CN118109540A - 一种提高SAMe产量的方法 - Google Patents
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Abstract
本发明属于基因工程领域,具体涉及一种提高SAMe产量的方法及其工程菌和应用。本发明提供的提高SAMe产量的方法包括在表达SAM2的基础菌株中过表达apt1‑prs128基因,摇瓶发酵后筛选出SAMe产量提高的基因工程菌株。此方法操作简单,成本较低,且生产速率快,易于扩大规模。
Description
技术领域
本发明属于基因工程及微生物发酵领域,具体涉及一种提高SAMe产量的方法。
背景技术
S-腺苷-L-甲硫氨酸(Ademetionine、S-adenosyl-L-methionine,SAMe)是一种重要的代谢中间体,存在于所有生物体内,并在各种生物反应中发挥关键作用。SAMe可作为辅助因子为甲基转移酶提供甲基供体[1]。SAMe还被用作亚甲基、氨基、核糖基部分和氨基丙基的来源[2]。SAMe是第二常用的酶底物,仅次于ATP[3]。SAMe存在于所有的真核细胞中,是人体组织和体液中普遍存在的一种生理活性分子,主要适用于肝硬化前和肝硬化所致肝内胆汁淤积、妊娠期肝内胆汁淤积,能使各种原因导致的肝功能异常转为正常。
SAMe的生产方法主要有化学合成法、酶促合成法和微生物发酵法等。化学合成法制备SAMe主要有3条途径:(1)利用5′-甲硫腺苷和DL-2-氨基-4-溴丁基酸化学合成消旋的SAMe,合成的SAMe有50%具有生物活性;(2)通过S-腺苷同型半胱氨酸(S-Adenosylhomocysteine,SAH)和甲基供体CH3I或(CH3)3S催化合成,反应底物SAH价格昂贵,而且产物中含有20%-30%没有生物活性的(R,S)-SAMe异构体;(3)以价格低廉的腺苷(Adenosine)为底物和亚硫酰氯进行反应制备5-(氯甲基)腺苷盐酸;以L-甲硫氨酸为底物在-30至-40℃的条件下生成L-同型半胱氨酸;以5-(氯甲基)腺苷盐酸盐和L-同型半胱氨酸为底物在70-80℃的条件下进行缩合反应得到S-腺苷同型半胱氨酸(SAH);以三甲基氧鎓四氟硼酸盐为甲基供体对SAH进行甲基化得到终产物SAMe。该法合成SAMe存在产率低、反应条件苛刻、反应产物异构体多、分离纯化困难以及环境污染等不足,现已很少使用。
酶促合成法是利用SAMe合成酶催化底物L-甲硫氨酸和ATP生成SAMe,且SAMe合成酶有三磷酸酶活性,将三磷酸分解为焦磷酸和磷酸,该法具有明确的工艺,提取出的SAMe纯度高,但生产过程操作复杂,耗时长,成本高。
微生物发酵法可以利用廉价的生产原料,通过特定的微生物代谢合成SAMe,操作简单,成本较低,且生产速率快,易于扩大规模。因此,发酵法生产SAMe受到越来越多的关注。一些微生物尤其是酵母属(Saccharomyces)的菌株,在含有L-甲硫氨酸的培养基中生长时,可以在细胞内积累较高浓度的SAMe。通过这些微生物的大规模发酵及提取精制,可获得有生物活性的SAMe。随着SAMe需求量不断提升,使用基因工程手段改造生产SAMe的研究也越来越多。但现有微生物发酵法生产存在产量相对较低的问题。因此本发明为解决该问题提供了一种提高SAMe产量的方法。
[1]Rudenko AY,Mariasina SS,Sergiev PV,Polshakov VI.(2022).Analogs ofS-Adenosyl-L-Methionine in Studies of Methyltransferases.Mol Biol,56(2):229-250.
[2]Fontecave M.,Atta M.,Mulliez E.(2014).S-Adenosylmethionine:nothing goes to waste.Trends Biochem.Sci,29:243-249.
[3]Cantoni GL.(1975).Biological methylation:selected aspects.AnnuRev.Biochem.,44:435-451.
发明内容
本发明提供的提高SAMe产量的方法包括在表达SAMe的基础菌株中过表达apt1-prs128基因,摇瓶发酵后筛选出SAMe产量提高的基因工程菌株。此方法操作简单,成本较低,且生产速率快,易于扩大规模。
术语
本发明中,SAMe:S-腺苷-L-甲硫氨酸,是一种重要的代谢中间体,存在于所有生物体内,并在各种生物反应中发挥关键作用。
本发明中,重组菌株:指经基因工程改造后得到的菌株,其可用作特异性表达某种功能。
本发明中,摇瓶发酵法:一种发酵方法,通常用于小规模生产或测试。在这种方法中,含有微生物和营养物质的瓶子在控制条件下摇动,以促进生长和代谢。
一方面,本发明提供了一种提高SAMe产量的方法。
所述的方法包括在表达SAMe的基础菌株中过表达apt1-prs128基因。
具体地,所述的apt1-prs128基因的核苷酸序列包括SEQ ID No.15或其功能片段。
具体地,所述的基础菌株为酵母菌(Saccharomyces)。
优选地,所述的基础菌株为酿酒酵母菌。
另一方面,本发明还提供了一种基因工程菌株,用于表达SAMe。
所述的基因工程菌株中过表达apt1-prs128基因;所述的apt1-prs128基因的核苷酸序列包括SEQ ID No.15或其功能片段。
具体地,所述的基因工程菌株包括表达apt1-prs128基因。
具体地,所述的基因工程菌株为酵母菌SHF16G或酵母菌SHF16。
具体地,所述的酵母菌SHF16的保藏号为CGMCC No.28947。
再一方面,本发明还提供了一种过表达apt1-prs128基因在提高SAMe产量中的应用。
具体地,所述的应用包括在表达SAMe的基础菌株中过表达apt1-prs128基因;所述的apt1-prs128基因的核苷酸序列包括SEQ ID No.15或其功能片段。
又一方面,本发明还提供了前述的基因工程菌株在SAMe生产中的应用。
具体地,所述的基因工程菌株用于提高SAMe的产量。
再一方面,本发明还提供了一种SAMe的生产方法。
具体地,所述的生产方法,包括以下步骤:
(1)活化前述的基因工程菌株;
(2)发酵;
(3)SAMe的提取。
具体地,所述的步骤(1)包括:取单菌落接种YPD中,过夜活化;
所述的步骤(2)包括:将活化的种子转接至发酵培养基中,发酵;
所述的步骤(3)包括:取发酵液,添加高氯酸,水浴,离心,上清液用水相膜过滤后,HPLC检测。
SEQ ID No.1:
CCGGTCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTCCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTATTAAATGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCGCATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCGGCCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGCCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGCGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATTCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGGCATTGCTACAGGCATCGTGGTGTCACTCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATAGTGTATCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATGGGTAATAACTGATATAATTAAATTGAAGCTCTAATTTGTGAGTTTAGTATACATGCATTTACTTATAATACAGTTTTTTAGTTTTGCTGattaagggttctcgagagctcgttttcgacactggatggcggcgttagtatcgaatcgacagcagtatagcgaccagcattcacatacgattgacgcatgatattactttctgcgcacttaacttcgcatctgggcagatgatgtcgaggcgaaaaaaaatataaatcacgctaacatttgattaaaatagaacaactacaatataaaaaaactatacaaatgacaagttcttgaaaacaagaatctttttattgtcagtactgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttgccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgaaacgtgagtcttttccttacccatggttgtttatgttcggatgtgatgtgagaactgtatcctagcaagattttaaaaggaagtatatgaaagaagaacctcagtggcaaatcctaaccttttatatttctctacaggggcgcggcgtggggacaattcaacgcgtctgtgaggggagcgtttccctgctcgcaggtctgcagcgaggagccgtaatttttgcttcgcgccgtgcggccatcaaaatgtatggatgcaaatgattatacatggggatgtatgggctaaatgtacgggcgacagtcacatcatgcccctgagctgcgcacgtcaagactgtcaaggagggtattctgggcctccatgtcgctggccgggtgacccggcggggacgaggcaagctaaacagatctctagacctaCTGGGTCCTTTTCATCACGTGCTATAAAAATAATTATAATTTAAATTTTTTAATATAAATATATAAATTAAAAATAGAAAGTAAAAAAAGAAATTAAAGAAAAAATAGTTTTTGTTTTCCGAAGATGTAAAAGACTCTAGGGGGATCGCCAACAAATACTACCTTTTATCTTGCTCTTCCTGCTCTCAGGTATTAATGCCGAATTGTTTCATCTTGTCTGTGTAGAAGACCACACACGAAAATCCTGTGATTTTACATTTTACTTATCGTTAATCGAATGTATATCTATTTAATCTGCTTTTCTTGTCTAATAAATATATATGTAAAGTACGCTTTTTGTTGAAATTTTTTAAACCTTTGTTTATTTTTTTTTCTTCATTCCGTAACTCTTCTACCTTCTTTATTTACTTTCTAAAATCCAAATACAAAACATAAAAATAAATAAACACAGAGTAAATTCCCAAATTATTCCATCATTAAAAGATACGAGGCGCGTGTAAGTTACAGGCAAGCGATCCGTCCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATACCACAGCTTTTCCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGgttagcatatctacaattgggtgaaatggggagcgatttgcaggcatttgctcggcatgccggtagaggtgtggtcaataagagcgacctcatgctatacctgagaaagcaacctgacctacaggaaagagttactcaagaataagaattttcgttttaaaacctaagagtcactttaaaatttgtatacacttattttttttataacttatttaataataaaaatcataaatcataagaaattcgcCCCGGGTCGACAGATCTTTGTTTGTTTATGTGTGTTTATTCGAAACTAAGTTCTTGGTGTTTTAAAACTAAAAAAAAGACTAACTATAAAAGTAGAATTTAAGAAGTTTAAGAAATAGATTTACAGAATTACAATCAATACCTACCGTCTTTATATACTTATTAGTCAAGTAGGGGAATAATTTCAGGGAACTGGTTTCAACCTTTTTTTTCAGCTTTTTTCAAATCAGAGAGAGCAGAAGGTAATAGAAGGTGTAAGAAAATGAGATAGATACATGCGTGGGTCAATTGCCTTGTGTCATCATTTACTCCAGGCAGGTTGCATCACTCCATTGAGGTTGTGCCCGTTTTTTGCCTGTTTGTGCCCCTGTTCTCTGTAGTTGCGCTAAGAGAATGGACCTATGAACTGATGGTTGGTGAAGAAAACAATATTTTGGTGCTGGGATTCTTTTTTTTTCTGGATGCCAGCTTAAAAAGCGGGCTCCATTATATTTAGTGGATGCCAGGAATAAACTGTTCACCCAGACACCTACGATGTTATATATTCTGTGTAACCCGCCCCCTATTTTGGGCATGTACGGGTTACAGCAGAATTAAAAGGCTAATTTTTTGACTAAATAAAGTTAGGAAAATCACTACTATTAATTATTTACGTATTCTTTGAAATGGCAGTATTGATAATGATAAACTCGAACTGACCGGT;
SEQ ID No.2:
TCATTTTTTCAACGCTTCCTTTTGAGCGTTCAGTAAAGTGAACACTGGAGCATTCAACTTACTCCTGCCTTTCAAGAAATCCAACTCCATAACAAAGTTATATTCCAAAAGGTTGGCTTCGAGTTGTTCAACTAATTCGCCGGCTGCAGCAGCAGAACCACCAGTGGCAATAATGTCATCAACAATGATAACGTTGGAACCTGCTGGAATAGCGTTTTTCTGTATCTCAAAAAGATCAGAACCGTACTCCTTTTCGTACGTAGCCTTAAAACATTCGCCAGGTAGCTTACCTGCCTTCCTGACTGGAACGAAACCAACACCTAGGGCCAAAGCTAAAGTTGGTCCGAACAAGAACCCACGGGATTCCAACCCGACGATATAATCAATTTTAACTTCTGGAAAAGCTTCTTCTAAATGCAGTTTGAAAGCATCGATCAACTTCTGGAAAAGACCTGGGTTCCTGAAAATGGGTAAGAAATCTTCGAAGAGAATGCCTTCACTAGGGAAGTTTGGATATTGATGTAAAGCCAACTTCAACTCTTGGGCATAACTTGCTATAGACAT;
SEQ ID No.3:
ACCGGTCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGAAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAATGTCTATAGCAAGTTATGCCCAAGAGTTGAAGTTGGCTTTACATCAATATCCAAACTTCCCTAGTGAAGGCATTCTCTTCGAAGATTTCTTACCCATTTTCAGGAACCCAGGTCTTTTCCAGAAGTTGATCGATGCTTTCAAACTGCATTTAGAAGAAGCTTTTCCAGAAGTTAAAATTGATTATATCGTCGGGTTGGAATCCCGTGGGTTCTTGTTCGGACCAACTTTAGCTTTGGCCCTAGGTGTTGGTTTCGTTCCAGTCAGGAAGGCAGGTAAGCTACCTGGCGAATGTTTTAAGGCTACGTACGAAAAGGAGTACGGTTCTGATCTTTTTGAGATACAGAAAAACGCTATTCCAGCAGGTTCCAACGTTATCATTGTTGATGACATTATTGCCACTGGTGGTTCTGCTGCTGCAGCCGGCGAATTAGTTGAACAACTCGAAGCCAACCTTTTGGAATATAACTTTGTTATGGAGTTGGATTTCTTGAAAGGCAGGAGTAAGTTGAATGCTCCAGTGTTCACTTTACTGAACGCTCAAAAGGAAGCGTTGAAAAAATGAACCCGGGgcgaatttcttatgatttatgatttttattattaaataagttataaaaaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttattcttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggtcgctcttattgaccacacctctaccggcatgccgagcaaatgcctgcaaatcgctccccatttcacccaattgtagatatgctaacCCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGGAAAAGCTGTGGTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGGACGGATCGCTTGCCTGTAACTTACACGCGCCTCGTATCTTTTAATGATGGAATAATTTGGGAATTTACTCTGTGTTTATTTATTTTTATGTTTTGTATTTGGATTTTAGAAAGTAAATAAAGAAGGTAGAAGAGTTACGGAATGAAGAAAAAAAAATAAACAAAGGTTTAAAAAATTTCAACAAAAAGCGTACTTTACATATATATTTATTAGACAAGAAAAGCAGATTAAATAGATATACATTCGATTAACGATAAGTAAAATGTAAAATCACAGGATTTTCGTGTGTGGTCTTCTACACAGACAAGATGAAACAATTCGGCATTAATACCTGAGAGCAGGAAGAGCAAGATAAAAGGTAGTATTTGTTGGCGATCCCCCTAGAGTCTTTTACATCTTCGGAAAACAAAAACTATTTTTTCTTTAATTTCTTTTTTTACTTTCTATTTTTAATTTATATATTTATATTAAAAAATTTAAATTATAATTATTTTTATAGCACGTGATGAAAAGGACCCAGtaggtctagagatctgtttagcttgcctcgtccccgccgggtcacccggccagcgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaccatgggtaaggaaaagactcacgtttcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggcaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagtactgacaataaaaagattcttgttttcaagaacttgtcatttgtatagtttttttatattgtagttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctcgacatcatctgcccagatgcgaagttaagtgcgcagaaagtaatatcatgcgtcaatcgtatgtgaatgctggtcgctatactgctgtcgattcgatactaacgccgccatccagtgtcgaaaacgagctctcgagaacccttaatCAGCAAAACTAAAAAACTGTATTATAAGTAAATGCATGTATACTAAACTCACAAATTAGAGCTTCAATTTAATTATATCAGTTATTACCCATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGATACACTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGAGTGACACCACGATGCCTGTAGCAATGCCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGAATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCGCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGGGCCGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATGCGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTTGTAATTAAAACTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGCAAGAAGTAAAAAAAGTTGTAATAGAACAAGAAAAATGAAACTGAAACTTGAGAAATTGAAGACCATTTAATAACTTAAATATCAATGGGAGGTCATCGAAAGAGAAAAAAATCAAAAAAAAAAATTTTCAAGAAAAAGAAACGTGATAAAAATTTTTATTGCCTTTTTCGACGAAGAAAAAGAAACGAGGCGGTCTCTTTTTTCTTTTCCAAACCTTTAGTACGGGTAATTAACGACACCCTAGAGGAAGAAAGAGGGGGAATTTAGTATGCTGTGCTTGGGTGTTTTGAAGTGGTACGGCGATGCGCGGAGTCCGAGAAAATCTGGAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGTGTGTGACCGG;
SEQ ID No.4:
TTTGTTTGTTTATGTGTGTTTATTCGAAACTAAG;
SEQ ID No.5:ACCCGGGgcgaatttcttatg;
SEQ ID No.6:CTTAGTTTCGAATAAACACACATAAACAAACAAAATGTCTATAGCAAGTTATGCCCAAG;
SEQ ID No.7:cataagaaattcgcCCCGGGTTCATTTTTTCAACGCTTCCTTTTGAGC;
SEQ ID No.8:
TTTGTAATTAAAACTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGCAAGAAGTAAAAAAAGTTGTAATAGAACAAGAAAAATGAAACTGAAACTTGAGAAATTGAAGACCATTTAATAACTTAAATATCAATGGGAGGTCATCGAAAGAGAAAAAAATCAAAAAAAAAAATTTTCAAGAAAAAGAAACGTGATAAAAATTTTTATTGCCTTTTTCGACGAAGAAAAAGAAACGAGGCGGTCTCTTTTTTCTTTTCCAAACCTTTAGTACGGGTAATTAACGACACCCTAGAGGAAGAAAGAGGGGGAATTTAGTATGCTGTGCTTGGGTGTTTTGAAGTGGTACGGCGATGCGCGGAGTCCGAGAAAATCTGGAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGTGTGTGACCGGACCGGTCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGAAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAATGTCTATAGCAAGTTATGCCCAAGAGTTGAAGTTGGCTTTACATCAATATCCAAACTTCCCTAGTGAAGGCATTCTCTTCGAAGATTTCTTACCCATTTTCAGGAACCCAGGTCTTTTCCAGAAGTTGATCGATGCTTTCAAACTGCATTTAGAAGAAGCTTTTCCAGAAGTTAAAATTGATTATATCGTCGGGTTGGAATCCCGTGGGTTCTTGTTCGGACCAACTTTAGCTTTGGCCCTAGGTGTTGGTTTCGTTCCAGTCAGGAAGGCAGGTAAGCTACCTGGCGAATGTTTTAAGGCTACGTACGAAAAGGAGTACGGTTCTGATCTTTTTGAGATACAGAAAAACGCTATTCCAGCAGGTTCCAACGTTATCATTGTTGATGACATTATTGCCACTGGTGGTTCTGCTGCTGCAGCCGGCGAATTAGTTGAACAACTCGAAGCCAACCTTTTGGAATATAACTTTGTTATGGAGTTGGATTTCTTGAAAGGCAGGAGTAAGTTGAATGCTCCAGTGTTCACTTTACTGAACGCTCAAAAGGAAGCGTTGAAAAAATGAACCCGGGgcgaatttcttatgatttatgatttttattattaaataagttataaaaaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttattcttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggtcgctcttattgaccacacctctaccggcatgccgagcaaatgcctgcaaatcgctccccatttcacccaattgtagatatgctaacCCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGGAAAAGCTGTGGTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGGACGGATCGCTTGCCTGTAACTTACACGCGCCTCGTATCTTTTAATGATGGAATAATTTGGGAATTTACTCTGTGTTTATTTATTTTTATGTTTTGTATTTGGATTTTAGAAAGTAAATAAAGAAGGTAGAAGAGTTACGGAATGAAGAAAAAAAAATAAACAAAGGTTTAAAAAATTTCAACAAAAAGCGTACTTTACATATATATTTATTAGACAAGAAAAGCAGATTAAATAGATATACATTCGATTAACGATAAGTAAAATGTAAAATCACAGGATTTTCGTGTGTGGTCTTCTACACAGACAAGATGAAACAATTCGGCATTAATACCTGAGAGCAGGAAGAGCAAGATAAAAGGTAGTATTTGTTGGCGATCCCCCTAGAGTCTTTTACATCTTCGGAAAACAAAAACTATTTTTTCTTTAATTTCTTTTTTTACTTTCTATTTTTAATTTATATATTTATATTAAAAAATTTAAATTATAATTATTTTTATAGCACGTGATGAAAAGGACCCAGtaggtctagagatctgtttagcttgcctcgtccccgccgggtcacccggccagcgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaccatgggtaaggaaaagactcacgtttcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggcaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagtactgacaataaaaagattcttgttttcaagaacttgtcatttgtatagtttttttatattgtagttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctcgacatcatctgcccagatgcgaagttaagtgcgcagaaagtaatatcatgcgtcaatcgtatgtgaatgctggtcgctatactgctgtcgattcgatactaacgccgccatccagtgtcgaaaacgagctctcgagaacccttaatCAGCAAAACTAAAAAACTGTATTATAAGTAAATGCATGTATACTAAACTCACAAATTAGAGCTTCAATTTAATTATATCAGTTATTACCCATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGATACACTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGAGTGACACCACGATGCCTGTAGCAATGCCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGAATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCGCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGGGCCGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATGCGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTC;
