CN117247883A - 来自Taxus chinensis的L-苯丙氨酸变位酶在合成β-丙氨酸中的应用 - Google Patents
来自Taxus chinensis的L-苯丙氨酸变位酶在合成β-丙氨酸中的应用 Download PDFInfo
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- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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Abstract
本发明为来自Taxus chinensis的L‑苯丙氨酸变位酶在合成β‑丙氨酸中的应用，公开了一种丙酮酸合成β‑丙氨酸的新途径，所述方法包括在酶催化下L‑丙氨酸生成β‑丙氨酸的反应，所述的酶选自脱氢酶和氨基变位酶。本发明的途径步骤短、无碳损、无CO2释放再固定、无转氨反应参与，并且发现四种不同物种来源的氨基变位酶可以催化关键反应L‑丙氨酸生成β‑丙氨酸。本发明解决了现有β‑丙氨酸合成途径碳损大、途径复杂、能耗高等诸多问题，并且制备方法可在全细胞催化、补料发酵、连续发酵等可控制底物水平的生产过程中会发挥更为明显的优势，为绿色、高效、经济的β‑丙氨酸生物制造过程奠定基础。
Description
本申请是申请号202111477590.3，发明名称为：利用丙酮酸合成β-丙氨酸的方法，申请日为2021年12月6日的中国专利发明专利申请的分案申请。
技术领域
本发明涉及生物医药技术领域，具体涉及一种利用丙酮酸合成β-丙氨酸的方法。
背景技术
β-丙氨酸(β-alanine)是天然存在的唯一β型氨基酸，是维生素B5、胍基丙酸、巴柳氮和肌肽等药物的合成原料，也可以作为食品添加剂用于改善食物风味，在医药、化工、食品等领域具有广泛应用。目前，β-丙氨酸的主要生产方法为化学合成法，包括丙稀晴法、丙烯酸法、亚氨基二丙烯法等，反应过程依赖高温高压等极端环境，部分工艺使用含腈原料，对环境和生物具有潜在危害。
近年来，生物法合成β-丙氨酸因其工艺简单、条件温和且对环境友好等优势受到广泛关注。生物法通过丙酮酸合成β-丙氨酸主要通过下述几条途径(1)三羧酸循环(TCA)还原臂生成富马酸，富马酸加氨生成天冬氨酸，通过天冬氨酸脱羧生成β-丙氨酸(Qian Y,LiuJ,Song W,Chen X,Luo Q,Liu L.Production ofβ-alanine from fumaric acid using adual-enzyme cascade.ChemCatChem,2018,10(21):4984-4991)；此合成途径基于天冬氨酸脱羧反应，原子经济性差，β-丙氨酸理论碳得率低。(2)乙醛酸循环和三羧酸循环(TCA)氧化臂途径导向β-丙氨酸的合成途径(Song CW,Lee J,Ko Y-S,Lee SY.Metabolicengineering of Escherichia coli for the production of 3-aminopropionicacid.Metabolic Engineering,2015,30:121-129)；由于途径中多步反应需要释放CO2，β-丙氨酸实际生产得率仅为0.14C-mol/C-mol葡萄糖，远低于理论得率1.00C-mol/C-mol葡萄糖。(3)草酰乙酸转氨、天冬氨酸脱羧合成β-丙氨酸途径(Piao X,Wang L,Lin B,Chen H,Liu W,Tao Y.Metabolic engineering of Escherichia coli for production of L-aspartate and its derivativeβ-alanine with high stoichiometricyield.Metabolic Engineering,2019,54:244-254.)；该途径中由L-天冬氨酸脱羧酶(ADC)脱去的CO2可以通过磷酸烯醇式丙酮酸羧化酶(PEPCx)重新固定，理论得率可以达到1.00C-mol/Cmol。