CN117660469A - 一种合成虫草素的转录因子、工程菌、构建方法和应用 - Google Patents
一种合成虫草素的转录因子、工程菌、构建方法和应用 Download PDFInfo
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- CN117660469A CN117660469A CN202410144112.8A CN202410144112A CN117660469A CN 117660469 A CN117660469 A CN 117660469A CN 202410144112 A CN202410144112 A CN 202410144112A CN 117660469 A CN117660469 A CN 117660469A
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- Prior art keywords
- cordycepin
- seq
- ania03334
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- aspergillus nidulans
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
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Abstract
本发明涉及微生物工程技术领域,尤其涉及一种合成虫草素的转录因子、工程菌、构建方法和应用,所述转录因子为ANIA03334,所述ANIA03334参与构巢曲霉合成虫草素,所述ANIA03334的核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示。本发明发现了能够调控构巢曲霉中虫草素生物合成的转录因子,对其进行敲除获得能够高产虫草素的构巢曲霉工程菌,在28℃条件下发酵5天,大大缩短了虫草素的生产周期。
Description
技术领域
本发明涉及微生物工程技术领域,尤其涉及一种合成虫草素的转录因子、工程菌、构建方法和应用。
背景技术
虫草素(cordycepin),又称冬虫夏草素,即3′-脱氧腺苷。1950 年,Cunningham等首次从药食两用真菌蛹虫草(Cordyceps militaris)的发酵液中分离出虫草素。该化合物具有多种药理活性,如抗菌、抗病毒、抗炎、抗氧化、促凋亡、抗血栓和抗肿瘤等,尤其对多种肿瘤细胞有较强的抑制效果,如肝癌、肺癌、乳腺癌、胃癌、结肠癌细胞等,是食品、药品及化妆品行业的珍贵原料,其纯品价格在国际市场一度高达每克2200 美元。虫草素主要可以通过化学合成法和生物法获得,虫草素的真菌发酵产量有限,而化学合成周期较长且极易污染环境、合成工艺繁琐、产物收率低且副产物多,限制其应用。生物法生产虫草素主要有两种途径:一是直接从天然或人工培育的蛹虫草子实体中提取,二是经过液体发酵,从蛹虫草菌丝体和发酵液中提取。这两种途径都具有一定的局限性,如子实体培养周期长(约60天)、培养条件复杂、不稳定,产量较低,效率低等,不能满足工业原料需求。因此,探索更高效环保的虫草素合成方法是当下需要解决的热点问题。
据报道,天然虫草素存在于部分丝状真菌中,包括蛹虫草菌、九州虫草菌、蝉虫草、构巢曲霉和白囊耙齿菌等。其中,构巢曲霉作为丝状真菌,是近年来研究较多的模式真菌,培养操作简单、生长周期短、结构及遗传背景相对简单等优点。现有技术中通过蛹虫草基因组数据分析,鉴定出虫草素的生物合成途径,而且发现构巢曲霉中含有虫草素合成的完整基因簇,研究人员也尝试改造对构巢曲霉虫草素基因簇进行改造使得虫草素含量提高了1.72~1.84倍,最高产量达到277mg/L,这也为利用基因工程或挖掘虫草素合成调控因子提高虫草素含量提供了可能。
发明内容
本发明的第一个方面提供了一种合成虫草素的转录因子,所述转录因子为ANIA03334,所述ANIA03334参与构巢曲霉合成虫草素,所述ANIA03334的核苷酸序列如SEQID NO.1所示,氨基酸序列如SEQ ID NO.2所示。
SEQ ID NO.1:
