CN113832041A - 高产赤霉素ga3的藤仓赤霉基因工程菌、构建方法及应用 - Google Patents
高产赤霉素ga3的藤仓赤霉基因工程菌、构建方法及应用 Download PDFInfo
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Abstract
本发明涉及一种高产赤霉素GA3的藤仓赤霉基因工程菌、构建方法及应用。本发明采用CRISPR‑Cas9基因编辑技术敲除筛选标签的相关基因(pyrG)和代谢路径的调节基因(meaB),并且过表达前体供应基因hmgR的催化基团编码序列thmgR,构建得到高产菌株F.fujikuroi RE:pyrG GpdA‑tHmgRΔmeaB,该菌株经过168h发酵培养基发酵后能积累2.35g/L的GA3产物。本发明提供了高产GA3的藤仓赤霉菌基因工程菌及构建方法,经过改造后的藤仓赤霉菌削弱了前体合成的反馈抑制,提高了代谢过程的前体供给,解除了GA3相关合成基因的转录抑制,相对于出发菌株能更高效的进行GA3的发酵生产,并且产量从2.0g/L提高到2.35g/L,为工程菌株的构建提供了可行的路线。
Description
(一)技术领域
本发明属于代谢工程领域,具体涉及一种高产赤霉素GA3的藤仓赤霉基因工程菌、构建方法及应用。
(二)背景技术
赤霉素(Gibberellin Acid,GA3)是由植物、部分真菌合成的一种植物生长激素。因其特殊的刺激植物生长的能力被广泛应用于农业、园艺与酿酒生产。藤仓赤霉菌最早由日本的植物病理学家在研究水稻恶苗病发现,后续研究证实藤仓赤霉菌天然具有完整的GA3合成能力。GA3是一种四环二萜羧酸,分子式C19H22O6,分子量346.37,熔点233~235℃,易溶于醇类、丙酮、乙酸乙酯、碳酸氢钠溶液以及pH为6.2的磷酸缓冲液,难溶于水和乙醚。GA3作为一种次级代谢产物,一般通过液态发酵、固态发酵与植物提取的方式获得。目前GA3的生产主要依赖液体深层发酵或固态发酵。
藤仓赤霉菌中GA3的合成场所在细胞质,其生物合成由TCA循环合成的乙酰辅酶A(Acetyl-CoA)出发,经甲羟戊酸途径合成异戊烯焦磷酸(IPP)及其异构体二甲烯丙基二磷酸(DMAPP),后续由合成酶催化经由基焦磷酸(GPP),法尼基焦磷酸(FPP)合成前体二基焦磷酸(GGPP)。在赤霉素合成基因簇的作用下,两分子GGPP环化合成焦磷酸古巴酯(CPP)后,产生了贝壳杉烯。在C-19上由P450单加氧酶逐步氧化产生贝壳杉烯酸,后续通过在C-7α与C-6β位置的氧化产生GA12-半醛,P450单加氧酶在C-3β与C-7的氧化产生GA14,GA14通过氧化转化为GA4,经去饱和作用生产GA7,经羟基化转化为GA3。
现有对藤仓赤霉菌合成GA3的技术方案,主要以不同方式的诱变为主,包括化学诱变,辐射诱变和ARTP诱变等。中国发明专利(公开号CN104892554A、CN201910304928.1与CN201910438160.7)分别描述了GA3的制备方法、诱变结果与ARTP的诱变技术。但通过传统诱变方案获得的菌株产量达到了2g/L后再难得到提升。现有对藤仓赤霉菌基因组编辑的技术方案主要有两种:(一)依赖传统的同源双交换,通过PEG-CaCl2介导的化学转化法,使用较长同源臂触发自发的双交换机制达到编辑基因组的目的;(二)采用近几年发明的CRISPR/Cas9技术,通过sgRNA的定位引导Cas9蛋白精确的切割靶点,在基因组上制造双链断裂缺口,并且在修复时引入或敲除目的基因。这两种方案都依赖筛选标记的验证,目前主要以潮霉素抗性,G418抗性,博来霉素抗性等。在现有技术方案中,外源质粒、片段、筛选标记由于整合到基因组上导致只能一次性使用。
尿嘧啶合成中的营养缺陷型筛选标签。初始菌株F.fujikuroi本身携带有在尿嘧啶合成过程中的必需基因乳清酸核苷-5'-磷酸脱羧酶pyrG基因。尿嘧啶生物合成途径非常保守,pyrG基因编码的产物乳清酸核苷-5′-磷酸脱羧酶是负责乳清苷-5′-磷酸向尿嘧啶生物合成途径中的最后一步。同时,乳清酸核苷-5′-磷酸脱羧酶可以催化含氟类似物5-氟乳清酸合成含氟核苷酸类似物,该含氟类似物使含pyrG基因的初始菌株无法存活。敲除pyrG基因使乳清酸核苷-5′-磷酸脱羧酶失活,菌株无法合成尿嘧啶。对于尿嘧啶营养缺陷型菌株,则需通过外源补加尿嘧啶进行补救途径。
(三)发明内容
本发明的目的是通过代谢工程技术,提供一种高产赤霉素GA3的藤仓赤霉基因工程菌、构建方法及应用。
为实现本发明的上述目的,本发明采用的技术方案:
一种高产赤霉素GA3的藤仓赤霉基因工程菌,由如下方法构建获得:
(1)将藤仓赤霉菌(F.fujikuroi)的乳清酸核苷-5'-磷酸脱羧酶pyrG基因敲除,构建得到营养缺陷型菌株F.fujikuroi-ΔpyrG;
(2)将pyrG基因表达框导入F.fujikuroi-ΔpyrG,同时敲除bZIP型转录抑制因子meaB基因,构建得到菌株F.fujikuroi-RE:pyrGΔmeaB;
(3)用来源于pAN7-1的gpdA启动子增强表达hmgR基因的催化区域,构建truncated-hmgR基因表达框,导入菌株F.fujikuroi-RE:pyrGΔmeaB中,构建得到菌株F.fujikuroi-RE:pyrG GpdA-tHmgRΔmeaB,即所述高产赤霉素GA3的藤仓赤霉菌基因工程菌。
所述来源于pAN7-1的gpdA启动子序列如SEQ ID No.3所示。
本发明还涉及构建所述高产赤霉素GA3的藤仓赤霉基因工程菌的方法,所述方法如下:
(1)运用CRISPR-Cas9基因编辑技术,将藤仓赤霉菌(F.fujikuroi)的pyrG基因敲除,构建得到营养缺陷型菌株F.fujikuroi-ΔpyrG;
(2)运用CRISPR-Cas9基因编辑技术,将pyrG基因表达框导入F.fujikuroi-ΔpyrG,同时敲除meaB基因,构建得到菌株F.fujikuroi-RE:pyrGΔmeaB;乳清酸核苷-5'-磷酸脱羧酶pyrG基因在藤仓赤霉菌F.fujikuroi中天然存在,所述pyrG基因及其原始启动子终止子序列如SEQ ID NO.1所示;
(3)运用CRISPR-Cas9基因编辑技术,用来源于pAN7-1的gpdA启动子增强表达hmgR基因的催化区域,构建thmgR基因表达框,导入菌株F.fujikuroi-RE:pyrGΔmeaB中,构建得到菌株F.fujikuroi-RE:pyrG GpdA-tHmgRΔmeaB,即所述高产赤霉素GA3的藤仓赤霉菌基因工程菌。具体是以藤仓赤霉菌F.fujikuroi基因组为模板,扩增不含反馈抑制区的3-羟基-3-甲基戊二酸单酰辅酶A还原酶hmgR基因编码区。以来源于pAN7-1的gpdA启动子,增强表达不含反馈抑制调控区域的thmgR基因,并导入F.fujikuroi-RE:pyrGΔmeaB,构建得到菌株F.fujikuroi RE:pyrG GpdA-tHmgRΔmeaB。所述thmgR基因的核苷酸序列如SEQ IDNO.2所示。
所述“反馈抑制”指的是3-羟基-3-甲基戊二酸单酰辅酶A还原酶的部分区域由于结合到内质网膜区域从而受到HMGR催化的下游产物的抑制。术语“增强表达”指的是增加由对应的多核苷酸编码的酶的活性,通过替换基因组上该基因的表达调控序列(启动子替换等)。
藤仓赤霉菌因具有相对较高的GA3合成能力而被广泛应用于工业化的液体深层发酵。GA3的合成场所在细胞质,在藤仓赤霉菌中GA3的主要合成路径如图1所示:其生物合成由TCA循环合成的乙酰辅酶A(Acetyl-CoA)出发,经甲羟戊酸途径合成异戊烯焦磷酸(IPP)及其异构体二甲烯丙基二磷酸(DMAPP),后续由合成酶催化经由基焦磷酸(GPP),法尼基焦磷酸(FPP)合成前体二基焦磷酸(GGPP)。在赤霉素合成基因簇的作用下,两分子GGPP环化合成焦磷酸古巴酯(CPP)后,产生了贝壳杉烯。在C-19上由P450单加氧酶逐步氧化产生贝壳杉烯酸,后续通过在C-7α与C-6β位置的氧化产生GA12-半醛,P450单加氧酶在C-3β与C-7位置的氧化产生GA14,GA14通过氧化转化为GA4,经去饱和作用生产GA7,经羟基化转化为GA3。
本发明采用CRISPR-Cas9基因编辑技术(重组表达质粒构建示意图参见图2)敲除筛选标签的相关基因(pyrG)和代谢路径的调节基因(meaB),并且过表达前体供应基因hmgR的催化基团编码序列thmgR,构建得到高产菌株F.fujikuroi RE:pyrG GpdA-tHmgRΔmeaB,该菌株经过168h发酵培养基发酵后能积累2.35g/L的GA3产物。
本发明还涉及所述藤仓赤霉基因工程菌在微生物发酵制备赤霉素GA3中的应用。
具体的,所述应用为:将所述藤仓赤霉基因工程菌接种至发酵培养基,25~30℃(优选28℃)、200~300rpm(优选250rpm)条件下发酵培养12~48h,获得含赤霉素GA3的发酵液,发酵液经分离纯化获得赤霉素GA3。
所述发酵培养基组成如下:玉米淀粉60~90g/L,米粉70~100g/L,大豆粉3~7g/L,花生粉3~7g/L,K2SO4 0.3~0.7g/L,KH2PO4 0.3~0.7g/L,溶剂为水,pH自然,121度灭菌30min。
通常,所述藤仓赤霉基因工程菌先接种至斜面培养基(25~30℃培养4~7天),活化后的菌株接种至种子培养基经种子培养(25~30℃、250rpm培养过夜)获得种子液再接种至发酵培养基,所述斜面培养基组成如下:土豆150~200g/L、蔗糖10~30g/L、硫酸镁0.1~0.3g/L、碳酸钙0.1~0.3g/L、琼脂20g/L,pH自然,115℃灭菌30min;所述种子培养基组成如下:玉米淀粉10~30g/L、蔗糖10~30g/L、花生粉10~30g/L、大豆粉10~30g/L、磷酸二氢钾0.5~1.5g/L、硫酸镁0.5~1.5g/L,pH自然,121℃灭菌30min。
