CN1329506C - 一种具有颗粒状甲烷单加氧酶活性的重组菌及其应用 - Google Patents
一种具有颗粒状甲烷单加氧酶活性的重组菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种具有甲烷单加氧酶活性的重组菌及其应用。本发明所提供的具有颗粒状甲烷单加氧酶活性的重组菌，是将由颗粒状甲烷单加氧酶的编码基因及脱硫基因的启动子序列组成的融合基因导入红球菌，筛选得到表达颗粒状甲烷单加氧酶的菌株即为具有颗粒状甲烷单加氧酶活性的重组菌；所述脱硫基因的启动子序列位于所述颗粒状甲烷单加氧酶的编码基因的上游。该重组菌彻底避开甲醇脱氢酶基因的影响，使甲烷氧化停留在甲醇这一步反应，克服了野生型甲烷氧化菌的缺陷，更易培养。该重组菌能将天然气的烷烃液化成为相应的醇类，能将沼气转化为甲醇，能将丙烯氧化成1，2-环氧丙烷。
Description
技术领域
本发明涉及一种具有颗粒状甲烷单加氧酶活性的重组菌及其应用。
背景技术
甲醇是一种重要的化工原料，年消费量仅次于乙烯、丙烯和苯，居第四位。从甲醇出发可以制备丙烯(MTP)、烯烃(MTO)、二甲基甲酰胺、甲酸甲酯、甲胺、碳酸二甲酯和乙二醇等产品。随着石油资源的逐步枯竭，甲醇在许多方面开始替代传统的化石燃料，被用来生产汽油燃料、二甲醚燃料和汽油添加剂，因此，甲醇的市场需求越来越大。目前，制备甲醇主要是通过氧化甲烷得到。由于甲烷分子相当稳定，C-H键能为104 kcalmol-1，因此，氧化甲烷生成甲醇需要非常苛刻的条件。经过多年的工艺改进和催化剂改良，化工生产中仍需255℃的温度和5～15MP的压力，才能使天然气转化为甲醇。而自然界的甲烷氧化菌依靠体内的甲烷单加氧酶，在常温常压下就能将甲烷氧化成为甲醇。甲烷单加氧酶不仅可以将甲烷转化成为甲醇，而且还可以催化其它一系列的单加氧反应，可以广泛地应用于生物转化及环境治理。如果能将甲烷单加氧酶进行表达，开发出一种细胞形态生物催化剂，那么在常温常压下就能将天然气转化为甲醇。这将大大降低甲醇的生产成本，是一种高度节能的、环境友好的甲醇生产工艺。同时，以可再生资源为原料通过厌氧甲烷发酵可以提供廉价的甲烷，是实现循环工业的极具潜力的一条途径。因此甲烷的资源化利用技术的研发具有重要的工业应用价值。
甲烷单加氧酶(EC.1.14.13.25，Methane Monooxygenase，简称MMO)分两种，一种是可溶性的甲烷单加氧酶(soluble methane monooxygenase，简称sMMO)，另一种是发现较晚的颗粒状甲烷单加氧酶(particulate methane monooxygenase，简称pMMO)。尽管sMMO和pMMO都能氧化甲烷生成甲醇，但二者还是存在很多的不同。sMMO存在于细胞质中，它底物范围广，许多的烃类化合物和芳香族化合物都能被氧化。与sMMO相比，pMMO的底物选择性相对较窄，只能氧化五碳以内的烷烃和烯烃，不能氧化芳香烃。但是pMMO却具有很多sMMO不具备的优势。pMMO存在于细胞膜上而不是细胞质中。该性质有利于底物与酶的接触和反应产物及时排出，意味着该酶催化的反应传质阻力更低，因而有利于在生物转化中应用。pMMO存在四级结构，由三个亚基组成(分别是23kDa的pmoC、27kDa的pmoA和45kDa的pmoB，Genbank U31650 AF186586)，pMM0是α3β3γ3的三聚体结构，铜离子位于pmoB中。尽管在pMMO酶分子研究方面取得了一系列的进展，而且Methyllococcus capsulatus(Bath)和OB3b的pmo基因已经克隆测序，但pMMO的重组异源表达却进展甚微，至今在大肠菌及其他宿主细胞中没有表达成功。
尽管利用野生型甲烷氧化菌能够将甲烷转化甲醇，但是其在大规模的工业应用还存在一系列难以克服的技术问题。首先，甲烷氧化菌的培养比较困难，与其他工业应用的微生物相比增殖速度极慢，而且能够得到的细胞密度低。尽管通过添加特殊底物(氯甲烷、有机酸、维生素或甲醇等)能促进甲烷氧化菌生长，但其生长速度还是远远低于其它菌类。其次，利用野生甲烷氧化菌进行甲烷转化甲醇的过程中，甲醇的积累不易控制。在这些菌中都含有甲醇脱氢酶，因此甲醇会进一步地被氧化成为甲醛，进一步进入的细胞合成代谢。如果加入特殊的化学试剂，选择性地抑制甲醇脱氢酶的活性，这样虽然暂时促进甲醇的积累，但细胞的代谢途径却被中断，严重影响细胞的代谢活性，进而阻止甲烷氧化反应。这主要是因为对于甲烷氧化菌而言，MMO氧化甲烷所需的NADH只能由甲醛进入相应的代谢途径后才能产生，因此如果将细胞控制在生产甲醇这一步的话，胞内原有的NADH被耗竭时，反应就停止了。
发明内容
本发明的目的是提供一种具有颗粒状甲烷单加氧酶活性的重组菌。
本发明所提供的具有颗粒状甲烷单加氧酶活性的重组菌，是将由颗粒状甲烷单加氧酶(pMMO)的编码基因及脱硫基因的启动子序列组成的融合基因导入红球菌，筛选得到表达颗粒状甲烷单加氧酶的菌株即为具有颗粒状甲烷单加氧酶活性的重组菌；所述脱硫基因的启动子序列位于所述颗粒状甲烷单加氧酶(pMMO)的编码基因的上游。
