CN114480239A - 一种利用wlp途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌及其应用 - Google Patents
一种利用wlp途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌及其应用 Download PDFInfo
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Abstract
一种利用WLP途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌及其应用。重构嗜甲基丁酸杆菌中包括天然WLP途径同化甲醇;所述重构嗜甲基丁酸杆菌中导入的基因包括来源于大肠杆菌的甘氨酸裂解系统氨基甲基转移酶(GcvT)、甘氨酸裂解系统H蛋白(GcvH)、甘氨酸脱羧酶(GcvP),过表达内源的丝氨酸羟甲基转移酶(glyA)。重构的嗜甲基丁酸杆菌在以甲醇为唯一碳源中发酵验证发现较原始菌株的最大生物量提高了55.26%,甲醇同化量提高了34.47%,同时将重构菌株发酵生产合成丁酸的发酵条件进行优化,其最终最大生物量提高了55.26%,甲醇同化量提高了57.14%,丁酸合成量提高了77.78%。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种利用WLP途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌及其应用。
背景技术
甲醇(Methanol,CH3OH)为结构最简单的饱和一元醇,是重要的碳一化合物平台之一。随着人类对化石燃料勘探、挖掘技术不断提高,自20世纪50年代以来生产甲醇的方法逐渐由干馏法发展为化学合成法,包括加压催化从化石燃料而来的合成气、直接氧化甲烷、还原大气中二氧化碳和氢气。丰富的原料和生产项目大量涌现,使得甲醇产能持续快速增长,2018年全球生产甲醇总量可达1.1亿吨,而当年甲醇需求量为8-9千万吨。甲醇还原力较高,其每个碳还原度比葡萄糖每个碳还原度高2,可用于增强某些还原性产物的生产,如羧酸、醇和脂肪酸。利用来源丰富、产能过剩的甲醇进行微生物法生产化学品,较传统发酵工艺有着更为经济且可持续发展优势,作为非粮食原料甲醇可提供较高还原力,对减轻生物制造业对粮食生物质的依赖具有重要意义。
嗜甲基丁酸杆菌(Butyribacterium methylotrophicum,BM),细菌学分类为梭菌属,是一种专性厌氧革兰氏阳性芽孢杆菌。嗜甲基丁酸杆菌可同时利用多种碳一原料进行发酵,包括甲醇、CO2和CO等。作为天然利用甲醇菌株,嗜甲基丁酸杆菌与好氧甲基营养菌有所不同,甲醇代谢途径还原力消耗较少,主要用于代谢产物的积累,同时菌株具有对甲醇的耐受性高,代谢甲醇速率较快等优势,其发酵主要产物是羧酸、丁酸和相应的醇类等,在生物制造业上有一定研究价值。
目前的研究主要集中在嗜甲基丁酸杆菌的发酵条件和培养基筛选等方面,并未涉及到相关基因工程改造等,限制菌株发展和应用。在嗜甲基丁酸杆菌中存在一条能够天然同化甲醇的代谢途径:WLP途径,但是其甲醇消耗速率以及丁酸合成效率仍然有进一步提高的空间,通过人工构建一条甲醇同化途径,还原性甘氨酸途径能够与WLP协同作用,进一步提高甲醇同化及丁酸合成效率,同时还原性甘氨酸途径还能固定CO2,符合绿色可持续发展理念。
发明内容
针对现有技术的不足,本发明的目的在于提供了一种利用WLP途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌及其应用,该构建首次提出了在嗜甲基丁酸杆菌中利用人工构建的甲醇同化途径还原性甘氨酸途径与天然甲醇同化途径WLP途径系统利用,提高了甲醇同化及产物丁酸合成效率,为未来嗜甲基丁酸杆菌生产化合物奠定了基础,具有深远意义。
一种利用WLP途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌,包括以下步骤:
步骤1,构建重组质粒:pXY3-thl2-rRGS和pXY3-thl2-glyA;
步骤2,构建重组质粒:pXY3-rRGS-thl2-glyA
步骤3,将重组质粒pXY3-rRGS-thl2-glyA甲基化修饰后转化至嗜甲基丁酸杆菌,即得重构嗜甲基丁酸杆菌BM/pXY3-rRGS-thl2-glyA。
