CN115029371B - 一种微生物生产的天然活性产物的高效分离方法 - Google Patents
一种微生物生产的天然活性产物的高效分离方法 Download PDFInfo
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Abstract
本发明属于生物工程技术领域,公开了一种微生物生产的天然活性产物的高效分离方法,针对传统分离工艺中路线复杂、收率低、高生产成本等问题,本发明基于天然活性产物的外排和内运系统,提出建立天然活性产物的“合成‑外排‑摄取”系统,是一种适用于微生物中完全或部分分布于胞内的微量天然活性产物的高效分离系统,先后通过构建天然活性产物的“合成‑外排”细胞,构建产物C的“摄取”细胞,菌株A液体发酵生产产物C,菌株A发酵液的收集,菌株B高密度培养及产物C的富集,产物C的分离纯化完成分离,不仅能够实现天然活性产物与微生物培养液中残留组分的高效分离,还能实现天然活性产物与其结构类似物的高效分离。
Description
技术领域
本发明属于生物工程技术领域,涉及一种微生物生产的天然活性产物的高效 分离方法,更具体地,属于一种基于小分子化合物外排和内运转运系统高效分离 微生物生产的天然活性产物的方法。
背景技术
天然活性产物是指动物、植物提取物或昆虫、海洋生物和微生物体内的组成 成分或其代谢产物以及人和动物体内许多内源性的化学成分,其中来源于真核生 物丝状真菌与藻类等微生物的代谢产物是具有天然生物活性成分的重要物质来 源,主要包括多糖类、酶类、抗生素类、皂苷类、生物碱、萜类、黄酮类等。自 发现天然活性产物具有特殊生理活性以来,利用天然活性产物已开发出许多具有治疗和保健作用的药物及化妆品。例如,从青霉和曲霉中发现的天然活性产物分 子康帕丁(Compactin)和洛伐他汀(Lovastatin)是可以预防和治疗冠心病的他 汀类药物的重要成分;从海绵黑松枝菌分离出来的酚吡咯(Phenolic Pyrrole)、 新阿米拉林(Neolamellarin A)可以抑制HIV-1整合酶和MCV拓扑异构酶进而 具有抗肿瘤的功能,可作为抗肿瘤药物的活性成分;发现于麦角真菌的麦角硫因(Ergothioneine)于2014年已被国家列入化妆品原料目录,作为化妆品美白、 抗氧化和抗衰老成分。由于天然活性产物所具有的特殊生理活性及功能,造就了 各类天然活性产物的广阔市场需求,大多数天然活性产物由细胞产生后保留在胞 内,需要释放到到胞外再经分离纯化后为人们所利用。
传统的天然活性产物分离方法有浸渍法、渗漉法、煎煮法、沉淀法、盐析法、 吸附法、回流提取法等,这些方法往往分离步骤多、耗时长,分离效率低且成本 高。近年来新兴的天然活性产物分离技术,如膜分离法、酶解技术、超声辅助提 取技术、微波辅助提取技术等,相对传统分离技术过程简单、对环境污染较少,但仍存在天然活性产物有效成分分离效率低、细胞无法循环利用,进而成本仍然 较高的问题。随着市场对天然活性产物需求的增加,工业化生产天然活性产物有 效成分行业迅猛发展,因此亟需开发一种实现从胞内高效率、低成本、稳定分离 出天然活性产物的方法。
发明内容
微生物发酵生产天然活性产物的工艺中,针对完全或部分分布于胞内的微量 天然活性产物,运用传统的分离技术进行分离纯化工艺路线复杂、收率低,造成 生产成本昂贵。为解决上述问题,本发明基于天然活性产物的外排和内运系统, 提出建立天然活性产物的“合成-外排-摄取”系统,包含菌株A和菌株B,建立一种适用于微生物生产的天然活性产物的高效分离系统,应用产物外排及内运系 统高效分离天然活性产物,所述产物外排及内运系统均由膜转运蛋白组成。
为实现上述目的,本发明的技术路线如下:
一种微生物生产的天然活性产物的高效分离方法,包括如下步骤:
S1:构建天然活性产物的“合成-外排”细胞:对于来源于微生物代谢的天 然活性产物,以下简称“产物C”,获得高效生产产物C的微生物细胞;挖掘负 责或参与产物C外排的转运蛋白,即外排蛋白Pexporter的基因序列信息,基于此 应用基因工程技术表达外排蛋白Pexporter,构建兼具产物合成及产物外排功能的 “合成-外排”细胞,以下简称“菌株A”;
S2:构建产物C的“摄取”细胞:基于基因组学、蛋白质组、转录组分析 等生物信息学手段,挖掘产物C的专一性内运转运蛋白,即内运蛋白Pimporter, 应用基因工程技术表达内运蛋白Pimporter,构建能专一性摄取产物C的“摄取” 细胞,以下简称“菌株B”;
S3:菌株A液体发酵生产产物C:对菌株A进行液体发酵,优化发酵条件, 积累A细胞的同时大量合成产物C,产物C在外排蛋白Pexporter作用下由胞内外 排进入发酵液中;
S4:菌株A发酵液的收集:选择合适孔径和材料的纳滤膜,对菌株A的培 养液进行固液分离,经膜过滤处理后,菌株A细胞被截留的同时循环回菌株A 的原发酵系统,即培养系统I,膜分离透过液为含产物C的清液;
S5:菌株B高密度培养及产物C的富集:对菌株B进行高密度液体培养, 即培养系统II,通过离心等手段收集B细胞;将步骤S4中含产物C的清液与收 集的B细胞进行混合,并在机械搅拌式发酵罐等发酵设备,即培养系统III中搅 拌培养,直至其发酵液中无法检测到产物C,此过程中B细胞在内运蛋白Pimporter作用下专一性摄取并富集产物C;
S6:产物C的分离纯化:利用纳滤膜收集S5中富集产物C后的B细胞, 对其进行超声破碎等处理,释放B细胞内的产物C,通过高速离心等手段进行固 液分离,收集富含产物C的清液,进而经过色谱分离,收集获得高纯度的产物C。
进一步的,步骤S1所述获得高效生产产物C的微生物细胞的技术手段为诱 变育种或基因工程改造。
进一步的,步骤S1所述挖掘负责或参与产物C外排的转运蛋白,即外排蛋 白Pexporter的基因序列信息以及步骤S2所述挖掘产物C的专一性内运转运蛋白, 即内运蛋白Pimporter,应用基因工程技术表达内运蛋白Pimporter的生物信息学手段 为基因组学、蛋白质组和转录组分析。
进一步的,步骤S3所述对菌株A进行液体发酵以及步骤S5所述对菌株B 进行高密度液体培养,所用的发酵设备为利用机械搅拌式发酵罐。
进一步的,步骤S4所述选择合适孔径和材料的纳滤膜依据产物C的分子量、 菌体细胞大小和发酵液pH值。
进一步的,步骤S5中,所述含产物C的清液与收集的B细胞搅拌培养的时 间根据目的天然活性产物的专一性内运蛋白Pimporter的摄取能力而定,培养时间 不宜过长,避免产物C被B细胞代谢利用。
与现有技术相比,本发明提出的产物分离策略,具备以下优点:
(1)能够实现目标天然活性产物与微生物培养液中残留组分,特别是高浓 度组分的高效分离;
(2)能够实现目标天然活性产物与微生物培养液中结构类似的组分的高效 分离;
(3)通过菌株B富集能够提高目标天然活性产物在分离组分中的丰度;
(4)将目标产物由胞外转入胞内,使得分离后的产物无培养液成分残留, 更加安全,可直接应用于医药、食品、饲料等产品生产。
附图说明
图1为本发明所提出的微生物生产的天然活性产物的高效分离策略的示意 图,菌株A为天然活性产物的“合成-外排”细胞;菌株B为天然活性产物的专 一性摄取细胞;产物C为微生物生产的天然活性产物。
具体实施方式
下面结合实施例对本发明做进一步阐述,但不构成对本发明的任何限制。如 无特殊说明,本发明所采用的实验方法均为常规方法,所用实验器材、材料、试 剂等均可从商业途径获得。
本发明中天然活性产物可以是氨基酸、多糖类、酶类、抗生素类、皂苷类、 生物碱、萜类、黄酮类、核苷酸等,由于原理相同,本实施例以个别天然活性产 物为例对本发明进行详细说明。
