CN113564195B - 一种果糖胺脱糖酶毕赤酵母表达载体、基因工程菌及构建方法和蛋白表达方法 - Google Patents
一种果糖胺脱糖酶毕赤酵母表达载体、基因工程菌及构建方法和蛋白表达方法 Download PDFInfo
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- CN113564195B CN113564195B CN202110878379.6A CN202110878379A CN113564195B CN 113564195 B CN113564195 B CN 113564195B CN 202110878379 A CN202110878379 A CN 202110878379A CN 113564195 B CN113564195 B CN 113564195B
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- fructosamine
- pichia pastoris
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- genetically engineered
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Abstract
本发明涉及一种果糖胺脱糖酶毕赤酵母表达载体、基因工程菌及构建方法和蛋白表达方法，属于蛋白表达技术领域。本发明所述果糖胺脱糖酶毕赤酵母表达载体包括骨架载体和编码果糖胺脱糖酶的基因；所述编码果糖胺脱糖酶的基因的核苷酸如SEQIDNO.1所示。本发明所述载体有利于该酶的工业化生产，生产方法绿色，安全，环保，无污染，可控，对氨基葡萄糖的大规模制备有益，会产生较大的社会价值和经济效益。
Description
技术领域
本发明涉及蛋白表达技术领域，具体涉及一种果糖胺脱糖酶毕赤酵母表达载体、基因工程菌及构建方法和蛋白表达方法。
背景技术
氨基葡萄糖（Glucosamine，GlcN），即2-氨基-2-脱氧-D-葡萄糖，又称葡萄糖胺、氨基葡糖或简称为葡糖胺、氨糖，是葡萄糖的一个羟基被氨基取代后的化合物。研究表明，氨基葡萄糖具有一些特殊的生理活性功能，如消炎镇痛，修复软骨损伤，治疗风湿性关节炎；强化白细胞增殖和分化，促进细胞因子分泌，改善免疫调节、抗肿瘤；恢复线粒体内酶表达，提高线粒体谷胱甘肽抗氧化能力，增强免疫功能；促进内质网相关蛋白降解，强化蛋白酶活性，改善蛋白稳态水平，延长细胞寿命等。
果糖胺脱糖酶作为一种新发现的生物酶类，它可以利用丙氨酸或谷氨酸作为氨基供体，将果糖-6-磷酸转化为氨基葡萄糖，同时产生丙酮酸或α-酮戊二酸。因此，果糖胺脱糖酶在酶法制备氨基葡萄糖及丙酮酸或α-酮戊二酸的联产技术中发挥着关键的作用。
果糖胺脱糖酶目前构建于大肠杆菌中，不利于该酶在食品药品领域的大规模工业生产。
发明内容
本发明的目的在于提供一种果糖胺脱糖酶毕赤酵母表达载体、基因工程菌及构建方法和蛋白表达方法。本发明所述载体有利于该酶的工业化生产，生产方法绿色，安全，环保，无污染，可控，对氨基葡萄糖的大规模制备有益，会产生较大的社会价值和经济效益。
本发明提供了一种果糖胺脱糖酶毕赤酵母表达载体，包括骨架载体和编码果糖胺脱糖酶的基因；所述编码果糖胺脱糖酶的基因的核苷酸如SEQ ID NO.1所示。
优选的是，所述骨架载体包括pPIC9K。
本发明还提供了上述技术方案所述果糖胺脱糖酶毕赤酵母表达载体的构建方法，包括以下步骤：
使用双酶切法将编码果糖胺脱糖酶的基因与骨架载体相连接，得到表达载体。
优选的是，所述双酶切法，每40~60μL的双酶切体系包括：10×quitcut buffer 4~6μL、pPIC9K或含编码果糖胺脱糖酶的基因的核酸28~32μL，4~6 U/μL的SnaBI和NotI各0.8~1.5μL和余量的水。
优选的是，所述双酶切法的双酶切条件为36~38℃，1~3h。
