CN110656097B - 一种蔗糖非发酵型蛋白激酶调节亚基及其应用 - Google Patents
一种蔗糖非发酵型蛋白激酶调节亚基及其应用 Download PDFInfo
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- CN110656097B CN110656097B CN201910870749.4A CN201910870749A CN110656097B CN 110656097 B CN110656097 B CN 110656097B CN 201910870749 A CN201910870749 A CN 201910870749A CN 110656097 B CN110656097 B CN 110656097B
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Abstract
本发明公开了一种蔗糖非发酵型蛋白激酶调节亚基及其应用，属于基因工程以及微生物工程技术领域。本发明的氨基酸序列如SEQ ID No.1所示的蔗糖非发酵型蛋白激酶调节亚基具有响应胞外营养水平并调控脂质代谢的功能，参与了菌体对胞外葡萄糖水平的响应。该基因对生长和脂质积累均有负调控作用，在葡萄糖限制的条件下可通过抑制高山被孢霉生长和增强脂肪酸的分解代谢为菌体供能。
Description
技术领域
本发明涉及一种蔗糖非发酵型蛋白激酶调节亚基及其应用，尤其是在响应胞外营养水平和调控脂质代谢中的应用，属于基因工程和微生物工程技术领域。
背景技术
高山被孢霉(Mortierella alpina)是一株具有很强脂质积累能力的产油丝状真菌，可合成多种具有生物活性的多不饱和脂肪酸(PUFAs)，具备很好的工业化开发价值。碳、氮源水平是影响高山被孢霉脂质积累的重要因素，高糖或氮限制均可促进其脂质积累，这一现象在产油微生物中广泛存在。前期对于产油微生物的研究多集中于发酵条件的优化，或对脂质代谢相关基因进行遗传改造，以上研究在提高产油微生物脂质积累方面均取得了理想的研究成果，但也遇到了基因操作选择型局限、脂质产量无法进一步突破的瓶颈。多组学研究发现，产油微生物脂质积累涉及细胞全局性生理生化响应，由多种途径共同参与完成。因此，在营养调控和上游信号通路进行更为深入的研究，通过全局调控研究产油微生物脂质积累机制，是突破其产脂瓶颈的创新型研究方向。
AMP激活的蛋白激酶(AMPK)是一种具有广泛调控功能的上游激酶，在所有真核生物中广泛存在且高度保守，是由起催化作用的α亚基和具有调节作用的β、γ亚基组成的异源三聚体。AMPK复合体α亚基N末端包含蛋白激酶的激活及催化结构域，其中Thr172残基对AMPK的激活起关键作用。AMPK最初于1973年在哺乳动物细胞中纯化得到，之后又有研究发现脂肪酸代谢通路的关键酶乙酰辅酶A羧化酶(ACC)能够被一种蛋白激酶磷酸化而失活。ACC是一个多亚基酶，具有两种亚型：定位于细胞质中负责催化长链脂肪酸合成的ACC1和定位于线粒体膜上负责脂肪酸β-氧化的ACC2。AMPK可在Ser1157位点抑制ACC1活性，最终导致脂肪酸合成减缓。后续研究表明，该蛋白激酶的活性受上游激酶的磷酸化以及AMP水平的调控，因而被统一命名为AMP激活的蛋白激酶[Trends in Plant Science，2007，12(1):20-28]。
蔗糖非发酵型蛋白激酶(SNF1)是哺乳动物AMPK蛋白激酶的同系物，也是由α、β亚基(催化亚基)和γ亚基(调节亚基)组成的异源三聚体，最先发现于无法利用蔗糖的突变菌株，这也是蔗糖非发酵型蛋白激酶这一名称的由来[Journal of Biological Chemistry,1994,269(4):2361-2364.]。关于酵母SNF1与脂质代谢方面的研究最早可追溯至1994年。研究证实脂肪酸合成过程中的关键限速酶乙酰-CoA羧化酶1(ACC1)受SNF1途径调控。SNF1可在Ser1157位点磷酸化ACC1使其酶活力降低，最终导致脂肪酸合成速率减缓，而脂肪酸氧化速率增快[Journal of Biological Chemistry,1994,269(30):19509-19515.]。
目前，针对酵母SNF1复合体同源蛋白已展开了较多研究，研究内容多集中于复合体功能或具有催化功能的α亚基和β亚基，且发现该复合体功能在物种间存在较大的差异。然而，SNF1复合体中的调节亚基，即γ亚基的功能，特别是在响应营养信号和调控胞内代谢之间的介导作用仍未被揭示，而在丝状真菌特别是产油丝状真菌中，SNF1复合体的研究及其功能解析也较少。
发明内容
[技术问题]
本发明要解决的技术问题是提供一种可响应胞外营养水平并调控脂质代谢的蔗糖非发酵型蛋白激酶调节亚基。本发明还应用所述蔗糖非发酵型蛋白激酶调节亚基来调控高山被孢霉合成脂质的能力。
[技术方案]
为解决上述问题，本发明提供了一种蔗糖非发酵型蛋白激酶调节亚基，所述蔗糖非发酵型蛋白激酶调节亚基为：
(a)由SEQ ID No.1所示的氨基酸序列组成的蛋白质；或者，
(b)在(a)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有蔗糖非发酵型蛋白激酶调节亚基活性的由(a)衍生的蛋白质。
本发明还提供了一种基因、重组质粒、细胞，所述基因编码上述蔗糖非发酵型蛋白激酶调节亚基。在本发明的一种实施方式中，所述基因的核苷酸序列如SEQ ID No.2所示。所述重组质粒携带上述基因。在本发明的一种实施方式中，所述重组质粒的载体为pBIG2-ura5s-ITs载体。所述pBIG2-ura5s-ITs载体记载于公开号为CN103571762A的专利申请文本中。所述细胞携带上述基因或上述重组质粒。在本发明的一种实施方式中，所述细胞为高山被孢霉、大肠杆菌或根癌农杆菌。
本发明还提供了上述蔗糖非发酵型蛋白激酶调节亚基在调节宿主细胞合成脂质方面的应用。所述脂质为磷脂、甘油三酯、花生四烯酸或游离脂肪酸。当高山被孢霉处于碳氮比＜6或葡萄糖浓度＜15g/L的营养条件中时，限制所述蔗糖非发酵型蛋白激酶调节亚基的表达，或者敲除所述蔗糖非发酵型蛋白激酶调节亚基，能够使高山被孢霉重组菌合成脂质的能力接近或超过高山被孢霉处于碳氮摩尔比＞70或葡萄糖浓度＞30g/L的营养条件中时的合成脂质的能力。
