CN117603952A - 细菌来源补身醇合酶及大肠杆菌生产补身醇的方法 - Google Patents
细菌来源补身醇合酶及大肠杆菌生产补身醇的方法 Download PDFInfo
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- CN117603952A CN117603952A CN202211007024.0A CN202211007024A CN117603952A CN 117603952 A CN117603952 A CN 117603952A CN 202211007024 A CN202211007024 A CN 202211007024A CN 117603952 A CN117603952 A CN 117603952A
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Abstract
本发明公开了细菌来源补身醇合酶及大肠杆菌生产补身醇的方法。本发明鉴定的三个补身醇合酶的核苷酸序列如列表SEQ ID NO:1‑3所示,氨基酸序列如列表SEQ ID NO:4‑6所示。一种用于生产补身醇的基因工程菌,在大肠杆菌中共同转染了本发明所述的表达载体以及含有法尼基焦磷酸合酶IspA编码基因、异戊烯基二磷酸异构酶IDI编码基因,高产萜类前体的磷酸化酶PhoN、IPK的表达载体所得。本发明还提供了利用大肠杆菌基因工程菌生产补身醇的方法,对基因工程生产补身醇提供新的思路。
Description
技术领域
本发明属于酶工程领域,涉及细菌来源补身醇合酶及大肠杆菌生产补身醇的方法。
背景技术
补身醇自1959年从Drimys winteri Forst中分离得到后,持续受到人们的关注,其反式十氢化萘(A/B)环常作为手性合成子合成活性优良的补身烷型萜类化合物,如水蓼二醛、warburganal等。目前,补身醇主要来源于植物提取,存在产率低、资源有限且不环保等因素,此外化学合成步骤繁琐,导致该化合物不适合大规模工业化生产。因此,寻找一种替代生产补身醇的方法对其开发和利用具有极大的意义和价值。
目前自然界中主要存在两种萜类天然产物生物合成途径,分别为MVA途径和MEP途径,通过在大肠杆菌中重构两种途径,能够促使其产生更多的萜类生物合成的前体物质DMAPP和IPP。目前,这些途径存在繁琐、限速、产量低等问题,至少需要7-8步才能生成最终的前体物质DMAPP和IPP,导致生产调控复杂。Moonhyuk Kwon等人利用酿酒酵母产生补身醇构建了相应的工程菌,然而,目前还没有在大肠杆菌中工程化生产补身醇的报道,而原核表达体系大肠杆菌相较于真核表达体系酿酒酵母具有操作更简便,成本更低等优势。因此,大肠杆菌生产补身醇将更具有工业化应用潜力。同时,由于目前鉴定的补身醇合酶较少,阻碍了其工程改造和运用的发展,这说明目前对补身醇合酶的研究还不成熟,阻碍了基因工程生产补身醇向工业化迈进的脚步。
发明内容
本发明的目的是针对现有技术的上述不足,提供细菌来源补身醇合酶及其编码基因。
本发明的第二个目的在于提供含有补身醇合酶编码基因的表达载体和基因工程菌。
本发明的第三个目的是提供所述的基因工程菌在制备补身醇合酶中的应用。
本发明的目的可通过以下技术方案实现:
一种补身醇合酶,选自氨基酸序列如SEQ ID NO.4-SEQ ID NO.13所示的任意一种。
本发明鉴定的三个补身醇合酶的核苷酸序列如列表SEQ ID NO:1-3所示,氨基酸序列如列表SEQ ID NO:4-6所示。
以补身醇合酶WP_009998890.1(即SEQ ID NO.6)为探针进行SSN分析参见图1,通过SSN网络图分析可得,三个补身醇合酶被归类于两个小簇内,同时这簇中还拥有七个与其氨基酸序列具有高度相似的酶,推测为补身醇合酶,其氨基酸序列如列表SEQ ID NO:7-13所示,并对其进行归纳总结,并将这些补身醇合酶进行归纳总结,见表1。