实施例1:天然活性产物麦角硫因的高效分离策略
本实施例以氨基酸麦角硫因为例,做出以下说明:
(1)构建天然产物麦角硫因的“合成-外排”细胞,本例为菌株Aegt:应用 基因工程技术表达麦角硫因(Ergothioneine,EGT)生物合成基因EGT1和EGT2, 构建表达质粒pYES2-Egt1-Egt2,基因EGT1具有如SEQ IDNO.1所示序列,基 因EGT2具有如SEQ IDNO.2所示序列。基于底盘细胞酿酒酵母Saccharomyces cerevisiae S88c,导入表达质粒pYES2-Egt1-Egt2,构建获得高产麦角硫因的酿酒 酵母工程菌。基于生物信息学挖掘到参与麦角硫因外排的有机阳离子外排蛋白 Ypq1p,进一步,构建过表达具有如SEQ IDNO.3所示序列的基因YPQ1的表达 质粒pYES2-Egt1-Egt2-YPQ1,构建兼具麦角硫因生物合成及外排功能的“合成- 外排”细胞,即菌株Aegt。
(2)构建天然产物麦角硫因的“摄取”细胞,本例为菌株Begt:基于文献 报道及生物信息学分析,挖掘出动物细胞中负责麦角硫因摄取的专一性内运蛋白 Octn1p,基于其氨基酸序列,经酿酒酵母密码子优化后获得适用于酵母细胞的内 运蛋白的具有如SEQ IDNO.4所示序列的编码基因ETT1。应用基因工程技术构 建表达质粒pYES2-ETT1,表达麦角硫因的专一性内运蛋白Ett1p,基于底盘细胞酿酒酵母S.cerevisiae S88c构建具有专一性摄取麦角硫因的“摄取”细胞,即 菌株Begt。
(3)菌株Aegt液体发酵生产麦角硫因:应用机械搅拌式发酵罐对菌株Aegt进行液体发酵,发酵温度30℃,发酵时间72h,转速为300rpm,pH 5.0,积累 Aegt细胞的同时大量合成麦角硫因,在外排转运蛋白Ypq1p的作用下,麦角硫因 从胞内排出进入发酵液中。
(4)菌株Aegt发酵液收集:根据麦角硫因生物分子量、菌体大小和发酵液 pH值选择膜面积为0.25m2的聚酰胺材质纳滤膜,对菌株Aegt细胞培养液进行固 液分离,经膜过滤处理,操作压力0.35MPa,压差0.25MPa,流量5~8m3/h,使 Aegt细胞被截留,回流进入原发酵系统,透过液为含麦角硫因的清液。
(5)菌株Begt高密度培养及麦角硫因的富集:利用机械搅拌式发酵罐对菌 株Begt进行高密度培养,使菌株Begt细胞大量扩增,收集Begt细胞;将步骤(4) 中收集到的含有麦角硫因的清液与收集的Begt细胞进行混合,并在机械搅拌式发 酵罐中30℃搅拌培养12-16h,即Begt细胞专一性摄取发酵液中麦角硫因,直至 发酵液中无法检测到麦角硫因。
(6)基于菌株Begt细胞的产物分离纯化工艺:利用膜面积为0.25m2的聚酰 胺材质纳滤膜过滤并收集步骤(5)中富集麦角硫因的Begt细胞,对Begt细胞进 行超声破碎,超声功率400W,每次辐射时间11S,超声总时间14.5min,破碎 处理后,1200rpm常温离心2min收集含麦角硫因的上清液,进一步经色谱分离, 色谱柱:XCharge C18 5μm 100A 4.6mm×250mm;流动相:0.1%甲酸;进样量: 50μL;柱温:25℃;等度洗脱;流速:0.5mL/min;检测波长:254nm,收集 得到较高纯度(>90%)麦角硫因。
实施例2:天然活性产物L-半胱氨酸的高效分离策略
本实施例以氨基酸L-半胱氨酸为例,做出以下说明:
(1)构建L-半胱氨酸的“合成-外排”细胞,本例为菌株ACys:应用基因工 程技术表达L-半胱氨酸(L-cysteine)生物合成基因丝氨酸乙酰转移酶编码基因 CysE,构建表达质粒pET22b-CysE,转入大肠杆菌Escherichia coli BL21(DE3) 中,同时敲除L-半胱氨酸脱巯基酶编码基因tanA和metC、谷氨酸半胱氨酸连接 酶编码基因gshA以及磷酸泛酸半胱氨酸连接酶编码基因dfp,构建获得高产L- 半胱氨酸的大肠杆菌工程菌,基因CysE具有如SEQIDNO.5所示序列。基于生 物信息学分析,得到参与L-半胱氨酸外排的转运蛋白EamBp,进一步,构建过 表达具有如SEQ IDNO.6所示序列的基因EamB的表达质粒pET22b-CysE-EamB,构建兼具L-半胱氨酸生物合成及外排功能的“合成-外排”细胞,即菌株ACys。
(2)构建L-半胱氨酸的“摄取”细胞,本例为菌株BCys:基于文献报道及 生物信息学分析,挖掘出酿酒酵母中负责L-半胱氨酸的专一性内运蛋白Yct1p,基因YCT1具有如SEQIDNO.7所示序列。应用基因工程技术构建表达质粒 pYES2-YCT1,表达L-半胱氨酸的专一性内运蛋白Yct1p,基于底盘细胞酿酒酵 母S.cerevisiae S88c构建具有专一性摄取L-半胱氨酸的“摄取”细胞,即菌株 BCys。
(3)菌株ACys液体发酵生产L-半胱氨酸:应用机械搅拌式发酵罐对菌株 ACys进行液体发酵,发酵温度37℃,发酵时间48h,转速为200rpm,pH 7.0, 积累ACys细胞的同时大量合成L-半胱氨酸,在外排转运蛋白EamBp的作用下, L-半胱氨酸从胞内排出进入发酵液中。
(4)菌株ACys发酵液收集:根据L-半胱氨酸生物分子量、菌体大小和发酵 液pH值选择膜面积为35m2的PVDF材质纳滤膜,对菌株ACys细胞培养液进行 固液分离,经膜过滤处理,操作压力-0.02MPa,使细胞ACys被截留,回流到原 发酵系统,过滤液为含L-半胱氨酸的清液。
(5)菌株BCys高密度培养及L-半胱氨酸的富集:利用机械搅拌式发酵罐对 菌株BCys进行高密度培养,使菌株BCys细胞大量扩增,收集BCys细胞,将步骤 (4)中收集到的含有L-半胱氨酸的清液与收集的BCys细胞进行混合,并在机械 搅拌式发酵罐中30℃搅拌培养12-16h,即BCys细胞专一性摄取发酵液中的L- 半胱氨酸,直至发酵液中无法检测到L-半胱氨酸。
(6)基于菌株BCys细胞的产物分离纯化工艺:利用膜面积为0.25m2的聚酰 胺材质纳滤膜过滤并收集步骤(5)中富集L-半胱氨酸的BCys细胞,对BCys细胞 进行超声破碎,超声功率400W,每次辐射时间11S,超声总时间14.5min,破 碎处理后,1200rpm常温离心2min收集含麦角硫因的上清液,进一步经色谱分离,色谱柱:ZORBAX SB-C18(250mm×4.6mm,5μm);流动相:0.1%磷酸 (pH 2.0)-乙腈(80:20),流速:0.6mL/min;检测波长:210nm;进样量:5μL;柱温:室温,收集得到较高纯度(>96%)的L-半胱氨酸。
实施例3:天然活性产物β-烟酰胺单核苷酸的高效分离策略
本实施例以核苷酸β-烟酰胺单核苷酸(Nicotinamide mononucleotide,NMN) 为例,做出以下说明:
(1)构建天然产物NMN的“合成-外排”细胞,本例为菌株ANMN:应用基 因工程技术构建表达NMN生物合成关键酶烟酰胺磷酸核糖转移酶编码基因Nampt,构建表达质粒pQE-Nampt,基因Nampt具有如SEQ IDNO.8所示序列。 基于底盘细胞大肠杆菌Escherichia coliM15,导入表达质粒pQE-Nampt,添加乳 糖或IPTG诱导重组烟酰胺磷酸核糖转移酶的表达,再通过添加底物烟酰胺,获 得目标产物NMN。基于生物信息学挖掘到参与NMN外排的转运蛋白PnuCp, 进一步,构建过表达具有如SEQ IDNO.9所示序列的基因PnuC的表达质粒 pQE-Nampt-PnuC,构建兼具NMN生物合成及外排功能的“合成-外排”细胞, 即菌株ANMN。
