CN110066802B - 具有提高出芽短梗霉代谢产物的启动子及其应用 - Google Patents
具有提高出芽短梗霉代谢产物的启动子及其应用 Download PDFInfo
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Abstract
本发明公开了具有提高出芽短梗霉代谢产物的启动子及其应用，启动子包括内源性启动子和外源性启动子，利用标签潮霉素筛选比较启动子强弱，构建了一个在出芽短梗霉中可应用的启动子库；为利用不同表达强度的启动子调控出芽短梗霉生成聚苹果酸途径中的关键限速基因，调节更多的碳流量流向目标代谢产物具有重要意义。
Description
技术领域
本发明涉及生物技术领域，具体涉及具有提高出芽短梗霉代谢产物的启动子，还涉及启动子提高出芽短梗霉代谢产物的应用。
背景技术
出芽短梗霉(Aureobasidium pullulans)是一种类酵母真菌，可以代谢产生聚苹果酸、普鲁兰多糖、短梗霉素A、重油、酶制剂等多种代谢产物。其中聚苹果酸(Polymalicacid，PMA)是一类水溶性脂肪聚酯类化合物，由其重复结构单位单体苹果酸通过分子间酯化形成的新型聚酯化合物，具有优良的生物降解性、高度水溶性、生物相容性等优良性质，在组织工程、食品包装、生物医药材料及药物载体等方面有着巨大的潜力。
启动子是RNA聚合酶特异性识别和结合的一段DNA序列，是基因表达的重要调控元件，在代谢工程改造和途径优化中发挥重要作用。因此，筛选能在出芽短梗霉中表达，并鉴定不同启动子在出芽短梗霉中的表达活性，对调控出芽短梗霉生成聚苹果酸途径中的关键限速基因，并控制碳流量流向特定代谢产物合成途径，如聚苹果酸合成途径，具有重要意义。
发明内容
有鉴于此，本发明的目的之一在于提供一种具有提高出芽短梗霉代谢产物的启动子，通过构建的启动子系统中内源性启动子和外源性启动子，内源性启动子分别是来自于出芽短梗霉中的6-磷酸葡萄糖异构酶启动子(glucose 6-phosphate isomerasepromoter，PGI)、1,6-二磷酸醛缩酶启动子(fructose 1,6-bisphosphatealdolasepromoter，FBA)、磷酸甘油酸激酶启动子(phosphoglycerate kinase promoter，PGK)，三组启动子基因在文中依次缩写为Asp.PGI、Asp.PGK、Asp.FBA，外源性启动子来源于红冬孢酵母菌(Rhodosporidium toruloides)的6-磷酸葡萄糖异构酶启动子与1,6-二磷酸醛缩酶启动子在文中依次缩写为Rt.PGI、Rt.FBA。五组启动子基因序列分别如SEQ ID NO:1～NO:5所示。本发明目的是利用内外源不同启动子的表达强度差异，调控出芽短梗霉代谢途径中的关键限速基因，从而调节更多的碳流量流向目标代谢产物合成途径，如聚苹果酸合成途径，实现提高菌株产酸水平；本发明的目的之二在于提供所述启动子在提高出芽短梗霉代谢产物含量中的应用；本发明的目的之三在于提供含有所述启动子的重组表达载体；本发明的目的之四在于提供所述重组表达载体在提高出芽短梗霉代谢产物含量中的应用；本发明的目的之五在于提供一种提高出芽短梗霉代谢产物的方法。
为达到上述目的，本发明提供如下技术方案：
1、具有提高出芽短梗霉代谢产物的启动子，所述启动子为出芽短梗霉6-磷酸葡萄糖异构酶启动子Asp.PGI、出芽短梗霉1,6-二磷酸醛缩酶启动子Asp.FBA、出芽短梗霉磷酸甘油酸激酶启动子Asp.PGK、红冬孢酵母菌6-磷酸葡萄糖异构酶启动子Rt.PGI和红冬孢酵母菌1,6-二磷酸醛缩酶启动子Rt.FBA中的至少一种；所述出芽短梗霉6-磷酸葡萄糖异构酶启动子Asp.PGI的核苷酸序列如SEQ ID NO.1所示；所述出芽短梗霉1,6-二磷酸醛缩酶启动子Asp.FBA的核苷酸序列如SEQ ID NO.2所示；所述出芽短梗霉磷酸甘油酸激酶启动子Asp.PGK的核苷酸序列如SEQ ID NO.3所示；所述红冬孢酵母菌6-磷酸葡萄糖异构酶启动子Rt.PGI的核苷酸序列如SEQ ID NO.4所示；所述红冬孢酵母菌1,6-二磷酸醛缩酶启动子Rt.FBA的核苷酸序列如SEQ ID NO.5所示。
2、所述启动子在提高出芽短梗霉代谢产物含量中的应用。
优选的，所述代谢产物为聚苹果酸、普鲁兰多糖或短梗霉素中的至少一种。
3、含有所述启动子的重组表达载体。
优选的，所述重组表达载体为pBARGPEI质粒的SmaI与EcoRI酶切位点处连入含启动子和潮霉素编码区的片段。
优选的，所述重组表达载体由以下方法构建：以SEQ ID NO.16和SEQ ID NO.17所示序列为引物，含潮霉素基因的质粒为模板扩增获得潮霉素编码区，然后将潮霉素编码区分别与出芽短梗霉6-磷酸葡萄糖异构酶启动子Asp.PGI、出芽短梗霉1,6-二磷酸醛缩酶启动子Asp.FBA、出芽短梗霉磷酸甘油酸激酶启动子Asp.PGK、红冬孢酵母菌6-磷酸葡萄糖异构酶启动子Rt.PGI和红冬孢酵母菌1,6-二磷酸醛缩酶启动子Rt.FBA混合作为模板，分别以SEQ ID NO.19与SEQ ID NO.18、SEQ ID NO.20与SEQ ID NO.18、SEQ ID NO.21与SEQ IDNO.18、SEQ ID NO.22与SEQ ID NO.18、SEQ ID NO.23与SEQ ID NO.18作为引物扩增获得含启动子和潮霉素编码区的重叠片段，然后连入pBARGPEI质粒SamI与EcoRI酶切位点处，获得重组表达载体。
4、所述重组表达载体在提高出芽短梗霉代谢产物含量中的应用。
5、一种提高出芽短梗霉代谢产物的方法，使用至少一个启动子启动至少一个代谢途径限速酶基因在出芽短梗霉中超量表达，即获得高产代谢产物的出芽短梗霉。
优选的，所述代谢途径限速酶基因为异柠檬酸裂解酶ICL基因或/和MLS基因，所述异柠檬酸裂解酶ICL基因的核苷酸序列如SEQ ID NO.34所示；所述苹果酸合成酶MLS基因的核苷酸序列如SEQ ID NO.35所示。
更优选的，所述异柠檬酸裂解酶ICL基因由红冬孢酵母菌1,6-二磷酸醛缩酶启动子Rt.FBA启动表达；所述MLS基因由出芽短梗霉1,6-二磷酸醛缩酶启动子Asp.FBA启动表达。
本发明中过表达ICL基因和MLS基因的重组载体由以下方法制备：克隆Rt.FBA与Asp.FBA启动子，然后ICL与MLS基因，再克隆出Tcyc1与Tsup4终止子，两两相连得到重叠片段Rt.FBA-ICL-Tcyc1表达盒与Asp.FBA-MLS-Tsup4表达盒。然后将pK2-hyg质粒进行EcoRI酶切，回收线性质粒载体后，同时将重叠片段与pK2-hyg质粒的载体骨架连接，连接产物转化至DH5α感受态细胞，进行转化子筛选后即获得启动子Rt.FBA与Asp.FBA基因串联组合表达载体。
Rt.FBA启动子过表达ICL的重组载体由以下方法制备：根据出芽短梗霉CCTCCM2012223的异柠檬酸裂解酶ICL序列克隆获得如SEQ ID NO.34所示的异柠檬酸裂解酶ICL基因序列，然后以SEQ ID NO.14和SEQ ID NO.15所示序列为引物，扩增获得SEQ ID NO.5所示所示Rt.FBA启动子，然后通过PCR扩增将异柠檬酸裂解酶ICL基因与Rt.FBA启动子连接，然后连入pBARGPEI质粒的SamI与EcoRI酶切位点处，再将含有Rt.FBA启动子、异柠檬酸裂解酶ICL基因和Trpc终止子组成完整表达盒扩增出来，连入pK2-gus质粒的EcoRI酶切位点处，获得Rt.FBA启动子过表达ICL的重组载体。
本发明的有益效果在于：本发明通过筛选出芽短梗霉内源启动子和外源启动子，利用标签潮霉素筛选比较启动子强弱，构建了一个在出芽短梗霉中可应用的启动子库。利用启动子库中强启动子调控异柠檬酸裂解酶基因构建重组载体，将重组载体转化出芽短梗霉后，最优的Rt.FBA启动子株可提高聚苹果酸发酵产量40％以上，此单基因调控案例证明该发明中的启动子系统可应用于出芽短梗霉菌株代谢途径限速靶点调控，不同强度启动子的组合调控也可以大幅度提供聚苹果酸及其他代谢产物的产量。本发明筛选的启动子也可用于出芽短梗霉中其他代谢产物普鲁兰多糖、短梗霉素等的高效合成。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为pK2-promoters-hyg质粒验证图(A：pK2-Asp.PGI-hyg；B：pK2-Asp.PGK-hyg；C：pK2-Asp.FBA-hyg；D：pK2-Rt.PGI-hyg；E：pK2-Rt.FBA-hyg)。
图2为出芽短梗霉五组启动子转化子的细胞形态图(A：Rt.FBA；B：Rt.PGI；C：Asp.PGK；D：Asp.FBA；E：Rt.PGI；F：pK2-gus)。
图3为五组启动子启动潮霉素相对表达水平。
图4为优化菌株的摇瓶发酵结果图(WT表示野生型；A3、B13、C5、C7、D1、D4、D7、E5、F1、I5分别为不同转化子的发酵结果)。
具体实施方式
下面结合附图和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的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。
本发明在已公开的红冬孢酵母菌的全基因组中找到Rt.PGI、Rt.FBA两组启动子基因序列，确定引用外源启动子基因序列为SEQ ID NO.4～SEQ ID NO.5所示，并通过对公开号为CN102827778A的中国专利公开的高产聚苹果酸出芽短梗霉(Aureobasidiumpullulans)菌株CCTCC M2012223的全基因组中找到Asp.PGI、Asp.PGK、Asp.FBA三组启动子基因序列，并通过验证优选确定应用的基因序列为SEQ ID NO.1～SEQ ID NO.3所示。
通过设计引物从出芽短梗霉中成功克隆得到Asp.PGI、Asp.PGK、Asp.FBA三组启动子的基因序列，以及合成的外源的Rt.PGI、Rt.FBA启动子基因序列，设计引物分别连上潮霉素后，在pBARGPEI质粒SmaI与EcoRI酶切点处插入序列，利用pBARGPEI质粒上的Trpc终止子组成完整表达盒。再设计引物将表达盒连入pK2-gus质粒的EcoRI酶切位点处构建重组载体，通过农杆菌转化至出芽短梗霉，以潮霉素为标签筛选上述启动子强弱，最终择其中强启动子构建异柠檬酸裂解酶(Isocitrate lyases,ICL)表达盒，用上述相同方法转入出芽短梗霉，后用摇瓶发酵试验表明可有效提高聚苹果酸的发酵产量。
实施例1、扩增启动子基因
根据在出芽短梗霉菌株(Aureobasidium pullulans CCTCC M2012223)的全基因组中找到Asp.PGI、Asp.PGK、Asp.FBA三组启动子基因序列，截取基因ATG前2000bp处设计引物，反向引物与ATG完全接上，两组外源性启动子Rt.PGI、Rt.FBA从合成的pUC57载体中扩增得到，具体引物如表1所示。
表1、扩增启动子基因所用引物
然后分别以Asp.PGI-F与HYG-Asp.PGI-R，Asp.FBA-F与HYG-Asp.FBA-R，Asp.PGK-F与HYG-Asp.PGK-R为引物对，出芽短梗霉基因组DNA为模板；Rt.PGI-F与HYG-Rt.PGI-R，Rt.FBA-F与HYG-Rt.FBA-R为引物对，红冬孢酵母菌基因组为模板进行PCR扩增，PCR扩增的退火温度为55-65℃，延伸时间为2min。扩增产物得到后进行琼脂糖凝胶电泳，结果显示成功得到预期长度的DNA片段。
实施例2、pBARGPEI质粒的构建
为了构建pBARGPEI-promoters-hyg载体，按照启动子基因和潮霉素基因序列，设计相关引物，使其能两两相连得到重叠片段。随后对pBARGPEI质粒(见公开号为CN104926930A的中国专利)进行SmaI与EcoRI双酶切，最后将重叠片段插入(各片段连接部位要与连接的片段有20bp的同源序列，插入的重叠片段首尾分别要有pBARGPEI载体的SmaI和EcoRI酶切位点对应两端的同源序列)，具体相关引物如表2所示。
表2、pBARGPEI质粒的构建有关引物
| 引物名称 | 序列5’-3’ |
| HYG-F | atgaaaaagcctgaactcaccgc(SEQ ID NO.16) |
| HYG-R | ctatttctttgccctcggacg(SEQ ID NO.17) |
| Trpc-HYG-R | <u>gataagcttgatatcgaatt</u>ctatttctttgccctcggacg(SEQ ID NO.18) |
| pBAR-Asp.PGI-F | <u>tcgactctagaggatccccc</u>gggcagaaaagatagagaaaaggc(SEQ ID NO.19) |
| pBAR-Asp.FBA-F | <u>tcgactctagaggatccccc</u>gcactgtcattgcaacctactg(SEQ ID NO.20) |
| pBAR-Asp.PGK-F | <u>tcgactctagaggatccccc</u>gtgcgaagtaaaaccaaacagc(SEQ ID NO.21) |
| pBAR-Rt.PGI-F | <u>tcgactctagaggatccccc</u>tggccgtctgccatttcatcgtcg(SEQ ID NO.22) |
| pBAR-Rt.FBA-F | <u>tcgactctagaggatccccc</u>ctctgctctcgctcgctgtggcttg(SEQ ID NO.23) |
以HYG-F、HYG-R为引物对，以pk2-hyg质粒(参见农杆菌介导的出芽短梗霉遗传转化及高效筛选聚苹果酸高产菌株.涂光伟等.生物工程学报.2015.5.25)为PCR扩增模板扩增得出hyg片段，然后再分别用pBAR-Asp.PGI-F与Trpc-HYG-R、pBAR-Asp.FBA-F与Trpc-HYG-R、pBAR-Asp.PGK-F与Trpc-HYG-R、pBAR-Rt.PGI-F与Trpc-HYG-R、pBAR-Rt.FBA-F与Trpc-HYG-R为引物对，以hyg和实施例1所得五组启动子片段分别组合为模板进行PCR扩增，最终得到五组重叠片段，同时将pBARGPEI质粒用SamI与EcoRI进行酶切，回收载体骨架。同时将重叠片段与pBARGPEI质粒的载体骨架连接，连接产物转化至DH5α感受态细胞，进行转化子筛选后获得五组重组表达载体pBARGPEI-Asp.PGI-hyg、pBARGPEI-Asp.PGK-hyg、pBARGPEI-Asp.FBA-hyg、pBARGPEI-Rt.PGI-hyg、pBARGPEI-Rt.FBA-hyg，简称为pBARGPEI-promoters-hyg。
实施例3、pK2-gus质粒中潮霉素表达盒的构建及启动子强度筛选
利用pBARGPEI质粒上的Trpc终止子组成完整表达盒，分别使用pK2-Asp.PGI-F与pK2-Trpc-R、pK2-Asp.FBA-F与pK2-Trpc-R、pK2-Asp.PGK-F与pK2-Trpc-R、pK2-Rt.PGI-F与pK2-Trpc-R、pK2-Rt.FBA-F与pK2-Trpc-R五组引物对，在pBARGPEI-promoters-hyg载体中扩增出五组潮霉素完整表达盒，然后分别连入pK2-gus质粒(pK2-gus为pk2-hyg的hyg基因替换为gus基因)的EcoRI酶切位点处，最终得到pK2-Asp.PGI-hyg、pK2-Asp.PGK-hyg、pK2-Asp.FBA-hyg、pK2-Rt.PGI-hyg、pK2-Rt.FBA-hyg，简称为pK2-promoters-hyg，相关引物如表3所示。连接产物转化至DH5α感受态细胞，于含有卡那霉素的LB平板上进行筛选，挑取转化子进行目的片段PCR再验证，结果如图1。将构建好的pK2-promoters-hyg质粒提取后送公司测序无误后，即获得五组重组表达载体pK2-promoters-hyg。
表3 pK2-promoters-hyg质粒构建相关引物
| 引物名称 | 序列5’-3’ |
| pK2-Asp.PGI-F | <u>ctatgacatgattacgaatt</u>gggcagaaaagatagagaaaaggc(SEQ ID NO.24) |
| pK2-Asp.FBA-F | <u>ctatgacatgattacgaatt</u>gcactgtcattgcaacctactg(SEQ ID NO.25) |
| pK2-Asp.PGK-F | <u>ctatgacatgattacgaatt</u>gtgcgaagtaaaaccaaacagc(SEQ ID NO.26) |
| pK2-Rt.PGI-F | <u>ctatgacatgattacgaatt</u>tggccgtctgccatttcatcgtc(SEQ ID NO.27) |
| pK2-Rt.FBA-F | <u>ctatgacatgattacgaatt</u>ctctgctctcgctcgctgtggcttg(SEQ ID NO.28) |
| pK2-Trpc-R | agtggatcccccggggaattcaggtattgctgttatctga(SEQ ID NO.29) |
实施例4、农杆菌遗传转化出芽短梗霉菌株
(1)pK2-promoters-hyg转化出芽短梗霉
将上述质粒分别转入根癌农杆(A.tumefacien，AGL-1)感受态细胞中，获得含有pK2-promoters-hyg载体的农杆菌，然后将目的农杆菌转入出芽短梗霉中，最终在含有头孢他啶和潮霉素筛选压力的M100平板上长出转化子，分别再进行验证引物PCR扩增，最终得到验证成功的过表达hyg出芽短梗霉菌株(菌株形态见如附图2)。
(2)潮霉素基因表达水平检测
