CN116732081A - 一种提高枯草芽孢杆菌合成七烯甲萘醌的方法、获得重组菌株及其应用 - Google Patents
一种提高枯草芽孢杆菌合成七烯甲萘醌的方法、获得重组菌株及其应用 Download PDFInfo
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Abstract
本发明公开了一种提高枯草芽孢杆菌合成七烯甲萘醌的方法、获得重组菌株及其应用。本发明通过敲除枯草芽孢杆菌的nudF和yclB基因减少DMAPP消耗的同时强化细胞电子传递链的电子供应，促进MK‑7的前体合成并且增强电子供应量，达到提高七烯甲萘醌高效合成的目的。实验表明，本发明分别敲除或同时敲除nudF和yclB得到的重组菌株，七烯甲萘醌的产量分别提升至野生型BS168的204%、183%、260%，因此具有较大的实用价值，有利于降低生产七烯甲萘醌的成本。
Description
技术领域
本发明涉及属于生物技术领域，具体涉及一种提高枯草芽孢杆菌（Bacillus subtilis）合成七烯甲萘醌的方法、获得重组菌株及其应用。
背景技术
七烯甲萘醌（MK-7）由甲基萘醌母环和母环 C-3 位置上连接的 7 个异戊二烯单元的异戊二烯侧链组成。MK-7对人体具有多种生理功能，在保护骨骼健康、预防心血管疾病等方面具有良好的效果，并且在糖尿病、慢性肾脏疾病、免疫紊乱和阿尔茨海默氏病等疾病的治疗方案中起着关键作用。作为维生素K2（Vitamin K2）的一种亚型，MK-7在人体中具有亲缘性高、半衰期长等优点。
枯草芽孢杆菌（Bacillus subtilis）作为一种食品安全级的模式生物，具有非致病性、遗传背景清晰、无密码子偏好性和基因操作工具齐全等优点，目前已广泛应用于生物合成和代谢工程领域的研究当中。枯草芽孢杆菌是MK-7的主要生产者，MK-7在枯草芽孢杆菌中的代谢过程主要分为四个模块：甘油异化途径、SA途径、MEP途径和MK-7途径。枯草芽孢杆菌摄入甘油后经甘油异化途径和糖酵解途径合成甘油醛-3磷酸（G3P）、丙酮酸（PYR）及磷酸烯醇式丙酮酸（PEP），PYR与G3P经MEP途径的合成七异戊二烯基焦磷酸（HDP）为MK-7的合成提供异戊二烯侧链。PEP和赤藓糖-4-磷酸（E4P）进入SA途径合成分支酸（CHA），再通过MK-7途径七步酶催化反应合成1, 4-二羟基-2-萘甲酸（DHNA）为MK-7的合成提供甲萘醌母环，最后HDP和DHNA通过1,4-二羟基-2-萘酸庚二烯基转移酶（menA）和甲基转移酶（menG）的催化最终生成MK-7。MEP途径是细菌中萜烯类化合物的主要合成途径，MK-7的关键前体HDP需要依靠IPP的级联放大反应来合成。在异戊烯醇合成途径当中，nudF基因编码的核分布蛋白催化IPP和DMAPP分别合成两种互为同分异构体的异戊烯醇，这与MK-7的合成途径形成竞争关系，通过敲除nudF来减少关键中间代谢产物DMAPP的消耗，有助于MK-7关键前体HDP的合成。MK-7在枯草芽孢杆菌的电子传递链中作为电子受体承担运输电子的功能，对枯草芽孢杆菌的生命活动具有重要意义。强化细胞电子传递链的电子供应来促进MK-7合成是枯草芽孢杆菌合成MK-7的工程改造方向之一，还原形式的黄素辅酶FMNH2通过将氢原子转移到MK-7上来实现电子在细胞膜上的传递，从而形成枯草芽孢杆菌一系列的有氧呼吸活动。而yclB基因编码的酚酸脱羧酶催化FMNH2的戊基化过程，FMNH2的消耗削减了电子传递链中的电子供应量，yclB基因的敲除有助于增强细胞电子传递链中的电子供应。
虽然在MK-7的研究中阻断产物合成途径中的分支途径是常用的改造策略，但是nudF和yclB两个基因并未直接出现在MK-7的合成途径中，且通过敲除nudF和yclB基因来促进MK-7的合成也未有相关研究报道。
发明内容
因此，本发明通过对菌株中公用中间代谢前体和电子传递共有受体进行综合分析，选择基因nudF和yclB为改造靶点，采用了分别敲除或同时敲除分支代谢途径基因nudF和yclB的方法来增加MK-7合成途径代谢通量和强化细胞电子传递链的电子供应，从而促进枯草芽孢杆菌中MK-7的合成。
本发明首先提供一种提高枯草芽孢杆菌合成七烯甲萘醌的方法，所述的方法是在出发枯草芽孢杆菌中敲除nudF和yclB基因中一个或两个实现。
优选地，所述的方法是在出发枯草芽孢杆菌中同时敲除nudF和yclB基因实现。
在一个具体实施方式中，所述nudF基因的Genebank ID为938719，所述yclB基因的Genebank ID为938296。
优选实施方式中，所述出发枯草芽孢杆菌是野生型Bacillus subtilis. 168（简写BS168）的基础上敲除莽草酸途径的分支代谢途径中的dhbB、trpE和aroH基因，并敲除甘油异化途径的分支代谢途径中的mgsA基因。