作为改进的是,步骤1中重组质粒pXY3-thl2-rRGS构建方法为:选取引物,在实验室已有质粒: pTrc99a-rGCS上复制来源于大肠杆菌的氨基甲基转移酶、甘氨酸裂解系统H蛋白、甘氨酸脱羧酶利用BamHI/Xba I酶切位点与载体pXY3连接。
作为改进的是,步骤1中重组质粒pXY3-thl2-glyA构建方法为:将来源于嗜甲基丁酸杆菌中的丝氨酸羟甲基转移酶利用BamHI/Xba I酶切位点与载体pXY3连接。
作为改进的是,步骤2中重组质粒pXY3-rRGS-thl2-glyA构建方法为:选取引物,通过PCR以操纵子形式复制质粒pXY3-thl2-glyA中thl2启动子以及丝氨酸羟甲基转移酶,再利用XbaI /Xho I酶切位点与载体pXY3-thl2-rRGS连接。
进一步改进的是,步骤3中pXY3-rRGS-thl2-glyA甲基化修饰并转化的方法为:将质粒pXY3-rRGS-thl2-glyA转化至养含有pMCljs的大肠杆菌Top10中,培养并提取甲基化后的质粒pXY3-rRGS-thl2-glyA,随后通过电转的方式转化至嗜甲基丁酸杆菌中,获得重构的嗜甲基丁酸杆菌BM/pXY3-rRGS-thl2-glyA。
上述构嗜甲基丁酸杆菌在甲醇同化发酵生产丁酸上的应用。
作为改进的是,在平板上挑取BM/pXY3-rRGS-thl2-glyA的单菌落,接种至含有红霉素的2ml YTF培养基中,培养12-16h后,将离心管中的菌液全部转接至安剖瓶中,生长至OD600为1-1 .2,将菌液倒入50ml离心管中,4000rpm离心10min,弃去上清,用PB培养基重悬后以OD600=0 .1的接种量接种至50ml PB培养基中,再加入100mM~500mM甲醇、0~60mM碳酸氢钠 ,每隔一定时间,吸取2ml菌液,离心后将上清液转移至新的离心管保存,用于高效液相色谱检测甲醇、丁酸,用2ml超纯水重悬后检测其OD600。
进一步改进的是,添加100mM甲醇及20mM碳酸氢钠。
有益效果:
与现有技术相比,本发明一种利用WLP途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌及其应用,具体优势如下:
1、该方法在传统工业微生物主要以糖基类原料作为发酵底物的基础上,实现了以更为廉价的甲醇为底物,经济环保;
2、该方法首次提出在嗜甲基丁酸杆菌中利用天然和人工构建甲醇途径,实现了甲醇同化效率及最高生物量显著提高。
3、通过对嗜甲基丁酸杆菌的发酵底物浓度进行优化,进一步提高了甲醇同化及丁酸合成效率。
附图说明
图1为质粒pXY3-rRGS-thl2-glyA构建示意图;
图2为双途径共利用对菌株生长及甲醇同化的影响,其中,(a)为菌株生长曲线图,(b)甲醇消耗图;
图3为甲醇添加浓度对菌株BM/pXY3-rRGS-thl2-glyA的影响,其中,(a)为菌株生长曲线图,(b)甲醇消耗图,(c)丁酸合成图;
图4为碳酸氢钠添加浓度对菌株BM/pXY3-rRGS-thl2-glyA的影响,其中,(a)为菌株生长曲线图,(b)甲醇消耗图,(c)丁酸合成图;
图5为最优条件下,菌株BM/pXY3-rRGS-thl2-glyA与原始菌株BM的生长、甲醇同化、丁酸合成的情况,其中 (a)为菌株生长曲线图,(b)甲醇消耗图,(c)丁酸合成图。
具体实施方式
以下通过具体实施方式的描述对本发明作进一步说明,但这并非是对本发明的限制,本领域技术人员根据本发明的基本思想,可以做出各种修改或改进,但是只要不脱离本发明的基本思想,均在本发明的范围之内。
实施例中未提及到的技术均为本领域常规技术,另外,使用的嗜甲基丁酸杆菌购自ATCC菌种保藏中心、Trans1-T1、pXY3、Top10等材料都是商业化产品,可直接购买。
实施例1 pXY3-rRGS-thl2-glyA质粒的构建
1、pXY3-thl2-rRGS质粒的构建
以实验室现存质粒pTrc99a-rGCS(其构建方法见公开号为CN112920984 A)为模板,通过常规PCR以操纵子形式扩增大肠杆菌来源的氨基甲基转移酶(GcvT)、甘氨酸裂解系统H蛋白(GcvH)、甘氨酸脱羧酶(GcvP)编码序列。
上游引物带有BamHI酶切位点,序列如SEQ ID No .1所示:
CGCGGATCCATGGCACAACAGACTCCTTTG;
下游引物带有XbaI酶切位点,序列如SEQ ID No .2所示:
CGCGGATCCTTACTGGTATTCGCTAATCGGTACGC。