各菌株发酵70h后，用Rnase-free离心管收集菌体，液氮速冻后与-80℃保存，继而用Trizol+Fungal RNA Kit(Omega USA)试剂盒提取RNA。采用商业反转试剂盒和qRT-PCR试剂盒反转录得到cDNA并采用CFX connect Real-time system(BIO-RAD公司)进行荧光定量表达。以β-actin作为内参基因，采用
法计算目标基因相对内参基因的相对表达量，并以Rt.PGI作为对照得出相对表达量，五组启动子启动潮霉素基因表达水平大小依次为Asp.PGI<Rt.PGI<Asp.FBA<Rt.FBA<Asp.PGK，如下附图3，相关引物如下表4。
表4、用于qRT-PCR相应的引物
| 引物名称 | 序列5’-3’ |
| q-HYG-F | tgtcctgcgggtaaatagc(SEQ ID NO.30) |
| q-HYG-R | tgtgaacggcgggagatg(SEQ ID NO.31) |
| q-ACT-F | gaagtgcgatgtcgatgtcaga(SEQ ID NO.32) |
| q-ACT-R | ggagcaagggcggtgatt(SEQ ID NO.33) |
实施例5、启动子调控ICL活性提高聚苹果酸产量
(1)构建启动子调控异柠檬酸裂解酶(Isocitrate lyases,ICL)基因的质粒构建与转化
按照实施例1～4的方法构建启动子调控异柠檬酸裂解酶ICL的质粒，区别为潮霉素基因替换为异柠檬酸裂解酶ICL基因(操作如实施例1-4)，转化出芽短梗霉CCTCCM2012223，获得含异柠檬酸裂解酶ICL转化子，简称OE::ICL。其中异柠檬酸裂解酶ICL基因的核苷酸序列如SEQ ID NO.34所示。
(2)含异柠檬酸裂解酶ICL转化子(OE::ICL)发酵
将OE::ICL转化子接种于含葡萄糖90g/L、NH4NO3 2g/L、KH2PO4 0.1g/L、KCI 0.5g/L、ZnSO4 0.1g/L、MgSO4 0.1g/L、玉米浆0.5g/L的发酵培养基中，然后在25℃、220rpm，摇瓶发酵96小时，取样生物量与发酵上清液；同时以出芽短梗霉CCTCC M2012223在相同的条件下进行发酵作为对照。结果显示，其中由Rt.FBA启动子过表达ICL转化子生物量为19.9g/L，对照组生物量为19.1g/L，OE::ICL转化子的聚苹果酸产量最高可达45.5g/L，比对照株提高了42.6％，糖酸转化率最高达到0.61g/g，较出芽短梗霉提高了48.9％。
看出用Rt.FBA启动子过表达ICL的出芽短梗霉菌株能够有效提高聚苹果酸的产量。
实施例5、启动子组合调控MLS和ICL活性提高聚苹果酸产量
(1)质粒构建与转化：将Asp.FBA启动子与苹果酸合成酶(Malate synthase，MLS)基因连接，Rt.FBA启动子与ICL连接，构建基因串联组合表达载体，具体操作如下所示，引物序列如表5所示：使用引物对pk2-Rt.FBA-F(SEQ ID NO.28)/Rt.FBA-R(SEQ ID NO.14)与Asp.FBA-F/Asp.FBA-R，以pUC57-Rt.FBA质粒与出芽短梗霉基因组DNA为模板扩增出相应的Rt.FBA与Asp.FBA启动子基因片段；使用引物对icl-Rt.FBAtail-F/icl-R与mls-Asp.FBAtail-F/mls-R，以出芽短梗霉基因组DNA为模板扩增出相应的ICL与MLS基因(SEQID NO.35)序列片段；使用引物对Tcyc1-icltail-F/Tcyc1-Asp.FBAtail-R与Tsup4-mlstail-F/Tsup4-pk2tail-R，以p414-TEF1p-Cas9-CYC1t质粒为模板扩增出相应的Tcyc1与Tsup4终止子基因序列片段。再分别进行两两相连得到重叠片段Rt.FBA-ICL-Tcyc1表达盒与Asp.FBA-MLS-Tsup4表达盒。然后将pK2-hyg质粒进行EcoRI酶切，回收线性质粒载体后，同时将重叠片段与pK2-hyg质粒的载体骨架连接，连接产物转化至DH5α感受态细胞，进行转化子筛选后即获得启动子Rt.FBA与Asp.FBA基因串联组合表达载体。
表5、用于构建基因串联组合表达相应的引物
| 引物名称 | 序列5’-3’ |
| Asp.FBA-F | gcactgtcattgcaacctactg(SEQ ID NO.36) |
| Asp.FBA-R | gtgattgtagttgtctggttgctg(SEQ ID NO.37) |
| icl-Rt.FBAtail-F | <u>ttctaacactaactagctac</u>aatgcgtcgagcaaccattc(SEQ ID NO.38) |
| icl-R | ctagaaacttccttccgtgtt(SEQ ID NO.39) |
| mls-AspFBAtail-F | <u>aaccagacaactacaatcac</u>atgtcttcttcttctctcga(SEQ ID NO.40) |
| mls-R | tcagtggttctcctcagcct(SEQ ID NO.41) |
| Tcyc1-icltail-F | <u>acggaaggaagtttctag</u>tcatgtaattagttatgtcacgct(SEQ ID NO.42) |
| Tcyc1-Asp.FBAtail-R | <u>taggttgcaatgacagtgc</u>gcaaattaaagccttcgagc(SEQ ID NO.43) |
| Tsup4-mlstail-F | <u>tgaggagaaccactga</u>catgtttcggcgttcgaaac(SEQ ID NO.44) |
| Tsup4-pk2tail-R | <u>aatgtcatcttctgtcgacg</u>agggacctagacttcaggttgtc(SEQ ID NO.45) |
将获得的串联组合表达载体转化出芽短梗霉CCTCC M2012223，获得含MLS和异柠檬酸裂解酶ICL转化子，简称OE::MLS+ICL。
(2)双基因载体构建过程
(2)OE::MLS+ICL转化子发酵
将OE::MLS+ICL转化子接种于含葡萄糖90g/L、NH4NO3 2g/L、KH2PO4 0.1g/L、KCI0.5g/L、ZnSO4 0.1g/L、MgSO4 0.1g/L、玉米浆0.5g/L的发酵培养基中，然后在25℃、220rpm，摇瓶发酵96小时，取样生物量与发酵上清液；同时以出芽短梗霉CCTCC M2012223在相同的条件下进行发酵作为对照，结果如图4所示。结果显示，其中OE::MLS+ICL转化子生物量为20.5g/L，对照组生物量为19.1g/L，OE::MLS+ICL转化子的聚苹果酸产量最高可达48.9g/L，比对照株提高了45.8％，糖酸转化率最高达到0.67g/g，较出芽短梗霉提高了52.4％。
由上述实例可以看出采用不同强度启动子，组合表达MLS和ICL的出芽短梗霉菌株能够有效提高聚苹果酸的产量。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
<110> 西南大学
<120> 具有提高出芽短梗霉代谢产物的启动子及其应用
<160> 45
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1948
<212> DNA
<213> 出芽短梗霉(Aureobasidium pullulans)
<400> 1
cttgatgttt gttgggaaaa gttggggtgg ggtgatgcgt gtactacacg tgggagcagc 60
aaggtcagtc tgatagtcgg agagaggtgt agtgtccaag acgacagaga tacatacgag 120
gattgagtga taaacccagg ggtgggaaag tgtgtgagag caaaaaagga atagtgatgg 180
tcaagtcaca atttgaagac gtgatgggga cacgagctgc caggccgacg agctccgggt 240
gaatacagct tatgacgaac tcaggcgggg cttcaatcag acggaacgtg cacaagccag 300
catcaaaagc cgatcaagct gcttctccgg cccaaacaaa ctctgttcgg ccggccttac 360
ttgttatgat aacaattcga ccttgtctcc gtcacatctc ctgaagtaaa agtgcacgca 420
tagacaaata ctcgtgtgta agactgttat ttctgatact gttgtcacgc gagggaaccc 480
atatcgtcca gtccaaagtc tacctagcca ggttgctgtc attctttgtt gcagtcctgc 540
tgcgtcgagc aaatgtacac gaccagcccg gcccttctat tgtaaatcct atcagttagt 600
