CN111304105A - 利用甲醇和木糖共底物产脂肪酶的基因工程菌及其应用 - Google Patents
利用甲醇和木糖共底物产脂肪酶的基因工程菌及其应用 Download PDFInfo
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Abstract
本发明涉及利用甲醇和木糖共底物产脂肪酶的基因工程菌及其应用,向宿主菌中导入甲醇氧化酶基因aox1、二羟基丙酮合酶基因das、过氧化氢酶基因cta和二羟基丙酮激酶基因dak;所述宿主菌为可利用木糖产脂肪酶的南极假丝酵母。本发明利用合成生物学的方法将甲醇代谢途径引入南极假丝酵母中,从而实现南极假丝酵母以非食品级原料甲醇和木糖作为共底物生产脂肪酶,在一定的程度上降低了生产成本,具有重大的意义和经济价值。
Description
技术领域
本发明属于生物工程领域,特别涉及一株利用甲醇和木糖共底物产脂肪酶的基因工程菌及其应用。
背景技术
微生物脂肪酶种类繁多,广泛存在于生物体内,因具有比动物脂肪酶更广的作用pH 、作用温度范围等优点,以及在酶理论研究和实际应用中的作用而得到广泛研究,脂肪酶可催化酯类化合物的分解、合成、酯交换等多种反应,具有高度的化学、区域和立体选择性,近几年,在有机合成、制药、去污剂、生物表面活性剂等领域得到广泛应用。
南极假丝酵母脂肪酶是一种重要的脂肪酶,具有许多优良的特性,由于它是一种新型的非特异性酶,它无论在溶液中还是以固定化形式存在时均具有很强的稳定性,在进行热处理时酶活最初下降很快,但在一段时间后酶活力便稳定下来,即使进行进一步的热处理也不会有明显变化,其在水解反应和有机合成反应中具有很高的立体选择性,在糖脂的合成中也具有潜在的应用价值。目前,生产南极假丝酵母脂肪酶的培养基成本较高,使南极假丝酵母脂肪酶生产成本较高,因此,若能以廉价的还原性的底物为原料,可以在一定程度上降低成本。
甲醇是煤化工产业中的重要产品,近年来随着甲醇工艺的发展,使得甲醇的价格持续走低,因而利用甲醇作为发酵原料成为生物转化过程降低成本的重要突破口。因此,若能通过合成生物学的手段,将甲醇代谢模块引入南极假丝酵母中,以甲醇和木糖为共底物生产脂肪酶,为利用廉价碳资源生产脂肪酶的研究奠定了基础。
发明内容
本发明的目的在于提供一种利用合成生物学的方法构建可以利用甲醇和木糖共底物进行代谢的菌株,并利用该菌株发酵产脂肪酶,解决了传统产脂肪酶成本高的问题。
为解决上述问题,本发明采用如下方案:
一株利用甲醇和木糖共底物产脂肪酶的基因工程菌,通过向宿主菌中导入甲醇氧化酶基因aox1、二羟基丙酮合酶基因das、过氧化氢酶基因cta和二羟基丙酮激酶基因dak;所述宿主菌为可利用木糖产脂肪酶的南极假丝酵母。
其中甲醇氧化酶基因将甲醇氧化成甲醛,消耗大量氧气伴随生产过氧化氢;过氧化氢在过氧化氢酶cta的催化下转化成氧气和水;甲醛和木酮糖-5磷酸在二羟基丙酮合酶das的催化下转化成二羟基丙酮,同时外源添加木糖为其提供木酮糖-5磷酸加速二羟丙酮的产生;随后二羟基丙酮在二羟基丙酮激酶dak的催化下转化成磷酸二羟丙酮,然后进一步代谢成果糖-6磷酸,进而进入糖酵解途径参与物质循环和有机酸代谢,如图1。南极假丝酵母可以利用木糖作为碳源,木糖代谢的木酮糖-5磷酸可作为甲醇代谢的前体,因此木糖可以加速甲醇的代谢从而提高构建菌株脂肪酶的相关酶活。