本发明提供了一种利用低碳氮比培养基生产脂质的方法，是以敲低或敲除所述蔗糖非发酵型蛋白激酶调节亚基的高山被孢霉为菌株，利用低碳氮比培养基培养菌株，培养结束后，收集菌体并从中提取脂质。所述低碳氮比培养基是碳氮比维持在3～6的培养基。例如：初始葡萄糖含量为5～15g/L，初始酒石酸铵浓度为5.0～10.0g/L，1.5g/L酵母提取物、7g/L磷酸二氢钾、2.0g/L磷酸氢二钠、1.5g/L七水硫酸镁、0.008g/L二水氯化钙以及微量元素,其中，微量元素浓度为：0.001g/L七水氯化铁、0.0001g/L七水硫酸锌、0.0001g/L五水硫酸铜、0.0001g/L硝酸钴、0.0001g/L五水硫酸锰，在发酵过程中，根据葡萄糖消耗情况适当补充葡萄糖，使之维持在5～15g/L的范围内。所述脂质包括了富含花生四烯酸(ARA)的油脂。
本发明还提供了一种重组高山被孢霉，是敲除了SEQ ID No.2所示的基因的高山被孢霉，或者干扰了SEQ ID No.2所示的基因的表达的高山被孢霉。
本发明还提供了干扰高山被孢霉的蔗糖非发酵型蛋白激酶调节亚基的表达的方法，是合成SEQ ID No.5所示的RNA干扰片段，并插入到pBIG2-ura5s-ITs载体中，然后利用根癌农杆菌将重组载体转化到高山被孢霉中，利用SEQ ID No.5所示的RNA干扰片段干扰蔗糖非发酵型蛋白激酶调节亚基的表达。
[有益效果]
本发明提供了氨基酸序列如SEQ ID No.1所示的蔗糖非发酵型蛋白激酶调节亚基(高山被孢霉SNF1复合体γ亚基，即Masnf4)，该蔗糖非发酵型蛋白激酶调节亚基具有以下特性：(1)响应胞外营养水平并调控微生物脂质代谢的功能，且对碳源(葡萄糖)饥饿的响应更为敏感，在葡萄糖限制的条件下(＜5g/L)，Masnf4敲低(SNF1复合体γ亚基的表达受到干扰)菌株M.alpina-Masnf4-Si生长速度未受限，菌球形态与营养充足条件下一样呈松散毛球状，并且能够积累脂质，生物量、脂肪酸含量分别是Masnf4过表达菌株M.alpina-Masnf4的2.2倍和1.5倍；(2)在葡萄糖受限的情况下，尤其是当葡萄糖完全耗尽后Masnf4敲低菌株中花生四烯酸(ARA)含量比Masnf4过表达菌株提高50.9％。(3)高山被孢霉的脂质积累通常需要在较高葡萄糖浓度下进行，本发明显示，在较低碳氮比的条件下(4.6)，培养4天后Masnf4敲低的菌株脂质积累量与同时间内高碳氮比(46.5)条件下对照组脂质积累量达到同等水平，这一特点可降低大批量发酵过程中的原料成本，减少葡萄糖的浪费。
本发明提供的高山被孢霉Masnf4能够用于调控菌体对胞外葡萄糖水平的响应，该基因对生长和脂质积累均有负调控作用，下调或敲除Masnf4基因，能够帮助菌株在低葡萄糖浓度的条件下积累脂质。本发明的成果可用于降低利用高山被孢霉合成脂质时的碳源消耗量，能够减少生产成本。
本发明通过解析高山被孢霉SNF1复合体调节亚基响应碳、氮源信号并调控脂质积累的规律，能够进一步研究产油微生物产脂机制提供理论基础，也为基于全局调控构建脂质高产菌株提供新颖的改造途径。
附图说明
图1：Masnf4与NCBI中已鉴定功能的蔗糖非发酵型蛋白激酶调节亚基的氨基酸序列比对结果。
图2A：重组高山被孢霉M.alpina-Masnf4基因组DNA验证的琼脂糖凝胶电泳结果；其中，M表示Marker，空表示以ddH2O代替DNA模板的阴性对照，NC表示原养型高山被孢霉，编号1-4分别表示4个转化子。
图2B：重组高山被孢霉M.alpina-Masnf4-Si基因组DNA验证的琼脂糖凝胶电泳结果；其中，M表示Marker，空表示以ddH2O代替DNA模板的阴性对照，NC表示原养型高山被孢霉，编号1-4分别表示4个转化子。
图3重组高山被孢霉M.alpina-Masnf4和M.alpina-Masnf4-Si转化子在Broth发酵培养基中生长和产脂情况。
图4重组高山被孢霉M.alpina-Masnf4和M.alpina-Masnf4-Si在碳源限制(图4A、图4C、
图4E)和氮源限制(图4B、图4D、图4F)培养条件下生长和产脂情况。
图5重组高山被孢霉M.alpina-Masnf4和M.alpina-Masnf4-Si在碳源限制和氮源限制条件下菌落形态。
具体实施方式
下面结合具体实施例，对本发明进行进一步的阐述。
下述实施例中涉及的高山被孢霉(Mortierella alpina)ATCC 32222购自美国标准生物品收藏中心(ATCC)；下述实施例中涉及的根癌农杆菌(Agrobacteriumtumefaciens)AGL-1购自北京华越洋生物；下述实施例中涉及的大肠杆菌(Escherichiacoli)DH5α购自Invitrogen公司；下述实施例中涉及的pBIG2-ura5s-ITs载体记载于公开号为CN103571762A的专利申请文本中；下述实施例中涉及的高山被孢霉尿嘧啶营养缺陷型菌株记载于公开号为CN103468581A的专利申请文本中。
下述实施例中涉及的KOD plus高保真DNA聚合酶购自日本Toyobo公司；下述实施例中涉及的Taq DNA聚合酶购自CWBIO公司；下述实施例中涉及的反转录试剂盒(PrimeScript RT regent Kit with gDNA Eraser RR047A&R6110A)购自Takara公司；下述实施例中涉及的质粒提取试剂盒购自北京天根生化科技有限公司；下述实施例中涉及的真菌基因组DNA提取试剂盒购自BioFlux公司；下述实施例中涉及的限制性内切酶、T4连接酶、Trizol、PCR产物纯化试剂盒、凝胶回收试剂盒、GeneRuler DNA Ladder Mix、PageRulerPrestained Protein Ladder购自Thermo