SEQ ID No.9:
AtgcctgatatgaagctttttgctggtaacgccaccccggaactagcacaacgtattgccaaccgcctgtacacttcactcggcgacgccgctgtaggtcgctttagcgatggcgaagtcagcgtacaaattaatgaaaatgtacgcggtggtgatattttcatcatccagtccacttgtgcccctactaacgacaacctgatggaattagtcgttatggttgatgccctgcgtcgtgcttccgcaggtcgtatcaccgctgttatcccctactttggctatgcgcgccaggaccgtcgcgtccgttccgctcgtgtaccaatcactgcgaaagtggttgcagacttcctctccagcgtcggtgttgaccgtgtgctgacagtggCtctgcacgctgaacagattcagggtttcttcgacgttccggttgataacgtatttggtagcccgatcctgctggaagacatgctgcagctgaatctggataacccaattgtggtttctccggacatcggcggcgttgtgcgtgcccgcgctatcgctaagctgctgaacgataccgatatggcaatcatcgacaaacgtcgtccgcgtgcgaacgtttcacaggtgatgcatatcatcggtgacgttgcaggtcgtgactgcgtactggtcgatgatatgatcgacactggcggtacgctgtgtaaagctgctgaagctctgaaagaacgtggtgctaaacgtgtatttgcgtacgcgactcacccgatcttctctggcaacgcggcgaacaacctgcgtaactctgtaattgatgaagtcgttgtctgcgataccattccgctgagcgatgaaatcaaatcactgccgaacgtgcgtactctgaccctgtcaggtatgctggccgaagcgattcgtcgtatcagcaacgaagaatcgatctctgccatgttcgaacactaa;
SEQ ID No.10:
ACCGGTCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGAAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAATGTCTATAGCAAGTTATGCCCAAGAGTTGAAGTTGGCTTTACATCAATATCCAAACTTCCCTAGTGAAGGCATTCTCTTCGAAGATTTCTTACCCATTTTCAGGAACCCAGGTCTTTTCCAGAAGTTGATCGATGCTTTCAAACTGCATTTAGAAGAAGCTTTTCCAGAAGTTAAAATTGATTATATCGTCGGGTTGGAATCCCGTGGGTTCTTGTTCGGACCAACTTTAGCTTTGGCCCTAGGTGTTGGTTTCGTTCCAGTCAGGAAGGCAGGTAAGCTACCTGGCGAATGTTTTAAGGCTACGTACGAAAAGGAGTACGGTTCTGATCTTTTTGAGATACAGAAAAACGCTATTCCAGCAGGTTCCAACGTTATCATTGTTGATGACATTATTGCCACTGGTGGTTCTGCTGCTGCAGCCGGCGAATTAGTTGAACAACTCGAAGCCAACCTTTTGGAATATAACTTTGTTATGGAGTTGGATTTCTTGAAAGGCAGGAGTAAGTTGAATGCTCCAGTGTTCACTTTACTGAACGCTCAAAAGGAAGCGTTGAAAAAATGAACCCGGGgcgaatttcttatgatttatgatttttattattaaataagttataaaaaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttattcttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggtcgctcttattgaccacacctctaccggcatgccgagcaaatgcctgcaaatcgctccccatttcacccaattgtagatatgctaacCCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGGAAAAGCTGTGGTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGGACGGATCGCTTGCCTGTAACTTACACGCGCCTCGTATCTTTTAATGATGGAATAATTTGGGAATTTACTCTGTGTTTATTTATTTTTATGTTTTGTATTTGGATTTTAGAAAGTAAATAAAGAAGGTAGAAGAGTTACGGAATGAAGAAAAAAAAATAAACAAAGGTTTAAAAAATTTCAACAAAAAGCGTACTTTACATATATATTTATTAGACAAGAAAAGCAGATTAAATAGATATACATTCGATTAACGATAAGTAAAATGTAAAATCACAGGATTTTCGTGTGTGGTCTTCTACACAGACAAGATGAAACAATTCGGCATTAATACCTGAGAGCAGGAAGAGCAAGATAAAAGGTAGTATTTGTTGGCGATCCCCCTAGAGTCTTTTACATCTTCGGAAAACAAAAACTATTTTTTCTTTAATTTCTTTTTTTACTTTCTATTTTTAATTTATATATTTATATTAAAAAATTTAAATTATAATTATTTTTATAGCACGTGATGAAAAGGACCCAGtaggtctagagatctgtttagcttgcctcgtccccgccgggtcacccggccagcgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaccatgggtaaggaaaagactcacgtttcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggcaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagtactgacaataaaaagattcttgttttcaagaacttgtcatttgtatagtttttttatattgtagttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctcgacatcatctgcccagatgcgaagttaagtgcgcagaaagtaatatcatgcgtcaatcgtatgtgaatgctggtcgctatactgctgtcgattcgatactaacgccgccatccagtgtcgaaaacgagctctcgagaacccttaatCAGCAAAACTAAAAAACTGTATTATAAGTAAATGCATGTATACTAAACTCACAAATTAGAGCTTCAATTTAATTATATCAGTTATTACCCATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGATACACTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGAGTGACACCACGATGCCTGTAGCAATGCCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGAATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCGCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGGGCCGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATGCGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCttagtgttcgaacatggcagagatcgattcttcgttgctgatacgacgaatcgcttcggccagcatacctgacagggtcagagtacgcacgttcggcagtgatttgatttcatcgctcagcggaatggtatcgcagacaacgacttcatcaattacagagttacgcaggttgttcgccgcgttgccagagaagatcgggtgagtcgcgtacgcaaatacacgtttagcaccacgttctttcagagcttcagcagctttacacagcgtaccgccagtgtcgatcatatcatcgaccagtacgcagtcacgacctgcaacgtcaccgatgatatgcatcacctgtgaaacgttcgcacgcggacgacgtttgtcgatgattgccatatcggtatcgttcagcagcttagcgatagcgcgggcacgcacaacgccgccgatgtccggagaaaccacaattgggttatccagattcagctgcagcatgtcttccagcaggatcgggctaccaaatacgttatcaaccggaacgtcgaagaaaccctgaatctgttcagcgtgcagaGccactgtcagcacacggtcaacaccgacgctggagaggaagtctgcaaccactttcgcagtgattggtacacgagcggaacggacgcgacggtcctggcgcgcatagccaaagtaggggataacagcggtgatacgacctgcggaagcacgacgcagggcatcaaccataacgactaattccatcaggttgtcgttagtaggggcacaagtggactggatgatgaaaatatcaccaccgcgtacattttcattaatttgtacgctgacttcgccatcgctaaagcgacctacagcggcgtcgccgagtgaagtgtacaggcggttggcaatacgttgtgctagttccggggtggcgttaccagcaaaaagcttcatatcaggcaTTTTGTAATTAAAACTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGCAAGAAGTAAAAAAAGTTGTAATAGAACAAGAAAAATGAAACTGAAACTTGAGAAATTGAAGACCATTTAATAACTTAAATATCAATGGGAGGTCATCGAAAGAGAAAAAAATCAAAAAAAAAAATTTTCAAGAAAAAGAAACGTGATAAAAATTTTTATTGCCTTTTTCGACGAAGAAAAAGAAACGAGGCGGTCTCTTTTTTCTTTTCCAAACCTTTAGTACGGGTAATTAACGACACCCTAGAGGAAGAAAGAGGGGGAATTTAGTATGCTGTGCTTGGGTGTTTTGAAGTGGTACGGCGATGCGCGGAGTCCGAGAAAATCTGGAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGTGTGTGACCGG;
SEQ ID No.11:TTTGTAATTAAAACTTAGATTAGATTGCTATGC;
SEQ ID No.12:GAATTCTGCAGATATCCATCACACTG;
SEQ ID No.13:CTAATCTAAGTTTTAATTACAAAAtgcctgatatgaagctttttgctgg;
SEQ ID No.14:CAGTGTGATGGATATCTGCAGAATTCttagtgttcgaacatggcagagatcg;
SEQ ID No.15:
GTATAATGTTACATGCGTACACGCGTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATGCGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCttagtgttcgaacatggcagagatcgattcttcgttgctgatacgacgaatcgcttcggccagcatacctgacagggtcagagtacgcacgttcggcagtgatttgatttcatcgctcagcggaatggtatcgcagacaacgacttcatcaattacagagttacgcaggttgttcgccgcgttgccagagaagatcgggtgagtcgcgtacgcaaatacacgtttagcaccacgttctttcagagcttcagcagctttacacagcgtaccgccagtgtcgatcatatcatcgaccagtacgcagtcacgacctgcaacgtcaccgatgatatgcatcacctgtgaaacgttcgcacgcggacgacgtttgtcgatgattgccatatcggtatcgttcagcagcttagcgatagcgcgggcacgcacaacgccgccgatgtccggagaaaccacaattgggttatccagattcagctgcagcatgtcttccagcaggatcgggctaccaaatacgttatcaaccggaacgtcgaagaaaccctgaatctgttcagcgtgcagaGccactgtcagcacacggtcaacaccgacgctggagaggaagtctgcaaccactttcgcagtgattggtacacgagcggaacggacgcgacggtcctggcgcgcatagccaaagtaggggataacagcggtgatacgacctgcggaagcacgacgcagggcatcaaccataacgactaattccatcaggttgtcgttagtaggggcacaagtggactggatgatgaaaatatcaccaccgcgtacattttcattaatttgtacgctgacttcgccatcgctaaagcgacctacagcggcgtcgccgagtgaagtgtacaggcggttggcaatacgttgtgctagttccggggtggcgttaccagcaaaaagcttcatatcaggcaTTTTGTAATTAAAACTTAGATTAGATTGCTATGCTTTCTTTCTAATGAGCAAGAAGTAAAAAAAGTTGTAATAGAACAAGAAAAATGAAACTGAAACTTGAGAAATTGAAGACCATTTAATAACTTAAATATCAATGGGAGGTCATCGAAAGAGAAAAAAATCAAAAAAAAAAATTTTCAAGAAAAAGAAACGTGATAAAAATTTTTATTGCCTTTTTCGACGAAGAAAAAGAAACGAGGCGGTCTCTTTTTTCTTTTCCAAACCTTTAGTACGGGTAATTAACGACACCCTAGAGGAAGAAAGAGGGGGAATTTAGTATGCTGTGCTTGGGTGTTTTGAAGTGGTACGGCGATGCGCGGAGTCCGAGAAAATCTGGAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGTGTGTGACCGGACCGGTCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGAAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAATGTCTATAGCAAGTTATGCCCAAGAGTTGAAGTTGGCTTTACATCAATATCCAAACTTCCCTAGTGAAGGCATTCTCTTCGAAGATTTCTTACCCATTTTCAGGAACCCAGGTCTTTTCCAGAAGTTGATCGATGCTTTCAAACTGCATTTAGAAGAAGCTTTTCCAGAAGTTAAAATTGATTATATCGTCGGGTTGGAATCCCGTGGGTTCTTGTTCGGACCAACTTTAGCTTTGGCCCTAGGTGTTGGTTTCGTTCCAGTCAGGAAGGCAGGTAAGCTACCTGGCGAATGTTTTAAGGCTACGTACGAAAAGGAGTACGGTTCTGATCTTTTTGAGATACAGAAAAACGCTATTCCAGCAGGTTCCAACGTTATCATTGTTGATGACATTATTGCCACTGGTGGTTCTGCTGCTGCAGCCGGCGAATTAGTTGAACAACTCGAAGCCAACCTTTTGGAATATAACTTTGTTATGGAGTTGGATTTCTTGAAAGGCAGGAGTAAGTTGAATGCTCCAGTGTTCACTTTACTGAACGCTCAAAAGGAAGCGTTGAAAAAATGAACCCGGGgcgaatttcttatgatttatgatttttattattaaataagttataaaaaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttattcttgagtaactctttcctgtaggtcaggttgc;
SEQ ID No.16:
CGCGGATCCTTAActgcaggtcgacaacccttaatataacttcgtataatgtatgctatacgaagttattaggtctagagatctgtttagcttgcctcgtccccgccgggtcacccggccagcgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaccatgggtaaggaaaagactcacgtttcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggcaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagtactgacaataaaaagattcttgttttcaagaacttgtcatttgtatagtttttttatattgtagttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctcgacatcatctgcccagatgcgaagttaagtgcgcagaaagtaatatcatgcgtcaatcgtatgtgaatgctggtcgctatactgctgtcgattcgatactaacgccgccatccagtgtcgaaaacgagctctcgagaacccttaatataacttcgtataatgtatgctatacgaagttattaggtgatatcagatccactagtgATTTAAATTGCAGGCCCgttagcatatctacaattgggtgaaatggggagcgatttgcaggcatttgctcggcatgccggtagaggtgtggtcaataagagcgacctcatgctatacctgagaaagcaacctgacctacaggaaagagttactcaagaataagaattttcgttttaaaacctaagagtcactttaaaatttgtatacacttattttttttataacttatttaataataaaaatcataaatcataagaaattcgcCCCGGGTTCATTTTTTCAACGCTTCCTTTTGAGCGTTCAGTAAAGTGAACACTGGAGCATTCAACTTACTCCTGCCTTTCAAGAAATCCAACTCCATAACAAAGTTATATTCCAAAAGGTTGGCTTCGAGTTGTTCAACTAATTCGCCGGCTGCAGCAGCAGAACCACCAGTGGCAATAATGTCATCAACAATGATAACGTTGGAACCTGCTGGAATAGCGTTTTTCTGTATCTCAAAAAGATCAGAACCGTACTCCTTTTCGTACGTAGCCTTAAAACATTCGCCAGGTAGCTTACCTGCCTTCCTGACTGGAACGAAACCAACACCTAGGGCCAAAGCTAAAGTTGGTCCGAACAAGAACCCACGGGATTCCAACCCGACGATATAATCAATTTTAACTTCTGGAAAAGCTTCTTCTAAATGCAGTTTGAAAGCATCGATCAACTTCTGGAAAAGACCTGGGTTCCTGAAAATGGGTAAGAAATCTTCGAAGAGAATGCCTTCACTAGGGAAGTTTGGATATTGATGTAAAGCCAACTTCAACTCTTGGGCATAACTTGCTATAGACATTTTGTTTGTTTATGTGTGTTTATTCGAAACTAAGTTCTTGGTGTTTTAAAACTAAAAAAAAGACTAACTATAAAAGTAGAATTTAAGAAGTTTAAGAAATAGATTTACAGAATTACAATCAATACCTACCGTCTTTATATACTTATTAGTCAAGTAGGGGAATAATTTCAGGGAACTGGTTTCAACCTTTTTTTTCAGCTTTTTTCAAATCAGAGAGAGCAGAAGGTAATAGAAGGTGTAAGAAAATGAGATAGATACATGCGTGGGTCAATTGCCTTGTGTCATCATTTACTCCAGGCAGGTTGCATCACTCCATTGAGGTTGTGCCCGTTTTTTGCCTGTTTGTGCCCCTGTTCTCTGTAGTTGCGCTAAGAGAATGGACCTATGAACTGATGGTTGGTGAAGAAAACAATATTTTGGTGCTGGGATTCTTTTTTTTTCTGGATGCCAGCTTAAAAAGCGGGCTCCATTATATTTAGTGGATGCCAGGAATAAACTGTTCACCCAGACACCTACGATGTTATATATTCTGTGTAACCCGCCCCCTATTTTGGGCATGTACGGGTTACAGCAGAATTAAAAGGCTAATTTTTTGACTAAATAAAGTTAGGAAAATCACTACTATTAATTATTTACGTATTCTTTGAAATGGCAGTATTGATAATGATAAACTCGAACTGACCGGTCCGGTCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTCCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTATTAAATGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAAtgcctgatatgaagctttttgctggtaacgccaccccggaactagcacaacgtattgccaaccgcctgtacacttcactcggcgacgccgctgtaggtcgctttagcgatggcgaagtcagcgtacaaattaatgaaaatgtacgcggtggtgatattttcatcatccagtccacttgtgcccctactaacgacaacctgatggaattagtcgttatggttgatgccctgcgtcgtgcttccgcaggtcgtatcaccgctgttatcccctactttggctatgcgcgccaggaccgtcgcgtccgttccgctcgtgtaccaatcactgcgaaagtggttgcagacttcctctccagcgtcggtgttgaccgtgtgctgacagtggCtctgcacgctgaacagattcagggtttcttcgacgttccggttgataacgtatttggtagcccgatcctgctggaagacatgctgcagctgaatctggataacccaattgtggtttctccggacatcggcggcgttgtgcgtgcccgcgctatcgctaagctgctgaacgataccgatatggcaatcatcgacaaacgtcgtccgcgtgcgaacgtttcacaggtgatgcatatcatcggtgacgttgcaggtcgtgactgcgtactggtcgatgatatgatcgacactggcggtacgctgtgtaaagctgctgaagctctgaaagaacgtggtgctaaacgtgtatttgcgtacgcgactcacccgatcttctctggcaacgcggcgaacaacctgcgtaactctgtaattgatgaagtcgttgtctgcgataccattccgctgagcgatgaaatcaaatcactgccgaacgtgcgtactctgaccctgtcaggtatgctggccgaagcgattcgtcgtatcagcaacgaagaatcgatctctgccatgttcgaacactaaGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCGCATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCAACCCATGCGGGTGAATATAAACATATAAAATGATGataataatatttatagaattgtgtagaattgcagattcccttttatggattcctaaatccttgaggagaacttctagtatattctgtatacctaatattatagcctttatcaacaatggaatcccaacaattatCTCAACATTCACATATTTCTCAGTTTAAACGCGGCCGCgttccatcccaatggcgcgccgagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattgacgcgtattgggatggaacGCGGCCGCGTTTAAACTGTTGGAATAGAAATCAACTATCatctactaactagtatttacattactagtatattatcatatacggtgttagaagatgacgcaaatgatgagaaatagtcatctaaattagtggaagctgaaacgcaaggattgataatgtaataggatcaaCCTGCAGG;
SEQ ID No.17:
CTGTTGGAATAGAAATCAACTATCatctactaactagtatttacattactagtatattatcatatacggtgttagaagatgacgcaaatgatgagaaatagtcatctaaattagtggaagctgaaacgcaaggattgataatgtaataggatcaaCCTGCAGGCGCGGATCCTTAActgcaggtcgacaacccttaatataacttcgtataatgtatgctatacgaagttattaggtctagagatctgtttagcttgcctcgtccccgccgggtcacccggccagcgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaccatgggtaaggaaaagactcacgtttcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggcaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagtactgacaataaaaagattcttgttttcaagaacttgtcatttgtatagtttttttatattgtagttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctcgacatcatctgcccagatgcgaagttaagtgcgcagaaagtaatatcatgcgtcaatcgtatgtgaatgctggtcgctatactgctgtcgattcgatactaacgccgccatccagtgtcgaaaacgagctctcgagaacccttaatataacttcgtataatgtatgctatacgaagttattaggtgatatcagatccactagtgATTTAAATTGCAGGCCCgttagcatatctacaattgggtgaaatggggagcgatttgcaggcatttgctcggcatgccggtagaggtgtggtcaataagagcgacctcatgctatacctgagaaagcaacctgacctacaggaaagagttactcaagaataagaattttcgttttaaaacctaagagtcactttaaaatttgtatacacttattttttttataacttatttaataataaaaatcataaatcataagaaattcgcCCCGGGTTCATTTTTTCAACGCTTCCTTTTGAGCGTTCAGTAAAGTGAACACTGGAGCATTCAACTTACTCCTGCCTTTCAAGAAATCCAACTCCATAACAAAGTTATATTCCAAAAGGTTGGCTTCGAGTTGTTCAACTAATTCGCCGGCTGCAGCAGCAGAACCACCAGTGGCAATAATGTCATCAACAATGATAACGTTGGAACCTGCTGGAATAGCGTTTTTCTGTATCTCAAAAAGATCAGAACCGTACTCCTTTTCGTACGTAGCCTTAAAACATTCGCCAGGTAGCTTACCTGCCTTCCTGACTGGAACGAAACCAACACCTAGGGCCAAAGCTAAAGTTGGTCCGAACAAGAACCCACGGGATTCCAACCCGACGATATAATCAATTTTAACTTCTGGAAAAGCTTCTTCTAAATGCAGTTTGAAAGCATCGATCAACTTCTGGAAAAGACCTGGGTTCCTGAAAATGGGTAAGAAATCTTCGAAGAGAATGCCTTCACTAGGGAAGTTTGGATATTGATGTAAAGCCAACTTCAACTCTTGGGCATAACTTGCTATAGACATTTTGTTTGTTTATGTGTGTTTATTCGAAACTAAGTTCTTGGTGTTTTAAAACTAAAAAAAAGACTAACTATAAAAGTAGAATTTAAGAAGTTTAAGAAATAGATTTACAGAATTACAATCAATACCTACCGTCTTTATATACTTATTAGTCAAGTAGGGGAATAATTTCAGGGAACTGGTTTCAACCTTTTTTTTCAGCTTTTTTCAAATCAGAGAGAGCAGAAGGTAATAGAAGGTGTAAGAAAATGAGATAGATACATGCGTGGGTCAATTGCCTTGTGTCATCATTTACTCCAGGCAGGTTGCATCACTCCATTGAGGTTGTGCCCGTTTTTTGCCTGTTTGTGCCCCTGTTCTCTGTAGTTGCGCTAAGAGAATGGACCTATGAACTGATGGTTGGTGAAGAAAACAATATTTTGGTGCTGGGATTCTTTTTTTTTCTGGATGCCAGCTTAAAAAGCGGGCTCCATTATATTTAGTGGATGCCAGGAATAAACTGTTCACCCAGACACCTACGATGTTATATATTCTGTGTAACCCGCCCCCTATTTTGGGCATGTACGGGTTACAGCAGAATTAAAAGGCTAATTTTTTGACTAAATAAAGTTAGGAAAATCACTACTATTAATTATTTACGTATTCTTTGAAATGGCAGTATTGATAATGATAAACTCGAACTGACCGGTCCGGTCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTCCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTATTAAATGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAAtgcctgatatgaagctttttgctggtaacgccaccccggaactagcacaacgtattgccaaccgcctgtacacttcactcggcgacgccgctgtaggtcgctttagcgatggcgaagtcagcgtacaaattaatgaaaatgtacgcggtggtgatattttcatcatccagtccacttgtgcccctactaacgacaacctgatggaattagtcgttatggttgatgccctgcgtcgtgcttccgcaggtcgtatcaccgctgttatcccctactttggctatgcgcgccaggaccgtcgcgtccgttccgctcgtgtaccaatcactgcgaaagtggttgcagacttcctctccagcgtcggtgttgaccgtgtgctgacagtggCtctgcacgctgaacagattcagggtttcttcgacgttccggttgataacgtatttggtagcccgatcctgctggaagacatgctgcagctgaatctggataacccaattgtggtttctccggacatcggcggcgttgtgcgtgcccgcgctatcgctaagctgctgaacgataccgatatggcaatcatcgacaaacgtcgtccgcgtgcgaacgtttcacaggtgatgcatatcatcggtgacgttgcaggtcgtgactgcgtactggtcgatgatatgatcgacactggcggtacgctgtgtaaagctgctgaagctctgaaagaacgtggtgctaaacgtgtatttgcgtacgcgactcacccgatcttctctggcaacgcggcgaacaacctgcgtaactctgtaattgatgaagtcgttgtctgcgataccattccgctgagcgatgaaatcaaatcactgccgaacgtgcgtactctgaccctgtcaggtatgctggccgaagcgattcgtcgtatcagcaacgaagaatcgatctctgccatgttcgaacactaaGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCGCATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCAACCCATGCGGGTGAATATAAACATATAAAATGATGataataatatttatagaattgtgtagaattgcagattcccttttatggattcctaaatccttgaggagaacttctagtatattctgtatacctaatattatagcctttatcaacaatggaatcccaacaattatCTCAACATTCACATATTTCTCAG;
SEQ ID No.18:GTATAATGTTACATGCGTACACGCGTCTG;
SEQ ID No.19:gcaacctgacctacaggaaagagttac;
SEQ ID No.20:CAGACGCGTGTACGCATGTAACATTATAC;
SEQ ID No.21:gtaactctttcctgtaggtcaggttgc;
SEQ ID No.22:CTGAGAAATATGTGAATGTTGAGataattg;
SEQ ID No.23:CTGTTGGAATAGAAATCAACTATCatctac。
本发明的有益效果:
本发明通过在表达SAM2的基础菌株中过表达相应的基因,使SAMe产量有所提高,基础菌株发酵的SAMe产量为1.4-1.6g/L,过表达apt1-prs128基因的菌株发酵的SAMe产量为1.7-2.3g/L。此方法操作简单,成本较低,且生产速率快,易于扩大规模。
保藏证明
保藏菌株:SHF16;
分类命名:酿酒酵母Saccharomyces cerevisiae;
保藏编号:CGMCC No.28947;
保藏时间:2023年11月13日;
保藏单位:中国微生物菌种保藏管理委员会普通微生物中心。
附图说明
图1为HPLC出峰对照图。
图2为质粒过表达结果。
图3为SHF16G发酵初筛结果。
图4为SHF16G发酵复筛结果。
图5为SHF16消抗克隆筛选结果。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
以下实施例中:
YPD液体培养基:20g/L葡萄糖、10g/L酵母粉、20g/L大豆蛋白胨FP410,pH值为6.5。
YPD平板:YPD液体培养基基础上添加0.2%琼脂。
YPG(2%半乳糖):2mL不加葡萄糖的YPD,加160μL的半乳糖。