但若要达到理论值，PEPCx必须将ADC脱去的CO2进行100％再固定。而在实际过程中，即使结合一系列工艺优化，如采取好氧富集菌体-厌氧高密度发酵的两阶段发酵方式，在密闭的厌氧发酵罐中周期性补给NH4HCO3，β-丙氨酸的厌氧阶段实际得率仍然只有0.76C-mol/C-mol葡萄糖。除了CO2再固定的效率问题，该途径还受到转氨过程(由AspC催化)循环代谢物再生效率的制约，每分子草酰乙酸生成一分子天冬氨酸，需消耗一分子L-谷氨酸。(4)草酰乙酸直接加氨生成天冬氨酸，由天冬氨酸脱羧生成β-丙氨酸(Zou X,Guo L,HuangL,Li M,Zhang S,Yang A,Zhang Y,Zhu L,Zhang H,Zhang J,Feng Z.Pathwayconstruction and metabolic engineering for fermentative production ofβ-alanine in Escherichia coli.Applied Microbiology and Biotechnology,2020,104(6):2545-2559)；该途径避免了L-谷氨酸再生效率约束，但仍未解决CO2再固定效率问题，且实际得率仅为0.42C-mol/C-mol。
现有的β-丙氨酸生物合成法仍然面临着CO2回收效率低、碳损大、途径复杂、能耗高等诸多挑战。因此，找到途径简单、无碳损失、无CO2释放再固定、无转氨反应参与的β-丙氨酸合成新途径对于β-丙氨酸的生物合成至关重要。
发明内容
针对现有β-丙氨酸合成途径存在的问题，采用前期开发的途径设计新算法comb-FBA[PMID：31491544]，以包含6566个MetaCyc已知反应的网络模型作为主反应集，以人工设计的28个理论可行非天然醛缩酶新反应和12个天然甲醛利用反应作为组合子集，进行了15,225次途径搜索计算。最终预测并通过实际实验验证了1条步骤短、无碳损、无还原力消耗的无碳损的乙酰磷酸/乙酰辅酶A合成新途径，即由丙酮酸生成L-丙氨酸，再由L-丙氨酸生成β-丙氨酸(图1)。本发明的目的是提供一种β-丙氨酸的合成方法，从丙酮酸到β-丙氨酸的合成途径简单、无碳损失、能耗低，并且通过大量研究筛选发现4个不同物种来源的氨基变位酶可以催化关键反应L-丙氨酸到β-丙氨酸。上述方法包括催化丙酮酸生成L-丙氨酸的脱氢酶和催化L-丙氨酸生成β-丙氨酸的氨基变位酶。
本发明提供一种生产β-丙氨酸基因工程菌，其特征在于，导入有催化丙酮酸生成L-丙氨酸的脱氢酶基因和催化L-丙氨酸生成β-丙氨酸的氨基变位酶基因；所述的氨基变位酶为来自Clostridium subterminale的L-赖氨酸-2,3-氨基变位酶(EC 5.4.3.2)；所述的氨基变位酶为来自Desulfofarcimen acetoxidans的L-赖氨酸-2,3-氨基变位酶(EC5.4.3.2)；或所述的氨基变位酶为来自Taxus chinensis的L-苯丙氨酸变位酶(EC5.4.3.10)；或所述的氨基变位酶为Clostridioides difficile的L-谷氨酸-2,3-氨基变位酶(EC 5.4.3.9)。
优选地，所述的基因工程菌的出发菌株为大肠杆菌，更优选为EscherichiacoliBL21(DE3)。
在一个具体实施方式，所述的脱氢酶为L-丙氨酸脱氢酶(EC 1.4.1.1)。
更优选地，所述来自Clostridium subterminale的L-赖氨酸-2,3-氨基变位酶基因经过密码子优化，其核苷酸序列如SEQ ID NO.1所示；所述来自Desulfofarcimenacetoxidans的L-谷氨酸-2,3-氨基变位酶基因经过密码子优化，其核苷酸序列如SEQ IDNO.2所示；所述来自Taxus chinensis的L-苯丙氨酸-2,3-氨基变位酶基因经过密码子优化，其核苷酸序列如SEQ ID NO.3所示；所述来自Clostridioides difficile的L-谷氨酸-2,3-氨基变位酶基因经过密码子优化，其核苷酸序列如SEQ ID NO.4所示。