Scientific公司；下述实施例中涉及的正十五烷酸(C15:0)、20％(w/w)盐酸甲醇购自Sigma公司；下述实施例中涉及的DEPC水、卡那霉素(Kanamycin,Kana)、利福平(Rifampicin，Rif)、壮观霉素(Spectinomycin,Spe)、头孢噻肟钠(Cefotaxime,Cef)、无氨基酵母氮源(YNB)和各类氨基酸购自上海生物工程有限公司；下述实施例中涉及的酵母提取物、胰蛋白胨购自Oxoid公司；下述实施例中涉及的低吸附型无酶枪头、无酶离心管、无酶PCR管、2mL棕色气相瓶及瓶盖购自苏州科晴生物公司；下述实施例中涉及的诱导转化剂乙酰丁香酮(Acetosuringone，AS，CAS#[2478-38-8])、2-(N-吗啡啉)乙磺酸(MES缓冲液，CAS#[145224-94-8])、尿嘧啶(Urail)、酵母氮源(Yeast NitrogenBase,without amino acids CAS#[A610507-0500]Lot:C418BA0040)和各类氨基酸购自生工生物工程(上海)股份有限公司；其他试剂购自国药集团。
下述实施例中涉及的载体构建和细菌感受态细胞制作均参考《分子克隆手册》。
下述实施例中涉及的引物由上海桑尼公司合成，测序工作由上海华大基因公司完成。RNA干扰片段由亦欣生物科技(上海)有限公司合成。
(高山被孢霉(Mortierella alpina)ATCC 32222、根癌农杆菌(Agrobacteriumtumefaciens)AGL-1以及大肠杆菌(Escherichia coli)DH5α均可以购买得到，不需要进行用于专利程序的保藏)
下述实施例中涉及的培养基如下：
Broth培养基：20g/L(活化用)/50g/L(产脂用)葡萄糖、5g/L酵母提取物、1g/L磷酸二氢钾、0.25g/L七水硫酸镁、10g/L硝酸钾。
Kendrick培养基：20g/L(活化用)/50g/L(产脂用)葡萄糖、3.3g/L(活化用)/2.0g/L(产脂用)酒石酸铵、1.5g/L酵母提取物、7g/L磷酸二氢钾、2.0g/L磷酸氢二钠、1.5g/L七水硫酸镁、0.008g/L二水氯化钙以及微量元素；其中，微量元素浓度为：0.001g/L七水氯化铁、0.0001g/L七水硫酸锌、0.0001g/L五水硫酸铜、0.0001g/L硝酸钴、0.0001g/L五水硫酸锰。在制备葡萄糖限制或氮源限制的培养基时，根据下表调整葡萄糖和酒石酸铵的添加浓度，其他内容不变：
GY培养基：20g/L葡萄糖、10g/L酵母提取物、2g/L硝酸钾、1g/L磷酸二氢钠、3g/L七水硫酸镁。
MM基础培养基：1.74g/L磷酸氢二钾、1.37g/L磷酸二氢钾、0.146g/L氯化钠、0.49g/L七水硫酸镁、0.078g/L氯化钙、0.53g/L硫酸铵、1.8g/L葡萄糖、10mL/L七水硫酸铁(100X)、5mL/L甘油，灭菌后加入已滤菌的MES缓冲液至终浓度为7.8g/L。
IM诱导培养基：在MM培养基的基础上稍作调整，额外添加0.1g/L的尿嘧啶，葡萄糖改为0.9g/L，其余不变，使用前添加100μg/mL的乙酰丁香酮(AS)和7.8g/L的MES，用于固体培养基时添加20g/L的琼脂条，添加AS的IM培养基需避光保存。
SC-CS培养基：20g/L葡萄糖、5g/L无氨基酵母氮源、1.7g/L硫酸铵、10mL/L氨基酸母液(100X)、20g/L琼脂，倒平板前添加100μg/mL的头孢噻圬和100μg/mL的壮观霉素；其中，氨基酸母液：60mg/L异亮氨酸、60mg/L亮氨酸、60mg/L苯丙氨酸、50mg/L苏氨酸、40mg/L赖氨酸、30mg/L酪氨酸、20mg/L腺嘌呤、20mg/L精氨酸、20mg/L组氨酸、10mg/L甲硫氨酸。
SOC复苏培养基：20g/L胰蛋白胨、5g/L酵母提取物、0.5g/L氯化钠、0.186g/L氯化钾、0.95g/L氯化镁、3.6g/L葡萄糖。
LB液体培养基：10g/L胰蛋白胨、5g/L酵母提取物、10g/L氯化钠，使用前添加100μg/mL的卡那霉素。
LB固体培养基：10g/L胰蛋白胨、5g/L酵母提取物、10g/L氯化钠、20g/L琼脂，使用前添加100μg/mL的卡那霉素。
YEP液体培养基：10g/L酵母提取物、10g/L胰蛋白陈、5g/L氯化钠，使用前添加100μg/mL的卡那霉素和100μg/mL的利福平，避光保存。
YEP固体培养基：10g/L酵母提取物、10g/L胰蛋白陈、5g/L氯化钠、20g/L琼脂，使用前添加100μg/mL的卡那霉素和100μg/mL的利福平，避光保存。
实施例1：编码蔗糖非发酵型蛋白激酶调节亚基的基因的筛选
根据NCBI中已鉴定功能的蔗糖非发酵型蛋白激酶调节亚基的基因序列为模板，在已完成测序的M.alpina ATCC 32222菌株的基因库中进行BLAST比对，获得备选的目的基因；然后将备选的目的基因在NCBI库中进行二次比对筛选，将最终得到的目的蛋白命名为Masnf4(氨基酸序列如SEQ ID No.1所示)，其编码基因命名为Masnf4(核苷酸序列如SEQ IDNo.2所示)。
Masnf4对应cDNA的全长为1596bp，编码531个氨基酸。为了进一步判断筛选出的Masnf4是否属于蔗糖非发酵型蛋白激酶调节亚基，将其与NCBI中已鉴定功能的AMPK/SNF1复合体调节亚基(γ亚基)进行氨基酸同源性和保守结构分析。如图1和表1所示，比对结果表明，Masnf4与NCBI中已鉴定功能的酵母蔗糖非发酵性蛋白激酶调节亚基具有一定的同源性和功能结构域，且具备该调节亚基的保守序列。对功能结构域进行分析，发现高山被孢霉Masnf4中存在两组串联的CBS对，每个CBS对由两个CBS结构域组成，这一结构最初是在一种代谢酶胱硫醚合酶中发现。CBS对上的AMP结合位点与AMP结合后可启动该复合体的别构激活机制，对该复合体活性具有关键调控作用，并可调控氧化还原平衡。CBS结构域是AMPK/SNF1复合体在进化过程中的保守结构域，从酵母到哺乳动物中这种串联的CBS结构域普遍存在，尽管这一结构的作用仍未被解析，但研究显示CBS区域具有重要的调控功能，其突变或缺失会导致哺乳动物遗传性疾病的发生，如先天性肌强直，特发性全身性癫痫，高钙尿结石和巴特氏症候群等。因此，可根据这一结构判断Masnf4属于AMPK/SNF1复合体家族蛋白激酶的调节亚基。
