CN106062177B - 具有改进的产生l-赖氨酸的能力的棒状杆菌微生物和用于使用其产生l-赖氨酸的方法 - Google Patents
具有改进的产生l-赖氨酸的能力的棒状杆菌微生物和用于使用其产生l-赖氨酸的方法 Download PDFInfo
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Abstract
具有改进的产生赖氨酸的能力、其中隔膜形成起始蛋白失活的棒状杆菌属微生物，和用于使用该微生物产生L‑赖氨酸的方法。
Description
发明领域
本公开涉及具有改进的产生L-赖氨酸的能力的棒状杆菌微生物和用于使用其产生L-赖氨酸的方法。
发明背景
棒状杆菌属(Corynebacterium)的微生物是革兰氏阳性细菌且广泛用于生产L-氨基酸。L-氨基酸，特别是L-赖氨酸，用在动物饲料、用于人的药物和化妆品领域中，并且通过使用棒状杆菌菌株发酵产生。
已经多次尝试改进使用棒状杆菌菌株产生L-氨基酸的方法。在尝试中，研究了通过使用重组DNA技术破坏或减弱特定基因的表达来改进产生L-氨基酸的棒状杆菌菌株。另外，关于扩增与每种L-氨基酸的生物合成有关的基因对L-氨基酸生产的影响和关于产生L-氨基酸的棒状杆菌菌株的改进进行了大量研究。此外，甚至可以引入源自其他细菌的外源基因。
然而，仍然需要超出常规方法的具有改进的产生L-赖氨酸的能力的菌株。
发明详述
技术问题
一个方面提供棒状杆菌微生物，其具有改进的产生L-赖氨酸的能力。
另一个方面提供用于使用该棒状杆菌微生物产生L-赖氨酸的方法。
技术方案
一个方面提供具有改进的产生L-赖氨酸的能力的棒状杆菌微生物，其中隔膜形成起始蛋白(septum formation initiator protein)是失活的。
术语“隔膜形成起始子(initiator)”如本文使用的指启动隔膜(即在棒状杆菌微生物的细胞分裂过程期间分开细胞内部的膜结构)的形成必需的因子。细胞分裂可以在存在隔膜形成起始子的情况下进行。在存在隔膜形成起始子的情况下，隔膜可以被缩入细胞中心周围的外周部分以将细胞质一分为二。之后可以进行外部膜的合成，继之以细胞分裂。隔膜形成起始蛋白可以是选自表1和2的一种蛋白。
[表1]
表2
隔膜形成起始子可以包括丝状温度敏感性(Fts)蛋白。Fts蛋白是涉及分裂体(divisome)形成的蛋白，其在微生物的细胞分裂过程中可能是必要的。Fts 蛋白可包括FtsB、FtsA、FtsZ、FtsEX、FtsK、FtsQ、FtsW、FtsI、或其组合，具体地，FtsB。FtsB可以具有SEQID NO:1的氨基酸序列或其70％同源性，具体地，其80％同源性，更具体地，其90％同源性，最具体地，其95％同源性序列。术语“同源性”指两个氨基酸序列之间的同一性程度，其可通过使用以下方法来确定，该方法利用BLAST 2.0来计算参数如得分、同一性或相似性，这是本领域普通技术人员广泛公知的。
编码Fts蛋白的基因可以是编码ftsA,ftsB,ftsZ,ftsEX,ftsK,ftsQ,ftsW,ftsI或其组合的核酸。
涉及棒状杆菌微生物的细胞分裂的ftsB基因可以与ftsZ,ftsEX,ftsK,ftsQ,ftsW和B类高分子量(HMW)-PBP一样好地形成分裂体。ftsB基因可以是编码 SEQ ID NO:1的氨基酸序列的核酸。ftsB基因可以是例如NCBI登录号 NCg10936基因。术语“NCBI登录号NCg10936基因”指具有源自谷氨酸棒状杆菌菌株的SEQ ID NO:2的核苷酸序列的基因。另外，基因可以存在于棒状杆菌属的微生物中并产生与NCBI登录号NCg10936基因产生的产物实质相同的产物。表述“实质相同”如本文使用的意指与NCBI登录号NCg10936基因的产物有相同活性和调控机制。