本发明还提供了一种含上述技术方案所述表达载体的基因工程菌，所述基因工程菌的宿主菌包括毕赤酵母。
优选的是，所述毕赤酵母包括毕赤酵母GS115。
本发明还提供了上述技术方案所述基因工程菌的构建方法，包括以下步骤：
将果糖胺脱糖酶毕赤酵母表达载体线性化，电转化毕赤酵母的感受态细胞，得到基因工程菌。
本发明还提供了基于上述技术方案所述基因工程菌的果糖胺脱糖酶表达方法，包括以下步骤：
将所述基因工程菌接种到BMGY液体培养基中，进行第一振荡培养至OD600在2~6之间，离心，弃上清，得到培养菌体；
用BMGY液体培养基重悬所述培养菌体，在100~300 r/min进行第二振荡培养84~108 h；所述第二振荡培养的期间，每10~14 h向培养基中加甲醇至甲醇的终体积百分含量为0.5%~1.0%。
优选的是，所述甲醇为100%的甲醇或甲醇的体积百分含量为80%以上的甲醇水溶液。
本发明提供了一种果糖胺脱糖酶毕赤酵母表达载体。本发明首次提供果糖胺脱糖酶毕赤酵母表达载体，并将其构建于真核生物毕赤酵母菌中，实现果糖胺脱糖酶的成功表达。本发明具有操作简单，成本低，周期短和易于实现的优点。本发明所述表达载体能够成功表达具有良好的热稳定性的果糖胺脱糖酶。本发明技术方案使用的原料来源丰富易取，成本低，无环境污染，所用设备、试剂价格便宜，便于规模化生产。
进一步的，本发明利用毕赤酵母GS115成功表达了高可溶性的果糖胺脱糖酶，并对表达的生物酶蛋白进行了纯化。
本发明对所获得的重组果糖胺脱糖酶进行了生物活性验证，发现依次通过上述果糖胺脱糖酶，可以将淀粉，葡萄糖，果糖和果糖-6-磷酸合成氨基葡萄糖，并且同时利用丙氨酸生成丙酮酸，谷氨酸生成酮戊二酸，天门冬氨酸生成羰基丁二酸，谷氨酰胺生产谷氨酸、酮戊二酸。通过数据表明上述重组生物酶具有很好地合成氨基葡萄糖和丙酮酸，酮戊二酸，羰基丁二酸的重要功能，具有较大应用潜力。
附图说明
图1为本发明提供的平板筛选结果图；
图2为本发明提供的果糖胺脱糖酶FrIB的SDS-PAGE电泳结果图。
具体实施方式
本发明提供了一种果糖胺脱糖酶（Fructosamine deglycase，FrIB）毕赤酵母表达载体，包括骨架载体和编码果糖胺脱糖酶的基因；所述编码果糖胺脱糖酶的基因的核苷酸如SEQ ID NO.1所示：
TTATATAACATTATAGTCTAATGCATAATGGTTCTTCATTTTCAGATCAATACTCAACTTTCCACATGTATCTTCTATGAGATAAAGGATGATTTCTCTCCTCTGCCAGCTCGTCTGCATAGCTTCTCAGCACACGATTGAGAACGAGCGGAGCAAGATAGCCTTTAACTGAATCGTCAATTGCAGTGAAGTCGTAAGATGCAGCATCAAGCACAGTGAGCTTTTTGCCATACTTTTTCGAGAAGGTAAGCGCCCGCTCTTCAAGAGGTCTTGTTTCATCTAAACCGAGCAGGATGATAAACGGCACGGATTCATCAATAATTTCAAACGGTCCGTGAAAATATTCTCCGGCATGAATGGCGTGGGAATGAATCCATTGCATTTCCATGAGAATGCAGATGCTGTAGGAGTAAGCGACACCGTAGTTTGCACCGCTTGCCATGGTATAAATAATACTTTCTTTTTCATGGGCTTTTGCAAATTGCTTGGCGTTGTCAGCTTCCTGCTTAAGGGCTTTTTCATATACAGCCTGCAATTGATCTAAGCCTTCAATTGCTTGTTGGAATTTCGTATTGTTTTCTAATACTTGCAGGGTTCCAAAAACGATTTGATACAAAACGCCATAGTTTGTATTGATCGCAAGCGCCTCATCACCCCAATCGTACTGGGCAACATATTGCGCTTCCTGCGCTAAAGGAGACTCCGGTTTAAACGTCATCGCAATCGTAAGTGCACCCTTGCCCCTTGCAAACGCAGCAGCTTTGACTGTCTCCGGGGTATTTCCCGAATGCGAGCACAAAATAACAAGAGACTTTTCACCAAGCTGAACAGGGTTGCGCTGAATAAATTCGTTGGCGCTGTAGAGGTCGGAGTTTATTGATTTTGACTCTCTGTCAAACACATACTTACTCGGATACATAATGGCAGAAGACCCTCCGCATGCGACAAAGAATACATGATCAATGGTTTTCCCTTTCAAATCCTGCAAGAAAGCTTGAACCTCACGATTTACTTTTGCTGTGGCCTGACTCAAATCCTTCACTCCCCGTTTTTATTATATAACGTTATATAACATTATATAT。
在本发明中，所述果糖胺脱糖酶的氨基酸序列如SEQ ID NO.2所示：MSQATAKVNREVQAFLQDLKGKTIDHVFFVACGGSSAIMYPSKYVFDRESKSINSDLYSANEFIQRNPVQLGEKSLVILCSHSGNTPETVKAAAFARGKGALTIAMTFKPESPLAQEAQYVAQYDWGDEALAINTNYGVLYQIVFGTLQVLENNTKFQQAIEGLDQLQAVYEKALKQEADNAKQFAKAHEKESIIYTMASGANYGVAYSYSICILMEMQWIHSHAIHAGEYFHGPFEIIDESVPFIILLGLDETRPLEERALTFSKKYGKKLTVLDAASYDFTAIDDSVKGYLAPLVLNRVLRSYADELAEERNHPLSHRRYMWKVEY。本发明所述果糖胺脱糖酶的原始序列来自NCBI（National Center forBiotechnology Information）数据库，所表达的氨基酸序列编号为GenBank: AOR99552.1，来源为枯草芽孢杆菌。本发明对果糖胺脱糖酶的获得方法没有特殊限定，采用本领域技术人员公知的人工合成方法即可。获得果糖胺脱糖酶的编码基因后，本发明优选将基因建于质粒pCold Ⅱ中，转化入大肠杆菌JM109中进行保存。本发明对所述构建和转化的方法没有特殊限定，采用常规方法即可。在本发明中，所述骨架载体优选包括pPIC9K。本发明对所述骨架载体的来源没有特殊限定，采用本领域技术人员公知的常规市售骨架载体即可。
本发明还提供了上述技术方案所述果糖胺脱糖酶毕赤酵母表达载体的构建方法，包括以下步骤：
使用双酶切法将编码果糖胺脱糖酶的基因与骨架载体相连接，得到表达载体。
在本发明中，所述双酶切法，每40~60μL的双酶切体系优选包括：10×quitcutbuffer 4~6μL、pPIC9K或含编码果糖胺脱糖酶的基因的核酸28~32μL，4~6 U/μL的SnaBI和NotI各0.8~1.5μL和余量的水。本发明对10×quitcut buffer、pPIC9K、SnaBI和NotI的来源没有特殊限定，采用本领域技术人员熟知的常规市售产品即可。
在本发明中，所述双酶切法的双酶切条件优选为36~38℃，1~3h，更优选为37℃，3h。
在本发明中，所述双酶切前，优选以含编码果糖胺脱糖酶的基因的质粒pCold Ⅱ的大肠杆菌JM109为模板，进行PCR扩增，扩增用引物优选包括正向引物：5’-TACGTAAATATATTGTAATATCAGATTACGT-3’（SEQ ID NO.3）和反向引物：5’-GCGGCCGCGTTATATAACATTATAGTCTAATGCA-3’（SEQ ID NO.4），其中，下划线为酶切位点，所用酶为SnaBⅠ和NotⅠ。本发明对所述引物的来源没有特殊限定，采用本领域技术人员熟知的常规人工合成方法即可。
本发明对编码果糖胺脱糖酶的基因与骨架载体相连接的方法没有特殊限定，采用常规连接体系进行连接即可。
本发明还提供了一种含上述技术方案所述表达载体的基因工程菌，所述基因工程菌的宿主菌包括毕赤酵母。在本发明中，所述毕赤酵母优选包括毕赤酵母GS115。本发明对毕赤酵母的来源没有特殊限定，采用本领域技术人员熟知的常规市售毕赤酵母GS115即可。
