在过表达内源MVA合成途径中的甲羟戊酸焦磷酸脱羧酶基因IDI和截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因tHMG1的酵母菌宿主中，采用组成型启动子表达人凝血酶基因F2，并采用在启动子前10BP处带有凝血酶适配体的诱导型启动子分别进行如下基因表达：共表达法尼基焦磷酸合酶突变体基因ERG20WW和橙花二磷酸合酶NPPS；以及，表达截短的柠檬烯合成酶基因LIS、柠檬烯羟基化酶基因L3H和细胞色素P450还原酶基因CPR；以及，表达反式异胡椒醇脱氢酶基因IPDH、异薄荷二烯酮还原酶基因IPR和类固醇异构酶基因KSI；以及，表达长叶薄荷酮还原酶基因PGR和薄荷醇还原酶基因MMR；以及，过表达融合蛋白tLIS-L3H基因。
进一步地，所述的甲羟戊酸焦磷酸脱羧酶的NCBI编号是NP_015208.1，所述的3-羟基-3-甲基戊二酰辅酶A还原酶的NCBI编号是NP_013636.1；所述的截短的3-羟基-3-甲基戊二酰辅酶A还原酶是将3-羟基-3-甲基戊二酰辅酶A还原酶前530个氨基酸序列删除得到。
进一步地，所述的人凝血酶的氨基酸序列如SEQ ID NO.1所示。
进一步地，所述的在启动子前10BP处带有凝血酶适配体的诱导型启动子的核苷酸序列如SEQ ID NO.2所示。
进一步地，所述的法尼基焦磷酸合酶突变体的氨基酸序列如SEQ ID NO.3所示，所述的橙花二磷酸合酶的氨基酸序列如SEQ ID NO.4所示。
进一步地，所述的柠檬烯合成酶的NCBI编号是AAC37366.1，柠檬烯羟基化酶的NCBI编号是AAD44151.1，细胞色素P450还原酶的NCBI编号是ABB88839.2，反式异胡椒醇脱氢酶的NCBI编号是AAU20370.1，异薄荷二烯酮还原酶的NCBI编号是AAQ75422.1，类固醇异构酶的NCBI编号是AFY18860.1，长叶薄荷酮还原酶的NCBI编号是AAQ75423.1，薄荷醇还原酶的NCBI编号是AAQ55960.1；所述的截短的柠檬烯合成酶是将柠檬烯合成酶前56个氨基酸序列删除得到。
进一步地，所述的酵母菌宿主为酿酒酵母、异常汉逊氏酵母、粟酒裂殖酵母、黏红酵母、热带假丝酵母、产朊假丝酵母、解脂假丝酵母或巴斯德毕赤酵母。
本发明的第二个目的是提供一种酵母菌工程菌，所述的酵母菌工程菌是在过表达甲羟戊酸焦磷酸脱羧酶基因IDI和截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因tHMG1的酵母菌宿主中，采用组成型启动子表达人凝血酶基因F2，并采用在启动子前10BP处带有凝血酶适配体的诱导型启动子分别进行如下基因表达：共表达法尼基焦磷酸合酶突变体基因ERG20WW和橙花二磷酸合酶NPPS；以及，表达截短的柠檬烯合成酶基因LIS、柠檬烯羟基化酶基因L3H和细胞色素P450还原酶基因CPR；以及，表达反式异胡椒醇脱氢酶基因IPDH、异薄荷二烯酮还原酶基因IPR和类固醇异构酶基因KSI；以及，表达长叶薄荷酮还原酶基因PGR和薄荷醇还原酶基因MMR；以及，过表达融合蛋白tLIS-L3H基因。
本发明的第三个目的是提供所述的酵母菌工程菌在发酵生产L-薄荷醇中的应用。
进一步地，所述的应用是将所述的酵母菌工程菌的种子液接种至发酵培养基中，在25～35℃，150～300rpm条件下培养，培养至15～20h时，添加5～15％半乳糖水溶液，至发酵结束，提取发酵液中的L-薄荷醇。
借由上述方案，本发明至少具有以下优点：
本发明以酿酒酵母工程菌WMT1为出发菌株，采用组成型启动子表达人凝血酶基因F2，构建酿酒酵母工程菌WMT10。以酿酒酵母工程菌WMT10为出发菌株，采用在启动子前10BP处带有凝血酶适配体的诱导型启动子共表达法尼基焦磷酸合酶突变体基因ERG20WW和橙花二磷酸合酶NPPS，构建酿酒酵母工程菌WMT11。以酿酒酵母工程菌WMT11为出发菌株，采用在启动子前10BP处带有凝血酶适配体的诱导型启动子表达截短的柠檬烯合成酶基因LIS、柠檬烯羟基化酶基因L3H和细胞色素P450还原酶基因CPR，构建酿酒酵母工程菌WMT12。以酿酒酵母工程菌WMT12为出发菌株，采用在启动子前10BP处带有凝血酶适配体的诱导型启动子表达反式异胡椒醇脱氢酶基因IPDH、异薄荷二烯酮还原酶基因IPR和类固醇异构酶基因KSI，构建酿酒酵母工程菌WMT13。以酿酒酵母工程菌WMT13为出发菌株，采用在启动子前10BP处带有凝血酶适配体的诱导型启动子表达长叶薄荷酮还原酶基因PGR和薄荷醇还原酶基因MMR，构建酿酒酵母工程菌WMT14，发酵产量达到33.63mg/L。以酿酒酵母工程菌WMT14为出发菌株，利用在启动子前10BP处带有凝血酶适配体的诱导型启动子过表达融合蛋白tLIS-L3H基因，提高L-薄荷醇代谢途径的催化效率，构建得到酵母酿酒酵母工程菌WMT15，L-薄荷醇的发酵产量达到51.84mg/L。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细说明如后。