通常，AMPK/SNF1复合体的调节亚基仅包含300余个氨基酸，由两组串联的CBS结构域组成，但是，与其他哺乳动物或酵母等同源基因不同的是，高山被孢霉的Masnf4的N端还包括一段约200个氨基酸组成的DBP(Duffy-antigen binding protein，达菲抗原结合蛋白)家族蛋白。这一DBP家族蛋白存在于真核生物中。在哺乳动物中，该蛋白被鉴定为能与红细胞的Duffy糖蛋白受体互补结合，与抵抗间日疟原虫的入侵的血型有关。但是在微生物中，这一段蛋白并非广泛存在且功能未被鉴定，而这段蛋白的存在正是导致高山被孢霉Masnf4与其他物种同源基因在序列和功能上存在差异的主要原因。
表1高山被孢霉蔗糖非发酵型蛋白激酶调节亚基序列比对结果
SEQ ID No.1：
MSDNKDGTIASEPAETAAHGDAVQSGVDSHPQGSELMQGAPASEDASEEQPQGQSSTDYFSPQEPTVNAFTPQQQDDAVGFASEIGQPDQTTVNPEPDTTHPKDSGGLSNTQAPPASSLTAALNNNTNHSNTAGSDRPGPDAAPPGSATPSSQTTDVPATENIDPVASYSAWRKKKDTEMLQGTQAVMESFLKQHSAYDVLPVSYRQIVLDTTLLIKKALSVLMQYSIVSAPLWDSTKRKFAGMLTATDFINLIQFYYSHSSYTAAIHEMDQFQISQLRDVEKVIGVPPPELFSLHPLQSLYDACQLLVESRAHRLPLVDMDSETGEEMIVSVLTQYRILKFIAMNYQDEKTLRQPLSELKIGIYTDLAVADLSTPVMTVIDMFVKRRISSVPIVDEDGVVLNVFETVDVMTLVKAGVYRDLDLCVAEALLRRPEDFAGVHTCTLNDSLSSIFGTIRKSQVHRLIVVDSGNKLKGIVSLSDIMRYLIGGTRETKEPAGGNVEEAEVHVEDIEKETKSTHSGDGDDNDAGDL
SEQ ID No.2：
ATGAGCGACAACAAGGACGGTACCATTGCTTCCGAGCCAGCTGAAACAGCCGCGCATGGGGACGCGGTGCAAAGCGGTGTTGACTCTCACCCACAAGGGTCAGAGCTAATGCAAGGAGCACCGGCATCAGAGGATGCCAGCGAGGAGCAACCGCAGGGCCAGTCCTCAACCGACTACTTTTCGCCTCAAGAACCCACAGTCAATGCTTTTACTCCGCAACAGCAGGACGACGCTGTGGGGTTTGCGTCAGAAATAGGCCAGCCTGATCAAACAACAGTGAACCCCGAACCCGATACCACTCACCCTAAAGACTCAGGAGGCCTGTCGAATACGCAAGCACCCCCGGCCTCGTCGCTGACGGCAGCCCTCAACAATAACACCAACCACTCTAATACGGCTGGGTCTGACAGGCCGGGACCGGATGCTGCACCACCAGGATCGGCTACCCCATCCTCACAAACAACGGATGTGCCTGCGACGGAAAATATTGACCCTGTTGCATCGTACTCTGCATGGCGCAAGAAGAAAGACACGGAAATGTTGCAAGGAACCCAGGCTGTGATGGAGTCGTTTCTAAAGCAGCACTCCGCCTACGATGTGTTACCCGTCAGCTATCGGCAAATTGTTTTGGATACCACGCTCTTGATCAAGAAGGCCCTTTCGGTCCTGATGCAGTACAGCATAGTGTCTGCTCCGTTGTGGGATTCAACCAAGCGCAAGTTTGCAGGCATGCTGACGGCCACAGACTTTATCAATTTGATTCAGTTTTACTACTCCCATTCATCCTACACCGCCGCAATTCATGAGATGGACCAATTCCAGATATCCCAGCTCAGAGATGTCGAGAAGGTAATAGGAGTGCCACCGCCCGAGCTGTTCTCGCTACACCCACTGCAATCGCTCTACGACGCCTGTCAGTTGCTGGTCGAATCAAGAGCGCATCGGTTACCCTTGGTGGATATGGACTCTGAAACAGGAGAGGAGATGATCGTGTCAGTCTTGACTCAGTACAGAATCCTCAAGTTCATTGCCATGAACTATCAGGACGAGAAGACACTTCGTCAACCGCTTTCGGAGCTCAAGATTGGGATCTATACGGACCTTGCCGTGGCAGATCTTTCCACGCCCGTGATGACCGTGATTGACATGTTCGTGAAGCGGAGGATATCCTCTGTGCCCATTGTCGATGAGGACGGTGTGGTTCTGAACGTCTTTGAGACTGTCGATGTGATGACGCTGGTCAAGGCAGGCGTGTACAGGGATTTGGATCTCTGCGTCGCAGAGGCGTTACTGCGGAGACCGGAAGACTTTGCAGGAGTTCATACCTGCACGCTCAACGACAGCCTCTCGTCCATTTTCGGAACCATTCGCAAGTCTCAAGTCCATCGCCTGATCGTCGTTGACAGCGGCAATAAGCTGAAGGGAATCGTCAGTCTCTCGGATATCATGCGTTATCTCATCGGTGGAACACGTGAGACCAAGGAGCCTGCAGGTGGCAATGTCGAAGAGGCGGAAGTCCACGTCGAGGACATCGAGAAGGAAACCAAATCGACGCACAGTGGCGATGGCGATGATAATGATGCGGGAGACCTATGA
实施例2：Masnf4的克隆