具体地，所述敲除是采用CpfⅠ基因编辑或cre/lox重组系统实现。
本发明也提供所述的方法获得的高效合成七烯甲萘醌的枯草芽孢杆菌重组菌株。
本发明进一步提供所述的高效合成七烯甲萘醌的枯草芽孢杆菌重组菌株在生产七烯甲萘醌中的应用。
本发明因此提供一种生产七烯甲萘醌的方法，其通过培养所述的高效合成七烯甲萘醌的枯草芽孢杆菌重组菌株以产生七烯甲萘醌。
具体地，所述培养条件是在38-42℃，150-250 rpm的摇床中连续发酵2-6天。任选地，进一步包括通过对发酵液进行细胞破碎和萃取收集七烯甲萘醌。
本发明通过敲除枯草芽孢杆菌的nudF和yclB基因减少DMAPP消耗的同时强化细胞电子传递链的电子供应，促进MK-7的前体合成并且增强电子供应量，达到提高七烯甲萘醌高效合成的目的。实验表明，本发明分别敲除或同时敲除nudF和yclB得到的重组菌株，七烯甲萘醌的产量分别提升至野生型BS168的204%、183%、260%，因此具有较大的实用价值，有利于降低生产七烯甲萘醌的成本。
附图说明
图1是发酵96h后，BS168、BS1、BS2、BS3、BS4重组菌株发酵生产MK-7的产量。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例，进一步阐述本发明。但需要说明的是，实施例并不构成对本发明要求保护范围的限制。
下述实施例中的方法，如无特别说明，均为常规方法。
菌株生长条件：枯草芽孢杆菌于37 ℃，200 rpm生长于LB 液体培养基，于40 ℃，200 rpm生长于发酵培养基。LB培养基成分包括5 g/L酵母浸粉，10 g/L蛋白胨，10 g/LNaCl，固体培养基在此基础上添加了17.5 g/L的琼脂；发酵培养基成分包括甘油30 g/L，大豆蛋白胨60 g/L，酵母浸粉5 g/L，K2HPO43 g/L，MgSO47H2O 0.5 g/L。液体培养基和琼脂平板分别补充了50 μg/ml 卡那霉素或100 μg/ml 壮观霉素。
细胞破碎和MK-7的萃取：取5 mL发酵液于15 mL离心管中，加入500 μl的溶菌酶缓冲液后将溶菌酶水溶液添加至20 mg/L的最终浓度，置于摇床37 ℃，200 rpm孵育1小时。之后加入5 ml 15% HCl进一步破碎细胞，在沸水中煮5 min，稍冷却后加入异丙醇和正己烷萃取剂（1:2，V/V）。
UPLC检测MK-7产量：采用C18分离柱，柱温箱25 ℃，流动相使用甲醇，流速0.5 mL/min，检测波长254 nm，进样量3 μL。
实施例1：枯草芽孢杆菌重组菌株BS2和BS3的构建
通过cre/lox重组系统将出发菌株BS1上的nudF基因和yclB基因分别敲除，构建枯草芽孢杆菌重组菌株BS2和BS3。具体构建方法如下：
所述出发菌株BS1是Bacillus subtilis. 168ΔdhbBΔTrpEΔaroHΔmgsA，该出发菌株是在实验室保藏的野生型Bacillus subtilis. 168的基础上使用CpfⅠ基因编辑工具分别敲除莽草酸途径的分支代谢途径基因dhbB、TrpE和aroH，使用cre/lox重组系统敲除甘油异化途径的分支代谢途径基因mgsA得到的重组菌株。在出发菌株BS1的基础上，使用枯草芽孢杆菌的基因操作工具cre/lox重组系统对分支途径基因nudF和yclB分别进行敲除，方法可参见文章（Radeck J, Kraft K, Bartels J, et al. The Bacillus BioBrickBox: generation and evaluation of essential genetic building blocks forstandardized work with Bacillus subtilis. [J]J Biol Eng. 2013 Dec 2;7(1):29.），具体方法如下：以枯草芽孢杆菌BS168基因组作为模板分别扩增nudF和yclB基因的上游同源臂F1和下游同源臂F3，得到基因片段nudF-F1、nudF-F3和yclB-F1、yclB-F3；以p7S6质粒作为模板扩增lox71-Spe-lox66基因敲除盒，得到nudF-F2和yclB-F2。分别将nudF-F1、nudF-F2、nudF-F3和yclB-F1、yclB-F2、yclB-F3进行重叠延伸PCR，将连接产物纯化回收后得到两个融合基因克隆片段nudF-F1-lox71-spe-lox66-nudF-F3和yclB-F1-lox71-spe-lox66-yclB-F3。将两个融合基因片段分别进行磷酸化自连后转入BS1的感受态并涂布到壮观霉素抗性的平板上筛选阳性转化子。BS1感受态通过Spizizen两步法制备，具体方法参见文章（Spizizen J. Transformation of biochemically deficient strains ofBacillus subtilis by deoxyribonucleate[J]. Proc Natl Acad Sci USA, 1958, 44:1072-1075.）。