所述红球菌可为红球菌属的红平红球菌(Rhodococcus erythropolis)、Rhodococcus aichiensis、Rhodococcus chlorophenolicus、Rhodococcuscoprophilus、Rhodococcus equi、Rhodococcus fascians、Rhodococcus globerulus、Rhodococcus luteus、Rhodococcus matinonascens、Rhodococcus Maris、Rhodococcus rhodnii、Rhodococcus rhodochrous或Rhodococcus tuber。
所述红平红球菌(Rhodococcus erythropolis)优选为红平红球菌(Rhodococcuserythropolis)LSSE8-1。
所述颗粒状甲烷单加氧酶的编码基因在Genbank序列号为U31650 AF186586，并可按照以下方法获得：以Methylosinus trichosporium OB3b的基因组DNA为模板，由引物5’ATGAATTCAACAACAGAGACAACAG 3’和5’AAAGCTTCCAAACGCCGCATTCTATC 3’进行PCR扩增得到。
所述脱硫基因的启动子的核苷酸序列为Genbank中序列号L37363中自5′端第1位-768位脱氧核苷酸；所述脱硫基因的启动子可按照以下方法获得：以红平红球菌LSSE8-1的基因组DNA为模板，用引物1：5’ACAAGCTTGCACGGCTCCGGGCAGTTC 3’和引物2：5’ACGAATTCATCGCG TATGCGTCCTTTA 3’进行PCR扩增得到。
所述由颗粒状甲烷单加氧酶(pMMO)的编码基因及脱硫基因的启动子序列组成的融合基因，形成序列表中SEQ ID №：1的DNA序列。
序列表中的序列1由4084个脱氧核苷酸组成，自5′端第1位至768位脱氧核苷酸为脱硫基因dsz的启动子序列，来源于红平红球菌；自5′端第769位至4084位脱氧核苷酸为颗粒状甲烷单加氧酶(pMMO)的编码基因序列，来源于丝孢甲烷弯菌(Methylosinus trichosporium)OB3b。
所述由颗粒状甲烷单加氧酶(pMMO)的编码基因及脱硫基因的启动子序列组成的融合基因，可通过插入现有的原核细胞表达载体得到的含有所述的重组表达载体导入红平红球菌。
所述原核细胞表达载体可为pBS305、pMVS301或pRESQ等可以在红球菌中复制的质粒载体。
含有所述融合基因的重组表达载体为pBS305-dszpmo；所述pBS305-dszpmo是将由颗粒状甲烷单加氧酶的编码基因及脱硫基因的启动子序列组成的融合基因插入到pBS305的HindIII酶切位点之间得到的。
本发明的第二个目的是提供一种利用上述重组菌表达颗粒状甲烷单加氧酶的方法。
本发明所提供的表达颗粒状甲烷单加氧酶方法，是培养上述具有颗粒状甲烷单加氧酶活性的重组菌，进行诱导表达，得到颗粒状甲烷单加氧酶。
所述进行诱导表达的时期为所述具有甲烷单加氧酶活性的重组菌在含有硫元素的培养基中培养至对数期或对数后期。
所述含有硫元素的培养基可为培养红平红球菌LSSE8-1的含有硫元素的常用培养基，具体可为含有如下成份的培养基：去离子水1000毫升，KH2PO4 2.44克；Na2HPO4·12H2O 14.03克；NH4Cl 2.00克；MgCl2·6H2O 0.36克；CaCl2·2H2O 0.001克FeCl3·6H2O 0.001克MnCl2·4H2O 0.004克；葡萄糖10克，Na2SO4 1mmol/L。
所述方法中，可将培养至对数期或对数后期的菌体在无硫元素培养基中培养2-4小时，进行诱导表达。
本发明选择适于工业应用的微生物红球菌，构建了具有颗粒状甲烷单加氧酶活性的重组菌，彻底避开甲醇脱氢酶基因的影响，使甲烷氧化停留在甲醇这一步反应，克服了野生型甲烷氧化菌的缺陷，更易培养(能用含葡萄糖等普通碳源的培养基培养)。本发明还利用具有颗粒状甲烷单加氧酶活性的重组菌成功地解决了颗粒状甲烷单加氧酶的异源表达问题。该重组菌能将天然气的烷烃液化成为相应的醇类，能将沼气转化为甲醇，能将丙烯氧化成1，2-环氧丙烷。可以用于环境中难降解化合物如石油中烃类化合物的羟化分解，因而可用于环境的生物治理。
附图说明
图1为pMD-T-dsz和pMD-T-pmoCAB的酶切分析电泳图
图2为pBS305-dszpmo的HindIII单酶切分析结果电泳图
图3为乙烷为唯一碳源培养LSSE8-1/pBS305-dszpmo6小时后的气相色谱分析结果
具体实施方式
下述实施例中的实验方法，如无特别说明，均为常规方法。
实施例1、具有颗粒状甲烷单加氧酶活性的重组菌的获得