其中所述宿主菌为南极假丝酵母(Candida antarctica)ZJB09193,保藏编号为CCTCC M 2010263,该菌株已在文献Liu et.al. Cloning, expression andcharacterization of a lipase gene from the Candida antarctica ZJB09193 andits application in biosynthesis of vitamin A esters. MicrobiologicalResearch. Volume 167, Issue 8, 6 September 2012, Pages 452-460中公开。本申请人在此声明,保证从本申请日起20年内免费对公众发放本菌株的生物材料。
其中所述甲醇氧化酶基因aox的GenBank登记号为XM-002494226.1;
所述二羟丙酮合酶基因das的GenBank登记号为FJ752551.1;
所述过氧化氢酶基因cta的GenBank登记号为AB472085.1;
所述二羟丙酮激酶基因dak的GenBank登记号为XM-002493026.1。
其中所述甲醇氧化酶基因aox、二羟丙酮合酶基因das、过氧化氢酶基因cta、二羟丙酮激酶基因dak的启动子序列选自TEF、PGPD、PDC1P中的一种。
甲醇氧化酶基因aox、二羟丙酮合酶基因das、过氧化氢酶基因cta、二羟丙酮激酶基因dak的终止子选自CYC1t、TDH2t中的一种。
上述利用甲醇生产脂肪酶的基因工程菌的构建方法如下:
(1)构建TEF-aox1-CYC1t、TEF-das-tCYC1、PDC1p-cta-TDH2t、pGPD-dak-TXPR2表达框,通过多片段克隆的方法,将TEF-das-tCYC1、pGPD-dak-TXPR2两个基因片段与113质粒连接,将TEF-aox1-CYC1t、PDC1p-cta-TDH2t两个基因片段与Pki质粒连接,转化至E.coli DH5α中;
(2)将测序正确的质粒通过酶切得到基因重组片段,将基因重组片段电转化至宿主菌,再通过G418抗性筛选得到所述基因工程菌。
本发明的另一目的在于提供上述基因工程菌在发酵产脂肪酶中的应用。
本发明提供了一种具体的应用方法,如下:
(1a)试管种子培养:将基因工程菌接种至试管的种子培养基中培养;
(2a)摇瓶种子培养:将试管种子培养液接种至摇瓶的种子培养基中培养;
(3a)发酵产脂肪酶:将摇瓶种子培养液接种到发酵培养基中发酵培养,获取脂肪酶。
作为本发明的进一步改进,所述试管种子培养和摇瓶种子培养的培养温度为24℃,培养时间18-22h。
其中,所述种子培养基配方如下:蛋白胨 6 g/L,水解酪蛋白4 g/L,酵母粉3 g/L,牛肉膏1.5 g/L,葡萄糖1 g/L;
其中,所述发酵培养基配方如下:大豆粉40 g/L,豆油5 ml/L,磷酸氢二钾4 g/L,硫酸镁1 g/L,吐温80ml/L,酵母粉5 g/L,木糖15 g/L,甲醇0~15 g/L;优选甲醇浓度7 g/L。
进一步的,将摇瓶种子培养液按照10%的接种量接种到发酵培养基中发酵培养;培养温度24℃。
本发明利用合成生物学的方法将甲醇代谢途径引入南极假丝酵母中,从而实现南极假丝酵母以非食品级原料甲醇和木糖作为共底物碳源生产脂肪酶,在一定的程度上降低了生产成本,具有重大的意义和经济价值。
附图说明
图1是基因工程菌代谢图。
图2是原始质粒113和pki。
图3是重组质粒113-DAS-DAK,Pki-AOX-CTA构建图。
图4是基因组PCR验证。
图5是添加不同浓度的甲醇,脂肪酶酶活的变化。
图6是不同浓度的甲醇对基因表达水平的影响。
具体实施方式
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径中获得。
