本发明属于生物催化
技术领域：
，涉及一种微生物转化法制备α-酮戊二酸的方法，具体地说，涉及一种新的微生物酶催化法合成α-酮戊二酸的方法。
背景技术：
：α-酮戊二酸(α-ketoglutaricacid)是自然界中一种重要的代谢中间产物，参与机体内氨基酸、糖类、蛋白质和脂肪等多种物质代谢。α-酮戊二酸还是一种重要的化学合成原料和中间体，在医药上是合成氨基酸和肽类的重要原料，被广泛应用于饲料、保健食品、精细化工等领域。目前，α-酮戊二酸的生产方法主要有化学合成法，微生物发酵法和酶转化法。化学合成法需要用到有机溶剂，腈化物等有毒原料，污染环境，步骤繁多，副反应多，成本高，不符合环保和工业化成本的要求。微生物发酵法生产周期长，杂酸种类多且含量高，产物分离提取工艺复杂，导致产品收率低，成本高。酶转化法选择性好，杂质少，产品浓度高，并且能耗低，周期短，已经逐渐成为α-酮戊二酸生产工艺的主流，并且仍在不断改进中。目前，α-酮戊二酸的酶法工艺包含l-谷氨酸氧化酶法，l-氨基酸(谷氨酸)脱氨酶法和l-谷氨酸脱氢酶法。cn103911400a采用l-氨基酸脱氨酶突变体转化l-谷氨酸生产α-酮戊二酸，转化率最高为85.25％。cn106834366a以l-谷氨酸及其盐为原料，经l-谷氨酸脱氢酶催化制备α-酮戊二酸，该工艺需要用到辅因子，辅因子通过有机小分子再生催化剂再生，该工艺是一种有益的探索，但由于产物浓度太低，无法工业应用。辅因子的再生以及催化剂的成本都会造成工业应用的困难。cn109988786a采用l-谷氨酸或l-谷氨酸盐为原料，经l-谷氨酸氧化酶催化制备α-酮戊二酸，并通过流加过氧化氢酶控制丁二酸含量在0.1-1.0g/l，产物浓度可以做到135.5g/l，转化率为99.9％。cn106047913a采用经过优化的l-谷氨酸氧化酶和过氧化氢酶共表达菌株，催化l-谷氨酸制备α-酮戊二酸，可以达到127.1g/l的产物浓度和96.9％的转化率。cn110283800a以l-谷氨酸盐为底物，采用l-谷氨酸氧化酶突变体和过氧化氢酶共表达的重组菌株，通过酶反应可以得到205g/l的产物浓度，转化率可以达到97.5％。从这些专利可以看出，l-氨基酸脱氨酶法和l-谷氨酸脱氢酶法的工业化仍有较大困难，l-氨基酸脱氨酶产生的氨在高浓度下也会对反应有抑制，l-谷氨酸脱氢酶需要用到nad+或者nadp+辅因子，这些辅因子的成本或者再生成本较高，由于这些原因，l-谷氨酸氧化酶法是最具优势的酶法，该法在反应中产生的过氧化氢，如果不及时除去，不仅会降低酶的活力，还会将α-酮戊二酸氧化为丁二酸，丁二酸的存在不利于α-酮戊二酸的分离纯化，专利cn109988786a采用流加过氧化氢酶的方法增加了成本，并且需要时刻监控丁二酸浓度，操作较复杂。cn106047913a和cn110283800a采用l-谷氨酸氧化酶和过氧化氢酶共表达的方法，降低了过氧化氢酶的成本，但收率偏低，究其原因，可能是共表达的过氧化氢酶表达量不够，对过氧化氢的亲和力不够或者稳定性不好，导致反应产生的过氧化氢酶不能及时被清除。因此过氧化氢酶是提高产品收率的一个至关重要的因素。技术实现要素：为了改进现有技术的微生物表达l-谷氨酸氧化酶和过氧化氢酶来催化制备α-酮戊二酸的方法，发明人对于l-谷氨酸氧化酶和过氧化氢酶的种类分别进行了广泛筛选，并且构建了多种过氧化氢酶与l-谷氨酸氧化酶共表达菌种，这些菌种通过微生物发酵得到包含l-谷氨酸氧化酶和过氧化氢酶的菌体，采用这些菌体转化l-谷氨酸钠来制备α-酮戊二酸，得到了较高产品浓度和转化率，并且副产物丁二酸含量低，从而提供了一种简单高效的α-酮戊二酸制备方法。具体地，本发明包括如下技术方案：一种共表达l-谷氨酸氧化酶和过氧化氢酶的基因工程菌，其中所述l-谷氨酸氧化酶的氨基酸序列是seqidno.1，所述过氧化氢酶的氨基酸序列选自seqidno.3、seqidno.5、seqidno.7、seqidno.9、seqidno.11、seqidno.13。