具体实施方式
气相-质谱联用检测L-薄荷醇：
检测方法：使用带有FID检测器和Chirasil-DEX-CB色谱柱(Agilent；25m，0.32mm，0.25μm)的Agilent Technologies 7890A仪器进行检测。进样器温度为180℃，载气为氦气，流速为1mL/min，压力为5.8psi。程序从70℃开始，以20℃/min的速率将温度升至150℃，保持3分钟，然后以2℃/min的速度将温度升至190℃，保持3分钟。
实施例1：重构酿酒酵母工程菌WMT10
(a)人工合成基因片段PGPD-F2-TADH1，以酿酒酵母S288C基因组为模版，采用引物1309a-U-F、1309a-U-R扩增得到基因1309a-U，采用引物1309a-D-F、1309a-D-R扩增得到基因1309a-D。
(b)将步骤(a)中获得的三个片段PGPD-F2-TADH1、1309a-U、和1309a-D基因片段进行重叠延伸PCR，PCR条件：98℃，5min预变性，然后98℃，变性10s，55℃，退火5s，72℃，延伸2min，总共30个循环，切胶回收大小正确的片段，得到融合基因片段1309a-U-PGPD-F2-TADH1-1309a-D基因片段。
(c)以pML104质粒为模板，采用引物1309a-F，1309a-R扩增后得到片段1309a，热击转入大肠杆菌JM109感受态后，采用引物1309a-YZ-F与L4440菌落PCR验证，并进行测序，测序正确的单菌落接入2mL LB培养基内培养16h后利用质粒提取试剂盒提取得到质粒pML104-1309a。
(d)制备酿酒酵母工程菌WMT1感受态，并将构建好的融合基因片段1309a-U-PGPD-F2-TADH1-1309a-D与质粒pML104-1309a共同转入WMT1感受态中，待SD Ura筛选固体平板上长出单菌落后采用引物1309a-U-F，1309a-D-R进行菌落PCR验证。
(e)将步骤(d)中菌落PCR验证正确的单菌落接入YPD液体培养基内培养16h，之后划线于含有5-FOA的YPD固体平板上，30℃培养3d，之后将长出的单菌落分别转板于YPD固体平板与SD Ura筛选固体平板上对比验证，YPD平板上正常生长但SD Ura筛选平板无法生长的单菌落即为正确的基因工程菌，并命名为WMT10。
引物序列：
1309a-U-F cagaaaaacagatgtgcccaaatc
1309a-D-F tattctttgaaatggcgagtattgataatgaaggtctactactccatcgtaaagc
1309a-D-R tgaggaatttacaataaggtggttcct
1309a-U-R gaaacttactatgacgcagtttaggatcgagcgacctcatgctatacctgag
1309a-YZ-F cctgtggtgactacgtatcc
1309a-F ggatacgtagtcaccacagggatcatttatctttcactgcggagaag
1309a-R cctgtggtgactacgtatccgttttagagctagaaatagcaagttaaaataagg
L4440 agcgagtcagtgagcgag
实施例2：重构酿酒酵母工程菌WMT11
(a)人工合成基因片段TCYC1-ERG20WW-P10mG-P10mG-NPPS-TADH1，以酿酒酵母S288C基因组为模版，采用引物911b-U-F、911b-U-R扩增得到基因911b-U，采用引物911bD-F、911b-D-R扩增得到基因911b-D。