本发明还提供一种产β-丙氨酸的方法，其特征在于，所述方法L-丙氨酸通过氨基变位酶催化生成β-丙氨酸，所述的氨基变位酶为来自Clostridium subterminale的L-赖氨酸-2,3-氨基变位酶(EC 5.4.3.2)；或所述的氨基变位酶为来自Desulfofarcimenacetoxidans的L-赖氨酸-2,3-氨基变位酶(EC 5.4.3.2)；或所述的氨基变位酶为来自Taxus chinensis的L-苯丙氨酸变位酶(EC 5.4.3.10)；或所述的氨基变位酶为Clostridioides difficile的L-谷氨酸-2,3-氨基变位酶(EC 5.4.3.9)；任选地，还包括丙酮酸通过L-丙氨酸的脱氢酶催化生成L-丙氨酸的前序步骤。
优选地，所述的脱氢酶为L-丙氨酸脱氢酶(EC 1.4.1.1)。
在一个具体实施方式中，通过全细胞催化的方式实现；所述丙酮酸是由葡萄糖经过糖酵解制备得到。
本发明还提供一种来自Clostridium subterminale的L-赖氨酸-2,3-氨基变位酶或其基因、来自Desulfofarcimen acetoxidans的L-谷氨酸-2,3-氨基变位酶或其基因、来自Taxus chinensis的L-苯丙氨酸-2,3-氨基变位酶或其基因、来自Clostridioidesdifficile的L-谷氨酸-2,3-氨基变位酶或其基因在制备β-丙氨酸中的用途。
在优选实施方式中，来自Clostridium subterminale的L-赖氨酸-2,3-氨基变位酶基因经过密码子优化，其核苷酸序列如SEQ ID NO.1所示；所述来自Desulfofarcimenacetoxidans的L-谷氨酸-2,3-氨基变位酶基因经过密码子优化，其核苷酸序列如SEQ IDNO.2所示；所述来自Taxus chinensis的L-苯丙氨酸-2,3-氨基变位酶基因是经过密码子优化的，其核苷酸序列如SEQ ID NO.3所示；所述来自Clostridioides difficile的L-谷氨酸-2,3-氨基变位酶基因经过密码子优化，其核苷酸序列如SEQ ID NO.4所示。
更优选地，其是通过导入所述来自Clostridium subterminale的L-赖氨酸-2,3-氨基变位酶、来自Clostridium subterminale的L-赖氨酸-2,3-氨基变位酶(EC 5.4.3.2)、来自Desulfofarcimen acetoxidans的L-赖氨酸-2,3-氨基变位酶(EC 5.4.3.2)、来自Taxus chinensis的L-苯丙氨酸变位酶(EC 5.4.3.10)的编码基因的重组菌的全细胞催化的方式实现，任选地，所述重组菌中还导入L-丙氨酸脱氢酶。
本发明提供的β-丙氨酸的制备途径，经验证催化速率较高，反应路线的碳理论得率为100％，没有碳损失。上述制备方法可在全细胞催化、补料发酵、连续发酵等可控制底物水平的生产过程中会发挥更为明显的优势。
附图说明
图1为β-丙氨酸的生物合成途径示意图。
图2为实施例2、3、4、5的GC-MS检测结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
采用前期开发的途径设计新算法comb-FBA[PMID：31491544]，以包含6566个MetaCyc已知反应的网络模型作为主反应集，以人工设计的28个理论可行非天然醛缩酶新反应和12个天然甲醛利用反应作为组合子集，进行了15,225次途径搜索计算，预测了1条步骤短、无碳损、无还原力消耗的无碳损的乙酰磷酸/乙酰辅酶A合成新途径，即由丙酮酸生成L-丙氨酸，再由L-丙氨酸生成β-丙氨酸(图1)。后续对此进行了验证。
根据文献报道来自Clostridium subterminale的L-赖氨酸-2,3-氨基变位酶(NCBI:WP_191740046.1)明确具有催化L-丙氨酸生成β-丙氨酸的活性(Ruzicka FJ,FreyPA(2010)Kinetic and spectroscopic evidence of negative cooperativity in theaction of lysine 2,3-aminomutase.Journal of Physical Chemistry B 114:16118-16124)，但活性较低。
