CN111393514B - 过氧化物转录抑制因子突变体、突变基因及其在制备维生素b2中的应用 - Google Patents
过氧化物转录抑制因子突变体、突变基因及其在制备维生素b2中的应用 Download PDFInfo
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Abstract
本发明公开了一种过氧化物转录抑制因子突变体、突变基因及其在制备维生素B2中的应用。其中所述突变体的氨基酸序列相对于SEQ ID No.3所示序列存在下述突变：第71位氨基酸替换为V。对枯草芽孢杆菌染色体上的过氧化物转录抑制因子基因中第71位氨基酸编码核苷酸进行定点突变而编码氨基酸V，而获得的基因工程菌，其生产维生素B2的能力得到了较大幅度的提升，并且对菌的生长有利，因而具有较大的应用推广价值。
Description
技术领域
本发明属于生物技术领域，具体涉及过氧化物转录抑制因子的突变体，其基因工程菌，以及在制备维生素B2中的应用。
背景技术
核黄素（Riboflavin）又称维生素B2，分子式为C17H20O6N4，是维生素B族中的一种水溶性维生素，在生物体内以黄素单核苷酸（FMN）和黄素腺嘌呤二核苷酸（FAD）两种形式存在，作为机体内一些重要的氧化还原酶的辅酶参与氧化还原反应，起到递氢的作用。核黄素的缺乏会导致生物体代谢发生障碍，但人和动物均不能自身合成，只能从食物中摄取，因此核黄素的生产在食品、饲料以及医药行业都具有非常广阔的市场。
目前核黄素的工业生产方法有植物萃取法、化学合成法、半合成法以及微生物合成法。其中，微生物合成法以其成本低、环境友好以及能源可再生等优点逐步占据主导地位，成为大部分工业化生产的主要方法。在众多可生产核黄素的微生物中，枯草芽孢杆菌（B.subtilis）作为一种非病原微生物，生理代谢及遗传背景清楚，便于确定代谢靶点及基因工程改造，并具有可靠的安全性，它们的发酵产物在食品与饲料业中已有长期的应用，对环境、医药和工业发酵生产都非常重要。其次枯草芽孢杆菌基因工程菌株能够过量合成叶酸、肌苷或鸟苷，具有为核黄素过量合成提供足够前体物的潜力，因此枯草芽孢杆菌基因工程菌在核黄素的微生物发酵生产中逐渐显示出了强大的生命力，成为主要生产菌株。
枯草芽孢杆菌（B.subtilis）中核黄素的合成需要5-磷酸核酮糖（Ru5P）和鸟嘌呤-5’ -三磷酸（GTP）两个前体物质，其中Ru5P来源于磷酸戊糖途径，GTP来源于嘌呤的从头合成途径。最终5-磷酸核酮糖和GTP在核黄素操纵子编码的酶的催化下经过7步反应生成核黄素。
过氧化物转录抑制因子（PerR）主要存在于革兰氏阳性细菌中，其功能为控制抗氧化基因的表达（Wang X,Tong H,Dong X. PerR-regulated manganese ion uptakecontributes to oxidative stress defense in an oral streptococcus.[J].Appliedand environmental microbiology,2014,80(8):2351-2359.），是许多细菌中过氧化物胁迫防御的主要调节剂。在有氧代谢过程中过氧化物的感应对细胞存活尤为重要，在氧化条件的刺激下PerR会在体内降解（Ahn Bo-Eun,Baker Tania A. Oxidization withoutsubstrate unfolding triggers proteolysis of the peroxide-sensor, PerR.[J].Proceedings of the National Academy of Sciences of the United States ofAmerica,2016,113(1):23-31.）。细菌通过控制胞内PerR的表达水平来调控过氧化物抵御因子基因的表达，以此种方式应对过氧化物胁迫压力。在枯草芽孢杆菌中，PerR主要作用方式是经由过氧化物的胁迫来改变蛋白构象，进而调节过氧化物抗性基因（Kebouchi M,SaulF,Taher R,et al.Structure and function of the Leptospira interrogans peroxidestress regulator (PerR), an atypical PerR devoid of a structural metal-binding site.