术语“失活”如本文使用的意指细胞或酶的活性水平不显现 (nonexhibition)，这可在与其可比的同一类型的细胞或初始酶中测量。与其可比的同一类型的细胞可以是未被操作如重组或修饰的细胞。在微生物中，隔膜形成起始蛋白的失活可以指去除多肽的活性，该多肽编码所述隔膜形成起始子。此外，在微生物中，可以使隔膜形成起始蛋白失活至足以产生L-赖氨酸的程度。
失活可以通过重组方法导致。重组方法可包括同源重组方法。同源重组方法可以通过以下进行：将包含基因的序列部分的载体转化到微生物，并在存在选择标志物的产物的情况下培养该微生物，由此基因的序列部分和微生物中的内源基因可经历同源同组。载体可以是pDZ-ΔftsB W140*载体，其包含由SEQ ID NO:4代表的NCBI登录号NCg10936基因片段，或pDZ-ftsB W140*载体，其包含由SEQ ID NO:6代表的NCBI登录号NCg10936基因片段。通过同源重组，微生物中的内源基因可以重组，且从重组的基因中，可通过选择标志物选择包含标志物的重组体。通过同源重组方法，可以获得棒状杆菌属的微生物，其中内源NCBI登录号NCg10936基因失活。
在微生物中，蛋白活性的失活可由基因的碱基、核苷、核苷酸或其组合的缺失、取代、添加、倒置、或其组合产生。详细而言，使蛋白活性失活的方法的例子可以是基因敲除办法、反义技术或RNAi技术。使蛋白活性失活的方法可进一步包括每个基因的初始拷贝的缺失、将初始拷贝用突变体取代、或从弱启动子表达初始拷贝。另外，还可以利用编码蛋白的基因的启动子的取代，通过随机或定点诱变的突变转移，或基因破坏或敲除。此外，还可以使用通过基因修饰将不稳定元件引入mRNA或异常化(aberrating)RNA 的核糖体结合位点(RBS)的方法。
基因的失活可通过以下实现：将包含NCBI登录号NCg10936基因的部分和选择标志物的载体转化到棒状杆菌属的微生物，接着在存在抗生素的情况下培养并选择。
选择标志物可用于选择用载体转化的细胞。选择标志物可以是赋予可选择表型，如药物抗性、营养缺陷、细胞毒性药物抗性或表面蛋白表达的标志物。
棒状杆菌微生物可以选自下组：谷氨酸棒状杆菌、产氨棒状杆菌(Corynebacterium ammoniagenes)、假结核棒状杆菌、有效棒状杆菌、 Corynebacteriumpseudogenitalium、Corynebacterium genitalium、拥挤棒状杆菌、无枝菌酸棒状杆菌、马氏棒状杆菌、Corynebacterium casei、 Corynebacterium lipophiloflavum、Corynebacterium tuberculostearicum、杰氏棒状杆菌、稍变棒状杆菌，和从其野生型产生的L-赖氨酸生产性突变体。具体地，所述棒状杆菌微生物可以是谷氨酸棒状杆菌。谷氨酸棒状杆菌可以是具有登录号KCCM11016P,KCCM10770P,KCCM11347P,或CJ3P的谷氨酸棒状杆菌。
术语“棒状杆菌微生物”如本文使用的指具有产生L-赖氨酸的能力的棒状杆菌属的微生物。棒状杆菌微生物的例子可包括具有改进的产生L-赖氨酸的能力的棒状杆菌属的微生物，对其引入了突变以使编码隔膜形成起始蛋白的基因失活以用于改进产生L-赖氨酸的能力。表述“具有产生L-赖氨酸的能力”意为，当在培养基中培养微生物时，微生物具有产生和在培养基中分泌 L-赖氨酸的能力。所述棒状杆菌微生物可以是以下微生物，与其野生型或亲本菌株相比，其可在培养基中以大量产生和积累L-赖氨酸。
根据一个实施方案，使编码棒状杆菌微生物中隔膜形成起始蛋白，例如 FtsB的基因失活，由此可以抑制棒状杆菌微生物的细胞分裂，同时可以相对改进产生L-赖氨酸的能力。
另一个方面提供用于产生L-赖氨酸的方法，该方法包括培养根据本发明的微生物从而在培养基中产生L-赖氨酸；和从所述微生物或培养基回收L-赖氨酸。所述微生物与上文的描述相同。