本发明的有益效果主要体现在,本发明提供了高产GA3的藤仓赤霉菌基因工程菌及构建方法,经过改造后的藤仓赤霉菌削弱了前体合成的反馈抑制,提高了代谢过程的前体供给,解除了GA3相关合成基因的转录抑制,相对于出发菌株能更高效的进行GA3的发酵生产,并且产量从2.0g/L提高到2.35g/L,为工程菌株的构建提供了可行的路线。
(四)附图说明
图1为工程菌株中GA3的合成路径;;
图2为重组表达质粒构建示意图;
图3为改造菌株和初始菌株的产物积累曲线。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例中的实验方法,如无特殊说明,均为常规方法。
实施例中所用的试验材料,如无特殊说明,均为常规生化试剂。
实施例1:F.fujikuroi-ΔpyrG的获得
pyrG基因的敲除:
为了降低阴性转化子的干扰,使转化挑选效率提高,因此对藤仓赤霉菌的pyrG基因进行了敲除,参见Van Hartingsveldt W,Mattern I E,van Zeijl C M J,etal.Development of a homologous transformation system for Aspergillus nigerbased on the pyrG gene.Molecular and General Genetics MGG,1987,206(1):71-75.
通过CRISPR-Cas9系统敲除藤仓赤霉菌(Fusarium fujikuroi)CCTCC NO:M2019378(已在CN110527630A中披露)基因组中编码乳清酸核苷-5'-磷酸脱羧酶的pyrG基因。通过PCR使用引物1和引物2,以sgRNA表达框作为模板扩增得到sgRNA-ΔpyrG-1片段,PCR反应条件如下:98℃3min;98℃15s,60℃15s,72℃15s,重复30个循环;72℃继续延续3min。
按同样的方法使用引物3和引物4扩增得到sgRNA-ΔpyrG-2片段,PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段。将回收的两个DNA片段使用引物1和引物4进行融合PCR,PCR反应条件如下:98℃3min;98℃15s,60℃15s,72℃30s,重复30个循环;72℃继续延续3min。PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段(核苷酸序列为SEQID NO.4所示)。以pFC332-Ffcas9载体作为模板,通过一步克隆试剂盒ClonExpress II OneStep Cloning Kit(购自南京诺唯赞vazyme)连接,构建能够表达靶定目的基因pyrG的sgRNA的pFC332-Ffcas9-ΔpyrG突变质粒载体。连接产物转化至E.coli DH5α受体菌中,涂布于含终浓度100mg/L氨苄霉素抗性的LB固体平板上,37℃培养12h。随机挑取单菌落至含终浓度100mg/L氨苄霉素抗性的LB液体培养基中,37℃培养12h,收集菌体并提取质粒获得pFC332-Ffcas9-ΔpyrG载体。
通过PCR使用引物5和引物6,以藤仓赤霉菌(Fusarium fujikuroi)CCTCC NO:M2019378基因组为模板扩增得到pyrG基因上游同源片段,PCR反应条件如下:98℃5min;98℃30s,60℃30s,72℃30s,重复30个循环;72℃继续延续5min。按同样的方法使用引物7和引物8扩增得到pyrG基因下游同源片段,PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段。将回收的两个DNA片段使用引物5和引物8进行融合PCR,PCR反应条件如下:98℃5min;98℃30s,60℃30s,72℃30s,重复30个循环;72℃继续延续5min。PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段(核苷酸序列为SEQ ID NO.5所示)。将pFC332-Ffcas9-ΔpyrG载体和回收的DNA片段一同转化至藤仓赤霉菌的原生质体。
为了制备藤仓赤霉菌的原生质体用于转化,将藤仓赤霉菌接种于YPD液体培养基,28℃培养48h。收集菌丝加入含有终浓度15mg/L复合酶Yatalase(购自大连宝生物)和15mg/L崩溃酶Driselase(购自sigma公司)的破壁缓冲液中,30℃孵育3h。使用Miracloth过滤缓冲液后,收集下层液体,2500rpm离心6分钟,弃上清,用1mL 1M山梨醇将沉淀细胞充分悬浮,原生质体制备成功。
将转化后的菌液涂至含有1mg/L的5-氟乳清酸的MYG平板上,28℃培养4天,挑取单菌落作为模板,以引物9和引物10进行PCR,观察到在1.0%琼脂糖凝胶中存在1200bp的DNA条带以确认pyrG基因的缺失。将通过此确认的菌株在含有5-氟乳清酸的YPD液体培养基中在28℃下培养2天以去除pFC332-Ffcas9-ΔpyrG载体,如此构建的菌株记为Fusariumfujikuroi-ΔpyrG。
表1:引物序列
引物1 | ttgattgagcaagagggcagCACATACGACCAAAGGTAGT |
引物2 | gtcaactggacgaggatagcCATACAACAGCGGGGATTCG |
引物3 | gctatcctcgtccagttgacGTTTTAGAGCTAGAAATAGC |
引物4 | cttgaatcgcgcattggatcAAAAAAAAGCACCGACTCGG |
引物5 | gaagtgcattgtaagcacacgcaag |
引物6 | cttcgaacaagctttaccgtatcgaagcgaaaacgactggaagtcg |
引物7 | cgacttccagtcgttttcgcttcgatacggtaaagcttgttcgaag |
引物8 | acattccacccatttacgcctcaca |
引物9 | gaaggtctatgctgatctcg |
引物10 | gcactagcacctaacttacc |
实施例2 F.Fujikuroi-RE:pyrGΔmeaB的获得
(1)pyrG基因的回补
pyrG缺陷型的藤仓赤霉菌中由于不能合成尿嘧啶导致不能存活,通过回补pyrG基因使赤霉菌重新获得合成尿嘧啶的能力,导致回复突变菌株可以在无外源添加尿嘧啶的平板上存活。通过PCR使用引物11和引物12,以藤仓赤霉菌(Fusarium fujikuroi)CCTCC NO:M2019378基因组为模板扩增得到完整pyrG表达框,PCR反应条件如下:98℃5min;98℃30s,60℃30s,72℃1min30s,重复30个循环;72℃继续延续5min。PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段。
(2)meaB基因上下游同源臂扩增
bZIP型转录抑制因子meaB抑制了赤霉素合成基因簇的表达,因此以同源双交换的方式将该基因敲除。通过PCR使用引物13和引物14,以藤仓赤霉菌(Fusarium fujikuroi)CCTCC NO:M 2019378基因组为模板扩增得到meaB基因上游同源片段,PCR反应条件如下:98℃5min;98℃30s,60℃30s,72℃30s,重复30个循环;72℃继续延续5min。PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段。按同样的方法使用引物15和引物16扩增得到meaB基因下游同源片段,PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段。
(3)外源转化片段连接
将回收的三个DNA片段以meaB上游,pyrG表达框,meaB下游的顺序使用引物13和引物16进行融合PCR,PCR反应条件如下:98℃5min;98℃30s,60℃30s,72℃4min,重复30个循环;72℃继续延续5min。PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段(核苷酸序列为SEQ ID NO.6所示)。
通过CRISPR-Cas9系统敲除藤仓赤霉菌(Fusarium fujikuroi)CCTCC NO:M2019378基因组中编码bZIP转录抑制因子的meaB基因。通过PCR使用引物17和引物18,以sgRNA表达框作为模板扩增得到sgRNA-ΔmeaB-1片段,PCR反应条件如下:98℃3min;98℃15s,60℃15s,72℃15s,重复30个循环;72℃继续延续3min.按同样的方法使用引物19和引物20扩增得到sgRNA-ΔmeaB-2片段,PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段。将回收的两个DNA片段使用引物17和引物20进行融合PCR,PCR反应条件如下:98℃3min;98℃15s,60℃15s,72℃30s,重复30个循环;72℃继续延续3min。PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段(核苷酸序列为SEQ ID NO.7所示)。以pFC332-Ffcas9质粒作为模板,通过一步克隆试剂盒ClonExpress II One Step Cloning Kit(购自南京诺唯赞vazyme)连接构建能够表达靶向目的基因meaB的sgRNA的pFC332-Ffcas9-ΔmeaB突变载体质粒。连接产物转化至E.coli DH5α受体菌中,涂布于含终浓度100mg/L氨苄霉素抗性的LB固体平板上,37℃培养12h。随机挑取单菌落至含终浓度100mg/L氨苄霉素抗性的LB液体培养基中,37℃培养12h,收集菌体并提取质粒获得pFC332-Ffcas9-ΔmeaB载体。将pFC332-Ffcas9-ΔmeaB载体和回收的DNA片段一同转化至藤仓赤霉菌Fusariumfujikuroi-ΔpyrG的原生质体。
