CN116240185B - 高丝氨酸脱氢酶突变体及其在生产l-高丝氨酸中的应用 - Google Patents
高丝氨酸脱氢酶突变体及其在生产l-高丝氨酸中的应用 Download PDFInfo
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- CN116240185B CN116240185B CN202310394817.0A CN202310394817A CN116240185B CN 116240185 B CN116240185 B CN 116240185B CN 202310394817 A CN202310394817 A CN 202310394817A CN 116240185 B CN116240185 B CN 116240185B
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- homoserine
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- corynebacterium glutamicum
- homoserine dehydrogenase
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Abstract
本发明属于生物工程领域，具体公开酶活提高的高丝氨酸脱氢酶突变体及其在制备L‑高丝氨酸或其衍生物中的应用。所述突变体的氨基酸序列相较于二穗短柄草（Brachypodium distachyon）来源的野生型高丝氨酸脱氢酶，存在T186A、N283K、A137T/I188V中一个突变类型。在具体实施中，经诱导表达纯化的酶活性测定分析，所述突变体的酶活性比野生型提高了1.26‑1.35倍。因此，本发明提供的有益突变体可以为工业化高效生产L‑高丝氨酸及下游代谢产物奠定了良好的基础。
Description
技术领域
本发明属于生物工程领域，具体公开酶活提高的高丝氨酸脱氢酶突变体及其在制备L-高丝氨酸或其衍生物中的应用。
背景技术
L-高丝氨酸是一种少数的没有被大规模生产的非蛋白质类氨基酸，是一种非必需的手性氨基酸，是苏氨酸、甲硫氨酸、赖氨酸等天冬氨酸家族氨基酸的重要前体。在制药，化妆品，农业以及香料方面有重要应用。L-高丝氨酸由于其具有与农药L-草铵膦相同的化学骨架，已被考虑为L-草铵膦的合成前体，相比化学法具有更好的经济性。
此外，L-高丝氨酸及其衍生物也是生产许多重要化合物的潜在平台，包括1,4 -丁二醇、异丁醇、2,4-二羟基丁酸酯、1,3-丙二醇。
在大肠杆菌、谷氨酸棒杆菌等微生物中，L-高丝氨酸的生物合成是从天冬氨酸支路途径开始的。葡萄糖经过糖酵解途径、磷酸戊糖途径和TCA循环等生成草酰乙酸，随后进入天冬氨酸支路途径。然后天冬氨酸前体在天冬氨酸激酶（Aspartokinase，EC: 2.7.2.4）、天冬氨酸半醛脱氢酶（Aspartate-semialdehyde dehydrogenase，EC: 1.2.1.11）和高丝氨酸脱氢酶（Homoserine dehydrogenase，EC: 1.1.1.3）等催化下最终生成L-高丝氨酸。其中高丝氨酸脱氢酶是高丝氨酸、苏氨酸、赖氨酸、异亮氨酸、甲硫氨酸等天冬氨酸家族氨基酸合成途径中的共同关键酶，其催化活性常受到末端产物或中间代谢物的反馈抑制或阻遏效应。
因此，通过筛选或分子改造高丝氨酸脱氢酶，获得具有优良的催化性能的关键酶突变体，对于天冬氨酸家族氨基酸的高效生产都具有非常重要的意义。
发明内容
基于上述需求，本发明的首要目的在于提供高丝氨酸脱氢酶突变体，以使得其催化性能得到提升，有利于微生物发酵生产L-高丝氨酸等代谢产物。
本发明通过以下技术思路实现，以B.distachyon野生型Hom片段为模板，利用易错PCR随机突变方法获得Hom编码基因突变文库，采用基于Goldengate技术的载体构建策略将其亚克隆至表达载体上，获得含有高丝氨酸脱氢酶Hom突变体编码基因的重组质粒文库。随后将重组质粒文库导入BL21（DE3）感受态细胞中，通过96孔板初筛和试管、摇瓶复筛，最终筛选获得酶活性提高的突变体，其突变位点分别为T186A、N283K、A137T/I188V。