微生物的培养可在适当培养基中在本领域公知的培养条件下实施。此类培养过程可以随着要选择的微生物容易地调整。培养方法可包括选自下组的至少一种：分批培养、连续培养和补料分批培养。
培养中使用的培养基可以满足具体微生物的要求。培养基可选自下组：碳源、氮源、微量元素及其组合。
碳源可以选自下组：碳水化合物、脂质、脂肪酸、醇、有机酸及其组合。碳水化合物可以是葡萄糖、蔗糖、乳糖、果糖、麦芽糖、淀粉、纤维素或其组合。脂质可以是大豆油、向日葵油、蓖麻油、椰子油或其组合。脂肪酸可以是棕榈酸、硬脂酸、亚油酸或其组合。醇可以是甘油或乙醇。有机酸可以包括乙酸。
氮源可以包括有机氮源、无机氮源、或其组合。有机氮源可以选自下组：胨、酵母提取物、肉膏、麦芽抽提物、玉米浸渍液(corn steep liquid,CSL)、大豆粉(soybean meal)及其组合。无机氮源可以选自下组：尿素、硫酸铵、氯化铵、磷酸铵、碳酸铵、硝酸铵及其组合。
培养基可以包含选自下组的一种：磷、金属盐、氨基酸、维生素、前体及其组合。磷源可以包括磷酸二氢钾、磷酸氢二钾(dipotassium phosphate)、其相应的含钠盐。金属盐可以是硫酸镁和硫酸亚铁(iron sulfate)。
可以将培养基或个别组分以分批模式、连续模式、或补料-分批模式添加到培养基。
在培养方法中，可以调节培养物的pH。可以通过对培养物添加氢氧化铵、氢氧化钾、氨水(ammonia)、磷酸或硫酸实施pH调节。此外，培养方法可以包括防止气泡产生。可以通过使用消泡剂进行气泡产生的防止。消泡剂可以包括脂肪酸聚乙二醇酯(fatty acidpolyglycol ester)。此外，培养方法可以包括将气体注射入培养物中。气体可以包括能够维持培养物的需氧条件的任何气体。气体可以是氧气或含氧气体。含氧气体可以包括空气。在培养中，培养物的温度可以在20至45℃，例如22至42℃或25至40℃的范围中。可以继续培养，直到L-赖氨酸的生产达到期望的水平。
在根据本发明的方法中，培养可以是连续培养或分批培养，如分批、补料-分批和重复的补料-分批培养。此类培养方法是本领域公知的，且可以使用任何适宜的方法。可以通过阴离子交换层析和茚三酮衍生化来分离和分析 L-氨基酸。
本申请的有利效果
根据一个方面的编码隔膜形成起始蛋白的基因失活的微生物，可以改进该微生物的生产L-赖氨酸的能力。
根据一个方面的用于生产L-赖氨酸的方法，可以以高生产力(productivity) 生产L-赖氨酸。
实施例
在下文中，本申请会参考实施例更为详细地描述。然而，这些实施例仅仅用于例示目的，并且本申请的范围并不意图受限于这些实施例。
实施例1：通过缺失制备ftsB基因失活的重组载体
基于美国国家卫生研究院的GenBank(NIH Genbank)中的碱基序列获得 ftsB基因的核苷酸序列(SEQ ID NO:2)。为了制备其中ftsB的开放阅读框内部消失的基因片段，基于SEQ ID NO:2，构建了引物0936F1,09369R1,0936F2, 和0936R2并分别称为SEQ ID NO:7至10。
为了制备由于ftsB基因缺失所致的失活重组载体，使用pDZ载体(参见韩国专利登记号10-0924065)。然后，如上文描述的，将具有ftsB基因的经修饰序列的构建用于失活的核酸分子插入pDZ载体的多克隆位点中，从而制备包含SEQ ID NO:4的核酸序列的pDZ-ΔftsB载体。SEQ ID NO:4的核酸序列编码具有SEQ ID NO:3的序列的氨基酸序列。
使用谷氨酸棒状杆菌ATCC13032基因组DNA作为模板和使用引物 0936F1-0936R1和0936F2-0936R2来实施PCR。使用PfuUltraTM高保真DNA聚合酶(Stratagene)作为聚合酶。PCR条件如下：在96℃变性30秒，在58℃退火 30秒，在72℃聚合2分钟，30个循环。
