一种抗酸胁迫重组乳酸菌及其应用的制作方法

本发明涉及一种抗酸胁迫重组乳酸菌及其应用，属于生物工程技术领域。

背景技术：

当乳酸菌被用作工业化生产之后，在其发酵过程中，随着菌体的代谢生长过程，酸性物质也随之产生并积累，导致细胞面临严重的酸胁迫。为维持发酵生产的稳定性并提高生产效率，工业上通常在发酵的过程中通过添加外源中和剂来维持ph处于稳定的范围。例如通过添加碱性物质(氨水或naoh)来控制发酵环境的ph值。然而碱性物质的添加往往会导致副产物的积累。而副产物中形成的盐类会再次导致细胞处于高渗的环境，从而造成渗透压胁迫的产生，再次影响菌体的生长与代谢。

在低ph的环境条件下，微生物细胞活性显著降低，从而导致发酵产物的生产效率显著下降。因此提高乳酸菌的酸胁迫耐受性对于其在发酵生产中的应用有着重要的意义。目前提高乳酸菌的酸胁迫耐受性的方法主要有：(1)诱变育种，该方法具有简便、类型繁多等特点，但工作量大、效率低是其主要缺点；(2)生化工程策略，已有报道用过外源添加天冬氨酸以提高乳酸菌的酸胁迫耐受性，但该方法的使用造成了生产成本的增加；(3)代谢工程策略，目前利用代谢工程策略提高乳酸菌环境胁迫的方法主要包括构建新的代谢途径、拓展已有代谢途径和削弱已有代谢途径。上述方法或存在成本问题，或存在成功率低的问题。

技术实现要素：

本发明的目的是提供一种抗酸胁迫元器件以提高乳酸乳球菌酸胁迫抗性。

本发明首先提供了抗酸胁迫元器件，其氨基酸序列如seqidno.1所示。

本发明还提供了一株酸胁迫抗性提高的重组乳酸乳球菌，过量表达转运蛋白trag。

在本发明的一种实施方式中，所述trag的氨基酸序列是seqidno.1所示的序列。

在本发明的一种实施方式中，编码所述trag的核苷酸序列是如seqidno.2所示的序列。

在本发明的一种实施方式中，编码所述trag的核苷酸序列来源于lactococcuslactisnz9000。

在本发明的一种实施方式中，所述重组菌的宿主为乳酸乳球菌lactococcuslactisnz9000。

本发明解决还提供了一种所述重组菌的构建方法，是将编码seqidno.1所示氨基酸序列的基因连接到表达质粒上获得重组质粒，再分别转化到宿主菌中获得重组菌。

在本发明的一种实施方式中，所述表达质粒为pnz8148。

在本发明的一种实施方式中，所述宿主菌是lactococcuslactisnz9000。

在本发明的一种实施方式中，所述构建方法具体是：将seqidno.2所示的核苷酸序列克隆到表达质粒pnz8148上，获得重组质粒pnz8148/trag，再将重组质粒转化到宿主菌lactococcuslactisnz9000中，得到重组菌株lactococcuslactisnz9000(pnz8148/trag)。

本发明还提供一种提高乳酸乳球菌酸胁迫抗性的方法，是在乳酸乳球菌中过量表达转运蛋白trag。

在本发明的一种实施方式中，所述表达转运蛋白trag的氨基酸序列是seqidno.1所示的序列。

在本发明的一种实施方式中，所述方法具体是：将seqidno.2所示的核苷酸序列克隆到表达质粒pnz8148上，获得重组质粒pnz8148/trag，再将重组质粒转化到宿主菌lactococcuslactisnz9000中，得到重组菌株lactococcuslactisnz9000(pnz8148/trag)，诱导表达trag。

本发明还提供所述重组乳酸乳球菌在食品、饲料、精细化学品领域的应用。

本发明通过在乳酸乳球菌中过量表达trag转运蛋白，得到了一株酸胁迫抗性显著提高的重组乳酸菌lactococcuslactisnz9000(pnz8148/trag)。在酸胁迫条件下，ph4.0胁迫3h后，重组菌株lactococcuslactisnz9000(pnz8148/trag)的存活率为对照的61.3倍。