用含如下成分的培养基培养红平红球菌LSSE8-1(该菌购自中国微生物菌种保藏管理委员会普通微生物中心)：每升溶液中含有2.44g KH2PO4，14.03g Na2HPO4·12H2O，0.3589g MgCl2·6H2O，0.001g CaCl2·2H2O，0.001g FeCl3·6H2O，0.004g MnCl2·4H2O，1.45ml甘油和2.00g NH4Cl，0.1mmol/L二甲基亚砜，pH7.0。其中，0.1mmol/L二甲基亚砜作为唯一的硫源。培养2-4天之后，取菌液3毫升，离心收集菌体，提取基因组DNA。以该基因组DNA为模板，用引物1：5’-AC
AAGCTTGCACGGCTCCGGGCAGTTC 3’(带下划线的核苷酸序列为HindIII的识别序列)和引物2 5’-AC
GAATTCATCGCGTATGCGTCCTTTA 3’(带下划线的核苷酸序列为EcoRI的识别序列)进行PCR扩增。其中，扩增反应条件如下：先94℃，预变性4分钟后进行30个循环的扩增反应；循环反应条件是：94变性1分钟，54℃退火1分钟，72℃延伸1分钟；30个循环结束后再72℃补充延伸4分钟。得到了预期的0.8kb的片段，即为含有脱硫基因dsz的启动子片段。
取13ml甲烷氧化菌培养基装入100ml的培养瓶中灭菌后接入Methylosinustrichosporium OB3b(Sullivan JP et al.Methanotrophs，Methylosinustrichosporium OB3b，sMMO，and their application to bioremediation，Crit.Rev.Microbiol.，1998，4(24)：335-373；Takeguchi M et al.，Propertiesof the membranes containing the part iculate methane monooxygenase fromMethylosinus trichosporium OB3b，Biometals，1998，11：229-234)种子液。每天用注射器通过0.2μm的过滤器加入20毫升的甲烷气体，30℃ 150rpm培养两周后收集菌体，提取总DNA。以此DNA为模板，由引物5’AT
GAATTCAACAACAGAGACAACAG 3’(带下划线的核苷酸序列为EcoRI的识别序列)和5’A
AAGCTTCCAAACGCCGCATTCTATC 3’(带下划线的核苷酸序列为HindIII的识别序列)进行PCR扩增反应。反应条件如下：先94℃预变性5分钟；然后以循环条件为94℃变性1分钟，54℃退火1分钟，72℃延伸3分钟进行30个循环的扩增反应；循环完成后72℃再补充延伸7分钟，得到3.5kb大小的片段即含有颗粒状甲烷单加氧酶的编码基因的片段。
两段PCR得到的DNA片段经过纯化后分别与克隆载体pMD-T simple载体(购自TaKaRa公司)连接后转化入大肠菌DH5α，经蓝白斑筛选，得到阳性克隆，提质粒用HindIII和EcoR I双酶切鉴定质粒，结果如图1所示，含有脱硫基因dsz的启动子片段与克隆载体pMD-T simple得到的重组质粒经HindIII和EcoR I双酶切后，得到了0.8kb的条带(椭圆示目的条带)；含有颗粒状甲烷单加氧酶的编码基因的片段与克隆载体pMD-T simple得到的重组质粒经HindIII和EcoR I双酶切后，得到了3.5kb的条带(椭圆示目的条带)，证明了扩增的片段插入到了质粒中。将重组正确的含有脱硫基因dsz的启动子片段的重组质粒命名pMD-T-dsz，含有颗粒状甲烷单加氧酶的编码基因的片段的重组质粒命名pMD-T-pmoCAB。图1中，M为DNA分子量标准，1为pMD-T-dsz重组质粒的酶切分析，2为pMD-T-pmoCAB重组质粒的分析结果。对质粒pMD-T-pmoCAB和pMD-T-dsz分别进行测序，结果表明pMD-T-pmoCAB含有GenBank号为U31650AF186586的自5′端第769位至4084脱氧核苷酸的颗粒状甲烷单加氧酶的编码基因(pmoCAB)序列；pMD-T-dsz含有Genbank序列号为L37363中自5′端第1位一768位脱氧核苷酸的脱硫基因dsz的启动子序列。
pMD-T-dsz和pMD-T-pmcCAB分别用HindIII和EcoR I双酶切后，得到具有HindIII和EcoRI粘性末端的含有脱硫基因dsz的启动子的DNA片段和具有HindIII和EcoR I粘性末端的含有颗粒状甲烷单加氧酶的编码基因的片段，把这两种DNA片段与pBS305质粒(Z.Shao，W.A.Dick and R.M.Behki，An improved Escherichia coli-Rhodococcusshuttle vector and plasmid transformation in Rhodococcus spp.Usingelectroporation，letters of Applied Microbiology，1995，21，261-266.)