因此，获得一对ftsB-A和ftsB-B DNA片段，各自分别包含74bp和95bp 的ftsB基因。使用Infusion克隆试剂盒(Invitrogen)将扩增的产物克隆到pDZ载体中，引起pDZ-ΔftsB载体的构建。pDZ-ΔftsB载体包含135bp ftsB的XbaI末端和没有ftsB的408bp内部区的片段两者。
实施例2：通过引入终止密码子来制备ftsB基因失活的重组载体
为了通过引入终止密码子来制备ftsB基因失活的重组载体，使用pDZ载体，并构建引物来将ftsB基因开放阅读框中第140位氨基酸(即色氨酸)的反密码子取代为终止密码子。使用引物对0936F1和0936R3和引物对0936F3和 0936R2，以与实施例相同的方式来实施用于扩增的PCR，并将扩增产物克隆到pDZ载体中，引起pDZ-ftsB W140*载体的构建。引物对0936F3和0936R3 的序列分别如SEQ ID NO:11和12所示。克隆到构建的载体中的fts基因的氨基酸序列为SEQ ID NO:5，且其核酸序列为SEQ ID NO:6(核苷酸序列)。
实施例3：构建ftsB基因失活的菌株
通过电脉冲方法将L-赖氨酸生产菌株谷氨酸棒状杆菌KCCM11016P((旧) 登录号KFCC10881，参见韩国专利登记号10-0159812和10-0397322)用实施例 1和2中构建的重组载体转化(使用Appl.Microbiol.Biotechnol.(1999) 52:541-545的转化方法)。然后，从含有25mg/L卡那霉素的选择培养基中选出具有通过基因同源性在染色体上插入的靶基因的菌株。载体在染色体中的成功插入通过观察菌落在含有X-gal(5-溴-4-氯-3-吲哚基-β-D-半乳糖苷)的固体培养基上是否是蓝色来确认。将染色体有插入的初步菌株在营养培养基中振摇培养(在温度30℃8小时)，然后将其从10-4稀释为10-10，接着在含有X-gal 的固体培养基上涂布。虽然大多数菌落为蓝色，但有一些菌落是白色。选择那些低比率的白色菌落，其进一步用于选择其中ftsB基因通过二次交叉 (secondary crossover)被失活的菌株。
为了选出其中ftsB基因通过缺失被失活的菌株，将一对基因特异性引物 SEQ IDNO:7和SEQ ID NO:10，0936F1-0936R2用作引物来实施PCR。分析关于靶位点的碱基序列用于最终确认，从而引起失活的菌株 KCCM11016P-ΔftsB的构建，其由ftsB基因的缺失所致。为了选出其中由于经修饰的终止密码子的转移导致ftsB基因失活的菌株，将一对引物SEQID NO: 7和SEQ ID NO:10，0936F1-0936R2用作引物来实施PCR。分析关于靶位点的碱基序列用于最终确认，从而引起ftsB基因失活的菌株KCCM11016P-ftsB W140*的构建，其由终止密码子的引入所致。
为了检查在属于谷氨酸棒状杆菌属的其他菌株中的效果，使用 KCCM10770P(参见韩国专利登记号10-0924065),KCCM11347P(参见韩国专利登记号10-1994-0001307)和CJ3P(Binder et al.Genome Biology 2012, 13:R40)作为亲本菌株，以与上文方法中相同的方式构建ftsB基因缺失的菌株和引入终止密码子的菌株，即KCCM10770P-ΔftsB,KCCM11016P-ftsB W140*, KCCM11347P-ΔftsB,KCCM11347P-ftsB W140*,CJ3P-ΔftsB,CJ3P-ftsB W140*。
实施例4：ftsB基因失活的菌株中的L-赖氨酸生产
培养实施例3种构建的L-赖氨酸生产菌株谷氨酸棒状杆菌 KCCM11016P-ΔftsB,KCCM11016P-ftsB W140*,KCCM10770P-ΔftsB, KCCM10770P-ftsB W140*,KCCM11347P-ΔftsB,KCCM11347P-ftsB W140, CJ3P-ΔftsB,和CJ3P-ftsB W140*用于L-赖氨酸产生，如在以下方法中的。