(2)构建天然产物NMN的“摄取”细胞,本例为菌株BNMN:基于文献报 道及生物信息学分析,挖掘出小鼠细胞中NMN的专一性内运蛋白Slc12a8p,其编码基因具有如SEQIDNO.10所示序列。应用基因工程技术构建酵母表达质粒 pYES2-Slc12a8,表达NMN的专一性内运蛋白基因Slc12a8,基于底盘细胞酿酒 酵母S.cerevisiae S88c构建具有专一性摄取NMN的“摄取”细胞,即菌株BNMN。
(3)菌株ANMN液体发酵生产NMN:应用机械搅拌式发酵罐对菌株ANMN进行液体发酵,发酵温度为37℃,发酵时间48h,转速为200rpm,pH 7.0,积 累ANMN细胞的同时大量合成NMN,在外排转运蛋白PunCp的作用下,NMN从 胞内排出进入发酵液中。
(4)菌株ANMN发酵液收集:根据NMN生物分子量,菌体大小和发酵液 pH值选择膜面积为35m2的PVDF材质纳滤膜,对菌株ANMN细胞培养液进行固 液分离,经膜过滤处理,操作压力-0.02MPa,使细胞ANMN被截留,回流到原发 酵系统,过滤液为含NMN的清液。
(5)菌株BNMN高密度培养及NMN的富集:利用机械搅拌式发酵罐对菌株 BNMN进行高密度培养,使菌株BNMN细胞大量扩增,收集BNMN细胞;将步骤(4) 收集到的含有NMN的清液与收集的BNMN细胞进行混合,在机械搅拌式发酵罐 中30℃搅拌培养12-16h,即BNMN细胞专一性摄取发酵液中的NMN,直至发 酵液中无法检测到NMN。
(6)基于菌株BNMN细胞的产物分离纯化工艺:利用膜面积为0.25m2的聚 酰胺材质纳滤膜过滤并收集步骤(5)中富集NMN的BNMN细胞,对BNMN细胞 进行超声破碎,超声功率400W,每次辐射时间11S,超声总时间14.5min,破 碎处理后,1200rpm常温离心2min收集含NMN的上清液,进一步经色谱分离, 色谱柱:Welch Xtimate C18(4.6×250mm,5μm)或等效色谱柱;柱温:30℃, 流速:0.8mL/min,进样量:5μL,检测波长:254nm;流动相A:50mmol/L磷酸二氢钾,流动相B:乙腈,收集得到较高纯度(>99%)的NMN。
以上所述实施方式仅为本发明的优选实施例,而并非本发明可行实施的全部 实施例。对于本领域一般技术人员而言,在不背离本发明原理和精神的前提下对 其所作出的任何显而易见的改动,都应当被认为包含在本发明的权利要求保护范 围之内。
序列表
<110> 大连工业大学
<120> 一种微生物生产的天然活性产物的高效分离方法
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2631
<212> DNA/RNA
<213> 单核非血红素亚砜铁合酶编码基因(EGT1)
<400> 1
atgccatctg ctgaatctat gacaccttca tctgctttgg gtcaattaaa agctacaggt 60
caacatgttt tgtctaaatt acaacaacaa acttctaatg ctgatattat tgatattagg 120
agagttgcag ttgaaattaa tttaaaaact gaaattactt ctatgtttag acctaaagat 180
ggtccaaggc aattaccaac tttgttgttg tataatgaaa ggggtttgca attgtttgaa 240
aggattacat atttggaaga atattatttg acaaatgatg aaattaaaat tttaactaaa 300
catgcaacag aaatggcttc ttttattcca tcaggtgcta tgattattga attgggttca 360
ggtaatttga ggaaagttaa tttgttgtta gaagcattag ataatgcagg taaagctatt 420
gattattatg ctttagattt gtctagggaa gaattggaaa ggactttagc acaagttcca 480
tcttataaac atgttaaatg tcatggtttg ttaggtactt acgatgatgg tagagattgg 540
ttaaaagcac ctgaaaatat taataaacaa aaatgtattt tgcatttggg ttcatctatt 600
ggtaatttta atagatcaga tgcagctaca tttttaaaag gttttacaga tgttttgggt 660
ccaaatgata aaatgttgat tggtgttgat gcatgtaatg atccagctag agtttatcat 720
gcttataatg ataaagttgg tattactcat gaatttattt taaatggttt gaggaatgca 780
aatgaaatta ttggtgaaac tgcatttatt gaaggtgatt ggagagttat tggtgaatat 840
gtttatgatg aagaaggtgg tagacatcaa gcattttatg ctccaactag ggatactatg 900
gttatgggtg aattgattcg ctcacatgat aggattcaaa ttgaacaatc tttaaaatat 960
tctaaagaag aatcagaaag gttgtggtct acagctggtt tggaacaagt ttctgaatgg 1020
acttatggta atgaatatgg tttacatttg ttagctaaat ctaggatgtc tttttctttg 1080
attccatcag tttatgcaag gtctgcattg cctacattag atgattggga agcattgtgg 1140
gctacttggg atgttgttac taggcaaatg ttaccacaag aagaattgtt ggaaaaacca 1200
attaaattga ggaatgcttg tattttttat ttaggtcata ttcctacatt tttggatatt 1260
caattgacta aaactactaa acaagctcca tctgaaccag cacatttttg taaaattttt 1320
gaaaggggta ttgatccaga tgttgataat ccagaattgt gtcatgctca ttcagaaatt 1380
cctgatgaat ggcctcctgt tgaagaaatt ttaacttatc aagaaactgt tagatcaagg 1440
ttaaggggtt tgtatgcaca tggtattgct aatattccaa ggaatgttgg tagggctatt 1500
tgggttggtt ttgaacatga attaatgcat attgaaactt tgttgtatat gatgttacaa 1560
tcagataaaa ctttgattcc aactcatatt cctagaccag attttgataa attagctagg 1620
aaagcagaat ctgaaagagt tccaaatcaa tggtttaaaa ttccagcaca agaaattact 1680
attggtttgg atgatcctga agatggttca gatattaata aacattatgg ttgggataat 1740
gaaaaaccac ctaggagagt tcaagttgct gcatttcaag ctcaaggtag acctattaca 1800
