CN116376995A - 一种利用苏氨酸制备甘氨酸、乙酰辅酶a及乙酰辅酶a衍生物的方法 - Google Patents
一种利用苏氨酸制备甘氨酸、乙酰辅酶a及乙酰辅酶a衍生物的方法 Download PDFInfo
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- CN116376995A CN116376995A CN202210352016.3A CN202210352016A CN116376995A CN 116376995 A CN116376995 A CN 116376995A CN 202210352016 A CN202210352016 A CN 202210352016A CN 116376995 A CN116376995 A CN 116376995A
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Abstract
本发明公开了一种利用苏氨酸制备甘氨酸的方法,发酵过程中,醛缩酶将苏氨酸分解为甘氨酸和乙醛。本发明还公开了一种同时制备甘氨酸和乙酰辅酶A的方法,发酵过程中,乙酰化乙醛脱氢酶将乙醛还原为乙酰辅酶A或乙酰辅酶A衍生物;或苏氨酸脱氢酶将苏氨酸脱氢得到2‑氨‑3酮丁酸,再经2‑氨‑3酮丁酸CoA连接酶连接得到乙酰辅酶A,在发酵条件的不同的情况下,乙酰辅酶A衍生物能转化为乙酰辅酶A衍生物。本发明构思独特,能制备得到甘氨酸、乙酰辅酶A及乙酰辅酶A衍生物,转化效率高,耗时较短且产量高,产物丰富,易于实现工业化加工和生产。
Description
技术领域
本发明涉及基因工程和发酵工程技术领域,具体涉及一种利用苏氨酸制备甘氨酸、乙酰辅酶A及乙酰辅酶A衍生物的方法。
背景技术
乙酰辅酶A(Acetyl-coA)由辅酶A(CoA)和乙酸以硫酯键连接而成,是细胞代谢的重要中间代谢产物之一,参与多种细胞生命过程。Acetyl-coA由糖、脂肪、氨基酸等分解代谢产生,最终通过三羧酸循环和氧化磷酸化,彻底氧化生成二氧化碳和水,释放能量用以ATP的合成。此外,它还可以作为许多重要生物大分子的前体,包括脂肪酸、1-丁醇、聚羟基烷酸、聚酮酸和异戊二烯类等,其中脂肪酸可用于膳食补充剂、药物和生物柴油等,聚酮酸可用于制药,异戊二烯类可用于制备香精、香料、生物燃料、膳食补充剂、维生素和药物等。然而,这些重要的生物大分子的有效生物合成往往受到Acetyl-coA供应不足的阻碍,因此如何提高Acetyl-coA的产量保证供应极为重要。
在有氧条件下,糖酵解产生丙酮酸经脱羧是合成Acetyl-coA的一个主要途径;此外外源脂肪酸通过β-氧化不断缩短碳链,最终形成Acetyl-coA;或者某些氨基酸代谢也是提供Acetyl-coA的途径之一。在大肠杆菌等微生物中,提高Acetyl-coA产量最直接方法之一是通过过表达Acetyl-coA合成酶和敲除竞争路径实现,利用多种基因的过表达以及代谢路径的优化,可以合成Acetyl-coA的多种衍生物如萜类、1-丁醇、聚羟基烷酸等。Kang Zhou等在大肠杆菌中利用Dickeya zeae的乙酰化乙醛脱氢酶(ada)、Saccharomycescerevisiae的乙醇脱氢酶(adh2)在不消耗ATP的情况下将乙醇转化为Acetyl-coA,并以此为平台合成PHB,产量为1.1g/L。Javier Cardenas等通过阻断戊糖磷酸途径,并修饰大肠杆菌中的丙酮酸脱氢酶加强Acetyl-coA以及NADPH/NADP+的合成,并以此提高了聚酮化合物的产量,达到1.6g/L。Menghao Cai等利用短而强的乙醇同化途径,在Crabtree-negativeyeast直接产生乙酰辅酶A,该方法可用于合成Acetyl-coA的衍生药物莫纳克林,产率达2g/L。然而,目前来说,通过微生物代谢工程提高细菌内Acetyl-coA的方法相对单一,而且转化效率普遍较低,耗时较长且产量不高,无法真正从工业量级上克服由于Acetyl-coA供应不足导致的许多重要大分子合成产量受限的问题,亟待改进。