将谷氨酸棒状杆菌亲本菌株和KCCM11016P-ΔftsB,KCCM11016P-ftsB W140*,KCCM10770P-ΔftsB,KCCM10770P-ftsB W140*, KCCM11347P-ΔftsB,KCCM11347P-ftsBW140*,CJ3P-ΔftsB,和CJ3P-ftsB W140*各自接种到含有25ml种子培养基的250ml三角振荡烧瓶 (corner-baffled flask)中，然后以200次旋转每分钟(rpm)在30℃摇动培养20小时。然后，将1ml的每种种子培养物接种入含有24ml生产培养基的250ml三角振荡烧瓶中，接着通过以200rpm在30℃摇动培养120小时。种子培养基和生产培养基的组成如下。
种子培养基(pH 7.0)
20g粗糖、10g胨(pepton)、5g酵母提取物、1.5g尿素、4g KH2PO4、8g K2HPO4、0.5gMgSO4·7H2O、100μg生物素、1,000μg硫胺素HCl、2,000μg 泛酸钙、2,000μg烟酰胺(基于1L蒸馏水)
生产培养基(pH 7.0)
100g葡萄糖、40g(NH4)2SO4、2.5g大豆蛋白、5g玉米浸渍固体、3g 尿素、1g KH2PO4、0.5g MgSO4·7H2O、100μg生物素、1,000μg盐酸硫胺素、 2,000μg泛酸钙、3,000μg烟酰胺、30g CaCO3(基于1L蒸馏水)
在完成培养后，通过使用HPLC的方法来测量L-赖氨酸生产。L-赖氨酸在谷氨酸棒状杆菌亲本菌株和KCCM11016P-ΔftsB,KCCM11016P-ftsB W140*,KCCM10770P-ΔftsB,KCCM10770P-ftsB W140*, KCCM11347P-ΔftsB,KCCM11347P-ftsB W140,CJ3P-ΔftsB,和CJ3P-ftsB W140*的培养溶液中的浓度显示于表3。显示于表3的结果是通过重复实验的平均值。
[表3]
如表3中显示的，当ftsB基因缺失或终止密码子引入亲本菌株 KCCM11016P中时，赖氨酸生产在它们所有之中增加了约4％。这些结果表明赖氨酸浓度增加不是通过ftsB基因本身的结构变化，而是通过作为隔膜形成起始子的FtsB蛋白的缺失。另外，来自其他亲本菌株KCCM10770P, KCCM11347P和CJ3P的FtsB失活菌株的赖氨酸浓度增加了约4％，如与具有野生型FtsB活性的亲本菌株相比的。在另一方面，FtsB失活菌株的细胞体积降低了约5％，如与亲本菌株相比的。该结果意味着可以通过控制菌株的量来改进生产赖氨酸的能力，这是由于对赖氨酸生产菌株的细胞分裂的抑制所致。KCCM11016P-ftsB W140*(CA01-2274)在2013年9月27日以保藏号 KCCM11454P保藏在位于韩国首尔Urim bd.,Hongje-1-dong,Seodaemun-gu的韩国微生物保藏中心。
保藏机构：韩国微生物保藏中心(国际)
保藏号：KCCM 11454P
保藏日期：2013年9月27日
Claims (4)
1.一种具有改进的产生L-赖氨酸的能力的棒状杆菌微生物，其与隔膜形成起始蛋白未被失活的亲本菌株相比隔膜形成起始蛋白失活，其中隔膜形成起始蛋白是丝状温度敏感性B(FtsB)蛋白。
2.根据权利要求1的微生物，其中所述隔膜形成起始蛋白具有SEQ ID NO:1的氨基酸序列。
3.根据权利要求1的微生物，其中所述棒状杆菌微生物是谷氨酸棒状杆菌(Corynebacterium glutamicum)。
4.一种用于产生L-赖氨酸的方法，所述方法包括：
培养权利要求1的微生物以在培养基中产生L-赖氨酸；和
从所述微生物或所述培养基回收L-赖氨酸。
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