其中l-谷氨酸氧化酶seqidno.1来源于链霉菌streptomycesspx-119-6，本文中简称为lgox；过氧化氢酶seqidno.3来源于百日咳博德特氏菌bordetellapertussis，本文中简称为kata；过氧化氢酶seqidno.5来源于大肠杆菌escherichiacoli，本文中简称为kate；过氧化氢酶seqidno.7来源于大肠杆菌escherichiacoli，本文中简称为katg；过氧化氢酶seqidno.9来源于嗜树木甲烷短杆菌methanobrevibacterarboriphilus，本文中简称为makat；过氧化氢酶seqidno.11来源于闪烁古生球菌archaeoglobusfulgidusdsm4304，本文中简称为pera；过氧化氢酶seqidno.13来源于褐色嗜热裂孢菌thermobifidafusca，本文中简称为tfucat。本发明的第二个方面提供了一种编码上述l-谷氨酸氧化酶和过氧化氢酶的基因。例如，编码l-谷氨酸氧化酶seqidno.1的基因的核苷酸序列为seqidno.2；编码过氧化氢酶seqidno.3的基因的核苷酸序列为seqidno.4；编码过氧化氢酶seqidno.5的基因的核苷酸序列为seqidno.6；编码过氧化氢酶seqidno.7的基因的核苷酸序列为seqidno.8；编码过氧化氢酶seqidno.9的基因的核苷酸序列为seqidno.10；编码过氧化氢酶seqidno.11的基因的核苷酸序列为seqidno.12；编码过氧化氢酶seqidno.13的基因的核苷酸序列为seqidno.14。优选地，上述过氧化氢酶的氨基酸序列是seqidno.9，且其编码基因的核苷酸序列为seqidno.10。本发明的第三个方面提供了一种构建上述基因工程菌的方法，例如，可以采用质粒转化、同源重组技术或者基因编辑技术将编码所述l-谷氨酸氧化酶的基因和编码所述过氧化氢酶的基因整合入宿主细胞的基因组中，得到基因工程菌。上述基因编辑技术可以采用crispr-cas9系统、crispr-cpf1系统、crispr-cas相关的转座系统integrate系统或者cast系统。上述integrate系统是指samsternberg研究组开发的基因编辑工具(insertionoftransposableelementsbyguiderna-assistedtargeting，引导rna辅助靶向的转座元件插入)；cast系统是指张锋研究组开发的基因编辑工具(crispr-associatedtransposase，crispr相关转座酶)。上述基因工程菌的宿主优选是细菌，比如大肠杆菌、谷氨酸棒杆菌、枯草芽孢杆菌等。优选大肠杆菌，包括但不限于大肠杆菌escherichiacolibl21(de3)、escherichiacolibl21(de3)plyss或者escherichiacolibl21(de3)star。更优选是大肠杆菌bl21(de3)。作为最传统的方法，采用质粒转化法将l-谷氨酸氧化酶基因和过氧化氢酶基因整合入宿主细胞的基因组中。典型地，所述质粒转化包括如下步骤：(1)全基因合成编码所述l-谷氨酸氧化酶的基因和编码所述过氧化氢酶的基因；(2)分别将编码所述l-谷氨酸氧化酶的基因和编码所述过氧化氢酶的基因通过ndei和bamhi酶切位点克隆到质粒pet24a(+)上，分别得到pet24a-谷氨酸氧化酶质粒和pet24a-过氧化氢酶质粒；或者再通过酶切和连接的方式得到pet24a-谷氨酸氧化酶+过氧化氢酶质粒；或者将编码所述l-谷氨酸氧化酶的基因与编码所述过氧化氢酶的基因通过自剪切多肽2a相融合从而形成融合基因，然后将融合基因通过ndei和bamhi酶切位点克隆到质粒pet24a(+)上，得到pet24a-谷氨酸氧化酶+过氧化氢酶质粒；(3)通过电转法或者化学转化法(氯化钙法)将步骤1中得到的质粒(pet24a-谷氨酸氧化酶质粒和pet24a-过氧化氢酶质粒、或者pet24a-谷氨酸氧化酶+过氧化氢酶质粒)转化到bl21(de3)感受态细胞中，筛选出酶切验证正确的阳性克隆株，得到正确的表达l-谷氨酸氧化酶和表达各个过氧化氢酶的菌种。