本发明还提供了上述技术方案所述基因工程菌的构建方法，包括以下步骤：
将果糖胺脱糖酶毕赤酵母表达载体线性化，电转化毕赤酵母的感受态细胞，得到基因工程菌。
具体的，本发明优选先将所述果糖胺脱糖酶毕赤酵母表达载体转化大肠杆菌感受态细胞，培养筛选单克隆，挑取单克隆扩大培养，提取质粒，得到重组质粒pPIC9K-FrIB；然后用Sacl线性化重组质粒pPIC9K-FrIB，电转化毕赤酵母的感受态细胞，得到基因工程菌。本发明对所述转化、筛选、扩大培养、提取质粒和线性化的方法没有特殊限定，采用常规方法即可。
本发明还提供了基于上述技术方案所述基因工程菌的果糖胺脱糖酶表达方法，包括以下步骤：
将所述基因工程菌接种到BMGY液体培养基中，进行第一振荡培养至OD600在2~6之间，离心，弃上清，得到培养菌体；
用BMGY液体培养基重悬所述培养菌体，在100~300 r/min进行第二振荡培养84~108 h；所述第二振荡培养的期间，每10~14 h向培养基中加甲醇至甲醇的终体积百分含量为0.5%~1.0%。
本发明将所述基因工程菌接种到BMGY液体培养基中，进行第一振荡培养至OD600在2~6之间，离心，弃上清，得到培养菌体。在本发明中，所述离心的条件优选为2000~4000 r/min离心3~8 min。在本发明中，所述第一振荡培养的时间优选为12~20h，温度优选为26~30℃。
弃上清后，本发明用BMGY液体培养基重悬所述培养菌体，在100~300 r/min进行第二振荡培养84~108 h；所述第二振荡培养的期间，每10~14 h向培养基中加甲醇至甲醇的终体积百分含量为0.5%~1.0%。在本发明中，所述第二振荡培养的温度优选为26~30℃。在本发明中，所述甲醇优选为100%的甲醇或甲醇的终体积百分含量为80%以上的甲醇水溶液。
本发明所述果糖胺脱糖酶能够用于合成或转化氨基葡萄糖。大肠杆菌在发酵过程会产生内毒素等，本发明采用毕赤酵母能够实现氨基葡萄糖食品级生产。在本发明中，所述合成或转化的底物优选包括淀粉，葡萄糖，果糖，果糖-6-磷酸中的一种或两种以上。氨基供体为丙氨酸，谷氨酸，天门冬氨酸或谷氨酰胺。在本发明中，所述合成或转化的方法为在底物和氨基供体存在的环境下，控制pH为6~8，温度为30~50℃进行合成或转化。在本发明中，所述pH更优选为6.7~7.5，温度更优选为40℃~50℃。本发明所述合成和/或转化过程，还能同时得到酮酸产物，如以丙氨酸为底物时，可以获得丙酮酸，以谷氨酸为底物时，能够获得α-酮戊二酸。本发明所述合成或转化的反应为可逆反应，本发明也可以利用酮酸和氨基葡萄糖作为反应原料，制备氨基酸和糖类。在本发明中，利用酮酸和氨基葡萄糖为反应原料时，果糖胺脱糖酶能够催化得到酮酸对应的氨基酸，以及果糖-6-磷酸。利用果糖-6-磷酸可以采用常规方法合成果糖、葡萄糖和淀粉，实现其它糖类的获得。
下面结合具体实施例对本发明所述的一种果糖胺脱糖酶毕赤酵母表达载体、基因工程菌及构建方法和蛋白表达方法做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。
实施例1
果糖胺脱糖酶基因的酵母表达载体的构建
（1）引物设计：从信号肽之后的成熟肽序列开始起设计引物（如SEQ ID NO.3和SEQID NO.4所示）；
（2）PCR反应，以克隆载体pCold Ⅱ-FrIB为模板（本发明对pCold Ⅱ-FrIB的构建方法没有限定，采用常规构建方法将FrIB构建于pCold Ⅱ-FrIB中即可），62℃退火，35cycles。
（3）SnaBI或NotI双酶切的相应生物酶基因的PCR产物和质粒pPIC9K。
表1 双酶切体系
| 成分 | 使用量 |
| 纯化PCR产物/质粒 | 30μL |
| 10*quitcut buffer | 5 μL |
| QuitCut SnaBI | 1μL |
| QuitCut NotI | 1μL |
| ddH2O | 13μL |
