CN114107358B - 一种增加应激性海藻糖含量的耐热性黑曲霉工程菌的构建方法 - Google Patents
一种增加应激性海藻糖含量的耐热性黑曲霉工程菌的构建方法 Download PDFInfo
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- CN114107358B CN114107358B CN202011411844.7A CN202011411844A CN114107358B CN 114107358 B CN114107358 B CN 114107358B CN 202011411844 A CN202011411844 A CN 202011411844A CN 114107358 B CN114107358 B CN 114107358B
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Abstract
本发明公开了一种增加应激性海藻糖含量的耐热性黑曲霉工程菌的构建方法，属于基因工程技术领域。本发明公开的耐热性黑曲霉工程菌的构建方法如下：首先构建异源表达tpsA和tpsB基因的重组质粒，tpsA和tpsB基因序列片段由cox启动子以及TtrpC终止子控制；同时利用同源重组技术敲除海藻糖降解基因ATH1，从而获得高耐热性的黑曲霉工程菌。本发明经过实验证实，与野生型菌株相比，该黑曲霉基因工程菌株内应激性海藻糖含量显著升高，43℃条件下工程菌可正常生长和产酶，葡萄糖酸产量未见降低，为高温条件下利用微生物发酵制备葡萄糖酸钠提供了优良菌种。
Description
技术领域
本发明涉及基因工程技术领域，更具体的说是涉及一种增加应激性海藻糖含量的耐热性黑曲霉工程菌的构建方法。
背景技术
海藻糖广泛存在于低等植物、藻类、细菌、真菌、昆虫及无脊椎动物中，既是一种贮藏性糖类，又是应激代谢的重要产物。由于海藻糖是由特殊双糖分子构成的非还原性糖，性质非常稳定，能够在高温、高寒、干燥失水等恶劣的条件下在细胞表面形成特殊的保护膜，有效的保护生物分子结构不被破坏，从而维持生命体的生命过程和生物特征。真核生物中海藻糖合成分为两步：首先在6-磷酸海藻糖合成酶(由TPSA编码)的作用下，由尿苷二磷酸-D-葡萄糖和葡萄糖-6-磷酸合成海藻糖-6-磷酸；然后在海藻糖磷酸合酶(由TPSB编码)的催化下，海藻糖-6-磷酸脱去磷酸生成海藻糖。
当对数生长期的细胞面临高温、氯化钠、过氧化氢、硫酸铜、高浓度酒精(体积分数>7％)、弱有机酸等胁迫时，细胞内海藻糖浓度会有不同程度的上升。Hottiger等认为海藻糖参与热耐受的机制是：(1)增加热环境下蛋白的稳定性；(2)抑制热应激所致的蛋白凝集。Singer等通过研究酵母细胞提出，适当的海藻糖含量可维持变性蛋白的半折叠状态，有利于分子伴侣的进一步加工。这些研究表明海藻糖在提高细胞热耐受性方面具有重要意义。
葡萄糖酸钠是一种多羟基有机酸盐，在化工、食品、医药、轻工等行业有广泛的用途。目前葡萄糖酸钠的生产多采用黑曲霉发酵法，并且建立了先进的发酵设备和完善的工艺控制流程，企业的生产能力主要受制于生产菌种的活力及性能。目前葡萄糖酸钠发酵生产利用最好的菌株是黑曲霉，但菌株耐高温性能差是相关企业普遍面临的难题。黑曲霉生长的最适温度一般在33℃-37℃，当发酵罐温度超过40℃时，菌株生长和产酶速率显著下降。每当夏季生产车间温度最高可达40℃以上，为保证正常生产必需消耗大量的冷却水给发酵罐降温，由此消耗大量能量。基于此，构建高耐热性产葡萄糖酸钠的黑曲霉工程菌的任务迫在眉睫。
因此，提供一种增加应激性海藻糖含量的耐热性黑曲霉工程菌的构建方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种增加应激性海藻糖含量的耐热性黑曲霉工程菌的构建方法。
为了实现上述目的，本发明采用如下技术方案：
一种增加应激性海藻糖含量的耐热性黑曲霉工程菌的构建方法，具体步骤如下：
(1)以黑曲霉FG-2基因组为模板，PCR扩增cox启动子序列；以pAN7-1质粒为模板，PCR扩增TtrpC终止子序列；采用多片段的一步克隆试剂盒将上述2段基因片段和线性化的pAN7-1载体进行连接，获得重组载体pAN7-1-cox-TtrpC；所述cox启动子的核苷酸序列如SEQ ID NO.1所示；所述TtrpC终止子的核苷酸序列如SEQ ID NO.4所示；
(2)根据黑曲霉密码子偏好性设计的柔性肽W1、W2和W3序列，刚性肽P1、P2和P3序列作为基因tpsA和tpsB的linker；以黑曲霉FG-2基因组为模板，分别PCR扩增tpsA和tpsB基因片段；采用重叠PCR的方式获得不同的tpsA-linker-tpsB基因片段；
(3)利用一步克隆的方法，将tpsA-linker-tpsB基因片段与线性化的pAN7-1-cox-TtrpC载体连接，获得重组质粒pAN7-1-cox-tpsA-linker-tpsB-TtrpC；所述基因tpsA和tpsB序列片段由启动子cox和终止子TtrpC控制；所述tpsA基因的核苷酸序列如SEQ IDNO.29所示，所述tpsB基因的核苷酸序列如SEQ ID NO.30所示；并将上述重组质粒采用原生质体转化的方式转入黑曲霉宿主菌FG-2，经转化子筛选和潮霉素抗性基因PCR验证获得过表达tpsA和tpsB基因的工程菌株FG(tpsAB-linker+)；
(4)PCR扩增Bar基因及ATH1基因上下游400bp序列；利用重叠PCR将上述3个基因片段连接，获得序列Bar-UD；将序列Bar-UD通过原生质体转化的方式转入过表达tpsA和tpsB基因的黑曲霉FG(tpsAB-linker+)细胞中，筛选获得敲除了ATH1基因的工程菌ΔATH1-FG(tpsAB-linker+)。
进一步，所述的耐热性黑曲霉工程菌在高温条件下生产葡萄糖酸钠的应用。
经由上述的技术方案可知，与现有技术相比，本发明公开提供了一种增加应激性海藻糖含量的耐热性黑曲霉工程菌的构建方法，本发明基于黑曲霉产生有机酸的天然特性，通过遗传重组改造黑曲霉的生理特性，获得了一株黑曲霉基因工程菌株，经过实验证实，与野生型菌株相比，该黑曲霉基因工程菌株内应激性海藻糖含量显著升高，43℃条件下工程菌可正常生长和产酶，葡萄糖酸钠产量未见降低，为微生物发酵法制备葡萄糖酸钠提供了优良菌种。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1附图为本发明PCR验证的载体pAN7-1中已插入cox+TtrpC片段的核酸凝胶电泳图；
其中，M为DNA Marker；1为菌落PCR筛选获得的阳性克隆中cox+TtrpC片段；2为菌落PCR筛选获得的阳性克隆中cox片段；
图2附图为本发明PCR验证的黑曲霉FG-2基因组中已插入cox-tpsA-linker-tpsB-TtrpC片段的核酸凝胶电泳图；
其中，M为DNA Marker；1为菌落PCR筛选获得的阳性克隆中cox-tpsA-linker-tpsB-TtrpC片段；2为菌落PCR筛选获得的阳性克隆中tpsA-linker-tpsB片段；
图3附图为本发明PCR验证的FG(tpsAB-linker+)中的ATH1基因已敲除核酸凝胶电泳图；
其中，M为DNA Marker；CK为黑曲霉FG-2基因组PCR产物；1-2为抗性筛选的2个菌落基因组PCR产物，仅1为菌落PCR筛选获得的阳性克隆。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1构建异源表达tpsA和tpsB基因的质粒
以黑曲霉FG-2基因组为模板，cox-F/R为引物，PCR扩增启动子cox序列(cox基因的核苷酸序列见SEQ ID NO.1)；PCR反应体系：2 x Phanta Max Buffer 25μl；dNTP Mix(10mMeach)1μl；Phanta Max Super-Fidelity DNA Polymerase 0.6μl；模板1μl；上下游引物(100μM)各0.1μl；ddH2O补足至50μl。反应程序：95℃预变性10min；95℃变性30s，59℃退火30s，72℃延伸1min，30个循环；72℃延伸10min；4℃保温。具体引物序列如下：
cox-F：5’-CCTGCAGGCATGCAAGCTTGAGGAAAACAAGAACTTGACGTGG-3’；SEQ ID NO.2；
cox-R：5’-AGTAACGTTAAGTGGATCCTGTCCTGGTGGGTGGGTTGCA-3’；SEQ ID NO.3。