表1 SSN分析可能的补身醇合酶
含有本发明所述的补身醇合酶编码基因的表达载体,所述的补身醇合酶编码基因的核苷酸序列如SEQ ID NO.1-SEQ ID NO.3中任一项所示。
含有补身醇合酶编码基因的宿主细胞,所述的补身醇合酶编码基因的核苷酸序列如SEQ ID NO.1-SEQ ID NO.3中任一项所示;所述的宿主细胞优选大肠杆菌,进一步优选大肠杆菌E.coli BL21(DE3)。
本发明所述的补身醇合酶编码基因、所述的表达载体、所述的宿主细胞在制备补身醇合酶中的应用。
一种用于生产补身醇的基因工程菌,其特征在于,在大肠杆菌中共同转染了本发明所述的表达载体以及含有法尼基焦磷酸合酶IspA编码基因、异戊烯基二磷酸异构酶IDI编码基因,高产萜类前体基因PhoN、IPK的表达载体所得。
本发明所述的基因工程菌在生产补身醇中的应用。
一种利用大肠杆菌生产补身醇的方法,包含以下步骤:
(1)发酵权利要求5所述的基因工程菌;
(2)发酵液离心取沉淀,沉淀用有机溶剂超声裂解浸提,再离心获得有机溶剂层,旋蒸得补身醇粗品。
作为本发明的一种优选,所述的方法还包括步骤(3)对补身醇粗品进行纯化的步骤:补身醇粗品称重,用1.5-2倍质量的200-300目硅胶拌样,干法上样,石油醚:乙酸乙酯=40:1进行洗脱,纯化得到补身醇。
作为本发明的一种优选,步骤(1)中发酵所述的基因工程菌方法为:将权利要求5所述的基因工程菌接种到LB培养基中进行培养,待OD600生长到在1.5-2.5作为种子液接种到1L LB发酵培养基,接种量0.5%,添加0.1%kanamycin和ampicillin抗生素,发酵培养基在37℃,200-220rpm摇床条件培养,待OD600在0.6-0.8范围内进行冰浴降温,准备诱导,诱导加入0.2-0.5mM的异丙基-β-D-硫代半乳糖苷,3-10mM的3-甲基-2-丁烯-1-醇和3-甲基-3-丁烯-1-醇,1-5ml的50%甘油,放入18℃摇床,220rpm发酵48-72h。
作为本发明的一种优选,步骤(2)中所述的有机溶液为丙酮,超声浸泡的条件为频率40kHz,功率200W,浸泡10~12h。
有益效果:
本发明发现并鉴定三个细菌来源的补身醇合酶,并通过SSN分析推测出其余七个补身醇合酶;基于发现的三个补身醇合酶构建了补身醇合酶大肠杆菌表达体系,对补身醇合酶KKZ71921.1进行体外酶学表征,对补身醇合酶的研究起到推动作用。同时本发明还提供了利用大肠杆菌基因工程菌生产补身醇的方法,对基因工程生产补身醇提供新的思路。
附图说明
图1补身醇合酶的序列相似性网络(SSN)分析
A图为以Streptomyces clavuligerus的补身醇合酶WP_009998890.1为模板建立SSN分析,三个环化酶分散在两个小簇内,
B图为将两个小簇放大,其中绿色代表WP_009998890.1,蓝色代表Streptomycescattleya来源AEW97977.1,红色代表Streptomyces showdoensis来源KKZ71921.1
图2补身醇大肠杆菌生产菌株
图3三个补身醇菌株发酵产物的HPLC图(202nm)
图4补身醇1H NMR图(A图,溶剂:CDCl3)和补身醇13C NMR图(B图,溶剂:CDCl3)
图5补身醇质谱图
图6补身醇标准曲线图
图7补身基焦磷酸1H NMR图(A图,溶剂D2O)和补身基焦磷酸13C NMR图(B图,溶剂D2O)
图8 KKZ71921.1体外酶反应液相图
具体实施方式
实施例1 Streptomyces showdoensis来源补身醇合酶构建