ATGTCCCGCAACCTCGTCATTACCAGCATAGAAAGCCTCACGGGGTCCCAAATTGCCAAAACCATCCTCGCATCGCGCAGTTTCGCCAAAGGCATCAAAAAGATCACAGGCTTAACCCTTTACCCAGATTCAGATGCATGCGCGGAGCTTAAAGAAGAGCATGGCGTGCAAATTGTTGAACACAAACCCGGCAATCTAGATGCCATGGTTAGCACACTGCAAGAAACGGGCGCCGACACAATCTGCCTCCTGCCACCCGGCCACAAGGACGCATTTAACATCACAACTGAGCTTATAACCGCCACGAAGAAGGCCGGTATCCCTAACGTATGCTTTATTTCGTCCGCTGGGTGTGACCTTGCCGAGCGTGAAGCGCAGCCGCTGCTGAGGAGTGTGATTGATCTCGAAGCGATGGTTATGGAGGCAAAGGGCGATGCCAGCACGGAGACGGGACATAGTCCGGTTGTGATTCGTCGCGGCTTCTACGCAGAACACCTCCTCTTGTACTCGCGACAAGCGCAAGAAGAGGGCAAATTGCCGCTCCCTATCGGCACATCGCACAAATTCGCACCGATGGCGTTATCTGATGTTGTGGAAGTAGTTGCACACGTGCTAACAGGCCACGGCAAACACGGGTTTTCCGATAAACATCGCGGTCAGCTGATGGTTCTTACGGGGCCCCAGCTTACGGCTGGCGACGAGCTCGCAACAGCCGCAAGCCAGGCGCTCGGCAAGGAATTGAAATTCGAAGATATATCAGAGTCTGAAGCCAAGAAGGTCCTCAAGGACGATGCCGGTAGTTCCGAGGGAGAGATCGCGTATCTCCTTGAGTACTACGCACTTGTCCGCGAAGGCAAGACGAACTACATATCGACGACTGCATTCCACGATGTGACCGGAAAGCATCCCGTAGAGCCAACGGAGTTCTTTAAGCAGTATGCCGAGAACTTGCGTCCGAAAGCCAACAAGAAGAGGAAGACTCAGTGA。
SEQ ID NO.2:
MSRNLVITSIESLTGSQIAKTILASRSFAKGIKKITGLTLYPDSDACAELKEEHGVQIVEHKPGNLDAMVSTLQETGADTICLLPPGHKDAFNITTELITATKKAGIPNVCFISSAGCDLAEREAQPLLRSVIDLEAMVMEAKGDASTETGHSPVVIRRGFYAEHLLLYSRQAQEEGKLPLPIGTSHKFAPMALSDVVEVVAHVLTGHGKHGFSDKHRGQLMVLTGPQLTAGDELATAASQALGKELKFEDISESEAKKVLKDDAGSSEGEIAYLLEYYALVREGKTNYISTTAFHDVTGKHPVEPTEFFKQYAENLRPKANKKRKTQ*。
本发明的第二个方面提供了一种合成虫草素的工程菌,所述工程菌为ANIA03334基因敲除的构巢曲霉突变菌株。
本发明的第三个方面提供了一种合成虫草素的工程菌的构建方法,包括如下步骤:
S1.构建ANIA03334基因敲除盒子;
S2. 将构建好的ANIA03334基因敲除盒子转化到构巢曲霉原生质体中,得到合成虫草素的工程菌。
在一些实施方式中,所述构建ANIA03334基因敲除盒子具体包括:快速融合多个片段基因,选择PyrG基因作为筛选标签替换目标基因ANIA03334,其中,构建敲除盒子的引物序列如SEQ ID NO.3-10所示,载体鉴定的引物序列如SEQ ID NO.11-16所示,定量分析的引物序列如SEQ ID NO.17-28所示。
本发明的第四个方面提供了一种合成虫草素的方法,包括:将上述工程菌接种到PDB液体培养基中,进行发酵培养,对发酵培养液进行分离纯化,获得虫草素。
在一些实施方式中,所述发酵温度为25-30℃,发酵时间为3-10天。
进一步地,所述发酵温度为28℃,发酵时间为5天。
本发明的第五个方面提供了一种合成虫草素的工程菌在生产虫草素及其制剂中的应用。
与现有技术相比,本发明具有以下有益效果:
1.本发明发现了能够调控构巢曲霉中虫草素生物合成的转录因子,对其进行敲除获得能够高产虫草素的构巢曲霉工程菌,在28 ℃条件下发酵5天,大大缩短了虫草素的生产周期。
2. 本发明所提供的构巢曲霉氮源合成抑制转录因子的敲除有助于构巢曲霉中虫草素的高效生产,同时,本发明为虫草素产量的提高提供了重要的工程改造策略。