以pAN7-1为模板，TtrpC-F/R和pct-F/R为引物，分别PCR扩增终止子TtrpC序列(TtrpC基因的核苷酸序列见SEQ ID NO.4)和线性化载体pAN7-1；PCR反应体系：2 x PhantaMax Buffer 25μl；dNTP Mix(10mM each)1μl；Phanta Max Super-Fidelity DNAPolymerase 0.6μl；100x模板1μl；上下游引物(100μM)各0.1μl；ddH2O补足至50μl。反应程序：95℃预变性5min；95℃变性30s，59℃退火30s，72℃延伸1min30s，30个循环；72℃延伸10min；4℃保温。具体引物序列如下：
TtrpC-F：5’-TGCAACCCACCCACCAGGACAGGATCCACTTAACGTTACT-3’；SEQ ID NO.5；
TtrpC-R：5’-TGTAAAACGACGGCCAGTGCTCGAGTGGAGATGTGGAGTG-3’；SEQ ID NO.6；
pct-F：5’-GCACTGGCCGTCGTTTTACA-3’；SEQ ID NO.7；
pct-R：5’-CAAGCTTGCATGCCTGCAG-3’；SEQ ID NO.8；
然后使用诺唯赞一步克隆试剂盒将片段cox、TtrpC和线性化载体pAN7-1进行重组反应，获得重组载体pAN7-1-cox-TtrpC。重组反应体系：线性化载体pAN7-1 3.2μl，插入片段cox 0.6μl，插入片段TtrpC 0.5μl，2xClonExpress Mix 5μl，ddH2O补足至10μl。将重组产物转化到DH5ɑ感受态细胞中，以氨苄(Amp+)进行抗性筛选，通过菌落PCR筛选阳性克隆，并提取质粒进行PCR验证，分别采用引物对cox-F/TtrpC-R、cox-F/R进行PCR验证，PCR反应体系：2 x Phanta Max Buffer 10μl；dNTP Mix(10mM each)1μl；Phanta Max Super-Fidelity DNA Polymerase 0.6μl；模板1μl；上下游引物(100μM)各0.1μl；ddH2O补足至20μl。反应程序：95℃预变性5min；95℃变性30s，59℃退火30s，72℃延伸2min，30个循环；72℃延伸10min；4℃保温。PCR验证结果见图1，泳道1为cox+TtrpC片段1691bp，泳道2为cox片段921bp。
实施例2异源表达tpsA和tpsB基因菌株的获得
根据黑曲霉密码子偏好性设计了柔性肽W1、W2和W3序列，刚性肽P1、P2和P3序列作为基因tpsA和tpsB的linker。
具体引物序列如下：
W1：5’-GAAGCTGCGGCAAAA-3’；SEQ ID NO.9；
W2：5’-GAAGCTGCGGCAAAAGAAGCAGCGGCTAAA-3’；SEQ ID NO.10；
W3：5’-GAAGCTGCGGCAAAAGAAGCAGCGGCTAAAGAAGCGGCGGCAAAA-3’；SEQ ID NO.11；
P1：5’-GGTGGCGGTGGCTCGGGCGGTGGTGGGTCG-3’；SEQ ID NO.12；
P2：5’-GGTGGCGGTGGCTCGGGCGGAGGTGGGTCGGGTGGCGGCGGATCC-3’；SEQ ID NO.13；
P3：5’-GGTGGCGGTGGCTCGGGTGGCGGTGGCTCGGGCGGAGGTGGGTCGGGTGGCGGCGGATCG-3’；SEQ ID NO.14；
tpsA基因的上游引物序列为tpsA-F：
5’-GCAACCCACCCACCAGGACAATTTTCTTCATTGGTCGCGT-3’；SEQ ID NO.15；
扩增含linker的tpsA基因的下游引物序列如下：
tpsA-W1-R：5’-TTTTGCCGCAGCTTCTCAACGCCCGCCTTCCAC-3’；SEQ ID NO.16；
tpsA-W2-R：5’-GCTTCTTTTGCCGCAGCTTCTCAACGCCCGCCTTCCAC-3’；SEQ ID NO.17；
tpsA-W3-R：5’-TTTAGCCGCTGCTTCTTTTGCCGCAGCTTCTCAACGCCCGCCTTCCAC-3’；SEQID NO.18；
tpsA-P1-R：5’-CCGCCCGAGCCACCGCCACCTCAACGCCCGCCTTCCAC-3’；SEQ ID NO.19；
tpsA-P2-R：5’-CGACCCACCTCCGCCCGAGCCACCGCCACCTCAACGCCCGCCTTCCAC-3’；SEQID NO.20；
tpsA-P3-R：5’-CACCTCCGCCCGAGCCACCGCCACCCGAGCCACCGCCACCTCAACGCCCGCCTTCCAC-3’；SEQ ID NO.21；
扩增含linker的tpsB基因的上游引物序列如下：
tpsB-W1-F：5’-GAAGCTGCGGCAAAAATGACCATCTACATCGCTTC-3’；SEQ ID NO.22；
tpsB-W2-F：5’-CAAAAGAAGCAGCGGCTAAAATGACCATCTACATCGCTTC-3’；SEQ IDNO.23；
tpsB-W3-F：5’-GAAGCAGCGGCTAAAGAAGCGGCGGCAAAAATGACCATCTACATCGCTTC-3’；SEQ ID NO.24；
tpsB-P1-F：5’-GCTCGGGCGGTGGTGGGTCGATGACCATCTACATCGCTTC-3’；SEQ IDNO.25；
tpsB-P2-F：5’-GGCGGAGGTGGGTCGGGTGGCGGCGGATCCATGACCATCTACATCGCTTC-3’；SEQ ID NO.26；
tpsB-P3-F：5’-CGGTGGCTCGGGCGGA GGTGGGTCGGGTGGCGGCGGATCGATGACCATCTACATCGCTTC-3’；SEQ ID NO.27；
tpsB基因的下游引物序列为tpsB-R：
5’-GGATTGGAGATTTTGCGTGTTTATGTAGCATTAATTGATT-3’；SEQ ID NO.28。
以黑曲霉FG-2基因组为模板，分别采用引物对tpsA-F/tpsA-n-R PCR扩增tpsA基因片段(tpsA基因的核苷酸序列见SEQ ID NO.29)，引物对tpsB-n-F/tpsB-R扩增tpsB基因片段(tpsB基因的核苷酸序列见SEQ ID NO.30)。PCR反应体系：2 x Phanta Max Buffer 25μl；dNTP Mix(10mM each)1μl；Phanta Max Super-Fidelity DNA Polymerase 0.6μl；模板1μl；上下游引物(100μM)各0.1μl；ddH2O补足至50μl。反应程序：95℃预变性10min；95℃变性30s，55℃退火30s，72℃延伸2min，30个循环；72℃延伸10min；4℃保温。
利用一步克隆的方法，将基因片段tpsA和tpsB与线性化的pAN7-1-cox-TtrpC载体连接，重组反应体系：线性化载体pAN7-1-cox-TtrpC 2.6μl，插入片段tpsA 1.3μl，插入片段tpsB 0.9μl，2xClonExpress Mix 5μl，ddH2O补足至10μl，将重组产物转化到DH5ɑ感受态细胞中，以氨苄(Amp+)进行抗性筛选，通过菌落PCR筛选阳性克隆，并提取质粒进行PCR验证，分别采用引物对cox-F/TtrpC-R和tpsA-F/tpsB-R进行PCR验证，PCR反应体系：2 xPhanta Max Buffer 10μl；dNTP Mix(10mM each)1μl；Phanta Max Super-Fidelity DNAPolymerase 0.6μl；模板1μl；上下游引物(100μM)各0.1μl；ddH2O补足至20μl。反应程序：95℃预变性5min；95℃变性30s，57.5℃退火30s，72℃延伸4min30s，30个循环；72℃延伸10min；4℃保温。
以原生质体转化的方式将上述重组质粒pAN7-1-cox-tpsA-linker-tpsB-TtrpC转入黑曲霉FG-2细胞中，以潮霉素(Hygromycin，250μg/mL)进行抗性筛选，通过菌落PCR筛选阳性克隆，并提取基因组进行PCR验证，分别采用引物对cox-F/TtrpC-R和tpsA-F/tpsB-R进行PCR验证，PCR反应体系：2 x Phanta Max Buffer 10μl；dNTP Mix(10mM each)1μl；Phanta Max Super-Fidelity DNA Polymerase 0.6μl；模板1μl；上下游引物(100μM)各0.1μl；ddH2O补足至20μl。反应程序：95℃预变性10min；95℃变性30s，57.5℃退火30s，72℃延伸4min 30s，30个循环；72℃延伸10min；4℃保温。PCR验证结果见图2，cox-tpsA-linker-tpsB-TtrpC片段约6.6kb，tpsA-linker-tpsB片段约4.9kb。所获得的过表达tpsA和tpsB基因的工程菌株命名为FG(tpsAB-linker+)。