设计引物(Forward:GCGATCGCTGACGTCGGTACCGTGAACGCATCACCGACGC,Reverse:TTTACCAGACTCGAGGGTACCTCATCGGGAGCAGCCTTCG)从Streptomyces showdoensis基因组中通过PCR克隆出补身醇合酶KKZ71921.1(核苷酸序列如SEQ ID NO.1,氨基酸序列如SEQ IDNO.4),KpnI(Takara公司)单酶切pET-Duet-1载体并将合酶基因通过ClonExpress II OneStep Cloning Kit同源重组到载体上,并转化到E.coli DH5α中,通过PCR鉴定挑选出阳性克隆,并通过测序鉴定(测序引物:YZ-1:TTGTACACGGCCGCATAATC,YZ-2(T7 terminator):GCTAGTTATTGCTCAGCGG)。
实施例2 Streptomyces cattleya来源补身醇合酶构建
设计引物(Forward:GCGATCGCTGACGTCGGTACCATGATCACCTCCTCACTGCT GTCCC,Reverse:TTTACCAGACTCGAGGGTACCTCAGACACGCGGGCGGGC)从Streptomyces cattleya基因组中通过PCR克隆出补身醇合酶AEW97977.1(核苷酸序列如SEQ ID NO.2,氨基酸序列如SEQID NO.5),KpnI单酶切pET-Duet-1载体并将合酶基因通过ClonExpress II One StepCloning Kit同源重组到载体上,并转化到E.coli DH5α中,通过PCR鉴定挑选出阳性克隆,并通过测序鉴定(测序引物:YZ-1:TTGTACACGGCCGCATAATC,YZ-2(T7 terminator):GCTAGTTATTGCTCAGCGG)。
实施例3 Streptomyces clavuligerus来源补身醇合酶构建
设计引物(Forward:GCGATCGCTGACGTCGGTACCATGCGTCCCGACCTGATCG,Reverse:TTTACCAGA CTCGAGGGTACCTCACACGAAGCTGAGCCCC)从Streptomyces clavuligerus基因组中通过PCR克隆出补身醇合酶WP_009998890.1(核苷酸序列如SEQ ID NO.3,氨基酸序列如SEQID NO.6),KpnI单酶切pET-Duet-1载体并将合酶基因通过ClonExpress II One StepCloning Kit同源重组到载体上,并转化到E.coli DH5α中,通过PCR鉴定挑选出阳性克隆,并通过测序鉴定(测序引物:YZ-1:TTGTACACGGCCGCATAATC,YZ-2(T7 terminator):GCTAGTTATTGCTCAGCGG)。
实施例4倍半萜表达载体的构建
通过将法尼基焦磷酸合酶IspA、异戊烯基二磷酸异构酶IDI,高产萜类前体基因PhoN、IPK构建到pRSF-Duet-1质粒上设计引物(Forward:TCATCACCACAGCCAATCCATGGACTTTCCGCAGCAACTC,Reverse:GATTATGCGGCCGTGTACAATTATTTATTACGCTGGATGATGTAGTC;Forward:TAATTGTACACGGCCGCATAATC,Reverse:GCCGAGCTCGAATTCGGATCCTTATTTAAGCTGGGTAAATGCAGATAA),以E.coli DH5α基因组为模板,对法尼基焦磷酸合酶基因ispA,异戊烯基二磷酸异构酶基因idi进行克隆,使用诺维赞ClonExpress Ultra One Step Cloning