CN116555054B - 一株高产dha的重组裂殖壶菌、其构建方法及应用 - Google Patents
一株高产dha的重组裂殖壶菌、其构建方法及应用 Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供高产DHA的重组裂殖壶菌,所述重组裂殖壶菌是在裂殖壶菌Schizochytrium sp.ATCC20888中通过敲除lipR蛋白同时过表达甘油‑3‑磷酸酰基转移酶基因(GPAT)、二酰甘油酰基转移酶基因(DGAT)、柠檬酸裂解酶基因(ACL)和6磷酸葡萄糖脱氢酶基因(G6PDH)构建而成的。本发明还提供上述重组裂殖壶菌的构建方法,并验证了该重组裂殖壶菌发酵产DHA的产量,通过发酵合成的总油含量达到16.6g/L,DHA含量达到10.3g/L,在原始菌株ATCC20888的基础上提高了2.1倍,显著高于现有技术水平。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种高产DHA的重组工程菌株,还涉及所述重组菌株的构建方法,及其在生产DHA中的应用。
技术背景
二十二碳六烯酸(decosahexaeonic acid,DHA)是一种重要的ω-3长链多不饱和脂肪酸,传统的DHA生产原料主要来源于鱼油,但难以实现高价值产品在食品和医药行业中的广泛应用。为满足DHA不断增长的市场需求,近年来,科学工作者开展了利用海洋微生物发酵生产DHA的研究。裂殖壶菌因其生长速度快、油含量高、DHA比例高,是当前DHA产业化的主要生产菌。
裂殖壶菌是一种含油量极高的海洋微藻,菌体含油量最高可达到50%以上,且其脂肪酸组成简单,DHA可占总脂肪酸含量的35%以上。裂殖壶菌发酵生产DHA的工艺已相对成熟,如何进一步提高DHA生产效率、降低生产成本是近年来研究的重点。裂殖壶菌发酵生产DHA的工艺过程可分为菌种筛选、发酵控制、高密度发酵、细胞获取、油脂提取和纯化。为了提高裂殖壶菌的发酵水平,目前的研究主要集中在菌种性能的改良、结合DHA合成途径借助分子操作进行代谢调控、发酵过程的优化和油脂的提取纯化等方面。
本发明通过在裂殖壶菌中敲除lipR蛋白的同时过表达甘油-3-磷酸酰基转移酶(GPAT)和二酰甘油酰基转移酶(DGAT)达到提高裂殖壶菌中三酰甘油含量的目的,然后再过表达柠檬酸裂解酶(ACL)和6磷酸葡萄糖脱氢酶(G6PDH)以提高乙酰辅酶A和NADPH的含量,从而提高DHA含量,得到一株能高产DHA的裂殖壶菌重组菌株。
发明内容
本发明的目的是提高裂殖壶菌中DHA含量,通过对三酰甘油合成途径中关键基因的过表达提高裂殖壶菌中油脂含量,进一步过表达乙酰辅酶A和NADPH合成途径中关键基因从而提高DHA合成所需要的前体物质,提供一种高产DHA的重组工程菌株,以及该重组菌株的构建方法和应用。
本发明的思路是通过在裂殖壶菌中调控关键基因的表达来调节总油脂和前体物质乙酰辅酶A和NADPH的合成,敲除lipR蛋白的同时过表达甘油-3-磷酸酰基转移酶(GPAT)和二酰甘油酰基转移酶(DGAT)达到提高裂殖壶菌中三酰甘油含量的目的,然后再过表达柠檬酸裂解酶(ACL)和6磷酸葡萄糖脱氢酶(G6PDH)以提高乙酰辅酶A和NADPH的含量,从而提高裂殖壶菌中DHA的含量,得到一株能高产DHA的裂殖壶菌重组菌株,其合成路线如图1所示。