实施例3黑曲霉中海藻糖降解基因ATH1的敲除
以黑曲霉FG-2基因组为模板，分别采用引物对AB-F/R和CB-F/R进行PCR扩增以获得片段AB和CB；两者PCR反应体系和反应程序一致，PCR反应体系：2 x Phanta Max Buffer25μl；dNTP Mix(10mM each)1μl；Phanta Max Super-Fidelity DNA Polymerase 0.6μl；模板1μl；上下游引物(100μM)各0.1μl；ddH2O补足至50μl。反应程序：95℃预变性10min；95℃变性30s，63℃退火30s，72℃延伸1min，30个循环；72℃延伸10min；4℃保温。
具体引物序列如下：
AB-F：5’-CATTCCAGGGCCTTCCGTACCC-3’；SEQ ID NO.31；
AB-R：5’-TAGGCTCCCGGCCTTTGCAAGAATTCGGTCGAAAAAAGAAAA-3’；SEQ ID NO.32；
CB-F：5’-TGCCCGTCACCGAGATCTGATATGAATTACGATCGATCGA-3’；SEQ ID NO.33；
CB-R：5’-GAGTGGGGAGGGCGCGTTGC-3’；SEQ ID NO.34。
以pBARGPE1质粒为模板，采用引物对Bar-F/R进行PCR扩增获得Bar基因片段(Bar基因的核苷酸序列见SEQ ID NO.35)；PCR反应体系：2 x Phanta Max Buffer 25μl；dNTPMix(10mM each)1μl；Phanta Max Super-Fidelity DNA Polymerase 0.6μl；100x模板1μl；上下游引物(100μM)各0.1μl；ddH2O补足至50μl。反应程序：95℃预变性5min；95℃变性30s，63.5℃退火30s，72℃延伸1min30s，30个循环；72℃延伸10min；4℃保温。
具体引物序列如下：
Bar-F：5’-TAGGCTCCCGGCCTTTGCAAGAATTCGGTCGAAAAAAGAAAA-3’；SEQ ID NO.36；
Bar-R：5’-TCGATCGATCGTAATTCATATCAGATCTCGGTGACGGGCA-3’；SEQ ID NO.37。
使用引物AB-F/CB-R进行重叠PCR将片段AB、CB和Bar连接，获得序列Bar-UD。PCR反应体系：2 x Phanta Max Buffer 25μl；dNTP Mix(10mM each)1μl；Phanta Max Super-Fidelity DNA Polymerase 0.6μl；三种片段模板各1μl；上下游引物(100μM)各0.1μl；ddH2O补足至50μl。PCR反应程序：95℃预变性10min；95℃变性45s，61℃退火45s，72℃延伸2min，30个循环；72℃延伸10min；4℃保温。将上述片段Bar-UD以黑曲霉原生质体转化的方式转入黑曲霉FG(tpsAB-linker+)细胞中，利用同源重组的方法敲除ATH1基因(ATH1基因的核苷酸序列见SEQ ID NO.38)。以草铵膦(Glufosinate-ammonium，200μg/mL)进行抗性筛选，通过菌落PCR筛选阳性克隆，并提取基因组进行PCR验证，引物分别为AB-F/CB-R，PCR反应体系：2 x Phanta Max Buffer 10μl；dNTP Mix(10mM each)1μl；Phanta Max Super-Fidelity DNA Polymerase 0.6μl；模板1μl；上下游引物(100μM)各0.1μl；ddH2O补足至20μl。反应程序：95℃预变性10min；95℃变性30s，61℃退火30s，72℃延伸2min，30个循环；72℃延伸10min；4℃保温。PCR验证结果见图3，ATH1基因若已敲除条带大小约为1.9k；若未敲除则条带大小应为4.2k左右。所构建的敲除ATH1基因的新菌株命名为ΔATH1-FG(tpsAB-linker+)。
实施例4新构建工程菌株内海藻糖含量的检测
挑取野生型FG-2、6种构建的FG(tpsAB-linker+)的菌株及6种工程菌ΔATH1-FG(tpsAB-linker+)分别将孢子数为1.2x107的孢子悬液按10％的接种量接种到含100mL发酵培养基(葡萄糖20g/L，磷酸二氢钾0.013g/L，玉米浆0.1g/L，尿素0.02g/L，硫酸镁0.002g/L，碳酸钙4g/L)的500ml三角瓶中，37℃、250rpm条件下培养48h。
将发酵液进行离心(10000rpm/min，8min)，获得的菌体沉淀洗涤多次后进行超声破碎，破碎后得到的液体过滤后采用HPLC液相检测海藻糖，使用Xbridge-NH2色谱柱，流动相为乙腈/水＝4：1加0.1％的氨水，流速1.0mL/min，柱温35℃，控制质量浓度在0.1～4.0g/100mL范围。不同菌株海藻糖产量见表1。
表1 不同菌株的海藻糖产量
实施例5工程菌耐高温性能检测
挑取野生型FG-2、多次重复试验筛选出的优势菌株FG(tpsAB-W2+)菌株及工程菌ΔATH1-FG(tpsAB-W2+)分别将孢子数为1.2x107孢子悬液按10％的接种量接种到含100mL发酵培养基(葡萄糖20g/L，磷酸二氢钾0.013g/L，玉米浆0.1g/L，尿素0.02g/L，硫酸镁0.002g/L，碳酸钙4g/L)的500ml三角瓶中，分别在37℃、40℃、43℃和48℃条件下250rpm培养48h。
发酵液测pH，过滤取上清测残糖含量，同时取1ml上清稀释后采用伍丰LC-100紫外液相检测葡萄糖酸钠含量，使用Amethyst C18-H色谱柱，流动相为50ml甲醇+1％磷酸500ml，流速1.0ml/min，控制质量浓度在2x10-2-10-5g/ml范围。不同菌株葡萄糖酸钠产量见表2。
表2 不同菌株的葡萄糖酸钠产量
实施例6工程菌15L发酵罐培养验证
取一支黑曲霉工程菌ΔATH1-FG(tpsAB-W2+)，刮取上层孢子，接入培养基中，在37℃培养至长满孢子后配制成菌悬液备用，所用培养基为青岛海博生物技术有限公司购买的改良马丁琼脂培养基。
在5L培养罐中加入葡萄糖0.6kg、麸皮0.66g、磷酸二氢钾0.6g、硫酸镁0.6g、磷酸氢二铵1.86g、消泡剂0.78ml，定容至3L，115℃灭菌20min，然后降至43℃，加入孢子数为1.2x107的菌悬液75ml；保持种子液的pH为5.2；控制溶解氧(DO)≥30％，发酵20h，得到种子液。
在15L发酵罐中(一级发酵罐)加入葡萄糖3.5kg、磷酸二氢钾0.79g、硫酸镁0.53g、尿素0.41g、磷酸氢二铵0.012g、消泡剂2.6ml，定容至10L，115℃灭菌20min，然后降至43℃，接入菌浓为1.6g/L种子液，接种量为10％，用浓度为350g/L的NaOH调节pH控制在5.2，控制发酵液的温度为43℃，使黑曲霉菌丝长度控制在10～15pm的范围内，溶氧控制在20％～30％之间，每2h测定酶活、葡萄糖及葡萄糖酸钠含量，当发酵罐中的葡萄糖含量低于3g/L的时候结束发酵。结果表明：43℃条件下，工程菌株ΔATH1-FG(tpsAB-W2+)发酵20h左右，葡萄糖酸钠产量就可达到32.7g/100mL发酵液。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
序列表
<110> 德州汇洋生物科技有限公司
<120> 一种增加应激性海藻糖含量的耐热性黑曲霉工程菌的构建方法
<160> 38
<170> SIPOSequenceListing 1.0
<210> 1
<211> 921
<212> DNA
<213> Artificial Sequence
<400> 1
aggaaaacaa gaacttgacg tggaagcaaa ttgcagactt cttcccgggc cgaacgagcg 60
gcaccttgca agtccgatac tgcaccaagc tgaaggctaa ggatgtagct tggagtgacg 120