连接(该载体使用之前需用HindIII单酶切并脱磷酸化)，将得到的连接产物转化大肠杆菌DH5α，经蓝白斑筛选得到阳性克隆，提质粒，进行HindIII单酶切鉴定，酶切结果如图2(1为DNA分子量标准，2为pBS305-dsz-pmoCAB)所示，表明得到4.2kb的重组片段和约7.9kb的载体片段，酶切结果与预期一致。将酶切鉴定正确的质粒命名为pBS305-dsz-pmoCAB。对pBS305-dsz-pmoCAB进行测序，结果表明该质粒含有具有序列表中序列1的由脱硫基因dsz的启动子和颗粒状甲烷单加氧酶的编码基因组成的融合基因序列。将pBS305-dsz-pmoCAB经电穿孔转化法导入红平红球菌LSSE8-1，将转化菌涂在培养基1中，进行筛选，得到了可在培养基1中生长的含有pBS305-dsz-pmoCAB的菌株，命名为LSSE8-1/pBS305-dsz-pmoCAB。
培养基1的组成如下：每升培养基中含有K2HPO4 2.44g，Na2HPO4·12H2O 14.03g，NH4Cl 2g，MgCl2·6H2O 0.3589g，DMSO 60μL，0.1-50mL微量元素贮存液，0.1-100mLCuCl2贮存液，FeCl2 80μmol，1.5％的琼脂，pH7.0。
其中，微量元素贮存液的成分为：
ZnCl2 0.2042克 MnCl2·4H2O 0.1575g
H3BO3 0.0631g Na2MoO4·2H2O 0.0488g
CoCl2·6H2O 0.049g KI 0.0834g
CaCl2·2H2O 3.5014g 去离子水定容至1L
CuCl2贮存液的成分为：
称取CuCl2·2H2O 0.85mg溶于1L水中，终浓度为5mM。
收集LSSE8-1/pBS305-dsz-pmoCAB的新鲜菌体，用无硫的培养基冲冼干净后，在该培养基中诱导后，用下列三个5’修饰有FAM荧光染料的探针进行荧光原位杂交，然后用荧光显微镜进行观察，可见有绿色荧光，说明该基因都已在脱硫基因(dsz)的启动子下被转录出相应的RNA。
| pmoC探针 | 5’CCAGCGGAATCTCGGTCCAAAG 3’ |
| pmoA探针 | 5’CACCAGAGCGGACGGGAACACC 3’ |
| pmoB探针 | 5’CGATGTCCCAACGGGCGTCCTG 3’ |
实施例2、具有颗粒状甲烷单加氧酶活性的重组菌细胞的获得与诱导
挑取营养斜面(培养基1)培养的上述重组菌株LSSE8-1/pBS305-dsz-pmoCAB，接入25毫升的基础培养基中。该基础培养基的成份：去离子水1000毫升，KH2PO4 2.44克；Na2HPO4·12H2O 14.03克；NH4Cl 2.00克；MgCl2·6H2O 0.36克；CaCl2·2H2O 0.001克FeCl3·6H2O 0.001克MnCl2·4H2O 0.004克；葡萄糖10克，Na2SO41mmol/L，pH7.0。30℃，150转/分培养24-48小时后，再将接入500毫升的基础培养基中，48-72小时后，离心获得菌体。用生理盐水冲洗菌体后，用无硫元素培养基(去离子水1000毫升，KH2PO42.44克；Na2HPO4·12H2O 14.03克；NH4Cl 2.00克；MgCl2·6H2O 0.36克；CaCl2·2H2O0.001克FeCl3·6H2O 0.001克MnCl2·4H2O 0.004克；葡萄糖10克)在摇床上培养2-4小时，诱导即完成。离心收集菌体。
实施例3、重组菌将乙烷转化为乙醇
用实施例2的LSSE8-1/pBS305-dsz-pmoCAB菌体(菌体处于对数期，OD600＞20时收集菌体)加入到5mM MgCl2-20mM磷酸缓冲液10毫升中，按乙烷∶空气＝1∶1的比例补入气体，30℃下150转/分进行反应6小时，离心取上清进行气相色谱分析，结果如图3所示。图3中，虚线为标准的1％的乙醇水溶液，实线为LSSE8-1/pBS305-dsz-pmoCAB反应液的上清，该结果显示LSSE8-1/pBS305-dsz-pmoCAB反应液中有乙醇存在。
实施例4、具有pMMO活性的重组菌株应用于天然气的液化。
用实施例2的LSSE8-1/pBS305-dsz-pmoCAB菌体(OD600＞20时收集菌体)，分别加入到5mM MgCl2-20mM磷酸缓冲液10毫升，该缓冲液中保持60微摩尔的葡萄糖，按天然气∶空气＝1∶1的比例补入气体，30℃下150转/分进行反应12小时，天然气即被转化为甲醇、乙醇、丙醇和丁醇等。
序列表
<160>1
<210>1
<211>4084
<212>DNA
<213>人工序列
<220>
<223>
<400> 1
acaagcttgc acggctccgg gcagttctcg cggcgctgga ggcacggatg ggcaccctca 60
acgaactcac ccaaaccacg ccgatagcga tcctcgccga aaccctcggc tacagccctc 120
