林肯链霉菌的推理选育和代谢分析
摘要
本文以花生粉和棉籽蛋白取代了原培养基中的黄豆饼粉,并采用响应面法对林肯霉素产生菌的发酵培养基进行优化。首先采用Plackett-Burman实验分析各因素的主效应,选出对响应值影响较大的3个因素,即花生粉、K2HP04和玉米浆。对这些因素做爬坡实验,确定三个重要因素的中心点浓度,并运用响应面分析法确定了这3个重要因素的最优水平,分别为熟花生粉18g／L、K2HP040.4g／L、玉米浆1.2g／L。采用优化后的培养基进行摇瓶发酵,林肯霉素摇瓶生物效价为4268μg／ml,比优化前提高了12-17%。最后分别比较优化前后培养基中金属离子和氨基酸含量的差别,可知金属离子和氨基酸对林肯霉素生物合成有较大贡献。
使用紫外照射结合NTG处理的复合诱变处理,并选用具有糖苷结构的IPTG(异丙基-β-D-硫代半乳糖苷)和林肯霉素本身作为筛选因子,取得很好的结果,效价提高了12-30%。在发酵Oh添加0.02%的IPTG,使效价提高了10%左右。实验证明IPTG具有诱导作用,即诱导p-半乳糖苷酶将乳糖分解为半乳糖和葡萄糖,提供营养物质或者合成抗生素的前体。实验发现,乳糖和半乳糖对林肯霉素的生产具有积极意义,尤其是半乳糖,几乎在发酵任何时间段添加都有利于抗生素的合成。多次实验证实在发酵前期(0-48h)添加0.01%的半乳糖,效价可提高20%左右。
通过对莽草酸途径的代谢流分析,找到途径中关键酶氨基苯甲酸合成酶的抑制剂三甲胺,通过三甲胺抑制氨基苯甲酸合成酶的活性,使代谢流更多地流向酪氨酸的合成方向。实验证明,在48h添加0.08%的三甲胺对林肯霉素的生物合成有积极的意义。使效价提高了20%左右。
The soybean flour was replaced by cottonseed protein and peanut meal as main nitrogen sources for lincomycin production. The response surface methodology was used to optimize the biosynthesis medium of Streptomyces linconinensis. First, peanut meal, KH2PO4 and corn steep liquor were found to be the important factors by Plackett-Burman design. Then center concentration of the three important factors was determined by the steepest ascent search. And the optimal concentration of the three important factors was defined by response surface methodology. The result were 18 g/L peanut meal,0.4 g/L KH2PO4 and 1.2 g/L corn steep liquor. The average potency of lincomycin could reach 4268μg/ml by using the optimal medium, which was increased by 12%-17%compared with the original medium. Finally, We found metal ions and amino acid had effects on lincomycin production by comparison the differents between the original culture and the optimal one.
Based on the structure of lincomycin, mutant strains, with resistance of feedback suppression of Isopropyl-β-D-thiogalactopyranoside (IPTG),which is an analog of methyl thiolincosamide, were thus selected and lincomycin production increased by 12-30%. IPTG was proved to be an inducer ofβ-D-galactosidase when it was added at the beginning of the culture. The production of lincomycin was increased by 10%with the addition of 2 mg/L IPTG at 0 h directly in the medium. IPTG can induce the degradation of the lactose to galactose, the addition of galactose was better than lactose, which can increase the yield of lincomycin by 20%,with the addition of 0.01%galactose at 0-48 h directly in the medium.
The inhibitor of anthranilate synthetase—trimethylamine was found by shikimic acid pathway analysis, which change the metabolite flux, so that more metabolite flux turned to the direction of tyrosine synthesis. Experiments showed the potency was increased by 20%or so via adding 0.08%trimethylamine at 48h directly in the medium.
使用紫外照射结合NTG处理的复合诱变处理,并选用具有糖苷结构的IPTG(异丙基-β-D-硫代半乳糖苷)和林肯霉素本身作为筛选因子,取得很好的结果,效价提高了12-30%。在发酵Oh添加0.02%的IPTG,使效价提高了10%左右。实验证明IPTG具有诱导作用,即诱导p-半乳糖苷酶将乳糖分解为半乳糖和葡萄糖,提供营养物质或者合成抗生素的前体。实验发现,乳糖和半乳糖对林肯霉素的生产具有积极意义,尤其是半乳糖,几乎在发酵任何时间段添加都有利于抗生素的合成。多次实验证实在发酵前期(0-48h)添加0.01%的半乳糖,效价可提高20%左右。
通过对莽草酸途径的代谢流分析,找到途径中关键酶氨基苯甲酸合成酶的抑制剂三甲胺,通过三甲胺抑制氨基苯甲酸合成酶的活性,使代谢流更多地流向酪氨酸的合成方向。实验证明,在48h添加0.08%的三甲胺对林肯霉素的生物合成有积极的意义。使效价提高了20%左右。
The soybean flour was replaced by cottonseed protein and peanut meal as main nitrogen sources for lincomycin production. The response surface methodology was used to optimize the biosynthesis medium of Streptomyces linconinensis. First, peanut meal, KH2PO4 and corn steep liquor were found to be the important factors by Plackett-Burman design. Then center concentration of the three important factors was determined by the steepest ascent search. And the optimal concentration of the three important factors was defined by response surface methodology. The result were 18 g/L peanut meal,0.4 g/L KH2PO4 and 1.2 g/L corn steep liquor. The average potency of lincomycin could reach 4268μg/ml by using the optimal medium, which was increased by 12%-17%compared with the original medium. Finally, We found metal ions and amino acid had effects on lincomycin production by comparison the differents between the original culture and the optimal one.
Based on the structure of lincomycin, mutant strains, with resistance of feedback suppression of Isopropyl-β-D-thiogalactopyranoside (IPTG),which is an analog of methyl thiolincosamide, were thus selected and lincomycin production increased by 12-30%. IPTG was proved to be an inducer ofβ-D-galactosidase when it was added at the beginning of the culture. The production of lincomycin was increased by 10%with the addition of 2 mg/L IPTG at 0 h directly in the medium. IPTG can induce the degradation of the lactose to galactose, the addition of galactose was better than lactose, which can increase the yield of lincomycin by 20%,with the addition of 0.01%galactose at 0-48 h directly in the medium.
The inhibitor of anthranilate synthetase—trimethylamine was found by shikimic acid pathway analysis, which change the metabolite flux, so that more metabolite flux turned to the direction of tyrosine synthesis. Experiments showed the potency was increased by 20%or so via adding 0.08%trimethylamine at 48h directly in the medium.
引文
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[2]Chang F N. Lincomycin. Antibiotics, Hahn E.E, ed.New York,1979
[3]Wright J L C. The lincomycin-celesticetin-anthramycin group, Biochemistry and Genetic Regulation of Commercially Important Antibiotics, Vining L.C, ed.London,1983
[4]陈春福.林可霉素产生菌的推理选育[J].中国抗生素杂志,2002,27(7)：394.
[5]储炬,李友荣.现代工业,发酵调控学[M].北京：化学工业出版社,2002.
[6]J.Spizek, T.Rezanka.Lincomycin,cultivation of producing strains and biosynthesis.Appl Microbiol Biotechnol,2004年,63,510-519
[7]Hoeksema H, Bannister B, Birkenmeyer RD, Kagan F, Magerlein BJ, MacKellar FA, Schroeder W, Slomp G, Herr RR (1964) