CN112680468A - 一种提高菌株的甲醇生物耐受性和生物转化效率的方法 - Google Patents
一种提高菌株的甲醇生物耐受性和生物转化效率的方法 Download PDFInfo
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- CN112680468A CN112680468A CN201910996363.8A CN201910996363A CN112680468A CN 112680468 A CN112680468 A CN 112680468A CN 201910996363 A CN201910996363 A CN 201910996363A CN 112680468 A CN112680468 A CN 112680468A
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Abstract
本发明公开了通过突变菌株中的cgl2365基因和/或cgl2857基因来构建甲醇耐受性提高的菌株的方法。本发明还公开了所构建的甲醇耐受性提高的甲醇生物转化菌株以及利用所述菌株进行甲醇生物转化的方法。本发明构建的菌株在利用甲醇和其它辅助碳源组成的混合碳源时,甲醇的利用比例显著提高,从而实质性提高了菌株对于甲醇的利用效率。
Description
技术领域
本发明涉及生物技术领域。具体地说,本发明涉及一种提高菌株的甲醇生物耐受性和生物转化率的方法、这种方法获得的菌株以及这种方法和获得的菌株在生物转化甲醇中的应用。
背景技术
甲醇是煤炭化工、页岩气化工和工农业废弃物气化的初级平台产品,我国甲醇产能已达8000万吨/年,占国际总量50%以上,呈现过剩趋势,甲醇经济的发展急需配套的甲醇转化利用技术【陈继军,陈光达,2016;张世新等,2013】。生物转化具有产品多样、产品选择性高和过程绿色环保等特点。因此,改造常用的平台菌株,开发甲醇生物转化方法,发展基于甲醇的生物制造产业,具有重要意义。目前已经发现多条甲醇生物转化途径,例如核酮糖单磷酸途径(RuMP途径)、木酮糖单磷酸途径、核酮糖二磷酸途径和丝氨酸循环等。其中,RuMP途径是目前研究和应用较多的甲醇生物转化途径。在RuMP途径中,甲醇首先被甲醇脱氢酶(Mdh)氧化成甲醛,甲醛与核酮糖-5-磷酸(Ru5P)在3-己酮糖-6-磷酸合成酶(Hps)的催化下生成己酮糖-6-磷酸(H6P),H6P被6-磷酸-3-己酮糖异构酶(Phi)催化生成果糖-6-磷酸(F6P),F6P进入糖酵解途径被利用,或通过戊糖磷酸途径进行碳重排再生Ru5P(Whitaker,W.B.,Sandoval,N.R.,Bennett,R.K.,Fast,A.G.,Papoutsakis,E.T.,2015.Syntheticmethylotrophy:engineering the production of biofuels and chemicals based onthe biology of aerobic methanol utilization.Curr.Opin.Biotechnol.33,165-175)。
现有技术主要是通过在平台菌株如大肠杆菌(Escherichia coli)和谷氨酸棒杆菌(Corynebacterium glutamicum)中阻断甲醛氧化生成甲酸和二氧化碳的途径,减少碳损失,同时表达Mdh、Hps和Phi,构建RuMP途径,赋予菌株生物转化甲醇的能力。例如,Witthoff等研究者在谷氨酸棒杆菌中失活了负责氧化甲醛生成甲酸的关键酶AdhE和Ald,同时表达了Mdh、Hps和Phi。所获得的基因工程菌株在甲醇作为唯一碳源的无机盐培养基中无法生长,在含有葡萄糖和甲醇的无机盐培养基中可以生长,最终消耗55mM葡萄糖和约80mM甲醇,甲醇与葡萄糖利用量的比例约为1.45:1(Witthoff,S.,Schmitz,K.,Niedenfuhr,S.,Noh,K.,Noack,S.,Bott,M.,Marienhagen,J.,2015.Metabolic engineering ofCorynebacterium glutamicum for methanol metabolism.Appl.Environ.Microbiol.81,2215-2225)。Bennett等研究者在大肠杆菌中失活了负责氧化甲醛生成甲酸的关键酶FrmA,同时表达了Mdh、Hps和Phi,以及来源于天然甲醇利用菌中的戊糖磷酸途径关键酶。所获得的基因工程菌株在甲醇作为唯一碳源的无机盐培养基中无法生长,在含有葡萄糖和甲醇的无机盐培养基中可以生长,最终消耗约275mM葡萄糖和38.3mM甲醇,甲醇与葡萄糖利用量的比例约为0.14:1(Bennett,R.K.,Gonzalez,J.E.,Whitaker,W.B.,Antoniewicz,M.R.,Papoutsakis,E.T.,2018.Expression of heterologous non-oxidative pentosephosphate pathway from Bacillus methanolicus and phosphoglucose isomerasedeletion improves methanol assimilation and metabolite production by asynthetic Escherichia coli methylotroph.Metab.Eng.45,75-85)。Tuyishime等研究者在谷氨酸棒杆菌中失活了负责氧化甲醛生成甲酸的关键酶AdhE和Ald,失活了戊糖磷酸途径的关键酶核糖磷酸异构酶RpiB,同时表达了Mdh、Hps和Phi,所获得的基因工程菌株在甲醇作为唯一碳源的无机盐培养基中无法生长,在含有木糖和甲醇的无机盐培养基中可以生长,进一步通过适应性进化筛选得到生长速度和甲醇利用速度大幅提升的突变株MX-11,该突变株生长过程中消耗甲醇96.9mM和木糖25.32mM,甲醇和木糖的利用比例为3.83:1(Tuyishime,P.,Wang,Y.,Fan,L.,Zhang,Q.,Li,Q.,Zheng,P.,Sun,J.,Ma,Y.,2018.Engineering Corynebacterium glutamicum for methanol-dependent growth andglutamate production.Metab.Eng.49,220-231)。