将转化后的菌液涂至MYG平板,28℃培养4天,挑取单菌落作为模板,以引物21和引物22进行PCR,观察到在1.0%琼脂糖凝胶中存在4600bp的DNA条带以确认meaB基因的缺失与pyrG表达框的插入。将通过确认的菌株在YPD液体培养基中在28℃下培养2天以去除pFC332-Ffcas9-ΔmeaB载体质粒,如此构建的菌株命名为Fusarium fujikuroi-RE:pyrGΔmeaB。
表2:引物序列
实施例3:F.fujikuroi-RE:pyrG GpdA-tHmgRΔmeaB的获得
(1)thmgR基因的增强表达
hmgR基因编码的3-羟基-3-甲基戊二酸单酰辅酶A还原酶在GA3的合成中,在甲羟戊酸途径中起到限速酶的作用。由于3-羟基-3-甲基戊二酸单酰辅酶A还原酶的C端能结合到内质网膜上,导致催化合成的产物触发反馈抑制。通过PCR使用引物23和引物24,以质粒pAN7-1为模板扩增得到gpdA启动子序列,PCR反应条件如下:98℃5min;98℃30s,60℃30s,72℃30s,重复30个循环;72℃继续延续5min。
通过PCR使用引物25和引物26,以藤仓赤霉菌(Fusarium fujikuroi)CCTCC NO:M2019378基因组为模板扩增得到负责催化的3-羟基-3-甲基戊二酸单酰辅酶A还原酶N端编码区,PCR反应条件如下:98℃5min;98℃30s,60℃30s,72℃60s,重复30个循环;72℃继续延续5min。PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段。将回收的两个DNA片段使用引物24和引物25进行融合PCR,PCR反应条件如下:98℃5min;98℃30s,60℃30s,72℃1min30s,重复30个循环;72℃继续延续3min。PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段。
(2)RE:pyrG末端与出发菌株meaB终止子作为上下游同源臂的扩增
出发菌株的meaB编码区下游序列为野生型终止子,具有终止表达的能力。因此将gpdA启动子增强表达的thmgR基因插入到该段序列之前,可以在基因组上构成完整的thmgR表达框。通过PCR使用引物27和引物28,以Fusarium fujikuroi-RE:pyrGΔmeaB的基因组为模板,PCR反应条件如下:98℃5min;98℃30s,60℃30s,72℃60s,重复30个循环;72℃继续延续5min扩增得到pyrG表达框下游片段作为同源臂上游,按同样的方法使用引物29和引物30扩增得到meaB终止子起始片段作为同源臂下游,PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段。将回收的三个DNA片段以pyrG表达框下游片段、gpdA-tHmgR、meaB终止子起始片段的顺序使用引物27和引物30进行融合PCR,PCR反应条件如下:98℃5min;98℃30s,60℃30s,72℃3min,重复30个循环;72℃继续延续3min。PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段(核苷酸序列为SEQ ID NO.8所示)。
通过CRISPR-Cas9系统编辑藤仓赤霉菌(Fusarium fujikuroi)CCTCC NO:M2019378基因组中编码bZIP转录抑制因子的meaB基因的终止子。通过PCR使用引物31和引物32,以sgRNA表达框作为模板扩增得到sgRNA-ΔTmeaB-1片段,PCR反应条件如下:98℃3min;98℃15s,60℃15s,72℃15s,重复30个循环;72℃继续延续3min.按同样的方法使用引物33和引物34扩增得到sgRNA-ΔTmeaB-2片段,PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段。将回收的两个DNA片段使用引物31和引物32进行融合PCR,PCR反应条件如下:98℃3min;98℃15s,60℃15s,72℃30s,重复30个循环;72℃继续延续3min。PCR产物用1.0%琼脂糖凝胶电泳检测并切胶回收纯化片段(核苷酸序列为SEQ ID NO.9所示)。以pFC332-Ffcas9载体作为模板,通过一步克隆试剂盒ClonExpress II One Step Cloning Kit(购自南京诺唯赞vazyme)连接构建能够表达靶向目的基因meaB的sgRNA的pFC332-Ffcas9-ΔTmeaB突变载体。连接产物转化至E.coli DH5α受体菌中,涂布于含终浓度100mg/L氨苄霉素抗性的LB固体平板上,37℃培养12h。随机挑取单菌落至含终浓度100mg/L氨苄霉素抗性的LB液体培养基中,37℃培养12h,收集菌体并提取质粒获得pFC332-Ffcas9-ΔTmeaB载体。将pFC332-Ffcas9-ΔTmeaB载体和回收的DNA片段一同转化至藤仓赤霉菌Fusariumfujikuroi-ΔpyrG的原生质体。
将转化后的菌液涂至MYG平板,28℃培养4天,挑取单菌落作为模板,以引物21和引物22进行PCR,观察到在1.0%琼脂糖凝胶中存在7400bp的DNA条带以确认tHmgR表达框的插入。将通过此确认的菌株在YPD液体培养基中在28℃下培养2天以去除pFC332-Ffcas9-ΔTmeaB载体质粒,如此构建的菌株记为Fusarium fujikuroi-RE:pyrG GpdA-tHmgRΔmeaB。
表3:引物序列
引物23 | ctgatatggacgatccaaagcttgagtacagtgaccggtgactctttctg |
引物24 | taggtcggttgttggacttggcCATggtgatgtctgctcaagcggggtag |
引物25 | ctaccccgcttgagcagacatcaccATGgccaagtccaacaaccgaccta |
引物26 | CCAGAGGTTTGTTCGAGCTCCAAGTctatcgctttgacctctgaatagca |
引物27 | gatacggtaaagcttgttcgaag |
引物28 | cagaaagagtcaccggtcactgtactcaagctttggatcgtccatatcag |
引物29 | tgctattcagaggtcaaagcgatagACTTGGAGCTCGAACAAACCTCTGG |
引物30 | CCAGTCCAAATCTTGCTTGGTTCCTTGT |
引物31 | ttgattgagcaagagggcagCACATACGACCAAAGGTAGT |
引物32 | tatcagtgctattctagccaCATACAACAGCGGGGATTCG |
引物33 | tggctagaatagcactgataGTTTTAGAGCTAGAAATAGC |
引物34 | cttgaatcgcgcattggatcAAAAAAAAGCACCGACTCGG |
实施例4:不同基因型菌株的摇瓶发酵测试
将实施例2,3构建的改造菌株和初始菌株分别接种到25mL种子培养基中,28℃、250rpm培养用作预培养物。48h后,接种2.8mL预培养物到装有40mL发酵培养基的500mL摇瓶。然后在28℃、250rpm培养7天,发酵过程中,其发酵液中GA3含量变化如图3。
种子培养基按以下方法制得:玉米淀粉20g/L、蔗糖20g/L、花生粉20g/L、大豆粉20g/L、磷酸二氢钾1.0g/L、硫酸镁1.0g/L,溶剂为自来水,pH自然,121℃灭菌30min。
发酵培养基组成:玉米淀粉80g/L,米粉80g/L,大豆粉5g/L,花生粉5g/L,K2SO40.5g/L,KH2PO4 0.5g/L,溶剂为自来水,pH自然,121度灭菌30min。
不同基因型的菌株发酵产量如表4所示。
表4:不同基因型工程菌株的发酵结果
经代谢工程改造后的菌株Fusarium fujikuroi-RE:pyrG GpdA-tHmgRΔmeaB GA3产量最高,达到2.35g/L。
序列表
<110> 浙江工业大学
<120> 高产赤霉素GA3的藤仓赤霉基因工程菌、构建方法及应用
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3101
<212> DNA
<213> F. fujikuroi
<400> 1
gtatccgaat aagtcggcgt tgtttttgtt gtttgcttgc tctgtttggc cgttggagct 60
cctgtttcat aaatacaagc tgactctttg tttcgagatt tgcaattgcc gcatggctgt 120
tcgcggtcgc actagatatg tattcagtca aatatttctt gtatcagaca aatgtatcgt 180
agtctcatag ttacggaccc tctgctttct tcgatggcat tcagtgcacg atatgacaat 240