aatgaagaat atgctcaata tttgttggaa aaaaatattg ataaattgcc agcttcttgg 1860
gctaggttag ataatgaaaa tatttctaat ggtactacta attcagtttc aggtcatcat 1920
tctaatagga cttctaaaca acaattgcca tcatcatttt tagaaaaaac tgctgttagg 1980
acagtttatg gtttagttcc tttaaaacat gctttagatt ggccagtttt tgcttcttat 2040
gatgaattag caggttgtgc tgcatatatg ggtggtagga ttcctacatt tgaagaaact 2100
aggtctattt atgcctacgc tgatgcttta aaaaaaaaaa aagaagcaga aaggcaatta 2160
ggtaggacag ttccagcagt taatgcacat ttaactaata atggtgttga aattacacct 2220
ccttcttcac cttcttcaga aacaccagca gaatcttcat caccatctga ttctaatact 2280
actttgatta ctacagaaga tttgttttca gatttagatg gtgcaaatgt tggttttcat 2340
aattggcatc ctatgcctat tacttctaaa ggtaatactt tagttggtca aggtgaattg 2400
ggtggtgttt gggaatggac ttcttcagtt ttgaggaaat gggaaggttt tgaacctatg 2460
gaattgtatc ctggttatac agcagatttt tttgatgaaa aacataatat tgttttaggt 2520
ggttcttggg caactcatcc taggattgca ggtaggaaat cttttgttaa ttggtatcaa 2580
aggaattatc cttatgcatg ggttggtgct agagttgtta gggatttatg a 2631
<210> 2
<211> 1581
<212> DNA/RNA
<213> PLP结合型C-S裂解酶编码基因(EGT2)
<400> 2
atgggtttat tagaaggtga agaattagtt ttgaggggta ggggtcaagg tggtgaacct 60
aggccagaaa gagaaccaga attaaaattg gaacatgttc ctgaaagggc tcctgatggt 120
gaaccagaaa ctgaaggtca attgggtcct aggaaagaac cagaacataa attggaagca 180
gaatcagaac ctttacaaga aactccacaa agggaagttt tggcatttgg tagagcttgg 240
aaatcagaat ttttatttga tccagcatgg aggaatttaa atcatggttc atttggtact 300
tatccattgt atattaggga taaattaaga gcatatcaag atcaagcaga agctaggcct 360
gatcatttta ttaggtatga agaatctaaa ttattacata ggagtcgagc ggcggttgct 420
aaaattgtta atgctccatt agatacagtt gtttttgttg gtaatgcaac tgaaggtgtt 480
aatacagttt tgaggaattt gaggtgggat tctttggaaa aaggtggtca aaaagatgtt 540
attttgtctt tttctacagt ttatgaagca tgtggtaatg ctgctgatta tattgttgaa 600
tattttgcag gtaaagttga acataggact attgaattgg aatatccagt tgaagatgct 660
gatgttattg cagctttgag gggtgcagca acacaagttg ctagagaagg taaaagggca 720
agattggcta tgatggatgt tgttacttca aggccaggtg ttgtttttcc ttgggaagca 780
gcggttagag tttgtagaga attgggtatt ttatctttag ttgatggtgc acaaggtgtt 840
ggtatggtta gattagattt aacagcagcc gatcctgatt tttttgtttc taattgtcat 900
aaatggttat tagttcctag gggttgtgca atgttatata caccagctag gactcaatgt 960
ttattaagga cagcattggc tacatctcat ggttatgttc caccttcagc agctcctgca 1020
ccacctggtt ctaaatcccg ctatgttgct aattttgaat ttgttggtac tagagataat 1080
ggtccatatt tgtgtgttgc tgatgcaatt gcatggaggg aaagagtttg tggtggtgaa 1140
gaaaatattt taaggtattt gtgggcatta aataaaaaag gtattaggat tgttgctagg 1200
gcattgggta ctactcattt ggataatgaa acagaaactt taactaattg tgctatgggt 1260
aatgttgctt tacctatgag agttgatgat gaagatgctt ctactgcatt ggatgcagca 1320
ccatctgctg caattgctgc acctgatgtt gttgttgcta gggaaaatgt tgctttagtt 1380
gataaatgga tgagagaaag gttatttgat gattataaaa cttttatgac attgtttgtt 1440
atgcaagata gatattgggt taggttatca gctcaaattt atttagatga acaagattat 1500
gaagcagctg gtgatatttt aaaggctttg tgtgaaagga ttaggaggag ggaatattta 1560
gttccacaac cagttgaata a 1581
<210> 3
<211> 927
<212> DNA/RNA
<213> 液泡膜阳离子氨基酸转运蛋白编码基因(YPQ1)
<400> 3
atgcaacttg tgccgctaga attgaataga tccactctaa gtgggatatc tgggtccatc 60
tccatctcct gctggatcat tgtgtttgtc cctcaaattt acgagaactt ttaccggaag 120
tcgtctgatg ggctgtcttt attgttcgtg gtactttggt tggccggcga tgtctttaac 180