基于PDB数据库(https://www.rcsb.org/)，对于拥有蛋白质三维结构的氨基变位酶，分析其活性中心、底物产物进出通道与电子传递通道等筛选潜在的具备L-丙氨酸-2,3-氨基变位酶活性的候选酶。最终，从几十种候选酶中成功筛选到3种具备L-丙氨酸-2,3-氨基变位酶活性的酶，分别为：来自Desulfofarcimen acetoxidans的L-谷氨酸-2,3-氨基变位酶(NCBI:WP_015758379.1)、来自Taxus chinensis的L-苯丙氨酸-2,3-氨基变位酶(NCBI:Q68G84.1)、来自Clostridioides difficile的L-谷氨酸-2,3-氨基变位酶(NCBI:WP_003430434.1)。这些酶与已报道的L-赖氨酸-2,3-氨基变位酶(NCBI:WP_191740046.1)的氨基酸序列同源性均较低。根据其氨基酸序列，针对大肠杆菌进行密码子优化，合成了表达相关酶的基因。
4种酶的基因密码子优化后序列如下：
来自Clostridium subterminale的L-赖氨酸-2,3-氨基变位酶基因kam_cls，经过密码子优化后的序列(SEQ ID NO.1)：
ATGATCAACCGTCGTTACGAACTGTTCAAAGACGTTTCTGACGCTGACTGGAACGACTGGCGTTGGCAGGTTCGTAACCGTATCGAAACCGTTGAAGAACTGAAAAAATACATCCCGCTGACCAAAGAAGAAGAAGAAGGTGTTGCTCAGTGCGTTAAATCTCTGCGTATGGCTATCACCCCGTACTACCTGTCTCTGATCGACCCGAACGACCCGAACGACCCGGTTCGTAAACAGGCTATCCCGACCGCTCTGGAACTGAACAAAGCTGCTGCTGACCTGGAAGACCCGCTGCACGAAGACACCGACTCTCCGGTTCCGGGTCTGACCCACCGTTACCCGGACCGTGTTCTGCTGCTGATCACCGACATGTGCTCTATGTACTGCCGTCACTGCACCCGTCGTCGTTTCGCTGGTCAGTCTGACGACTCTATGCCGATGGAACGTATCGACAAAGCTATCGACTACATCCGTAACACCCCGCAGGTTCGTGACGTTCTGCTGTCTGGTGGTGACGCTCTGCTGGTTTCTGACGAAACCCTGGAATACATCATCGCTAAACTGCGTGAAATCCCGCACGTTGAAATCGTTCGTATCGGTTCTCGTACCCCGGTTGTTCTGCCGCAGCGTATCACCCCGGAACTGGTTAACATGCTGAAAAAATACCACCCGGTTTGGCTGAACACCCACTTCAACCACCCGAACGAAATCACCGAAGAATCTACCCGTGCTTGCCAGCTGCTGGCTGACGCTGGTGTTCCGCTGGGTAACCAGTCTGTTCTGCTGCGTGGTGTTAACGACTGCGTTCACGTTATGAAAGAACTGGTTAACAAACTGGTTAAAATCCGTGTTCGTCCGTACTACATCTACCAGTGCGACCTGTCTCTGGGTCTGGAACACTTCAGAACTCCGGTAAGCAAAGGTATCGAAATCATCGAAGGTCTGCGTGGTCACACCTCTGGTTACTGCGTTCCGACCTTCGTTGTTGACGCTCCGGGTGGTGGTGGTAAAACCCCGGTTATGCCGAACTACGTTATCTCTCAGTCTCACGACAAAGTTATCCTGCGTAACTTCGAAGGTGTTATCACCACCTACTCTGAACCGATCAACTACACCCCGGGTTGTAATTGCGACGTATGCACCGGGAAAAAAAAAGTTCACAAAGTTGGTGTTGCTGGTCTGCTGAACGGTGAAGGTATGGCTCTGGAACCGGTTGGTCTGGAACGTAACAAACGTCACGTTCAGGAATAA