[J]. The Journal of biological chemistry,2018,293(2):497-509.），其编码基因perR的转录水平并不受过氧化氢的影响，编码过氧化氢酶（KatA）、烷基过氧化氢还原酶（AhpC和F）以及铁储存蛋白（MrgA）的基因都由PerR调控（Lilliam C, Adam D,Barbara S,et al.Lack of a signi¢cant role for the PerR regulator in Bacillus subtilis spore resistance.[J].FEMS Microbiology Letters,2000,188: 203-208.）。调控基因perR的转录表达水平可以在一定程度上有助于菌株的生长，进而提高所需目标物的产量。目前，尚未有过氧化物转录抑制因子突变基因perR用于维生素B2合成的报道。
发明内容
本发明人前期筛选出了一株高产维生素B2的枯草芽孢杆菌CGMCC NO. 16132菌株（见中国专利申请201910604170.3），可发酵生产维生素B2。本发明人对其作了进一步研究以发现对产维生素B2能力有影响的基因。经过研究发现一种过氧化物转录抑制因子编码基因的突变，通过验证在枯草芽孢杆菌中将过氧化物转录抑制因子编码基因定点突变为所述突变基因则能够提高所述菌生产维生素B2的能力。
首先，本发明提供一种过氧化物转录抑制因子的突变体，其特征在于，其多肽氨基酸序列在SEQ ID No.3所示原始序列的基础上存在下述突变：第71位氨基酸替换为V。
优选地，其氨基酸序列如SEQ ID No.4所示。
其次，本发明提供所述的过氧化物转录抑制因子的突变体的编码基因。
优选地，所述核苷酸序列如SEQ ID No.2所示。
相应地，本发明第三方面还提供含有所述过氧化物转录抑制因子的突变体的编码基因的表达盒、重组载体。所述重组载体对出发载体并没有特别的限制，可为本领域中已知的任何载体，只要它能够在宿主中进行复制即可。例如，所述载体包括但不限于质粒，噬菌体。一旦转化入合适的宿主之后，所述载体可以复制并独立于宿主基因组发挥功能，或者在某些情况下整合入基因组本身。
更优选地是重组表达载体，更优选原核重组表达载体。最优选是适合于在枯草芽孢杆菌中进行表达的表达载体。
第四方面，本发明提供含有所述过氧化物转录抑制因子的突变体的编码基因的重组宿主细胞。其中所述“宿主细胞”是具有本领域通常理解的含义，其能够导入本发明的突变体的编码基因的细胞，导入之后称为重组宿主细胞。本发明的宿主细胞可以是原核细胞或真核细胞，优选为原核细胞，更优选为枯草芽孢杆菌。
第五方面，本发明提供过氧化物转录抑制因子突变体编码基因在制备维生素B2中的应用。
第六方面，本发明提供一种增强枯草芽孢杆菌产维生素B2的方法，其是将染色体上的过氧化物转录抑制因子编码基因进行定点突变，获得产维生素B2的能力增强的枯草芽孢杆菌，其中所述定点突变是将所述编码基因的第71位氨基酸编码核苷酸替换为编码氨基酸V编码核苷酸，更具体的是所述编码基因的第211位核苷酸由G置换了T。其中的定点突变可以采用本领域已知的各种方法来实现。
优选地，所述枯草芽孢杆菌的原始菌株是枯草芽孢杆菌BS168 ribCm的菌株。
本发明第六方面，提供一种利用上述第五方面的方法获得的枯草芽孢杆菌制备维生素B2的方法，其包括培养所述枯草芽孢杆菌，以及收集维生素B2的步骤，更优选还包括纯化维生素B2的步骤。
其中枯草芽孢杆菌的培养可以根据本领域的常规方法进行，例如摇瓶培养、批次培养、连续培养和分批补料培养等，并可以根据实际情况选择合适的培养条件如温度、时间和培养基的pH值等。此外，可以从细胞或培养基中回收或纯化维生素B2的方法可采取本领域常规的方法，例如过滤、阴离子交换色谱、结晶和HPLC等方法。