(b)将步骤(a)中获得的三个片段TCYC1-ERG20WW-P10mG-P10mG-NPPS-TADH1、911b-U、和911b-D基因片段进行重叠延伸PCR，PCR条件：98℃，5min预变性，然后98℃，变性10s，55℃，退火5s，72℃，延伸2min，总共30个循环，切胶回收大小正确的片段，得到融合基因片段911b-U-TCYC1-ERG20WW-P10mG-P10mG-NPPS-TADH1-911b-D基因片段。
(c)以pML104质粒为模板，采用引物911b-F，911b-R扩增后得到片段911b，热击转入大肠杆菌JM109感受态后，采用引物911b-YZ-F与L4440菌落PCR验证，并进行测序，测序正确的单菌落接入2mL LB培养基内培养16h后利用质粒提取试剂盒提取得到质粒pML104-911b。
(d)制备酿酒酵母工程菌WMT10感受态，并将构建好的融合基因片段911b-U-TCYC1-ERG20WW-P10mG-P10mG-NPPS-TADH1-911b-D与质粒pML104-911b共同转入WMT10感受态中，待SDUra筛选固体平板上长出单菌落后采用引物911b-U-F，911b-D-R进行菌落PCR验证。
(e)将步骤(d)中菌落PCR验证正确的单菌落接入YPD液体培养基内培养16h，之后划线于含有5-FOA的YPD固体平板上，30℃培养3d，之后将长出的单菌落分别转板于YPD固体平板与SD Ura筛选固体平板上对比验证，YPD平板上正常生长但SD Ura筛选平板无法生长的单菌落即为正确的基因工程菌，并命名为WMT11。
引物序列：
911b-U-F cactcatcaaacagccttaacaat
911b-D-F aggtatagcatgaggtcgctctggagaagtaaatgaaaaatgaaatagcatac
911b-D-R aatatgggtacgagagaaactctcg
911b-U-R aaggctttaatttgcggccttatatatacatttatatttatgcccattcaacatccg
911b-YZ-F cctgtggtgactacgtatcc
911b-F gggaaacaagacaatattacgatcatttatctttcactgcggaga
911b-R gccttattttaacttgctatttctagctctaaaacgggaaacaagacaatattac
L4440 agcgagtcagtgagcgag
实施例3：重构酿酒酵母工程菌WMT12
(a)人工合成基因片段TCYC1-tLIS-P10mG-P10mG-L3H-TADH1-TTDH3-CPR-P10mG，以酿酒酵母S288C基因组为模版，采用引物YPRC15-U-F、YPRC15-U-R扩增得到基因YPRC15-U，采用引物YPRC15-D-F、YPRC15-D-R扩增得到基因YPRC15-D。
(b)将步骤(a)中获得的三个片段TCYC1-tLIS-P10mG-P10mG-L3H-TADH1-TTDH3-CPR-P10mG、YPRC15-U、和YPRC15-D基因片段进行重叠延伸PCR，PCR条件：98℃，5min预变性，然后98℃，变性10s，55℃，退火5s，72℃，延伸2min，总共30个循环，切胶回收大小正确的片段，得到融合基因片段YPRC15-U-TCYC1-tLIS-P10mG-P10mG-L3H-TADH1-TTDH3-CPR-P10mG-YPRC15-D基因片段。
(c)以pML104质粒为模板，采用引物YPRC15-F，YPRC15-R扩增后得到片段YPRC15，热击转入大肠杆菌JM109感受态后，采用引物YPRC15-YZ-F与L4440菌落PCR验证，并进行测序，测序正确的单菌落接入2mL LB培养基内培养16h后利用质粒提取试剂盒提取得到质粒pML104-YPRC15。
(d)制备酿酒酵母工程菌WMT11感受态，并将构建好的融合基因片段YPRC15-U-TCYC1-tLIS-P10mG-P10mG-L3H-TADH1-TTDH3-CPR-P10mG-YPRC15-D与质粒pML104-YPRC15共同转入WMT11感受态中，待SD Ura筛选固体平板上长出单菌落后采用引物YPRC15-U-F，YPRC15-D-R进行菌落PCR验证。
(e)将步骤(d)中菌落PCR验证正确的单菌落接入YPD液体培养基内培养16h，之后划线于含有5-FOA的YPD固体平板上，30℃培养3d，之后将长出的单菌落分别转板于YPD固体平板与SD Ura筛选固体平板上对比验证，YPD平板上正常生长但SD Ura筛选平板无法生长的单菌落即为正确的基因工程菌，并命名为WMT12。