来自Desulfofarcimen acetoxidans的L-谷氨酸-2,3-氨基变位酶基因eam_dea，经过密码子优化后的序列(SEQ ID NO.2)：ATGTTCCTGAACGTGACGTTTGTTACGTTCCAGACCAACCGGTTCCAGAGCCATACCTTCACCGTTCAGCAGACCAGCAACACCAACTTTGTGAACTTTTTTTTTCCCGGTGCATACGTCGCAATTACAACCCGGGGTGTAGTTGATCGGTTCAGAGTAGGTGGTGATAACACCTTCGAAGTTACGCAGGATAACTTTGTCGTGAGACTGAGAGATAACGTAGTTCGGCATAACCGGGGTTTTACCACCACCACCCGGAGCGTCAACAACGAAGGTCGGAACGCAGTAACCAGAGGTGTGACCACGCAGACCTTCGATGATTTCGATACCTTTGCTTACCGGAGTTCTGAAGTGTTCCAGACCCAGAGACAGGTCGCACTGGTAGATGTAGTACGGACGAACACGGATTTTAACCAGTTTGTTAACCAGTTCTTTCATAACGTGAACGCAGTCGTTAACACCACGCAGCAGAACAGACTGGTTACCCAGCGGAACACCAGCGTCAGCCAGCAGCTGGCAAGCACGGGTAGATTCTTCGGTGATTTCGTTCGGGTGGTTGAAGTGGGTGTTCAGCCAAACCGGGTGGTATTTTTTCAGCATGTTAACCAGTTCCGGGGTGATACGCTGCGGCAGAACAACCGGGGTACGAGAACCGATACGAACGATTTCAACGTGCGGGATTTCACGCAGTTTAGCGATGATGTATTCCAGGGTTTCGTCAGAAACCAGCAGAGCGTCACCACCAGACAGCAGAACGTCACGAACCTGCGGGGTGTTACGGATGTAGTCGATAGCTTTGTCGATACGTTCCATCGGCATAGAGTCGTCAGACTGACCAGCGAAACGACGACGGGTGCAGTGACGGCAGTACATAGAGCACATGTCGGTGATCAGCAGCAGAACACGGTCCGGGTAACGGTGGGTCAGACCCGGAACCGGAGAGTCGGTGTCTTCGTGCAGCGGGTCTTCCAGGTCAGCAGCAGCTTTGTTCAGTTCCAGAGCGGTCGGGATAGCCTGTTTACGAACCGGGTCGTTCGGGTCGTTCGGGTCGATCAGAGACAGGTAGTACGGGGTGATAGCCATACGCAGAGATTTAACGCACTGAGCAACACCTTCTTCTTCTTCTTTGGTCAGCGGGATGTATTTTTTCAGTTCTTCAACGGTTTCGATACGGTTACGAACCTGCCAACGCCAGTCGTTCCAGTCAGCGTCAGAAACGTCTTTGAACAGTTCGTAACGACGGTTGATTAA
来自Taxus chinensis的L-苯丙氨酸-2,3-氨基变位酶基因fam-tac，经过密码子优化后的序列(SEQ ID NO.3)：
ATGGGTTTCGCTGTTGAATCTCGTTCTCACGTTAAAGACATCCTGGGTCTGATCAACGCTTTCAACGAAGTTAAAAAAATCACCGTTGACGGTACCACCCCGATCACCGTTGCTCACGTTGCTGCTCTGGCTCGTCGTCACGACGTTAAAGTTGCTCTGGAAGCTGAACAGTGCCGTGCTCGTGTTGAAACCTGCTCTTCTTGGGTTCAGCGTAAAGCTGAAGACGGTGCTGACATCTACGGCGTTACTACCGGCTTCGGTGCTTGCTCGTCTCGTCGTACCAACCGTCTGTCTGAACTGCAGGAATCTCTGATCCGTTGCCTGCTGGCTGGTGTTTTCACCAAAGGTTGCGCTCCGTCTGTTGACGAACTGCCGGCTACCGCTACCCGTTCTGCTATGCTGCTGCGTCTGAACTCTTTCACCTACGGTTGCTCTGGTATCCGTTGGGAAGTTATGGAAGCTCTGGAAAAACTGCTGAACTCTAACGTTTCTCCGAAAGTTCCGCTGCGTGGTTCTGTTTCTGCTTCTGGTGACCTGATCCCGCTGGCTTACATCGCTGGTCTGCTGATCGGTAAACCGTCTGTTATCGCTCGTATCGGTGACGACGTTGAAGTTCCGGCTCCGGAAGCTCTGTCTCGTGTTGGTCTGCGTCCGTTCAAACTGCAGGCTAAAGAAGGTCTGGCTCTGGTTAACGGTACCTCTTTCGCTACCGCTGTTGCTTCTACCGTTATGTACGACGCTAACGTTCTGCTGCTGCTGGTTGAAACCCTGTGCGGTATGTTCTGCGAAGTTATCTTCGGTCGTGAAGAATTTGCTCACCCGCTGATCCACAAAGTTAAACCGCACCCGGGTCAGATCGAATCTGCTGAACTGCTGGAATGGCTGCTGCGTTCTTCTCCGTTCCAGGAACTGTCTCGTGAATACTACTCTATCGACAAACTGAAAAAACCGAAACAGGACCGTTACGCTCTGCGTTCTTCTCCGCAGTGGCTGGCTCCGCTGGTTCAGACCATCCGTGACGCTACCACCACCGTTGAAACCGAAGTTAACTCTGCTAACGACAACCCGATCATCGACCACGCTAACGACCGTGCTCTGCACGGTGCTAACTTCCAGGGTTCTGCTGTTGGTTTCTACATGGACTACGTTCGTATCGCTGTTGCTGGTCTGGGTAAACTGCTGTTCGCTCAGTTCACCGAACTGATGATCGAATACTACTCTAACGGTCTGCCGGGTAACCTGTCTCTGGGTCCGGACCTGTCTGTTGACTACGGTCTGAAAGGTCTGGACATCGCTATGGCTGCTTACTCTTCTGAACTGCAGTACCTGGCTAACCCGGTTACCACCCACGTTCACTCTGCTGAACAGCACAACCAGGACATCAACTCTCTGGCTCTGATCTCTGCTCGTAAAACCGAAGAAGCTCTGGACATCCTGAAACTGATGATCGCTTCTCACCTGACCGCTATGTGCCAGGCTGTTGACCTGCGTCAGCTGGAAGAAGCTCTGGTTAAAGTTGTTGAAAACGTTGTTTCTACCCTGGCTGACGAATGCGGTCTGCCGAACGACACCAAAGCTCGTCTGCTGTACGTTGCTAAAGCTGTTCCGGTTTACACCTACCTGGAATCTCCGTGCGACCCGACCCTGCCGCTGCTGCTGGGTCTGAAACAGTCTTGCTTCGACACCATCCTGGCTCTGCACAAAAAAGACGGTATCGAAACCGACACCCTGGTTGACCGTCTGGCTGAATTTGAAAAACGTCTGTCTGACCGTCTGGAAAACGAAATGACCGCTGTTCGTGTTCTGTACGAAAAAAAAGGTCACAAAACCGCTGACAACAACGACGCTCTGGTTCGTATCCAGGGTTCTAAATTCCTGCCGTTCTACCGTTTCGTTCGTGAAGAACTGGACACCGGTGTTATGTCTGCTCGTCGTGAACAGACCCCGCAGGAAGACGTTCAGAAAGTTTTCGACGCTATCGCTGACGGTCGTATCACCGTTCCGCTGCTCCACTGCCTGCAGGGCTTCCTGGGTCAGCCAAACGGTTGCGCTAACGGTGTTTAA。
