CN112300977B - 合成α-熊果苷的基因工程菌株及其构建方法与应用 - Google Patents
合成α-熊果苷的基因工程菌株及其构建方法与应用 Download PDFInfo
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Abstract
本发明公开了一种合成α‑熊果苷的基因工程菌株及其构建方法与应用,属于基因工程技术领域。本发明利用表达蔗糖磷酸化酶(SmSP)的重组枯草芽孢杆菌全细胞催化生产α‑熊果苷,为α‑熊果苷的生产提供了新的思路和方法。本发明通过优化蔗糖磷酸化酶(SmSP)的表达载体以及启动子,提高了SmSP的表达量,α‑熊果苷的产量由原始菌株的10.6g/L提升至41.0g/L。由于质粒表达不稳定,本发明将优化后的基因表达框P43‑gtfA整合至B.subtilis WB600基因组,获得基因工程菌株BSP43‑SmSP。当底物HQ的浓度为50g/L,蔗糖浓度为310.9g/L,菌体浓度OD600=20,催化体系在摇瓶中30℃、220rpm催化20h时,该菌株α‑熊果苷的产量稳定,且相比对照提升至61.1g/L,底物对苯二酚的摩尔转化率为49.4%。
Description
技术领域
本发明涉及一种合成α-熊果苷的基因工程菌株及其构建方法与应用,属于基因工程技术领域。
背景技术
α-熊果苷(4-氢醌-α-D-吡喃葡萄糖苷)是一种天然糖苷,包含1个葡萄糖基和1个酚基,之间通过α-1,4糖苷键相连。近年来,由于α-熊果苷有良好的对光辐射的耐褐变性和对酪氨酸酶的抑制活性,其在化妆品工业中被广泛用作美白剂。此外,α-熊果苷不仅可以减少皮肤色素的沉积,同时还有杀菌、消炎的作用,是一种新型的无刺激、无过敏的天然美白活性物质。目前合成α-熊果苷的方法有化学合成法、酶法合成以及发酵法。其中,化学合成法立体选择性差,产物大多都为混和物,不能制备单一的产物,并且反应过程所用的化学物质极易造成环境污染,也会存在消费者对产品有过敏反应等问题;发酵法生产α-熊果苷由于前期底物投入较多,底物转化率低,发酵周期较长,需要多种酶参与,且产量较低,因此不适用于大规模生产;而酶合成法副产物少,产物单一且易于分离提取,环境污染小,是目前生产α-熊果苷最常用的方法。
蔗糖磷酸化酶是一种同型二聚体的细胞内酶,属于糖基水解酶13家族。蔗糖磷酸化酶可在一步反应中将蔗糖转化为相应的糖基化产物,即将蔗糖的糖基转移至对应受体上,形成相应的产物。有研究表明,蔗糖磷酸化酶主要以蔗糖、1-磷酸-葡萄糖为糖基供体,多羟基的糖和糖醇、酚羟基、羧基等多类物质均可以作为该酶的糖基受体。蔗糖磷酸化酶由于其广泛的受体特异性,因而在高效生产α-熊果苷方面具有很大的潜力。迄今为止,在许多细菌中都发现了蔗糖磷酸化酶的存在,其中来自变异链球菌(Streptococcus mutans)和青春双歧杆菌(Bifidobacterium adolescentis)的蔗糖磷酸化酶最稳定。
枯草芽孢杆菌作为一种对环境无害的(GRAS)革兰氏阳性细菌,由于其具有非致病性、分泌蛋白能力强的特性和良好的发酵基础及生产技术,是目前原核表达系统中表达和分泌外源蛋白的理想宿主及重要的模式菌株。
本实验室前期选用来源于变异链球菌的蔗糖磷酸化酶(SmSP)在枯草芽孢杆菌中进行异源表达,以蔗糖和对苯二酚(HQ)为底物,全细胞转化合成α-熊果苷(如图1所示)。然而现有的构建的异源表达蔗糖磷酸化酶的枯草芽孢杆菌转化合成熊果苷的产量较低,不利于工业化生产。
因此,如何利用表达蔗糖磷酸化酶(SmSP)的枯草芽孢杆菌高效的催化合成α-熊果苷,是本领域亟待解决的问题。
发明内容
为解决上述技术问题,本发明提供了一种合成α-熊果苷的基因工程菌株及其构建方法与应用,该基因工程菌株可以稳定且较高效的合成α-熊果苷。
本发明的第一个目的是提供一种合成α-熊果苷的基因工程菌株,所述的基因工程菌株是在枯草芽孢杆菌宿主中,以P43为启动子,异源表达蔗糖磷酸化酶。
进一步地,所述的异源表达为游离表达或整合表达。
进一步地,所述的游离表达是以pP43NMK-P43质粒为载体进行表达。
进一步地,所述的整合表达是将连接有P43启动子的蔗糖磷酸化酶基因片段整合至整合位点ganA基因上。
进一步地,所述的蔗糖磷酸化酶的氨基酸序列如SEQ ID NO.1所示。
进一步地,所述的枯草芽孢杆菌宿主为枯草芽孢杆菌B.subtilis WB600、枯草芽孢杆菌B.subtilis 168或枯草芽孢杆菌B.subtilis WB800。