现有技术虽然实现了甲醇与另一种辅助碳源的共利用,但是甲醇相对辅助碳源的利用比例仍较低。由于甲醇的价格低于常用碳源如葡萄糖的价格,因此提高菌株利用混合碳源时甲醇的利用比例,对于降低原料成本十分重要。此外,甲醇作为有机溶剂,对微生物细胞具有一定的毒性,因此亟需提高菌株对高浓度甲醇的耐受性。
发明内容
本发明的目的在于提供一种能够提高菌株对甲醇的耐受性和利用率的方法;
本发明的另一目的在于提供所述方法获得的菌株以及本发明的方法和获得的菌株在甲醇的生物转化中的应用。
在第一方面,本发明提供一种甲醇耐受性和利用率提高的菌株的构建方法,所述方法包括以下步骤:
使得所述菌株中以下基因突变:cgl2365基因和/或cgl2857基因。
在优选的实施方式中,所述方法还包括检测所得菌株的甲醇耐受性。
在具体的实施方式中,所述cgl2365基因是野生型cgl2365基因,其核苷酸序列如SEQ ID NO:5所示,编码的氨基酸序列如SEQ ID NO:6所示;
所述cgl2857基因是野生型cgl2857基因,其核苷酸序列如SEQ ID NO:11所示,编码的氨基酸序列如SEQ ID NO:12所示。
在优选的实施方式中,所述突变包括使得所述菌株中本身包含的野生型cgl2365基因和/或cgl2857基因突变,或者在本身不含野生型cgl2365基因和/或cgl2857基因的菌株中外源性地导入突变型cgl2365基因和/或cgl2857基因。
在优选的实施方式中,所述突变通过以下方法之一或组合实现:基因的错义突变、基因的同义突变等。
在具体的实施方式中,所述cgl2365基因突变为其核苷酸序列如SEQ ID NO:5所示,且第542位由C突变为G;或者,所述cgl2365基因突变为其编码的氨基酸序列如SEQ IDNO:6所示,且第181位由丙氨酸突变为甘氨酸;
所述cgl2857基因突变为其核苷酸序列如SEQ ID NO:11所示,且第183位的G突变为A。
在优选的实施方式中,所述菌株包括但不限于肠杆菌属家族(Escherichia)、棒杆菌属家族(Corynebacterium)、芽孢杆菌属家族(Bacillus);优选棒杆菌属家族(Corynebacterium)。
在优选的实施方式中,所述菌株包括但不限于谷氨酸棒杆菌(Corynebacteriumglutamicum)、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis);优选谷氨酸棒杆菌(Corynebacterium glutamicum)。
在优选的实施方式中,所述方法构建的菌株的甲醇耐受性为300mM以上;优选400mM以上;更优选500mM以上,最优选600mM以上。在一优选例中,所述方法构建的菌株利用甲醇和木糖组成的混合碳源时,甲醇和木糖的消耗量之比大于4:1;优选大于5:1;更优选大于6:1;最优选大于7:1。
在优选的实施方式中,所述方法利用甲醇作为唯一碳源进行甲醇的生物转化。
在第二方面,本发明提供一种甲醇耐受性和利用率提高的菌株,所述菌株中的以下基因发生突变:cgl2365基因和/或cgl2857基因。
在具体的实施方式中,所述cgl2365基因突变为其核苷酸序列如SEQ ID NO:5所示,且第542位由C突变为G;或者,所述cgl2365基因突变为其编码的氨基酸序列如SEQ IDNO:6所示,且第181位由丙氨酸突变为甘氨酸;
所述cgl2857基因突变为其核苷酸序列如SEQ ID NO:11所示,且第183位的G突变为A。
在具体的实施方式中,所述菌株采用第一方面任一项所述的方法构建。
在优选的实施方式中,所述菌株包括但不限于肠杆菌属家族(Escherichia)、棒杆菌属家族(Corynebacterium)、芽孢杆菌属家族(Bacillus);优选棒杆菌属家族(Corynebacterium)。
在优选的实施方式中,所述菌株包括但不限于谷氨酸棒杆菌(Corynebacteriumglutamicum)、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis);优选谷氨酸棒杆菌(Corynebacterium glutamicum)。
在优选的实施方式中,所述方法构建的菌株利用甲醇和木糖组成的混合碳源时,甲醇和木糖的消耗量之比大于4:1;优选大于5:1;更优选大于6:1;最优选大于7:1。
在一优选例中,所述方法利用甲醇作为唯一碳源进行甲醇的生物转化。
在第三方面,本发明提供第一方面所述方法构建的菌株或第二方面所述菌株在生物转化甲醇以及利用甲醇生产后续产物中的应用。
在优选的实施方式中,所述利用甲醇生产后续产物是指利用甲醇生产氨基酸、有机酸、有机醇等。
在优选的实施方式中,所述利用甲醇生产氨基酸是指生产谷氨酸、赖氨酸、苏氨酸、蛋氨酸等氨基酸及其衍生物;优选谷氨酸。
在第四方面,本发明提供以下突变型基因或其编码的蛋白,
突变型cgl2365基因,其核苷酸序列如SEQ ID NO:5所示,且第542位由C突变为G;所述突变型cgl2365基因编码的氨基酸序列如SEQ ID NO:6所示,且第181位由丙氨酸突变为甘氨酸;或
突变型cgl2857基因,其核苷酸序列如SEQ ID NO:11所示,且第183位的G突变为A。
在第五方面,本发明提供第四方面所述的突变型基因或其编码的蛋白在提高菌株的甲醇耐受性或构建甲醇耐受性的菌株中的用途。
在第六方面,本发明提供一种表达载体,所述表达载体包含第四方面所述的突变型基因。
在第七方面,本发明提供一种宿主细胞,所述宿主细胞包含第五方面所述的表达载体或基因组上整合有第四方面所述的突变型基因。
在第八方面,本发明提供一种生物转化甲醇的方法,包括利用第一方面所述构建方法构建的菌株或第二方面所述菌株或第四方面所述的基因或编码蛋白或第六方面所述的表达载体或第七方面所述的宿主细胞进行甲醇的生物转化。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
具体实施方式
发明人经过广泛而深入的研究,出乎意料地发现使得菌株中的cgl2365基因和/或cgl2857基因发生突变,能够显著提高该菌株的甲醇耐受性以及该菌株利用甲醇和其它辅助碳源组成的混合碳源时的甲醇利用比例,从而实质性提高了菌株对于甲醇的利用效率。在此基础上完成了本发明。