acgtcttctc ttcctcagac gcggaccatc agcatcctca gttgctgctg tcatgtcggc 300
ttcttcggaa acggatatga atgattgatc acttgggaat actaattagg agcaaatatg 360
caacgttatg gtaaaaggac ctagtatgag atccagaaat ttggagacta gaggtcaata 420
tttaggtgga gtatgggaag gtctatgctg atctcggcgg acgaggatca gctttcaaag 480
tcctcttcac ttaactccgg acttccaaga aaacggcgtc actttgattg tggggcatcc 540
attacaagaa tggaattcag ccctgtgcat ctttgaatgg acccacgtta ttgaaggaag 600
aaatggaaat gcgcggttgg agtggattag ctagctctaa cggtcgacaa gaagtgcatt 660
gtaagcacac gcaaggaacc gaattattcc gccattgcaa tatatatttc gtatcttttc 720
cgataatcat tctcgtaatg ttgtgttgaa cttggtctca ttgatgcatg tactgtatag 780
agcaaccacg cttatgcagt atgaggaggt ctagtgctta gatgatgttc ccaatcaggg 840
aaccaagtct catttctctt gtaagtgagt tactccatgg atactgcatg tagtcatgat 900
attcacgtca gacccactag atgtctcgat aactccagtg cagcctgtct accgcgcgaa 960
aaacctcaag gccgtcccga cttccagtcg ttttcgcttc ttaacgaacc cgctccgtgt 1020
aagccttcca tgccgcggac cgatatcgct cagcttcctc ctcgggatcg cttgccttga 1080
tgataccgcg tccaacaatg gcaatgtccg caccggcaat accaatgatc ttctgtggcg 1140
tgttgtactg ttgacccttg ccatcaccct tgatggcttc attctcatcc gcgccctcag 1200
ggggcaattg gcagccgggg gtcatgtgga tgaagtcatc ctcgggttct gtgttgaggc 1260
actcctgtga gacaaagccc atgacgaagc tcttgtgttc ccgtgcggcc tctacacatg 1320
cttgtgtata ttccttattc atgaagttgc cttgactcga catctgtgcc agaataagca 1380
ggcctctctc gatcggcgct tcgtcgatgc cggggaatac cgtctcatcg ccctcaggaa 1440
tcgtcttgac gaggcgcggt gaatcggccg actcgtactg ctgggtgacg gtggtggtgg 1500
aaacaatgct accttttcgg ccgctgtcag cgcgagcagg ttccggcgtg tccttctggt 1560
tctcgttgtc tgaacgctcg ctatcctcgt ccagttgact ggcggtgggt gttccaatac 1620
tgatagatgt cttgacctcg tagtggtacc gctcgagcca tcgattggca gcgtgggcca 1680
gagatgcaac cgatgccttg cctggaacca tatttacatt ggtaatgtgt gcccactcga 1740
tgatgcgcgc agtaccgcta gtatactgca actcgaccgt attgccaatg tcgccaaact 1800
tgcgatcctc aaagatgagg aaaccatgct tgcgcgctag tgaggcgagc ttggcaccag 1860
tacctgtcct agggtcaaag tcccagccgg caaccatgtc ataatgcgtc ttgagaacga 1920
cgatggaggg accaatcttg tcggcgaagt agagcagttc gcgagcggtt gcgacatcgg 1980
cgctgaggca gagattcgag gccttgaggt ccatgagctt gtagagatgt cgactgagag 2040
gatgggtagc tgtctcagct cgactggcga aagtcgcctt gagggtagga tgcggcgaca 2100
tgatacggta aagcttgttc gaagatcaat ttgatatgaa caaaggtatc ggaaaagacg 2160
gtgacccgaa ctggggagcg agttcagata cactaactaa atcgtatgtg gatctaagtc 2220
gcaagttttg ggcaacctcg agactaggcc aattcacaaa gaagttcaca acgatctagt 2280
caacctggcg caggtggcgg tttcgcttga tagaggggaa gctggggggg atcgaaaaaa 2340
acggacacaa gagccacaga taggaacgag gggtgaagat cttattagct tcggccgaat 2400
gggtctcgac tgaaagtgtc gtccaatgtg aggcgtaaat gggtggaatg tcgatcggga 2460
gtgggaggag aattatgagg agagagagga gaggagagga gagaggagag gaaagagagc 2520
gagtcaagag gaaggaggag gaggtttgaa gtaaaagacc tttcgcagaa tgacacggga 2580
tgggatggga tgggggtagt taagaagaga ctgtaggtga gaggtgaaag gtaaaaagtg 2640
aaaagtgacg atccacagct cgacttaaac aaaagtgacc tatggaacca accaactaaa 2700
aggcactgaa ggtgaggcga gagggtcact accacctaag gtaagttagg tgctagtgct 2760
agggtacctt agcttagctc caacgtgaag cgaaaaggcg cctgaacctc gagactcgta 2820
acaagctaac acctttttaa ggcccgaggc tcaaacgcag ccagaaagta agtaccaagg 2880
taggcattcg gaggttgaag gtaggtactg tacaggcgtg gcgtcaaaca gggatcaaca 2940
agagtgtctc ttttttactg taacaggcgg caaatgcaac atgtacgcac taggtagagg 3000
cactactgta tgtactcaga caggaacagg ggcaaggatg tttgacagca attcaaagaa 3060
aatggctaga atagcactga tatggacgat ccaaagcttg a 3101
<210> 2
<211> 1967
<212> DNA
<213> F. fujikuroi
<400> 2
atggccaagt ccaacaaccg acctacattg gtcactgtct tgactcccat caagtacgag 60
ctcgagtacc cttcgatcca ctacgctctt ggttctgccg ccagcaaccc tgcctacaac 120
gatgctttcc accatcactt ccagggctac ggtgtcggcg gccgcatggt tggcggcatt 180
ctcaagtctc tcgaggatcc cgtcctctcc aagtggattg tcattgccct tgctctcagc 240
gtcgctctta acggctacct cttcaacgtt gcccgctggg gaatcaagga ccccaatgtt 300
cctgagcaca acatcgaccg aaacgaactt gcccgtgcgc agcagtttaa cgacaccggc 360
tccgctactc ttcctctcgg cgaatacgtg cctcccactc ccatgcgaac ccaacccagc 420
actcctgcta ttaccgacga cgaggccgaa ggtcttcaca tgacaaaggc tcgacctgcc 480
aacctgccca accgaagcaa cgaagaactt gagaagctct tgtctgagaa gcgcgtccgc 540
gagatgaccg acgaggaggt tatttctctg tctatgcgtg gcaagatccc tggttacgca 600
cttgaaaaga ccctcggtga cttcactcgc gctgtcaaga ttcgacgaag cattattgct 660