使用Trizol法提取高山被孢霉(Mortierella alpina)ATCC 32222的总RNA，按照Takara反转录试剂盒说明书进行反转录获得cDNA，在高山被孢霉(Mortierella alpina)ATCC 32222的cDNA文库中通过PCR反应扩增Masnf4，扩增Masnf4所用引物见表2。
所用PCR仪为BIO-RAD T100 Thermal Cycler，使用KOD plus高保真DNA聚合酶，反应体系为50μL，体系内容按照该DNA聚合酶说明书内容进行；反应过程如下：95℃预变性5min，然后95℃变性30s，58℃退火30s，68℃延伸1.5min，重复上述三步骤30次，然后68℃充分延伸7min，最后降到4℃保持10min后停止。
反应结束，得到扩增产物，对扩增产物进行纯化后通过1％琼脂糖凝胶电泳验证扩增产物条带大小，获得Masnf4。
表2引物序列及其用途
实施例3：Masnf4在高山被孢霉中的表达和RNA干扰
(1)高山被孢霉表达载体的构建
使用限制性内切酶HindⅢ和SmaⅠ对实施例2获得的Masnf4以及表达载体pBIG2-ura5s-ITs进行酶切，然后利用T4连接酶将经酶切、纯化后的DNA进行连接，获得连接产物，具体酶切体系(20μL)如表3。
表3酶切体系
| 试剂 | 用量 |
| 10×cutmart buffer | 2μL |
| 限制性内切酶 | 1μL |
| PCR产物或载体 | 200ng或1μg |
| ddH<sub>2</sub>O | 补至20μL |
将获得的连接产物于4～16℃下过夜连接后，转化至大肠杆菌DH5α感受态细胞，转化方法如下：无菌状态下取100μL感受态细胞，加入5～8μL连接产物，吹吸混匀；将混匀的感受态细胞移入预冷过的电转杯中，避免产生气泡；将电转柄放入Bio-Rad电转仪，调到合适预设程序档位，电转，电压条件为1.8kv；加入1mL SOC复苏培养基于电转后的感受态细胞中，混匀转移至1.5mL离心管中，37℃、150rpm孵育1h；取200μL涂布含有100μg/mL卡那霉素的LB固体培养基平板，37℃倒置培养过夜；挑取阳性转化子，提取质粒，测序验证结果表明连接成功，获得重组质粒pBIG2-ura5s-Masnf4。
(2)高山被孢霉干扰载体的构建
根据文献报道，选择Masnf4的活性位点上的片段150bp，以正向序列-ITs-反向互补序列的顺序合成RNA干扰片段，并使用HindⅢ和XhoⅠ内切酶进行酶切，插入pBIG2-ura5s-ITs载体，质粒构建方法、转化方法同上述“(1)高山被孢霉表达载体的构建”，构建好的干扰载体命名为pBIG2-ura5s-Masnf4Silent。
RNA干扰片段序列如SEQ ID No.5：所示。
CCCAAGCTTGGGCTGTTCTCGCTACACCCACTGCAATCGCTCTACGACGCCTGTCAGTTGCTGGTCGAATCAAGAGCGCATCGGTTACCCTTGGTGGATATGGACTCTGAAACAGGAGAGGAGATGATCGTGTCAGTCTTGACTCAGTACAGAATCCTCAAGTCCCAAGCGAATTTGTCATCTCGACTGGTGCAAACTGCGCAAACGGCTGACTCATTGCCCATGCTTTTCTTCTCCACGCCTATCGTCTGTTACTGCATCTCTGTCGTGTTGAATGCGTAACTGATGGACTTCCGTATGCTCTTGAGGATTCTGTACTGAGTCAAGACTGACACGATCATCTCCTCTCCTGTTTCAGAGTCCATATCCACCAAGGGTAACCGATGCGCTCTTGATTCGACCAGCAACTGACAGGCGTCGTAGAGCGATTGCAGTGGGTGTAGCGAGAACAGCCGCTCGAGCGG
(3)高山被孢霉转化子的验证
将获得的重组质粒pBIG2-ura5s-Masnf4和pBIG2-ura5s-Masnf4Silent通过电击转化法转入根癌农杆菌AGL-1，获得分别携带重组质粒pBIG2-ura5s-Masnf4和pBIG2-ura5s-Masnf4Silent的根癌农杆菌；使用生理盐水刮取高山被孢霉尿嘧啶营养缺陷型菌株的孢子，于4℃～28℃培养箱放置6～24h，获得萌发的孢子液；将分别携带重组质粒pBIG2-ura5s-Masnf4和pBIG2-ura5s-Masnf4Silent的根癌农杆菌分别在YEP培养基中活化、MM培养基中培养和IM培养基中诱导，取经IM培养基诱导培养的根癌农杆菌测定OD660，用IM培养基梯度稀释为OD660＝0.2～1.2，得到分别携带重组质粒pBIG2-ura5s-Masnf4和pBIG2-ura5s-Masnf4Silent的根癌农杆菌菌液；分别取携带重组质粒pBIG2-ura5s-Masnf4和pBIG2-ura5s-Masnf4Silent的根癌农杆菌菌液与孢子液100～200μL于无菌EP管中上下颠倒混匀，涂布于贴有玻璃纸的IM固体培养基中于16～28℃的条件下避光共培养12～48h；共培养结束后，将带有共培养体系的玻璃纸转移至含有壮观霉素(Spe)和头孢噻肟(Cef)的SC-CS培养基，于16～28℃的条件下进行培养，直至有菌落长出；菌落长出后，挑取菌落边缘新长出的菌丝，连续在新的SC-CS培养基上于28℃的条件下培养12～48h进行传代培养；传代培养后，选取可以稳定生长的菌落，挑取至Broth液体活化培养基中于28℃的条件下培养2d，获得菌液；取菌液提取真菌基因组DNA进行PCR验证，认为扩增得到目的条带的转化子为正确的阳性转化子(PCR结果如图2所示)；PCR验证共获得Masnf4过表达和干扰菌株阳性转化子各4个，将这些阳性转化子进行测序。结果表明，各转化子中过表达的Masnf4的碱基序列与高山被孢霉基因组中的序列数据一致，干扰载体中，由于形成发卡结构，该片段无法被扩增出来，因此仅可在800bp附近看到筛选标记ura5s的条带，说明在高山被孢霉体内该发卡结构可以正确形成，证实所选8个阳性转化子在基因组水平验证正确，获得携带Masnf4基因或该基因干扰片段的重组高山被孢霉M.alpina-Masnf4和M.alpina-Masnf4-Si。
