CN102978148B - 一株氧化葡糖酸杆菌工程菌及其构建方法和在制备木糖醇中的应用 - Google Patents
一株氧化葡糖酸杆菌工程菌及其构建方法和在制备木糖醇中的应用 Download PDFInfo
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Abstract
本发明公开了一种氧化葡糖酸杆菌工程菌构建方法,该方法是通过过量表达控制磷酸戊糖途径的关键限制酶葡萄糖-6-磷酸脱氢酶(G6PDH),提高该途径的代谢通量,进而实现辅酶NADH的循环再生。本发明还公开了上述工程菌的发酵工艺及其在转化制备木糖醇中的应用。本发明所述的工程菌转化D-阿拉伯糖醇产木糖醇的效果较出发菌株具有明显优势,木糖醇对D-阿拉伯糖醇的转化率可达60-90%。该技术在一定程度上解决了生物转化法制备木糖醇存在的转化率不高,木糖醇浓度低的难题,并且具有经济适用、操作简便、节能环保的优点。
Description
技术领域
本发明属于生物工程技术领域,涉及一株氧化葡糖酸杆菌工程菌及其构建方法和在制备木糖醇中的应用。
背景技术
木糖醇由于其甜度与蔗糖相当,可抑制形成龋齿的变形杆菌活力,代谢不依赖胰岛素,并有降低血脂、抗酮体等功能而备受到越来越多的重视。木糖醇是一种优良的功能性甜味剂和重要的平台化合物,在食品、医药、国防和轻工业中具有广泛的应用前景。据预测,国际市场上木糖醇总需求量将达10万吨以上,市场缺口高达40%,可见市场发展空间极大。
目前工业上主要采用化学法生产木糖醇,该工艺以玉米芯为原材料,经过酸水解后分离得到木糖,然后利用化学加氢获得木糖醇。该合成工艺对资源和能源消耗大、环境污染极为严重,不符合当前提倡的绿色环保可持续发展的生产理念。
近年来以来源丰富价格低廉的葡萄糖为底物生产木糖醇引起人们的广泛关注,但是到目前为止在自然界中还没有发现直接发酵葡萄糖产木糖醇的微生物。1969年,Onishi等首先报道了通过三步发酵将葡萄糖转化为木糖醇。2000年前后Shunichi等提出通过两步工艺将葡萄糖转化为木糖醇,即首先利用酵母菌将葡萄糖转化为D-阿拉伯糖醇,之后利用氧化葡萄糖酸杆菌(Gluconobacter oxydans)将D-阿拉伯糖醇氧化还原为木糖醇。葡萄糖发酵生产D-阿拉伯糖醇的工艺已经成熟,转化率可达45%(理论转化率50%),而由于氧化葡萄糖酸杆菌转化D-阿拉伯糖醇产木糖醇的低得率限制了该工艺的推广。
研究者发现D-阿拉伯糖醇转化生成木糖醇是两个酶催化的过程,第一个为G.oxydans细胞质外膜上的D-阿拉伯糖醇脱氢酶,该酶快速的把D-阿拉伯糖醇转化为D-木酮糖,转化率接近100%;第二个为木糖醇脱氢酶(xylitol dehydrogenase,XDH),该酶负责把前一步生成的木酮糖还原为木糖醇。由于XDH对NADH的专一辅酶依赖性,造成XDH催化木酮糖产木糖醇的效率低下,限制了催化转化流程的高效运转。因此为XDH提供足量的辅酶成为解决这一难题的首选方案。
如上说提到的氧化葡萄糖酸杆菌(G.oxydans)是一种专性好氧的革兰氏阴性菌,属于醋酸杆菌科(Acetobacterceae),其最大的特点是细胞质外膜空间含有众多脱氢酶,能够不完全氧化一系列的醇及糖醇类化合物生成相应的醛、酮或酸,产物不需要通过透膜运输而直接分泌到胞外,从而大大提高了氧化葡萄糖酸杆菌的工业应用价值。氧化葡萄糖酸杆菌不存在糖酵解途径,三羧酸循环途径也不完整,导致了菌体细胞内作为转移磷酸化电子供体的还原力和辅酶NADH的浓度和总量偏低,进而造成催化转化过程中依赖辅酶NADH的XDH活力不高,木糖醇转化率低下。