cttatgggtg ccgttatgca acatcttttg tctaccatga tcattcttgc tgcttactat 240
acggtggcag atatcatttt actaggtcaa tgtctatggt acgataatga ggaaaaacca 300
gcagtagacc ctattcatct ctcccctgcc aatccaataa acgaaaacgt tctgcacgat 360
gtgttcaatg aacaacaacc gcttttgaat tcccaaggtc agccaaatcg tattgatgaa 420
gaaatggctg ctccttcatc cgacggaaac gctggtgatg ataatctccg tgaagtcaat 480
tcaagaaatt tgataaaaga catatttatt gttagtggtg tagtttttgt aggtttcatc 540
tcgtggtatg taacctactg cgtaaactac acgcaacctc ctcccgtgga ggatccatca 600
ctgcctgttc ccgaactgca gatcaattgg atggctcaga tattcggtta cttaagtgcc 660
cttttgtatc tgggttcaag aattcctcag atattactga attttaagag aaagtcttgt 720
gaaggtatca gtttcctatt ctttttgttc gcctgtttgg gtaataccac atttattttc 780
tctgtgattg tcatttcttt agactggaag tatctaatta tgaatgcttc ctggttggtt 840
ggaagcatag gtactttatt catggatttc gtcatatttt cccagttttt catttacaaa 900
agaaataaaa aatttatact gaattaa 927
<210> 4
<211> 1656
<212> DNA/RNA
<213> 有机阳离子转运蛋白Octn1p编码基因(ETT1)
<400> 4
atgagagatt acgatgaagt tattgctttt ttgggtgaat ggggtccatt tcaaagattg 60
attttttttt tgttgtccgc tagtattatt ccaaatggtt tcaatggtat gagtgttgtt 120
tttttggctg gtactccaga acatagatgt agagttcctg atgctgctaa tttgtcttct 180
gcttggagaa ataattctgt tccattgaga ttgagagatg gtagagaagt tccacattct 240
tgtagtagat atagattggc tactattgct aatttctctg ctttgggttt ggaacctggt 300
agagatgttg atttgggtca attggaacaa gaatcttgtt tggatggttg ggaatttagt 360
caagatgttt atttgtctac cgttgttact gaatggaatt tggtttgtga agataattgg 420
aaggttccat tgactacttc tttgtttttt gttggtgttt tgttgggttc ttttgtttct 480
ggtcaattgt ctgatagatt tggtagaaag aatgttttgt tcgctactat ggctgttcaa 540
actggttttt cttttttgca aattttctcc atttcctggg aaatgtttac tgttttgttt 600
gttattgttg gtatgggtca aatttctaat tacgttgttg cttttatttt gggtactgaa 660
attttgggta agtctgttag aattattttc tctactttgg gtgtttgtac tttttttgct 720
gttggttata tgttgttgcc tttgtttgct tatttcatta gagattggag aatgttgttg 780
ttggctttga ctgttcctgg tgttttgtgt gttcctttgt ggtggtttat tcctgaatct 840
cctagatggt tgatttctca aagaagattt agagaagctg aagatattat tcaaaaggct 900
gctaaaatga acaacattgc tgttcctgct gttattttcg attctgttga agaattgaac 960
ccattgaagc aacaaaaagc atttattttg gatttgttca gaaccagaaa cattgctatt 1020
atgaccatta tgtctttgtt gttgtggatg ttgacttctg ttggttattt tgctttgtct 1080
ttggatgctc caaatttgca tggtgatgct tatttgaatt gttttttgtc tgctttgatt 1140
gaaattccag cttatattac tgcttggttg ttgttgagaa ctttgcctag aagatatatt 1200
attgctgctg ttttgttctg gggtggtggt gttttgttat ttattcaatt ggttcctgtt 1260
gattactact ttttgtctat tggtttggtt atgttgggta agtttggtat tacttctgct 1320
ttttctatgt tgtatgtttt cactgctgaa ttgtatccta ctttggttag aaatatggct 1380
gttggtgtta cttctactgc ttctagggtt ggttctatta ttgctccata ttttgtttac 1440
ttgggtgctt ataatagaat gttgccatat attgttatgg gttctttgac tgttttgatt 1500
ggtattttga ctttgttctt cccagaatct ttgggtatga ctttgccaga aactttggaa 1560
caaatgcaaa aagttaagtg gtttagaagt ggtaaaaaga ctagagattc tatggaaact 1620
gaagaaaacc caaaggtttt gattactgca ttttaa 1656
<210> 5
<211> 822
<212> DNA/RNA
<213> 丝氨酸乙酰转移酶编码基因(CysE)
<400> 5
atgtcgtgtg aagaactgga aattgtctgg aacaatatta aagccgaagc cagaacgctg 60
gcggactgtg agccaatgct ggccagtttt taccacgcga cgctactcaa gcacgaaaac 120
cttggcagtg cactgagcta catgctggcg aacaagctgt catcgccaat tatgcctgct 180
attgctatcc gtgaagtggt ggaagaagcc tacgccgctg acccggaaat gatcgcctct 240
gcggcctgtg atattcaggc ggtgcgtacc cgcgacccgg cagtcgataa atactcaacc 300