术语定义
本文所用的术语“外源性”是指某体系中包含了原来不存在的物质。例如,通过转化等方式在某菌株中引入该菌株中原本不存在的基因,则该基因对于该菌株是“外源性”的。
本文所述的“基因突变”是指改变基因的序列,这种改变可以体现在核苷酸序列水平,也可以同时体现在所编码的氨基酸序列水平;即,所述突变可以是错义突变或同义突变。
cgl2365基因和/或cgl2857基因及其突变体
现有技术中,本文所述的cgl2365基因也被称为NCgl2282,cg2598,编码假定的膜蛋白;cgl2857基因也被称为NCgl2760,cg3164,编码脂甘露聚糖成熟与脂阿拉伯素合成所需的膜蛋白,在棒杆菌属微生物中天然存在。
基于本发明的教导,本领域知晓cgl2365基因和/或cgl2857基因可以来自棒杆菌属的微生物,来自棒杆菌属的cgl2365基因和/或cgl2857基因可以具有一定的同源性,例如80%以上、90%以上、95%以上、96%以上、97%以上、98%以上、99%以上的同源性。例如,在具体的实施方式中,所述cgl2365基因是野生型cgl2365基因,其核苷酸序列如SEQ IDNO:5所示,编码的氨基酸序列如SEQ ID NO:6所示;所述cgl2857基因是野生型cgl2857基因,其核苷酸序列如SEQ ID NO:11所示,编码的氨基酸序列如SEQ ID NO:12所示。
本文所述的突变型cgl2365基因或突变的cgl2365基因为其核苷酸序列如SEQ IDNO:5所示,且第542位由C突变为G;或者,其编码的氨基酸序列如SEQ ID NO:6所示,且第181位由丙氨酸突变为甘氨酸;所述cgl2857基因突变为其核苷酸序列如SEQ ID NO:11所示,且第183位的G突变为A。
本发明的甲醇耐受性提高的菌株的构建方法
本文所述的“甲醇耐受性提高的菌株”是指该菌株对甲醇的耐受性较出发菌株(即未使用本发明方法改造前的菌株)有所提高,或能够在高甲醇环境下生长,进而对于甲醇的利用效率显著提高。
具体地说,本发明通过使得菌株中的cgl2365基因和/或cgl2857基因发生突变来显著提高菌株对甲醇的耐受性。然而,本领域技术人员应该明白,本发明的方法并不限于对菌株中本身包含的野生型cgl2365基因和/或cgl2857基因进行突变。当在本身不含cgl2365基因和/或cgl2857基因的菌株中外源性地导入经突变的cgl2365基因和/或cgl2857基因时,该菌株对甲醇的耐受性同样得到显著提高。换言之,本发明的方法可以适用于本身含有野生型cgl2365基因和/或cgl2857基因的菌株,例如本身具备一定程度甲醇耐受性的菌株,从而进一步提高其甲醇耐受性;本发明的方法也可以适用于本身不具备甲醇耐受性的菌株,例如在该菌株中外源性导入经突变的cgl2365基因和/或cgl2857基因,从而使得其具备甲醇耐受性。
对于突变cgl2365基因和/或cgl2857基因的具体技术手段,本领域技术人员可以采取本领域熟知的各种方法,只要能够实现所述cgl2365基因和/或cgl2857基因的突变即可。在获得cgl2365基因和/或cgl2857基因突变的或包含经突变的cgl2365基因和/或cgl2857基因的菌株后,本领域技术人员还可任选检测所得菌株的甲醇耐受性。
本文所用的术语“生物转化”具备本领域技术人员常规理解的意义,即,使用酶、休止细胞或活细胞,将甲醇转化为对环境无害的产物(如二氧化碳),或菌体和各种化学品等产品。
本文所述的“甲醇生物转化菌株”、“生物转化甲醇的菌株”或“本发明菌株”具备相同的含义,均是指通过生物学途径转化甲醇的微生物,从而能够借助该甲醇生物转化菌株将甲醇转化为对环境无害的产物(如二氧化碳),或将甲醇转化成菌体、氨基酸、有机酸、多元醇等产品。
本发明的菌株或者本发明方法构建的菌株对甲醇表现出优异的耐受性,例如甲醇耐受性达到300mM以上;优选400mM以上;更优选500mM以上,最优选600mM以上。然而,基于本发明的教导,本领域技术人员会知晓,本发明菌株的甲醇耐受性提高的实质在于其在利用甲醇和其它辅助碳源组成的混合碳源时,对甲醇的利用率大大提高。例如,本发明的菌株利用甲醇和木糖组成的混合碳源时,甲醇和木糖的消耗量之比大于4:1;优选大于5:1;更优选大于6:1;最优选大于7:1。甚至可以以甲醇为唯一碳源进行生长。因此,本发明的菌株或者本发明方法构建的菌株是优异的甲醇生物转化菌株。
采用本发明的方法构建的菌株可以是任何可通过上述方法构建的甲醇生物转化菌株,包括但不限于包括但不限于肠杆菌属家族(Escherichia)、棒杆菌属家族(Corynebacterium)、芽孢杆菌属家族(Bacillus);优选棒杆菌属家族(Corynebacterium)。在进一步的实施例中,本发明构建的甲醇耐受性菌株或甲醇生物转化菌株可以是谷氨酸棒杆菌(Corynebacterium glutamicum)、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis),优选谷氨酸棒杆菌(Corynebacterium glutamicum)。
本发明的优点:
1.本发明为构建甲醇耐受性提高的菌株,进而构建甲醇生物转化效率提高的菌株提供了全新的思路;
2.本发明的菌株在利用甲醇和其它辅助碳源组成的混合碳源时,甲醇的利用比例显著提高;和
3.本发明的方法能够进一步提高本身已经具有相当甲醇耐受性的菌株的甲醇生物转化效率,从而能够具备显著的经济价值和社会价值,为实现甲醇生物转化和甲醇化工奠定了基础,使甲醇真正有望成为生物发酵和生物制造产业的新原料平台。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。
本发明所用试剂和原料均市售可得。
实施例
实施例1在谷氨酸棒杆菌MX-11中引入cgl2365基因错义突变
菌株C.glutamicum MX-11出自文献(Tuyishime,P.,Wang,Y.,Fan,L.,Zhang,Q.,Li,Q.,Zheng,P.,Sun,J.,Ma,Y.,2018.Engineering Corynebacterium glutamicum formethanol-dependent growth and glutamate production.Metab.Eng.49,220-231)。
(1)构建pK18mobsacB-tet-cgl2365C542G质粒