Kit构建到BamHI酶切的pRSF-Duet-1载体的第一个单克隆位点;再设计引物(Forward:GCGATCGCTGACGTCGGTACCATGAAGCGCCAGCTGTTTACC,Reverse:TTTACCAGACTCGAGTTAACGAATAACGGTGCCAATAAA),以合成的含PhoN,IPK基因(通用生物公司合成,碱基序列见SEQ ID NO.14)的质粒为模板,对PhoN-IPK基因进行克隆(基因合成),构建到KpnI酶切、含有ispA和idi基因的重组pRSF-Duet-1载体的第二个单克隆位点。并通过测序引物(YZ-2,YZ-3:tggatgtggtgggagatact,YZ-4:GGGCTATCTTTGGCGGTAAT)确定构建成功。
实施例5补身醇合酶KKZ71921.1蛋白表达载体的构建
设计引物(Forward:CAGCAAATGGGTCGCGGATCCGTGAACGCATCACCGACGC,Reverse:TTGTCGACGGAGCTCGAATTCTCATCGGGAGCAGCCTTCG)从Streptomyces showdoensis基因组中通过PCR克隆出补身醇合酶KKZ71921.1,EcoRI单酶切pET28a(+)载体并将合酶基因通过ClonExpress II One Step Cloning Kit同源重组到载体上,并转化到E.coli DH5α中,通过PCR鉴定挑选出阳性克隆,并通过测序鉴定(测序引物:YZ-1(T7 promoter:TAATACGACTCACTATAGG,YZ-2(T7terminator):GCTAGTTATTGCTCAGCGG),表达补身醇合酶KKZ71921.1的表达载体pET28a(+)-KKZ71921.1构建成功。
实施例6产补身醇基因工程菌的构建及发酵
将实施例4构建成功的倍半萜表达载体分别同实施例1-3中构建的三个补身醇重组载体共同转化到大肠杆菌表达载体E.coli BL21(DE3)上得到三个产补身醇的基因工程菌,并分别命名为Strdrime-1(Streptomyces showdoensis),Strdrime-2(Streptomycescattleya)和Strdrime-3(Streptomyces clavuligerus),挑选单个转化后的单克隆到LB培养基中进行培养,待OD600生长到在0.8作为种子液接种到1L发酵培养基,接种量0.5%,添加0.1%kanamycin(浓度:0.05mg/ml)和ampicillin(浓度:0.1mg/ml)抗生素,发酵培养基在37℃,220rpm摇床条件培养,待OD600生长到0.8,进行冰浴降温,准备诱导。诱导加入0.25mM的异丙基-β-D-硫代半乳糖苷(IPTG,生工生物公司),5mM的3-甲基-2-丁烯-1-醇和1.6mM3-甲基-3-丁烯-1-醇(阿拉丁试剂),2ml的50%甘油,放入18℃摇床,220rpm发酵72h。
实施例7发酵液后处理及补身醇的分离提取
发酵液用3700rpm低速离心机进行离心,收集菌体,用等体积的丙酮超声浸泡10h,之后再用3700rpm离心机进行离心处理,收集丙酮层,旋转蒸发仪蒸干即为发酵产物粗品。粗品称重,用1.5倍质量的200-300目硅胶拌样,干法上样,石油醚:乙酸乙酯=40:1进行洗脱,纯化得到补身醇,经1H和13C NMR和质谱鉴定为(-)-补身醇(图4、5),测得旋光值(c=0.235,CH3OH)。
实施例8补身醇标准曲线的建立
准确称取2mg的补身醇标准品,配置浓度为2mg/ml的标准品母液,分别用甲醇稀释至1mg/ml、0.5mg/ml、0.25mg/ml、0.1mg/ml、0.05mg/ml、0.025mg/ml、0.0125mg/ml七种不同浓度梯度,分别用HPLC进行分析(55%乙腈,45%超纯水等度洗脱15min,补身醇标准品在8.6min出现202nm吸收信号),根据不同峰面积y对应不同浓度x,建立标准曲线,y=2056.26x,R2=0.9998(图6)。