研究发现作为氧化葡糖酸杆菌核心代谢的磷酸戊糖途径可以促进NADH的再生循环和积累,而与之相关的两个关键酶分别由基因gox0145(亦记做zwf)和gox1705(或记做gnd)编码,其中zwf合成的葡糖-6-磷酸脱氢酶G6PDH是整个途径的限速酶与代谢阀门。这两个酶都具有对NADP/NAD的双辅酶依赖性,但是在氧化葡糖酸杆菌生理条件下,G6PDH倾向与依赖NADP产生NADPH,6PGDH倾向于依赖辅酶NAD产生NADH。另有研究表明氧化葡糖酸杆菌胞内存在可以将NADPH转化为NADH由gox0310-0312编码的氢转移酶。以上所述是解决氧化葡糖酸杆菌催化制备木糖醇过程中的辅酶NADH不足的理论基础和实验设计指南。
发明内容
本发明所要解决的技术问题是提供一种可以利用廉价辅底物产生充足辅酶供给的氧化葡糖酸杆菌工程菌。
本发明还要解决的技术问题是提供上述氧化葡糖酸杆菌工程菌的构建方法。
本发明最后要解决的技术问题是提供上述氧化葡糖酸杆菌工程菌的应用。
为解决上述技术问题,本发明采用的技术方案如下:
一株氧化葡糖酸杆菌工程菌,它是导入了葡糖6-磷酸脱氢酶基因gox0145与启动子PtufB的氧化葡糖酸杆菌,所述的葡糖6-磷酸脱氢酶基因gox0145位于启动子PtufB的下游。
本发明氧化葡糖酸杆菌工程菌是通过过量表达控制磷酸戊糖途径的关键限制酶葡萄糖-6-磷酸脱氢酶(G6PDH),提高该途径的代谢通量,进而实现辅酶NADH的循环再生
其中,所述的葡糖6-磷酸脱氢酶基因gox0145的核苷酸序列如SEQ IDNo.1所示。
其中,所述的启动子PtufB是tufB基因(the elongation factor EF-Tu)的启动子,核苷酸序列如SEQ IDNo.2。
其中,所述的氧化葡糖酸杆菌为氧化葡糖酸杆菌Gluconobacter oxydans NH-10。该菌株在中国专利CN101948878A中已经公开,现保藏于中国微生物菌种保藏管理委员会普通微生物中心,编号CGMCCNo.2709,登记日期2008年10月14日。
上述氧化葡糖酸杆菌工程菌的构建方法,包括如下步骤:
(1)设计引物克隆启动子PtufB、葡糖6-磷酸脱氢酶基因gox0145;
(2)将启动子PtufB序列连接到广宿主型表达载体上,以构建带有启动子的表达载体;
(4)将基因gox0145片段连接到启动子的下游构建重组表达载体;
(4)将步骤(3)得到的重组表达载体转化氧化葡糖酸杆菌即得重组的辅酶循环途径加强型氧化葡糖酸杆菌菌株Gluconobacter oxydans pnadh-5。
步骤(2)中,所述的广宿主型表达载体为pBBR1MCS-5。
本发明所述的工程菌的主要代谢途径如图4所示。
上述氧化葡糖酸杆菌工程菌在转化D-阿拉伯糖醇制备木糖醇中的应用。
具体方法是,将氧化葡糖酸杆菌工程菌经种子培养后接种于摇瓶或发酵罐,发酵后离心收获菌体;将所得菌体加入到D-阿拉伯糖醇转化体系中,利用菌体静息细胞实现转化D-阿拉伯糖醇制备木糖醇。
对于摇瓶转化体系,D-阿拉伯糖醇转化体系中,D-阿拉伯糖醇浓度为20~50g/L,0.1M pH6.0的磷酸钾缓冲液作为反应介质,菌体加入量为5~15(w/v)%,温度25~35℃;采用两阶段通气法,第一阶段,摇瓶转速200~240rpm,反应8~14h,第二阶段加入5~20g/L葡萄糖作为辅底物,控制pH在5.5~6.5,摇瓶转速降低到40~80rpm,反应50~70h。
对于发酵罐转化体系,D-阿拉伯糖醇转化体系中,D-阿拉伯糖醇浓度为40~60g/L,0.1M pH6.0的磷酸钾缓冲液作为反应介质,并全程控制pH在5.5~6.5,菌体加入量为10~15(w/v)%,温度25~35℃;采用两阶段通气法,第一阶段氧化反应,搅拌桨转速400~600rpm通气量1~1.2vvm时长5~9h;第二阶段还原反应,补加5~20g/L葡萄糖作为辅底物提供还原力,搅拌桨转速150~200rpm,停止通入空气或者改通入一定量氮气,该阶段反应40~60h。
氧化葡糖酸杆菌工程菌的发酵培养条件如下:
摇瓶发酵:分别以RSM/YPG培养基,在摇瓶中加入适量培养基,优化调节温度和摇床转速,YPG培养基中加入碳酸钙调控pH。发酵罐发酵:YPG培养基,发酵温度28~33°C,pH5.9~6.1。
种子培养基和斜面培养基(g/L):葡萄糖20,酵母粉5,蛋白胨3;固体培养基中琼脂添加量1.5%。YPG培养基(g/L):葡萄糖30,酵母粉10,山梨醇10,硫酸铵、磷酸二氢钾、七水硫酸镁微量。培养基调节pH6.0~6.5后,121°C高温灭菌;用来培养重组菌的培养基温度降至50~60°C时加入相应浓度的庆大霉素50μg/L和头孢霉素25μg/L。
关键酶酶活测定方法:酶活单位定义为25°C时,每分钟生成1μmol的NADH或者NAD所需的酶量。酶活测定反应体系(1ml):5mmo l/L的NAD+溶液,0.1mol/L的底物溶液及100mmol/L pH7.0磷酸钠缓冲液。
D-阿拉伯糖醇、D-木酮糖、木糖醇的含量测定:高效液相色谱法,色谱条件:AgilentHPLC,示差折光检测器(Shodex RI-101),色谱柱为Rezex:RCM-MonosaccharideCa2+(Phenomenex,USA),柱温80°C,流动相为纯水,流速0.5mL/min。
显著效果:研究数据显示本发明的工程菌静息细胞制备木糖醇的转化率可以达到60%-90%,而原始菌细胞制备效率不足25%。本发明方法较好地解决了生物转化法制备木糖醇存在的转化率不高,木糖醇浓度低的难题,并且具有操作简便、经济适用、节能环保等诸多优点。
附图说明
图1重组表达载体构建流程示意图。图中所示的质粒pRtB-zwf所指即构建的表达载体pBBR-PtufB-gox0145。
图2关键酶基因gox0145基因克隆与质粒验证。Lane M:5000bp DNA标准marker;Lane1:gox0145PCR片段;Lane2&3:gox0145连接pMD-18T载体克隆验证。
图3克隆和双酶切验证表达载体的构建。Lane M:2000bp和15000bp标准DNAmarker;克隆验证:Lane1,PtufB;Lane2,gox0145;Lane3,PtufB+gox0145;双酶切验证:Lane4,KpnⅠ和BamHⅠ酶切表达质粒pBBR-PtutB-gox0145。
图4氧化葡糖酸杆菌工程菌的磷酸戊糖途径辅酶循环再生过程与木糖醇转化制备的关系示意图。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1:表达载体的构建。
根据NCBI数据库Gluconobacter oxydans621H基因组注释的PtufB、gox0145基因序列设计引物克隆各基因片段,所采用的引物序列见表1;
表1
反应体系如下:10×PCR buffer5μL,Mg2+4μL,dNTP4μL,上下游引物各2μL,exTaq DNA polymerase0.5μL,基因组模板1μL,加ddH2O至反应总体积为50μL。
PCR反应条件为:95℃2min;94℃30s;55℃30s;72℃1min;循环30次;72℃10min;10℃保存。
将克隆得到的各基因片段和提取得到的质粒pBBR1MCS-5纯化后回收。以限制酶KpnⅠ和XhoⅠ双酶切质粒pBBR1MCS-5和启动子片段PtufB,切胶回收后用T4ligase恒温孵育进行连接反应,所得连接液转化到感受态细胞E.coli JM109,挑选阳性转化子孵化并保存从而得到带有强启动子PtufB的质粒pBBR-PtufB。提取该质粒分辨进行酶切和PCR验证,酶切出现4700和300bp两条带,PCR验证结果出现300bp条带,从而证明确实将PtufB连接到质粒pBBR1MCS-5上获得了pBBR-PtufB。提取该质粒和目的片段用限制酶XhoⅠ和BamHⅠ双酶切,然后以T4连接酶进行连接获得含有启动子的广宿主表达质粒pBBR-PtufB-gox0145,转化到感受态细胞E.coli BL21(DE3),利用带有抗性的培养基平板挑取阳性转化子。提取质粒并经KpnⅠ和BamHⅠ双酶切,分别出现4700和1800bp两条带,验证结果无误,证明成功构建了含有启动子的广宿主表达质粒pBBR-PtufBgox0145。