甘氨酸是化工、医药、农药等行业的重要原料,产量大、用途广。目前生产甘氨酸的方法大都采用α-氯乙酸或Strecker法制备甘氨酸。专利CN101270061以氯乙酸、氨为原料,在乌洛托品和有机胺存在下也可以分离出含量较高的甘氨酸,但为了回收利用母液中的有效成分需要消耗大量的醇钠或醇钾,经济价值低,同时过量的醇需要回收将导致能耗的升高和投资的增加,并且产生了盐泥,后续处理较为麻烦。因此,本发明旨在开发一种联产甘氨酸和Acetyl-coA或Acetyl-coA衍生物的方法,以更好地满足实际需要。
发明内容
本发明所解决的技术问题在于提供一种利用苏氨酸制备甘氨酸、乙酰辅酶A及乙酰辅酶A衍生物的方法,以提高甘氨酸和乙酰辅酶A或乙酰辅酶A衍生物的产量及转化率,更适于工业化生产。
本发明所解决的技术问题采用以下技术方案来实现:
一种利用苏氨酸制备甘氨酸的方法,以苏氨酸为底物、加入含有醛缩酶基因的重组微生物进行发酵,发酵过程中,苏氨酸分解为甘氨酸和乙醛。
进一步地,所述醛缩酶基因为苏氨酸缩醛酶基因或苯丝氨酸醛缩酶基因,包括ItaE、TdcB、glyA、Sdsl、bhcC、psald、pdxA或vrtJ中的一种或几种。优选为上述酶基因种类,本发明亦可采用其它具有同样功能的酶基因。
进一步地,包括通过基因工程方法构建所述重组微生物,所述基因工程方法包括质粒表达或基因组整合。
进一步地,重组微生物通过质粒表达的方法进行构建,构建方法为:通过PCR扩增获得醛缩酶编码基因,将获得的基因连接至质粒载体上并转化至感受态细胞中,测序后获得重组表达质粒载体;将重组表达质粒载体转化至微生物中即得到重组微生物。
进一步地,所述质粒为pZAlac、pZElac中的一种或两种,感受态细胞为E.colidh5a感受态细胞。
进一步地,重组表达质粒载体为pZE-ItaE,质粒pZE-ItaE构建方法为:以恶臭假单胞菌(Pseudomonas putida)的基因组为模板通过PCR扩增得ItaE基因,将ItaE基因连接至含有IPTG诱导型启动子的pZElac载体上并转化至大肠杆菌E.coli dh5a感受态细胞中,测序后得到质粒pZE-ItaE。
进一步地,发酵过程中,将重组微生物的菌种接入含有氨苄青霉素和氯霉素的2-xyT培养基中,培养3-6h后加入IPTG至终浓度0.2~0.4mM,继续培养15~30h,离心得到菌液;将菌液加入Tris-HCl缓冲液中,以15~30g/L的量加入苏氨酸作为底物,发酵得到含甘氨酸和乙醛的发酵液。
一种利用苏氨酸制备甘氨酸、乙酰辅酶A的方法,以苏氨酸为底物,加入含有醛缩酶基因和乙酰化乙醛脱氢酶基因的重组微生物进行发酵,发酵过程中,苏氨酸分解为甘氨酸和乙醛,乙醛直接转化为乙酰辅酶A或乙醛转化为乙酸再转化成乙酰辅酶A;
或以苏氨酸为底物,加入含有苏氨酸脱氢酶基因和2-氨-3酮丁酸CoA连接酶基因的重组微生物进行发酵,发酵过程中,苏氨酸脱氢得到2-氨-3酮丁酸,2-氨-3酮丁酸在2-氨-3酮丁酸CoA连接酶的作用下得到乙酰辅酶A。
进一步地,所述醛缩酶基因包括ItaE、TdcB、glyA、Sdsl、bhcC、psald、pdxA或vrtJ中的一种或几种;所述乙酰化乙醛脱氢酶基因包括eutE、bphJ、TTHB247、mhpF、ADH2或hsaG中的一种或几种;所述苏氨酸脱氢酶基因包括tdh、ydfG、pdxA、asd、AKHSDH2或trmG中的一种或几种;所述2-氨-3酮丁酸CoA连接酶基因包括kbI、GCAT、Gcat或TTHA1582中的一种或几种。优选为上述酶基因种类,本发明亦可采用其它具有同样功能的酶基因。
进一步地,包括通过基因工程方法构建重组微生物,所述基因工程方法包括质粒表达或基因组整合。
进一步地,重组微生物通过质粒表达的方法进行构建,构建方法为:通过PCR扩增获得所述醛缩酶编码基因、乙酰化乙醛脱氢酶编码基因,或通过PCR扩增获得苏氨酸脱氢酶编码基因、2-氨-3酮丁酸CoA连接酶编码基因,将获得的基因连接至质粒载体上并转化至感受态细胞中,测序后获得重组表达质粒载体;将重组表达质粒载体转化至微生物中即得到重组微生物。