实施例9三株菌发酵产物产量鉴定
对三株菌Strdrime-1、Strdrime-2和Strdrime-3的50ml发酵液进行3700rpm离心,收取沉淀,用2ml丙酮浸泡沉淀并超声10-12h,3700rpm离心20min后取丙酮层,过滤后进行HPLC分析,用55%乙腈,45%超纯水等度洗脱15min,设置进样量为3μL,提取8.6min,202nm吸收波长下的峰面积,根据补身醇标准曲线计算50ml发酵液的产量。对三株菌发酵产物进行定量分析,发现Streptomyces showdoensis来源的补身醇合酶工程菌产量最高,为26.9mg/L,Streptomyces clavuligerus来源的补身醇合酶工程菌产量为19.6mg/L,Streptomyces cattleya来源的补身醇合酶工程菌产量为14.7mg/L。
实施例10补身醇合酶KKZ71921.1的制备与纯化
将pET28a(+)-KKZ71921.1质粒转化至E.coli BL21(DE3),挑选单克隆至LB液体培养基中培养12h作为种子液,0.5%种子液及0.1%的卡那霉素抗生素(浓度:0.05mg/1ml)接入1L的液体LB培养基中,于37℃恒温摇床(230rpm)中空气浴振摇至OD值为0.6将其取出,冰浴30min后,于超净台中加入IPTG至终浓度0.25mM,转移至18℃摇床(200rpm)中空气浴振摇18h。
发酵结束后,将发酵液于台式低速冷冻离心机(4℃,3750rpm)离心20min收取菌体,利用Wash Buffer(50mM Tris(pH 7.5),500mM NaCl)对菌体进行溶解,于细胞破碎仪(6号变幅杆,65%功率)破碎30min,将破碎液转移到50ml超速离心管中,于高速离心机(4℃,15000rpm)离心30min,取35ml的Wash Buffer(50mM Tris-HCl,500mM NaCl溶于1L超纯水)对菌体进行溶解,之后加1mg Lysozyme并破碎获得上清及沉淀。利用10ml注射器将上清于0.45μm滤膜进行过滤后,由AKTA蛋白纯化仪的样品泵处将样品吸入镍柱内,之后根据所设置的镍柱程序(表2)进行走样。待程序结束后,根据镍柱的出峰情况针对性的收集样品并进行浓缩到2.5ml,准备进行脱盐保存。于4℃冰箱内取出脱盐柱,先用10个柱体积的超纯水冲洗,随后用5个柱体积的Stock Buffer(50mM Tris(pH=7.5),100mM NaCl)平衡,制备完成后放置于4℃冰箱保存。将浓缩后的样品加入已制备好的脱盐柱中,在4℃环境下用3ml的Stock Buffer冲洗得目标蛋白KKZ71921.1溶液,最后将溶液分装至200μL的EP管中,放入液氮冷冻,并最后保存于-80℃冰箱内备用。
表2.AKTA Pure镍柱程序
B buffer(50mM Tris-HCl,500mM NaCl,500mM Imidazole溶于1L超纯水)
实施例11补身醇合酶的体外反应鉴定
对纯化后的Streptomyces showdoensis来源的补身醇合酶KKZ71921.1,进行体外酶反应,在50mM Tris buffer,1mM MgCl2,10%甘油反应体系中投入2mM法尼基焦磷酸,100nM纯酶,反应总体积100μL,于30℃水浴孵育10min,反应结束后加等体积甲醇淬灭,于离心机(13000rpm)离心20min,取上清进行高效液相分析,使用YMC-Pack ODS-A色谱柱,以25mM碳酸氢铵为流动相A,以纯乙腈为流动相B,按表3进行线性梯度洗脱,检测波长210nm,柱温30℃,流速0.8ml/min,精密吸取样品20μL,产物补身基焦磷酸在5.6min出峰,底物法尼基焦磷酸在7.1min出峰,液相图见图8。