ccgttgttat acctgaaggg ttttcatgcc ttgcaggcct atcgcatcgg tcactggttg 360
tggaatcagg ggcgtcgcgc actggcaatc tttctgcaaa accaggtttc tgtgacgttc 420
caggtcgata ttcacccggc agcaaaaatt ggtcgcggta tcatgcttga ccacgcgaca 480
ggcatcgtcg ttggtgaaac ggcggtgatt gaaaacgacg tatcgattct gcaatctgtg 540
acgcttggcg gtacgggtaa atctggtggt gaccgtcacc cgaaaattcg tgaaggtgtg 600
atgattggcg cgggcgcgaa aatcctcggc aatattgaag ttgggcgcgg cgcgaagatt 660
ggcgcaggtt ccgtggtgct gcaaccggtg ccgccgcata ccaccgccgc tggcgttccg 720
gctcgtattg tcggtaaacc agacagcgat aagccatcaa tggatatgga ccagcatttc 780
aacggtatta accatacatt tgagtatggg gatgggatct aa 822
<210> 6
<211> 588
<212> DNA/RNA
<213> L-半胱氨酸外排蛋白编码基因(EamB)
<400> 6
gtgacaccga cccttttaag tgctttttgg acttacaccc tgattaccgc tatgacgcca 60
ggaccgaaca atattctcgc ccttagctct gctacgtcgc atggatttcg tcaaagtacc 120
cgcgtgctgg cagggatgag tctgggattt ttgattgtga tgttactgtg tgcgggcatt 180
tcattttcac tggcagtgat tgacccggca gcggtacacc ttttgagttg ggcgggggcg 240
gcatatattg tctggctggc gtggaaaatc gccaccagcc caacaaagga agacggactt 300
caggcaaaac caatcagctt ttgggccagc tttgctttgc agtttgtgaa cgtcaaaatc 360
attttgtacg gtgttacggc actgtcgacg tttgttctgc cgcaaacaca ggcgttaagc 420
tgggtagttg gcgtcagcgt tttgctggcg atgattggga cgtttggcaa tgtgtgctgg 480
gcgctggcgg ggcatctgtt tcagcgattg tttcgccagt atggtcgcca gttaaatatc 540
gtgcttgccc tgttgctggt ctattgcgcg gtacgcattt tctattaa 588
<210> 7
<211> 1596
<212> DNA/RNA
<213> L-半胱氨酸内运蛋白编码基因(YCT1)
<400> 7
atgtcaaaag ttgacgtaaa aattggagca gactcgatct cctcttctga tgaaatccta 60
gttccttcga gactcgctga tgttacgcta gcattcatgg aggagaatga cgcagcagtt 120
ccagaaatca cgcctgaaca agaaaaaaaa ttaaagagaa agctttttct cacaatattc 180
acctttgtct ctgccattaa ccttttactt tacatggaca aagccacttt atcctatgat 240
tcgattctag gcttctttga agatacaggt cttacccaaa atacttacaa tactgtaaat 300
acgctgtttt acgttggttt tgcaatcggc caatttcctg gacaatactt ggctcaaaag 360
ttaccacttg ggaaattctt gggtgggttg ttggccacat ggactatact tattttccta 420
agttgtaccg catacaactt ttccggtgtc gttgcgttga gatttttctt ggggctaaca 480
gagagtgttg ttatcccgat attaattacc actatgggta tgttcttcga tgcttcagaa 540
agagctgctg ctcagccatt tttctttgca gcatgtatgg ggtctccaat tccaactggg 600
tttattgctt atggtgttct tcatataaca aatcccagca tttcgttatg gaaaatattc 660
actatcatca ttggtggttt gacttttatc atgacggttg ttgtaattct gtggtttcct 720
aataatcctg ctgatgtgaa attcttttca atacaagaaa gggtatggat tatcaggaga 780
gttcaggcat ccacaggctc ttccattgaa caaaaagtct tcaaaaagag tcaattcaga 840
gaggcaatga aagattatat aacctggtta tttggattgt tttttcttct tcaacagtta 900
gccaacaatc tgccctatca acagaacctg ctatttgaag gaatgggtgg agttgatgct 960
ctaggttcga cattggtatc agttgccggt gctggtttcg ccgtcgtttg tgccttcatc 1020
gctacgttga tgttagcaaa atggaaaaat atttcagctt taacagccat cttttggact 1080
ttaccagcat tggtgggatc catcgctgca gctgctttac catgggacaa taagattggt 1140
atcttagcaa atatctgtat ggcaggacaa atatttggta ttccttttat tatagctctt 1200
agctgggcaa gttcaagtgc atctgggtac accaaaaaac tcacaagaag ttcggtgtcc 1260
ttatttgcga tgggaattgc taatatcata tcaccacaaa tatggagaga gaaggactct 1320
cctcgctttt tacctgcctg gattgttcaa atcgttttat cattctctct tgcaccagcc 1380
attttgttac tgatccattt catactaaaa agaaggaata atcaaagact aaaaaattat 1440
gacgaaaatt tacaaaatta tttggacaga attcaactca ttgaaagcga aaatccttct 1500