实施例中,脂肪酶酶活检测方法如下:以对硝基苯乙酸酯(p-nitrophenylacetate,p-NPA)为底物,利用多功能酶标仪测定脂肪酶的活性。反应体系如下:900 uL 20mMTris-HCl缓冲液,50 uL 10 mM p-NPA乙腈溶液,50 uL酶液,30℃反应2 min (此时处于反应初速度时间范围内)。测定反应液在405 nm处的吸光值变化。酶活单位(U):在30℃,pH8.0条件下,1min内催化生成1umol对硝基苯酚所需要的酶量定义为1 U。
实施例1:获取表达基因
以毕赤酵母Pichia pastoris基因组为模板,设计引物扩增甲醇氧化酶aox1、二羟丙酮合酶基因das、过氧化氢酶基因cta、二羟丙酮激酶基因dak。
实施例2:利用合成生物学的方法构建Candida-aox1-das-cta-dak
为了能够快速有效的实现多基因组的共同表达并保证基因表达的稳定性,利用DNA集合的方法将基因表达框整合进南极假丝酵母基因组中。
(1)设计引物进行扩增,分别在每个基因两端加上启动子、终止子的同源臂,设计上下游引物,获得表达框的,基因与引物序列见表1。
表1 基因与引物序列
(2)进行多片段克隆,组成启动子-基因-终止子的表达框TEF-aox1-CYC1t、TEF-das-tCYC1、PDC1p-cta-TDH2t、pGPD-dak-TXPR2;上述启动子-基因-终止子的表达框就是将所示的基因使用多片段克隆的方法组合连接组成表达片段。
(3)将das、dak两个基因片段与113质粒通过多片段克隆的方法连接,转化至E.coli DH5α中,将aox1、cta两个基因片段与Pki质粒通过多片段克隆的方法连接,转化至E.coli DH5α中,质粒。如图3所示,经质粒酶切和菌落PCR验证,将验证正确的质粒送至测序公司进行测序。
(4)将测序正确的质粒,通过NotI酶切,将有目的基因的片段通过试剂盒胶回收后将该基因重组片段电转化进入南极假丝酵母中,具体为:
南极假丝酵母感受态制备方法:
(1)将出发菌株接种于5 mL的种子培养基中,24℃,12 h,转500 µL培养物于50 mL 种子培养基中,24℃培养至菌体浓度OD600=0.8~1.0。
(2)冰浴15 min,停止细胞生长,菌液移至50 mL离心管中,4000 rpm、5 min,去上清。
(3)使用30 mL预冷的灭菌水重悬细胞,离心4000 rpm、5 min,去除上清。
(4)使用20 mL预冷的1 M山梨醇洗涤重悬细胞沉淀,4000 rpm、5 min,去除上清,重复2遍。
(5)使用200 µL~250 µL 1 M山梨醇重悬细胞,并转移至预冷的离心管中,即为酵母感受态。
电转化:
(1)取10 µL重组质粒和40 µL感受态加入到预冷的1.5 mL的离心管中。
(2)将目的片段和感受态在离心管中轻轻吹打混合后,转移至电转杯中,在冰中预冷5 min。
(3)将电转杯***的水用吸水纸擦干净,使用1500 V电压点击。
(4)往点击后的电转杯中加入1 mL 种子培养基,轻轻悬浮细胞移至1.5 mL 离心管中,24℃培养箱复苏2 h。
复苏后的细胞用灭菌水清洗两遍,涂布在G418抗性筛选培养基上,1 g/L G418筛选得到重组南极假丝酵母,进行PCR验证如图4所示。以实现基因的同时表达,进而实现甲醇的代谢。
实施例3:重组菌株的发酵实验
(1)试管种子培养:将重组南极假丝酵母按1%(v/v)接种量从冻存管接种到试管种子培养基中,试管装液量5 mL,24℃有氧培养18~22 h,获得试管种子培养液。
其中,所述种子培养基配方如下:蛋白胨 6 g/L,水解酪蛋白4 g/L,酵母粉3 g/L,牛肉膏1.5 g/L,葡萄糖1 g/L。
(2)摇瓶种子培养:将试管种子培养液按1%(v/v)接种量从试管培养基接种到摇瓶种子培养基中,250 mL 三角瓶装液量50 mL,24℃有氧培养18~22h,获得摇瓶种子培养液。