反应条件为:95℃ 2min ,95℃ 20s,55℃ 20s,72℃ 180s,共30个循环;72℃5min;得到的序列经1%琼脂糖凝胶电泳后回收相应片段;将该序列与表达载体pXY3用Takara 公司的BamH I和XbaI酶切,酶切反应体系为:10×buffer H 2μl,XbaI0.5μl,BamH I 0.5μl,基因片段及pXY3载体3μl,H2O 14μl。
酶切体系于37℃条件下反应2小时。将酶切产物连接,反应体系为:10×Ligasebuffer 1μl,T4 DNA Ligase(Takara)1μl,基因片段7μl,载体1μl。连接于25℃下反应3小时。将连接产物转化大肠杆菌Trans1-T1。PCR筛选阳性菌株Trans1-T1-pXY3-thl2-rRGS并进行DNA测序,验证重组质粒构建正确。
将阳性菌株接种至5ml LB/Amp 液体培养基,LB/Amp 液体培养基组成为蛋白胨10g/L、酵母粉5g/L、氯化钠5g/L,于37℃、200rpm 条件下振荡培养过夜。24小时后按照天根质粒提试剂盒操作说明提取质粒pXY3-thl2-rRGS。
2、pXY3-thl2-glyA质粒的构建
以嗜甲基丁酸杆菌全基因组为模板,通过常规PCR扩增丝氨酸羟甲基转移酶编码序列。
所用上游引物带有BamH I酶切位点,序列如SEQ ID No .3所示:
CGGGATCCatgaattttgaacacgtgaaaagag。
下游引物带有Xba I酶切位点,序列如SEQ ID No .4所示:
GCTCTAGAttattcgtataaaggatatttcttggtcagg。
反应条件为:95℃ 2min ,95℃ 20s,55℃ 20s,72℃ 35s,共30个循环;72℃5min;得到的序列经1%琼脂糖凝胶电泳后回收相应片段;将该序列与表达载体pXY3用Takara 公司的BamH I和Xba I酶切,酶切反应体系为:10×buffer H 2μl,Xba I 0.5μl,BamH I 0.5μl,基因片段及pXY3载体3μl,H2O 14μl。酶切体系于37℃条件下反应2小时。将酶切产物连接,反应体系为:10×Ligase buffer 1μl,T4 DNA Ligase(Takara)1μl,基因片段7μl,载体1μl。连接于25℃下反应3小时。将连接产物转化大肠杆菌Trans1-T1。PCR筛选阳性菌株Trans1-T1-pXY3-thl2-glyA并进行DNA测序,验证重组质粒构建正确。
将阳性菌株接种至5ml LB/Amp 液体培养基,LB/Amp 液体培养基组成为蛋白胨10g/L、酵母粉5g/L、氯化钠5g/L,于37℃、200rpm 条件下振荡培养过夜。24小时后按照天根质粒提试剂盒操作说明提取质粒pXY3-thl2-glyA。
3、pXY3-rRGS-thl2-glyA质粒的构建
以质粒为pXY3-thl2-glyA模板,通过常规PCR扩增出thl2启动子以及丝氨酸羟甲基转移酶编码序列。
所用上游引物带有XbaI酶切位点,序列如SEQ ID No .5所示:
GCTCTAGAtttttaacaaaatatattgataaaaataataatagtgg;
下游引物带有Xho I酶切位点,序列如SEQ ID No .6所示:
CCGCTCGAGttattcgtataaaggatatttcttggtcagg。
反应条件为:95℃ 2min ,95℃ 20s,55℃ 20s,72℃ 37s,共30个循环;72℃5min;得到的序列经1%琼脂糖凝胶电泳后回收相应片段;将该序列与表达载体pXY3-thl2-rRGS用Takara 公司的XbaI /Xho I酶切,酶切反应体系为:10×buffer H 2μl,Xba I 0.5μl,Xho I 0.5μl,基因片段及载体pXY3-thl2-rRGS3μl,H2O 14μl。酶切体系于37℃条件下反应2小时。将酶切产物连接,反应体系为:10×Ligase buffer 1μl,T4 DNA Ligase(Takara)1μl,基因片段7μl,载体1μl。连接于25℃下反应3小时。将连接产物转化大肠杆菌Trans1-T1。PCR筛选阳性菌株Trans1-T1-pXY3-rRGS-thl2-glyA并进行DNA测序,验证重组质粒构建正确。
将阳性菌株接种至5ml LB/Amp 液体培养基,LB/Amp 液体培养基组成为蛋白胨10g/L、酵母粉5g/L、氯化钠5g/L,于37℃、200rpm 条件下振荡培养过夜。24小时后按照天根质粒提试剂盒操作说明提取质粒pXY3-rRGS-thl2-glyA。其构建示意图如图1所示。
实施例2 BM/pXY3-rRGS-thl2-glyA菌株的构建及发酵表型
将质粒pMCljs热激转化进入大肠杆菌Top10中,得到重组菌株Top10/pMCljs ,将该重组菌株制备成感受态细胞,具体操作方法见专利公开号为:CN 113106047 A。
将重组质粒pXY3-rRGS-thl2-glyA转化至感受态Top10/pMCljs,得到重组菌株Top10/pMCljs/pXY3-rRGS-thl2-glyA。将重组菌株接种于5ml含有终浓度为50ug/mL氨苄抗性和100ug/mL盐酸壮双霉素的LB培养基中,37℃培养 12小时后提取质粒,即得到甲基化后质粒pXY3-rRGS-thl2-glyA,最后将甲基化的质粒pXY3-rRGS-thl2-glyA转化至嗜甲基丁酸杆菌中,即获得重构菌株:BM/pXY3-rRGS-thl2-glyA。
在平板上挑取BM/pXY3-rRGS-thl2-glyA的单菌落,接种至含有终浓度为30ug/mL红霉素的2ml YTF培养基中,培养12-16h后,将离心管中的菌液全部转接至安剖瓶中,生长至OD600为1-1.2,将菌液倒入50ml离心管中,4000rpm离心10min,弃去上清,用PB培养基重悬后以OD600=0.1的接种量接种至50ml PB培养基中,再加入100mM甲醇,每隔24 h,吸取2ml菌液,离心后将上清液转移至新的离心管保存. 甲醇含量测定:安捷伦高效液相色谱,色谱柱为Biorad HPX-87H,流动相为0.8 g/L的H2SO4溶液,柱温为60℃,进样体积为20 μL,流速为0.5 mL/min,检测器为示差检测器。丁酸含量测定,检测器为紫外检测器。最后用2ml超纯水重悬后检测其OD600。其发酵表型如图2所示,重构菌株较原始菌株相比,大生物量提高了55.26%,甲醇同化量提高了34.47%。
实施例3 BM/pXY3-rRGS-thl2-glyA菌株发酵条件优化
在平板上挑取BM/pXY3-rRGS-thl2-glyA的单菌落,接种至含有终浓度为30ug/mL红霉素的2ml YTF培养基中,培养12-16h后,将离心管中的菌液全部转接至安剖瓶中,生长至OD600为1-1 .2,将菌液倒入50ml离心管中,4000rpm离心10min,弃去上清,用PB培养基重悬后以OD600=0.1的接种量接种至50ml PB培养基中,再加入100mM-500mM甲醇(如图3所示,100mM甲醇的添加浓度其生长情况最佳,同时400mM甲醇时甲醇消耗最多,200mM甲醇时丁酸合成比最佳,但是差距微弱,因此100mM甲醇浓度为最佳浓度)、0-60mM碳酸氢钠 (如图4所示,在添加100mM甲醇+20mM碳酸氢钠时菌株的生长、甲醇消耗以及丁酸合成都是最佳的),每隔24 h,吸取2ml菌液,离心后将上清液转移至新的离心管保存。甲醇含量测定:安捷伦高效液相色谱,色谱柱为Biorad HPX-87H,流动相为0.8 g/L的H2SO4溶液,柱温为60℃,进样体积为20 μL,流速为0.5 mL/min,检测器为示差检测器。丁酸含量测定,检测器为紫外检测器。最后用2ml超纯水重悬后检测其OD600。
实施例4 BM/pXY3-rRGS-thl2-glyA菌株最佳条件下催化合成丁酸
在平板上挑取BM/pXY3-rRGS-thl2-glyA的单菌落,接种至含有终浓度为30ug/mL红霉素的2ml YTF培养基中,培养12-16h后,将离心管中的菌液全部转接至安剖瓶中,生长至OD600为1-1.2,将菌液倒入50ml离心管中,4000rpm离心10min,弃去上清,用PB培养基重悬后以OD600=0.1的接种量接种至50ml PB培养基中,再加入100mM甲醇+20mM碳酸氢钠,每隔24 h,吸取2ml菌液,离心后将上清液转移至新的离心管保存。甲醇含量测定:安捷伦高效液相色谱,色谱柱为Biorad HPX-87H,流动相为0.8 g/L的H2SO4溶液,柱温为60℃,进样体积为20 μL,流速为0.5 mL/min,检测器为示差检测器。丁酸含量测定,检测器为紫外检测器。最后用2ml超纯水重悬后检测其OD600。其催化合成情况如图5所示,其最终最大生物量提高了55.26%,甲醇同化量提高了57.14%,丁酸合成量提高了77.78%。
提高量计算公式为:(样品量-对照量)/对照量
综上所述,本发明首次提出通过人工构建甲醇同化途径与嗜甲基丁酸杆菌内源甲醇途径协同作用,提高了嗜甲基丁酸杆菌同化甲醇能力以及产品丁酸合成能力,为未来利用嗜甲基丁酸杆菌生产化合物奠定了基础,具有深远意义。