tccattgaag aagggaaagt ggtaacccac gagaacaatt tggcagtctt tgatttgact 1560
gatttagaaa acgaaacttt tatatatcct ttgtaa 1596
<210> 8
<211> 1467
<212> DNA/RNA
<213> 烟酰胺磷酸核糖转移酶编码基因(Nampt)
<400> 8
atggatagcc tgttaaatca ttatagtcgt gctagtgcta ttccatcatt attatgcgat 60
ttttacaaaa catctcatcg tatcatgtat ccggaaggtt cacaaattat ttatagtaca 120
tttacacctc gtagcaatga acaagcgcct tatttaacac aagttgtgtc atttggtttt 180
caagccttta tcattaaata tttaattcat tattttaatg ataacttttt ttctcgtgat 240
aaacatgatg ttgtgactga atactctgca tttattgaga aaaccttaca gttagaggat 300
acgggtgaac acattgcaaa attacatgag ttgggttatt tgcctatccg tattaaagct 360
attcctgaag gaaaaacggt ggcaattaaa gttccggtga tgacgattga aaatacgcat 420
ccggatttct gttggctgac taactattta gaaacattaa ttaatgtatc actgtggcag 480
ccgatgactt ctgcctcgat tgcttttgct tatcgtacag cattaattaa atttgctaat 540
gaaacttgtg ataatcaaga acatgtgcca tttcaatcgc atgatttttc aatgcgtggt 600
atgagttctt tagaatccgc agaaacttca ggtgctggcc atttaacttc ttttttaggt 660
acagacacta ttcctgcact gtcttttgtt gaagcgtatt atggttcaag cagtctgatt 720
ggcacgtcta ttccggcttc tgagcattca gtaatgagtt cacatggtgt cgatgaatta 780
tcaacatttc gttatttaat ggcaaaattt ccgaatagta tgttgtcaat tgtgtcagat 840
actacagact tttggcataa cattaccgtt aatttgccgt tattaaagca agaaattatt 900
gcacgtccag aaaatgcccg tttagtcatt cgtccagata gcggtaactt ttttgcgatt 960
atttgtggtg atccaaccgc tgatactgag catgaacgta aaggactgat tgaatgttta 1020
tgggatattt ttggtggtac agttaatcag aaaggttata aagtgatcaa tccacatatt 1080
ggggcaattt atggtgatgg cgtgacttat gaaaaaatgg ttaagatctt agaaggatta 1140
aaagccaaag gatttgcctc aagtaatatt gtgtttggcg ttggtgcaca aacctatcaa 1200
cgtaatacac gtgatacgtt gggctttgcg ctgaaagcga catctatcac tattaatggc 1260
gaagaaaaag ctattttcaa aaatcctaaa accgataatg gtctgaaaaa atcgcaaaaa 1320
ggtcgtgtta aactgctgtc ttatgatact taccttgatg gtttaactgc aaaggatgat 1380
tttagtgatg atttattaga gctgttattt gaaaatggta agttattacg ccgtacagac 1440
tttgatcaga ttcgtcaaaa cttgtaa 1467
<210> 9
<211> 720
<212> DNA/RNA
<213> 烟酰胺核苷转运蛋白编码基因(PnuC)
<400> 9
atggattttt ttagtgtgca gaatatcctg gtacatatac caataggggc aggcggttat 60
gatctctcat ggatcgaagc ggtaggcacg atcgccgggt tgctgtgtat tggccttgcc 120
agtctggaga agatcagcaa ctacttcttt ggcctgatca acgtcacctt gtttggcatt 180
attttctttc agattcagct gtatgccagc ctgctattac aggtgttttt ctttgccgcg 240
aatatttacg gttggtatgc gtggtcgcga caaaccagtc agaacgaggc ggagttgaaa 300
attcgctggt tgccattgcc gaaggcactc agctggttgg cggtttgcgt tgtttcgatt 360
ggtctgatga cggtatttat caatccggtg tttgcatttt tgacccgcgt ggcagtcatg 420
atcatgcaag cattaggatt acaggttgtg atgcctgaac tgcaaccgga cgctttcccg 480
ttctgggatt catgcatgat ggtgttatct atcgtggcaa tgattctgat gacgcgtaag 540
tatgtggaaa actggctgtt gtgggtgatt attaacgtga ttagcgtcgt tatttttgca 600
cttcagggcg tttacgccat gtctctggag tacatcatcc tgacctttat tgcgctcaac 660
ggcagccgga tgtggatcaa cagcgcacgt gaaagaggct cacgcgcgct gtcccattaa 720
<210> 10
<211> 2118
<212> DNA/RNA
<213> 溶质载体家族12成员8编码基因(Slc12a8)
<400> 10
atggcccaga ggtctccgca agaactcttc cacgaggcag cccagcaggg catcctggcc 60
cagccccagc cctggtggaa gatccagctg ttcatgtggg agccggtgct gtttgggacc 120
tgggatggtg tgttcacatc ctgcatgatc aacatttttg gcgttgtcct tttcttgagg 180
accggctggc tggtgggaaa cacaggtgtg ctcctgggct tgctcctggt gtccttcgtc 240
gtcctcgtgg ccctcatcac cgtgctgtcg ggcattggtg tcgcagagca tggcgggatc 300
agcagtggcg gtgtctactc catgatctcc tcggtgcttg gtgggcagat gggaggcact 360