3. 本发明获得的能够调控构巢曲霉虫草素生物合成的转录因子,为实现虫草素的高效生物合成提供了新选择。
附图说明
图1为虫草素生物合成途径示意图。
图2为构巢曲霉中虫草素合成的基因簇示意图。
图3A为构巢曲霉基因敲除盒子构建策略示意图,图3B为本发明中所用的ANIA03334敲除盒子示意图。
图4为敲除盒子阳性转化子PCR验证电泳图。其中1和9为阳性转化子,M为2000 bpmaker,34-5'F1和maker-R,maker-F和34-3'-R2分别为两对敲除盒子上的引物,用于筛选目标基因是否被成功替换,34-F和34-R为转录因子ANIA03334全长引物,其电泳分析无条带说明该基因已被敲除。
图5为虫草素标准曲线图。
图6A为260nm检测PDB培养后的HPLC谱图,图6B为虫草素含量测定结果图。根据虫草素含量分析发现ANIA03334敲除菌株中虫草素含量高于对照菌株。
图7为虫草素合成关键酶基因的相对定量分析。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例,都属于本发明保护的范围。
如图1所示,现有技术中虫草素生物合成途径均存在子实体培养周期长、培养条件复杂、不稳定,产量较低,效率低等局限性。
如图2所示,利用构巢曲霉真菌基因组分析虫草素合成基因簇,并对一个基因簇上的未知基因用uniprot分析筛选出一个氮源物质合成抑制转录因子ANIA03334,其中cns1、cns2、cns3及cns4为构巢曲霉中参与虫草素合成的基因簇。
实施例1
本实施例提供了合成虫草素的工程菌的构建方法,包括如下步骤:
1. 溶液配制
LETS buffer(1L,用超纯水定容):
6.04 g 氯化锂水溶液(0.1M);
40 mL 乙二胺四乙酸(20 mM,pH8.0);
10 mL 1 M Tris-盐酸(10 mM,pH8.0);
5 g SDS(0.5%);
TE buffer(100 mL,用超纯水定容):
1 mL 1 M Tris-盐酸(10 mM,pH8.0);
0.2 mL 0.5 M 乙二胺四乙酸(1mM);
PCI(苯酚:氯仿:异戊醇=25mL:24mL:1mL);
0.1% 吐温 80(V/V):100 µL 吐温 80加入100 mL超纯水,121 ℃灭菌 20 min;
1 M Tris-盐酸(1 L):121.14 g Tris,加入950 mL超纯水,盐酸调节pH到(7.0、7.5、8.0),加水定容至1 L;
20×Nitrate Salts: 120 g 硝酸钠,10.4 g 氯化钾,10.4 g 七水合硫酸镁,30.4 g 磷酸二氢钾,加水定容到1L,121 ℃灭菌 20 min;
1000×Trace Elements:1.1 g 硼酸,2.2 g 七水合硫酸锌,0.50 g 四水合氯化锰,0.16 g 七水合硫酸铁, 5g 乙二胺四乙酸四钠,0.16 g 五水合氯化钴,0.11 g四水合钼酸铵,0.16 g 五水合硫酸铜,加 80 mL水,充分溶解后,用氯化钾 调节pH至6.5,定容到100 mL,121 ℃灭菌 20 min;
Osmotic medium:10 mM 磷酸氢二钠/磷酸二氢钠缓冲液(pH 5.8),1.2 M 硫酸镁,定容到500 mL,0.22 μm 滤膜过滤除菌,4 ℃保存。
Trapping Buffer:109.3 g 山梨醇,100 mL 1 M Tris-盐酸(pH 7.0)(0.1 M),加水定容至 1L,121 ℃灭菌20 min,4 ℃保存;
STC buffer:218.6 g 山梨醇,10 mL 1 M Tris-盐酸(pH 7.5),终浓度为0.01 M,1.47 g 二水合氯化钙,加水定容至 1L,121℃灭菌 20 分钟,4℃保存;
200 mL:43.72 g 山梨醇,2 mL 1 M Tris-盐酸(pH 7.5),0.22 g 氯化钙,加水定容至 100 mL,121℃灭菌 20 分钟,4℃保存;
PEG solution:500mL烧杯,加入10mL超纯水,加入0.5549 g 氯化钙(50 mM),溶解后加入60 g PEG 6000,微波加热40 s多次,加入pH=7.5,1 M Tris-盐酸 5 mL(0.05 M),微波加热至透明,转移至量筒中,清洗3次,定容至100 mL,121 ℃灭菌 20 分钟。