其中，PCR使用Taq酶体系进行，所用引物为质粒载体pBIG2-ura5s-ITs的通用引物，具体序列如下：
上游引物Hispro F1：CACACACAAACCTCTCTCCCACT(SEQ ID No.6)；
下游引物TrpC R1：CAAATGAACGTATCTTATCGAGATCC(SEQ ID No.7)。
由图2可知，通过使用通用引物进行PCR验证，携带重组质粒pBIG2-ura5s-Masnf4和pBIG2-ura5s-Masnf4Silent的根癌农杆菌的T-DNA区域的尿嘧啶回补标记ura5s和目的基因Masnf4可被成功扩增出来，且条带大小符合理论值(因引物设计在载体上，所获得的片段比实际片段大156bp)，说明目的基因被成功转入高山被孢霉中。干扰菌株中，由于形成发卡结构，该片段无法被扩增出来，但T-DNA区域的尿嘧啶回补标记ura5s，说明在高山被孢霉体内该发卡结构可以正确形成，理论上可对目的基因进行RNA干扰。
取重组高山被孢霉M.alpina-Masnf4的单孢子接种于Broth培养基(含有20g/L葡萄糖)中，于28℃的条件下培养2d进行活化；连续活化三代，离心，收集活化后的菌体；将菌体打碎至均匀菌絮状；将打碎的菌体以1％(v/v)的接种量接种到Broth培养基(含有50g/L葡萄糖)中，于28℃、200rpm摇床培养7d后，离心，收集菌体；将菌体进行破碎后离心，弃去沉淀，获得上清液，上清液中含有蔗糖非发酵型蛋白激酶调节亚基的蛋白。
实施例4：重组高山被孢霉在常规营养条件下的生长和产脂分析
生长分析：以高山被孢霉原养型菌株为阴性对照，取实施例3构建的重组高山被孢霉M.alpina-Masnf4和M.alpina-Masnf4-Si以及高山被孢霉原养型菌株的单孢子接种于Broth种子培养基中，于28℃的条件下培养2d进行活化；连续活化三代，收集活化后的菌体；将菌体打碎至均匀菌絮状；将打碎的菌体以1％(v/v)的接种量接种到Broth发酵培养基中，于28℃、200rpm摇床培养168h时收集菌体，过滤并洗涤后，于真空冷冻干燥至恒重，称量菌体重量，计算生物量，计算结果见图3-A,C。
产脂分析：将菌体研磨成粉末，称取50mg，精确加入100μL 2mg/mL的C15:0作为内标，加入2mL 4mol/L的盐酸充分混匀；80℃水浴1h，-80℃放置15min；重复3次后冷却至室温，加入1mL甲醇和1mL氯仿，混匀，震荡2min；3000g离心10min；收集氯仿层于新的提脂瓶中；重复此步骤两次；合并氯仿层，氮吹干燥后加入1mL 10％的盐酸-甲醇，60℃水浴3h进行甲酯化处理；之后在上述甲酯化处理体系中加入1mL饱和氯化钠和1mL正己烷，混匀，3000g离心10min，重复此步骤两次；收集正己烷层于新瓶，剩余液体继续加入1mL正己烷，震荡混匀1min后于3000g离心10min；合并正己烷层，氮吹干燥，加入1mL正己烷回溶，即获得脂肪酸甲酯；使用GC-MS检测菌体中脂肪酸的组成及含量，检测结果见图3-B,D；其中，脂肪酸甲酯分析采用GC2010(Shimadzu Co.，Japan)，色谱柱为DB-Waxetr(30m×0.32m，0.22μm)；氢火焰离子检测器检测，汽化室和检测器温度分别为240℃和260℃，分流方式进样1uL，分流比10:1，载气为氮气；程序升温：初始温度120℃保持3min，以5℃/min升到190℃，再以4℃/min升到220℃，保持20min；通过与商业化的脂肪酸甲酯标准品(37种脂肪酸甲酯混标，Supelco，USA)和加入内标C15:0的质量比较，定性、定量分析样品中脂肪酸组分，总脂肪酸含量用单位菌体中总脂肪酸的质量表示。
由图3可知，在营养丰富的Broth种子培养基中，重组高山被孢霉M.alpina-Masnf4和M.alpina-Masnf4-Si各个转化子与对照组在生物量和脂肪酸含量上并无显著差别(不同字母表示数据具有显著性，ns表示无显著差别，P＜0.05)，其原因是Broth种子培养基中葡萄糖和氮源均充足，可能未能激活高山被孢霉SNF1复合体的活性，之后，挑选可以稳定生长的转化子分别在限碳和限氮的培养基中进行后续实验。
实施例5：重组高山被孢霉在营养胁迫条件下生长和产脂分析
以高山被孢霉原养型菌株为阴性对照，取重组高山被孢霉M.alpina-Masnf4和M.alpina-Masnf4-Si以及高山被孢霉原养型菌株的单孢子接种于Kendrick种子培养基中，于28℃的条件下培养2d进行活化；连续活化三代，收集活化后的菌体；将菌体打碎至均匀菌絮状；将打碎的菌体以1％(v/v)的接种量接种到Kendrick限碳或限氮培养基中，于28℃、200rpm摇床培养，分别于36h，96h和168h时收集菌体，根据实施例3所述，进行生物量、脂肪酸和脂肪酸组分进行分析，结果如图4所示，对菌体形态进行分析，结果如图5所示。
由图4可以看出，选择发酵前期(36h，低碳/氮)、发酵中期(96h，碳/氮耗尽)及发酵末期(168h碳/氮饥饿)样品分析生长和产脂情况。相较于氮源，重组菌对碳源水平响应更敏感。碳源水平较低时(36h)M.alpina-Masnf4-Si生物量和脂肪酸含量分别是M.alpina-Masnf4的2.2倍和1.5倍，说明对高山被孢霉Masnf4进行干扰使得重组菌在较低碳源水平下即可合成较多脂肪酸。随着碳源逐渐耗尽(96h之后)，重组菌生物量无明显改变，脂肪酸含量开始降低，而对照组则无明显变化，说明该基因对菌株生长和脂质积累有负调控作用(图4,A-D)。由于菌体在96h后一直处于碳饥饿的环境中，TCA循环被抑制，胞内能量失衡，因此脂肪酸被分解用于产生能量并提供碳骨架。发酵末期的脂肪酸组分结果显示，M.alpina-Masnf4-Si中花生四烯酸(ARA)比例分别比对照组和过表达菌株提高21.1％和50.9％(图4,E、F)，由于长链脂肪酸的β-氧化主要发生在过氧化物酶体中，说明Masnf4影响了高山被孢霉过氧化物酶体中脂质代谢。上述结果为通过基因工程手段进一步提升高山被孢霉等产油微生物产脂肪酸(尤其是不饱和脂肪酸)的能力，进而为提高脂肪酸(尤其是不饱和脂肪酸)的生物合成能力提供了充实的理论支持。