(3)发酵产脂肪酶:将摇瓶种子培养液按10%(v/v)接种量从摇瓶培养基接种到发酵培养基中,250 mL 三角瓶装液量50 mL,24℃有氧培养24h,提取RNA,通过荧光定量PCR,测其在发酵10h的甲醇代谢相关基因表达水平以及南极假丝酵母木糖表达的相关基因表达水平。
其中,所述发酵培养基配方如下:大豆粉40 g/L,豆油5 ml/L,磷酸氢二钾4 g/L,硫酸镁1 g/L,吐温80ml/L,酵母粉5 g/L,木糖15 g/L,甲醇0、5 g/L、7 g/L、15 g/L。
结果如图5所示,在甲醇浓度为7g/L时,脂肪酶的酶活最高。7g/L时的脂肪酶A的基因表达水平相较于其他甲醇浓度的结果如图6所示,得出结论,适当的甲醇会提高脂肪酶酶活,das的表达水平提高表明,添加的15 g/L的木糖为甲醇的代谢提供了木酮糖-5磷酸。7g/L的甲醇的添加会提高脂肪酶A的基因表达,同时木糖代谢的相关基因的表达也有所提高,因此甲醇的代谢模块引入促进了木糖的利用,并对产脂肪酶的相关基因的表达有提高。
序列表
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Claims (10)
1.一株利用甲醇和木糖共底物产脂肪酶的基因工程菌,其特征在于,向宿主菌中导入甲醇氧化酶基因aox1、二羟基丙酮合酶基因das、过氧化氢酶基因cta和二羟基丙酮激酶基因dak;所述宿主菌为可利用木糖产脂肪酶的南极假丝酵母。
2.根据权利要求1所述的基因工程菌,其特征在于,所述宿主菌为南极假丝酵母Candida antarctica)ZJB09193。
3.根据权利要求1所述的基因工程菌,其特征在于,构建方法如下:
(1)构建TEF-aox1-CYC1t、TEF-das-tCYC1、PDC1p-cta-TDH2t、pGPD-dak-TXPR2表达框,通过多片段克隆的方法,将TEF-das-tCYC1、pGPD-dak-TXPR2两个基因片段与113质粒连接,将TEF-aox1-CYC1t、PDC1p-cta-TDH2t两个基因片段与Pki质粒连接,转化至E.coli DH5α中;
(2)将测序正确的质粒通过酶切得到基因重组片段,将基因重组片段电转化至宿主菌,再通过G418抗性筛选得到所述基因工程菌。
4.权利要求1~3任一项所述基因工程菌在发酵产脂肪酶中的应用。
5.根据权利要求4所述的应用,其特征在于,包括如下步骤:
(1a)试管种子培养:将基因工程菌接种至试管的种子培养基中培养;
(2a)摇瓶种子培养:将试管种子培养液接种至摇瓶的种子培养基中培养;
(3a)发酵产脂肪酶:将摇瓶种子培养液接种到发酵培养基中发酵培养,获取脂肪酶。
6.根据权利要求5所述的应用,其特征在于,所述试管种子培养和摇瓶种子培养的培养温度为24℃;培养时间18-22h。
7.根据权利要求5所述的应用,其特征在于,所述种子培养基配方如下:蛋白胨 6 g/L,水解酪蛋白4 g/L,酵母粉3 g/L,牛肉膏1.5 g/L,葡萄糖1 g/L。
8.根据权利要求5所述的应用,其特征在于,所述发酵培养基配方如下:大豆粉40 g/L,豆油5 ml/L,磷酸氢二钾4 g/L,硫酸镁1 g/L,吐温80ml/L,酵母粉5 g/L,木糖15 g/L,甲醇0~15 g/L。
9.根据权利要求8所述的应用,其特征在于,甲醇浓度为7 g/L。
10.根据权利要求5所述的应用,其特征在于,将摇瓶种子培养液按照10%的接种量接种到发酵培养基中发酵培养。
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