酶解液:30 mg Yatalase(TAKARA Bio Inc.),50 mg Lysing Enzyme(Sigma),溶于10 mL Osmotic medium,过滤灭菌;
LMM 液体培养基:
5 mL 20×Nitrate Salts;
100 µL 1000×Trace Elements;
1 g 葡萄糖;
0.5 g 酵母提取物;
调节 pH 至 6.5(NaOH调)定容至100 mL,121 ℃灭菌20 min。
SMM 固体培养基:
5 mL 20×Nitrate Salts;
100 µL 1000×Trace Elements;
1 g 葡萄糖;
21.86 g 山梨醇;
1.6 g 琼脂;
SMM top 培养基:除琼脂减半,其他成分与SMM 固体培养基相同;
GMM固体培养基(100 mL, PH6.5):
1 g葡萄糖;
5 mL 20×Nitrate Salts;
100 µL 1000×Trace Elements;
1.6 g 琼脂;
如需添加尿苷和尿嘧啶(简写 UU,终浓度:50 mg/100 mL),需要在高压灭菌前加入。
PDB液体培养基:称取24 g 马铃薯葡萄糖肉汤粉末(美国BD公司)加入1 L超纯水,121 ℃灭菌 20 min;
补充物质:吡哆醇(1000×):15mg 吡哆醇,15 mL ddH2O,过滤除菌,4 ℃保存;
2. 敲除盒子的构建
利用Double-Joint方法快速融合多个片段基因,该方法利用末端互补的引物扩增获得具有重叠链的PCR产物,通过PCR产物重叠链的延伸,将不同的DNA 片段连接起来,该技术无需内切酶消化和连接酶处理就可以实现DNA片段的体外连接,为同源重组片段提供了快速有效的途径。选择PyrG基因作为筛选标签替换目标基因ANIA03334,其引物见表1。
表1
编号 | 引物名称 | 引物序列 |
SEQ ID NO.3 | 34-5'F1 | GCCCTACTGAAGCAGCTACTAT |
SEQ ID NO.4 | 34 nest-F | GAGACCCTAGTATCTGGCTCTA |
SEQ ID NO.5 | 34-5'R1 | TCGTCAGACACAGAATAACTCTCGGAGGATCGGCAGACTCCTGACTAT |
SEQ ID NO.6 | pyrG-F | GAGAGTTATTCTGTGTCTGACGA |
SEQ ID NO.7 | pyrG-R | ATTCTGTCTGAGAGGAGGCAC |
SEQ ID NO.8 | 34-3'-F2 | AGTGCCTCCTCTCAGACAGAATAGTGGTTGTTGTGTTTCAAACAGTGA |
SEQ ID NO.9 | 34 nest-R | AGCGTAGGGTTATCTGAC |
SEQ ID NO.10 | 34-3'-R2 | ATGGGAGATCATTATATTAGTGGT |
SEQ ID NO.11 | 34-5'F1 | GCCCTACTGAAGCAGCTACTAT |
SEQ ID NO.12 | maker-R | CTGTAGTCAGGTACAGCTAGAATGGG |
SEQ ID NO.13 | maker-F | GCTTATATGGCCAGAGTATGCG |
SEQ ID NO.14 | 34-3'-R2 | ATGGGAGATCATTATATTAGTGGT |
SEQ ID NO.15 | 34-F | ATGTCCCGCAACCTCGT |
SEQ ID NO.16 | 34-R | GCAGTTGATACAATGTCAATCTGCC |
SEQ ID NO.17 | cns1-QF | TGCTGCCCATCATGTTCTGT |
SEQ ID NO.18 | cns1-QR | TCGGACGCTGGATAACATCG |
SEQ ID NO.19 | cns2-QF | ACAAGTCGCTGCTCTTCTCC |
SEQ ID NO.20 | cns2-QR | TGGTTGATCCCATGTCTGGC |
SEQ ID NO.21 | cns3-QF | GAGGTCGTCTATGCTCGTGG |
SEQ ID NO.22 | cns3-QR | CGTGTACGATTCGGGGAGAG |
SEQ ID NO.23 | cns4-QF | CCCTTCAAAAGCGGGTCTCT |