在营养充足或氮源限制的液体培养条件下，高山被孢霉是蓬松、均匀的菌球状。由图5可知，在碳限制条件下，对照组和M.alpina-MaSnf4菌体为细碎的菌丝或紧实小球，这一形态多出现在营养状态不佳或不适宜生长的培养条件下。M.alpina-Masnf4-Si则仍为蓬松毛球状，可知该基因改变了重组菌对环境及营养胁迫的敏感度，影响了菌株对压力的响应进而改变菌体形态。
实施例6利用低碳氮比培养基合成脂质的方法
取实施例4构建的重组菌M.alpina-Masnf4-Si进行活化。之后，在Kendrick培养基的基础上，改变葡萄糖和酒石酸铵添加量，使得发酵培养基的碳氮比维持在3～6:1范围内。即，初始葡萄糖含量为5～15g/L，初始酒石酸铵浓度为5.0～10.0g/L，1.5g/L酵母提取物、7g/L磷酸二氢钾、2.0g/L磷酸氢二钠、1.5g/L七水硫酸镁、0.008g/L二水氯化钙以及微量元素，其中，微量元素浓度为：0.001g/L七水氯化铁、0.0001g/L七水硫酸锌、0.0001g/L五水硫酸铜、0.0001g/L硝酸钴、0.0001g/L五水硫酸锰。由此，将碳氮比维持在3～6之间。接种活化后的M.alpina-Masnf4-Si，于28℃、200rpm摇床培养。在发酵过程中，根据葡萄糖消耗情况适当补充葡萄糖，使葡萄糖浓度在发酵过程中维持在5～15g/L的范围内，以免因为碳源耗尽导致储存脂质进入分解代谢循环，同时也避免高碳氮比导致碳源的浪费从而提高生产成本。重组菌培养5～10天后收集菌体，根据实施例4记载的提取脂肪酸的方法提取得到脂质。
实施例7利用低碳氮比培养基合成花生四烯酸的方法
采用实施例6制备的碳氮比维持在3～6:1范围内的Kendrick培养基，培养并活化实施例4所述的敲低高山被孢霉蔗糖非发酵型蛋白激酶调节亚基的重组菌M.alpina-Masnf4-Si，于28℃、200rpm摇床发酵培养5～10天，在发酵过程中，根据葡萄糖消耗情况适当补充葡萄糖，使之维持在5～15g/L的范围内。发酵结束后，收集菌体，根据实施例4所述的方法提取脂质，即可获得富含花生四烯酸的油脂。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种蔗糖非发酵型蛋白激酶调节亚基及其应用
<130> BAA190819A
<160> 7
<170> PatentIn version 3.3
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<211> 531
<212> PRT
<213> 高山被孢霉
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Pro Asp Thr Thr His Pro Lys Asp Ser Gly Gly Leu Ser Asn Thr Gln
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Ala Pro Pro Ala Ser Ser Leu Thr Ala Ala Leu Asn Asn Asn Thr Asn
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His Ser Asn Thr Ala Gly Ser Asp Arg Pro Gly Pro Asp Ala Ala Pro
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Glu Asn Ile Asp Pro Val Ala Ser Tyr Ser Ala Trp Arg Lys Lys Lys
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Asp Thr Glu Met Leu Gln Gly Thr Gln Ala Val Met Glu Ser Phe Leu
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<210> 2
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<400> 2
atgagcgaca acaaggacgg taccattgct tccgagccag ctgaaacagc cgcgcatggg 60
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ccggcatcag aggatgccag cgaggagcaa ccgcagggcc agtcctcaac cgactacttt 180
tcgcctcaag aacccacagt caatgctttt actccgcaac agcaggacga cgctgtgggg 240
tttgcgtcag aaataggcca gcctgatcaa acaacagtga accccgaacc cgataccact 300
caccctaaag actcaggagg cctgtcgaat acgcaagcac ccccggcctc gtcgctgacg 360
gcagccctca acaataacac caaccactct aatacggctg ggtctgacag gccgggaccg 420
gatgctgcac caccaggatc ggctacccca tcctcacaaa caacggatgt gcctgcgacg 480
gaaaatattg accctgttgc atcgtactct gcatggcgca agaagaaaga cacggaaatg 540
ttgcaaggaa cccaggctgt gatggagtcg tttctaaagc agcactccgc ctacgatgtg 600
ttacccgtca gctatcggca aattgttttg gataccacgc tcttgatcaa gaaggccctt 660