本发明的有益效果在于：经研究证实，本发明中含有过氧化物转录抑制因子突变基因的基因工程菌生物安全，实验表明染色体上基因突变不仅未对菌体的生长造成影响，反而对菌体的生长有益，故可以有效的提高枯草芽孢杆菌生产维生素B2的能力。实验数据表明，将BS168 ribCm中过氧化物转录抑制因子编码基因perR变为突变基因perR（L71V）可以提高产维生素B2的能力达48.1%，因而在制备维生素B2中具有较大的应用价值。
附图说明
图1：不同枯草芽孢杆菌菌株发酵41h后的VB2产量。
图2：不同枯草芽孢杆菌菌株发酵41h后的生物量。
具体实施方式
本发明的以下实施例和附图仅说明实现本发明的具体实施方案，这些方案和附图不可以理解为对本发明的限制，任何在不脱离本发明的原理和实质的情况下所做的任何改变，均落在本发明的保护范围之内。
本实施例中所用到的实验技术与实验方法，如无特殊说明均为常规技术方法。本实施例中所使用的材料、试剂等，如无特殊说明，均可通过正规商业渠道获得。
培养基配方：
LB 培养基（g/L）：氯化钠 10，胰蛋白胨10，酵母提取物5，固体培养基加琼脂粉18。
发酵培养基（g/L）：玉米浆干粉10、蔗糖30、硫酸镁2、硫酸铵7、磷酸氢二钾3、磷酸二氢钾1，用NaOH调节pH至7.2～7.4。
维生素B2的检测方法
发酵液混匀，用0.01mol/L NaOH将其稀释到适当的倍数后，混匀，避光碱溶20min，12000rpm离心2min，取上清液以0.01mol/L NaOH做空白在444nm下测定吸光度（显示值控制在0.2-0.8之间），按以下公式计算核黄素的含量：FB（mg/L）=（稀释倍数*吸光度）/0.0321。
实施例1：菌株CGMCC NO. 16132与BS168 ribCm进行比较基因组分析
以菌株BS168 ribCm（具体构建过程参见中国专利申请201910604170.3）的基因组为参考基因组，将菌株CGMCC NO. 16132送到金唯智生物科技有限公司进行全基因组重测序及差异性分析，以发现对产维生素B2能力有影响的基因。通过序列比对分析，发现菌株CGMCC NO. 16132中发生了大量的突变，共包含338个突变位点：经过突变数据统计，突变共涉及72个基因。编码区域内的突变共涉及59个基因，其中同义突变8个、非同义突变41个、无义突变2个、移码突变5个、非移码突变3个；非编码区内的突变共涉及13个基因。
去除同义突变以及未知基因功能的基因突变，检索由葡萄糖到维生素B2生物合成涉及到的代谢途径，侧重考察途径富集分析相关代谢途径中参与酶催化、外排以及代谢调控的基因突变，初步筛选到一些可能对产维生素B2能力有影响的基因，包括维生素B2合成途径直接相关的突变基因、嘌呤外排泵突变基因、嘌呤合成途径调控突变基因、嘌呤降解途径突变基因等。其中，经分析发现过氧化物转录抑制因子编码基因perR发生了点突变，其第211位核苷酸由G置换了T（未突变的该基因核苷酸序列如SEQ ID No.1所示，突变后的核苷酸序列如SEQ ID No.2所示）。为了验证突变位点对维生素B2产量的影响，对出发菌株BS168ribCm中的perR基因进行了点突变。
实施例2：构建含perR突变体的菌株
以Bacillus subtilis 168染色体为模板，用引物UPperRm-F、UPperRm-R（含DR）扩增带接头的且带有点突变的UPperRm（含DR）片段，用引物araR-F、araR-R（含DR）扩增带接头的araR（含DR）片段；以pC194质粒为模板，用引物cat-F、cat-R扩增带接头的cat片段；以菌株CGMCC NO. 16132的染色体为模板，用引物DNperR-F、DNperR-R扩增下游同源臂片段DNperR，其中所述突变的perR的核苷酸序列如SEQ ID No.2所示，编码的氨基酸序列如SEQID No.4所示；相对于SEQ ID No.3所示原始氨基酸序列的基础上存在下述突变：第71位氨基酸替换为V。
以UPperRm片段、cat片段、araR片段和DNperR片段为模板，用引物UPperRm-F、DNperR-R进行融合PCR，得到组装片段UCR-perRm，经核酸电泳检测正确，胶回收后得到纯化的UCR-perRm片段。