aaatggttcg attttctcct gatgtatttc tacggctgtc tcacacatgc taatgaaggg 180
aataggtaca aaggctgcag cgggcaatgc acgagtacga gaacgatcgg tggcgcatca 240
ttgcagggaa ggttggaaat ggcttcaccc cagctgcttg ccgcgagaaa gccatgcagc 300
tccatgagta aaaccgttgg ggaattttca tatttatatc tactgtcgcc agattcggcc 360
ctgcttggac cctctgatct ccttactctc catattggtt caaatgtcgg gtctccgata 420
gggctggtgg tgcaggcttg ttgtaggcac gggaggatga tcagcataac tctgattcac 480
tatagggacg ggttgatgta aggtattaag tgatgtatga taattcattt tagcccgggg 540
gaacatatgg cgccggcatt tgttcgttcg caatgaaccg acactagcgt ccgctctcgc 600
agtttagcac cggctgatcc cgggctgaac gcggccattg ctcggccggg gcatgtgttc 660
cttaatctac ggcagaccgc agaagaccac tggcgcagat tgtagaccct aagccctaag 720
acggacaccc aatcgagtag gtctgcggac caggtcactg cgggcagccg gagaagctcc 780
gcaaccaatc aatccccggc gctgactaag ggcaggcgac cacgggccga agcggcttca 840
aactcacctc aacctccaaa ctccctcatc tccaaacgtc cttgccttgt ctgccgtcat 900
tgcaacccac ccaccaggac a 921
<210> 2
<211> 43
<212> DNA
<213> Artificial Sequence
<400> 2
cctgcaggca tgcaagcttg aggaaaacaa gaacttgacg tgg 43
<210> 3
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 3
agtaacgtta agtggatcct gtcctggtgg gtgggttgca 40
<210> 4
<211> 770
<212> DNA
<213> Artificial Sequence
<400> 4
ggatccactt aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt 60
tggtgtcgat gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg 120
ttcatttgtc caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag 180
aataaaacgc gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg 240
cattgactgc aacctagtaa cgccttncag gctccggcga agagaagaat agcttagcag 300
agctattttc attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg 360
agactgagga atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg 420
acatgctcct cttctttact ctgatagctt gactatgaaa attccgtcac cagcncctgg 480
gttcgcaaag ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc 540
atcgtaggta taaacctcga aatcanttcc tactaagatg gtatacaata gtaaccatgc 600
atggttgcct agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact 660
ctcctatgag tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt 720
ctagaagtcc tcgtgtactg tgtaagcgcc cactccacat ctccactcga 770
<210> 5
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 5
tgcaacccac ccaccaggac aggatccact taacgttact 40
<210> 6
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 6
tgtaaaacga cggccagtgc tcgagtggag atgtggagtg 40
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 7
gcactggccg tcgttttaca 20
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 8
caagcttgca tgcctgcag 19
<210> 9
<211> 15
<212> DNA
<213> Artificial Sequence
<400> 9
gaagctgcgg caaaa 15
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 10
gaagctgcgg caaaagaagc agcggctaaa 30
<210> 11
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 11
gaagctgcgg caaaagaagc agcggctaaa gaagcggcgg caaaa 45
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 12
ggtggcggtg gctcgggcgg tggtgggtcg 30
<210> 13
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 13
ggtggcggtg gctcgggcgg aggtgggtcg ggtggcggcg gatcc 45
<210> 14
<211> 60
<212> DNA
<213> Artificial Sequence
<400> 14
ggtggcggtg gctcgggtgg cggtggctcg ggcggaggtg ggtcgggtgg cggcggatcg 60
<210> 15
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 15
gcaacccacc caccaggaca attttcttca ttggtcgcgt 40
<210> 16
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 16
ttttgccgca gcttctcaac gcccgccttc cac 33
<210> 17
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 17
gcttcttttg ccgcagcttc tcaacgcccg ccttccac 38
<210> 18
<211> 48
<212> DNA
<213> Artificial Sequence
<400> 18
tttagccgct gcttcttttg ccgcagcttc tcaacgcccg ccttccac 48
<210> 19
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 19
ccgcccgagc caccgccacc tcaacgcccg ccttccac 38