附图说明

图1：重组质粒pnz8148/trag的结构图；

图2：重组菌株和对照菌株的生长曲线；

图3：ph4.0条件下重组菌株与对照菌株的存活率对比。

具体实施方式

下面结合具体实施例对本发明做更详细的说明。

实施例1重组菌株的构建

从ncbi数据库的l.lactisnz9000中得到如seqidno.2所示的trag的基因序列，并将其克隆到乳酸乳球菌表达质粒pnz8148上，获得重组质粒pnz8148/trag，再将其电转入宿主菌l.lactisnz9000中，得到重组菌株l.lactisnz9000(pnz8148/trag)。

具体如下：

根据trag的基因序列设计分别如seqidno.3、seqidno.4所示的引物trag-f、trag-r(表1)，以l.lactisnz9000的基因组为模板pcr扩增，获得seqidno.2所示的基因片段。将pcr产物和载体pnz8148分别用ncoⅰ和hindⅲ双酶切，酶切产物经纯化后，进行连接。连接产物转化大肠杆菌mc1061(商业化菌株)感受态，氯霉素平板上筛选阳性克隆，经菌落pcr验证和酶切验证，片段大小正确后再进行测序鉴定，最终获得含有正确序列的重组质粒pnz8148/trag(重组质粒结构如图1所示)。然后从重组mc1061中提取重组质粒，电转化l.lactisnz9000感受态细胞，氯霉素平板上筛选阳性克隆，经菌落pcr验证和酶切验证，片段大小正确后，最终获得含有正确重组质粒的菌株l.lactisnz9000(pnz8148/trag)。

电转化条件为：1μl质粒中与40μl的感受态细胞混合，移入预冷的电转杯中，冰上放置10min。电压2000v，电容25μf，电阻200ω。电击完毕后，立即向电转杯中加入1ml含有20mmmgcl2和2mmcacl2的gm17培养基(培养基配方：m17培养基+0.5％glucose)。然后置于30℃静置培养1.5h，涂布于含有氯霉素的gm17平板上，培养36h，挑取转化子验证。

表1引物

实施例2过量表达trag菌株的生长性能试验

对于考察菌株在过量表达trag时的生长情况，将菌株l.lactisnz9000(pnz8148/trag)和l.lactisnz9000(pnz8148)(对照)接种于加了10μg/ml氯霉素的gm17液体培养基(1ml)中进行活化，放在30℃培养箱中静置培养过夜。再以2％的接种量将种子液转接至新鲜的氯霉素(10μg/ml)gm17液体培养基中，30℃静置培养。每隔2小时取样，测定600nm波长下的od值。培养至od6000.4时加入10ng/ml的nisin诱导表达trag蛋白。以时间为横坐标，od600值为纵坐标，绘制生长曲线。

结果如图2所示。经生长性能试验分析，重组菌株的生物量和对照菌株无明显差异，说明在l.lactisnz9000中过量表达trag蛋白对菌株的生长性能没有影响。

实施例3酸胁迫条件下耐受性试验

对于考察菌株对酸的耐受性分析试验，分别测定了重组菌株和对照菌株在ph4.0条件下的存活率。

具体操作方式如下：将菌株诱导培养6h，离心收集细胞，经0.85％的生理盐水洗涤两次后重悬于等体积的新鲜的ph4.0(乳酸调节)的gm17(含有10μg/ml氯霉素)中，胁迫不同时间。胁迫后的菌悬液洗涤两次后重悬于等体积的生理盐水中，取10μl重悬液，稀释不同梯度点种于gm17氯霉素平板上测定活菌数和存活率。

经耐受性实验分析，在ph4.0的gm17中胁迫3h后，重组菌株l.lactisnz9000(pnz8148/trag)的存活率为对照的61.3倍，说明重组菌株对酸胁迫的耐受性显著提高。说明通过在l.lactisnz9000中过量表达trag的方法可以提高乳酸乳球菌酸胁迫抗性。

虽然本发明已以较佳的实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

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thrthrglyglycysthralaglyalaalaalathrcyscysala

170017051710

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thrthrthralaalacysthralathrcysglycyscysglythr

173017351740

thralacysalathrglyalaalathrglyalaalaglyalaala

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glyalaalaalaalaalathrthrglycysthrthrglyalathr

176017651770

glyalathrglythrglycysglyalacysglythrglyglycys

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glycysthralacysthralathrthrthrthralaalaalathr

179017951800

glyalathrcysalathrglyglyalaalathrglyalathrthr

180518101815

cysalaalathrcysthrglyalaalaalathrthrcyscysthr

182018251830

glycysthrthralaala

1835

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