进一步地,所述质粒为pZAlac、pZElac中的一种或两种,感受态细胞为E.colidh5a感受态细胞。
进一步地,所述重组表达质粒载体为pZE-ItaE_eutE,pZE-ItaE_eutE的构建方法为:以大肠杆菌MG1655基因组为模板通过PCR扩增得eutE基因,以恶臭假单胞菌(Pseudomonas putida)的基因组为模板通过PCR扩增得ItaE基因,将eutE基因及ItaE基因连接至含有IPTG诱导型启动子的pZElac载体上并转化至大肠杆菌E.coli dh5a感受态细胞中,测序后得到质粒pZE-ItaE_eutE。
进一步地,所述重组表达质粒载体为pZE-tdH_kbI,pZE-tdH_kbI构建方法为:以大肠杆菌MG1655基因组为模板通过PCR扩增得tdH、_kbI基因,将tdH、_kbI基因连接至含有IPTG诱导型启动子的pZElac载体上并转化至大肠杆菌E.coli dh5a感受态细胞中,测序后得到质粒pZE-tdH_kbI。
进一步地,发酵时,将所述重组微生物接种至含有氨苄青霉素和氯霉素、不含抗生素的2-xyT培养基中,以15~25g/L的量加入苏氨酸作为底物,在温度为30~35℃的条件下培养3~6h后,加入IPTG至终浓度0.2~0.4mM,继续培养10~12h,培养结束后将离心,保留上清液,得到甘氨酸和乙酰辅酶A。
进一步地,所述微生物选自大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌中的一种或几种。
进一步地,所述微生物选自大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、巨大芽孢杆菌(Bacillus megaterium)、解淀粉芽孢杆菌(Bacillusamyloliquefaciens)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Candida utilis)或毕赤酵母(Pichiapastoris)中的一种或几种。
一种利用苏氨酸制备甘氨酸和乙酰辅酶A衍生物的方法,采用如上所述的方法制备甘氨酸和乙酰辅酶A,乙酰辅酶A转化为乙酰辅酶A衍生物。
进一步地,所述乙酰辅酶A衍生物包括甲羟戊酸、唾液酸,N-乙酰化氨基葡萄糖,3-羟丁酸,脂肪酸中的一种或几种。产物优选为上述几种,本发明亦可产生其它乙酰辅酶A衍生物。
进一步地,所述乙酰辅酶A衍生物为甲羟戊酸,发酵时,将重组微生物接种至摇瓶中,摇瓶中含有氨苄青霉素且不含葡萄糖的M9培养基,以15~25g/L的量加入苏氨酸作为底物,于30~35℃、转速200~300rpm的条件下培养3~5h,然后加入IPTG至终浓度0.2~0.4mM,继续培养发酵20~30h,收集发酵液即可。
进一步地,所述乙酰辅酶A衍生物为甲羟戊酸,发酵时,将所述重组微生物扩大培养后接种入发酵罐中,发酵罐中为含有氨苄青霉素且不含葡萄糖的M9培养基,发酵培养6~8h后,加入IPTG至终浓度0.1~0.2mM,以100~150g/L的量加入苏氨酸作为底物,在30~35℃下于继续发酵培养15~20h,收集发酵液即可。
进一步地,所述微生物的乙醇脱氢酶表达活性被减弱或消除,所述乙醇脱氢酶的编码基因为yqhD、qbdA、adhE或mdH的一种或几种。
根据实际需要可通过碳酸钙、氢氧化钙、碳酸钠、碳酸氢钠、氢氧化钠、碳酸钾或氢氧化钾调节pH。
本发明的部分构思路径如下:
有益效果:本发明所述的利用苏氨酸制备甘氨酸和乙酰辅酶A的方法,能单独得到甘氨酸,或能同时得到甘氨酸、乙酰辅酶A或同时得到甘氨酸和乙酰辅酶A衍生物,转化效率高,耗时较短且产量高,产物丰富,经济价值高,易于实现工业化加工和生产。
本发明所述的利用苏氨酸制备甘氨酸的方法,其发酵液中产生的甘氨酸浓度高达11.2g/L,产量高,副产物少,经济价值高,后续处理成本低。