引物序列：
YPRC15-U-F gtagaaataccgattcaattttgggga
YPRC15-D-F cgcccgctcggcggcttctaatccgtctttctttgtcttgacgtgatttgga
YPRC15-D-R ctgcatactcactatcgtaaactgtc
YPRC15-U-R gcatatatttaagtttgtttgcgaaacccggccgcaaattaaagccttcgagcgt
YPRC15-YZ-F ctaaaactatgctctgttgttcggatt
YPRC15-F tatgctctgttgttcggattgatcatttatctttcactgcggaga
YPRC15-R ccttattttaacttgctatttctagctctaaaactatgctctgttgttcggatt
L4440 agcgagtcagtgagcgag
实施例4：重构酿酒酵母工程菌WMT13
(a)人工合成基因片段TCYC1-IPDH-P10mG-P10mG-IPR-TADH1-TTDH3-KSI-P10mG，以酿酒酵母S288C基因组为模版，采用引物208a-U-F、208a-U-R扩增得到基因208a-U，采用引物208a-D-F、208a-D-R扩增得到基因208a-D。
(b)将步骤(a)中获得的三个片段TCYC1-IPDH-P10mG-P10mG-IPR-TADH1-TTDH3-KSI-P10mG、208a-U、和208a-D基因片段进行重叠延伸PCR，PCR条件：98℃，5min预变性，然后98℃，变性10s，55℃，退火5s，72℃，延伸2min，总共30个循环，切胶回收大小正确的片段，得到融合基因片段208a-U-TCYC1-IPDH-P10mG-P10mG-IPR-TADH1-TTDH3-KSI-P10mG-208a-D基因片段。
(c)以pML104质粒为模板，采用引物208a-F，208a-R扩增后得到片段208a，热击转入大肠杆菌JM109感受态后，采用引物208a-YZ-F与L4440菌落PCR验证，并进行测序，测序正确的单菌落接入2mL LB培养基内培养16h后利用质粒提取试剂盒提取得到质粒pML104-208a。
(d)制备酿酒酵母工程菌WMT12感受态，并将构建好的融合基因片段208a-U-TCYC1-IPDH-P10mG-P10mG-IPR-TADH1-TTDH3-KSI-P10mG-208a-D与质粒pML104-208a共同转入WMT12感受态中，待SD Ura筛选固体平板上长出单菌落后采用引物208a-U-F，208a-D-R进行菌落PCR验证。
(e)将步骤(d)中菌落PCR验证正确的单菌落接入YPD液体培养基内培养16h，之后划线于含有5-FOA的YPD固体平板上，30℃培养3d，之后将长出的单菌落分别转板于YPD固体平板与SD Ura筛选固体平板上对比验证，YPD平板上正常生长但SD Ura筛选平板无法生长的单菌落即为正确的基因工程菌，并命名为WMT13。
引物序列：
208a-U-F ctgttaccaaatactcctcctctactc
208a-D-F cgcccgctcggcggcttctaatccgacggcaatgacaaaaactgaatatc
208a-D-R ttactttacatgttatcggaggcctg
208a-U-R tctctgatttttttttcaatgagtgcaggccgcaaattaaagccttc
208a-YZ-F aaaacagatcttttgtttagcggac
208a-F agatcttttgtttagcggacgatcatttatctttcactgcggaga
208a-R ccttattttaacttgctatttctagctctaaaacagatcttttgtttagcggac
L4440 agcgagtcagtgagcgag
实施例5：重构酿酒酵母工程菌WMT14
(a)人工合成基因片段TCYC1-PGR-P10mG-P10mG-MMR-TADH1，以酿酒酵母S288C基因组为模版，采用引物1622b-U-F、1622b-U-R扩增得到基因1622b-U，采用引物1622b-D-F、1622b-D-R扩增得到基因1622b-D。