其中用于形成融合基因的自剪切多肽2a(self-cleaving2apeptide，2a)或称可自切割2apeptide，其可以选自p2a、e2a、t2a、f2a，但不限于此。本发明的另一个方面提供了一种上述基因工程菌在制备α-酮戊二酸中的应用，即提供一种制备α-酮戊二酸的方法，其包括，在合适的反应条件(比如提供氧气或空气，合适的温度和ph)下，向反应体系中加入权利要求1-7中任一项所述的基因工程菌经过微生物产酶发酵得到的菌体，将反应底物l-谷氨酸钠催化转化为α-酮戊二酸。在一种优选的实施方式中，反应底物l-谷氨酸是以价格相对低廉的工业产品谷氨酸钠一水合物的形式提供。在反应过程中，控制反应体系ph6.0-8.0、优选ph6.5-7.5、更优选ph7.0左右；温度25-45℃、优选28-42℃、更优选30-40℃、更优选32-38℃、最优选35℃左右。上述反应可以在发酵罐或反应釜内进行，此时，控制转速600-1000rpm，通气量1-2vvm/min，罐压0.05-0.1mpa，溶氧30-40％。与现有技术相比，本发明进一步优化了特定的l-谷氨酸氧化酶与特定的过氧化氢酶的组合，显著提高了制备α-酮戊二酸的效率和收率，有效抑制了副产物丁二酸的产生，具有重要的工业应用价值。具体实施方式本发明提供的基因工程菌实现了优选的l-谷氨酸氧化酶和过氧化氢酶的共表达。为了在不同微生物中进行酶蛋白的最佳表达，可以针对特定的微生物比如大肠杆菌进行密码子优化。密码子优化是通过增加目的基因的翻译效率使生物体中蛋白质表达最大化的一种技术。不同的生物体由于突变倾向和天然选择而通常显示出对于编码相同氨基酸的一些密码子的特殊偏好性。例如，在生长快速的微生物如大肠杆菌中，优化密码子反映出其各自的基因组trna库的组成。因此，在生长快速的微生物中，氨基酸的低频率密码子可以被编码相同氨基酸的但高频率的密码子置换。因此，优化的dna序列的表达在快速生长的微生物中得以改良。例如，在本文中所提供的l-谷氨酸氧化酶seqidno.1的基因序列seqidno:2是经过密码子优化的核苷酸序列，但不限于此。过氧化氢酶seqidno.9(makat)的基因序列seqidno:10也是经过密码子优化的核苷酸序列。为了描述简便起见，在本文中，有时会将某种蛋白比如l-谷氨酸氧化酶与其编码基因名称混用，本领域技术人员应能理解它们在不同描述场合表示不同的物质。本领域技术人员根据语境和上下文容易理解它们的含义。为描述简单起见，有时将“l-谷氨酸氧化酶”简称为“谷氨酸氧化酶”；类似地，将“l-谷氨酸”简称为“谷氨酸”、或者其商品形式之一“谷氨酸钠”。本发明的反应体系是一种双酶联合催化反应体系，作为生物催化剂的菌体包括存活菌体和死亡菌体。菌体本身就是一种天然的酶固定化形式，而且不需要进行破碎处理、甚至提取纯化处理，就可以作为一种酶制剂用于催化反应。由于反应底物和反应产物都是小分子化合物，可以很方便地穿过菌体的生物屏障--细胞膜，因此不需要对菌体进行破碎处理，这在经济方面是有利的。另一方面，相比游离酶或固定化化酶的催化，本发明利用微生物的简单发酵就可以源源不断、取之不尽地提供两种酶(l-谷氨酸氧化酶、过氧化氢酶)同时地、按比例的供应，无需进一步提取、纯化分离两种酶等操作，也无需根据两种酶的酶活力进行比例调整，以工业品级的谷氨酸钠一水合物为原料底物，能够显著降低生产成本，经济效益明显，因此工业化应用前景广阔。以下结合具体实施例对本发明做进一步详细说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。实施例中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。实施例中如果对于实验操作温度没有做出具体说明，则该温度通常指室温(10-30℃)。