本发明的第二个目的是提供所述的基因工程菌株的构建方法,包括如下步骤:(1)将蔗糖磷酸化酶基因连接至含有P43的质粒上获得含有蔗糖磷酸化酶基因的表达载体,或将P43基因片段与蔗糖磷酸化酶编码基因片段相连后融合至整合框获得基因表达框,在表达框两端加上待整合位点上下游同源臂获得待同源重组的线性片段;
(2)将表达载体或线性片段转入枯草芽孢杆菌感受态中,筛选得到所述的基因工程菌株。
本发明的第三个目的是提供所述的基因工程菌株在全细胞催化合成α-熊果苷中的应用。
进一步地,所述的应用是将所述的基因工程菌株细胞作为催化剂,在25-35℃催化蔗糖和对苯二酚反应生产α-熊果苷;其中,催化体系包含OD600=10-100的基因工程菌细胞株,250-350g/L蔗糖,40-60g/L对苯二酚。
进一步地,所述的基因工程菌株细胞通过将所述的基因工程菌株接种至TB培养基中35-38℃培养10-15h得到。
本发明的有益效果是:
本发明利用表达蔗糖磷酸化酶(SmSP)的重组枯草芽孢杆菌全细胞催化生产α-熊果苷,为α-熊果苷的生产提供了新的思路和方法。本发明通过优化蔗糖磷酸化酶(SmSP)的表达载体以及启动子,提高了SmSP的表达量,α-熊果苷的产量由原始菌株的10.6g/L提升至41.0g/L。由于质粒表达不稳定,本发明将优化后的基因表达框P43-gtfA整合至B.subtilisWB600基因组,获得基因工程菌株BSP43-SmSP。当底物HQ的浓度为50g/L,蔗糖浓度为310.9g/L,菌体浓度OD600=20,催化体系在摇瓶中30℃、220rpm催化20h时,该菌株α-熊果苷的产量稳定,且相比对照提升至61.1g/L,底物对苯二酚(HQ)的摩尔转化率为49.4%。
附图说明
图1为蔗糖磷酸化酶的反应机制;
图2为基因工程菌株BS-HT-SmSP催化合成α-熊果苷产量;
图3为重组菌株BS-STOP-SmSP、BS-MA-SmSP和BS-NMK-SmSP催化合成α-熊果苷产量;
图4为重组菌株BS-NMK-SmSP、BS-spoVG-SmSP、BS-hbs-SmSP、BS-yvyD-SmSP、BS-srfA-SmSP和BS-abrB-SmSP催化合成α-熊果苷产量;
图5为重组菌BSP43-SmSP和BS-NMK-SmSP催化合成α-熊果苷产量。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
涉及的检测方法:
α-熊果苷的测定方法:
高效液相色谱(HPLC)检测法:Agilent 1200,UV检测器,Agilent SB-Aq色谱柱(4.6mm×250mm,5μm),流动相:10mmol/L的稀磷酸和甲醇(体积比为8:2),流速0.6mL/min,柱温35℃,进样体积为10μL。
实施例1:全细胞催化合成α-熊果苷
将待发酵的重组枯草芽孢杆菌于含有相应抗性的LB固体培养基上划线活化,37℃过夜培养。单菌落转接至添加有相应抗性的LB液体培养基(20mL),37℃摇床养10h,得到种子液。将种子液以1%(v/v)的接种量转接至30mL添加相应抗性的TB培养基中,37℃培养12h,即得发酵液。将发酵液离心(8,000rpm,10min,4℃)收集细胞,用20mmol/L的PB缓冲液(pH 7.0)洗涤两次。全细胞催化体系包含310.9g/L蔗糖,50g/L HQ,20mmol/L PB,细胞添加量为OD600=20。全细胞催化反应在50mL的密闭容器中进行,置于30℃摇床中震荡反应20h。全细胞催化反应结束后,12,000rpm离心10min,上清液置于4℃保存。
实施例2:基因工程菌株BS-HT-SmSP的构建
表1
根据枯草芽孢杆菌的密码子偏好性,人工合成来源于S.mutans UA159的gtfA基因,所述的基因序列如SEQ ID NO.1所示。以gtfA-F(BamH I)和gtfA-R(Sma I)为引物,PCR扩增gtfA基因,获得两端带有酶切位点的gtfA基因。选择大肠杆菌-枯草芽孢杆菌穿梭载体pHT01-Pgrac100作为表达载体,将gtfA基因通过酶切连接的方式,克隆到限制性酶切位点BamH I和Sma I之间,获得重组质粒pHT01-Pgrac100-gtfA,将该质粒转化入枯草芽孢杆菌B.subtilis WB600中,获得一种能够催化合成α-熊果苷的基因工程菌株BS-HT-SmSP。结果如图2所示,采用实施例1中的全细胞催化合成α-熊果苷的方法,α-熊果苷的产量在20h时基本达到最大值,为10.6g/L。
实施例3:蔗糖磷酸化酶(SmSP)表达载体的优化
表2