cgcaacaagg ccaccaccga cattacccac tcgctcgacc gatccaagct accttacgag 720
aactataact gggagcgcgt ttttggcgca tgctgtgaga acgttattgg atacatgcct 780
cttcccgtcg gtgttgctgg tcctcttgtc atcgatggac agagctactt catccccatg 840
gctactactg agggtgtctt ggttgccagt gccagtcgag gttgcaaggc catcaactct 900
ggtggtggtg ccatcactgt tctcactgct gatggtatga ctcgtggtcc ttgtgtcgct 960
ttcgagactc ttgagcgcgc tggtgctgcc aagctctggc ttgactctga agctggtcag 1020
gatatgatga agaaggcttt caactcaacc agtcgcttcg cccgcctcca gtccatgaag 1080
accgctcttg ctggtaccaa cctgtacatt cgattcaaga ccaccaccgg tgacgctatg 1140
ggtatgaaca tgatttccaa gggagtcgag cacgctctaa gcgtcatggc caacgatgga 1200
ggtttcgacg acatgcagat catctctgtc tctggcaact actgtacgga taagaaggcc 1260
gcggccctca actggatcga cggacgtggt aagggtgttg tcgctgaggc tatcattccc 1320
ggtgaggtcg tccgcagcgt tctcaagagc gatgtcgact ctcttgttga gctcaacgtt 1380
gctaagaact tgattggttc tgctatggct ggttcagttg gtggtttcaa cgcccacgct 1440
gccaacattg tcgctgctat tttcctggcc actggacagg accctgctca ggttgtcgag 1500
agcgccaatt gtatcaccat catgaagaag taagtttgtc tcgcacatca agtcggaaac 1560
atgctaacgt ccatagcctc aatggagctc tccagatctc cgtctctatg ccctcgctcg 1620
aggtcggaac tctcggcggt ggtaccatcc tcgagcccca gggcgccatg ctcgacatcc 1680
ttggtgtccg aggctctcac cccaccaacc ccggtgacaa cgcccgccgt ctcgcccgca 1740
tcatcggtgc agccgtcctc gccggcgagc tttctctctg cagtgccttg gccgccggtc 1800
atctcgtccg agctcacatg cagcataacc gaagtgccgc tccctctcgc agcaccactc 1860
ctgctcctcc catgacgccc gtctcactgg ccatgaccag tgcccaagag cgctcagcgt 1920
caacaacgtc gatgagcgcc gctgctattc agaggtcaaa gcgatag 1967
<210> 3
<211> 879
<212> DNA
<213> 未知(Unknown)
<400> 3
gtacagtgac cggtgactct ttctggcatg cggagagacg gacggacgca gagagaaggg 60
ctgagtaata agccactggc cagacagctc tggcggctct gaggtgcagt ggatgattat 120
taatccggga ccggccgccc ctccgccccg aagtggaaag gctggtgtgc ccctcgttga 180
ccaagaatct attgcatcat cggagaatat ggagcttcat cgaatcaccg gcagtaagcg 240
aaggagaatg tgaagccagg ggtgtatagc cgtcggcgaa atagcatgcc attaacctag 300
gtacagaagt ccaattgctt ccgatctggt aaaagattca cgagatagta ccttctccga 360
agtaggtaga gcgagtaccc ggcgcgtaag ctccctaatt ggcccatccg gcatctgtag 420
ggcgtccaaa tatcgtgcct ctcctgcttt gcccggtgta tgaaaccgga aaggccgctc 480
aggagctggc cagcggcgca gaccgggaac acaagctggc agtcgaccca tccggtgctc 540
tgcactcgac ctgctgaggt ccctcagtcc ctggtaggca gctttgcccc gtctgtccgc 600
ccggtgtgtc ggcggggttg acaaggtcgt tgcgtcagtc caacatttgt tgccatattt 660
tcctgctctc cccaccagct gctcttttct tttctctttc ttttcccatc ttcagtatat 720
tcatcttccc atccaagaac ctttatttcc cctaagtaag tactttgcta catccatact 780
ccatccttcc catcccttat tcctttgaac ctttcagttc gagctttccc acttcatcgc 840
agcttgacta acagctaccc cgcttgagca gacatcacc 879
<210> 4
<211> 263
<212> DNA
<213> 未知(Unknown)
<400> 4
ttgattgagc aagagggcag cacatacgac caaaggtagt ggaaaatacg ggatcccgtc 60
cgctctccca tagtcaagcc actaaccggc ggattagtag ttgggtcggt gacgaccagc 120
gaatccccgc tgttgtatgg ctatcctcgt ccagttgacg ttttagagct agaaatagca 180
agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 240
tttgatccaa tgcgcgattc aag 263
<210> 5
<211> 700
<212> DNA
<213> 未知(Unknown)
<400> 5
gaagtgcatt gtaagcacac gcaaggaacc gaattattcc gccattgcaa tatatatttc 60
gtatcttttc cgataatcat tctcgtaatg ttgtgttgaa cttggtctca ttgatgcatg 120
tactgtatag agcaaccacg cttatgcagt atgaggaggt ctagtgctta gatgatgttc 180
ccaatcaggg aaccaagtct catttctctt gtaagtgagt tactccatgg atactgcatg 240
tagtcatgat attcacgtca gacccactag atgtctcgat aactccagtg cagcctgtct 300
accgcgcgaa aaacctcaag gccgtcccga cttccagtcg ttttcgcttc gatacggtaa 360
agcttgttcg aagatcaatt tgatatgaac aaaggtatcg gaaaagacgg tgacccgaac 420
tggggagcga gttcagatac actaactaaa tcgtatgtgg atctaagtcg caagttttgg 480
gcaacctcga gactaggcca attcacaaag aagttcacaa cgatctagtc aacctggcgc 540
aggtggcggt ttcgcttgat agaggggaag ctggggggga tcgaaaaaaa cggacacaag 600
agccacagat aggaacgagg ggtgaagatc ttattagctt cggccgaatg ggtctcgact 660
gaaagtgtcg tccaatgtga ggcgtaaatg ggtggaatgt 700
<210> 6
<211> 4101
<212> DNA
<213> 未知(Unknown)
<400> 6
ctgagtgatc cccttctctg ttatcgcaac tgtccccccc aagaactcga cgcgaattac 60
agcccgtctt ttatctcttg gtagaaattc ttacgtttcc caaagaccct tacctgcgac 120
ctacaactga taggcgcttg ttagacgcag cgcgtatcat tgtcgctcag ccagtaccat 180