来自Clostridioides difficile的L-谷氨酸-2,3-氨基变位酶基因eam-cld，经过密码子优化后的序列(SEQ ID NO.4)：
ATGAACGAACAGACCCGTATCTCTCTGGAACGTGCTGCTGAACTGAAATCTAAAATCGACGACTACATCCAGGCTCGTAAAACCATCAACCGTGGTCTGGAAAAAGAAGAAGAAATCAACAAACGTAAACAGAAAATCCTGTCTATCCTGAACGGTACCGAAGAAGACTGGAACAACTACAAATGGCAGCTGTCTAACCGTATCACCGACGTTGACACCCTGTCTAAAATCATCACCCTGACCAAAAAAGAAAAAGAATACATCAAAGAAGTTGGTACCCAGTTCCGTTGGGCTATCTCTCCGTACTACCTGTCTCTGATCGACCCGGAAGACATCTGCGACCCGATCAAACTGCTGTCTATCCCGACCCACATCGAACTGGAAGACGAACAGGAAGACCTGGACCCGATGGGTGAAGAATACACCAACCCGGCTGGTTGCATCACCCGTCGTTACCCGGACCGTCTGATCATCAACGTTACCAACGAATGCGCTATGTACTGCCGTCACTGCCAGCGTCGTCGTAACATCGGTCAGCAGGACTCTCACAAATCTAAAGCTATCATCCAGGAATCTATCGACTACATCCGTGAAAACGAAGAAATCCGTGACGTTCTGGTTACCGGTGGTGACGCTCTGACCCTGAAAGACGACTACCTGGAATGGATCCTGTCTCAGCTGAAAGAAATCCCGCACGTTGACTACGTTCGTCTGGGTACCCGTACCCTGGTTACCATGCCGCAGCGTATCACCGACGAATTTTGCAACATGCTGAAAAAATACCACCCGGTTTACATCAACACCCACTTCAACCACCCGATGGAAATCACCAAAGAATCTAAAGAAGCTTGCGAAAAACTGGCTAACGCTGGTGTTCCGCTGGGTAACCAGGCTGTTCTGCTGAACGGTATCAACAACGACAAATTCGTTATGCGTTGCCTGAACCAGGAACTGCTGAAAATCCGTGTTAAACCGTACTACATCTTCCAGTCTAAACACGTTAAAGGTACCAAACACTTCAACACCTCTGTTGACGACGGTCTGGAAATCATGGAATACCTGCGTGGTTACACCTCTGGTATGGCTATCCCGACCTACATCGTTAACGCTCCGAAAGGTGGTGGTAAAACCCCGCTGCTGCCGCAATACCTGGTAAGCAAAGGTACCGACTACGTTATGCTGCGTACCTGGGAAGGTAAAGTTATCAAAATGGAAGACGAACCGGCTGTTGACATCAAAAAACTGATCAAAGAACAGGCTCAGGACTAA。
实施例2
将含有Clostridium subterminale的L-赖氨酸-2,3-氨基变位酶基因的质粒(pET-28a-kam_cls)转入到大肠杆菌Escherichia coli BL21(DE3)中，上述质粒选择IPTG诱导型启动子。将上述菌株在LB培养基中进行培养，培养2.5h后，菌体OD600值达到0.6-0.8时，添加IPTG至终浓度0.5mM，诱导(16℃，18h)表达上述质粒上的目标酶蛋白。离心(4℃、6000r/min、20min)收集菌体后，加入HEPES(PH为8.0)缓冲液，悬浮，重新离心收集菌体。将最终收集的菌体加入2mL HEPES缓冲液重新悬浮,得到全细胞悬浮液。
L-丙氨酸在10mM的初始浓度下，SAM浓度为1mM，PLP浓度为2.4mM，DTT浓度为1mM，加入252ul(OD600nm＝60)的上述全细胞悬浮液，进行β-丙氨酸的制备反应，反应4h后，将反应液离心(4℃、12000r/min、10min)，获得上清液；将上清液冷冻干燥，干燥的样品衍生处理，具体过程如下：