agacattgga agctcatgcg cgacgcatcc ggatcgacct ttgcacgcta cgtggcgacg 180
cggctggact gacgctggag gtccgacccg acgtgtgtgg tgtagcgccg cttaacgggt 240
gcgcacggcg ggacatcggc cagctggctt gcccctcctc cgcaggtagt cgaccacccc 300
ttcccgcagc ggtcggaggt gatcgaccgt tagggtcatt tgctcgcaga tcggctgatg 360
ttgccgatcg acgtggtcga cgggacacgc tcgcgattgg catggcgtcc ggtgcataca 420
cgacgatcta accagatcga cggttttgag cgtcggtcaa cgtcgactcg atgcgccgtg 480
cgagtgagat cctttgtggt gcttggctat tgacctcgac aaggatagag attcgaagga 540
cctcggatcg acccaaatgc ggacggccgg cagcggcgaa ggcggccaag tcatcggcac 600
cgtcaccgtc accttgaccc gacgtgcccc gtggttcaag gcctgaattt ggctggtgga 660
gcattgaaat caggtgaagt ttaacggtgg gcacaccccg ggggtggggg tgagactgct 720
tagcgacagg aatctagcca tgattgacat ttaaaggacg catacgcgat gaattcaaca 780
acagagacaa cagccggcgc agccgccggc tcggacgcga tcgttgatct gcgtggcatg 840
tgggtcggtg tcgccggcct gaacatcttc tatctgatcg tccgcattta cgagcagatc 900
tacggctggc gcgcgggcct cgactcgttc gctccggagt tccagacgta ttggctgtcg 960
tggaagaccc gtgaccgcaa cgtcgacgcg gtcgctccgc gcgaggagct gcgccgtcac 1020
gtggtcctgg tcgagtggct ggtggtctac gccgtcgcca tttactgggg cgcgagcttc 1080
ttcacggagc aggacggcac ctggcacatg acggtgattc gcgacacgga cttcacgccg 1140
tcgcacatca tcgagttcta catgagctac ccgatctact cgatcatggc ggtgggcgcg 1200
ttcttctatg cgaagacccg cattccgtat tttgctcatg gcttctcgct ggcgttcctg 1260
atcgtcgcca tcggcccgtt catgatcatc ccgaacgtcg gcctgaacga gtggggccac 1320
accttctggt tcatggaaga gctgttcgtc gctccgctgc attggggctt cgtgttcttc 1380
ggctggatgg cgctcggcgt gttcggcgtc gttctgcaga tcctgatggg cgtcaagcgc 1440
ctcatcggca aggactgcgt cgcggccctg gtcggctgaa gatgaattgg accgcccgac 1500
tttttttaag gggccgggcg gtccgcacgg acatctcgca atccgatagt ctcctgaacg 1560
ggaccttcgt ccgaacggcc gcgcaacgcg gggccggctc gggcggggtt cgggagagaa 1620
gggaacggga ggcacgaaag acaacccggc gcgagaagag tcggggactg gtcgacggct 1680
gcggcgggaa ccgccgggcg gaccgacacc aaggagaaga gtgatgttta catcgaagag 1740
cgggggggca atcgggcctt tccattcggt cgcggaagcg gcgggatgcg tcaagaccac 1800
cgattggatg tttctgacgc tgctgtttct ggcggtgctg ggcggctacc acattcactt 1860
catgctgacg gcgggcgact gggacttctg ggtcgactgg aaagaccgtc gtatgtggcc 1920