ctctgatagc caagcgtttt tgggtttgtc tgcgtttctg tgtctgataa gggccctttt 240
cacactccag tacgctgttg acccccgtac gacccccaat ccgggctgca tttcctatct 300
cttgcgcccg ctctccctac gcccgcaacc gcaaagcaaa aacccaacgg gcgcacactt 360
gcctgtctcg attctttgcg cccgtcgtcg tcttgacgtt ccacctcgtc acgtaacaga 420
attcccgttt tgcgtctctg gggcgtttgc cgctgcgcgg caaatcgtct agtagcccgg 480
tctgggtaat ggcagacaag gtatccgaat aagtcggcgt tgtttttgtt gtttgcttgc 540
tctgtttggc cgttggagct cctgtttcat aaatacaagc tgactctttg tttcgagatt 600
tgcaattgcc gcatggctgt tcgcggtcgc actagatatg tattcagtca aatatttctt 660
gtatcagaca aatgtatcgt agtctcatag ttacggaccc tctgctttct tcgatggcat 720
tcagtgcacg atatgacaat acgtcttctc ttcctcagac gcggaccatc agcatcctca 780
gttgctgctg tcatgtcggc ttcttcggaa acggatatga atgattgatc acttgggaat 840
actaattagg agcaaatatg caacgttatg gtaaaaggac ctagtatgag atccagaaat 900
ttggagacta gaggtcaata tttaggtgga gtatgggaag gtctatgctg atctcggcgg 960
acgaggatca gctttcaaag tcctcttcac ttaactccgg acttccaaga aaacggcgtc 1020
actttgattg tggggcatcc attacaagaa tggaattcag ccctgtgcat ctttgaatgg 1080
acccacgtta ttgaaggaag aaatggaaat gcgcggttgg agtggattag ctagctctaa 1140
cggtcgacaa gaagtgcatt gtaagcacac gcaaggaacc gaattattcc gccattgcaa 1200
tatatatttc gtatcttttc cgataatcat tctcgtaatg ttgtgttgaa cttggtctca 1260
ttgatgcatg tactgtatag agcaaccacg cttatgcagt atgaggaggt ctagtgctta 1320
gatgatgttc ccaatcaggg aaccaagtct catttctctt gtaagtgagt tactccatgg 1380
atactgcatg tagtcatgat attcacgtca gacccactag atgtctcgat aactccagtg 1440
cagcctgtct accgcgcgaa aaacctcaag gccgtcccga cttccagtcg ttttcgcttc 1500
ttaacgaacc cgctccgtgt aagccttcca tgccgcggac cgatatcgct cagcttcctc 1560
ctcgggatcg cttgccttga tgataccgcg tccaacaatg gcaatgtccg caccggcaat 1620
accaatgatc ttctgtggcg tgttgtactg ttgacccttg ccatcaccct tgatggcttc 1680
attctcatcc gcgccctcag ggggcaattg gcagccgggg gtcatgtgga tgaagtcatc 1740
ctcgggttct gtgttgaggc actcctgtga gacaaagccc atgacgaagc tcttgtgttc 1800
ccgtgcggcc tctacacatg cttgtgtata ttccttattc atgaagttgc cttgactcga 1860
catctgtgcc agaataagca ggcctctctc gatcggcgct tcgtcgatgc cggggaatac 1920
cgtctcatcg ccctcaggaa tcgtcttgac gaggcgcggt gaatcggccg actcgtactg 1980
ctgggtgacg gtggtggtgg aaacaatgct accttttcgg ccgctgtcag cgcgagcagg 2040
ttccggcgtg tccttctggt tctcgttgtc tgaacgctcg ctatcctcgt ccagttgact 2100
ggcggtgggt gttccaatac tgatagatgt cttgacctcg tagtggtacc gctcgagcca 2160
tcgattggca gcgtgggcca gagatgcaac cgatgccttg cctggaacca tatttacatt 2220
ggtaatgtgt gcccactcga tgatgcgcgc agtaccgcta gtatactgca actcgaccgt 2280
attgccaatg tcgccaaact tgcgatcctc aaagatgagg aaaccatgct tgcgcgctag 2340
tgaggcgagc ttggcaccag tacctgtcct agggtcaaag tcccagccgg caaccatgtc 2400
ataatgcgtc ttgagaacga cgatggaggg accaatcttg tcggcgaagt agagcagttc 2460
gcgagcggtt gcgacatcgg cgctgaggca gagattcgag gccttgaggt ccatgagctt 2520
gtagagatgt cgactgagag gatgggtagc tgtctcagct cgactggcga aagtcgcctt 2580
gagggtagga tgcggcgaca tgatacggta aagcttgttc gaagatcaat ttgatatgaa 2640
caaaggtatc ggaaaagacg gtgacccgaa ctggggagcg agttcagata cactaactaa 2700
atcgtatgtg gatctaagtc gcaagttttg ggcaacctcg agactaggcc aattcacaaa 2760
gaagttcaca acgatctagt caacctggcg caggtggcgg tttcgcttga tagaggggaa 2820
gctggggggg atcgaaaaaa acggacacaa gagccacaga taggaacgag gggtgaagat 2880
cttattagct tcggccgaat gggtctcgac tgaaagtgtc gtccaatgtg aggcgtaaat 2940
gggtggaatg tcgatcggga gtgggaggag aattatgagg agagagagga gaggagagga 3000
gagaggagag gaaagagagc gagtcaagag gaaggaggag gaggtttgaa gtaaaagacc 3060
tttcgcagaa tgacacggga tgggatggga tgggggtagt taagaagaga ctgtaggtga 3120
gaggtgaaag gtaaaaagtg aaaagtgacg atccacagct cgacttaaac aaaagtgacc 3180
tatggaacca accaactaaa aggcactgaa ggtgaggcga gagggtcact accacctaag 3240
gtaagttagg tgctagtgct agggtacctt agcttagctc caacgtgaag cgaaaaggcg 3300
cctgaacctc gagactcgta acaagctaac acctttttaa ggcccgaggc tcaaacgcag 3360
ccagaaagta agtaccaagg taggcattcg gaggttgaag gtaggtactg tacaggcgtg 3420
gcgtcaaaca gggatcaaca agagtgtctc ttttttactg taacaggcgg caaatgcaac 3480
atgtacgcac taggtagagg cactactgta tgtactcaga caggaacagg ggcaaggatg 3540
tttgacagca attcaaagaa aatggctaga atagcactga tatggacgat ccaaagcttg 3600
aacttggagc tcgaacaaac ctctggaatt ggtcacctag agggtccatt catcgacaga 3660
cgcctgaaga gctgctatca cggaagactc cacactcccg tcagctctcg ctcaagtctt 3720