SEQ ID NO.24 | cns4-QR | GTACATCGGCAGCGATCTGA |
SEQ ID NO.25 | ANIA03334-QF | CGGTAGTTCCGAGGGAGAGA |
SEQ ID NO.26 | ANIA03334-QR | ATGCTTTCCGGTCACATCGT |
SEQ ID NO.27 | actin-QF | TCAATCCCAAGTCCAACC |
SEQ ID NO.28 | actin-QR | TACGACCGGAAGCATACA |
构建方法包括如下步骤:
(1)Round I
以质粒载体pYH-WA-pyrG-KI为模板,用高保真 DNA 聚合酶 (TransStartFastPfu DNA Polymerase,TransGen Biotech )扩增 PyrG基因片段;
以构巢曲霉RJMP1.49菌株基因组 DNA为模板扩增用于构建ANIA03334敲除盒子的上游和下游同源臂片段(5F和3F,核苷酸序列如SEQ ID NO.29-30所示)。
5F: SEQ ID NO.29:
TAGGGCCCTACTGAAGCAGCTACTATACTACATAATAAATATACTAAAAAAGATAGAGGCTTATATTAATAGGATTCTACTCTCAGTAAGTATGCTTTAGCAAATAATATTGGAGATTATAGAATCCTTGCCGTGACTGTACATCTTGTTTGATGCCTTTAACAAGTGTGTTGATCTTGAGTTTATAAAAGACTATTATTAGTTTTTTTTGGGAGACCCTAGTATCTGGCTCTAAGGTTTGACTTCTCCTTACAAGTTGTCCATATATCCAAGAAACTCCCTCAGTATTTCCTTGCTGCCTATAGATTCTAGTATGCGCATCTGAGATAGACCTGGAAGCTATATATCGCAAGGGATAAGCTAAGCTAGAAATAATAATATAGTTAACACAGATTATATAACTAAGATTGCTAATATACCGATTTATTGAGCTAATAGGATGTCAGTACCTATATATTATTCTACCTTTTCTTGCTTGCAGGCTAACAAGGCTAAAGTTTTTTCCTGATAGTTCTTTGTACTAAAGCAGTCTTGAGAGAGCCAACCACGGGGGATATGGAAGACTTGCTGTCTATCCTAGCTAAAGATTTACCAGCTATATTTTAGGCAACCATATCTTGTATCCGCCATCTTTTAGAAGGATGAAAAAGACTTGGGATGATAACATTGATGTCTCTAGCGTATGCAAAACAGCTTATTACTACGAACAGGCTAACAGATTTCCTGTCAGTCCATCTCGGTCAAACAGTAGTGCGCCCCAATCATCAACCAGCGCCTCGGATAGTACTGGAATGCTCTCTGGGACTGTGACAATGGACCCCGTTACAGATATAATGCGCCTATCTCACTGCGCTGTCAATGAATACCTAATCAATAAGATTCACTGTGAGACTCTATTAAATTACATATGTGCAGTTGATACAATGTCAATCTGCCTATCTTCCCCTTGATCGTACATGCAGGTTCAATAGCTCGGACCTCACCACACGTATACTTCTTCCATCATCCGTCGCATCACGTCTTTAACAAAATTCAAATCCAGACTTCATATACTCATTAATGCTCTTAGTGTACATATGTCTGGTTAACTGATAGTCAGGAGTCTGCCGATCCTCC。
3F:SEQ ID NO.30:
AGTGGTTGTTGTGTTTCAAACAGTGATGTTGTATAGGCACAATTAGCTATTCCTTTTTTTTTTTTTGAGCTGTGAGCAAGGAAAGTGGTTGGCTTTTATCCCAGGTTGTCGGTTGCTTGCGTTGCTTGCGTCATTGTGATCTCAGTCTGCGGTGATCGAATCATTATAATGCGTACCTAGTTAAACCAAGTTTTAAACCAAAATCAATCGAGATCGAACTCGATGCAAACTCAATTGGCATCCATGGCAGACTCAGCTGATCATTGGCGCCTAAGGTGTAACCCTCTCTGGACTCGTCCGCAGCTATTGTCCAGGCACCGATGTGAGACCCTACGAACAGAACTTCTCAATCTCTAGAAGTAGAATAATAGCTAATACGGCTTAATCAAACGCCCGCTTATATCAATGATAGGTTGCACACCATTTGCCCTTCGTCGAGCTTATTCCAGCCGGGCGCTCAGCTAACCAGGGAACCATACTCTAGGGTATTATCAACATAACAAGCTTAATGATGCCATGGTTCCAGTACCGCCTTGAAGTCATACAAAGCAATCTCTACCTGAATATATATAGGAGCCAAAGGACGGATGACTCTGCAGCACAAATATCTAAGAGGTTGTCGAGCTATGATATGTGCCGAGCAGAAAAGGAACACGCTCTCACTGTAGCCCTACTCATCACAGTGCTGAGCTAAACAGTCTTATTGAGAATCTTTTCAACTAAGATCTCTGATTCTTTATTTTTTTTTTATTTTTTTTTTATTTTACTATATTTGAAATATATTTAGCTTGATATACGTATATCAGGTGTGTCGCAGCACGCAGGGAAGCTTCGTCAGATAACCCTACGCTTTTGCCCTACTTCTCATTATGTATGGCCTATCAGCGGCAGAACGCATATTCATATTCAAGCAGGCCTCTCGACAACTTCCAAGCGGCTGTGGTAGACAATTACACGACCTCGTCGATTTCTTGCCTCTTAAGCTGACAAAGATGAAGGGGCAAGCGTCCAGTCTAGGGGCAAGAATGCGAGGCCTTCGTCAGACGTTACAGCTGGCAGACCAAGTAAATATATTAGACTCTGACCACTAATATAATGATCTCCCATAGAT。
将PCR产物进行DNA片段纯化,根据北京全式金生物技术股份有限公司的DNA片段纯化试剂盒(EasyPure PCR Purification Kit,EP101)说明书进行操作。
(2)Round II(25 μL体系)
将纯化后的PCR产物进行连接反应,具体反应体系如下:
FastPfu DNA Polymerase Buffer 5 μL
2.5 mM dNTP 2.5 μL
5F 300ng
3F 300ng
PyrG 600ng
FastPFU 0.5μL
PCR反应温度程序:
95 ℃2 min
95 ℃30 s
58 ℃10 min
72 ℃5 min
72 ℃10 min
取5 μL PCR产物进行凝胶电泳检测。
(2)Round III(50 μL体系)
Round II products 1 μL
Buffer 10 μL
2.5dNTP 4 μL
F-nest 1 μL
R-nest 1 μL
FastPFU 1 μL
ddH2O 32 μL
取5 μL PCR产物进行凝胶电泳检测, 将连接成功的片段用巢式引物大量扩增,用北京全式金生物技术股份有限公司的DNA片段纯化试剂盒(EasyPure PCR PurificationKit,EP101)进行纯化,利用Nanodrop对纯化产物进行定量分析,积累5 ug以上的样品,用于敲除实验。具体原理和设计如图3A和3B所示。
3. 构巢曲霉的原生质体制备及敲除盒子转化
(1)用灭菌后的扁牙签蘸取5 mL 0.1% Tween 80 溶液,刮取平板上的构巢曲霉,用3层神奇滤布绵过滤洗出的液体,即为孢子悬液;
(2)取适量孢子悬液至10 mL LMM 培养基中(补充营养缺陷Pyridoxine+Uridineand Uracil);
(3)37 ℃,220 rpm,震荡培养 8 小时,镜检时孢子出芽的尾部约为萌发前细胞直径的3-5倍长;
(4)将萌发好的孢子溶液转移至50 mL离心管中,4 ℃,8000 rpm,离心15 min,去上清液;
(5)加入30 mL灭菌的去离子水,上下颠倒重悬,4℃,8000 rpm,离心 15 min,去上清液;
(6)将剩余部分吹打均匀,转入 1.5 mL EP管,常温离心,彻底去除上清;
(7)用10 mL酶解液将萌发的孢子混匀,转移至新的灭菌的三角瓶中;
(8)25℃,120 rpm震荡过夜,至形成无壁均匀的球状细胞;