本发明所述的利用苏氨酸制备甘氨酸、乙酰辅酶A的方法,其制备得到的发酵液中乙酰辅酶A浓度高达12~14ng/L,远远高于野生型大肠杆菌直接发酵得到的产量。
本发明所述的利用苏氨酸制备甘氨酸和乙酰辅酶A衍生物的方法,通过摇瓶发酵制备得到的发酵液中,甘氨酸浓度高达11g/L,作为乙酰辅酶A衍生物之一的甲羟戊酸,浓度高达3.8g/L,通过发酵罐发酵得到的发酵液中甘氨酸浓度高于60g/L,作为乙酰辅酶A衍生物之一的甲羟戊酸,浓度高于30g/L。
附图说明
图1为实施例2中乙酰辅酶A的检测结果示意图。
图2为实施例4中不同时期的发酵液中甘氨酸和甲羟戊酸的浓度检测结果示意图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施例进一步阐述本发明。
在本发明中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本文中所用的核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时,为了更好地理解本公开,下面提供相关术语的定义和解释。
也应理解本文使用的术语仅是为了描述具体实施方式的目的,并不意欲是限制性的。
本文中使用冠词“一”和“所述”来指代冠词的语法宾语中的一个或多于一个。
替代方案(例如,“或”)的使用应当被理解为意指替代方案中的一个、两个或其任何组合。术语“和/或”应当被理解为意指替代方案中的一个或两个。
如本文所用,术语“基因合成”,指利用重组DNA技术产生或利用本领域可用和公知的合成DNA或氨基酸序列技术获得。
“编码”指的是多核苷酸诸如基因、cDNA或mRNA中核苷酸的特异性序列用作模板合成在生物学过程中的其他多聚体和大分子的固有性质,所述多聚体和大分子具有核苷酸(即,rRNA、tRNA和mRNA)的限定序列或氨基酸的限定序列中的任一个和由其产生的生物学性质。因此,如果相应于那个基因的mRNA的转录和翻译在细胞或其他生物学系统中产生蛋白质,则基因编码蛋白质。核苷酸序列等同mRNA序列并通常提供在序列表中的编码链,和用作转录基因或cDNA的模板的非编码链两者,都可被称为编码那个基因或cDNA的蛋白质或其他产物。
如本文所用,术语“内源的”指的是来自有机体、细胞、组织或系统的或在有机体、细胞、组织或系统内产生的任何物质。
如本文所用,术语“外源的”指的是任何从有机体、细胞、组织或系统引入的或在有机体、细胞、组织或系统外产生的物质。
如本文所用,术语“表达”被定义为由它的启动子驱动的特定核苷酸序列的转录和/或翻译。
除非另有规定,“编码氨基酸序列的多核苷酸序列”包括为彼此简并版本并编码相同的氨基酸序列的所有的核苷酸序列。短语编码蛋白质或RNA的核苷酸序列也可包括内含子,其程度为编码该蛋白质的核苷酸序列可在某些版本中包含内含子(一个或多个)。
如本文所用的,术语“载体”为物质组合物,其包括分离的核酸,并且其可用于传递分离的核酸至细胞内部。转移的核酸通常连接到例如插入到载体核酸分子中。载体可以包含引导细胞中的自主复制的序列或可以包含足以允许整合到宿主细胞DNA中的序列。很多载体在本领域中是已知的,包含但不限于质粒、噬菌粒、人工染色体、细菌噬菌体以及动物病毒。因此,术语“载体”包括自主复制的质粒或病毒。
所述受体菌株可以是大肠杆菌(Escherichia coli)、谷氨酸棒杆菌(Corynebacteriumglutamicum)、枯草芽孢杆菌(Bacillus subtilis)、巨大芽孢杆菌(Bacillus megaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、需钠弧菌(Vibrio natriegens)或酿酒酵母(Saccharomyces cerevisiae)等。
实施例用到的重组表达质粒载体为pZE-ItaE,构建方法为:
以恶臭假单胞菌(Pseudomonas putida)的基因组为模板通过PCR扩增得ItaE基因:
ItaE-F(GAATTCATTAAAGAGGAGAAAGGTACCATGACAGACAAGAGCCAACAATTCG CCAGCG)/ItaE-R(CTTTCGTTTTATTTGATGCCTCTAGATCAGCCACCAATGATCGTGCG GATA)
通过非连接酶依赖型单片段快速克隆试剂盒将其连接到含有IPTG诱导型启动子的pZElac载体上,用标准方法将pZElac载体先热转化到E.coli dh5a感受态细胞中,涂布氨苄抗性平板过夜培养,挑阳性克隆进行测序验证,测序正确的重组质粒载体命名为质粒pZE-ItaE。
实施例用到的重组表达质粒载体为pZE-ItaE_eutE,构建方法为:
根据NCBI公布的恶臭假单胞菌(Pseudomonas putida)的基因组序列设计引物:
ItaE-F’(GAATTCATTAAAGAGGAGAAAGGTACCATGACAGACAAGAGCCAACAATTCGCCAGCG)/ItaE-R’(GATTCATACTAGTAGTTAATTTCTCCTTCAGCCACCAATGATCGTGCGGATATCCGC);
根据NCBI公布的大肠杆菌MG1655的基因组序列设计引物:
eutE-F(ATTGGTGGCTGAAGGAGAAATTAACTACTAGTATGAATCAACAGGATATTGAAC)/eutE-R(TTTCGTTTTATTTGATGCCTCTAGATTAAACAATGCGAAACGCATCGACTAATAC);
以恶臭假单胞菌(Pseudomonas putida)的基因组为模板通过PCR扩增得ItaE基因,以大肠杆菌MG1655基因组为模板通过PCR扩增得eutE基因,并通过非连接酶依赖型单片段快速克隆试剂盒将其连接到含有IPTG诱导型启动子的pZElac载体上,用标准方法将pZElac载体先热转化到E.coli dh5a感受态细胞中,涂布氨苄青霉素抗性平板过夜培养,挑阳性克隆进行测序验证,测序正确的重组质粒载体命名为pZE-ItaE_eutE。其中E.colidh5a感受态细胞经过基因编辑,可以充分地表达质粒,适合进行质粒测序和质粒扩增。
实施例用到的重组表达质粒载体为pZE-tdH_kbI,构建方法为:
根据NCBI公布的大肠杆菌MG1655的基因组序列设计引物:
tdH-F(GAATTCATTAAAGAGGAGAAAGGTACCATGAAAGCGTTATCCAAACTGAA AG)/tdH-R(TCTCCACGCATAGTTAATTTCTCCTTTAATCCCAGCTCAGAATAACTTTC);
kbI-F(GAAAGTTATTCTGAGCTGGGATTAAAGGAGAAATTAACTATGCGTGGAGA)/kbI-R(TTTCGTTTTATTTGATGCCTCTAGATCAGGCGATAACGCCCAGTTGTTTA);
以大肠杆菌MG1655基因组为模板通过PCR扩增得tdH、kbI基因,并通过非连接酶依赖型单片段快速克隆试剂盒将其连接到含有IPTG诱导型启动子的pZElac载体上,用标准方法将pZElac载体先热转化到E.coli dh5a感受态细胞中,涂布氨苄青霉素抗性平板过夜培养,挑阳性克隆进行测序验证,测序正确的重组质粒载体分别命名为pZE-tdH_kbI。
实施例1
将重组表达质粒载体pZE-ItaE转化至表达菌株大肠杆菌Escherichia coli BL21中,将菌种接入含有氨苄青霉素和氯霉素的2-xyT培养基中,在30℃下于50ml三角瓶中(装液量10mL)培养3-6个小时,转速240rpm,加入IPTG至终浓度0.3mM,继续培养20个小时,以诱导蛋白质表达。将细菌在4℃离心5分钟,转速8000rpm,倒去上清培养液,冰浴待用。
在一个2ml体系中,加入上述菌液,该体系中含有50mM Tris-HCl缓冲液(pH 7.5),20g/L的苏氨酸,在30℃摇床、240rpm的条件下振荡反应20h,得到含甘氨酸和乙醛的转化液。反应结束后,将所得的转化液用去离子水稀释20倍,12500转/分离心10~15分钟,吸取上清稀释进行高效液相色谱分析,测定催化体系中底物和产物的浓度。经测定,催化反应20h之后,本体系产生甘氨酸浓度为11.8g/L,产率达93.6%,乙醛为2.7g/L,在此过程中,乙醛部分被氧化还原。产率计算公式为:(实际产量/理论产量)×100%。