gaaatccatg gtcgagagtt tggggggttg agaaaccaac cactcctgac aaaaaacctc 3780
gcgcaccatt ggcatgcttc atgaagataa gaacacttaa cggtcgcact tgattgaaag 3840
gcaatttcga cgcgactgac ttgttgattc ttctgcggga tgtgcgatag atatatccca 3900
ccgcaacaga cttgagtaac aggctcacca cagcgaggcc tatagtcata cccattcccc 3960
agttaatatg gcgctgggca taagtagggc tatggtagca tccggatggc aaggcacgtc 4020
gaaggacttg acgactacaa gcaaagacag aacacttcag catggagagt atatcgtgtt 4080
gaacagctct agcggaacta t 4101
<210> 7
<211> 263
<212> DNA
<213> 未知(Unknown)
<400> 7
ttgattgagc aagagggcag cacatacgac caaaggtagt ggaaaatacg ggatcccgtc 60
cgctctccca tagtcaagcc actaaccggc ggattagtag ttgggtcggt gacgaccagc 120
gaatccccgc tgttgtatgc aagacgggta gtcctaactg ttttagagct agaaatagca 180
agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 240
tttgatccaa tgcgcgattc aag 263
<210> 8
<211> 4846
<212> DNA
<213> 未知(Unknown)
<400> 8
gatacggtaa agcttgttcg aagatcaatt tgatatgaac aaaggtatcg gaaaagacgg 60
tgacccgaac tggggagcga gttcagatac actaactaaa tcgtatgtgg atctaagtcg 120
caagttttgg gcaacctcga gactaggcca attcacaaag aagttcacaa cgatctagtc 180
aacctggcgc aggtggcggt ttcgcttgat agaggggaag ctggggggga tcgaaaaaaa 240
cggacacaag agccacagat aggaacgagg ggtgaagatc ttattagctt cggccgaatg 300
ggtctcgact gaaagtgtcg tccaatgtga ggcgtaaatg ggtggaatgt cgatcgggag 360
tgggaggaga attatgagga gagagaggag aggagaggag agaggagagg aaagagagcg 420
agtcaagagg aaggaggagg aggtttgaag taaaagacct ttcgcagaat gacacgggat 480
gggatgggat gggggtagtt aagaagagac tgtaggtgag aggtgaaagg taaaaagtga 540
aaagtgacga tccacagctc gacttaaaca aaagtgacct atggaaccaa ccaactaaaa 600
ggcactgaag gtgaggcgag agggtcacta ccacctaagg taagttaggt gctagtgcta 660
gggtacctta gcttagctcc aacgtgaagc gaaaaggcgc ctgaacctcg agactcgtaa 720
caagctaaca cctttttaag gcccgaggct caaacgcagc cagaaagtaa gtaccaaggt 780
aggcattcgg aggttgaagg taggtactgt acaggcgtgg cgtcaaacag ggatcaacaa 840
gagtgtctct tttttactgt aacaggcggc aaatgcaaca tgtacgcact aggtagaggc 900
actactgtat gtactcagac aggaacaggg gcaaggatgt ttgacagcaa ttcaaagaaa 960
atggctagaa tagcactgat atggacgatc caaagcttga gtacagtgac cggtgactct 1020
ttctggcatg cggagagacg gacggacgca gagagaaggg ctgagtaata agccactggc 1080
cagacagctc tggcggctct gaggtgcagt ggatgattat taatccggga ccggccgccc 1140
ctccgccccg aagtggaaag gctggtgtgc ccctcgttga ccaagaatct attgcatcat 1200
cggagaatat ggagcttcat cgaatcaccg gcagtaagcg aaggagaatg tgaagccagg 1260
ggtgtatagc cgtcggcgaa atagcatgcc attaacctag gtacagaagt ccaattgctt 1320
ccgatctggt aaaagattca cgagatagta ccttctccga agtaggtaga gcgagtaccc 1380
ggcgcgtaag ctccctaatt ggcccatccg gcatctgtag ggcgtccaaa tatcgtgcct 1440
ctcctgcttt gcccggtgta tgaaaccgga aaggccgctc aggagctggc cagcggcgca 1500
gaccgggaac acaagctggc agtcgaccca tccggtgctc tgcactcgac ctgctgaggt 1560
ccctcagtcc ctggtaggca gctttgcccc gtctgtccgc ccggtgtgtc ggcggggttg 1620
acaaggtcgt tgcgtcagtc caacatttgt tgccatattt tcctgctctc cccaccagct 1680
gctcttttct tttctctttc ttttcccatc ttcagtatat tcatcttccc atccaagaac 1740
ctttatttcc cctaagtaag tactttgcta catccatact ccatccttcc catcccttat 1800
tcctttgaac ctttcagttc gagctttccc acttcatcgc agcttgacta acagctaccc 1860
cgcttgagca gacatcacca tggccaagtc caacaaccga cctacattgg tcactgtctt 1920
gactcccatc aagtacgagc tcgagtaccc ttcgatccac tacgctcttg gttctgccgc 1980
cagcaaccct gcctacaacg atgctttcca ccatcacttc cagggctacg gtgtcggcgg 2040
ccgcatggtt ggcggcattc tcaagtctct cgaggatccc gtcctctcca agtggattgt 2100
cattgccctt gctctcagcg tcgctcttaa cggctacctc ttcaacgttg cccgctgggg 2160
aatcaaggac cccaatgttc ctgagcacaa catcgaccga aacgaacttg cccgtgcgca 2220
gcagtttaac gacaccggct ccgctactct tcctctcggc gaatacgtgc ctcccactcc 2280
catgcgaacc caacccagca ctcctgctat taccgacgac gaggccgaag gtcttcacat 2340
gacaaaggct cgacctgcca acctgcccaa ccgaagcaac gaagaacttg agaagctctt 2400
gtctgagaag cgcgtccgcg agatgaccga cgaggaggtt atttctctgt ctatgcgtgg 2460
caagatccct ggttacgcac ttgaaaagac cctcggtgac ttcactcgcg ctgtcaagat 2520
tcgacgaagc attattgctc gcaacaaggc caccaccgac attacccact cgctcgaccg 2580
atccaagcta ccttacgaga actataactg ggagcgcgtt tttggcgcat gctgtgagaa 2640
cgttattgga tacatgcctc ttcccgtcgg tgttgctggt cctcttgtca tcgatggaca 2700
gagctacttc atccccatgg ctactactga gggtgtcttg gttgccagtg ccagtcgagg 2760