注:下划线表示酶切位点,斜体字母代表同源臂序列
通过将gtfA基因一步克隆连接至pSTOP1622-PxylA、pP43NMK-P43质粒,以及酶切连接至pMA0911.1-PHpaII质粒获得了三个重组表达载体pSTOP1622-PxylA-gtfA、pMA0911.1-PHpaII-gtfA和pP43NMK-P43-gtfA。将上述三个质粒分别转入B.subtilisWB600中,获得BS-STOP-SmSP、BS-MA-SmSP和BS-NMK-SmSP三个重组菌株,采用实施例1中的全细胞催化合成α-熊果苷的方法,对这三株重组菌与BS-HT-SmSP菌株做催化验证,结果如图3所示,BS-NMK-SmSP催化生成的α-熊果苷的产量在不同时间均高于其他三种重组菌株,最高产量为40.2g/L。
实施例4:蔗糖磷酸化酶(SmSP)启动子的优化
以质粒pP43NMK-P43-gtfA为模板,选择另外5种枯草芽孢杆菌内源性启动子PspoVG、PyvyD、Phbs、PsrfA、PabrB通过PCR替换P43启动子,随后转入B.subtilis WB600中。结果如图4所示,由不同启动子控制下的重组菌株BS-NMK-SmSP,BS-spoVG-SmSP,BS-hbs-SmSP,BS-yvyD-SmSP,BS-srfA-SmSP,BS-abrB-SmSP采用实施例1中的全细胞催化合成α-熊果苷的方法,经全细胞催化,分别能产生41.0g/L、36.7g/L、32.0g/L、7.4g/L、4.4g/L和16.2g/L的α-熊果苷,其中P43启动子控制下的重组菌株BS-NMK-SmSP生成α-熊果苷的产量最高。
实施例5:基因工程菌株BSP43-SmSP的构建
使用Cre-loxP系统进行基因组整合。选取整合位点ganA基因。首先构建待同源重组的线性片段,将构建好的壮观霉素整合框(线性spc基因两端带有loxP序列)通过融合PCR与P43-gtfA基因表达框融合成为spc-gtfA,在spc-gtfA的两端通过融合PCR分别加上ganA基因的上下游同源臂(各800bp),获得待同源重组的线性片段。将该线性片段转入枯草芽孢杆菌感受态中,涂布于壮观霉素平板,将菌P验证整合成功的转化子制备成感受态,将实验室保藏的温敏型Cre质粒转化入该感受态,添加IPTG诱导表达Cre重组酶消除Spc抗性基因,随后升温至40℃消除温敏型Cre质粒,获得BSP43-SmSP菌株。如图5所示,采用实施例1中的全细胞催化合成α-熊果苷的方法,BSP43-SmSP的α-熊果苷产量相比游离表达的BS-NMK-SmSP提高至61.1g/L,底物对苯二酚(HQ)的摩尔转化率为49.4%。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
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Claims (4)
1.一种合成α-熊果苷的基因工程菌株在全细胞催化合成α-熊果苷中的应用,其特征在于,所述的基因工程菌株是在枯草芽孢杆菌宿主中,以P43为启动子,异源表达蔗糖磷酸化酶;
所述的异源表达为整合表达;
所述的整合表达是将连接有P43启动子的蔗糖磷酸化酶基因片段整合至整合位点ganA基因上;
所述的蔗糖磷酸化酶的氨基酸序列如SEQ ID NO.1所示;
所述的枯草芽孢杆菌宿主为枯草芽孢杆菌B. subtilis WB600;
所述的应用是将所述的基因工程菌株细胞作为催化剂生产α-熊果苷,其中,催化体系包含OD600=20的基因工程菌株细胞,310.9 g/L蔗糖,50 g/L 对苯二酚。
2.根据权利要求1所述的应用,其特征在于,基因工程菌株的构建方法包括如下步骤:
(1)将蔗糖磷酸化酶基因连接至含有P43的质粒上获得含有蔗糖磷酸化酶基因的表达载体,或将P43基因片段与蔗糖磷酸化酶编码基因片段相连后融合至整合框获得基因表达框,在表达框两端加上待整合位点上下游同源臂获得待同源重组的线性片段;
(2)将表达载体或线性片段转入枯草芽孢杆菌感受态中,筛选得到所述的基因工程菌株。
3.根据权利要求1所述的应用,其特征在于,所述的应用是将所述的基因工程菌株细胞作为催化剂,在25-35℃催化蔗糖和对苯二酚反应生产α-熊果苷。
4.根据权利要求1所述的应用,其特征在于,所述的基因工程菌株细胞通过将所述的基因工程菌株接种至TB培养基中35-38℃培养10-15 h得到。
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