<210> 20
<211> 48
<212> DNA
<213> Artificial Sequence
<400> 20
cgacccacct ccgcccgagc caccgccacc tcaacgcccg ccttccac 48
<210> 21
<211> 58
<212> DNA
<213> Artificial Sequence
<400> 21
cacctccgcc cgagccaccg ccacccgagc caccgccacc tcaacgcccg ccttccac 58
<210> 22
<211> 35
<212> DNA
<213> Artificial Sequence
<400> 22
gaagctgcgg caaaaatgac catctacatc gcttc 35
<210> 23
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 23
caaaagaagc agcggctaaa atgaccatct acatcgcttc 40
<210> 24
<211> 50
<212> DNA
<213> Artificial Sequence
<400> 24
gaagcagcgg ctaaagaagc ggcggcaaaa atgaccatct acatcgcttc 50
<210> 25
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 25
gctcgggcgg tggtgggtcg atgaccatct acatcgcttc 40
<210> 26
<211> 50
<212> DNA
<213> Artificial Sequence
<400> 26
ggcggaggtg ggtcgggtgg cggcggatcc atgaccatct acatcgcttc 50
<210> 27
<211> 60
<212> DNA
<213> Artificial Sequence
<400> 27
cggtggctcg ggcggaggtg ggtcgggtgg cggcggatcg atgaccatct acatcgcttc 60
<210> 28
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 28
ggattggaga ttttgcgtgt ttatgtagca ttaattgatt 40
<210> 29
<211> 1697
<212> DNA
<213> Artificial Sequence
<400> 29
attttcttca ttggtcgcgt tgatttacta cgtatcaact ctctccatcc agaatgacga 60
agcgcaacct catcgttgtt tcgaaccgtc tccctctgtc gctgaaaaag gtcgacgggg 120
gatacgaatc ttccctctcc agtggaggtc tagtcacatc tttatcggga ttgactaaaa 180
ccactacctt tagctggttc ggatggccag gcatcgacat caccgatgaa accgagcagg 240
gacagattcg caagagcttg gatgaacata acgctgtgcc gattttcttg gataatgaat 300
tggcaaataa tcactataat aacttctcta gtgcgattct tccgttcatt cggtttactg 360
agaataggga gattgctaat ggtttttctg gtatctagat gccatcctct ggcccatcct 420
ccactaccaa tccggcataa acttcgaaga aggcccctgg gaagcttacc aacgagtcaa 480
cgagatcttt gccgatacga tcgccgaggc agcccagccg gggtcattga tctgggtgca 540
cgattaccat cttatgctgt tgccgcggct gttgagagat cgattggcgg ggaagaattg 600
cgcgattggg ttctcgctgc atacgccgtt cccggcgggg gacttttggc ggaatctgcc 660
ggtgcagaag gacttgttga ggggactgct ggcgagtgat gttattggat tccatacgga 720
tgagtataaa agaaattttg tgctttgtgc ggagtctctg taggttactg tctggtgatc 780
tatggtggga tacgtgctga tatgaatgta ttagggccga tgcgaaggtg aaggagtcag 840
ggacgctgga gtacgagggt catgaggtgg ttgcggagaa gttcattgtc ggcatcgatc 900
cgcagaaatt tacggatgca ctgcagaatg aggaggtgca gaatcgcata agggacttgc 960
agcgcaggta tatggggatg aaggttattg ttggggtgga tcggctggat tacatcaagg 1020
ggctcgtgca gaagttgaag ggctatgatg ctttcttgga tcagtatccg gagctgagca 1080
ataaggtggt gctaatccag gtggcggtgc ccagccggga ggatgtgaag gagtaccagg 1140
atctggagac ggagttgagc acgctggcag gaagaatcaa tggaaaacac tgtgggtggc 1200
ctcctgcgtc gacatttgtt gtctactctg tgctgacggg tctagctacc gccgatggct 1260
gtccgctgat ctacatgcac cgatcagtta atttcagcga actgacagcg ctgtactcgg 1320
ttgccgatgc ctgcttgttg acttcaacgc gggatggaat gaaccttgtt tcgttcgagt 1380
acgttgcgtg ccaagcgcag cggcatggag tgctagtgct gtccgagttt gccggagcag 1440
cgtcttttat gaaggagggg tgtcttactt tccatccagc gaatatgtca gagatggttg 1500
atgcggttca tcaagcagtc acgatgagtg gtgatgatcg caagtctcga tatgaaacgt 1560
tgcggcactt tatcgagaca aacactaggt gagtttactg tcacggcacg atacttgggc 1620
tttgctgata atgtagtgca aaatggggcg agacgttcat cgagacattg accaagcatg 1680
tggaaggcgg gcgttga 1697
<210> 30
<211> 3220
<212> DNA
<213> Artificial Sequence
<400> 30
atgaccatct acatcgcttc actgtaagtg cctagcaatg tacacaacta ctagtgtttt 60
cttctgactg tcgcggggtt cagttttctg ccctacacgg ttaatttcca tcagaatgaa 120
cccgagattc gtccggcgga gggggcgagc cctcaaccaa ccaatcccgc tgagaattcc 180
actcctaacc caaacgcgac tctgagcttg ttcgagagga acaatggagc gccccaagtt 240
ggccttacac caggtgccac tactgagcat gaatgcattt tctccacaga catttccaag 300
gcggaacaag agcacaccgg gtttcctttc cccaagactg acggcgaagt gactctgttg 360
acggagagcg aagctcactc cccagcctgg ggctccacct tagccctgaa ccaacctcgc 420