ttgcaaggcc atcaactctg gtggtggtgc catcactgtt ctcactgctg atggtatgac 2820
tcgtggtcct tgtgtcgctt tcgagactct tgagcgcgct ggtgctgcca agctctggct 2880
tgactctgaa gctggtcagg atatgatgaa gaaggctttc aactcaacca gtcgcttcgc 2940
ccgcctccag tccatgaaga ccgctcttgc tggtaccaac ctgtacattc gattcaagac 3000
caccaccggt gacgctatgg gtatgaacat gatttccaag ggagtcgagc acgctctaag 3060
cgtcatggcc aacgatggag gtttcgacga catgcagatc atctctgtct ctggcaacta 3120
ctgtacggat aagaaggccg cggccctcaa ctggatcgac ggacgtggta agggtgttgt 3180
cgctgaggct atcattcccg gtgaggtcgt ccgcagcgtt ctcaagagcg atgtcgactc 3240
tcttgttgag ctcaacgttg ctaagaactt gattggttct gctatggctg gttcagttgg 3300
tggtttcaac gcccacgctg ccaacattgt cgctgctatt ttcctggcca ctggacagga 3360
ccctgctcag gttgtcgaga gcgccaattg tatcaccatc atgaagaagt aagtttgtct 3420
cgcacatcaa gtcggaaaca tgctaacgtc catagcctca atggagctct ccagatctcc 3480
gtctctatgc cctcgctcga ggtcggaact ctcggcggtg gtaccatcct cgagccccag 3540
ggcgccatgc tcgacatcct tggtgtccga ggctctcacc ccaccaaccc cggtgacaac 3600
gcccgccgtc tcgcccgcat catcggtgca gccgtcctcg ccggcgagct ttctctctgc 3660
agtgccttgg ccgccggtca tctcgtccga gctcacatgc agcataaccg aagtgccgct 3720
ccctctcgca gcaccactcc tgctcctccc atgacgcccg tctcactggc catgaccagt 3780
gcccaagagc gctcagcgtc aacaacgtcg atgagcgccg ctgctattca gaggtcaaag 3840
cgatagactt ggagctcgaa caaacctctg gaattggtca cctagagggt ccattcatcg 3900
acagacgcct gaagagctgc tatcacggaa gactccacac tcccgtcagc tctcgctcaa 3960
gtcttgaaat ccatggtcga gagtttgggg ggttgagaaa ccaaccactc ctgacaaaaa 4020
acctcgcgca ccattggcat gcttcatgaa gataagaaca cttaacggtc gcacttgatt 4080
gaaaggcaat ttcgacgcga ctgacttgtt gattcttctg cgggatgtgc gatagatata 4140
tcccaccgca acagacttga gtaacaggct caccacagcg aggcctatag tcatacccat 4200
tccccagtta atatggcgct gggcataagt agggctatgg tagcatccgg atggcaaggc 4260
acgtcgaagg acttgacgac tacaagcaaa gacagaacac ttcagcatgg agagtatatc 4320
gtgttgaaca gctctagcgg aactatatac aaaagcacct aattatcgct tctcacaact 4380
tttaacgaca aatcatttca agagcgagat ttacctgttg gcttaaaaca attgatgaga 4440
cagtggcgca gaaaggacac tggaaaaaag aaaacaagca ttatattacg atgtcgggat 4500
ttggtttgac tgaagacgca atgtttgagg ttttctaaaa gttatatatg atcagacgaa 4560
ggatctgttg aaacggtgtt tatttttaag atatcaaatc atacaatagt tggttataac 4620
gggacattgt atttgcacat agcaattgtg taatgaagca cgcattaaat actatcttgc 4680
gcaatatatt atgctctcca ttaactcttg atcatccccc ccagtcgatc gcaccgagtg 4740
ggacagcgcc tgaacctcta catagtcaat aacattaccc cagaaatgca cgatcatgga 4800
caaggcggat tggagccaac aaggaaccaa gcaagatttg gactgg 4846
<210> 9
<211> 263
<212> DNA
<213> 未知(Unknown)
<400> 9
ttgattgagc aagagggcag cacatacgac caaaggtagt ggaaaatacg ggatcccgtc 60
cgctctccca tagtcaagcc actaaccggc ggattagtag ttgggtcggt gacgaccagc 120
gaatccccgc tgttgtatgt ggctagaata gcactgatag ttttagagct agaaatagca 180
agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 240
tttgatccaa tgcgcgattc aag 263
Claims (6)
1.一种高产赤霉素GA3的藤仓赤霉基因工程菌,由如下方法构建获得:
(1)将藤仓赤霉菌(F.fujikuroi)的pyrG基因敲除,构建得到营养缺陷型菌株F.fujikuroi-ΔpyrG;
(2)将pyrG基因表达框导入F.fujikuroi-ΔpyrG,同时敲除meaB基因,构建得到菌株F.fujikuroi-RE:pyrGΔmeaB;
(3)用来源于pAN7-1的gpdA启动子增强表达hmgR基因的催化区域,构建thmgR基因表达框,导入菌株F.fujikuroi-RE:pyrGΔmeaB中,构建得到菌株F.fujikuroi-RE:pyrG GpdA-tHmgRΔmeaB,即所述高产赤霉素GA3的藤仓赤霉菌基因工程菌。
2.如权利要求1所述的藤仓赤霉基因工程菌,其特征在于所述来源于pAN7-1的gpdA启动子序列如SEQ ID No.3所示。
3.构建权利要求1所述高产赤霉素GA3的藤仓赤霉基因工程菌的方法,其特征在于所述方法如下:
(1)运用CRISPR-Cas9基因编辑技术,将藤仓赤霉菌(F.fujikuroi)的pyrG基因敲除,构建得到营养缺陷型菌株F.fujikuroi-ΔpyrG;
(2)运用CRISPR-Cas9基因编辑技术,将pyrG基因表达框导入F.fujikuroi-ΔpyrG,同时敲除meaB基因,构建得到菌株F.fujikuroi-RE:pyrGΔmeaB;
(3)运用CRISPR-Cas9基因编辑技术用来源于pAN7-1的gpdA启动子增强表达hmgR基因的催化区域,构建thmgR基因表达框,导入菌株F.fujikuroi-RE:pyrGΔmeaB中,构建得到菌株F.fujikuroi-RE:pyrG GpdA-tHmgRΔmeaB,即所述高产赤霉素GA3的藤仓赤霉菌基因工程菌。
4.权利要求1所述藤仓赤霉基因工程菌在微生物发酵制备赤霉素GA3中的应用。
5.如权利要求4所述的应用,其特征在于所述应用为:将所述藤仓赤霉基因工程菌接种至发酵培养基,25~30℃、200~300rpm条件下发酵培养12~48h,获得含赤霉素GA3的发酵液,发酵液经分离纯化获得赤霉素GA3。
6.如权利要求5所述的应用,其特征在于所述发酵培养基组成如下:玉米淀粉60~90g/L,米粉70~100g/L,大豆粉3~7g/L,花生粉3~7g/L,K2SO40.3~0.7g/L,KH2PO4 0.3~0.7g/L,溶剂为水,pH自然,121度灭菌30min。
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NL2032683B1 (en) * | 2022-07-18 | 2024-01-26 | Sestina Bio Llc | Bioproduction of isoprenoids |
WO2024055735A1 (zh) * | 2022-09-15 | 2024-03-21 | 浙江工业大学 | 一种高产赤霉素ga 3的基因工程菌、构建方法及应用 |
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