实施例12体外反应产物分离鉴定
对Streptomyces showdoensis来源的补身醇合酶KKZ71921.1纯化,进行体外酶反应,在50mM Tris buffer,1mM MgCl2,10%甘油反应体系中投入36mg法尼基焦磷酸,10mg纯酶,反应总体积20ml,于30℃水浴过夜孵育,反应完结束后加等体积甲醇淬灭,于台式冷冻低速离心机(4℃,13000rpm)离心20min,旋蒸浓缩至5ml,进行制备液相分离,使用安捷伦ZORBAX Eclipse XDB-C18色谱柱分离,以10mM醋酸铵为流动相A,以纯乙腈为流动相B,按表4进行线性梯度洗脱,检测波长210nm,产物出峰时间18.6min,底物出峰时间19.9min,对产物进行真空冷冻干燥,获得白色粉末,D2O溶解,1H,13C NMR进行结构鉴定,见图7。
表3补身醇合酶的体外反应HPLC分析方法
表4补身醇合酶的体外反应制备液相分离方法
Claims (10)
1.一种补身醇合酶,其特征在于,选自氨基酸序列如SEQ ID NO.4-SEQ ID NO.13所示的任意一种。
2.含有补身醇合酶编码基因的表达载体,其特征在于所述的补身醇合酶编码基因的核苷酸序列如SEQ ID NO.1-SEQ ID NO.3中任一项所示。
3.含有补身醇合酶编码基因的宿主细胞,其特征在于所述的补身醇合酶编码基因的核苷酸序列如SEQ ID NO.1-SEQ ID NO.3中任一项所示;所述的宿主细胞优选大肠杆菌,进一步优选大肠杆菌E.coli BL21(DE3)。
4.SEQ ID NO.1-SEQ ID NO.3中任一项所示的补身醇合酶编码基因、权利要求2所述的表达载体、权利要求3所述的宿主细胞在制备补身醇合酶中的应用。
5.一种用于生产补身醇的基因工程菌,其特征在于,在大肠杆菌中共同转染了权利要求2所述的表达载体以及含有法尼基焦磷酸合酶IspA编码基因、异戊烯基二磷酸异构酶IDI编码基因,高产萜类前体基因PhoN、IPK的表达载体所得。
6.权利要求5所述的基因工程菌在生产补身醇中的应用。
7.一种利用大肠杆菌生产补身醇的方法,其特征在于,包含以下步骤:
(1)发酵权利要求5所述的基因工程菌;
(2)发酵液离心取沉淀,沉淀用有机溶剂超声裂解浸提,再离心获得有机溶剂层,旋蒸得补身醇粗品。
8.根据权利要求7所述的方法,其特征在于,还包括步骤(3)对补身醇粗品进行纯化的步骤:补身醇粗品称重,用1.5-2倍质量的200-300目硅胶拌样,干法上样,石油醚:乙酸乙酯=40:1进行洗脱,纯化得到补身醇。
9.根据权利要求7所述的方法,其特征在于,步骤(1)中发酵所述的基因工程菌方法为:将权利要求5所述的基因工程菌接种到LB培养基中进行培养,待OD600生长到在1.5-2.5作为种子液接种到1L LB发酵培养基,接种量0.5%,添加0.1%kanamycin和ampicillin抗生素,发酵培养基在37℃,200-220rpm摇床条件培养,待OD600在0.6-0.8范围内进行冰浴降温,准备诱导,诱导加入0.2-0.5mM的异丙基-β-D-硫代半乳糖苷,3-10mM的3-甲基-2-丁烯-1-醇和3-甲基-3-丁烯-1-醇,1-5ml的50%甘油,放入18℃摇床,220rpm发酵48-72h。
10.根据权利要求7所述的方法,其特征在于,步骤(2)中所述的有机溶液为丙酮,超生浸泡的条件为频率40-50kHz,功率200-300W,浸泡10h。
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