CN102586140B - 一种提高微生物有机溶剂耐受性的基因及其编码蛋白质与应用 - Google Patents
一种提高微生物有机溶剂耐受性的基因及其编码蛋白质与应用 Download PDFInfo
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Abstract
一种提高微生物有机溶剂耐受性的基因及其编码蛋白质与应用，属于生物工程技术领域。本发明公开的来源于恶臭假单胞菌P.putidaCCTCCNO：M2011442的mmsB基因，序列长度为888bp，编码295个氨基酸，由该基因编码的3-羟基异丁酸脱氢酶具有促进微生物细胞有机溶剂耐受性的作用。通过蛋白质二维电泳和菌体耐溶剂性实验证明，mmsB基因在恶臭假单胞菌中表达水平的提高与其有机溶剂耐受性的提高相关；同时，mmsB基因在大肠杆菌宿主中的重组表达能显著提高大肠杆菌细胞等革兰氏阴性菌的有机溶剂耐受性。本发明为阐明微生物细胞溶剂耐受性提供了有效方法，并为构建适用于工业化应用的溶剂耐受性整体细胞生物催化剂提供了理论依据。
Description
技术领域
本发明涉及一种提高微生物有机溶剂耐受性的基因及其编码的蛋白质，属于生物工程技术领域。应用于通过基因工程手段提高恶臭假单胞菌和大肠杆菌等革兰氏阴性菌的有机溶剂耐受能力。
背景技术
大多数的有机溶剂对微生物都是有毒害作用的，有机溶剂能够破坏细胞膜表面的磷脂双分子层，使细胞失去结构和功能上的完整性，从而导致微生物细胞裂解死亡。1989年，Aono和Inoue研究表明，有机溶剂对细胞的毒性大小可以用溶剂在辛醇和水中的分配系数（Log P ow ）来表示，Log P ow 的值越低，表示该溶剂的疏水性越高，对微生物的毒害作用越大。然而在全细胞生物催化反应中，为了降低产物的抑制作用和实现生物转化与分离的耦合，提高转化效率，常需要采用水/有机相两相体系。虽然微生物自身具有一定的溶剂耐受性，但绝大多数微生物对有机溶剂的耐受性较低，甚至0.1%（v/v）的有机溶剂就可以导致细胞的裂解死亡，因此探究微生物的耐溶剂性机制，提高微生物细胞对有机溶剂的耐受性就显得尤为重要。
1989年，Inoue等在Nature上首次报道了筛选获得的Pseudomonas putida能够耐受50%（v/v）甲苯和高浓度的环己烷、二甲苯等。1991年，Aono和Inoue的研究小组报道了通过溶剂平板筛选和NTG诱变成功获得了大肠杆菌耐溶剂突变株OST系列菌株。该小组对大肠杆菌OST菌株的研究显示，对于细胞膜疏水性有明显影响的一些细胞膜组分的表达水平是影响大肠杆菌细胞溶剂耐受性的重要原因。细胞外膜上的脂多糖LPS（lipopolysaccharide）成分的增加导致了细胞外膜疏水性的降低，从而使细胞溶剂耐受性增加。在对具有溶剂耐受性重组菌株与其野生型亲本菌株的生长对比时发现，细胞外膜蛋白和应激反应蛋白与微生物细胞的耐溶剂性紧密相关。基因芯片技术（DNA microarray）的应用也使得更多的耐溶剂性相关基因被发现，Shimizu 等发现glpC基因编码的一种膜蛋白（厌氧型甘油-3-磷酸脱氢酶C亚基），能显著提高菌株的耐溶剂性，其原因可能是细胞外膜结构的改变导致其疏水性的降低。另外，甘露糖的磷酸转移酶系统ManXYZ在大肠杆菌中的表达可以改变细胞膜表面的性质，使其对糖类的亲和性提高，从而对溶剂（正己烷）的耐受性有显著提高。但是，微生物的耐溶剂性机制目前尚无明确完善的体系。
早在1970年，Bannerjee等人阐明了3-羟基异丁酸脱氢酶是NAD+依赖型的氧化还原酶，参与缬氨酸的代谢途径。但是目前并无报道涉及3-羟基异丁酸脱氢酶在提高恶臭假单胞菌和大肠杆菌等革兰氏阴性菌的有机溶剂耐受性方面的作用。
发明内容
本发明的目的是提供一种提高微生物耐有机溶剂性的方法。mmsB基因在恶臭假单胞菌中表达水平的提高与其有机溶剂耐受性的提高相关；同时，mmsB基因在大肠杆菌宿主中的重组表达能显著提高大肠杆菌细胞等革兰氏阴性菌的有机溶剂耐受性。从而为阐明微生物细胞溶剂耐受性提供了有效方法，并为构建适用于工业化应用的溶剂耐受性整体细胞生物催化剂提供了理论依据。
本发明的技术方案：由微生物菌株恶臭假单胞菌P. putida通过驯化培养获得耐有机溶剂突变菌株（Pseudomonas putida）JUCT1，已保藏于中国典型培养物保藏中心，保藏编号CCTCC NO：M 2011442。
提取该驯化菌株CCTCC NO：M 2011442的总蛋白，通过二维电泳技术，比对分析P. putida CCTCC NO：M 2011442在不同溶剂条件下蛋白组的表达差异，经扫描后，使用PDQuestTM 2-D Analysis Software（Bio-Rad公司）分析比对蛋白质点，采用基质辅助激光解吸电离串联飞行时间质谱（MALDI-TOF/TOF）鉴定不同溶剂条件下表达量差异大的蛋白，并将质谱结果与NCBI数据库中的已知蛋白序列进行比对，最终确定该CCTCC NO：M 2011442的蛋白质为3-羟基异丁酸脱氢酶，由mmsB基因编码。
采用引物：
上游引物：5'-ATCGGGATCCATGCGTATTGCATTCATTGG-3'（BamH Ⅰ）
下游引物：5'-CCCCAAGCTTTCAATCCTTCTTGCGATACC-3'（Hind Ⅲ）
提取恶臭假单胞菌CCTCC NO：M2011442基因组，并以该基因组为模板，PCR扩增基因mmsB。基因mmsB经PCR产物回收后，连接T载体pMD18，并转化大肠杆菌JM109；提取转化后经验证的重组大肠杆菌JM109/pMD18-T-mmsB的质粒，采用限制性内切酶BamH Ⅰ和Hind Ⅲ对pMD18-T-mmsB质粒和表达载体pQE-80L分别进行双酶切。双酶切产物通过T4连接酶连接过夜，重组质粒pQE-80L-mmsB转化后获得重组大肠杆菌JM109/pQE-80L-mmsB。
本发明公开的一种提高微生物有机溶剂耐受能力的mmsB基因，从CCTCC NO：M 2011442的在不同溶剂条件下蛋白组的表达差异，确认该mmsB基因具有提高微生物的有机溶剂耐受能力；其为：
（1）核苷酸序列为SEQ NO. 1所示；
或（2）与所述序列SEQ NO. 1同源性超过90%，具有相同生理功能的核苷酸序列。
本发明中所述的一种提高微生物有机溶剂耐受能力的mmsB基因编码蛋白质，
（1）其氨基酸序列为SEQ NO. 2所示；
或（2）SEQ NO. 2经过一个或几个氨基酸的取代、缺失或添加且具有相同功能的氨基酸序列。
所述mmsB基因的应用，在于对提高恶臭假单胞菌耐受有机溶剂性中的作用。
所述mmsB基因的应用，在于在包括大肠杆菌的革兰氏阴性菌中的重组表达显著提高宿主细胞的有机溶剂耐受性，及其在构建溶剂耐受性整体细胞生物催化剂中的应用。
所述mmsB基因编码蛋白质的应用，在于对提高恶臭假单胞菌耐受有机溶剂性中的作用。
所述mmsB基因编码蛋白质的应用，在于在包括大肠杆菌的革兰氏阴性菌中的重组表达显著提高宿主细胞的有机溶剂耐受性，及其在构建溶剂耐受性整体细胞生物催化剂中的应用。
恶臭假单胞菌的mmsB基因在该菌株中表达水平的提高与其有机溶剂耐受性的提高相关；同时，mmsB基因在大肠杆菌等革兰氏阴性菌中的重组表达可显著提高宿主细胞的有机溶剂耐受性，为构建适用于工业化应用的溶剂耐受性整体细胞生物催化剂提供了理论依据。
本发明的有益效果：本发明通过蛋白质组学技术鉴定获得了一个能够提高微生物耐溶剂能力的蛋白质及其编码基因mmsB，并通过基因工程方法进行基因的重组表达，将来源于恶臭假单胞菌的mmsB基因在大肠杆菌JM109中进行重组表达，经1 mmoL/L的IPTG（异丙基-β-D-硫代半乳糖吡喃糖苷）诱导表达后，重组大肠杆菌菌株在有机溶剂环己烷中的存活能力和生长能力均较对照菌株有大幅度的提高。
生物材料样品保藏：上述耐有机溶剂突变菌株恶臭假单胞菌（P. putida）JUCT1，现保藏于中国典型培养物保藏中心，简称CCTCC，保藏单位地址：中国武汉武汉大学，保藏编号：CCTCC NO：M 2011442，保藏日期2011年12月4日。
附图说明
图1 菌株CCTCC NO：M 2011442在不同有机溶剂培养条件下的全蛋白二维电泳图。A：培养基中不含有机溶剂；B：培养基中含有60%（v/v）环己烷；C：选取的蛋白质点，C1局部放大图。箭头符号表示A、B两图谱中蛋白质差异量超过50%的蛋白质点。
图2 基因mmsB在大肠杆菌JM109中重组表达的SDS-PAGE分析。M：Marker；1：E. coli JM109/pQE-80L 经IPTG诱导；2：E. coli JM109/pQE-80L-mmsB经IPTG诱导后可溶性蛋白；3：E. coli JM109/pQE-80L-mmsB经IPTG诱导后全细胞蛋白。
图3 重组E. coli JM109/pQE-80L-mmsB与原始菌株在有机溶剂存在下的菌落形成效率比对。
具体实施方式
实施例1：耐溶剂驯化菌株P. putida CCTCC NO：M2011442总蛋白的提取
实验菌种：通过对恶臭假单胞菌P. putida进行驯化培养获得的有机溶剂耐受性菌株P. putida CCTCC NO：M2011442；所使用的试剂均为市售分析纯或生化试剂；本实验使用的仪器主要包括超声波细胞粉碎仪、冷冻离心机、恒温摇床等；所用的培养基为营养肉汤培养基。
将P. putida CCTCC NO：M 2011442分别接种于两个装液量为30/250mL 的三角瓶中，其中一瓶含有50%（v/v）的环己烷，30℃、200 r/min培养12 h。分别12000 r/min离心1min收集菌体。并取湿菌体150 mg，在菌体中加入1.5 mL样品裂解液，其成分为8 moL/L尿素，2 moL/L硫脲，1% ASB-14两性离子去污剂，40 mmoL/L 的Tris base，0.001%溴酚蓝。同时加入15 μL TBP（磷酸三丁酯），重悬细胞，悬浮液于冰上进行超声破碎（超声功率为300 w，超声程序设置为超声3 s停3 s，30 min），使细胞完全破碎。将细胞破碎液16000 g离心20 min，沉淀细胞碎片，取上清至干净的离心管，即为细胞的全蛋白样品。蛋白的含量测定采用BIO-RAD公司生产的RC-DC Protein Assay Kit。测得的蛋白含量为：无有机溶剂存在：4.6674 mg/mL；有机溶剂存在：4.7808 mg/mL。
实施例2：总蛋白的二维电泳
所用仪器主要包括IEF系统、蛋白电泳仪和扫描仪。
所用的主要溶液配制：
胶条平衡缓冲液母液：尿素36 g，SDS 2 g，Tris-Hcl 25 mL（1.5 moL/L pH 8.8 Tris-HCl），甘油20 mL，溴酚蓝100 μL（1%溴酚蓝），用超纯水定容至100 mL。
胶条平衡缓冲液Ⅰ：胶条平衡缓冲液母液 10mL，DTT 0.2g。
胶条平衡缓冲液Ⅱ：胶条平衡缓冲液母液 10mL，碘乙酰胺 0.25g。
琼脂糖封胶液（GE公司）：琼脂糖0.5 g，Tris 0.303 g，甘氨酸1.44 g，SDS 1 mL（10% SDS），溴酚蓝100 μL（1%溴酚蓝），用超纯水定容至100 mL，加热溶解至澄清。
取蛋白样品180 μg，加入样品裂解液（成分同案例1）稀释至125 μL，再加入0.5 μL的IPG buffer（GE公司的等电聚焦缓冲液，线性，pH 4-10）。从水化盘中槽的边缘线性加入样品后，将IPG胶条（GE公司的等电聚焦胶条）胶面朝下置于水化盘中样品溶液上，20℃过夜放置。取出水化盘中的IPG胶条，转移到聚焦盘中，在聚焦盘的IPG胶条两端搭上湿润的盐桥，在每根IPG胶条上覆盖2-3 cm的矿物油，同时在胶条两边的泳道上也同样用矿物油覆盖。设置等点聚焦的程序为：50 V，30 min；150 V，30 min；250 V，30 min；500 V，30 min；1000 V，1 h；4000 V，3 h；4000 V，20000 V/h；500 V，保持。每根胶条的极限电流为50 μA/根，等电聚焦的温度设置为20℃。
胶条转移至水化盘中，在胶条上加入新配置的5 mL胶条平衡缓冲液Ⅰ，样品水化盘放在水平摇床上缓慢摇晃15 min；第一次平衡后，彻底倒掉样品水化盘中的胶条平衡缓冲液Ⅰ，再加入胶条平衡缓冲液Ⅱ，继续在水平摇床上缓慢摇晃15min。将平衡好的IPG胶条浸没在电泳缓冲液中，然后胶条胶面朝上放在凝胶的长玻璃板上，轻推胶条使之与聚丙烯酰胺凝胶面完全接触。将加热融化后的琼脂糖封胶液灌入凝胶上方，彻底凝固后，再转移至电泳槽中。SDS-PAGE电泳起始的电压为75 V，待样品完全走出IPG胶条，浓缩成一条线后，加大电压为150 V，继续电泳至溴酚蓝指示剂到达底部边缘。聚丙烯酰胺电泳胶采用考马斯亮蓝染色1 h后，脱色至无底色。聚丙烯酰胺电泳胶经扫描后，图像采用PDQuestTM 2-D Analysis Software 进行分析，结果如图1。
实施例3：3-羟基异丁酸脱氢酶的鉴定及克隆表达
切取图1所示的C1蛋白质点，采用基质辅助激光解吸电离串联飞行时间质谱（MALDI-TOF/TOF）鉴定（上海中科新生命生物科技有限公司测定），将鉴定后的结果与NCBI数据库比对，确定其蛋白质为3-羟基异丁酸脱氢酶，该酶由295个氨基酸组成，其编码基因为mmsB，基因长度为888 bp，如表1所示。
表 1 蛋白质点的MALDI-TOF/TOF 鉴定结果
A：未加有机溶剂的P. putida CCTCC NO：M 2011442总蛋白。
B：在50%（v/v）环己烷生长下的P. putida CCTCC NO：M 2011442总蛋白。
以恶臭假单胞菌CCTCC NO：M 2011442基因组为模板，PCR扩增基因mmsB。基因mmsB经PCR产物回收后，连接载体，并转化大肠杆菌JM109；提取转化后验证成功的重组大肠杆菌JM109/pMD18-T-mmsB的质粒，采用限制性内切酶BamH Ⅰ和Hind Ⅲ对pMD18-T-mmsB质粒和表达载体pQE-80L分别进行双酶切。双酶切产物采用T4连接酶进行连接反应过夜，重组质粒pQE-80L-mmsB转化后获得重组大肠杆菌E. coli JM109/pQE-80L-mmsB，提取菌株E. coli JM109/pQE-80L-mmsB质粒并进行PCR验证和双酶切验证。将E. coli JM109/pQE-80L-mmsB接种于LB-Kan培养基中，37℃，150 r/min培养至OD600为0.6左右时，加入终浓度为1 mmoL/L的IPTG，30℃，150 r/min继续培养3小时。将培养后的菌液12000 r/min离心收集菌体，按照按照1:8（w/v）加入20 mmol/L、pH 7.0的磷酸盐缓冲液，重悬菌体。悬浮液于冰上超声破碎（超声功率为300 w，超声程序设置为超声1 s停3 s，共20 min）。分别取4 μL未离心的破碎液和在4 ℃、8000 r/min条件下离心10 min的上清液进行12% SDS-PAGE表达验证。结果如图2所示，3-羟基异丁酸脱氢酶的分子量大小约为35 KDa。
实施例4：3-羟基异丁酸脱氢酶对微生物有机溶剂耐受能力的影响
将重组菌株E. coli JM109/pQE-80L-mmsB转接至含有0.02 mmol/L的IPTG的LBGMg（LB培养基中加入0.1% (w/v) 葡萄糖和10 mmoL/L MgSO4）培养基中培养至OD660=0.2后，加入3%的环己烷并考察有机溶剂的加入对菌株的生长产生影响。在OD660处检测菌体浓度，结果如表2所示。对照菌株E. coli JM109/pQE-80L在3%（v/v）环己烷中几乎不生长，培养8 h后OD660只增长0.06，而重组菌株的耐溶剂性显著优于对照菌株，重组表达3-羟基异丁酸脱氢酶的菌株OD660达到1.70。
表2 重组蛋白对微生物耐溶剂性的影响
| 培养时间（h） | 对照菌株（OD660） | 重组菌株（OD660） |
| 0 | 0.04 | 0.04 |
| 1 | 0.1 | 0.12 |
| 2 | 0.2 | 0.21 |
| 2.5 | 0.24 | 0.38 |
| 3 | 0.24 | 0.67 |
| 3.5 | 0.25 | 0.94 |
| 4 | 0.24 | 1.12 |
| 4.5 | 0.24 | 1.37 |
| 5 | 0.25 | 1.46 |
| 5.5 | 0.26 | 1.54 |
| 6 | 0.27 | 1.6 |
| 7 | 0.27 | 1.65 |
| 8 | 0.28 | 1.7 |
将重组菌株和对照菌株大肠杆菌JM109于含有0.02 mmol/L的IPTG的LBGMg培养基中培养过夜，按照菌落生长曲线，分别稀释菌液，使每微升菌体数分别为107、106、105、104、103个，分别取3 μL重组菌株和对照菌株点种于涂有50 μL 0.1 mmol/L IPTG的LBGMg-Kan固体培养基中，30℃培养1 h后，在培养基上覆盖2 mm厚度的萘烷。30℃继续培养12 h，菌落形成效率如图3。重组表达3-羟基异丁酸脱氢酶的菌株比对照菌株具有更好的菌落形成效率和有机溶剂耐受能力。
<160>2
<210> SEQ NO.1
<211>888
<212>DNA
<213> CCTCC NO：M 2011442
<400>1
atgcgtattg cattcattgg cctcggcaac atgggcgcgc ccatggcccg caacctgatc 60
aaggccgggc accaactgaa cctgttcgac ctgaacaaaa ccgtgctggc cgaactggca 120
gaactgggcg ggcagatcag cccgtcgccc aaggacgcgg cggccaacag cgagctggtt 180
atcaccatgc tgccggcggc ggcccatgtg cgcagcgtgt acctgaacga cgacggcgtg 240
ctggccggca ttcgccccgg cacgccaacc gtggactgca gcaccatcga cccgcagacc 300
gcgcgtgacg tgtccaaggc tgcagcggcc aagggcgtgg acatgggcga cgcgccggtg 360
tccggtggca ccggcggcgc ggcagcgggc accctgacct tcatggtcgg cgccagcgcc 420
gagctgttcg ccagcctcaa gccggtgctg gagcagatgg gccgcaacat cgtgcactgc 480
ggtgaagtcg gcaccgggca gatcgccaag atctgcaaca acctgctgct gggcatctcc 540
atgatcggcg tctccgaggc catggccttg ggtaacgcgc tgggcatcga caccaaggtg 600
ctggccggca tcatcaacag ctcgaccggg cgttgctgga gttcggacac ctacaacccg 660
tggccgggca ttatcgaaac ggcgccggca tcgcgtggct ataccggtgg ctttggcgcc 720
gaactgatgc tcaaggacct ggggctggcc accgaagcgg cgcgccaggc acaccagccg 780
gtgatcctcg gggccgtggc ccagcagctg tatcaggcca tgagcctgcg cggcgaaggg 840
ggcaaggact tctcggccat cgtcgagggg tatcgcaaga aggattga 888
<160>2
<210> SEQ NO.2
<211>295
<212>PRT
<213> CCTCC NO：M 2011442
<400>2
Met Arg Ile Ala Phe Ile Gly Leu Gly Asn Met Gly Ala Pro Met
5 10 15
Ala Arg Asn Leu Ile Lys Ala Gly His Gln Leu Asn Leu Phe Asp
20 25 30
Leu Asn Lys Thr Val Leu Ala Glu Leu Ala Glu Leu Gly Gly Gln
35 40 45
Ile Ser Pro Ser Pro Lys Asp Ala Ala Ala Asn Ser Glu Leu Val
50 55 60
Ile Thr Met Leu Pro Ala Ala Ala His Val Arg Ser Val Tyr Leu
65 70 75
Asn Asp Asp Gly Val Leu Ala Gly Ile Arg Pro Gly Thr Pro Thr
80 85 90
Val Asp Cys Ser Thr Ile Asp Pro Gln Thr Ala Arg Asp Val Ser
95 100 105
Lys Ala Ala Ala Ala Lys Gly Val Asp Met Gly Asp Ala Pro Val
110 115 120
Ser Gly Gly Thr Gly Gly Ala Ala Ala Gly Thr Leu Thr Phe Met
125 130 135
Val Gly Ala Ser Ala Glu Leu Phe Ala Ser Leu Lys Pro Val Leu
140 145 150
Glu Gln MET Gly Arg Asn Ile Val His Cys Gly Glu Val Gly Thr
155 160 165
Gly Gln Ile Ala Lys Ile Cys Asn Asn Leu Leu Leu Gly Ile Ser
170 175 180
Met Ile Gly Val Ser Glu Ala Met Ala Leu Gly Asn Ala Leu Gly
185 190 195
Ile Asp Thr Lys Val Leu Ala Gly Ile Ile Asn Ser Ser Thr Gly
200 205 210
Arg Cys Trp Ser Ser Asp Thr Tyr Asn Pro Trp Pro Gly Ile Ile
215 220 225
Glu Thr Ala Pro Ala Ser Arg Gly Tyr Thr Gly Gly Phe Gly Ala
230 235 240
Glu Leu Met Leu Lys Asp Leu Gly Leu Ala Thr Glu Ala Ala Arg
245 250 255
Gln Ala His Gln Pro Val Ile Leu Gly Ala Val Ala Gln Gln Leu
260 265 270
Tyr Gln Ala Met Ser Leu Arg Gly Glu Gly Gly Lys Asp Phe Ser
275 280 285
Ala Ile Val Glu Gly Tyr Arg Lys Lys Asp
290 295
Claims (2)
1.一种mmsB基因的应用，其特征在于在提高恶臭假单胞菌耐受有机溶剂性中的作用；
所述mmsB基因的核苷酸序列为SEQ ID NO. 1。
2.一种mmsB基因所编码的蛋白质的应用，其特征在于在提高恶臭假单胞菌耐受有机溶剂性中的作用；
所述mmsB基因所编码的蛋白质为：从恶臭假单胞菌（Pseudomonas putida）CCTCC No：M2011442中分离获得的氨基酸序列为SEQ ID NO. 2的mmsB蛋白，其具有提高恶臭假单胞菌耐受有机溶剂的功能。
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| Chowdhury,E.K.，等.Pseudomonas putida msdh and hibdh genes for methylmalonate-semialdehyde dehydrogenase and 3-hydroxyisobutyrate dehydrogenase, partial and complete cds.《Genbank登录号AB090855》.2004,全文. * |
| 彭仁,等.耐有机溶剂脂肪酶产生菌的筛选和鉴定.《江西师范大学学报(自然科学版)》.2011,第35卷(第2期),197-200. * |
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