a.使用BamHI内切酶线性化pK18mobsacB-tet空载体。
b.以C.glutamicum ATCC 13032的基因组DNA为模板,使用单链核苷酸cgl2365-F1(TATGACATGATTACGAATTCCAGCTGGGGCAGCGTTGAG;SEQ ID NO:1)和cgl2365-R1(CATCCCACCGTGGGCAAGCAGACA;SEQ ID NO:2)作为引物,扩增cgl2365的上半部分片段,同时引入cgl2365第542位碱基C到G的突变。
c.以C.glutamicum ATCC 13032的基因组DNA为模板,使用单链核苷酸cgl2365-F2(GCCCACGGTGGGATGGATGGCACTCGGGTGA;SEQ ID NO:3)和cgl2365-R2(CGACGGCCAGTGCCAAGCTTACGGACGGTTGGAACATTTGCG;SEQ ID NO:4)作为引物,扩增cgl2365的下半部分片段,同时引入cgl2365第542位碱基C到G的突变。
d.使用ClonExpress II One Step Cloning Kit(南京诺唯赞生物科技有限公司)将上述cgl2365上半部分片段、cgl2365下半部分片段和线性化的pK18mobsacB-tet质粒连接,构建pK18mobsacB-tet-cgl2365C542G质粒。
(2)在C.glutamicum MX-11中引入cgl2365 C542G突变
a.参照文献(Ruan,Y.,Zhu,L.,Li,Q.,2015.Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cellwall and increasing the cytoplasmic membrane fluidity.Biotechnol.Lett.37,2445-2452)的方法,制备C.glutamicum MX-11的感受态细胞。在感受态制备过程中,在文献中所述培养基中额外加入木糖和核糖作为C.glutamicum MX-11的碳源。
b.将上述pK18mobsacB-tet-cgl2365C542G质粒转化至C.glutamicum MX-11的感受态细胞中,参照文献(A.,Tauch,A.,W.,Kalinowski,J.,Thierbach,G.,Pühler,A.,1994.Small mobilizable multi-purpose cloning vectors derived from theEscherichia coli plasmids pK18 and pK19:selection of defined deletions in thechromosome of Corynebacterium glutamicum.Gene 145,69-73)的方法,在C.glutamicumMX-11中引入cgl2365 C542G突变,对应cgl2365编码蛋白质的第181位丙氨酸突变为甘氨酸,构建的突变株命名为MX-11-cgl2365 C542G。
cgl2365野生型基因序列如下:
GTGTCGGAAATCATGGGAGATTTAGCAAAGCACATGGGCAGTGAACCACCAGCATGGTGGAAGTTTTTACCGATGATTGTCCTCGCTGGAGCCACTCGAGTTACCTATGAAGTAGAACCTTGGCTGGCGATCCCATTATTCATTTTGGCTTTTGCATCGATATTGATCCCATTCCCGATCTCTAAGACAAAAGGACTCCGTGATATCGATGCCTGGAAAATCCACACCACGCAAGGCGATAAAAAGCGTGCCATCCGCCAACTGATCATTCCGGCTACGGCTTTGGCCATCGACATCATTGGGCTGCCGACATTATTTAATGCCCCTCCCCTTGCTTCCGCTGCACTTTTTGGCGGTGTTTACGGCGCTTCCCTAGCTTGGGCTGCGTACAGAGCTGATCAGCTTCCACGCATTCGAACGAAGGAACGCCTCGCAGAACTTTCACAAAATGCATCTCTGGATGATGTGCGCTCAGATGACTTAGATGTTCTAGAGCAGCCGGAATCCCGTGAATTAGTGCGCTGTCTGCTTGCCCACGGTGCGATGGATGGCACTCGGGTGATGGCCAGACAGGTCGCGCGAGTACTGGATACCGAGGTAGACGAAGTACATCAGGTAGCACGCTCACTAGAACAGCATGGTTTGGTTAGTCGCTCCACCATCATGCCGGGTGGGGATCCAGGAAAAGTATTCATCGAAGTTTCCCTGAAAGGGATCTCAGCCATCAAGGCACTTGAATCCGGACGCTAA(SEQ ID NO:5)
cgl2365野生型氨基酸序列如下:
VSEIMGDLAKHMGSEPPAWWKFLPMIVLAGATRVTYEVEPWLAIPLFILAFASILIPFPISKTKGLRDIDAWKIHTTQGDKKRAIRQLIIPATALAIDIIGLPTLFNAPPLASAALFGGVYGASLAWAAYRADQLPRIRTKERLAELSQNASLDDVRSDDLDVLEQPESRELVRCLLAHGAMDGTRVMARQVARVLDTEVDEVHQVARSLEQHGLVSRSTIMPGGDPGKVFIEVSLKGISAIKALESGR*(SEQ ID NO:6)
实施例2在谷氨酸棒杆菌MX-11中引入cgl2857基因同义突变
(1)构建pK18mobsacB-tet-cgl2857G183A质粒
a.使用BamHI内切酶线性化pK18mobsacB-tet空载体。
b.以C.glutamicum ATCC 13032的基因组DNA为模板,使用单链核苷酸cgl2857-F1(TATGACATGATTACGAATTCTGCCGAGCGTTTTCATCCAACTG;SEQ ID NO:7)和cgl2857-R1(CTTCGGAATCGTCCGCGCCTGACCAGTCAC;SEQ ID NO:8)作为引物,扩增cgl2857的上半部分片段,同时引入cgl2857第183位碱基G到A的突变。