实施例2
将质重组表达质粒载体pZE-ItaE_eutE转化至野生型大肠杆菌中,接入含有氨苄青霉素和氯霉素的2-xyT培养基中。以20g/L的量加入苏氨酸作为底物在30℃下于50ml三角瓶中(装液量10mL),培养3-6个小时,转速240rpm,加入IPTG至终浓度0.3mM,继续培养10个小时,以进行蛋白质表达。将细菌在4℃进行破壁,离心5分钟,转速8000rpm,保留上清培养液,得到甘氨酸和乙酰辅酶A。经检测,产生的甘氨酸浓度为10.8g/L,产率达85.7%,乙酰辅酶A浓度18.53ng/l。
实施例3
本实施例为实施例2的对照试验组。将重组表达质粒载体pZE-ItaE_eutE、pZE-tdH_kbI分别转化至敲除基因aceE的大肠杆菌中得到重组微生物,将上述两个菌种、野生型大肠杆菌和敲除基因aceE的大肠杆菌菌种分别接入含有氨苄青霉素和氯霉素、不含抗生素的2-xyT培养基中,基因aceE为大肠杆菌本身具有的与乙酰辅酶A表达相关的基因,敲除后能更直观地说明重组表达质粒载体的作用。以20g/L的量加入苏氨酸作为底物在30℃下于50ml三角瓶中(装液量10mL),培养3-6个小时,转速240rpm,加入IPTG至终浓度0.3mM,继续培养10个小时,以进行蛋白质表达。将细菌在4℃进行破壁,离心5分钟,转速8000rpm,保留上清培养液,冰浴待用。检测结果如图1所示,其中采用本发明所述的重组微生物进行发酵的,得到的乙酰辅酶A浓度分别为12.09ng/L和13.789ng/L,得到的甘氨酸浓度分别为10.5g/L和11.2g/L,产率分别为83.3%,88.9%。而采用野生型大肠杆菌进行发酵得到的乙酰辅酶A浓度仅为1.61ng/L,甘氨酸浓度为0.3g/L;采用敲除基因aceE的大肠杆菌菌种进行发酵得到的乙酰辅酶A浓度仅为1.38ng/L,甘氨酸浓度为0.2g/L。说明本发明所述的重组微生物在以苏氨酸为底物的发酵过程中,具有极大的优势。
实施例4
该实施例为摇瓶发酵。将重组表达质粒载体pZE-ItaE_eutE转化至敲除基因adh E的大肠杆菌得到重组微生物,大肠杆菌敲除该基因后,乙醇脱氢酶表达活性被减弱或消除,进而能减少对乙酰辅酶A的消耗,从而能极大提升产物的产量。
将重组微生物接入含有氨苄青霉素且不含葡萄糖的M9培养基中,以20g/L的量加入苏氨酸(即苏氨酸与培养基的比例,下同),于50ml三角瓶中(装液量10mL,三角瓶含0.5gCaCO3),转速240rpm、温度30℃的条件下培养3个小时后,加入IPTG至终浓度0.3mM,继续培养发酵24小时,收集发酵液。检测甘氨酸浓度为11g/L,产率为87.3%,甲羟戊酸的浓度为3.8g/L,产率为46.1%。
实施例5
该实施例为发酵罐发酵。将重组表达质粒载体质pZE-ItaE_eutE转化至敲除基因adh E的大肠杆菌得到重组微生物,本实施例中,大肠杆菌为大肠杆菌CMEV-1,大肠杆菌CMEV-1选自申请人之前发表过的文献中公开的菌株,文献号为DOI:10.1128/AEM.02178-16。大肠杆菌敲除该基因后,乙醇脱氢酶表达活性被减弱或消除,进而能减少对乙酰辅酶A的消耗,从而提升产物的产量。将菌种接种至50mL含有100μg/mL氨苄青霉素的LB液体培养基进行扩大培养,37℃,220rpm过夜培养(约14h),继续将菌种接入含有氨苄青霉素的M9培养基,并装入1L发酵罐中(装液量500mL),转速600rpm,发酵培养6小时,加入IPTG至终浓度0.1mM,然后以持续流加补料的方式加入总量约120g/L的量加入苏氨酸,该方式能在维持渗透压不受影响的情况下保持持续产出,从而提高产量。不同时间点收集发酵液,利用高效液相色谱检测甘氨酸和甲羟戊酸的浓度。检测结果如图2所示,可知,发酵过程中,甘氨酸和甲羟戊酸的产量在16h左右同时出现高峰,其中甘氨酸产品浓度高于60g/L,甲羟戊酸产品浓度高于30g/L,具有极为优异的效果。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (22)