gaccgtggtt ccgatcctgg gcgtgacctt cgccgctgcg gcgcaggcgt tcttctggga 1980
gaacttcaag ctgccgttcg gcgcgacctt cgcggtctcc ggcctgctga tcggggagtg 2040
gatcaaccgc tactgcaact tctggggctg gacctatttc ccgatcagcc tggtgttccc 2100
gtccgctctg gtggttccgg cgctgtggct ggacatcatc atgctgctgt cgggctccta 2160
tgtgatcacg gcggttgtgg gctcgctggg ctggggcctt ctgttctacc cgaacaactg 2220
gccggcgatc gcggctctgc atcaggcgac ggagcagcat ggtcagctga tgtccctcgc 2280
ggatctcgtc ggcttccact tcgtccgcac gtcgatgccg gaatatatcc gcatggtcga 2340
gcgcggcacg ctgcgcacct tcggtaagga ggtcgttccg gtggccgcgt tcttctcggg 2400
cttcgtgtcg atgatggtct acttcctgtg gtggttcgtc ggtaagtggt attcgaccac 2460
caaggtgatc cagaagatct gatcgaagag agcaattagc ttctctttgg ttcctcggaa 2520
aagacgagga gcggttcccg gatcggcgat gagatcgccg cgaaggggcc gctccggaga 2580
agacgagtga ggccgcctgg cgctccgcga tgcggacagg gcgggctcca agagaaaact 2640
gggaggttag attcacatga aagctctgga aagaatggcc gaactggcga ccggacgggt 2700
cggaaagctc ctcggcctga gcgttgcggc tgcggtcgcc gcgacggcgg cttcggtggc 2760
cccggcggaa gcgcacggcg agaagtcgca gcaggcgttt ctgcgcatgc gcacgctgaa 2820
ctggtatgac gtgaagtggt cgaagacctc gttgaacgtc aacgagtcga tggttctgtc 2880
gggcaaggtt cacgtcttct cggcgtggcc gcaggcggtc gccaatccga agtcgtcgtt 2940
cctgaacgcc ggcgagcccg gcccggtttt ggttcgcacg gcgcagttca tcggcgagca 3000
gttcgctccg cgctcggtgt cgctcgaggt cggcaaggac tatgcgttct cgatcgatct 3060
gaaggctcgc cgcgccgggc gctggcacgt ccatgctcag atcaacgtcg aaggcggcgg 3120
tccgatcatc ggacccggcc agtggatcga gatcaagggc gacatggccg acttcaagga 3180
tccggtcacg ctgctcgacg gcacgaccgt ggacctcgag acctatggca tcgatcgcat 3240
ctatgcctgg catttcccgt ggatgatcgc ggccgcggcc tggatcctct actggttctt 3300
caagaagggc atcatcgctt cttatcttcg catcagcgaa ggcaaggacg aggagcagat 3360
cggcgatgac gaccgtcgcg tgggcgcgat cgttctcgcg gtgacgatcc tggcgacgat 3420
catcggctat gcggtgacga acagcacctt cccgcgcacg atcccgctgc aggccggctt 3480
gcagaagccg ctgacgccga tcatcgagga aggcaccgcc ggcgttggtc cgcatgtggt 3540
gacggccgag ctcaagggcg gcgtctacaa ggtgccgggc cgtgagctga cgatccaagt 3600
gaaggtgacg aacaagaccg acgagccgct gaagctcggc gagtatacgg cggcgggtct 3660
gcgcttcctg aaccccgacg tgttcacgac caagccggag ttcccggact atctgctggc 3720