c.以C.glutamicum ATCC 13032的基因组DNA为模板,使用单链核苷酸cgl2857-F2(GCGCGGACGATTCCGAAGGATTTGGATCT;SEQ ID NO:9)和cgl2857-R2(CGACGGCCAGTGCCAAGCTTCGGCCAAAAACTTGGAAGGCC;SEQ ID NO:10)作为引物,扩增cgl2857的下半部分片段,同时引入cgl2857第183位碱基G到A的突变。
d.使用ClonExpress II One Step Cloning Kit(南京诺唯赞生物科技有限公司)将上述cgl2857上半部分片段、cgl2857下半部分片段和线性化的pK18mobsacB-tet质粒连接,构建pK18mobsacB-tet-cgl2857G183A质粒。
(2)在C.glutamicum MX-11中引入cgl2857G183A突变
a.参照文献(Ruan,Y.,Zhu,L.,Li,Q.,2015.Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cellwall and increasing the cytoplasmic membrane fluidity.Biotechnol.Lett.37,2445-2452)的方法,制备C.glutamicum MX-11的感受态细胞。在感受态制备过程中,在文献中所述培养基中额外加入木糖和核糖作为C.glutamicum MX-11的碳源。
b.将上述pK18mobsacB-tet-cgl2857G183A质粒转化至C.glutamicum MX-11的感受态细胞中,参照文献(A.,Tauch,A.,W.,Kalinowski,J.,Thierbach,G.,Pühler,A.,1994.Small mobilizable multi-purpose cloning vectors derived from theEscherichia coli plasmids pK18 and pK19:selection of defined deletions in thechromosome of Corynebacterium glutamicum.Gene 145,69-73)的方法,在C.glutamicumMX-11中引入cgl2857G183A突变,对应cgl2857编码蛋白质的第61位苏氨酸产生同义突变,构建的突变株命名为MX-11-cgl2857G183A。
cgl2857野生型基因序列如下:
ATGAATCCGCGATGGCGGATGGGTGCATATGATTGGGTAGACATTATTTCAACATGCGAGTTTAGCGGAAAGGTGTGGGCTGTTTTTATGAAGCGATCTGCAACGGTCCTCATTATTGCGGGCGTGCTGTTCCTCATTTTTGCCTTCACGGTACCGCCGTATGTGACTGGTCAGGCGCGGACGATTCCGAAGGATTTGGATCTGACGTTGGTGAGCGAAAGTCCGCAGGGGTTTGTGCGCACTGAACATATTGTGACTGCTCCGACGGAAAAGGTCGATGAGATCGCGACGCATGTGGATCAGACAGTTACGGATGTGCAGGGGAAAACTGTTGCGGAAATTTCGGATGATGTGGTGTTGATTGGACACTCTCGTTATCCGGTGATTAAGCCGACTGCCACCATTTCGGGTTCGCCGGCGGATAGTAGCAATGTGGTGCGGGAGGGGTTGCATTACTTCTTCCCGGCTAATACGTTGCGGAATTCTTATCCCTATTATGACATCGTATTGGGTGAGGATTCCCCGGTGGATTATGTCTCGCGCGAGGGCAATACTTATACCTTCTACCAGCATCTTCGTTATGTTCCATTGGATGATTCTCACACCTATTCGGTGGAGCGGACCCTGAAAGTGGATCGTTTTTCCGGCATCATTGTGGCTAAAGATGAGGCGATGACGTTTCATGGCCCAGACGGCGATGACACAGTAGAATTCACTTATACTGCGGATACGTTGAAGCTTCTGCAGGATCATGCGCATGATATTGATCAGCGGTTGTCGTGGGCTAAGGGGTTTGATTTCTTTTCTAAATTCTTAGGCCTGTCGTTGCTTGCGATTGGTGTGTTCCTCACGGGAATTTTCAAGCGCGGCCAGCTGATGAGCACTGTGAATAAACTCAGGAGTTAA(SEQ ID NO:11)
cgl2857野生型氨基酸序列如下:
MNPRWRMGAYDWVDIISTCEFSGKVWAVFMKRSATVLIIAGVLFLIFAFTVPPYVTGQARTIPKDLDLTLVSESPQGFVRTEHIVTAPTEKVDEIATHVDQTVTDVQGKTVAEISDDVVLIGHSRYPVIKPTATISGSPADSSNVVREGLHYFFPANTLRNSYPYYDIVLGEDSPVDYVSREGNTYTFYQHLRYVPLDDSHTYSVERTLKVDRFSGIIVAKDEAMTFHGPDGDDTVEFTYTADTLKLLQDHAHDIDQRLSWAKGFDFFSKFLGLSLLAIGVFLTGIFKRGQLMSTVNKLRS*(SEQ ID NO:12)
实施例3甲醇生物转化
(1)培养基
在不含葡萄糖的CGXII培养基中,添加甲醇和木糖作为碳源,额外添加1mM异丙基硫代半乳糖苷(IPTG)、5mg/L氯霉素和25mg/L卡那霉素。CGXII培养基配方参照文献(Keilhauer,C.,Eggeling,L.,Sahm,H.,1993.Isoleucine synthesis inCorynebacterium glutamicum:molecular analysis of the ilvB-ilvN-ilvCoperon.J.Bacteriol.175,5595-5603)。
(2)培养条件
将C.glutamicum菌株MX-11、MX-11-cgl2365 C542G、MX-11-cgl2857G183A分别接种于含有上述培养基的摇瓶中,初始OD600nm约为0.5(初始细胞干重约为0.153gCDW/L)。将摇瓶置于摇床中培养,温度为30℃,转速为220rpm,摇瓶为250mL,装液量为50mL,使用不透气封口膜封闭摇瓶以防止甲醇挥发。