catgaaggat ggatccttaa tactgcctcc tgcatcgccc aagccctcga ctggtcggtt 660
gtatgaccat cctgcgaatt gttgttgcat cgtggatgag agcattggtc cctcgacgta 720
cgaatctgta ggtgcttctg atgtgaactc gcgatcgaag ttggcagtat ccctgtcatc 780
aagctaaggt catgtcagca tgttttgttg cttgtcgtac agcaatcacg attactcacc 840
acgttgggct tgaagctagg ctcgtacttg cgatccaaaa gctttctcca gtcaatgcta 900
tggaagaatg gatgtgcctt gatttctgct gcaccgttga cacccaatct ctttgtcgga 960
tctctgtcga gtagcttctg caacaggtct cgtgctgcag gaggcacaat ttcggggccg 1020
gggaagtgta gaggctctgc cagaatcttg cggtacatgt cgttagtgtt ttcgtcgtag 1080
aacggcggca gtcctgtcag catctcgtag aggagaacac ccaacgtcca ccagtcaacc 1140
gtcttggtgt agccagtacc gtgcagcagc tcgggtgcga gatactcggg cgtaccacag 1200
aatgctacaa tcttgattaa tatgaagtcg ctgataccaa tttatagaaa cacatactgt 1260
ttgtcttgtc ctcatccttc atgtcgagtt tgcagagacc gaaatcgcac agagcaatat 1320
ggccagtgta gtcgagcaag atgttctcgg gcttcaagtc tctgtagatg acgttgaatc 1380
cgtgtagaca ctcaagcgcg cacaagagtt cagcagcgta gaaacgagat cggttgatgt 1440
cgaagcgctg ctctttttgc agatggtgga aaagctcacc gccgttgacg aaggccagca 1500
cgatgtagag cttctctggt gactggaagg agaacttcat gggaacaata aaaggattgt 1560
tgatctgagc cagcaccgaa cgctcggcca gagtgtgggc aacttcggaa cgtgagatga 1620
tgtgagcttt tctgattgtc ttcaaggcgt agattctgcg ggtatcgcgc tttctggatg 1680
tgagtgttag ttttaattcg cgacgggacg atagtgggca gagacgtact gcacttgcat 1740
gacctttccg aaactgcctt tgccaacaac cttgagcagt tcgaaatcct caatcttgag 1800
cgatctttgt ctgttctcga cgaatttgac gccacatctg attgatccag ttccgtactg 1860
gaggtcaacc cactcagtgc cagattgacc ttgagtcttt gccttctctg cgtgtgcgaa 1920
agcggccttt tctctatctt ttctgccc 1948
<210> 2
<211> 1988
<212> DNA
<213> 出芽短梗霉(Aureobasidium pullulans)
<400> 2
gtgattgtag ttgtctggtt gctgtggtat gagttgaagc ccgtctatcc tcaattaatg 60
ggaaagatca aattgaagag taaacagttt tcggaaaatg aacaggattg ttgttctgct 120
aatattcaag gatatataaa cgaggtcatt gccccgctat ataccccacc ggtcttcttc 180
ccgaccgttc cgcgttgccc accttgggac ttccgcaaca ttctgccgag acgtgatctc 240
ctcaagatcc ggagggtgta ccccgtggta tcggtgattt tgccggagag cctgttcggt 300
gtgatgcaag ccagtgccgg gagtcgtgtg tgatacgaga atagggaaat tgcatatatt 360
ctataaagat atgatctggt tactcggatc taaattgtga agtcgtcact cttcaacaaa 420
gagacgacag acaaaagagc ccaggaagag aaggaagtca gaagggaaaa agagtccaag 480
aaaaaaatgc ttttccatac atggcttcgt tcaatatttg atagcaaggc attaatcatt 540
gccgattgag cagtaagcca agtctctgcc aaaagtgttt tccgaaaggc gtacgcgcca 600
ttatgttgac tggctgggca tgcgtggaca ttgacctggc ggtctcagct agaccacaat 660
ctagtacaca gctccggttg gtgtcaccag tgtgcgctca tgagatgcgt ccgtctggta 720
gggcatactg gcgcgcatgc ccgtcttgag ctgctttggt gtttgctctg catgctgcat 780
ggaagtcgag gctgctcaga ggatccatgc aactataaga gggtcgaatt tcgagggtcg 840
tagtactgcc ttgtcctagt tggcatgtcc atttgtagag accttctctt caacttcgac 900
gtacttggcg caagcgaagt agcccttgtt ctcgaccaca cctcggttct tcacgactgc 960
agtgtatttg atgacccatt gaggaatgac ctgaccttcc atcttttgac cattctcttg 1020
cctctttctc ttctcgccaa tcttctccga ggcttgcgat aggactccag caccttcgtg 1080
atcgagtaga cccctgatag tgccacgagg catgatgttc accgagtcat tgttgacaag 1140
cagttcgaca tttttgcttt ttgccaagct agagagaggt tgaggcacac tgcagcagtc 1200
gcgcaggctg atctgattga ccgaaggctt cacacttgcc ttctcgagga aaggttgcaa 1260
gcggtttgct ccaaactcgg acacaccaag tcttcgcacc agaccgtcct tgtgcagctt 1320
ctcgagcagc ttccaggtct ccaattggtt gtccacaggc tcaggagaag attccttggt 1380
tcctctgctt ttgatcttgt ctggacagca ctcactctcc tcgtcgaagt agacgccggg 1440
aaaggagaca atgaggagat caacattagg catatgcaat tctttctgaa ccagatcgac 1500
agcttggcga agatggtcct ctcttgcaga agtgtcctcg tcggagagga agaagacctt 1560
tgcagttata tcgtattggt cccgctcttc gtgtagggag ctgttggaga agtcgagacg 1620
tgggatgtac atggtctttc cgtcatccga gtatgagatc caatcgacct gttcagcctt 1680
ctcttgagac agctccttgg cctcgtgcct tgcgccctcg aagttgtcgc gtaggctcca 1740
agtgagttcg gtatgggaat tgtcggcggc acccacgctt tgctgaagca tggtattgct 1800
aggagagata agcacaagct accatgacgc gacatagcaa gcaataccca gtggatagaa 1860