(b)将步骤(a)中获得的三个片段TCYC1-PGR-P10mG-P10mG-MMR-TADH1、1622b-U、和1622b-D基因片段进行重叠延伸PCR，PCR条件：98℃，5min预变性，然后98℃，变性10s，55℃，退火5s，72℃，延伸2min，总共30个循环，切胶回收大小正确的片段，得到融合基因片段1622b-U-TCYC1-PGR-P10mG-P10mG-MMR-TADH1-1622b-D基因片段。
(c)以pML104质粒为模板，采用引物1622b-F，1622b-R扩增后得到片段1622b，热击转入大肠杆菌JM109感受态后，采用引物1622b-YZ-F与L4440菌落PCR验证，并进行测序，测序正确的单菌落接入2mL LB培养基内培养16h后利用质粒提取试剂盒提取得到质粒pML104-1622b。
(d)制备酿酒酵母工程菌WMT13感受态，并将构建好的融合基因片段1622b-U-TCYC1-PGR-P10mG-P10mG-MMR-TADH1-1622b-D与质粒pML104-1622b共同转入WMT13感受态中，待SD Ura筛选固体平板上长出单菌落后采用引物1622b-U-F，1622b-D-R进行菌落PCR验证。
(e)将步骤(d)中菌落PCR验证正确的单菌落接入YPD液体培养基内培养16h，之后划线于含有5-FOA的YPD固体平板上，30℃培养3d，之后将长出的单菌落分别转板于YPD固体平板与SD Ura筛选固体平板上对比验证，YPD平板上正常生长但SD Ura筛选平板无法生长的单菌落即为正确的基因工程菌，并命名为WMT14。
引物序列：
1622b-U-F atatgtctctcctgcatcactaaatgt
1622b-D-F tcaggtatagcatgaggtcgctcctaattccgatgatggttgttatgacg
1622b-D-R actaccatacccctttcgagaaaata
1622b-U-R aaggtaacagcaaaaacaaatagttcacggccgcaaattaaagccttcgagc
1622b-YZ-F actttgcgatgtggtggcttta
1622b-F tttgcgatgtggtggctttagatcatttatctttcactgcggaga
1622b-R ccttattttaacttgctatttctagctctaaaactttgcgatgtggtggcttta
L4440 agcgagtcagtgagcgag
实施例6：重构酿酒酵母工程菌WMT15
(a)人工合成基因片段P10mG-tLIS-L3H-TADH1，以酿酒酵母S288C基因组为模版，采用引物106a-U-F、106a-U-R扩增得到基因106a-U，采用引物106a-D-F、106a-D-R扩增得到基因106a-D。
(b)将步骤(a)中获得的三个片段P10mG-tLIS-L3H-TADH1、106a-U、和106a-D基因片段进行重叠延伸PCR，PCR条件：98℃，5min预变性，然后98℃，变性10s，55℃，退火5s，72℃，延伸2min，总共30个循环，切胶回收大小正确的片段，得到融合基因片段106a-U-P10mG-tLIS-L3H-TADH1-106a-D基因片段。
(c)以pML104质粒为模板，采用引物106a-F，106a-R扩增后得到片段106a，热击转入大肠杆菌JM109感受态后，采用引物106a-YZ-F与L4440菌落PCR验证，并进行测序，测序正确的单菌落接入2mL LB培养基内培养16h后利用质粒提取试剂盒提取得到质粒pML104-106a。
(d)制备酿酒酵母工程菌WMT14感受态，并将构建好的融合基因片段106a-U-P10mG-tLIS-L3H-TADH1-106a-D与质粒pML104-106a共同转入WMT14感受态中，待SD Ura筛选固体平板上长出单菌落后采用引物106a-U-F，106a-D-R进行菌落PCR验证。
(e)将步骤(d)中菌落PCR验证正确的单菌落接入YPD液体培养基内培养16h，之后划线于含有5-FOA的YPD固体平板上，30℃培养3d，之后将长出的单菌落分别转板于YPD固体平板与SD Ura筛选固体平板上对比验证，YPD平板上正常生长但SD Ura筛选平板无法生长的单菌落即为正确的基因工程菌，并命名为WMT15。
引物序列：
106a-U-F cacttccatatttggaccaaatgaaaa
106a-D-F tcgctcttattgaccacacctctaccgggctaatttttccggcagaaagattttc