本发明提供一种来源于野生型二穗短柄草（Brachypodiumdistachyon）的高丝氨酸脱氢酶突变体，其特征在于，相对于SEQ ID No.2所示氨基酸序列而言，其特征在于，相对于SEQ ID No.2所示氨基酸序列而言，仅第T186位由苏氨酸T突变为丙氨酸A，或者第283位由天冬酰胺N突变为赖氨酸K中的一个位点突变；仅存在第137位由丙氨酸A突变为苏氨酸T，仅存在于第188位由异亮氨酸I突变为缬氨酸V的组合突变。本发明进一步提供所述高丝氨酸脱氢酶突变体的编码基因。优选地实施方式中，所述高丝氨酸脱氢酶Hom编码基因的核苷酸序列是在SEQ ID No.1所示核苷酸酸序列的基础进行突变获得的。本发明还提供含有所述的高丝氨酸脱氢酶Hom突变体编码基因的表达载体和宿主细胞。在一个优选实施方式中，所述表达载体包括但不限于pEC-K99E，通过构建含有所述Hom突变体编码基因的重组质粒，并将其导入微生物底盘细胞中，用于发酵生产L-高丝氨酸及下游衍生物等。在具体的实施方式中，所述微生物底盘细胞可选自棒杆菌属、肠杆菌属，优选地是大肠杆菌（Escherichia coli）、谷氨酸棒杆菌（Corynebacterium glutamicum）。
本发明还提供了高丝氨酸脱氢酶突变体在发酵生产L-高丝氨酸及衍生物中的应用。选用具有一定L-高丝氨酸生产能力的底盘工程菌进行应用性能测试，在一个具体的实施例中，所述工程菌Cg-HSE特征在于谷氨酸棒杆菌Corynebacterium glutamicumATCC13032中敲除了自身高丝氨酸降解途径基因thrB，同时过表达天冬氨酸激酶基因LysC。
本发明相应提供一种制备L-高丝氨酸或其衍生物的方法，其特征在于，将含有所述编码基因的重组微生物发酵生产所述L-高丝氨酸和/或其衍生物，所述微生物以具有L-高丝氨酸生产能力的底盘工程菌作为出发菌。
优选地，所述微生物是具有L-高丝氨酸生产能力的大肠杆菌、谷氨酸棒杆菌，更优选为具有L-高丝氨酸生产能力的大肠杆菌、谷氨酸棒杆菌。
进一步优选地，所述谷氨酸棒杆菌中敲除了自身高丝氨酸降解途径基因thrB，同时过表达天冬氨酸激酶LysC；尤其优选地，所述出发谷氨酸棒杆菌是Corynebacterium glutamicumATCC 13032。
本发明有益效果在于，通过对B.distachyon 来源的高丝氨酸脱氢酶编码基因Hom进行突变和筛选，获得了催化活性不同程度提升的高丝氨酸脱氢酶突变体。因此，本发明提供的所述高丝氨酸脱氢酶突变体能够为高效发酵生产L-高丝氨酸及下游其他天冬氨酸家族代谢产物奠定了良好基础，相较于未突变型高丝氨酸脱氢酶具有更好的工业化应用前景。
附图说明
图1、高丝氨酸脱氢酶BdHSD及其突变体酶活测定。
图2、表达不同高丝氨酸脱氢酶突变体对高丝氨酸产量的影响。
实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明作进一步的详细说明，但并不应理解为对本发明的限制。实施例中所使用的实验方法如无特殊说明，均为本领域技术人员所熟知的常规方法。下述实施例中所使用的材料、试剂等如无特殊说明，均可从商业途径得到。
实施例1、高丝氨酸脱氢酶Hom突变体文库的构建
二穗短柄草B.distachyon的高丝氨酸脱氢酶Hom基因野生型的编码序列（SEQ IDNO：1）：
ATGCCCCCACAGGCTCAAGCAAAATCAGTATTGCCTGTAGTACTACTTGGCTGCGGCGGTGTTGGTCGCCACCTGCTGCGCCACATCGTGTCTTGTCGTCCGCTGCATGCAAATCAGGGCGTGAGCATCCGCGTGGTCGGCGTCAGCGATAGTTCCAGCTTGTTGTTAGCGGCGGACGACCTCAGAGCGGGCGCGGGTCTGGACGACGCGCTGCTGGGAGATTTATGCGCAGCAAAGTCGGCGGGTTCCCCGCTGTCCTCGCTGCTGGCGCGTGGTCAGTGTCAGCTGTTCAACAAGCCGGAGGCAATGTCTAAAGTGATCGACGCTGCGACGATGCTGGGCCGCACCACCGGTCTGGTTCTGGTGGACTGTTCTGCCACCTATGATACCATCGGTGTGTTAAAAGACGCAGTTGACCAGGGTTGCTGCGTTGTGCTGGCGAACAAGAAACCGCTGACGAGCAGCTACGAAGATTTCCAAAAGCTGACGAGCAATTTTCGTCAAATTCGATTTGAGTCCACCGTTGGTGCGGGTTTACCAGTTATTGCGAGCGTTACCCGTATCATCGCCAGCGGCGACCCGATTTCACGTATTGTGGGCAGCCTCAGCGGCACCCTGGGCTACGTTATGTCCGAGCTGGAGGATGGTAAACGCTTCAGCGAGGTGGTGAAAACCGCAAAGAGCTTGGGTTACACCGAGCCGGACCCGCGTGATGACTTGTCAGGGATGGATGTTGCACGCAAAGCCCTTATTcTGGCTCGTCTGCTTGGCCGTCAGATTTCCATGGAAGACATCAACGTGGAATCCCTGTACCCGAGCGAACTCGGTCCGGAAGTCATCAGCACCAATGACTTCCTGGAATCCGGCTTGGCTCAACTGGATGAATCTATTGAGGAACGTGTGAAGGCTGCTAGCCTGCGTGGTAATGTTCTGCGTTATGTGGGTGTCATCGAGAGCACGGGCTGCCAGGTTGGCTTGCAAGAAGTTCCGAAAGATAGCGCGTTGGGTCGTCTGATGGGTTCCGACAACGTGGTGGAAATTTATAGCCGTTGCTACGAGAACAGCCCGCTGGTTATCCAGGGTGCGGGTGCGGGCAACGACACTACTGCCGCGGGCGTCCTGGCTGATATCGTTGATCTGCAAGATTTGTTTCAAAAGCGCGTT。
其编码野生型的氨基酸序列（SEQ ID NO：2）：
MPPQAQAKSVLPVVLLGCGGVGRHLLRHIVSCRPLHANQGVSIRVVGVSDSSSLLLAADDLRAGAGLDDALLGDLCAAKSAGSPLSSLLARGQCQLFNKPEAMSKVIDAATMLGRTTGLVLVDCSATYDTIGVLKDAVDQGCCVVLANKKPLTSSYEDFQKLTSNFRQIRFESTVGAGLPVIASVTRIIASGDPISRIVGSLSGTLGYVMSELEDGKRFSEVVKTAKSLGYTEPDPRDDLSGMDVARKALILARLLGRQISMEDINVESLYPSELGPEVISTNDFLESGLAQLDESIEERVKAASLRGNVLRYVGVIESTGCQVGLQEVPKDSALGRLMGSDNVVEIYSRCYENSPLVIQGAGAGNDTTAAGVLADIVDLQDLFQKRV。
本发明采用保真性低的EasyTaq DNA聚合酶（Beijing TransGen Biotech,China），以野生型B.distachyon基因组为模板，利用引物P1 (5’-CACCAGGTCTCAGGAGATATACATATGCCCCCACAGGCTCAAGCAA-3’）和P2（5’-CACCAGGTCTCAGGTGAACGCGCTTTTGAAACAAATCTTGCAGATC-3’），通过易错PCR方法获得Hom编码基因突变文库。通过在PCR反应体系中加入一定浓度的镁离子和锰离子，进一步降低PCR扩增过程中的保真性，控制获得的Hom编码基因中含有2-3个点突变。本发明采用的易错PCR体系为：5 μL 10 × EasyTaq缓冲液、0.2 μM上游引物P1、0.2 μM上游引物P2、200 μM dNTPs、0.8 mM MnCl2、6 mM MgSO4、50 ng模板DNA、1μLEasyTaq DNA聚合酶，加入无菌水补齐至50 μL体系。PCR反应程序为：95℃ 预变性3 min；95℃变性30 s；58℃退火30 s；72℃延伸2 min，循环35次；72℃ 延伸 5 min，4℃保存。
采用高保真性的Phusion High-Fidelity DNA 聚合酶，以pET21b质粒为模板，利用引物P3（5’-CACCAGGTCTCACACCACCACCACCACCACTGAGATC-3’）和P4（5’-CACCAGGTCTCACTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTG-3’），通过普通PCR方法获得pET21b质粒骨架。10 μL 5 × Phusion HF缓冲液、0.5 μM上游引物P3、0.5 μM上游引物P4，200 μMdNTPs、3% DMSO、50 ng模板DNA、0.5 μL Phusion High-Fidelity DNA聚合酶 (ThermoScientific, USA)，加入无菌的水补齐至50 μL体系。PCR反应程序为：98℃预变性1 min；98℃变性10 s；62℃退火20 s；72℃延伸3 min，循环35次；72℃ 延伸 5 min，4℃保存。
基于Goldengate方法将PCR获得的Hom编码基因突变文库和pET21b质粒骨架进行连接，获得含有Hom编码基因不同突变体的重组表达质粒pET21b-hommut。Goldengate 连接体系为：T4 DNA Ligase buffer 1.5 μL，T4 DNA Ligase 1 μL，BsaI 1 μL，10×BSA 1.5μL，质粒骨架、片段（摩尔比为1:1，共10μL），总体积为15μL。PCR连接条件：37℃ 3min， 22℃4 min（40个循环）；22℃ 30min；80℃5min；4℃ 10 min。然后将上述连接体系按照常规的大肠杆菌热激转化法，导入大肠杆菌DH5α感受态细胞，获得的重组质粒文库。
实施例2、高丝氨酸脱氢酶变体文库的筛选鉴定
转化至E. coli BL21（DE3）中。挑取单菌落至96孔板中37℃培养过夜，转接至新的LB培养基中加入0.4 mmol/L IPTG，16℃诱导16~20 h。离心后加入含溶菌酶的裂解缓冲液孵育3h后，离心收集上清获得粗酶液。通过测定反应体系中NADPH含量变化速率对酶突变体进行高通量评价和筛选。通过多轮筛选，共获得了10个粗酶活显著提升的突变体。随后对10个突变体进行蛋白纯化和酶活测定，结果表明mut-5、mut-8和mut-10 表现出较高的酶活性，比酶活达到10.3 U/mg，9.7U/mg，9.6U/mg，分别比野生型提高了35.5%，27.6%，26.3%。
所述LB培养基组分包括：1%酵母提取物，2%胰蛋白胨，1% NaCl。
实施例3、高丝氨酸脱氢酶突变体的酶活测定方法
收集诱导表达的菌体，离心去除培养基，菌体用10 mL预冷的裂解buffer重悬菌体沉淀（20 mM Na2HPO3，200 mM NaCl，pH 7.5）。使用超声波细胞破碎仪破碎细胞，设定200W功率，超声2 s间歇1 s，超声10 min；随后在高速冷冻离心机上8000×g离心10 min，收集上清用于后续蛋白纯化以及酶活性测定。
高丝氨酸脱氢酶酶活是通过检测340 nm处吸光度的变化测定的，摩尔消光系数为6220 M−1 cm−1。反应体系为100 mmol/L Tris缓冲液（pH 7.5），80 mmol/L L-Hse，400mmol/L NaCL和1 mmol/L NAD(P)+。高丝氨酸脱氢酶的酶活单位定义为单位时间内每生成1μmol NAD(P)H所消耗的酶量。
通过对高丝氨酸脱氢酶的活性筛选测定分析，结果表明T186A、N283K和A137T/I188V突变体比酶活达到10.3 U/mg，9.7U/mg，9.6U/mg，分别比野生型提高了35.5%，27.6%，26.3%（如图1所示）。
实施例4、表达高丝氨酸脱氢酶突变体提高工程菌发酵生产L-高丝氨酸
基于实施例3获得的高丝氨酸脱氢酶突变体，构建微生物细胞工厂用于发酵生产L-高丝氨酸及下游衍生产品。本实施例中优选取的质粒为大肠杆菌-谷氨酸棒杆菌pEC-XK99E穿梭诱导表达型载体，该质粒本身含有强启动子（包括操纵序列lacO），当加入IPTG或乳糖等诱导物时，会促使阻遏蛋白离开操纵序列从而起始基因表达。基于Goldengate方法将PCR获得的Hom编码基因突变片段和pEC-XK99E质粒骨架进行连接，构建表达质粒pEC-XK99E-WT、pEC-XK99E-T186A、pEC-XK99E-N283K和pEC-XK99E-A137T/I188V。随后将上述表达质粒分别电转化至谷氨酸棒杆菌合成菌株H101中，构建高丝氨酸生产菌株H101-BdHSDwt，H101-BdHSD1，H101-BdHSD2，H101-BdHSD3。在一个具体的实施例中，所述工程菌H101特征在于谷氨酸棒杆菌Corynebacterium glutamicumATCC 13032中敲除了自身高丝氨酸降解途径基因thrB，同时过表达天冬氨酸激酶LysC。
将上述获得的工程菌株分别接种至含有20 mL LBHIS培养基的三角瓶中，培养16h至对数中期。离心收集细胞，用新鲜的发酵培养基悬浮，接种至含有25 mL发酵培养基的500 mL三角瓶中，调整起始OD600为1.0。所述LBHIS培养基为：酵母粉5 g/L，蛋白胨10 g/L，氯化钠10 g/L，脑心浸液18.5 g/L，山梨醇91 g/L。
种子培养基：玉米浆30 g/L，尿素5 g/L，硫酸镁0.5 g/L，硫酸铵5 g/L，磷酸二氢钾0.25 g/L，葡萄糖25 g/L，苏氨酸0.4 g/L。
发酵培养基：玉米浆70 g/L，FeSO4-柠檬酸盐溶液0.55 ml/L（FeSO4-7H2O 20 g/L，柠檬酸18.14 g/L），硫酸铵35 g/L，硫酸镁0.5 g/L，85%磷酸0.225 ml/L，磷酸二氢钾0.25 g/L，一水葡萄糖50 g/L，维生素溶液7 ml/L（含生物素300 mg/L，维生素B1500 mg/L，泛酸钙盐2 g/L，烟酰胺600 mg/L），尿素5 g/L，苏氨酸0.4 g/L。
研究结果如图2所示经过36 h发酵培养后，表达BdHSD突变体的菌株高丝氨酸产量显著提升，其中含有BdHSD T186A突变体的重组菌株H101-BdHSD1的L-高丝氨酸产量最高，达到3.2 g/L，比对照菌株H101-BdHSDwt的L-高丝氨酸含量提高了28%。相较于未突变型高丝氨酸脱氢酶，在底盘工程菌中表达上述高丝氨酸脱氢酶Hom突变体可以明显提升菌株发酵生产L-高丝氨酸的能力。
Claims (11)
1.一种来源于二穗短柄草（Brachypodium distachyon）的高丝氨酸脱氢酶Hom突变体，其特征在于，所述突变体的氨基酸序列相对于SEQ ID No.2所示氨基酸序列而言，仅第186位由苏氨酸T突变为丙氨酸A；或者仅第283位由天冬酰胺N突变为赖氨酸K；或者仅存在第137位由丙氨酸A突变为苏氨酸T和第188位由异亮氨酸I突变为缬氨酸V的组合突变。
2.如权利要求1所述高丝氨酸脱氢酶Hom突变体的编码基因。
3.如权利要求2所述的编码基因，其特征在于，其核苷酸序列是在SEQ ID No.1所示核苷酸序列的基础上进行突变获得的。
4.含有如权利要求2或3所述编码基因的表达载体。
5.含有如权利要求2或3所述编码基因的重组微生物。
6.如权利要求5所述的重组微生物，其特征在于，其是大肠杆菌或谷氨酸棒杆菌。
7.如权利要求1所述的高丝氨酸脱氢酶Hom突变体或其编码基因在制备L-高丝氨酸中的应用。
8.一种制备L-高丝氨酸的方法，其特征在于，将含有如权利要求2或3所述编码基因的重组微生物发酵生产所述L-高丝氨酸，所述微生物以具有L-高丝氨酸生产能力的底盘工程菌作为出发菌。
9.如权利要求8所述的方法，其特征在于，所述微生物是具有L-高丝氨酸生产能力的大肠杆菌或谷氨酸棒杆菌。
10.如权利要求9所述的方法，其特征在于，所述谷氨酸棒杆菌是出发谷氨酸棒杆菌中敲除了自身高丝氨酸降解途径基因thrB，同时过表达天冬氨酸激酶LysC。
11.如权利要求10所述的方法，其特征在于，所述出发谷氨酸棒杆菌是Corynebacterium glutamicum ATCC 13032。
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| 谷氨酸棒杆菌高丝氨酸脱氢酶编码基因hom的敲除;饶志明等;中国生物工程杂志;20061231;第27卷(第1期);第59-63页 * |
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