实施例2:重组工程菌的转化。
重组质粒在大肠杆菌与氧化葡萄糖酸杆菌(G. oxydans)之间的转移可以通过帮助质粒pRK2013的作用实现接合转移的转化方法称之为三亲本接合。(1)菌体培养:将供体菌(带有重组质粒的E.coli BL21),帮助菌HB101/pRK2013接种在加有相应抗生素的LB中培养8h,受体菌NH-10在培养基中培养18h,进行接合实验。(2)三亲接合:取上述受体亲本菌液1.5mL,离心收集菌体,生理盐水洗2次,倒掉液体,加入0.5mL帮助菌,混匀,离心,加入1.5mL供体菌离心,用液体培养基洗一次,混匀,取一部分液体涂布至无抗性固体平板,30℃过夜培养。(3)接合子筛选:将培养过夜的结合菌体用无菌水从固体培养基上洗下来,涂布到含有Cef和Gm的固体培养基上,培养2-4天,筛选重组子获得表达菌G. oxydans pnadh-5。
实施例3:重组工程菌的发酵与收获。
种子培养基和斜面培养基(g/L):葡萄糖20,酵母粉5,蛋白胨3;固体培养基中琼脂添加量1.5%。YPG培养基(g/L):葡萄糖30,酵母粉10,山梨醇10,硫酸铵、磷酸二氢钾、七水硫酸镁微量(共0.1~0.5)。培养基调节pH6.0~6.5后,121℃高温灭菌;用来培养重组菌的培养基温度降至50~60℃时加入相应浓度的庆大霉素和头孢霉素。
将活化后的各菌株G. oxydans NH-10、G. oxydans pnadh-5接种到种子培养基中恒温孵育18h至对数生长中后期,以10%(v/v)的比例接种到发酵培养基中发酵培养。发酵罐发酵:YPG培养基,在7.5L发酵罐上,最终装液量4.5~5.0L。控制温度30°C,pH6.0~6.5左右。发酵结束后将发酵液在高速冷冻离心机上低温离心,然后用pH6.0100mM KPB重新悬浮并洗涤菌体三次,将获得的菌体置于-20°C低温保存备用。
实施例4:利用工程菌静息细胞转化D-阿拉伯糖醇产木糖醇。
不同底物浓度条件下木糖醇浓度和转化率的考察:G. oxydans NH-10、G. oxydanspnadh-5静息细胞转化D-阿拉伯糖醇生成木糖醇,转化过程的条件为:D-阿拉伯糖醇20~50g/L,0.1M pH6.0的磷酸钾缓冲液作为反应介质,细胞添加量10%(w/v),温度30°C,转速220rpm。反应9h时加入10g/L的葡萄糖提供还原力,转速调整为60rpm,5~10g/L碳酸钙控制pH在6.0,催化反应进行至转化平衡。液相检测重组菌催化转化产物溶液的组成和转化率发现:20~40g/L的底物溶液,D-阿拉伯糖醇几乎没有残余,但并未完全转化为木糖醇,转化率从90%逐步降为65%;50g/L的D-阿拉伯糖醇溶液有约8g/L的底物残留,木糖醇浓度约为35g/L,转化率约为70%。原始菌转化产物浓度最高为7.6g/L,转化率为25%。
不同细胞浓度对木糖醇产物和转化率的考察:G. oxydans pnadh-5静息细胞转化D-阿拉伯糖醇生成木糖醇,转化过程的条件为:D-阿拉伯糖醇30g/L,0.1M pH6.0的磷酸钾缓冲液作为反应介质,细胞添加量5-15%(w/v),温度30°C,转速220rpm。反应9h时加入10g/L的葡萄糖提供还原力,转速调整为60rpm,5g/L碳酸钙控制pH在6.0,催化反应进行至转化平衡。液相检测催化转化产物溶液的组成和转化率发现:随着静息细胞浓度的逐步提高,木糖醇浓度有所提高,转化率上升,而达到最高转化率所需时间呈递减趋势。5%细胞浓度时木糖醇转化率62%,10%细胞浓度转化率70%,15%细胞浓度转化率约75%。
转化率=木糖醇终浓度(g/L)/D-阿拉伯糖醇起始浓度(g/L)×100%