| 总体积 | 50μL |
37 ℃酶切2 h。
（4）如常规方法连接，转化，并进行双酶切鉴定。
对这个克隆抽提质粒，从AOX3和AOX5两端进行全长测序，进一步验证插入的目的基因的正确性。
实施例2
含有果糖胺脱糖酶基因的重组酵母菌的构建和诱导表达
1、毕赤酵母电转化感受态的制备
向25 mL YPD培养基中接种毕赤酵母GS115单菌落，30℃，250 r /min过夜培养至OD600约为2~6。转接1 mL培养物至100 mL YPD培养基中，继续培养至OD600约为1.4。6,000 r/min，离心10 min后，弃掉上清液。将细胞轻轻重悬于50 mL Solution I溶液中，室温静止30 min后，6, 000 r /min, 4℃离心10 min。弃掉上清后，将细胞轻轻重悬于10 mL 1 mo1/L山梨醇溶液中，将细胞洗涤4次后，再次将细胞轻轻重悬于0.5 mL 1 mo1/L山梨醇溶液中，每管80μL分装，-80℃冰箱冻存备用。
2、重组质粒pPIC9K-FrIB线性化
用SacⅠ线性化重组质粒pPIC9K-FrIB，体系50μL：10×Cut smart Buffer 5μL，重组质粒pPIC9K-FrIB 30μg，5U/μL的Q.cut SacⅠ2μL，补水到50μL。37℃金属浴消化5 min，65℃灭活10 min。加入体积量10%的3M pH 5.2乙酸钠水溶液，1倍体积量的100%乙醇沉淀，-20℃静置30 min，离心弃上清，再用75%乙醇洗1次，离心弃上清，干燥，加灭菌水溶解即可。
3、电转化毕赤酵母
将线性化质粒进行电泳以及胶回收，回收得到相应的片段后，电转化毕赤酵母GS115感受态。取2~3μL浓度为5μg/μL的线性化质粒与80μL感受态细胞混合均匀，转移至0.2cm预冷的电转杯，冰上放置5 min，电压1500 V，电容25μF，电阻200Ω进行电击，电击后立刻加入1 mL 1 mo1/L山梨醇复苏，30℃培养1 h。将转化液离心涂布于MD平板，30℃培养3~4d，长出肉眼可见的单菌落（转化子）。
4、重组质粒在酵母中的诱导表达
选择长出可分的单菌落的MD平板，并将MD平板上长出的单菌落编号，灭菌牙签挑取单菌落，复制到YPD平板上的划线格中，29℃培养1~2d。利用含遗传霉素G418的YPD平板筛选，获得抗2.0mg/ml遗传霉素G418的转化子，如图1所示，由图1可知，于平板中长出阳性克隆子，说明该重组质粒已经成功转入宿主酵母中。
挑取筛选出的四株转化子单菌落，分别接种到50 mL BMGY液体培养基中，28℃振荡培养16 h，测定其OD600在2~6之间。3000 r/min离心5 min，弃上清。用20 mL BMGY液体培养基重悬菌体，28℃、200 r/min振荡培养96 h。期间每12 h向培养基中加100%甲醇至终体积百分含量为0.5%~1.0%。
实施例3
果糖胺脱糖酶蛋白的纯化
重组酶的纯化及其SDS-PAGE凝胶电泳分析
蛋白质纯化参考GE Healthcare指南，SDS-PAGE分析按照《分子克隆实验指南》(第三版)，使用的凝胶浓度为12.5 %，上样量5~25 μL。蛋白质用考马斯亮蓝R-250染色。
其中native-SDS-PAGE实验步骤：
A、在5~10 μL酶液中加入5~10 μL样品缓冲液[0.1 mol/L三羟甲基氨基甲烷盐酸（Tris-HC1），pH 6.8；2 % SDS（重量︰体积），10%甘油（体积︰体积），0.01%溴酚蓝（重量︰体积）]在37℃水浴中放置5~10 min，再进行上样电泳分离。注：在提取样品时，样品提取液中不加巯基乙醇是为了在电泳过程中使蛋白酶适度变性，以便在电泳结束后能恢复这些蛋白酶的活性。β-巯基乙醇：用于打开二硫键的，使蛋白质的四级或三级结构被破坏。是一种具有特殊臭味的无色透明液体，易燃、易溶于水和醇、醚等多种有机溶剂。