taatagtttc ggccgtcatg attgttgagg tggatttcta agcgagaaaa agtaggaaag 1920
gatcaggacg cttaaaaaat gcaggaaatg tacaggaaac aaaaaacagt aggttgcaat 1980
gacagtgc 1988
<210> 3
<211> 1982
<212> DNA
<213> 出芽短梗霉(Aureobasidium pullulans)
<400> 3
tgtgactgaa ttggatgtgt tagacagttt ggtgtggaag acagcagcag agagaagcgt 60
accgagttat atgcaacaat gcttgatggg gtgttgtgat gtgtgagggg cttgaatctt 120
tccccaacag cttccggcgg tcggaacgag gggcagctca aaaaactgcg tcacgtcccg 180
ccgatgtgat tccgaggatc ttcataccct ctcgtgttat gcatcaactg atatcaccaa 240
tcgtgagcca tttctatgta cgaacgccgt gggatcacct gcttcactct aagtcatatc 300
atgcccgtct tcgagtcgca cgtcttctca ttcggtctat tatttacaat accttgcact 360
aaccgacgtg tttatcgatc ggaccgtaac gccccgtaaa gtcgccagaa cgcctgcctg 420
ttccgcagtc cgatggatgc atgaaggtaa tcaatacact ctcccgattt attccttgtg 480
aatggctttc taagcctcct cagccgagtt ggtcagagag aggtaggctg tccacgcaat 540
accaacagtc gacacgaaag gctgtaacag ttttagttgc tgatcttcgg tcgagttcag 600
gtaccactta caatctggaa ctgacgcggc atcagtctga agttgatcaa ctgtacagcg 660
ggccagacca agtagttggc cttgagcgaa ggaatgtaga tatcctggaa cttcttctgg 720
atctccttac gaccacctcc ttctgccaca gtcatgaaag taaagaaggc tccgagacca 780
accggtgcga agaggagttg gtcaaaagca gttctcttgg ctgctgcgct ggtaccagct 840
cccttggcga gagggaagat gcgattgagg aagccgaacc acttgtgttg gacgggggcc 900
atgatgaaac cgtatgccat aaatcttgtc aatctgccca gcgtatcagc acctttgaac 960
tcttgtctct tgctcttggt tgtctgtacc tctcgaaatc aaatggcgga ggcaggacct 1020
tagaatcggg aatgagctct tcctcagaga ttctgcgatc tatttcatgg atttcgatag 1080
ccaatcgatc gttcttaggg tcggggccac caggtttgcg aacggcgcgt tctctgactg 1140
ctgtgagagt ttgggcgaca gtgtcggcta caccaccaag cacctggagg aaacgtcaga 1200
tacacatatc agcacgtggt tgtagatgga catacggcgt tggtgaccat ggtcgtaagc 1260
actgatacga atgttagcac atagtcacga gatgatcaca aatgccaacc atacctggtc 1320
tcgcagcata gtagctgtta aacttggaca ccagaatgga aaccatgatt aattcggtga 1380
tgaatactaa ggtgtgtagg agaggctgtc gcggaaacaa acaggtcagc gttgaaaagt 1440
tatacaagta aggcaataga ccaagtgaga ctcacatatc gcccgtaaat gtatctcgag 1500
ttcaaattcc acaggacctg cctgcgtatg cttttgaaag aaggcatgat tgagagaagg 1560
aaaatgtgaa tgacgctcgc actctttctg ggggctctga cgtcgattca aagccgagtc 1620
gatcgggatc agggtcagct gcagtggggc acgcgcggaa gaacgtccta tcgccagaga 1680
gattctcagc gctttgttcc cttgtgctaa gccgctcaac agatgttatc aaggtaggag 1740
gttggagatg ttatagctaa tggcgaaact ggtcttgttg aaggagggtc taggtttgtc 1800
tctcccagcc agccgtcgtc agtcgccatc gatcagcacc ctgcgtgccg cgcttgttct 1860
ggtggccggc tgtgggatcc gagaggagga attgccgcga agctttcaat ccgcgaactg 1920
tccatcagcc aatggctgcc tcgtctcgaa gacgtttcat gctgtttggt tttacttcgc 1980
ac 1982
<210> 4
<211> 1500
<212> DNA
<213> 红冬孢酵母菌(Rhodosporidium toruloides)
<400> 4
ggttcgtagc gtggtgagtg aaagcagagc gaaagcgacg agcgaggcgg cccgatgagg 60
tcgtcattca gcaagtcgct ccgatgggct tgcttgcggt tccaagcagt ctagtatgtc 120
cttgcactgt cgccgacgct ggaccgaacg ggatcaggcg aggttaagag ggcaacattc 180
tcgagttcgt tccgacgaga gcaaccgtac gagctacaac aagggcagcg agccgtctat 240
gcgagagaaa ggtctatgtc tagtgtgtgt ctaagccgtc cccgtcagtc tatccgcctt 300
cgaagtcgta caatctgtcc gcggttcttc acgcctcgtt caagtcccgc atcttgaccg 360
caggactctc gaccggtgcg aggccgtcct gctgaagctg gagttggtga tgtggcggct 420
gctgcgggtc ggacgacgag ccttggtgct gctcgagctc gtgcagcgaa gagccgaggt 480
gttgctcctt gaaggcgtag aaataggcgg cggtatcctc gtcttggatg tcccgctcga 540
ggctgtgcat gggcggaaga cggtgtcagt gcacggtcgg agagcaggag agggaagacg 600
aaacgtacag ttcgcggcgg atcttggtcg cccgaatcgc gttgccggtc aggcggtaca 660
ggatgtcgac tgcgatagat cagcgcgtca gtcacctcgt ttgacaatgc gtacgagggt 720
gaatggaagg cggactttgt tcgagcgaga gacccttggt ctcgggaatg cagctgcgcg 780
agaagctcgt cagcctgtgt gtgccaaacc cgtcgaaaga tggcagtgac gcacaagtac 840
acaaacagaa tcgcgatgca cgacacaccg ccccactgcg caattcacat acctcgtcag 900
caaagctcat ccctccgcat gagcgaagga gggagcaaga acgggttggg acgcacgatg 960
aagaacacct tgccctgaag acccgcgttt cctttgccag tgttgacgag atctgcgcgc 1020
accgcctgct cagccttcga atatagcaaa tgagcagaga gggaggagac gaacaaggag 1080