实施例5:生物反应器中进行的催化转化D-阿拉伯糖醇产木糖醇。
为了进一步了解和控制催化反应过程中的各项指标参数,利用优化后的转化条件将转化过程放大到7.5L NBS发酵罐中进行。G. oxydans NH-10、G. oxydans pnadh-5静息细胞转化D-阿拉伯糖醇生成木糖醇,转化过程的条件为:D-阿拉伯糖醇50g/L,0.1MpH6.0的磷酸钾缓冲液作为反应介质,细胞添加量15%(即湿菌体150g/L),温度30℃;因为第一阶段反应为膜结合D-阿拉伯糖醇脱氢酶催化的氧化反应,对氧的需求很高,所以初期控制搅拌桨转速600rpm,通气量控制1~1.2vvm。待氧化反应结束后加入10g/L的辅底物葡萄糖,启动辅酶循环途径为XDH催化木酮糖还原为木糖醇提供足量的还原力,搅拌桨转速调整为150~200rpm,通入气体改为氮气排尽反应器和反应体系内的氧气,降低葡萄糖被G. oxydans的膜结合葡萄糖脱氢酶(m-GDH)氧化为葡糖酸的比例。其他条件不变进行催化转化直至反应平衡。检测可得D-阿拉伯糖醇已耗尽,木糖醇的浓度约为40g/L,转化率达到80%。将转化液离心收集菌体可重复使用5次。
Claims (7)
1.一种氧化葡糖酸杆菌工程菌,其特征在于,该菌是导入了启动子PtufB和葡萄糖-6-磷酸脱氢酶基因gox0145的氧化葡糖酸杆菌,所述基因gox0145位于启动子PtufB的下游,受其调控节制;
其中,所述的葡萄糖-6-磷酸脱氢酶基因gox0145的核苷酸序列如SEQ IDNo.1所示;
其中,所述的启动子PtufB是tufB基因的启动子,核苷酸序列如SEQ IDNo.2所示;
其中,所采用的起始受体菌株为氧化葡糖酸杆菌Gluconobacter oxydans NH-10。
2.权利要求1所述的氧化葡糖酸杆菌工程菌的构建方法,其特征在于如下步骤:
(1)设计引物克隆启动子PtufB、葡萄糖-6-磷酸脱氢酶基因gox0145;
(2)将启动子PtufB序列连接到广宿主型质粒上,以构建带有启动子的表达载体;
(3)将基因gox0145片段连接到启动子的下游构建重组表达载体;
(4)将步骤(3)得到的重组表达载体转化氧化葡糖酸杆菌即得。
3.根据权利要求2所述的氧化葡糖酸杆菌工程菌的构建方法,其特征在于,步骤(2)中,所述的广宿主型表达载体为pBBR1MCS-5。
4.权利要求1所述的氧化葡糖酸杆菌工程菌在转化D-阿拉伯糖醇高效制备木糖醇中的应用。
5.根据权利要求4所述的应用,其特征在于,将氧化葡糖酸杆菌工程菌经种子培养后接种于摇瓶或发酵罐,发酵后离心收获菌体;将所得菌体加入到D-阿拉伯糖醇转化体系中,利用菌体静息细胞实现转化D-阿拉伯糖醇制备木糖醇。
6.根据权利要求5所述的应用,其特征在于,对于摇瓶转化体系,D-阿拉伯糖醇转化体系中,D-阿拉伯糖醇浓度为20~50g/L,0.1M pH6.0的磷酸钾缓冲液作为反应介质,菌体加入质量体积比为5~15%,温度25~35℃;采用两阶段通气法,第一阶段,摇瓶转速200~240rpm,反应8~14h,第二阶段加入5~20g/L葡萄糖作为辅底物,控制pH在5.5~6.5,摇瓶转速降低到40~80rpm,反应50~70h。
7.根据权利要求5所述的应用,其特征在于,对于发酵罐转化体系,D-阿拉伯糖醇转化体系中,D-阿拉伯糖醇浓度为40~60g/L,0.1M pH6.0的磷酸钾缓冲液作为反应介质,并全程控制pH在5.5~6.5,菌体加入质量体积比为10~15%,温度25~35℃;采用两阶段通气法,第一阶段氧化反应,搅拌桨转速400~600rpm通气量1~1.2vvm时长5~9h;第二阶段还原反应,补加5~20g/L葡萄糖作为辅底物提供还原力,搅拌桨转速150~200rpm,停止通入空气或者改通入一定量氮气,该阶段反应40~60h。
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