(9)将裂解好的原生质体混合液缓慢转移至50 mL透明玻璃管中,缓慢滴加10 mLTrapping Buffer,此时可见管内分为清晰的两层;
(10)使用水平转子,4℃,5000 rpm,离心15 min,用移液枪轻轻地吸取中间的白色原生质体层至新的15 mL离心管;
(11)加入等体积的 STC buffer重悬;
(12)使用角转子,4 ℃,6000 rpm,离心8 min,去上清;
(13)将剩余部分吹打均匀,转入1.5 mL EP管,常温转速12000 rpm离心30 s,观察EP管内沉积的原生质体,若颜色有非白色细胞沉积用枪头轻轻吸出白色的原生质体至新的EP管内,重复上述步骤至管内沉积细胞均为白色;
(14)加入100 μL STC buffer,显微镜镜检,视野里应为均一的无壁球状原生质体;
(15)每100 µL原生质体加入10 µL的质粒(3-10 μg)轻轻混匀后冰上静置至少50min;
(16)加入1.25 mL 60 % PEG Solution,缓慢地加入在离心管内壁,让液体在内壁缓慢来回颠倒混匀,平放室温静置20 min;
(17)混合液中加5 mL STC buffer混匀,每个SMM筛选培养基(补充营养缺陷Pyridoxine)上加1 mL混匀后的混合液,倒入 5 mL 融化的 SMM top 培养基;
(18)37 ℃倒置培养 2-3天。
4. 构巢曲霉阳性转化子的筛选及鉴定
(1) 用灭菌枪头将挑选8-12个转化子转接至 GMM(补充营养缺陷Pyridoxine)平板上,37 ℃条件下培养 3天;
(3) 用灭菌枪头刮取少量孢子,接至 4 mL LMM (补充营养缺陷Pyridoxine)培养基中,37 ℃静置培养24 h;
(4) 提取转化子基因组DNA,用鉴定引物(表1)进行 PCR 验证及电泳分析,筛选出阳性转化子进行后续的次级代谢产物分析。
实验结论:根据图4 所示,其中34-5'F1和maker-R,maker-F和34-3'-R2分别为两对敲除盒子上的引物,根据条带大小显示该敲除盒子已经***构巢曲霉原生质体中,而34-F和34-R为转录因子ANIA03334全长引物,在1和9中未发现该基因电泳条带,说明该基因已经被成功敲除,这说明构巢曲霉阳性转化子中编号1和9为阳性转化子。
5. 构巢曲霉基因组的提取
(1)取出-20 ℃保菌的孢子+4 mL LMM(补充营养缺陷Pyridoxine),37 ℃培养24h;
(2)挑取菌体用擦手纸将水分吸干,转入1.5 mL EP管中,加入400 µL LETSbuffer和灭菌钢珠,70 Hz研磨30 s;
(3)补加LETS buffer 300µL,静置15 min;
(4)加入700 µL PCI,上下颠倒10-15次,静置5 min,4 ℃,12000 rpm离心10 min;
(5)取上清400 µL,加入800 µL 95%乙醇上下颠倒混匀,4 ℃,12000 rpm离心10min;
(6)弃上清,加入200 µL 75 %乙醇上下颠倒洗涤沉淀;
(7)室温13000 rpm离心2 min,弃上清,尽可能吸干残余的液体,室温放置1 h挥干残留液体;
(8)加入50 µL TE Buffer,RNase(终浓度为10 mg/mL),37 ℃孵育30 min;
(9)65 ℃处理5 min,使RNase失活,提取gDNA,-20 ℃保存。
6. 构巢曲霉敲除阳性转化子的培养及菌株发酵及产物提取
(1)菌株发酵
将构巢曲霉对照菌株RJMP1.49 和突变株∆ 34甘油菌于 GMM 固体培养板上划线活化,37 ℃培养 3 d ,用5 mL 0.1 % Tween 80收集孢子液接种于PDB液体培养基中,置于28 ℃,200 rpm震荡培养5 d,每个菌株3个重复。
(2)产物提取
将发酵产物上清过滤后直接转移至旋转蒸发仪中浓缩,即得发酵粗提物,将粗提物用甲醇溶解。
7. HPLC分析
利用Vanquish Core HPLC ***(Vanquish 二极管阵列检测器)(Thermo FisherScientific,美国)对发酵产物进行分析,比较突变菌株与野生菌株中虫草素含量积累差异,反相分析柱 (Ascentis®, C18, 5 μm, 2.1×150 mm)(美国Sigma-Aldrich公司) ,柱温:35 ℃,流速0.8 mL/min,进样10 μL,流动相A为水,流动相C为甲醇,梯度洗脱条件如下:0-50 min 5% C,50-55 min 5% C,55-65 min 100 % C,检测波长设置为210 nm。