ccacgagcag cattcccggc ctcaccctcg atcctcaagc atcaagaaat cggcccttca 480
gggacagagg cgcccaaaga aaagccacga tcggtcccga agacaccaac ctcgcatatc 540
cgggacagct gggcggacca cagtcgcaaa agctcattct cctacgccga ctggacgatc 600
gagacggccg agcagggcaa cggaggcttg cgcaatgctg tgcgatcggc cacggacgcc 660
ggacagctgg aagacaaggt ctgggtgggt acgttgggga tgcccactga tgcgctaccg 720
gagcacacca aggaggccat cgccgaaaag ctcgaagatg agtacggttc cgtgaccgtt 780
tacgtcagtg acggtgactt tgatggccat tacacgcact tctgcaagac tatcctctgg 840
cccgtgttcc attaccagat tccggacaac cccaagagca aggcttacga ggaccactcc 900
tgggtctact acgtcaagac gaaccaagcc tttgctgagc gcatcgcaaa gaactggaag 960
cgtggtgatt ccatctgggt gcaggattac cacctgctgc tggtcccggc catgctgcgg 1020
aaactgcttc ccgatgccca gattggcttt ttcctccaca ttgccttccc ctcatcggag 1080
gtgttccggt gcttggcgcc tcgcaaggag cttctcgagg gaatactggg tgctaacctg 1140
gtcgggtttc agacggatga gtattgtcga cacttcctcc agacgtgcag ccgcatcctc 1200
tgtgtggagg ccaccaatga tgggctgcag ctggaggatc gctttgtcaa cgttggcaaa 1260
ttctctattg gtattgaccc gacttcatgg gaccaacgcc ggcgggccgc agacgtggaa 1320
cgatggatca agaccatttc ggagcgttac gaggggaagc gactaatcgt gtcgcgggac 1380
aagatcgacc aggtgcgcgg aatccgacag aaactgctga gctacgaact cttcctcaat 1440
acatatcccg aatggcgaga ccaggtggtg ttgatccagg tggcgaccag caccaccgag 1500
cagccggagc tggaagcaac catttccgat atcgcgatgc ggatcaactc cactcactcc 1560
acgcttgcgc atcagccgtt ggtcttctta aagcaggatc tggctttccc ccaatatctc 1620
gccttgattt ccgtggcaga cgccctgatt atcaccagct tacgtgaagg catgaacttg 1680
accagccatg agtttgtcta ctgccaggac ggcaaatggg gcaacaaaaa gtacggatcc 1740
ctgatcctga gcgagttcac tggcagcgcg tcggtgtttg gcgaccatgc ccttctggtc 1800
aacccctggg actaccgtca atgtgcggaa gctatccata ccgccttgtc gagggatgag 1860
caggagcgcc aggaagtgtg gacgaagcta caccaggcag tactgcagaa ctctacccac 1920
aactgggtca agtcattcag tgagacgttg agccgggtct ggaacgagca gtcgtcccgg 1980
gagatcatgg ccgtgccccg gcttcagata aacaagttgg aggaaatgta tcatcgatcg 2040
tcgcgccggc tgatcatcgt ggactatgaa ggcactctcg cctcctgggg ctcaccaaag 2100
agtattattg tgacgactcc gcagcgtgcg atcacaacac tggcagaact aaccgaggat 2160
ccgcggaacg tggtgtatgt gatgagcgcc cgcatgcctg aggagatgga gcggctgttc 2220
cggctggtca ccggcttggg tctgattgcc gaaaacgggt gcttcgtgcg cgagcccaac 2280
tcggagacct ggctgaagtt gacggacaag gtccaaacgg acgcgtggaa ggcggcagtg 2340
tcacacatct tggaatacta ccaggaacgc gcggaaggta gctgggtcga acagcggcac 2400
tgctcgctca tgtttcacta cgagtcggcg gaggaccagg tggcggcgtc gcggctggcg 2460
tcagagtgtg ctggccacat caacgatgct tgtttgagcc aaggggtgca tgcagttccc 2520
gtggagcgtg cgctggtggt ggaaccggcg ggcatcaaca aggcatcggc agcggaagtg 2580
gcatggcggt catgtctcaa gcagagccaa cgggacgaga gtgtgccgcg gccggagttc 2640
ctgctggcga ttggagatgg tcgcgatgat gaatcagtgt tccgatgggc caacaagctc 2700
gacagcgcgc gagcagtcaa ctacgccatg acggtgacgc tcggatcccg tagcacggaa 2760
gcgaaggcga ctctgacaca gggagtgacc ggtaagctgc tctcaataaa ttcacgataa 2820
ggagtgaaag gatgctaact gcagggtgac gaataggagt cttatcgtgt ctggagaagc 2880
tggcggcaac ccgaactggg ccgtgacaag acaaacgatg cggaatgaga atatctacga 2940
gtacatgact tgaagcgagc gtacatgatg ggcaaaatta actcttcact tgcgttgcat 3000
gaaacgggca catatttcgg gcatatcagt acatttttat ttgaacatct tcaactacaa 3060
aagcaagaac aagcaaatag gcgtgcggac tggttttcaa tgtatcgttg ggggtaaaca 3120
gggagaatat agaatgagag aggaagagag agagggcagc tgtgggagtt caaggggggt 3180
cacatagacg aggccataat acacgcaaaa tctccaatcc 3220
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 31
cattccaggg ccttccgtac cc 22
<210> 32
<211> 42
<212> DNA
<213> Artificial Sequence
<400> 32
taggctcccg gcctttgcaa gaattcggtc gaaaaaagaa aa 42
<210> 33
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 33
tgcccgtcac cgagatctga tatgaattac gatcgatcga 40
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 34
gagtggggag ggcgcgttgc 20