(3)取样检测
定时取样,检测培养液中剩余的甲醇和木糖浓度。甲醇和木糖浓度检测方法为:将培养液样品在12,000×g离心10分钟,使用上清进行检测。使用岛津公司的高速液相色谱仪(Shimadzu Prominence UFLC)和Bio-Rad公司的色谱柱Aminex HPX-87H column(300×7.8mm),色谱柱柱温为55℃,检测器为示差折光检测器,检测器温度为55℃,流动相为5mMH2SO4,流速为0.5mL/min,进样量为10μL。当生物量不再升高时,停止培养。计算菌株对甲醇和木糖的消耗量,结果如表1所示。
当培养基中的初始甲醇浓度由125mM提高至460mM时,菌株MX-11的生长和甲醇利用受到明显抑制。采用本发明的方法构建的菌株MX-11-cgl2365 C542G和MX-11-cgl2857G183A,对高浓度甲醇的耐受性明显提高,在含有460mM甲醇的培养基中,甲醇和辅助碳源的利用比例分别提高至7.22:1和7.11:1,显著高于MX-11的甲醇和辅助碳源利用比例3.83:1。本发明的方法可显著提高菌株对高浓度甲醇的耐受性和甲醇利用效率,具有重要的应用价值。
表1.不同菌株的甲醇和木糖消耗量a
a数据为三次独立实验的平均值
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 中国科学院天津工业生物技术研究所
<120> 一种提高菌株的甲醇生物耐受性和生物转化效率的方法
<130> P2019-1554
<160> 12
<170> SIPOSequenceListing 1.0
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tatgacatga ttacgaattc cagctggggc agcgttgag 39
<210> 2
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
catcccaccg tgggcaagca gaca 24
<210> 3
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gcccacggtg ggatggatgg cactcgggtg a 31
<210> 4
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cgacggccag tgccaagctt acggacggtt ggaacatttg cg 42
<210> 5
<211> 750
<212> DNA
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 5
gtgtcggaaa tcatgggaga tttagcaaag cacatgggca gtgaaccacc agcatggtgg 60
aagtttttac cgatgattgt cctcgctgga gccactcgag ttacctatga agtagaacct 120
tggctggcga tcccattatt cattttggct tttgcatcga tattgatccc attcccgatc 180
tctaagacaa aaggactccg tgatatcgat gcctggaaaa tccacaccac gcaaggcgat 240
aaaaagcgtg ccatccgcca actgatcatt ccggctacgg ctttggccat cgacatcatt 300
gggctgccga cattatttaa tgcccctccc cttgcttccg ctgcactttt tggcggtgtt 360
tacggcgctt ccctagcttg ggctgcgtac agagctgatc agcttccacg cattcgaacg 420
aaggaacgcc tcgcagaact ttcacaaaat gcatctctgg atgatgtgcg ctcagatgac 480
ttagatgttc tagagcagcc ggaatcccgt gaattagtgc gctgtctgct tgcccacggt 540
gcgatggatg gcactcgggt gatggccaga caggtcgcgc gagtactgga taccgaggta 600
gacgaagtac atcaggtagc acgctcacta gaacagcatg gtttggttag tcgctccacc 660
atcatgccgg gtggggatcc aggaaaagta ttcatcgaag tttccctgaa agggatctca 720
gccatcaagg cacttgaatc cggacgctaa 750
<210> 6
<211> 249
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 6
Val Ser Glu Ile Met Gly Asp Leu Ala Lys His Met Gly Ser Glu Pro
1 5 10 15
Pro Ala Trp Trp Lys Phe Leu Pro Met Ile Val Leu Ala Gly Ala Thr
20 25 30
Arg Val Thr Tyr Glu Val Glu Pro Trp Leu Ala Ile Pro Leu Phe Ile
35 40 45
Leu Ala Phe Ala Ser Ile Leu Ile Pro Phe Pro Ile Ser Lys Thr Lys
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Gly Leu Arg Asp Ile Asp Ala Trp Lys Ile His Thr Thr Gln Gly Asp
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Lys Lys Arg Ala Ile Arg Gln Leu Ile Ile Pro Ala Thr Ala Leu Ala