HPLC结果显示在PDB液体培养基中振荡培养5天后,虫草素(COR)产量增加约2倍左右,虫草素最高产量为1.034 mg/mL。
实验结论:根据图5及图6的HPLC分析结果计算虫草素含量,其突变菌株中虫草素含量约为1.010036231 mg/mL,其中最高含量可达1.034 mg/mL,对照菌株中虫草素含量约为0.504384701 mg/mL。
8. qRT-PCR
(1)RNA提取
将构巢曲霉对照菌株RJMP1.49 和突变株∆ 34甘油菌于 GMM 固体培养板上(加上一层灭菌的玻璃纸)划线培养3 d,将孢子收集用液氮研磨,再根据Eastep® Super总RNA提取试剂盒(上海普洛麦格生物技术公司)说明书进行RNA提取。
(2)反转录反应
利用PrimeScript™ RT Master Mix (Perfect Real Time) (Takara Bio Inc,Beijing)试剂盒说明书进行反转录,具体体系如下:
5X PrimeScript RT Master Mix(Perfect Real Time) 2 μL
Total RNA 1μg
RNase Free dH2O up to 10 μL
混匀后进行反转录反应,条件如下: 37 ℃ 15 min,85℃ 5 s。
(3)qRT-PCR
利用PrimeScript™ RT Master Mix (Perfect Real Time) (北京宝日医生物技术公司) 试剂盒说明书进行基因定量分析,仪器为QuantStudio 5 Real-Tiem PCRInstrument(96-well 0.2mLBlock)(Thermo Fisher Scientific,美国),其定量引物见表1,具体体系如下:
TB Green Premix Ex Taq II(Tli RNaseH Plus)(2X) 10 μL
PCR Forward Primer(10 μM) 0.8 μL
PCR Reverse Primer(10 μM) 0.8 μL
ROX Reference Dye or Dye(50X)0.4 μL
RT 反应液(cDNA 溶液稀释10倍 1 μl
灭菌水 7 μL
PCR程序:
Stage 1:95℃ 30 s
Stage 2:95℃ 5 s
60℃ 30 s
40 cycles
Dissociation Stage:95℃ 15 s
60 ℃ 30 s 95℃ 15 s
如图7所示,cns1、cns 2、cns 3及cns 4为构巢曲霉中虫草素合成的关键酶编码基因,其表达量影响了虫草素的合成。本发明中发现的转录因子ANIA0334被敲除后,cns1、cns2、cns 3及cns 4的表达量显著上调,说明该转录因子能够调控虫草素合成的基因簇表达,从而影响虫草素的合成。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种合成虫草素的工程菌的构建方法,其特征在于:所述工程菌为ANIA03334基因敲除的构巢曲霉突变菌株;
所述ANIA03334的核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示;
所述构建方法包括如下步骤:
S1.构建ANIA03334基因敲除盒子;
S2. 将构建好的ANIA03334基因敲除盒子转化到构巢曲霉原生质体中,得到合成虫草素的工程菌。
2.根据权利要求1所述的构建方法,其特征在于:所述构建ANIA03334基因敲除盒子具体包括:快速融合多个片段基因,选择PyrG基因作为筛选标签替换目标基因ANIA03334,其中,构建敲除盒子的引物序列如SEQ ID NO.3-10所示,载体鉴定的引物序列如SEQ IDNO.11-16所示,定量分析的引物序列如SEQ ID NO.17-28所示。
3.一种合成虫草素的方法,其特征在于:包括:将权利要求1所述的工程菌接种到PDB液体培养基中,进行发酵培养,对得到的发酵培养液进行分离纯化,获得虫草素。
4.根据权利要求1所述的方法,其特征在于:所述发酵的温度为25-30℃,发酵的时间为3-10天。
5.一种如权利要求1所述的合成虫草素的工程菌在生产虫草素及其制剂中的应用。
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