基于此,本发明提供高产DHA的重组裂殖壶菌,所述重组裂殖壶菌是在裂殖壶菌Schizochytrium sp.ATCC20888中通过敲除lipR蛋白同时过表达甘油-3-磷酸酰基转移酶基因(GPAT)、二酰甘油酰基转移酶基因(DGAT)、柠檬酸裂解酶基因(ACL)和6磷酸葡萄糖脱氢酶基因(G6PDH)构建而成的。
在本发明中,所述甘油-3-磷酸酰基转移酶基因(GPAT)的序列如SEQ ID No.1所示,二酰甘油酰基转移酶基因(DGAT)的序列如SEQ ID No.2所示,柠檬酸裂解酶基因(ACL2)的序列如SEQ ID No.3所示,6磷酸葡萄糖脱氢酶基因(G6PDH2)的序列如SEQ ID No.4所示。
本发明还提供上述重组裂殖壶菌的构建方法,所述方法包括以下步骤:
(1)构建GPAT基因和DGAT基因过表达质粒
根据测序获得的lipR蛋白基因序列设计引物lipRup-F和lipRup-R以及lipRdown-F和lipRdown-R扩增得到裂殖壶菌(Schizochytriumsp.ATCC20888)的上下游同源臂并连接到pJN44载体上,分别得到重组质粒pJN44-lipRup和pJN44-lipRdown,然后通过酶切将胶回收的lipRup片段和lipRdown片段连接到PBS-Zeo载体上,得到重组质粒PBS-Zeo-lipR,经验证正确后用于后续实验。
获取并合成来源于解脂亚罗酵母的GPAT基因和DGAT基因序列,以及ccg1启动子和终止子片段,将ccg1启动子、GPAT基因片段、DGAT基因片段和ccg1终止子依次连接到载体pJN44的多酶切位点SpeI/SmaI、SmaI/MfeI、MfeI/BstEⅡ和BstEⅡ/BamHI上,构建得到含有GPAT和DGAT表达盒的质粒pJN44-GPAT/DGAT,然后将重组质粒pJN44-GPAT/DGAT中含目的基因的表达盒用SpeI/BamHI酶切并连接至载体PBS-Zeo-lipR上,得到重组质粒PBS-Zeo-GPAT/DGAT-lipR,经验证正确后用于后续实验。
其中,lipR基因序列如SEQ ID No.5所示;
(2)构建含有甘油-3-磷酸酰基转移酶基因(GPAT)和二酰甘油酰基转移酶基因(DGAT)的重组工程菌株
将上述验证正确的重组质粒PBS-Zeo-GPAT/DGAT-lipR线性化后取10μg转化到100μL裂殖壶菌感受态中,置于0.1cm间隙的电转杯中进行电穿孔(1.5KV,200Q,50uF,两次)。然后加入1mL种子培养基,28℃孵育4h,取300μL菌液均匀涂于含100μg/mL zeocin的博莱霉素种子筛选培养基,28℃避光培养48h待长出单菌落后做菌落PCR验证及测序验证,正确的重组菌命名为裂殖壶菌AT-GD。
裂殖壶菌感受态制备:裂殖壶菌细胞在种子培养基中培养24h,离心(5900g,4℃,5min)收获菌体细胞,用预冷的无菌水和1M山梨醇洗涤3次,随后在1M山梨醇中重悬。
(3)构建含有柠檬酸裂解酶基因(ACL)和6磷酸葡萄糖脱氢酶基因(G6PDH)的重组工程菌株
从NCBI上获取来源于裂殖壶菌、解脂亚罗酵母和希瓦氏菌的柠檬酸裂解酶基因(ACL)和6磷酸葡萄糖脱氢酶基因(G6PDH)序列信息,添加酶切位点送生工合成,将合成片段ACL1,ACL2,ACL3,G6PDH1,G6PDH2,G6PDH3与质粒pPICZαA分别经酶切连接获得重组质粒pPICZαA-ACL1,pPICZαA-ACL2,pPICZαA-ACL3,和pPICZαA-G6PDH1,pPICZαA-G6PDH2,pPICZαA-G6PDH3。经验证正确后用于后续实验。