实施例材料和方法实施例中的全基因合成、引物合成及测序皆由南京金斯瑞生物科技有限公司完成。所用谷氨酸钠一水合物、标准品α-酮戊二酸和丁二酸均购自上海素元化工有限公司。lb培养基：10g/l胰蛋白胨，5g/l酵母提取物，10g/l氯化钠，ph7.2。(lb固体培养基另加20g/l琼脂粉。)实施例中的分子生物学实验包括质粒构建、酶切、连接、感受态细胞制备、转化、培养基配制等等，主要参照《分子克隆实验指南》(第三版)，j.萨姆布鲁克，d.w.拉塞尔(美)编著，黄培堂等译，科学出版社，北京，2002)进行。必要时可以通过简单试验确定具体实验条件。pcr扩增实验根据质粒或dna模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。hplc检测方法：流动相配方：6mmol/lh2so4流动相配比(buffer＝100％)色谱条件：进样量：5μl；流速：0.6ml/min，采集时间15min色谱柱：aminexhpx-87hdeae离子排阻柱(300mm×7.8mm)柱温：60，检测波长：206nm各成分保留时间分别为：酮戊二酸：8.2min；丁酮酸：11.3min；丁二酸：12.4min；乳酸：13.4min；乙酸：16.0min。需说明的是，为描述方便起见，在实施例中，可将菌株编号、酶编号、酶编码基因编号共用一个编号，这是本领域技术人员容易理解的，即同一个编号在不同环境中可以指代不同的生物形式。比如kata既可以代表菌株bl21(de3)/pet24a-lgox-kata，也可以代表酶seqidno:3编号、酶编码基因seqidno:4编号。实施例1l-谷氨酸氧化酶和过氧化氢酶共表达菌株的构建筛选来源于微生物的l-谷氨酸氧化酶和过氧化氢。将l-谷氨酸氧化酶(lgox)氨基酸序列(seqidno:1)和六种过氧化氢的酶氨基酸序列(kata氨基酸序列seqidno:3，kate氨基酸序列seqidno:5，katg氨基酸序列seqidno:7，makat氨基酸序列seqidno:9，pera氨基酸序列seqidno:11，tfucat氨基酸序列seqidno:13)提供给南京金斯瑞生物科技公司，针对大肠杆菌bl21(de3)进行核苷酸序列优化，以利于在大肠杆菌中表达。经密码子优化，编码l-谷氨酸氧化酶seqidno.1的基因的核苷酸序列为seqidno.2；编码过氧化氢酶seqidno.3的基因的核苷酸序列为seqidno.4；编码过氧化氢酶seqidno.5的基因的核苷酸序列为seqidno.6；编码过氧化氢酶seqidno.7的基因的核苷酸序列为seqidno.8；编码过氧化氢酶seqidno.9的基因的核苷酸序列为seqidno.10；编码过氧化氢酶seqidno.11的基因的核苷酸序列为seqidno.12；编码过氧化氢酶seqidno.13的基因的核苷酸序列为seqidno.14。将这些编码基因进行全基因合成，并分别通过ndei和bamhi酶切位点克隆到pet24a(+)上。将加载了上述编码基因的质粒分别通过氯化钙法转化到bl21(de3)感受态细胞中。涂布含有50mg/l卡那霉素的lb琼脂平板，挑出单菌落提取质粒，进行酶切验证，得到正确的分别表达l-谷氨酸氧化酶和表达6个过氧化氢酶的基因工程菌种。将各过氧化氢酶菌种提取质粒并纯化后，用bglii和bamhi限制性内切酶双酶切，回收并纯化小片段。将l-谷氨酸菌种提取质粒后，用bamhi限制性内切酶单酶切，回收纯化，分别采用t4dna连接酶连接该片段和包含各过氧化氢酶的小片段。采用氯化钙法转化bl21(de3)感受态细胞，涂布含有50mg/l卡那霉素的lb琼脂平板，挑出单菌落提取质粒，进行酶切验证，分别得到正确共表达l-谷氨酸氧化酶和各个过氧化氢酶的菌种：bl21(de3)/pet24a-lgox-kata，bl21(de3)/pet24a-lgox-kate，bl21(de3)/pet24a-lgox-katg，bl21(de3)/pet24a-lgox-makat，bl21(de3)/pet24a-lgox-pera，bl21(de3)/pet24a-lgox-tfucat。