1.一种利用苏氨酸制备甘氨酸的方法,其特征在于,以苏氨酸为底物、加入含有醛缩酶基因的重组微生物进行发酵,发酵过程中,苏氨酸分解为甘氨酸和乙醛。
2.根据权利要求1所述的利用苏氨酸制备甘氨酸的方法,其特征在于,所述醛缩酶基因包括苏氨酸缩醛酶基因或苯丝氨酸醛缩酶基因,苏氨酸缩醛酶基因或苯丝氨酸醛缩酶基因包括ItaE、TdcB、glyA、Sdsl、bhcC、psald、pdxA或vrtJ中的一种或几种。
3.根据权利要求1所述的利用苏氨酸制备甘氨酸的方法,其特征在于,包括通过基因工程方法构建所述重组微生物,所述基因工程方法包括质粒表达或基因组整合。
4.根据权利要求3所述的利用苏氨酸制备甘氨酸的方法,其特征在于,重组微生物通过质粒表达的方法进行构建,构建方法为:通过PCR扩增获得醛缩酶编码基因,将获得的基因连接至质粒载体上并转化至感受态细胞中,测序后获得重组表达质粒载体;将重组表达质粒载体转化至微生物中即得到重组微生物。
5.根据权利要求4所述的利用苏氨酸制备甘氨酸的方法,其特征在于,所述质粒为pZAlac、pZElac中的一种或两种,感受态细胞为E.coli dh5a感受态细胞。
6.根据权利要求4所述的利用苏氨酸制备甘氨酸的方法,其特征在于,重组表达质粒载体为pZE-ItaE,质粒pZE-ItaE构建方法为:以恶臭假单胞菌(Pseudomonas putida)的基因组为模板通过PCR扩增得ItaE基因,将ItaE基因连接至含有IPTG诱导型启动子的pZElac载体上并转化至大肠杆菌E.coli dh5a感受态细胞中,测序后得到质粒pZE-ItaE。
7.根据权利要求1所述的利用苏氨酸制备甘氨酸的方法,其特征在于,发酵过程中,将重组微生物的菌种接入含有氨苄青霉素和氯霉素的2-xyT培养基中,培养3-6h后加入IPTG至终浓度0.2~0.4mM,继续培养15~30h,离心得到菌液;将菌液加入Tris-HCl缓冲液中,以15~30g/L的量加入苏氨酸作为底物,发酵得到含甘氨酸和乙醛的发酵液。
8.一种利用苏氨酸制备甘氨酸、乙酰辅酶A的方法,其特征在于,以苏氨酸为底物,加入含有醛缩酶基因和乙酰化乙醛脱氢酶基因的重组微生物进行发酵,发酵过程中,苏氨酸分解为甘氨酸和乙醛,乙醛直接转化为乙酰辅酶A或乙醛转化为乙酸再转化成乙酰辅酶A;
或以苏氨酸为底物,加入含有苏氨酸脱氢酶基因和2-氨-3酮丁酸CoA连接酶基因的重组微生物进行发酵,发酵过程中,苏氨酸脱氢得到2-氨-3酮丁酸,2-氨-3酮丁酸在2-氨-3酮丁酸CoA连接酶的作用下得到乙酰辅酶A。
9.根据权利要求8所述的利用苏氨酸制备甘氨酸、乙酰辅酶A的方法,其特征在于,所述醛缩酶基因包括ItaE、TdcB、glyA、Sdsl、bhcC、psald、pdxA或vrtJ中的一种或几种;所述乙酰化乙醛脱氢酶基因包括eutE、bphJ、TTHB247、mhpF、ADH2或hsaG中的一种或几种;所述苏氨酸脱氢酶基因包括tdh、ydfG、pdxA、asd、AKHSDH2或trmG中的一种或几种;所述2-氨-3酮丁酸CoA连接酶基因包括kbI、GCAT、Gcat或TTHA1582中的一种或几种。
10.根据权利要求8所述的利用苏氨酸制备甘氨酸、乙酰辅酶A的方法,其特征在于,包括通过基因工程方法构建重组微生物,所述基因工程方法包括质粒表达或基因组整合。
11.根据权利要求10所述的利用苏氨酸制备甘氨酸、乙酰辅酶A的方法,其特征在于,重组微生物通过质粒表达的方法进行构建,构建方法为:通过PCR扩增获得所述醛缩酶编码基因、乙酰化乙醛脱氢酶编码基因,或通过PCR扩增获得苏氨酸脱氢酶编码基因、2-氨-3酮丁酸CoA连接酶编码基因,将获得的基因连接至质粒载体上并转化至感受态细胞中,测序后获得重组表达质粒载体;将重组表达质粒载体转化至微生物中即得到重组微生物。