106a-D-R attcagaaaaaaaagccaacgaatatcg
106a-U-R aaaaaaaaaaaaaaaaaaaaatagccgccatgacggattagaagccgccgag
106a-YZ-F gcgctaccctgaccgtat
106a-F gggcgctaccctgaccgtatgatcatttatctttcactgcggagaag
106a-R atacggtcagggtagcgcccgttttagagctagaaatagcaagttaaaataagg
L4440 agcgagtcagtgagcgag。
实施例7：重构酿酒酵母工程菌的发酵培养
在2ml YPD培养基中接种固体YPD平板上的酿酒酵母工程菌单菌落，30℃，220rpm培养16-20h后，按接种量1％接入含有25mL YPD液体培养基的250mL圆底摇瓶内，30℃，220rpm培养96h。发酵至16h时，加入10％半乳糖水溶液。发酵结束后，取600μL菌液加入0.5mm玻璃珠与等体积乙酸乙酯破碎，之后高速离心取上层乙酸乙酯层进行气相质谱联用检测。
以酿酒酵母工程菌WMT1为出发菌株，采用组成型启动子表达人凝血酶基因F2，构建酿酒酵母工程菌WMT10。以酿酒酵母工程菌WMT10为出发菌株，采用在启动子前10BP处带有凝血酶适配体的诱导型启动子共表达法尼基焦磷酸合酶突变体基因ERG20WW和橙花二磷酸合酶NPPS，构建酿酒酵母工程菌WMT11。以酿酒酵母工程菌WMT11为出发菌株，采用在启动子前10BP处带有凝血酶适配体的诱导型启动子表达截短的柠檬烯合成酶基因LIS、柠檬烯羟基化酶基因L3H和细胞色素P450还原酶基因CPR，构建酿酒酵母工程菌WMT12。以酿酒酵母工程菌WMT12为出发菌株，采用在启动子前10BP处带有凝血酶适配体的诱导型启动子表达反式异胡椒醇脱氢酶基因IPDH、异薄荷二烯酮还原酶基因IPR和类固醇异构酶基因KSI，构建酿酒酵母工程菌WMT13。以酿酒酵母工程菌WMT13为出发菌株，采用在启动子前10BP处带有凝血酶适配体的诱导型启动子表达长叶薄荷酮还原酶基因PGR和薄荷醇还原酶基因MMR，构建酿酒酵母工程菌WMT14，发酵产量达到33.63mg/L。以酿酒酵母工程菌WMT14为出发菌株，利用在启动子前10BP处带有凝血酶适配体的诱导型启动子过表达融合蛋白tLIS-L3H基因，提高L-薄荷醇代谢途径的催化效率，构建得到酵母酿酒酵母工程菌WMT15，L-薄荷醇的发酵产量达到51.84mg/L。
以上仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
序列表
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<120> 一种提高酵母菌中L-薄荷醇产量的方法
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Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Glu Glu Gly Val
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Glu Asp Ser Asp Arg Ala Ile Glu Gly Arg Thr Ala Thr Ser Glu Tyr
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ggttggtgtg gttgggctgg agctccggat tagaagccgc cgagcgggcg acagccctcc 60
gacggaagac tctcctccgt taattaactt gtaatattct aatcaagctt ataaaagagc 120
actgttgggc gtgagtggag gcgccggaaa aaagcatcga aaaaa 165
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Claims (4)