tcgcgtaacc gataccgaaa tctgcgagcg tcaagcagtt cgtcagctca agtgaggcat 1140
gtagggaggg agagacgacg cactgaacag ccagttcgac gcgaccgaca tggacatctg 1200
tttcgcccga gtcgccgtcg cgtagatctc gctcgtgctg caaacgatgt ccgcgttagt 1260
ctcgctgtgt gagcggagca aggaaagttg acagcgtaca caacccatgc gatcggaccc 1320
ctgttgaagc aaacccatca gctcgcaaat caaccgatga ggaacagatg cgagctcacc 1380
aagtcgccgc aaagaacgcg ataaacacac acacgaaccc aatcaacacc ttctgacccg 1440
cctcgttgtc ctgcgacaag gccgtcgagc aacccgcgac gatgaaatgg cagacggcca 1500
<210> 5
<211> 1500
<212> DNA
<213> 红冬孢酵母菌(Rhodosporidium toruloides)
<400> 5
tgtagctagt tagtgttaga agtgaagtgg tgaagagcga ggagcaacga gagagcgagc 60
gagcaaggcc gtgccaaccg aaccgacatc atccacaacg cggggccact cgtacaaact 120
gcacagaaat tcttgcaaca ccgtaacaac cagctgcaag ccggagcgag cgtcatccag 180
ccaactccgc tctctcgcct ccctcgcgat atatggcatc gtgcgagcac caggtggcaa 240
gcaggtcaag cgaaagtaaa gcagctcctg tcgatgctgg acaacatcgc agccgcaaga 300
gtccgaagga acgaggcgaa agtgcggttg caggcggtgg aaaaggttgg aagcaggtga 360
agctggagga ggtgcgtcta catctacatc tgcagtgaga cgggcacgat gagagtcagc 420
gtgagaagca gaagagacag gagcaaccag cgagcgagcg aaagcgacgc acgaaatcct 480
cgtcctcgtc gaactgggcc gcggcgacat cgtcgtcgag cacctctccg aacgacgaca 540
agtcctccct gcccctcgcg accatcctcg cagggggctt cccaccgact gcgccgccct 600
tcgcgccagc cttctcgagc ttctgcttgt ccatcgcgag gttggccata ctcgcgcgca 660
ccttgcccac ctcgtccgac ttgagcggcg cggcgagcag gcgggcgagc tcgtcgacaa 720
aattgccgaa cagcttcgag ccggcgtgct tctggatcag agaatcggac agggcgacgg 780
cgagagccga aaagtcctcc tttgtccggc agttcatctc gagcgggttg tctgcgaaac 840
gagtaccgtc agctgcagat cttcttgatc atctgagtga agtgcgacgc accgttcaag 900
ctcgcctcgc caaagagaga cgccgcgctg tccatgtcgg accgcagttg cgccgcccgt 960
gcctttgctc gcaacgcagc cgggtcgttt tcgcgggcct gcagagcaag tactgtcagc 1020
tgtctccgca cgaatgagca gaatccggaa agcgtcgccc agcaggacaa ctctcggatc 1080
ctcctcgccg tgaaggcaga ctcaccaatc gctccgcctc ctcagccttc ttccgctcct 1140
ccgcctcctt ctgcgcaatc ttctgcttcg tagcgccctt gttcctcagc gacccaacca 1200
caacctgggg cttcggcttg tcctcctcgt cgctgacatc ccagtcgtcc ttgacctcgt 1260
catcggcgtc ttcgtctgcg aaccggcctt tcttgactgc tggtacgggg ggagggagag 1320
gcttcttcgt gtcctcttcg gaggaggagg cgtctgtgag caggagaagc gagagtgtga 1380
gcgagcggtg cgggaaggag gcgacgacag tgggagggaa gcgcacccca gtcactgccc 1440
attttcgagt tcaaggagga agtctagcag gcgaacaagc cacagcgagc gagagcagag 1500
<210> 6
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gggcagaaaa gatagagaaa aggc 24
<210> 7
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gtgagttcag gctttttcat cttgatgttt gttgggaaaa gttgg 45
<210> 8
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gcactgtcat tgcaacctac tg 22
<210> 9
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gtgagttcag gctttttcat gtgattgtag ttgtctggtt gctg 44
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gtgcgaagta aaaccaaaca gc 22
<210> 11
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gtgagttcag gctttttcat tgtgactgaa ttggatgtgt tagac 45
<210> 12
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tggccgtctg ccatttcatc gtcg 24
<210> 13
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gtgagttcag gctttttcat ggttcgtagc gtggtgagtg aaag 44
<210> 14
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ctctgctctc gctcgctgtg gcttg 25
<210> 15
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gtgagttcag gctttttcat tgtagctagt tagtgttaga agtg 44
<210> 16
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atgaaaaagc ctgaactcac cgc 23
<210> 17
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ctatttcttt gccctcggac g 21
<210> 18
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gataagcttg atatcgaatt ctatttcttt gccctcggac g 41
<210> 19
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
tcgactctag aggatccccc gggcagaaaa gatagagaaa aggc 44
<210> 20
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tcgactctag aggatccccc gcactgtcat tgcaacctac tg 42