将上述验证正确的重组质粒酶切线性化处理后,分别转化裂殖壶菌AT-GD感受态细胞(感受态制备方法同上),28℃孵育4h,取300μL菌液均匀涂于含100μg/mL zeocin的博莱霉素种子筛选培养基,28℃避光培养48h待长出单菌落后做菌落PCR验证及测序验证,正确的重组菌分别命名为AT-GDA1,AT-GDA2,AT-GDA3,AT-GDG1,AT-GDG2,AT-GDG3。
(4)含有柠檬酸裂解酶基因(ACL)和6磷酸葡萄糖脱氢酶基因(G6PDH)的重组工程菌株筛选
分别测定步骤(3)中各重组菌株生物量、总油含量以及DHA含量,筛选出生物量、总油含量及DHA含量较高的菌株,确定菌株对应的基因来源,得到柠檬酸裂解酶基因和6磷酸葡萄糖脱氢酶基因最佳来源。
(5)高产DHA的裂殖壶菌重组工程菌株构建
将步骤(4)中筛选到的来源于解脂亚罗酵母菌的柠檬酸裂解酶基因(ACL2)和6磷酸葡萄糖脱氢酶基因(G6PDH2)同时整合到裂殖壶菌AT-GD中,得到重组菌株AT-GDAG经发酵后测定其生物量、总油含量和DHA含量。
在本发明中,所述重组菌株是选用裂殖壶菌Schizochytrium sp.ATCC-20888为底盘细胞构建而成,可以从购买于上海研生实业有限公司购买获得。
在本发明中,PBS-Zeo载体是根据中国发明专利申请CN 201510417269.4公开的方法的构建得到的,质粒pPICZαA购于淼灵质粒平台。
本发明中的甘油-3-磷酸酰基转移酶(GPAT)和二酰甘油酰基转移酶(DGAT)均从解脂亚罗酵母中克隆所得。
本发明中的柠檬酸裂解酶基因(ACL)和6磷酸葡萄糖脱氢酶基因(G6PDH)分别来源于裂殖壶菌、解脂亚罗酵母和希瓦氏菌。
在本发明中,所述重组菌株转化方式为:将线性化后的重组质粒取10μg加入100μl裂殖壶菌感受态细胞,混匀后转移至预冷的0.1cm间隙电转杯中,冰上静置30min后进行电击,电击条件为1.5KV,200Q,50uF,两次,结束后立即加入1ml种子培养基,28℃孵育4h,取300μL菌液均匀涂于含100μg/mL zeocin的平板上(种子培养基添加2%琼脂),28℃避光培养48h待长出单菌落后做菌落PCR验证及测序验证。
在本发明中,所述重组菌株培养条件为:在种子培养基中200rpm、28℃恒温摇床里培养48h,然后接入发酵培养基中,200rpm、28℃恒温摇床里培养96h。
本发明通过调控关键基因的表达来调节总油脂和前体物质乙酰辅酶A和NADPH的合成,敲除lipR蛋白的同时过表达甘油-3-磷酸酰基转移酶(GPAT)和二酰甘油酰基转移酶(DGAT)达到提高裂殖壶菌中三酰甘油含量的目的,然后再过表达最佳来源的柠檬酸裂解酶(ACL)和6磷酸葡萄糖脱氢酶(G6PDH)以提高乙酰辅酶A和NADPH的含量,从而提高裂殖壶菌中DHA的含量,得到一株能高产DHA的裂殖壶菌重组菌株,采用本发明的裂殖壶菌重组菌株AT-GDAD进行发酵合成的总油含量达到16.6g/L,DHA含量达到10.3g/L,在原始菌株ATCC20888的基础上提高了2.1倍。
附图说明
图1为本发明的高产DHA的裂殖壶菌重组菌株的DHA合成路线。
具体实施方式
下面结合附图和实施例非限制性地解释本发明的技术方案。
在本发明中,如无特殊说明,用于说明浓度的“%”为重量百分比,“:”为重量比。
本发明涉及以下培养基:
种子培养基含有:葡萄糖40g/L、酵母膏2g/L、谷氨酸钠10g/L、KH2PO4 4g/L、NaCl15g/L、MgCl2 3g/L、CaCl2·2H2O 1g/L、KCl 2g/L、MgSO4·7H2O 5g/L、FeCl3 0.1g/L;