cgaccgtggc ctgtcgaccg atccgacccc gctcgccccc ggcgagacga agacgatcga 3780
agtcaaggtg caggacgccc gttgggacat cgagcgtctc tcggacctcg cctatgacac 3840
ggacagccag atcggcggcc tgctgatgtt cttcagcccg tcgggcaagc gctacgccac 3900
cgaaatcggc ggcccggtca ttccgaagtt cgtcgccggc gacatgccct gatcgacttc 3960
gaaatcatac cgaacaaaag ccggccggac cgaaaggtcc ggccggtttt cgttgcggca 4020
gtcatggaaa tgcgaaagat cacgtttata tcgcaatgcg atagaatgcg gcgtttggaa 4080
gctt 4084
Claims (11)
1、具有颗粒状甲烷单加氧酶活性的重组菌，是将由颗粒状甲烷单加氧酶的编码基因及脱硫基因的启动子序列组成的融合基因导入红球菌，筛选得到表达颗粒状甲烷单加氧酶的菌株即为具有颗粒状甲烷单加氧酶活性的重组菌；所述脱硫基因的启动子序列位于所述颗粒状甲烷单加氧酶的编码基因的上游。
2、根据权利要求1所述的重组菌，其特征在于：所述红球菌为红球菌属的红平红球菌(Rhodococcus erythropolis)、Rhodococcus aichiensis、Rhodococcuschlorophenolicus、Rhodococcus coprophilus、Rhodococcus equi、Rhodococcusfascians、Rhodococcus globerulus、Rhodococcus luteus、Rhodococcusmarinonascens、Rhodococcus Maris、Rhodococcus rhodnii、Rhodococcusrhodochrous或Rhodococcus ruber。
3、根据权利要求2所述的重组菌，其特征在于：所述红平红球菌为红平红球菌(Rhodococcus erythropolis)LSSE8-1。
4、根据权利要求1或2或3所述的重组菌，其特征在于：所述颗粒状甲烷单加氧酶的编码基因在Genbank序列号为U31650 AF186586，所述脱硫基因的启动子的核苷酸序列为Genbank中序列号L37363中自5′端第1位-768位脱氧核糖核苷酸。
5、根据权利要求1或2或3所述的重组菌，其特征在于：所述由颗粒状甲烷单加氧酶的编码基因及脱硫基因的启动子序列组成的融合基因，具有序列表中SEQ ID №：1的DNA序列。
6、根据权利要求2或3所述的重组菌，其特征在于：所述由颗粒状甲烷单加氧酶的编码基因及脱硫基因的启动子序列组成的融合基因，通过插入原核细胞表达载体pBS305、pMVS301或pRESQ得到的含有所述融合基因的重组表达载体导入红平红球菌。
7、根据权利要求6所述的重组菌，其特征在于：含有所述融合基因的重组表达载体为pBS305-dsz-pmoCAB；所述pBS305-dsz-pmoCAB是将由颗粒状甲烷单加氧酶的编码基因及脱硫基因的启动子序列组成的融合基因插入pBS305的HindIII识别位点得到的。
8、一种表达颗粒状甲烷单加氧酶的方法，是培养权利要求1-7中任一所述的具有颗粒状甲烷单加氧酶活性的重组菌，进行诱导表达，得到颗粒状甲烷单加氧酶。
9、根据权利要求8所述的方法，其特征在于：所述进行诱导表达的时期为所述具有甲烷单加氧酶活性的重组菌在含有硫元素的培养基中培养至对数期或对数后期。
10、根据权利要求9所述的方法，其特征在于：所述方法中，将培养至对数期或对数后期的菌体在无硫元素培养基中培养2-4小时，进行诱导表达。
11、权利要求1-7中任一所述的具有颗粒状甲烷单加氧酶活性的重组菌在天然气及沼气液化中的应用。
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US9611487B2 (en) | 2012-12-21 | 2017-04-04 | Greenlight Biosciences, Inc. | Cell-free system for converting methane into fuel and chemical compounds |
| US10006062B2 (en) | 2010-05-07 | 2018-06-26 | The Board Of Trustees Of The Leland Stanford Junior University | Methods for control of flux in metabolic pathways through enzyme relocation |