<210> 21
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
tcgactctag aggatccccc gtgcgaagta aaaccaaaca gc 42
<210> 22
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tcgactctag aggatccccc tggccgtctg ccatttcatc gtcg 44
<210> 23
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tcgactctag aggatccccc ctctgctctc gctcgctgtg gcttg 45
<210> 24
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ctatgacatg attacgaatt gggcagaaaa gatagagaaa aggc 44
<210> 25
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
ctatgacatg attacgaatt gcactgtcat tgcaacctac tg 42
<210> 26
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ctatgacatg attacgaatt gtgcgaagta aaaccaaaca gc 42
<210> 27
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ctatgacatg attacgaatt tggccgtctg ccatttcatc gtc 43
<210> 28
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
ctatgacatg attacgaatt ctctgctctc gctcgctgtg gcttg 45
<210> 29
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
agtggatccc ccggggaatt caggtattgc tgttatctga 40
<210> 30
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
tgtcctgcgg gtaaatagc 19
<210> 31
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
tgtgaacggc gggagatg 18
<210> 32
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
gaagtgcgat gtcgatgtca ga 22
<210> 33
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
ggagcaaggg cggtgatt 18
<210> 34
<211> 1797
<212> DNA
<213> Aureobasidium pullulans
<400> 34
atgcgtcgag caaccattca gagcctgcaa cggcgcccaa tgtcaagact ccatctcaca 60
gctcgcccta cttctcgctt catctcatcg agcttggttc gcatgtctgc gtcgataaaa 120
tccgtcgatc ctcctttacc ttcagcagag cctacctcat ctttccagtt gttgccaaca 180
gaacagaagg ttggggagta tgaagatgct gtatttgatg accaagtaaa acaggtccaa 240
gactggtggg cttcaaccag atacaaaggc atcaaaaggc cttattctgc agaagatgtc 300
gttagcaaga gaggcactct gcagcaaatc tacccttcct cgttgatggc tcgaaaactt 360
ttcaaccttc tggaggaacg ggctgctaag ggagaacctg tgcacacgat gggagccatt 420
gatcctgtgc aaatgacaca acaggcacct caccaggaga ttctctacgt ttctggatgg 480
gcctgctctt ctgttctgac cagtacaaac gaggtctcgc ctgattttgg tgactatccc 540
tataacacag tccccaacca agtacaacgt cttttcaaag cacagcaatt acacgacaga 600
aaacatttcg atgcacgcag gaagatgtct gcggatgaga gaaacaagac gccatacatt 660
gattaccttc gaccaattgt tgccgatggt gatactggac atggtggtct ttcggcagtt 720
ttgaagctgg caaaactctt tgccgaaaat ggtgctgcag ctgttcattt cgaggatcaa 780
ttgcatggag gcaagaaatg cggtcacttg gcaggcaagg ttttggtccc tgtcggtgaa 840
cacatcaatc gtcttgttgc tacacgtttc caatgggata tgatgggctg tgaaaacctc 900
atcatcgcac gcacagattc cgagagcggc aagttgttga gcagcgccat tgacgcacgc 960
gaccacgaat acattcttgg ctcgaccgag gatatagaac ctctggcaga gacgctccaa 1020
gtcatggaga tgaacggtgc gactggtgca gaaatcgatg cttacgaggc taagtgggtc 1080
aagcagcata aattggtcac tttcgatgaa gctgctatcg cccacatcaa ggccaagacg 1140
tccgactcga acaaggttgc caaatacgag agcgagatat ccgcgaatcg taacagacct 1200
ctaaacagcc gtcgccagct tgccgcatct attgcaggtt ctcctgtcca attcagttgg 1260
gacgtcccgc gaaccaagga aggtttctac cactaccgtg ccggacttcc tgcagcaacg 1320
aagcgagcta tcgagtttgc tccgtttgca gatttgctgt ggttggagac tggagatcca 1380
agcgtgcaaa aagctgcagg ctttgcccag gagattagag aggtgcatcc tggaaagaag 1440
ctcgtctaca atctcagccc cagcttcaac tggatgggac atggtttcac ggaggaccag 1500
ttgaaaaatt tcatctggga tctgggcaag gaaggatttg tacttcagct tatctccctc 1560
gccggcctgc acagtaccgc aacaatcact aacgagcttg ccaagggatt caagactgat 1620
ggcatgttgg cgtatgtgaa tttggttcaa cgtcgtgaac gtgaactggg ttgcgatgta 1680
ctcacacatc agaaatggag cggagcaagt taccttgacg ggattctggg cagtattcag 1740
agcggcagta gtagcagtaa gagtatgggc gagggtaaca cggaaggaag tttctag 1797
<210> 35
<211> 1668
<212> DNA
<213> 出芽短梗霉(Aureobasidium pullulans)
<400> 35
atgtcttctt cttctctcga cgccgaagag cagcaatacc aggacgaggt tgccgccatc 60
aagcaatggt ggacctcacc tcgctggaga tacaccaagc gtccttttac cgctgagcag 120
attgcaaaca agcgcggaaa tttgacaatt caacaccctg gcaatgccct ctccaagaag 180