挑取单菌落进行PCR验证、测序验证，确认融合基因片段成功整合到BS1中。转入p148cre质粒对整合成功的重组枯草芽孢杆菌进行壮观霉素抗性的消除，通过化学转化的方式将p148cre质粒转入整合成功的重组枯草芽孢杆菌中（Radeck J, Kraft K, BartelsJ, et al. The Bacillus BioBrick Box: generation and evaluation of essentialgenetic building blocks for standardized work with Bacillus subtilis. [J]JBiol Eng. 2013 Dec 2;7(1):29.），添加IPTG诱导cre基因表达使lox71和lox66两个位点重组，借助卡那霉素抗性平板筛选阳性克隆。p148cre质粒的消除通过挑取阳性转化子接种到LB液体培养基中，添加0.05%的SDS于51 ℃，200 rpm培养12 h，划线至LB平板上，挑取单菌落影印至LB平板和K50平板上确保p148cre质粒成功消除。最终得到在BS1基础上敲除nudF基因的枯草芽孢杆菌重组菌株BS2和在BS1基础上敲除yclB基因的枯草芽孢杆菌重组菌株BS3。
实施例2：枯草芽孢杆菌重组菌株BS4的构建
在实施例1得到的重组菌株BS2的基础上采用与实施例1类似的方法，将yclB-F1-lox71-spe-lox66-yclB-F3基因敲除盒整合到BS2的基因组上。经壮观霉素抗性筛选、菌落PCR验证、测序验证、转入p148cre质粒、卡那霉素抗性筛选、IPTG诱导、p148cre质粒消除、平板影印验证，最终得到在出发菌株BS1基因组上同时敲除nudF基因和yclB基因的枯草芽孢杆菌重组菌株BS4。
表1实施例中所用寡核苷酸序列
实施例3：重组菌株发酵生产MK-7
种子液的制备
将出发菌株BS168、BS1以及实施例1和实施例2中构建完成的重组菌株BS2、BS3、BS4划线至LB平板上，挑单菌落接种到LB培养基中活化14小时，得到种子液。
发酵培养
将步骤（1）中得到的种子液按0.1的初始OD接种至30 ml的发酵培养基中进行摇瓶发酵，以野生型菌株BS168和出发菌株BS1作为对照，每个菌株做三个平行。在40 ℃，200rpm的摇床中连续发酵4天后检测MK-7的产量，同时每隔24小时取发酵液稀释40倍后使用紫外-分光光度计测量菌株的生物量。96h发酵液经细胞破碎和萃取后使用UPLC测量MK-7的产量。
结果表明：相较于野生型菌株BS168，出发菌株BS1的产量提高至BS168的125%。此外，在BS1的基础上敲除nudF得到BS2重组菌株，敲除yclB得到BS3重组菌株，同时敲除nudF和yclB得到BS4重组菌株，使重组菌株BS2、BS3、BS4的MK-7产量分别提升至野生型菌株BS168的204%、183%、260%。
Claims (10)
1.一种提高枯草芽孢杆菌(Bacillus subtilis)合成七烯甲萘醌的方法，其特征在于，所述的方法是在出发枯草芽孢杆菌中敲除nudF和yclB基因中一个或两个实现。
2.如权利要求1所述的方法，其特征在于，所述的方法是在出发枯草芽孢杆菌中同时敲除nudF和yclB基因实现。
3.如权利要求1所述的方法，其特征在于，所述nudF基因的Genebank ID为938719，所述yclB基因的Genebank ID为938296。
4.如权利要求1或2所述的方法，其特征在于，所述出发枯草芽孢杆菌是野生型Bacillus subtilis. 168的基础上敲除莽草酸途径的分支代谢途径中的dhbB、trpE和aroH基因，并敲除甘油异化途径的分支代谢途径中的mgsA基因。
5.如权利要求4所述的方法，其特征在于，所述敲除是采用CpfⅠ基因编辑或cre/lox重组系统实现。
6.如权利要求1至5任一项所述的方法获得的高效合成七烯甲萘醌的枯草芽孢杆菌重组菌株。
7.如权利要求6所述的高效合成七烯甲萘醌的枯草芽孢杆菌重组菌株在生产七烯甲萘醌中的应用。
8.一种生产七烯甲萘醌的方法，其特征在于，培养如权利要求6所述的高效合成七烯甲萘醌的枯草芽孢杆菌重组菌株以产生七烯甲萘醌。
9.如权利要求8所述的方法，其特征在于，所述培养条件是在38-42℃，150-250 rpm的摇床中连续发酵2-6天。
10.如权利要求8或9所述的方法，其特征在于，进一步包括通过对发酵液进行细胞破碎和萃取收集七烯甲萘醌。
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