(1)肟化：将干燥后的样品溶于100μl含40mg/ml盐酸甲氧胺的吡啶溶液，在30℃下静置90min；
(2)硅烷化：将肟化后的反应液离心(室温、12000rpm/min、5min)，取10μl上清加入到含有90μl的1％三甲基氯硅烷的N-甲基-N-(三甲基甲硅烷基)三氟乙酰胺,在37℃下静置30min。
将衍生后的样品通过GC-MS定量检测到的β-丙氨酸的含量为0.015mM。
实施例3
将含有Desulfofarcimen acetoxidans的L-赖氨酸-2,3-氨基变位酶基因的质粒(pET-28a-eam_dea)转入到大肠杆菌Escherichia coli BL21(DE3)中，后续实验过程同实施例2，通过GC-MS定量检测到的β-丙氨酸的含量为0.027mM。
实施例4
将含有Taxus chinensis的L-苯丙氨酸变位酶基因的质粒(pET-28a-fam_tac)转入到大肠杆菌Escherichia coli BL21(DE3)中，后续实验过程同实施例2，通过GC-MS定量检测到的β-丙氨酸的含量为0.175mM。
实施例5
将含有Clostridioides difficile的L-谷氨酸-2,3-氨基变位酶基因的质粒(pET-28a-eam_cld)转入到大肠杆菌Escherichia coli BL21(DE3)中，后续实验过程同实施例2，通过GC-MS定量检测到的β-丙氨酸的含量为0.142mM。
实施例6
将含有来自枯草芽孢杆菌的L-丙氨酸脱氢酶基因(NCBI：NP_391071.1)及Taxuschinensis的L-苯丙氨酸变位酶基因fam_tac的质粒(pET-28a-fam_tac-alaDH)导入到大肠杆菌Escherichia coli BL21(DE3)，后续实验过程除了将10mM的L-丙氨酸替换为10mM的丙酮酸钠以外，其他操作同实施例2，通过GC-MS定量检测到的β-丙氨酸的含量为0.035mM。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种生产β-丙氨酸基因工程菌，其特征在于，导入有催化丙酮酸生成L-丙氨酸的脱氢酶基因和催化L-丙氨酸生成β-丙氨酸的氨基变位酶基因；所述的氨基变位酶为来自Taxus chinensis的L-苯丙氨酸变位酶。
2.如权利要求1所述的生产β-丙氨酸基因工程菌，其特征在于，所述的基因工程菌的出发菌株为大肠杆菌。
3.如权利要求2所述的生产β-丙氨酸基因工程菌，其特征在于，所述的基因工程菌的出发菌株为Escherichia coliBL21(DE3)。
4.如权利要求1所述的生产β-丙氨酸基因工程菌，其特征在于，所述的脱氢酶为L-丙氨酸脱氢酶。
5.如权利要求1所述的生产β-丙氨酸基因工程菌，其特征在于，所述来自Taxus chinensis的L-苯丙氨酸-2,3-氨基变位酶的基因经过密码子优化，其核苷酸序列如SEQ IDNO.3所示。
6.一种产β-丙氨酸的方法，其特征在于，所述方法L-丙氨酸通过氨基变位酶催化生成β-丙氨酸，所述的氨基变位酶为来自Taxus chinensis的L-赖氨酸-2,3-氨基变位酶；任选地，还包括丙酮酸通过L-丙氨酸的脱氢酶催化生成L-丙氨酸的前序步骤。
7.如权利要求6所述的方法，其特征在于，所述的脱氢酶为L-丙氨酸脱氢酶。
8.如权利要求6所述的方法，其特征在于，通过全细胞催化的方式实现；所述丙酮酸是由葡萄糖经过糖酵解制备得到。
9.来自Taxus chinensis的L-苯丙氨酸-2,3-氨基变位酶或其基因在制备β-丙氨酸中的用途。
10.如权利要求9所述的用途，其特征在于，所述来自Taxus chinensis的L-苯丙氨酸-2,3-氨基变位酶基因是经过密码子优化的，其核苷酸序列如SEQ ID NO.3所示；
具体地，其是通过导入所述来自Taxus chinensis的L-苯丙氨酸变位酶的编码基因的重组菌的全细胞催化的方式实现，且所述重组菌中还导入L-丙氨酸脱氢酶。
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