将UCR-perRm片段Spizizen转化BS168 ribCm，涂布于含有8mg/L氯霉素的LB固体平板上，培养24h后进行菌落PCR验证，核酸电泳正确的送金唯智测序，测序正确后，获得perR基因带有点突变的中间菌株BS168 UCR-perRm，其中所述突变的perR的核苷酸序列如SEQID No.2所示，编码的氨基酸序列如SEQ ID No.4所示。
挑取中间菌株BS168 UCR-perRm的单菌落于含有5mL LB的试管中，37℃振荡培养8h后，取200uL菌液涂布于含有40mg/L新霉素的LB固体平板上，培养24h后进行菌落PCR验证，核酸电泳正确的送金唯智测序，测序正确后，获得了染色体内部通过DR发生同源重组而去掉筛选标记cat-araR且perR基因带有点突变的菌株BS168 perRm，其中所述突变的perR的核苷酸序列如SEQ ID No.2所示，编码的氨基酸序列如SEQ ID No.4所示。
本部分所用引物如下：
表1 构建含perR突变体的菌株所用到的引物
本部分所用到的菌株及质粒如下：
表2 构建含perR突变体的菌株所用到的菌株和质粒
| 菌株与质粒 | 相关性质 |
| <i>B. subtilis </i>168菌株 | <i>trpC2</i> |
| BS168 ribC<sup>m</sup>菌株 | <i>trpC2</i>，质粒pGMBsub04过表达了<i>rib</i> operon，△<i>araR</i>：：Para-neo，Em<sup>r</sup>，Nm<sup>r</sup>，<i>ribC</i><sup>m</sup> |
| BS168 UCR-perR<sup>m</sup>菌株 | BS168 ribC<sup>m</sup>衍生菌株，<i>trpC2</i>，Em<sup>r</sup>，Nm<sup>s</sup>，Cm<sup>r</sup>，<i>ribC</i><sup>m</sup> |
| BS168 perR<sup>m</sup> | BS168 UCR-perR<sup>m</sup>衍生菌株，<i>trpC2</i>，Em<sup>r</sup>，Nm<sup>r</sup>，Cm<sup>s</sup>，<i>ribC</i><sup>m</sup>，<i>perR</i><sup>m</sup> |
| pC194质粒 | Cm<sup>r</sup>，Staphylococcus aureus plasmid |
实施例3：评价不同菌株的维生素B2生产能力
1、菌株培养条件：
将出发菌株BS168 ribCm、和工程菌株BS168 perRm在无菌条件下用接种针划线含有25mg/L红霉素的LB固体平板，在37℃培养箱中倒置培养24h，以获得新鲜活化的单菌落。用接种针挑取单菌落划线含有25mg/L红霉素的LB固体斜面，在37℃培养箱中培养48h。刮取斜面上1/3的菌苔接种含有70mL发酵培养基的500mL挡板三角瓶中（每株菌3个平行），37℃、200rpm振荡培养41h后取发酵液测定OD600和维生素B2产量。
、不同菌株OD600和维生素B2产量的比较
与出发菌株BS168 ribCm相比，含有过氧化物转录抑制因子突变基因perR（L71V）的工程菌株BS168 perRm的维生素B2产量提高了48.1%（参见下表3和图1）。
表3不同菌株OD600和维生素B2产量的比较
| 菌株 | OD<sub>600</sub> | 维生素B<sub>2</sub>产量（g/L） |
| 出发菌株BS168 ribC<sup>m</sup> | 15.82±0.357 | 1.04±0.043 |
| 工程菌株BS168 perR<sup>m</sup> | 18.98±0.405 | 1.54±0.032 |
因此，实验结果表明，本发明中将过氧化物转录抑制因子编码基因perR变为突变基因perR（L71V）的这种遗传修饰能提高菌株生产维生素B2的能力。另外，含有过氧化物转录抑制因子突变基因perR（L71V）的工程菌株BS168 perRm的生物量高于出发菌株BS168ribCm，说明perR基因的点突变对菌体的生长有益（见表3和图2）。
序列表
<110> 中国科学院天津工业生物技术研究所