发酵培养基含有:葡萄糖40g/L、酵母膏2g/L、谷氨酸钠10g/L、KH2PO4 4g/L、NaCl15g/L、MgCl2 3g/L、(NH4)2SO4 6g/L、KCl 2g/L、MgSO4·7H2O 5g/L、FeCl3 0.1g/L。
固体种子培养基:在种子培养基的基础上,添加1.5-2%琼脂,灭菌,在超净台内倒入灭菌的平板中。
博莱霉素种子筛选培养基:1.5%固体种子培养基灭菌后,当培养基降温至50℃,加入终浓度为100μg/ml的博莱霉素(Z-cin),立即倒平板,4℃冰箱避光保存。
在本发明中,裂殖壶菌ATCC20888来自购买于上海研生实业有限公司。
在本发明中,菌株的生物量检测方法如下:
准确量取搅拌均匀的发酵液200ml,倒入干净无水的离心筒中,在4000r/min的转速下离心10min,取出离心筒,弃去上清液,将离心筒底部的菌体用药勺全部取出,放入已恒重的称量盘中,均匀铺平,并划成有规则的方块;于105℃的干燥箱中烘干,期间不时翻动菌体,3h后取出,恒重称量并记录,生物量即为烘干菌体质量(W)除以发酵液体积(V)再乘1000。
计算公式为:
总油含量测定方法如下:
油脂含量的测定方法:取一定体积的发酵液(100ml),将其在50℃预热,用NaOH溶液将pH调至10-12之间,按3‰(g/l)的比例添加破壁酶,50℃下搅拌并保温2h;按1:1:1(发酵液:乙醇:正己烷)(v/v)比例分别添加乙醇、正己烷,搅拌、分层、萃取,连续萃取2-3次,取正己烷相,真空旋转蒸干溶剂,获取油脂。将烧瓶放在烘箱中105℃烘至恒重,冷却后称重。
油脂中DHA含量检测方法如下:
采用常规方法将提取的油脂进行甲酯化,然后利用日本岛津高效气相色谱仪检测油脂中DHA含量。气相分析条件:色谱柱:DB-23(60m*0.25mm*0.25μm);检测器:FID;载气:氮气;分流比:30/1;进样口温度:250℃;检测器温度:280℃;进样量:1μl;升温程序:初始柱温为100℃,先以25℃/min的速度升至196℃,再以2℃/min的速度升至220℃,保持12min。柱流速:3.0ml/min;尾吹流速:30ml/min;氢气流速:40ml/min;空气流速:400ml/min。
实施例1构建含有甘油-3-磷酸酰基转移酶基因(GPAT)和二酰甘油酰基转移酶基因(DGAT)的重组工程菌株
表1GPAT和DGAT基因序列信息
表2实施例1涉及的引物
1、构建GPAT基因和DGAT基因过表达质粒
根据测序获得的lipR蛋白基因序列(其核酸序列如SEQ ID No.5所示)设计带酶切位点引物lipRup-F和lipRup-R以及lipRdown-F和lipRdown-R,扩增得到裂殖壶菌ATCC20888的上下游同源臂片段,并以NotⅠ/XbaI为酶切位点双酶切载体PBS-Zeo,并将上游片段连接到质粒PBS-Zeo中Zeocin(博莱霉素)抗性基因片段的上游获得重组质粒PBS-Zeo-lipRup,同样以SalⅠ/XhoⅠ双酶切载体PBS-Zeo-lipRup,并将下游片段连接到质粒PBS-Zeo中Zeocin(博莱霉素)抗性基因片段的下游获得重组质粒pJN44-lipR;
从NCBI上获取各序列信息,添加酶切位点合成来源于解脂亚罗酵母的GPAT基因和DGAT基因,以及ccg1启动子和终止子片段,启动子序列如SEQ ID No.6所示,终止子序列如SEQ ID No.7所示,将ccg1启动子、GPAT基因片段、DGAT基因片段和ccg1终止子分别用SpeI/SmaI,SmaI/MfeI,MfeI/BstEⅡ,BstEⅡ/BamHI酶切后依次连接到载体pJN44的多克隆酶切位点上,构建得到含有GPAT和DGAT表达盒的质粒pJN44-GPAT/DGAT,然后将重组质粒pJN44-GPAT/DGAT中含目的基因的表达盒用SpeI/BamHI酶切并连接至载体PBS-Zeo-lipR上,得到重组质粒PBS-Zeo-GPAT/DGAT-lipR。将获得的重组质粒热激转化大肠杆菌感受态细胞中,在含有100μg/ml博来霉素抗性的LB平板上培养筛选单菌落,提取质粒进行测序验证。
2、制备裂殖壶菌感受态细胞
挑取平板上已活化好的裂殖壶菌ATCC20888单菌落至50ml种子培养基中28℃,200r/min培养48h;按4%的接种量再次转接至50ml种子培养基中28℃,200r/min培养48h取20ml菌液,5900g,4℃,离心5min,弃上清收集菌体,用预冷的无菌水和1M山梨醇洗涤3次,随后在1M山梨醇中重悬菌体,分装于1.5ml无菌离心管中。
3、重组菌株AT-GD构建
将上述验证正确的重组质粒PBS-Zeo-GPAT/DGAT-lipR酶切线性化处理后,取10μl加入100μl裂殖壶菌ATCC 20888感受态细胞,混匀后转移至预冷的电转杯,冰上静置30min后进行电击,电击条件为1.5KV,200Q,50uF,两次,结束后立即加入1ml种子培养基,28℃孵育4h,取300μL菌液均匀涂于含100μg/mL zeocin的博莱霉素种子筛选培养基平板上,28℃避光培养48h待长出单菌落后做菌落PCR验证及测序验证。
将平板上长出的单菌落接种至种子培养基,28℃,200rpm培养48h后提取基因组DNA进行PCR验证。结果显示抗性筛选标记已重组至裂殖壶菌的基因组,实现了外源基因的同源重组,获得重组菌株AT-GD。
实施例2构建含有柠檬酸裂解酶基因(ACL)和6磷酸葡萄糖脱氢酶基因(G6PDH)的重组工程菌株
表3ACL基因和G6PDH基因序列信息
表4实施例2涉及以下引物
1、构建ACL基因和G6PDH基因过表达质粒
从NCBI上获取来源于裂殖壶菌、解脂亚罗酵母和希瓦氏菌的柠檬酸裂解酶基因(ACL)和6磷酸葡萄糖脱氢酶基因(G6PDH)序列信息,添加酶切位点送生工合成,将合成片段ACL1,ACL2,ACL3,G6PDH1,G6PDH2,G6PDH3与质粒pPICZαA分别经KpnI/XbaI,酶切后进行连接获得重组质粒pPICZαA-ACL1,pPICZαA-ACL2,pPICZαA-ACL3,和pPICZαA-G6PDH1,pPICZαA-G6PDH2,pPICZαA-G6PD3H。将获得的重组质粒热激转化大肠杆菌感受态细胞中,在含有100μg/ml博来霉素的LB平板上培养筛选单菌落,提取质粒进行测序验证。
2、重组质粒的转化
将上述验证正确的重组质粒酶切线性化处理后,分别转化裂殖壶菌AT-GD感受态细胞(感受态制备方法同实施例1中步骤2),28℃,200rpm培养4h后,取300μL菌液均匀涂于含100μg/mL博来霉素的固体平板,置于28℃培养箱培养48h。将平板上长出的单菌落接种至种子培养基,28℃,200rpm培养48h后做菌落PCR验证。结果显示目的基因已转化至裂殖壶菌中游离表达,获得重组菌株AT-GDA1,AT-GDA2,AT-GDA3,AT-GDG1,AT-GDG2,AT-GDG3。
3、重组菌株的筛选
将上述重组菌株分别在28℃,200rpm培养48h后分别测定其生物量、总油含量以及DHA含量,筛选出生物量、总油含量及DHA含量较高的菌株,确定菌株对应的基因来源,得到柠檬酸裂解酶基因和6磷酸葡萄糖脱氢酶基因最佳来源。
检测各菌株的生物量、总有含量和DHA含量,结果如表5显示:重组菌株AT-GDA2和AT-GDG2的生物量、总油含量以及DHA含量均最高,分别达到22.5DWg/L、16.5g/L、9.2g/L和22.8DWg/L、16.7g/L、9.5g/L,因此确定解脂亚罗酵母菌是ACL基因和G6PDH的最佳来源。
表5各重组菌株发酵液中生物量、总油含量及DHA含量
5、高产DHA的裂殖壶菌重组工程菌株AT-GDAG构建
将步骤4中筛选到的来源于解脂亚罗酵母菌的柠檬酸裂解酶基因和6磷酸葡萄糖脱氢酶基因同时整合到裂殖壶菌AT-GD中,重新设计带酶切位点引物,从质粒pPICZαA-ACL2上扩增ACL2片段,利用限制性内切酶EcoRI和KpnI双酶切质粒pPICZαA-G6PDH2,然后将ACL2片段与pPICZαA-G6PDH2载体片段连接,获得含有ACL2和G6PDH2基因的重组质粒pPICZαA-ACL2/G6PDH2,然后将获得的重组质粒热激转化大肠杆菌感受态细胞中,在含有100μg/ml博来霉素抗性的LB平板上培养筛选单菌落,提取质粒进行测序验证。
将上述验证正确的重组质粒pPICZαA-ACL2/G6PDH2酶切线性化处理后,转化到裂殖壶菌AT-GD感受态细胞(感受态制备方法同实施例1中步骤2),28℃,200rpm培养4h后,取300μL菌液均匀涂于含100μg/mL博来霉素的固体平板,置于28℃培养箱培养48h。将平板上长出的单菌落接种至种子培养基,28℃,200rpm培养48h后提取基因组做PCR验证。结果显示目的基因ACL2和G6PDH2已已转化至裂殖壶菌中游离表达,获得重组菌株AT-GDAG。
实施例3重组裂殖壶菌AT-GDAG在产DHA中的应用
将得到的重组菌株AT-GDAG在发酵培养基中,220rpm、28℃条件下恒温摇床里培养96h,然后取样测定其生物量、总油含量以及DHA含量。
结果如表6显示,比较各菌株的生物量、总油含量以及DHA含量,发现重组菌株AT-GDAG的生物量达到22.2DWg/L,总油含量达到16.6g/L,DHA含量达到10.3g/L,在原始菌株ATCC20888的基础上提高了2.1倍。
表6重组菌株发酵液中生物量、总油含量及DHA含量
Claims (5)
1.一株高产DHA的重组裂殖壶菌,其特征在于所述重组裂殖壶菌是敲除lipR蛋白并过表达甘油-3-磷酸酰基转移酶基因GPAT、二酰甘油酰基转移酶基因DGAT、柠檬酸裂解酶基因ACL和6磷酸葡萄糖脱氢酶基因G6PDH的裂殖壶菌,
所述甘油-3-磷酸酰基转移酶基因GPAT、二酰甘油酰基转移酶基因DGAT、柠檬酸裂解酶基因ACL和6磷酸葡萄糖脱氢酶基因G6PDH来源于解脂亚罗酵母,
所述甘油-3-磷酸酰基转移酶基因GPAT的核酸序列如SEQ ID No.1所示,二酰甘油酰基转移酶基因DGAT的核酸序列如SEQ ID No.2所示,来源于解脂亚罗酵母的柠檬酸裂解酶基因ACL2的核酸序列如SEQ ID No.3所示,来源于解脂亚罗酵母的6磷酸葡萄糖脱氢酶基因G6PDH2的核酸序列如SEQ ID No.4所示;
所述重组裂殖壶菌的出发菌株是裂殖壶菌Schizochytrium sp. ATCC20888。
2. 权利要求1所述的高产DHA的重组裂殖壶菌的构建方法,所述方法包括以下步骤:
(1)构建GPAT基因和DGAT基因过表达质粒
根据lipR蛋白基因序列设计引物lipRup-F和lipRup-R以及lipRdown-F和lipRdown-R扩增得到裂殖壶菌的上下游同源臂并连接到pJN44载体上,分别得到重组质粒pJN44-lipRup和pJN44-lipRdown,然后通过酶切将胶回收的lipRup片段和lipRdown片段连接到PBS-Zeo载体上,得到重组质粒PBS-Zeo-lipR;
获取并合成来源于解脂亚罗酵母的GPAT基因和DGAT基因序列,以及ccg1启动子和终止子片段,将ccg1启动子、GPAT基因片段、DGAT基因片段依次连接到载体pJN44的多酶切位点上,构建得到含有GPAT和DGAT表达盒的重组质粒pJN44-GPAT/DGAT,然后将重组质粒pJN44-GPAT/DGAT中含目的基因的表达盒用SpeI/BamHI酶切并连接至载体PBS-Zeo-lipR上,得到重组质粒PBS-Zeo-GPAT/DGAT-lipR;
(2)构建含有甘油-3-磷酸酰基转移酶基因GPAT和二酰甘油酰基转移酶基因DGAT的重组工程菌株
经验证正确的重组质粒PBS-Zeo-GPAT/DGAT-lipR线性化后转化到裂殖壶菌感受态中,然后在28℃避光培养48h,待长出单菌落后,所得菌落经PCR验证及测序验证,得到验证正确的重组裂殖壶菌AT-GD,所述重组裂殖壶菌AT-GD是敲除lipR蛋白并过表达甘油-3-磷酸酰基转移酶基因GPAT和二酰甘油酰基转移酶基因DGAT的重组裂殖壶菌;
(3)构建含有柠檬酸裂解酶基因ACL和6磷酸葡萄糖脱氢酶基因G6PDH的重组工程菌株
分别获取来源于解脂亚罗酵母的柠檬酸裂解酶基因ACL与6磷酸葡萄糖脱氢酶基因G6PDH序列信息,添加酶切位点送生工合成,分别得到合成片段ACL2和G6PDH2,所得片段与质粒pPICZαA分别经酶切连接获得重组质粒pPICZαA-ACL2和pPICZαA-G6PDH2;所得重组质粒经验证正确;
上述重组质粒酶切线性化处理后,分别转化裂殖壶菌AT-GD感受态细胞,再在28℃避光培养48h,待长出单菌落后进行菌落PCR验证及测序验证,得到验证正确的重组菌AT-GDA2和AT-GDG2,所述重组菌AT-GDA2是敲除lipR蛋白并过表达甘油-3-磷酸酰基转移酶基因GPAT、二酰甘油酰基转移酶基因DGAT和来源于解脂亚罗酵母的柠檬酸裂解酶基因ACL2的重组裂殖壶菌,所述重组菌AT-GDG2是敲除lipR蛋白并过表达甘油-3-磷酸酰基转移酶基因GPAT、二酰甘油酰基转移酶基因DGAT和来源于解脂亚罗酵母的6磷酸葡萄糖脱氢酶基因G6PDH2的重组裂殖壶菌;
(4)高产DHA的裂殖壶菌重组工程菌株构建
将步骤(3)的柠檬酸裂解酶基因和6磷酸葡萄糖脱氢酶基因同时整合到步骤(2)得到的裂殖壶菌AT-GD中,得到重组菌株经验证正确后即为高产DHA的重组裂殖壶菌。
3. 根据权利要求2所述的构建方法,其特征在于步骤(1)的lipR基因序列如SEQ IDNo.5所示。
4.权利要求1所述的重组裂殖壶菌在生产DHA中的应用。
5.根据权利要求4所述的应用,其特征在于所得重组裂殖壶菌在种子培养基中,200rpm、28℃恒温摇床里避光培养48h,然后接种至发酵培养基中,200rpm、28℃恒温摇床里避光培养96h。
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