ttgtgggaca ttgtcgagaa gagatttgcc gagaaagatg ctagtttcac ctatggatgt 240
cttgaccccg tcatggtcac acagatggcc aagtacctag acactgtcta tgtctcggga 300
tggcaaagtt cctcgaccgc gtcatcaaca gacgaacccg gcccagatct tgcagactac 360
ccctacacaa ccgtgcccga caaggtccaa catcttttca tggctcaact tttccacgat 420
cgcaagcagc gtcatgaaag actctcgaca cccgcctctg agcgcaaaaa ccttcccaac 480
accgacttcc tccgcccaat cgttgccgat gctgacactg gtcacggtgg tctcacagcc 540
atcatgaagc tcaccaagct tttcgttgaa aaaggtgccg caggcattca cattgaagat 600
caagccccag gaacaaagaa gtgtggacac atggctggca aagttctcgt gcctatcagc 660
gaacacatca accgtcttgt cgctatccgt gcccaagcgg acatcatggg cagcgatctc 720
ctcgccgttg ctcgtaccga ctctgaggcc gcaaccctca tcacctcgac tatcgaccct 780
cgcgaccatg ccttcattgt tggcgccaca aaccctgctc ttcaaccctt gagcgaactc 840
atgggtgccg ccgaagcagc aggcaaatcc ggcgatgaac ttcaagccat tgaagacaca 900
tggactgagc aagcgcacct caagctcttc tccgaagccg taatcgacac catcaacgcc 960
ggtgttcacg tcaacaaggc agagttaatt gcccaattca ccgaacaggc caaaggcaag 1020
tccaacgcag agtcgcgcgc tattgccaag gctcttactg gtgtcgacgt ccacttcgac 1080
tgggagtccg ctcgcacaag agagggctac taccgctacc gcggcggctg ccaatgcgcc 1140
gtcaaccgcg cgatcgccta cgcaccctac tgcgacatga tctggatgga gtcaaaactc 1200
cctgactacg ctcaagccga ggagtttgcc actggtgttc acgccgtatg gcctgaacag 1260
aagttggcct acaacctttc gccatctttc aactggcaaa ctgccatgag cacttccgac 1320
caggaaacct acatcaagcg tctcgctaag ctcggctact gctggcaatt tatcactttg 1380
gctggtctgc atcaaactgc tttgatcgct gatactttca gtaaagacta tgctgctcgt 1440
ggtatgcgtg cttacggcga gaccattcag agcaaagaag aagcaaacgg agtcgaagtc 1500
ctcaaacacc agaaatggtc tggtgctaat tatgtcgacg agttgcttaa gatggtttcg 1560
ggtggtgtgt ccagtaccgc cgctatgggc aagggtgtca cagaagatca gttcaaagct 1620
gtttcccgtc caggcgagag tggctacaag gctgaggaga accactga 1668
<210> 36
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gcactgtcat tgcaacctac tg 22
<210> 37
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
gtgattgtag ttgtctggtt gctg 24
<210> 38
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ttctaacact aactagctac aatgcgtcga gcaaccattc 40
<210> 39
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
ctagaaactt ccttccgtgt t 21
<210> 40
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
aaccagacaa ctacaatcac atgtcttctt cttctctcga 40
<210> 41
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
tcagtggttc tcctcagcct 20
<210> 42
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
acggaaggaa gtttctagtc atgtaattag ttatgtcacg ct 42
<210> 43
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
taggttgcaa tgacagtgcg caaattaaag ccttcgagc 39
<210> 44
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
tgaggagaac cactgacatg tttcggcgtt cgaaac 36
<210> 45
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
aatgtcatct tctgtcgacg agggacctag acttcaggtt gtc 43
Claims (3)
1.红冬孢酵母菌1,6-二磷酸醛缩酶启动子Rt.FBA在出芽短梗霉菌株中调控出芽短梗霉菌株代谢途径限速酶基因表达提高出芽短梗霉代谢产物含量中的应用，其特征在于：所述红冬孢酵母菌1,6-二磷酸醛缩酶启动子Rt.FBA的核苷酸序列如SEQ ID NO.5所示的反向互补序列，所述限速酶基因为异柠檬酸裂解酶ICL基因，所述异柠檬酸裂解酶ICL基因的核苷酸序列如SEQ ID NO.34所示，所述代谢产物为聚苹果酸。
2.一种提高出芽短梗霉代谢产物的方法，其特征在于：使用红冬孢酵母菌1,6-二磷酸醛缩酶启动子Rt.FBA启动至少一个出芽短梗霉菌株代谢途径限速酶基因在出芽短梗霉中超量表达，即获得高产代谢产物的出芽短梗霉，所述红冬孢酵母菌1,6-二磷酸醛缩酶启动子Rt.FBA的核苷酸序列如SEQ ID NO.5所示的反向互补序列，所述代谢产物为聚苹果酸，所述限速酶基因为异柠檬酸裂解酶ICL基因，所述异柠檬酸裂解酶ICL基因的核苷酸序列如SEQ ID NO.34所示。
3.根据权利要求2所述提高出芽短梗霉代谢产物的方法，其特征在于：进一步包括使用出芽短梗霉1,6-二磷酸醛缩酶启动子Asp.FBA启动苹果酸合成酶MLS基因在出芽短梗霉中超量表达，所述出芽短梗霉1,6-二磷酸醛缩酶启动子Asp.FBA的核苷酸序列如SEQ ID NO.2所示的反向互补序列；所述苹果酸合成酶MLS基因的核苷酸序列如SEQ ID NO.35所示。
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Application publication date: 20190730 Assignee: Hunan Hongjian Biotechnology Co.,Ltd. Assignor: SOUTHWEST University Contract record no.: X2024980012125 Denomination of invention: The promoter with the ability to enhance the metabolic products of Aspergillus oryzae and its application Granted publication date: 20210302 License type: Exclusive License Record date: 20240815 |
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