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gacgatatat ataaagctct ggaagggaaa tttcctaaca tgagcgtagc gacggtatat 180
aacaatttgc gtgtgttccg ggaatcaggt ttggtaaaag agctcacata cggtgatgct 240
tccagcagat tcgattttgt cacatccgat cactatcacg cgatttgcga aaactgcggt 300
aaaattgtgg acttccacta cccgggcctt gatgaagtgg agcagctcgc tgcccacgtc 360
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atggctgcac atgaactaaa agaagcctta gaaacgttga aggaaaccgg agttcgcatt 60
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gacgatatat ataaagctct ggaagggaaa tttcctaaca tgagcgtagc gacggtatat 180
aacaatttgc gtgtgttccg ggaatcaggt gtggtaaaag agctcacata cggtgatgct 240
tccagcagat tcgattttgt cacatccgat cactatcacg cgatttgcga aaactgcggt 300
aaaattgtgg acttccacta cccgggcctt gatgaagtgg agcagctcgc tgcccacgtc 360
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Val Phe Arg Glu Ser Gly Val Val Lys Glu Leu Thr Tyr Gly Asp Ala
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Glu Asn Cys Gly Lys Ile Val Asp Phe His Tyr Pro Gly Leu Asp Glu
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Val Glu Gln Leu Ala Ala His Val Thr Gly Phe Lys Val Ser His His
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Claims (9)
1.一种过氧化物转录抑制因子的突变体，其特征在于，其多肽氨基酸序列相对于SEQID No.3所示序列仅存在下述突变：第71位氨基酸替换为V。
2.如权利要求1所述的过氧化物转录抑制因子的突变体的编码基因。
3.如权利要求2所述的编码基因，其特征在于，核苷酸序列如SEQ ID No.2所示。
4.含有权利要求2所述的过氧化物转录抑制因子的突变体的编码基因的重组载体。
5.含有权利要求2所述的过氧化物转录抑制因子的突变体的编码基因的重组宿主细胞。
6.如权利要求1所述的过氧化物转录抑制因子的突变体、或其编码基因在制备维生素B2中的应用。
7.一种增强枯草芽孢杆菌产维生素B2的方法，其特征在于，将染色体上的编码如SEQ IDNo.3所示氨基酸序列的过氧化物转录抑制因子编码基因进行定点突变，获得产维生素B2的能力增强的枯草芽孢杆菌，其中所述定点突变是将所述编码基因的第71位氨基酸L的编码核苷酸定点突变为编码氨基酸V。
8.如权利要求7所述的方法，其特征在于，所述枯草芽孢杆菌以枯草芽孢杆菌BS168ribCm菌株作为出发菌株。
9.一种利用权利要求7或8所述的方法获得的枯草芽孢杆菌制备维生素B2的方法，其特征在于，包括培养所述枯草芽孢杆菌，以及收集维生素B2的步骤。
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