1.一种提高酵母菌中L-薄荷醇产量的方法，其特征在于，所述方法是在过表达甲羟戊酸焦磷酸脱羧酶基因IDI和截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因tHMG1的酿酒酵母菌宿主中，采用组成型启动子表达人凝血酶基因F2，并采用在启动子前10BP处带有凝血酶适配体的诱导型启动子分别进行如下基因表达：共表达法尼基焦磷酸合酶突变体基因ERG20WW和橙花二磷酸合酶NPPS；以及，表达截短的柠檬烯合成酶基因LIS、柠檬烯羟基化酶基因L3H和细胞色素P450还原酶基因CPR；以及，表达反式异胡椒醇脱氢酶基因IPDH、异薄荷二烯酮还原酶基因IPR和类固醇异构酶基因KSI；以及，表达长叶薄荷酮还原酶基因PGR和薄荷醇还原酶基因MMR；以及，过表达融合蛋白tLIS-L3H基因；
所述的甲羟戊酸焦磷酸脱羧酶的NCBI编号是NP_015208.1，所述的3-羟基-3-甲基戊二酰辅酶A还原酶的NCBI编号是NP_013636.1；所述的截短的3-羟基-3-甲基戊二酰辅酶A还原酶是将3-羟基-3-甲基戊二酰辅酶A还原酶前530个氨基酸序列删除得到；
所述的人凝血酶的氨基酸序列如SEQ ID NO.1所示；
所述的在启动子前10BP处带有凝血酶适配体的诱导型启动子的核苷酸序列如SEQ IDNO.2所示；
所述的法尼基焦磷酸合酶突变体的氨基酸序列如SEQ ID NO.3所示，所述的橙花二磷酸合酶的氨基酸序列如SEQ ID NO.4所示；
所述的柠檬烯合成酶的NCBI编号是AAC37366.1，柠檬烯羟基化酶的NCBI编号是AAD44151.1，细胞色素P450还原酶的NCBI编号是ABB88839.2，反式异胡椒醇脱氢酶的NCBI编号是AAU20370.1，异薄荷二烯酮还原酶的NCBI编号是AAQ75422.1，类固醇异构酶的NCBI编号是AFY18860.1，长叶薄荷酮还原酶的NCBI编号是AAQ75423.1，薄荷醇还原酶的NCBI编号是AAQ55960.1；所述的截短的柠檬烯合成酶是将柠檬烯合成酶前56个氨基酸序列删除得到。
2.一种酵母菌工程菌，其特征在于，所述的酵母菌工程菌是在过表达甲羟戊酸焦磷酸脱羧酶基因IDI和截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因tHMG1的酿酒酵母菌宿主中，采用组成型启动子表达人凝血酶基因F2，并采用在启动子前10BP处带有凝血酶适配体的诱导型启动子分别进行如下基因表达：共表达法尼基焦磷酸合酶突变体基因ERG20WW和橙花二磷酸合酶NPPS；以及，表达截短的柠檬烯合成酶基因LIS、柠檬烯羟基化酶基因L3H和细胞色素P450还原酶基因CPR；以及，表达反式异胡椒醇脱氢酶基因IPDH、异薄荷二烯酮还原酶基因IPR和类固醇异构酶基因KSI；以及，表达长叶薄荷酮还原酶基因PGR和薄荷醇还原酶基因MMR；以及，过表达融合蛋白tLIS-L3H基因；
所述的甲羟戊酸焦磷酸脱羧酶的NCBI编号是NP_015208.1，所述的3-羟基-3-甲基戊二酰辅酶A还原酶的NCBI编号是NP_013636.1；所述的截短的3-羟基-3-甲基戊二酰辅酶A还原酶是将3-羟基-3-甲基戊二酰辅酶A还原酶前530个氨基酸序列删除得到；
所述的人凝血酶的氨基酸序列如SEQ ID NO.1所示；
所述的在启动子前10BP处带有凝血酶适配体的诱导型启动子的核苷酸序列如SEQ IDNO.2所示；
所述的法尼基焦磷酸合酶突变体的氨基酸序列如SEQ ID NO.3所示，所述的橙花二磷酸合酶的氨基酸序列如SEQ ID NO.4所示；
所述的柠檬烯合成酶的NCBI编号是AAC37366.1，柠檬烯羟基化酶的NCBI编号是AAD44151.1，细胞色素P450还原酶的NCBI编号是ABB88839.2，反式异胡椒醇脱氢酶的NCBI编号是AAU20370.1，异薄荷二烯酮还原酶的NCBI编号是AAQ75422.1，类固醇异构酶的NCBI编号是AFY18860.1，长叶薄荷酮还原酶的NCBI编号是AAQ75423.1，薄荷醇还原酶的NCBI编号是AAQ55960.1；所述的截短的柠檬烯合成酶是将柠檬烯合成酶前56个氨基酸序列删除得到。
3.权利要求2所述的酵母菌工程菌在发酵生产L-薄荷醇中的应用。
4.根据权利要求3所述的应用，其特征在于，所述的应用是将所述的酵母菌工程菌的种子液接种至发酵培养基中，在25~35℃，150~300rpm条件下培养，培养至15~20h时，添加5~15%半乳糖水溶液，至发酵结束，提取发酵液中的L-薄荷醇。
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