<210> 35
<211> 1130
<212> DNA
<213> Artificial Sequence
<400> 35
aattcggtcg aaaaaagaaa aggagagggc caagagggag ggcattggtg actattgagc 60
acgtgagtat acgtgattaa gcacacaaag gcagcttgga gtatgtctgt tattaatttc 120
acaggtagtt ctggtccatt ggtgaaagtt tgcggcttgc agagcacaga ggccgcagaa 180
tgtgctctag actcgacaga agatgatatt gaaggagcac tttttgggct tggctggagc 240
tagtggaggt caacaatgaa tgcctatttt ggtttagtcg tccaggcggt gagcacaaaa 300
tttgtgtcgt ttgacaagat ggttcattta ggcaactggt cagatcagcc ccacttgtag 360
cagtagcggc ggcgctcgaa gtgtgactct tattagcaga caggaacgag gacattatta 420
tcatctgctg cttggtgcac gataacttgg tgcgtttgtc aagcaaggta agtgaacgac 480
ccggtcatac cttcttaagt tcgcccttcc tccctttatt tcagattcaa tctgacttac 540
ctattctacc caagcaaagc ttcgattagg aagtaaccat gagcccagaa cgacgcccgg 600
ccgacatccg ccgtgccacc gaggcggaca tgccggcggt ctgcaccatc gtcaaccact 660
acatcgagac aagcacggtc aacttccgta ccgagccgca ggaaccgcag gagtggacgg 720
acgacctcgt ccgtctgcgg gagcgctatc cctggctcgt cgccgaggtg gacggcgagg 780
tcgccggcat cgcctacgcg ggcccctgga aggcacgcaa cgcctacgac tggacggccg 840
agtcgaccgt gtacgtctcc ccccgccacc agcggacggg actgggctcc acgctctaca 900
cccacctgct gaagtccctg gaggcacagg gcttcaagag cgtggtcgct gtcatcgggc 960
tgcccaacga cccgagcgtg cgcatgcacg aggcgctcgg atatgccccc cgcggcatgc 1020
tgcgggcggc cggcttcaag cacgggaact ggcatgacgt gggtttctgg cagctggact 1080
tcagcctgcc ggtaccgccc cgtccggtcc tgcccgtcac cgagatctga 1130
<210> 36
<211> 42
<212> DNA
<213> Artificial Sequence
<400> 36
taggctcccg gcctttgcaa gaattcggtc gaaaaaagaa aa 42
<210> 37
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 37
tcgatcgatc gtaattcata tcagatctcg gtgacgggca 40
<210> 38
<211> 3458
<212> DNA
<213> Artificial Sequence
<400> 38
atgcaagtca aggtatggtt gagagcctag cccccgtaaa cttgtcgctg atgcgcttgc 60
tagttcctgg caacgttgtt gccgcttttg ctgcatcttc ctgcggctgt ggacggtctg 120
ccaggaaaga atgcgcgtat ttcggcgtcc ctgaagagac acgctgggcg cgacgtgccc 180
caaacagctc ttaactccac caatgtctat cagaccaagt tctcgggcgt gacctgggat 240
gaggatcatt ggctgctcac caccaccacg cctgaccagg gccactacca gtcgcgtggg 300
tctgttgcaa acggatacct gggaatcaat gttgctaata ttggtccctt cttcgagctt 360
gacgaaccgg tcaatggcga tgtgatcaat ggctggcccc tctactcccg gcgccagagt 420
ttcgctacta tttccggttt ctgggataga caagcccaca ccaacggctc caatttcccc 480
tggctttcgc agtatggaga tgacagtgta atcagtggtg ttccgcactg gagtggcctg 540
attcttgact tgggcgatga cacgtacctc gacgctaccg tggataaccg cacgatctcg 600
aactttaagt ctacctatga cttcaagtcg ggtgtgctgt cctggtcgta tacctggaca 660
ccccaaggaa acaaaggctc ttatgcaatc acctaccgac tcttcgcgca caagctgtac 720
gtgaaccgag ccgtggtgga catggaaatc acccctctga cgaacggcaa tgctacggtg 780
gttaacgtgc tggatggcta tgcagcagtc cgcacagact ttgtcgcatc tggccaagag 840
gagggtgcga tcttctctgc agtgcggcct tggggggtca acaacgtcac ggcttacgtt 900
tatgcaactt tggatggctc cgacagtgtc gacttgtctt cgcgtaggat cgtcaccgac 960
aagccttatg tgagcacaaa cagctcgtcg gtcgcacagg cggtcgatgt tatgtttact 1020
gcgaatgaga ctgtccgcat caccaagttt gtgggtggcg ccaccacgga ctacttcctt 1080
gccacgcagg agacagccaa ggctgcatgc ctggctggct tggctgatgg ctacgtcaag 1140
tcgctgcagt cgcatgttgg ggaatgggcc acgattatgc acgaccactc ggtcgatcgc 1200
ttcacggatc cagcgaccgg taagctacct gaagacagcc acatcgtcga ctcggccatc 1260
attgctgtca cgaacaccta ctatttgctg cagaacaccg ccggaaccaa tgcgatcgtg 1320
gctgctggtg ggattcctgt gaatgttgac agttgtgctc ccggcggact tacctcggat 1380
tcgtacggtg gccagatctt ctgggatgcg gatctatgga tgcagcctgg tcttgtggcc 1440
tcccatcctg agtcagctca gcggttcacc aactaccgta ttgcattgca ctaccaagct 1500
caggccaaca tcgaaactgc attcactggc tcgaagaacc aaacttcgtt cagctcttcc 1560
gcagcgattt acccctggac tagcggcaga ttcggcaact gcaccgcaac cggaccctgc 1620
tgggactacc agtatcattt gaacggcgat attggcctgg cgatgatcaa ccaatgggtt 1680
gcaagtggtg acacggcatg gtttaagaac tacctgttcc cgatctatga cgcagcagcc 1740