实施例2基因工程菌催化制备α-酮戊二酸的比较考察实施例1中构建的共表达l-谷氨酸氧化酶和过氧化氢酶的重组菌转化谷氨酸制备α-酮戊二酸的能力，具体方法为，将上述6个菌种分别接种摇瓶(蛋白胨12g/l，酵母提取物24g/l，甘油5g/l，三水磷酸氢二钾16.43g/l，磷酸二氢钾2.31g/l，1000ml种子培养基装入到5l摇瓶，121℃灭菌20min。)，在摇床上37℃培养至od600约5时，降温至30℃，加入终浓度0.03mmiptg(过滤除菌)溶液诱导，继续培养16小时后，台式离心机3500rpm离心30分钟，收集菌体，分别得到bl21(de3)/pet24a-lgox-kata(简称lgox+kata)，bl21(de3)/pet24a-lgox-kate(简称lgox+kate)，bl21(de3)/pet24a-lgox-katg(简称lgox+katg)，bl21(de3)/pet24a-lgox-makat(简称lgox+makat)，bl21(de3)/pet24a-lgox-pera(简称lgox+pera)，bl21(de3)/pet24a-lgox-tfucat(简称lgox+tfucat)的发酵菌体。均采用下面的方法制备α-酮戊二酸。转化体系：在3l发酵罐中，加入适量水和300g谷氨酸钠一水合物(纯度99％)，控制温度35℃，ph7.0，加入10g发酵菌体，定容到1l。转化条件：ph7.0，转速600-1000rpm，通气量1-2vvm/min，35℃，罐压0.07mpa，溶氧控制在30-40％左右。转化30小时，补充水使转化体积为1l，搅拌均匀，采用hplc检测α-酮戊二酸和丁二酸的浓度。结果见表1。表1、六个菌株制备α-酮戊二酸的转化数据共表达菌种α-酮戊二酸浓度(g/l)丁二酸浓度(g/l)转化率(％)lgox+kata223.15.496.2lgox+kate223.84.596.5lgox+katg216.110.393.2lgox+makat231.00.3899.6lgox+pera224.53.896.8lgox+tfucat221.07.395.3由表1中数据可以看出，菌株lgox+makat催化谷氨酸转化制备α-酮戊二酸的效果是最好的，转化率最高，不仅产物α-酮戊二酸浓度最高，而且副产物丁二酸的产量最低，表明l-谷氨酸氧化酶lgox与过氧化氢酶makat的组合优于lgox与过氧化氢酶kata、kate、katg、pera或tfucat的组合。以下实验重点考察该菌株，并考察反应条件的影响。实施例3采用bl21(de3)/pet24a-lgox-makat制备α-酮戊二酸向1l发酵罐中，加入200ml水和180g谷氨酸钠一水合物(99％)，通过夹套通热水控制温度35℃，ph7.0，加入6g实施例2中制备的bl21(de3)/pet24a-lgox-makat发酵菌体，用定容到600ml。底部通入纯氧气，通过顶部的搅拌器搅拌反应22小时，补加水使反应体系为600ml，取样，使用hplc检测α-酮戊二酸为231.4g/l，丁二酸为0.22g/l，α-酮戊二酸转化率为99.8％。实施例4采用bl21(de3)/pet24a-lgox-makat制备α-酮戊二酸将菌种接种摇瓶(蛋白胨12g/l，酵母提取物24g/l，甘油5g/l，三水磷酸氢二钾16.43g/l，磷酸二氢钾2.31g/l，200ml种子培养基装入到1l摇瓶，121℃灭菌20min)，37℃，220rpm，摇床培养12小时，用于接种发酵罐。在3l发酵罐中，配制1.5l培养基，成分为：硫酸铵4g/l，磷酸二氢钾5g/l，甘油10g/l，玉米浆15g/l，七水合硫酸镁1g/l，泡敌1‰。用碱液调节ph为7.0，121℃灭菌30min。降温至37℃后，接入种子培养基，开启通气和搅拌，维持溶氧≥20％。