以上所述,仅为本发明较佳的具体实施方式,本发明的保护范围不限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
序列表
<110> 南京工业大学
<120> 一种利用WLP途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌及其应用
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<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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gctctagatt attcgtataa aggatatttc ttggtcagg 39
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Claims (8)
1.一种利用WLP途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌,其特征在于,包括以下步骤:
步骤1,构建重组质粒pXY3-thl2-rRGS和pXY3-thl2-glyA;
步骤2,构建重组质粒pXY3-rRGS-thl2-glyA
步骤3,将重组质粒pXY3-rRGS-thl2-glyA甲基化修饰后转化至嗜甲基丁酸杆菌,即得重构嗜甲基丁酸杆菌BM/pXY3-rRGS-thl2-glyA。
2.根据权利要求1所述的一种利用WLP途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌,其特征在于,步骤1中重组质粒pXY3-thl2-rRGS构建方法为:选取引物,在实验室已有质粒pTrc99a-rGCS上复制来源于大肠杆菌的氨基甲基转移酶、甘氨酸裂解系统H蛋白、甘氨酸脱羧酶利用BamHI/Xba I酶切位点与载体pXY3连接。
3.根据权利要求1所述的一种利用WLP途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌,其特征在于,步骤1中重组质粒pXY3-thl2-glyA构建方法为:将来源于嗜甲基丁酸杆菌中的丝氨酸羟甲基转移酶利用BamHI/Xba I酶切位点与载体pXY3连接。
4.根据权利要求1所述的一种利用WLP途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌,其特征在于,步骤2中重组质粒pXY3-rRGS-thl2-glyA构建方法为:选取引物,通过PCR以操纵子形式复制质粒pXY3-thl2-glyA中thl2启动子以及丝氨酸羟甲基转移酶,再利用XbaI /Xho I酶切位点与载体pXY3-thl2-rRGS连接。
5.根据权利要求1所述的一种利用WLP途径和还原性甘氨酸途径协同同化甲醇的重构嗜甲基丁酸杆菌,其特征在于,步骤3中pXY3-rRGS-thl2-glyA甲基化修饰并转化的方法为:将质粒pXY3-rRGS-thl2-glyA转化至养含有pMCljs的大肠杆菌Top10中,培养并提取甲基化后的质粒pXY3-rRGS-thl2-glyA,随后通过电转的方式转化至嗜甲基丁酸杆菌中,获得重构的嗜甲基丁酸杆菌BM/pXY3-rRGS-thl2-glyA。
6.基于权利要求1所述的重构嗜甲基丁酸杆菌在甲醇同化发酵生产丁酸上的应用。
7.根据权利要求6所述的应用,其特征在于,在平板上挑取BM/pXY3-rRGS-thl2-glyA的单菌落,接种至含有红霉素的2ml YTF培养基中,培养12-16h后,将离心管中的菌液全部转接至安剖瓶中,生长至OD600为1-1.2,将菌液倒入50ml离心管中,4000rpm离心10min,弃去上清,用PB培养基重悬后以OD600=0 .1的接种量接种至50ml PB培养基中,再加入100mM-500mM甲醇、0-60mM碳酸氢钠 ,每隔一定时间,吸取2ml菌液,离心后将上清液转移至新的离心管保存,用于高效液相色谱检测甲醇、丁酸,用2ml超纯水重悬后检测其OD600。
8.根据权利要求7所述的应用,其特征在于,添加100mM甲醇及20mM碳酸氢钠。
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