acactttact ctgaactggt ggagcggaat ggctcctcct ggacattgac taacatgact 1800
gatccggtag gtaatctgtc cctgtcacat gatttagctg acctccgcag gatgagtatg 1860
ccaacagcat caatgcgggc ggatacacaa tgccgttgat cgccgaaaca ctgcagaacg 1920
ccaacaagtt gcgcaagcag ttcggtcttg agccgaatga gacatgggac gagatcgcag 1980
aggacgttct gatcctccgt gagaacggtg tcaccctgga atatacgtcg atgaacggct 2040
ctgctgtcgt taagcaggct gacattgtgc tcaacacgtt tccgttgact tatgagtcag 2100
acaactacac ggccacgaat tcgctgaccg acttggacta cgtaagttgc aactccttat 2160
gtgacgtgct atgctaacaa ctacagtatg caaacaagca atccgcagat ggaccggcca 2220
tgacatatgc catcttcgcc attgttgcca gcgatgtttc tccgtccggc tgctctgcct 2280
tcacctatca ccagtactct tacgctccat atgcacgtgg cccgtggtac cagctgtctg 2340
agcagatgat cgacgatgcc agcatcaatg gtggaactca cccggcattc ccgttcctga 2400
ccggccacgg tggtgccaac caggtagccc tctatggtta ccttggtctc cgtctccacc 2460
cagatgacac catctacatc gacccgaatc tcccgcccca gatccctcat attacttacc 2520
ggaccttcta ctggcatggc tggccgatct ctgcgtggtc taactataca cacaccacca 2580
tccagcggga ttcgtctctt gcaccgctgg caagcgctga ccttctcttc tccaacgtta 2640
gcatcaaggt ccaagtcggc cagtccaccg cctcggccga tgaggcaact atttactacc 2700
ttccactctc cggagcactg actgtcccta atcgcatgat cgggtctgtt aacacgactc 2760
ctggcaatca agtccagtgc catccagttt actcaccgga tgcctacgaa ccgggccagt 2820
tccctatctc tgctgtggac ggcgctactt ccaccaagtg gcagccctcg acatctgacc 2880
tgacttcttt gacagtcact ttgtctacca cggcagaggc tggagccgag gaagtttccg 2940
gcttctactt cgactggtct caggcaccgc ccgagaacct gactgtcatc ttccacgatt 3000
cgcccatcgg gaacccctcc actgtgttcg ctgccgctgg ctcgaactct acaggatacc 3060
gggtgattac atcgatgtcc aacatcgtcc agtctaagcc gtacaacgcc atctctgcgg 3120
aggagctgaa cgttgtctct atccctaccg ccaacaccac caccatcaca ctcgatgcgc 3180
ccgtacagaa agcccggtac gccacacttc tgatcgcagg caaccaggcc aacgagactg 3240
ctggtgctac cgtggccgag tgggtcattc tgggacagaa cagcacttcc agctcctcgg 3300
cgcaggccaa gcggaagatg agtgcgcgga gcaaggccac tttggctcaa ctcagctgaa 3360
cagcttgctt gctctcgtat gagacgttcg cgtctgtatc ataggtatcc taccatatct 3420
ggaccagaag tacgtaataa tcaattaatg ctacataa 3458
Claims (2)
1.一种增加应激性海藻糖含量的耐热性黑曲霉工程菌的构建方法，其特征在于，具体步骤如下：
（1）利用一步克隆法，在质粒pAN7-1的基础上添加cox启动子和TtrpC终止子，获得重组载体pAN7-1-cox-TtrpC；所述cox启动子的核苷酸序列如SEQ ID NO.1所示；所述TtrpC终止子的核苷酸序列如SEQ ID NO.4所示；
（2）利用一步克隆法，构建基因tpsA和tpsB异源表达质粒pAN7-1-cox-tpsA-linker-tpsB-TtrpC；所述tpsA基因的核苷酸序列如SEQ ID NO.29所示，所述tpsB基因的核苷酸序列如SEQ ID NO.30所示；
（3）将所述质粒pAN7-1-cox-tpsA-linker-tpsB-TtrpC转化到黑曲霉宿主菌中，获得重组工程菌；
（4）利用同源重组技术敲除步骤（3）获得的重组工程菌中海藻糖降解基因ATH1，获得耐热性产葡萄糖酸钠的黑曲霉基因工程菌；
步骤（2）所述linker为根据黑曲霉密码子偏好性设计的柔性肽W1、W2和W3序列，刚性肽P1、P2和P3序列；W1、W2和W3序列分别如SEQ ID NO.9-11所示；P1、P2和P3序列分别如SEQ IDNO.12-14所示。
2.根据权利要求1所述的构建方法构建得到的耐热性黑曲霉工程菌在高温条件下生产葡萄糖酸钠的应用，其特征在于，所述高温条件为43℃。
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| CN103436454A (zh) * | 2013-07-31 | 2013-12-11 | 天津科技大学 | 高效表达α-葡萄糖转苷酶的基因工程菌及其构建方法 |
| CN105296524A (zh) * | 2015-11-23 | 2016-02-03 | 齐鲁工业大学 | 一种制备食品级海藻糖的黑曲霉工程菌的构建方法与应用 |
| CN110029068A (zh) * | 2019-04-10 | 2019-07-19 | 天津科技大学 | 一种低溶氧条件下高产有机酸的黑曲霉基因工程菌株及应用 |
| CN110734865A (zh) * | 2019-12-02 | 2020-01-31 | 天津科技大学 | 一种低pH条件下高产苹果酸的黑曲霉基因工程菌株及应用 |
| CN111218408A (zh) * | 2020-01-21 | 2020-06-02 | 天津科技大学 | 一株高效生产苹果酸的黑曲霉菌株、构建方法及应用 |
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| CN103436454A (zh) * | 2013-07-31 | 2013-12-11 | 天津科技大学 | 高效表达α-葡萄糖转苷酶的基因工程菌及其构建方法 |
| CN105296524A (zh) * | 2015-11-23 | 2016-02-03 | 齐鲁工业大学 | 一种制备食品级海藻糖的黑曲霉工程菌的构建方法与应用 |
| CN110029068A (zh) * | 2019-04-10 | 2019-07-19 | 天津科技大学 | 一种低溶氧条件下高产有机酸的黑曲霉基因工程菌株及应用 |
| CN110734865A (zh) * | 2019-12-02 | 2020-01-31 | 天津科技大学 | 一种低pH条件下高产苹果酸的黑曲霉基因工程菌株及应用 |
| CN111218408A (zh) * | 2020-01-21 | 2020-06-02 | 天津科技大学 | 一株高效生产苹果酸的黑曲霉菌株、构建方法及应用 |
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