O培养基:50g/L葡萄糖、5g/L酵母粉、10g/L蛋白胨、0.5g/L七水硫酸镁、2g/L磷酸氢二钾、4g/L磷酸二氢钾、1.5g/L L-甲硫氨酸。
G418:是一种氨基糖苷类抗生素,购买自翌圣生物,货号60220ES08。
BY4742酵母:购买自淼灵生物,货号T0081。
Phleo板:腐草霉素,购买自翌圣生物,货号60217ES20。
基础实施例1酿酒酵母基因操作方法
(1)酵母电转化感受态的制备:利用山梨醇法制备酵母电转感受态。
(2)质粒转化:0.5μg质粒加入感受态细胞中,混匀后加入2mm电转杯,放置5-10min,电击转化整合片段,立即加入0.5mL YPD液体培养基,于30℃复苏培养2小时,从中取出120μL涂布于添加0.5g/L G418的YPD平板,30℃培养,筛选转化子。
(3)δ整合片段转化:2-3μg整合片段约10μL加入感受态细胞中,混匀后加入2mm电转杯,放置5-10min,电击转化δ整合片段,立即加入0.5mL YPD液体培养基,于30℃复苏培养2小时,从中取出120μL涂布于添加不同浓度G418的YPD平板;G418浓度筛选浓度为1g/L、2g/L、3g/L。
基础实施例2酿酒酵母菌摇瓶发酵生产SAMe方法
(1)酿酒酵母摇瓶发酵生产SAMe
从含0.5g/L G418的YPD平板上挑取单菌落接种于含4mL YPD液体培养基的试管,30℃,250rpm活化过夜。次日下午两点,培养约20h的种子转接400μL到发酵底物为L-Met的20mL O培养基,30℃培养,250rpm,发酵24h补加0.5mL的0.75g/L L-Met(母液浓度为30g/L)继续发酵24h。
(2)SAMe的提取
取500μL发酵液,添加1mL 1.5mol/L高氯酸,于40℃水浴锅水浴半小时,8000r/min离心10min,上清液用0.22μm的水相膜过滤后,HPLC检测。
(3)HPLC检测
色谱柱为Welch Ultimate Aq-C18(4.6*250mm,5μm),紫外检测器检测,波长为254nm,初始流速1.0mL/min,7min时流速升到1.2mL/min,柱温30℃,进样量5μL,流动相A为10mM辛烷磺酸钠和50mM磷酸二氢钾,用磷酸调pH3.0,流动相B为乙腈,等度洗脱,洗脱比例为A:B=82:18,洗脱时间为11min。SAMe出峰的HPLC谱图见图1。
实施例1SHF16G整合片段
1.质粒过表达apt1-prs128
1.1pHF130(pHA180-pGPD-APT1)质粒的构建
1.1.1以pHA180(本实验室构建的基础质粒)为模板,其核苷酸序列如SEQ IDNo.1所示,以引物对GPD-homL和GPD-homR(表1)PCR得到5988bp的pHF130片段。
1.1.2以BY4742酵母基因组为模板,以引物对APT1-homL和APT1-homR(表1)PCR得到619bp的带同源臂的APT1片段,核苷酸序列如SEQ ID No.2所示。
1.1.3将以上得到的pHF130片段和带同源臂的APT1片段通过无缝克隆方式(GBclonart无缝克隆试剂盒,苏州神洲基因有限公司)重组到含有大肠杆菌ori和Amp,以及酿酒酵母的TEF promotor、GPD promotor和KanMX组件的载体上,得到质粒pHF130,pHF130的核苷酸序列如SEQ ID No.3所示。
表1
1.2pHF140(pHA180-pTEF1-prs128-pGPD-APT1)质粒的构建
1.2.1以pHF130为模板,以引物对TEF-homL和TEF-homR(表2)PCR得到6505bp的pHF140片段,其核苷酸序列如SEQ ID No.8所示。
1.2.2核糖磷酸焦磷酸激酶(Prs)催化5-磷酸核糖与ATP反应合成PRPP。但Prs受到ADP的反馈抑制,将Prs的第128位的天冬氨酸突变为丙氨酸获得的Prs128,就可以解除ADP的反馈抑制作用。
以pHX297(pEZ07-prs128)质粒为模板,以引物对prs128-homL和prs128-homR(表2)PCR得到997bp的带同源臂的prs128片段,其核苷酸序列如SEQ ID No.9所示。
1.2.3将以上得到的pHF140片段和带同源臂的prs128片段通过无缝克隆方式(GBclonart无缝克隆试剂盒,苏州神洲基因有限公司)重组到含有大肠杆菌ori和Amp,以及酿酒酵母的TEF promotor、GPD promotor和KanMX组件的载体上,得到质粒pHF140,pHF140的核苷酸序列如SEQ ID No.10所示。
1.2.4将pHF140质粒按照基础实施例1方法转化至SHF14(本实验构建的一株生产SAMe的二倍体酵母基因工程菌株,来源于SHF01)中,得到的转化子按照上述摇瓶发酵方法,进行发酵验证,结果见图2,SHF14发酵的SAMe产量为
1.4g/L,SHF14/pHF140发酵的SAMe产量为1.6g/L,过表达apt1-prs128,SAMe产量有提高。
表2
2.prs128-APT1基因的δ整合
2.1prs128-APT1整合质粒的构建
2.1.1以pHF140为模板,以引物对YHF279-homL和YHF280-homR PCR得到3026bp的prs128-APT1片段,该基因的核苷酸序列为SEQ ID NO.15。
2.1.2以pHF85为模板,以引物对YHF281-homL和YHF282-homR PCR得到4923bp的带同源臂的pHF143片段。
2.1.3将以上得到带同源臂的prs128-APT1片段和pHF143片段通过无缝克隆方式(GBclonart无缝克隆试剂盒,苏州神洲基因有限公司)重组到含有大肠杆菌ori、Amp以及Amp(Bacillus)组件的载体上,得到prs128-APT1整合质粒pHF143,pHF143的核苷酸序列如SEQ ID No.16所示。
2.1.4以pHF143质粒为模板,以引物对3delta-R和5delta-F(表3)PCR得到5187bp的SHF16G整合片段,核苷酸序列为SEQ ID No.17。
2.2prs128-APT1基因的多拷贝整合
2.2.1将2.1.4中的整合片段按照上述酿酒酵母基因操作方法转化至SHF14(本实验构建的一株生产SAMe的二倍体酵母基因工程菌株)中,得到的转化子。
2.2.2按照基础实施例2中的摇瓶发酵方法发酵筛选出的高产菌株,命名为SHF16G,如图3、4所示,SHF14发酵的SAMe产量为1.4-1.6g/L,SHF16G发酵的SAMe产量为1.7-2.3g/L,可见prs128-APT1基因δ整合后筛选出的高产菌株,SAMe产量有提高。
表3
实施例2SHF16G消抗
按照上述酵母基因工程操作方法将pSH65质粒(购买自翌圣生物,货号P1352)转化在SHF16G中进行消抗,转化子接种YPG(2%半乳糖),诱导培养2-3小时,稀释10-2倍(990μL的无菌水加上10μL的菌液),从中取出120μl涂布YPD,培养2-3天,选取单克隆,点15mg/LPhleo板和0.5g/L G418抗性板培养,选取两个抗性平板都不长的克隆,划线YPD纯化(根据实际情况选择是否要纯化)。
正确消抗的菌株命名为SHF16,分类命名:酿酒酵母菌Saccharomycescerevisiae,保藏编号:CGMCC No.28947,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏时间:2023年11月13日,保藏地址:北京市朝阳区北辰西路1号院3号。
按照上述摇瓶发酵方法发酵验证,结果见图5,SHF16G发酵的SAMe产量为1.68g/L,SHF16发酵的SAMe产量为1.7g/L,SHF16G消抗后,SAMe产量未下降。
Claims (10)
1.一种提高SAMe产量的方法,其特征在于,包括:在表达SAM2的基础菌株中过表达apt1-prs128基因,所述的apt1-prs128基因的核苷酸序列包括SEQ ID No.15,或其功能片段。
2.根据权利要求1所述的方法,其特征在于,所述的过表达包括通过游离质粒、定点整合或δ整合到基因组上实现。
3.根据权利要求2所述的方法,其特征在于,所述的过表达通过δ整合到基因组上实现。
4.一种基因工程菌株,其特征在于,用于表达SAMe,所述的基因工程菌株中过表达apt1-prs128基因;所述的apt1-prs128基因的核苷酸序列包括SEQ IDNo.15或其功能片段。
5.根据权利要求4所述的基因工程菌株,其特征在于,所述的基因工程菌株为酵母菌SHF16G或酵母菌SHF16,酵母菌SHF16保藏号为CGMCCNo.28947。
6.根据权利要求4所述的基因工程菌株,其特征在于,包括表达所述的apt1-prs128基因。
7.过表达apt1-prs128基因在提高SAMe产量中的应用,其特征在于,包括在表达SAM2的基础菌株中过表达apt1-prs128基因,所述的apt1-prs128基因的核苷酸序列包括SEQ IDNo.15或其功能片段。
8.权利要求4-6任一项所述的基因工程菌株在SAMe生产中的应用。
9.一种SAMe的生产方法,其特征在于,包括以下步骤:
(1)权利要求4-6任一项所述的基因工程菌株活化;
(2)发酵;
(3)SAMe的提取。
10.根据权利要求9所述的生产方法,其特征在于,
所述的步骤(1)包括:取单菌落接种YPD中,过夜活化;
所述的步骤(2)包括:将活化的种子转接至发酵培养基中,发酵;
所述的步骤(3)包括:取发酵液,添加高氯酸,水浴,离心,上清液用水相膜过滤后,HPLC检测。
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