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Ile Asp Ile Ile Gly Leu Pro Thr Leu Phe Asn Ala Pro Pro Leu Ala
100 105 110
Ser Ala Ala Leu Phe Gly Gly Val Tyr Gly Ala Ser Leu Ala Trp Ala
115 120 125
Ala Tyr Arg Ala Asp Gln Leu Pro Arg Ile Arg Thr Lys Glu Arg Leu
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Ala Glu Leu Ser Gln Asn Ala Ser Leu Asp Asp Val Arg Ser Asp Asp
145 150 155 160
Leu Asp Val Leu Glu Gln Pro Glu Ser Arg Glu Leu Val Arg Cys Leu
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Ser Leu Glu Gln His Gly Leu Val Ser Arg Ser Thr Ile Met Pro Gly
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Ala Ile Lys Ala Leu Glu Ser Gly Arg
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<213> 人工序列(Artificial Sequence)
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tatgacatga ttacgaattc tgccgagcgt tttcatccaa ctg 43
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<213> 人工序列(Artificial Sequence)
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Cys Thr Thr Cys Gly Gly Ala Ala Thr Cys Gly Thr Cys Cys Gly Cys
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Gly Cys Cys Thr Gly Ala Cys Cys Ala Gly Thr Cys Ala Cys
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<213> 人工序列(Artificial Sequence)
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gcgcggacga ttccgaagga tttggatct 29
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<212> DNA
<213> 人工序列(Artificial Sequence)
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cgacggccag tgccaagctt cggccaaaaa cttggaaggc c 41
<210> 11
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<212> DNA
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 11
atgaatccgc gatggcggat gggtgcatat gattgggtag acattatttc aacatgcgag 60
tttagcggaa aggtgtgggc tgtttttatg aagcgatctg caacggtcct cattattgcg 120
ggcgtgctgt tcctcatttt tgccttcacg gtaccgccgt atgtgactgg tcaggcgcgg 180
acgattccga aggatttgga tctgacgttg gtgagcgaaa gtccgcaggg gtttgtgcgc 240
actgaacata ttgtgactgc tccgacggaa aaggtcgatg agatcgcgac gcatgtggat 300
cagacagtta cggatgtgca ggggaaaact gttgcggaaa tttcggatga tgtggtgttg 360
attggacact ctcgttatcc ggtgattaag ccgactgcca ccatttcggg ttcgccggcg 420
gatagtagca atgtggtgcg ggaggggttg cattacttct tcccggctaa tacgttgcgg 480
aattcttatc cctattatga catcgtattg ggtgaggatt ccccggtgga ttatgtctcg 540
cgcgagggca atacttatac cttctaccag catcttcgtt atgttccatt ggatgattct 600
cacacctatt cggtggagcg gaccctgaaa gtggatcgtt tttccggcat cattgtggct 660
aaagatgagg cgatgacgtt tcatggccca gacggcgatg acacagtaga attcacttat 720
actgcggata cgttgaagct tctgcaggat catgcgcatg atattgatca gcggttgtcg 780
tgggctaagg ggtttgattt cttttctaaa ttcttaggcc tgtcgttgct tgcgattggt 840
gtgttcctca cgggaatttt caagcgcggc cagctgatga gcactgtgaa taaactcagg 900
agttaa 906
<210> 12
<211> 301