| US10036001B2 (en) | 2010-08-31 | 2018-07-31 | The Board Of Trustees Of The Leland Stanford Junior University | Recombinant cellular iysate system for producing a product of interest |
| US10421953B2 (en) | 2013-08-05 | 2019-09-24 | Greenlight Biosciences, Inc. | Engineered proteins with a protease cleavage site |
| US10858385B2 (en) | 2017-10-11 | 2020-12-08 | Greenlight Biosciences, Inc. | Methods and compositions for nucleoside triphosphate and ribonucleic acid production |
| US10954541B2 (en) | 2016-04-06 | 2021-03-23 | Greenlight Biosciences, Inc. | Cell-free production of ribonucleic acid |
| US11274284B2 (en) | 2015-03-30 | 2022-03-15 | Greenlight Biosciences, Inc. | Cell-free production of ribonucleic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104822824A (zh) * | 2012-10-15 | 2015-08-05 | 凯利斯塔公司 | 用于烃的生物氧化的遗传工程改造的微生物 |
| WO2018181774A1 (ja) * | 2017-03-31 | 2018-10-04 | 株式会社カネカ | 新規モノオキシゲナーゼおよびその利用 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10006062B2 (en) | 2010-05-07 | 2018-06-26 | The Board Of Trustees Of The Leland Stanford Junior University | Methods for control of flux in metabolic pathways through enzyme relocation |
| US10036001B2 (en) | 2010-08-31 | 2018-07-31 | The Board Of Trustees Of The Leland Stanford Junior University | Recombinant cellular iysate system for producing a product of interest |
| US9611487B2 (en) | 2012-12-21 | 2017-04-04 | Greenlight Biosciences, Inc. | Cell-free system for converting methane into fuel and chemical compounds |
| US10421953B2 (en) | 2013-08-05 | 2019-09-24 | Greenlight Biosciences, Inc. | Engineered proteins with a protease cleavage site |
| US11274284B2 (en) | 2015-03-30 | 2022-03-15 | Greenlight Biosciences, Inc. | Cell-free production of ribonucleic acid |
| US10954541B2 (en) | 2016-04-06 | 2021-03-23 | Greenlight Biosciences, Inc. | Cell-free production of ribonucleic acid |
| US10858385B2 (en) | 2017-10-11 | 2020-12-08 | Greenlight Biosciences, Inc. | Methods and compositions for nucleoside triphosphate and ribonucleic acid production |
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