12.根据权利要求11所述的利用苏氨酸制备甘氨酸、乙酰辅酶A的方法,其特征在于,所述质粒为pZAlac、pZElac中的一种或两种,感受态细胞为E.coli dh5a感受态细胞。
13.根据权利要求11所述的利用苏氨酸制备甘氨酸、乙酰辅酶A的方法,其特征在于,所述重组表达质粒载体为pZE-ItaE_eutE,pZE-ItaE_eutE的构建方法为:以大肠杆菌MG1655基因组为模板通过PCR扩增得eutE基因,以恶臭假单胞菌(Pseudomonas putida)的基因组为模板通过PCR扩增得ItaE基因,将eutE基因及ItaE基因连接至含有IPTG诱导型启动子的pZElac载体上并转化至大肠杆菌E.coli dh5a感受态细胞中,测序后得到质粒pZE-ItaE_eutE。
14.根据权利要求11所述的利用苏氨酸制备甘氨酸、乙酰辅酶A的方法,,其特征在于,所述重组表达质粒载体为pZE-tdH_kbI,pZE-tdH_kbI构建方法为:以大肠杆菌MG1655基因组为模板通过PCR扩增得tdH、_kbI基因,将tdH、_kbI基因连接至含有IPTG诱导型启动子的pZElac载体上并转化至大肠杆菌E.coli dh5a感受态细胞中,测序后得到质粒pZE-tdH_kbI。
15.根据权利要求8所述的利用苏氨酸制备甘氨酸、乙酰辅酶A的方法,其特征在于,发酵时,将所述重组微生物接种至含有氨苄青霉素和氯霉素、不含抗生素的2-xyT培养基中,以15~25g/L的量加入苏氨酸作为底物,在温度为30~35℃的条件下培养3~6h后,加入IPTG至终浓度0.2~0.4mM,继续培养10~12h,培养结束后将离心,保留上清液,得到甘氨酸和乙酰辅酶A。
16.根据权利要求1或8所述的方法,其特征在于,所述微生物选自大肠杆菌、芽孢杆菌、棒状杆菌、酵母或链霉菌中的一种或几种。
17.根据权利要求1或8所述的方法,其特征在于,所述微生物选自大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、巨大芽孢杆菌(Bacillusmegaterium)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Candida utilis)或毕赤酵母(Pichia pastoris)中的一种或几种。
18.一种利用苏氨酸制备甘氨酸和乙酰辅酶A衍生物的方法,其特征在于,采用权利要求8所述的方法制备甘氨酸和乙酰辅酶A,乙酰辅酶A转化为乙酰辅酶A衍生物。
19.根据权利要求18所述的利用苏氨酸制备甘氨酸和乙酰辅酶A衍生物的方法,其特征在于,所述乙酰辅酶A衍生物包括甲羟戊酸、唾液酸,N-乙酰化氨基葡萄糖,3-羟丁酸,脂肪酸中的一种或几种。
20.根据权利要求18所述的利用苏氨酸制备甘氨酸和乙酰辅酶A衍生物的方法,其特征在于,所述乙酰辅酶A衍生物为甲羟戊酸,发酵时,将重组微生物接种至摇瓶中,摇瓶中含有氨苄青霉素且不含葡萄糖的M9培养基,以15~25g/L的量加入苏氨酸作为底物,于30~35℃、转速200~300rpm的条件下培养3~5h,然后加入IPTG至终浓度0.2~0.4mM,继续培养发酵20~30h,收集发酵液即可。
21.根据权利要求18所述的利用苏氨酸制备甘氨酸和乙酰辅酶A衍生物的方法,其特征在于,所述乙酰辅酶A衍生物为甲羟戊酸,发酵时,将所述重组微生物扩大培养后接种入发酵罐中,发酵罐中为含有氨苄青霉素且不含葡萄糖的M9培养基,发酵培养6~8h后,加入IPTG至终浓度0.1~0.2mM,以100~150g/L的量加入苏氨酸作为底物,在30~35℃下于继续发酵培养15~20h,收集发酵液即可。
22.根据权利要求8或18所述的方法,其特征在于,所述微生物的乙醇脱氢酶表达活性被减弱或消除,所述乙醇脱氢酶的编码基因为yqhD、qbdA、adhE或mdH的一种或几种。
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