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 12
Met Asn Pro Arg Trp Arg Met Gly Ala Tyr Asp Trp Val Asp Ile Ile
1 5 10 15
Ser Thr Cys Glu Phe Ser Gly Lys Val Trp Ala Val Phe Met Lys Arg
20 25 30
Ser Ala Thr Val Leu Ile Ile Ala Gly Val Leu Phe Leu Ile Phe Ala
35 40 45
Phe Thr Val Pro Pro Tyr Val Thr Gly Gln Ala Arg Thr Ile Pro Lys
50 55 60
Asp Leu Asp Leu Thr Leu Val Ser Glu Ser Pro Gln Gly Phe Val Arg
65 70 75 80
Thr Glu His Ile Val Thr Ala Pro Thr Glu Lys Val Asp Glu Ile Ala
85 90 95
Thr His Val Asp Gln Thr Val Thr Asp Val Gln Gly Lys Thr Val Ala
100 105 110
Glu Ile Ser Asp Asp Val Val Leu Ile Gly His Ser Arg Tyr Pro Val
115 120 125
Ile Lys Pro Thr Ala Thr Ile Ser Gly Ser Pro Ala Asp Ser Ser Asn
130 135 140
Val Val Arg Glu Gly Leu His Tyr Phe Phe Pro Ala Asn Thr Leu Arg
145 150 155 160
Asn Ser Tyr Pro Tyr Tyr Asp Ile Val Leu Gly Glu Asp Ser Pro Val
165 170 175
Asp Tyr Val Ser Arg Glu Gly Asn Thr Tyr Thr Phe Tyr Gln His Leu
180 185 190
Arg Tyr Val Pro Leu Asp Asp Ser His Thr Tyr Ser Val Glu Arg Thr
195 200 205
Leu Lys Val Asp Arg Phe Ser Gly Ile Ile Val Ala Lys Asp Glu Ala
210 215 220
Met Thr Phe His Gly Pro Asp Gly Asp Asp Thr Val Glu Phe Thr Tyr
225 230 235 240
Thr Ala Asp Thr Leu Lys Leu Leu Gln Asp His Ala His Asp Ile Asp
245 250 255
Gln Arg Leu Ser Trp Ala Lys Gly Phe Asp Phe Phe Ser Lys Phe Leu
260 265 270
Gly Leu Ser Leu Leu Ala Ile Gly Val Phe Leu Thr Gly Ile Phe Lys
275 280 285
Arg Gly Gln Leu Met Ser Thr Val Asn Lys Leu Arg Ser
290 295 300
Claims (12)
1.一种甲醇耐受性和利用率提高的菌株的构建方法,所述方法包括以下步骤:
使得所述菌株中以下基因突变:cgl2365基因和/或cgl2857基因。
2.如权利要求1所述的构建方法,其特征在于,所述cgl2365基因是野生型cgl2365基因,其核苷酸序列如SEQ ID NO:5所示,编码的氨基酸序列如SEQ ID NO:6所示;
所述cgl2857基因是野生型cgl2857基因,其核苷酸序列如SEQ ID NO:11所示,编码的氨基酸序列如SEQ ID NO:12所示。
3.如权利要求2所述的构建方法,其特征在于,所述cgl2365基因突变为其核苷酸序列如SEQ ID NO:5所示,且第542位由C突变为G;或者,所述cgl2365基因突变为其编码的氨基酸序列如SEQ ID NO:6所示,且第181位由丙氨酸突变为甘氨酸;
所述cgl2857基因突变为其核苷酸序列如SEQ ID NO:11所示,且第183位的G突变为A。
4.一种甲醇耐受性和利用率提高的菌株,所述菌株中的以下基因发生突变:cgl2365基因和/或cgl2857基因。
5.如权利要求4所述的菌株,其特征在于,所述cgl2365基因突变为其核苷酸序列如SEQID NO:5所示,且第542位由C突变为G;或者,所述cgl2365基因突变为其编码的氨基酸序列如SEQ ID NO:6所示,且第181位由丙氨酸突变为甘氨酸;
所述cgl2857基因突变为其核苷酸序列如SEQ ID NO:11所示,且第183位的G突变为A。
6.如权利要求4或5所述的甲醇耐受性和利用率提高的菌株,其特征在于,所述菌株采用权利要求1-3中任一项所述的方法构建。
7.权利要求1-3中任一项所述方法构建的菌株或权利要求4-6中任一项所述菌株在生物转化甲醇以及利用甲醇生产后续产物中的应用。
8.以下突变型基因或其编码的蛋白,
突变型cgl2365基因,其核苷酸序列如SEQ ID NO:5所示,且第542位由C突变为G;所述突变型cgl2365基因编码的氨基酸序列如SEQ ID NO:6所示,且第181位由丙氨酸突变为甘氨酸;或
突变型cgl2857基因,其核苷酸序列如SEQ ID NO:11所示,且第183位的G突变为A。
9.权利要求8所述的突变型基因或其编码的蛋白在提高菌株的甲醇耐受性或构建甲醇耐受性的菌株中的用途。
10.一种表达载体,所述表达载体包含权利要求8所述的突变型基因。
11.一种宿主细胞,所述宿主细胞包含权利要求10所述的表达载体或基因组上整合有权利要求8所述的突变型基因。
12.一种生物转化甲醇的方法,包括利用权利要求1-3所述构建方法构建的菌株或权利要求4-6所述菌株或权利要求8的基因或编码蛋白或权利要求10的表达载体或权利要求11的宿主细胞进行甲醇的生物转化。
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