B、制胶与电泳：在制备分离胶的过程中加入0.2 %（体积百分含量） 的明胶，混合均匀后再进行灌胶，凝固后即为Gelatin-SDS-PAGE（底物胶）。“浓缩胶”的聚丙烯酰胺密度为5 %，“分离胶”中聚丙烯酰胺密度为12 %，厚度为1mm3。加样跑电泳。注：明胶因为是在制备凝胶时加入，所以已经交联在凝胶中，在电泳中不会在电场的作用下泳动。
C、去SDS：电泳完成后，将分离胶在复性缓冲液[2 %（体积百分含量） TritonX-100，50 mmol/L Tris-HC1，pH 7.5]中浸洗2~3次，每次5~10 min。
D、复性：将分离胶置于缓冲液[50 mmol/L Tris-HC1，pH7.5]中在37 ℃下放置进行酶反应3 h。
E、染色与脱色：用考马斯亮蓝染色30 min，然后数小时换一次脱色液（5%（体积百分含量） 乙酸+10%（体积百分含量） 甲醇），直至背景清晰。注：经染色和脱色处理的凝胶背景颜色为蓝黑色，蛋白酶反应部位颜色变浅。凝胶中呈现蛋白酶反应的区域大小和该部位的透光率与蛋白酶活性成正比。
由图2所示，泳道1为蛋白Marker；泳道2为果糖胺脱糖酶FrIB的SDS-PAGE电泳图。蛋白在39.7kD左右有明显条带，与理论值相符，证明该蛋白可以在酵母菌中成功表达并被纯化出来。
实施例4
纯化后的果糖胺脱糖酶的活性检测
反应体系：
1mL反应体系中含有15mM的丙氨酸，20mM的果糖-6-磷酸，0.2 mL的果糖胺脱糖酶，2.5mM的EDTA及100mM的不同pH值缓冲液（Na2HPO4-NaH2PO4，pH2.0~10.0），将以上反应体系放在1.5mL离心管中混匀，然后放入PCR仪中37℃下反应20min，反应结束后95℃下加热5min终止反应，离心取上清通过使用高效液相色谱法(HPLC)检测丙酮酸产量，从而计算出果糖胺脱糖酶酶活。
检测条件：
采用高效液相色谱法（HPLC）定量分析丙酮酸。所用色谱柱为C18柱，流动相为乙腈（含0.1%（体积百分含量） 二环已胺、0.1%（体积百分含量） 甲酸）的水溶液（体积比5：95），流速为0.6 mL/min，柱温40 ℃，检测波长为230 nm。此法以丙酮酸钠作对照品，按外标法以峰面积计算反应液中丙酮酸的含量。
酶活力定义：
在最适反应温度37℃条件下，每分钟催化1μmol丙氨酸转化为丙酮酸所需的酶量定义为一个酶活单位，即1U。
经过测试，发现酶活为2.49 U/mg。上述数据表明该酵母表达系统的重组果糖胺脱糖酶具有很好合成氨基葡萄糖和丙酮酸的功能，具有较大应用潜力。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
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Claims (1)
1.基于含果糖胺脱糖酶毕赤酵母表达载体的基因工程菌的果糖胺脱糖酶表达方法，包括以下步骤：
将含果糖胺脱糖酶毕赤酵母表达载体的基因工程菌接种到BMGY液体培养基中，进行第一振荡培养至OD600在2~6之间，离心，弃上清，得到培养菌体；
用BMGY液体培养基重悬所述培养菌体，在28℃、200 r/min进行第二振荡培养96 h；所述第二振荡培养的期间，每12 h向培养基中加100%甲醇至甲醇的终体积百分含量为0.5%~1.0%；
所述含果糖胺脱糖酶毕赤酵母表达载体的基因工程菌的宿主菌为毕赤酵母GS115；所述果糖胺脱糖酶毕赤酵母表达载体，包括骨架载体和编码果糖胺脱糖酶的基因；所述编码果糖胺脱糖酶的基因的核苷酸如SEQ ID NO.1所示；所述骨架载体为pPIC9K；
所述含果糖胺脱糖酶毕赤酵母表达载体的基因工程菌的制备方法包括如下步骤：取2~3μL浓度为5μg/μL的线性化质粒与80μL毕赤酵母GS115感受态细胞混合均匀，转移至0.2cm预冷的电转杯，冰上放置5min，电压1500V，电容25μF，电阻200Ω进行电击，电击后立刻加入1mL 1mo1/L山梨醇复苏，30℃培养1h；将转化液离心涂布于MD平板，30℃培养3~4d。
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