gtggggctgc tctatgtatt tggacagtgt gttgcaggtg ctatgtacat caccggcttt 420
gcggagtcca tctcagatct gctgggactt ggggacatct gggcagtgcg tggaatttca 480
gttgctgtgc ttctggcttt gctgggcatc aacctggcag gtgtcaagtg gattatccgc 540
ctccagctgc tgctgctgct cctgctggct gtctcgaccc tggactttgt ggtgggctct 600
ttcacccacc tggacccaga acatggcttt attggctact ccccagaact gctacagagc 660
aacattctgc cagagtacag ccccggggag tcattcttca ctgtgtttgg ggtgttcttc 720
cctgcagcta caggagtcat ggctggcttc aacatgggag gagacctgag agaccctgct 780
gacagtgtcc ccttaggctc cctagcagct gttggcgtct cgtggtttct ctacatcatc 840
tttgccttcc tgcttggtgc cgtctgtacc cgagaggccc tccgctctga cttcctgata 900
gctgaaaagg tgtctctggt tggtttcctc ttcctattgg gcctgtacat ctcatccctg 960
gcttcctgta tggggggact ctatggcgca ccccggatcc tgcagtgcat cgcccaggac 1020
aaagtcatcc ctgcactcgc ctttctggcg aatgggaaag ggccaaataa aacaccggta 1080
gcagccatct gcctgaccag cttggtgacc atggcctttg tcctggtggg tcaggtgaat 1140
gttctggcgc ccgttgtcac catcaatttc atgctgacct acatcatggt ggactactct 1200
tacttcgccc tctccatggc tcactgtggc ctcgccccat ctcctgagcc cgtccccaga 1260
caaggcccag atactctgca ctgctctgag cacctgctcc aggacagggc tcccagctac 1320
ggctctgatg tccctgccag aagcctctct gagggcaccc tgctggagtt caccaaggac 1380
atggatcagt tcctccagcc aatagaggaa ctggagagtc gtcagcttgg gtcaagagaa 1440
ggaaacaacc caaagaatca gaagcgcaag ggtaagaaag gcgccaagca aaccctacaa 1500
gatagcttcc tcttggaccc tgggtctcct ttgtcctttc ctacgaggac ttctgagagg 1560
ttgtctgttg ccttctgtgg ggagcaagag tcctatcaga agcagcagac ttctaggagt 1620
gaatcacatg accatcttgt tcctgatcta cgcaaccagc ctagagtgaa cagagaagat 1680
ttctttctga aatgcagact tcaggaacaa gagatccaga gaagaccaag tgttttctat 1740
gcttgcatgt gtaacccctg ggtctccctg ttaggggctc ttgcatccct gctcatcatg 1800
tttgtgatcc agtggctcta taccctagct agtatgggtg ttgctgccct tgtgtatttc 1860
tacattggcc aggcaagtcc aggcctttac ctcggatcag catcaaactt cagctttttc 1920
caatggatga agtccttctt ggtcccctcc tgcaggagcc tgaggtccgc ccaggagcaa 1980
atcatcttgg cgccatcacc agccaaggtt gacatggcaa tgactcagct tacccaggac 2040
aatgcagact tcgccacccg agatcgttac caccactcct ccttcctgag ccgggagcag 2100
ttgatgcctc cctactag 2118
Claims (1)
1.一种微生物生产的天然产物麦角硫因的高效分离方法,其特征在于,包括如下步骤:
(1)构建天然产物麦角硫因的“合成-外排”细胞,菌株Aegt:挖掘麦角硫因生物合成基因EGT1和EGT2、以及参与麦角硫因外排的有机阳离子外排蛋白Ypq1p的编码基因YPQ1,基因EGT1序列如SEQIDNO.1所示,基因EGT2序列如SEQIDNO.2所示,基因YPQ1的序列如SEQIDNO.3所示,应用基因工程技术构建表达基因EGT1、EGT2和YPQ1的表达质粒,向底盘细胞酿酒酵母导入表达质粒,构建兼具麦角硫因生物合成及外排功能的“合成-外排”细胞,即菌株Aegt;
(2)构建天然产物麦角硫因的“摄取”细胞,菌株Begt:基于文献报道及生物信息学分析,挖掘出动物细胞中负责麦角硫因摄取的专一性内运蛋白Octn1p,经密码子优化获得适用于酿酒酵母的序列如SEQIDNO.4所示的编码基因ETT1,应用基因工程技术构建表达基因ETT1的表达质粒,基于底盘细胞酿酒酵母构建具有专一性摄取麦角硫因功能的“摄取”细胞,即菌株Begt;
(3)菌株Aegt液体发酵生产麦角硫因:应用机械搅拌式发酵罐对菌株Aegt进行液体发酵,发酵温度30℃,发酵时间72h,转速为300rpm,pH5.0,积累Aegt细胞的同时大量合成麦角硫因,在外排蛋白Ypq1p的作用下,麦角硫因从胞内排出进入发酵液中;
(4)菌株Aegt发酵液收集:根据麦角硫因生物分子量、菌体大小和发酵液pH值选择膜面积为0.25m2的聚酰胺材质纳滤膜,对菌株Aegt细胞培养液进行固液分离,经膜过滤处理,操作压力0.35MPa,压差0.25MPa,流量5~8m3/h,使Aegt细胞被截留,回流进入原发酵系统,透过液为含麦角硫因的清液;
(5)菌株Begt高密度培养及麦角硫因的富集:利用机械搅拌式发酵罐对菌株Begt进行高密度培养,使菌株Begt细胞大量扩增,收集Begt细胞;将步骤(4)中收集到的含有麦角硫因的清液与收集的Begt细胞进行混合,并在机械搅拌式发酵罐中30℃搅拌培养12-16h,使Begt细胞专一性摄取发酵液中麦角硫因,直至发酵液中无法检测到麦角硫因;
(6)基于菌株Begt的产物分离纯化工艺:利用膜面积为0.25m2的聚酰胺材质纳滤膜过滤并收集步骤(5)中富集麦角硫因的Begt细胞,对Begt细胞进行超声破碎,超声功率400W,每次超声时间11s,超声总时间14.5min,破碎处理后,1200rpm常温离心2min收集含麦角硫因的上清液,进一步经色谱分离,收集得到高纯度麦角硫因。
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