tcggtcctga tgcagtacag catagtgtct gctccgttgt gggattcaac caagcgcaag 720
tttgcaggca tgctgacggc cacagacttt atcaatttga ttcagtttta ctactcccat 780
tcatcctaca ccgccgcaat tcatgagatg gaccaattcc agatatccca gctcagagat 840
gtcgagaagg taataggagt gccaccgccc gagctgttct cgctacaccc actgcaatcg 900
ctctacgacg cctgtcagtt gctggtcgaa tcaagagcgc atcggttacc cttggtggat 960
atggactctg aaacaggaga ggagatgatc gtgtcagtct tgactcagta cagaatcctc 1020
aagttcattg ccatgaacta tcaggacgag aagacacttc gtcaaccgct ttcggagctc 1080
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gttctgaacg tctttgagac tgtcgatgtg atgacgctgg tcaaggcagg cgtgtacagg 1260
gatttggatc tctgcgtcgc agaggcgtta ctgcggagac cggaagactt tgcaggagtt 1320
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gtccatcgcc tgatcgtcgt tgacagcggc aataagctga agggaatcgt cagtctctcg 1440
gatatcatgc gttatctcat cggtggaaca cgtgagacca aggagcctgc aggtggcaat 1500
gtcgaagagg cggaagtcca cgtcgaggac atcgagaagg aaaccaaatc gacgcacagt 1560
ggcgatggcg atgataatga tgcgggagac ctatga 1596
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cccaagcttg ggctgttctc gctacaccca ctgcaatcgc tctacgacgc ctgtcagttg 60
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gagatgatcg tgtcagtctt gactcagtac agaatcctca agtcccaagc gaatttgtca 180
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cctatcgtct gttactgcat ctctgtcgtg ttgaatgcgt aactgatgga cttccgtatg 300
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<212> DNA
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caaatgaacg tatcttatcg agatcc 26
Claims (10)
1.一种蔗糖非发酵型蛋白激酶调节亚基，其特征在于，其是由SEQ ID No.1所示的氨基酸序列组成的蛋白质。
2.一种基因，其特征在于，所述基因编码权利要求1所述的蔗糖非发酵型蛋白激酶调节亚基。
3.如权利要求2所述的一种基因，其特征在于，所述基因的核苷酸序列如SEQ ID No.2所示。
4.权利要求1所述蔗糖非发酵型蛋白激酶调节亚基在调控高山被孢霉脂质合成能力中的应用，其特征在于，当高山被孢霉处于碳氮比＜6或葡萄糖浓度＜15g/L的营养条件中时，限制所述蔗糖非发酵型蛋白激酶调节亚基的表达，或者敲除所述蔗糖非发酵型蛋白激酶调节亚基，能够使高山被孢霉重组菌合成脂质的能力接近或超过高山被孢霉处于碳氮摩尔比＞20或初始葡萄糖浓度＞30g/L的营养条件中时的合成脂质的能力。
5.一种利用低碳氮比培养基合成脂质的方法，其特征在于，以干扰了权利要求2所述基因表达或者敲除了权利要求2所述基因的高山被孢霉为生产菌株，以碳氮摩尔比＜6的培养基培养菌株，培养结束后，从菌体中提取得到脂质。
6.根据权利要求5所述的一种利用低碳氮比培养基合成脂质的方法，其特征在于，所述碳氮摩尔比＜6的培养基的组成是：初始葡萄糖浓度为5～15g/L，初始酒石酸铵浓度为5.0～10.0g/L,1.5g/L酵母提取物、7g/L磷酸二氢钾、2.0g/L磷酸氢二钠、1.5g/L七水硫酸镁、0.008g/L二水氯化钙以及微量元素,其中，微量元素浓度为：0.001g/L七水氯化铁、0.0001g/L七水硫酸锌、0.0001g/L五水硫酸铜、0.0001g/L硝酸钴、0.0001g/L五水硫酸锰，在发酵培养过程中控制培养基内葡萄糖浓度5～15g/L。
7.一种重组高山被孢霉，其特征在于，是敲除了权利要求2或3所述的基因的高山被孢霉，或者干扰了权利要求2或3所述的基因的表达的高山被孢霉。
8.根据权利要求7所述的一种重组高山被孢霉，其特征在于，利用SEQ ID No.5所述核苷酸片段干扰高山被孢霉的蔗糖非发酵型蛋白激酶调节亚基的表达。
9.干扰高山被孢霉的蔗糖非发酵型蛋白激酶调节亚基的表达的方法，其特征在于，合成SEQ ID No.5所示的RNA干扰片段，并插入到pBIG2-ura5s-ITs载体中，然后利用根癌农杆菌将重组载体转化到高山被孢霉中，利用SEQ ID No.5所示的RNA干扰片段干扰蔗糖非发酵型蛋白激酶调节亚基的表达。
10.权利要求7所述的一种重组高山被孢霉在脂质生产中的应用。
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