当溶氧上升后，以0.5ml/分钟的速度补加补料培养基(甘油400g/l，蛋白胨20g/l，121℃灭菌20min)。当od600至约20时，降温至22℃，加入终浓度0.03mm的iptg诱导18小时后放罐，台式离心机3500rpm离心30分钟收集菌体。在3l发酵罐中，加入适量水和300g谷氨酸钠一水合物(纯度99％)，控制温度35℃，ph7.0，加入10g发酵菌体，定容到1l，ph7.0，转速600-1000rpm，通气量1-2vvm/min，35℃，罐压0.07mpa，溶氧控制在30-40％左右。转化至20小时，溶氧回升后，补加谷氨酸钠一水合物50g，继续反应至40小时，补充水使转化体积恢复到1l，搅拌均匀后，取样，采用hplc检测α-酮戊二酸的浓度为268.4g/l，丁二酸浓度为0.44g/l，α-酮戊二酸转化率为99.2％。以上实施例仅用以说明本发明的技术方案，而非对其限制。尽管参照前述实施例对本发明进行了详细的说明，本领域技术人员应当理解，其对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换，这些修改或者替换，并不使相应技术方案的本质脱离本发明的技术方案的精神和范围。序列表<110>雅本化学股份有限公司湖州颐辉生物科技有限公司<120>微生物转化法制备α-酮戊二酸<130>shpi2010696<160>14<170>siposequencelisting1.0<210>1<211>847<212>prt<213>streptomycesspx-119-6<400>1metleuaspglnglnthrileasnileilelysalathrvalproval151015leulysgluhisglyvalthrilethrthrthrphetyrlysasnleu202530phealalyshisprogluvalargproleupheaspmetglyarggln354045gluserleugluglnprolysalaleualametthrvalleualaala505560alaglnasnilegluasnleuproalaileleuproalavallyslys65707580ilealavallyshiscysglnalaglyvalalaalaalahistyrpro859095ilevalglyglngluleuleuglyalailelysgluvalleuglyasp100105110alaalathraspaspileleuaspalatrpglylysalatyrglyval115120125ilealaaspvalpheileglnvalglualaaspleutyralaglnala130135140valglumetthrthraspthralaargarghisthrglyalagluarg145150155160alaasnglumetthrtyrgluglnleualaarggluleuleuleuval165170175glyproalaprothrasngluaspleulysleuargtyrleuaspval180185190leuileaspasnglyleuasnproproglyproprolysargileleu195200205ilevalglyalaglyilealaglyleuvalalaglyaspleuleuthr210215220argalaglyhisaspvalthrileleuglualaasnalaasnargval225230235240glyglyargilelysthrphehisalalyslysglygluproserpro245250255phealaaspproalaglntyralaglualaglyalametargleupro260265270serphehisproleuthrleualaleuileasp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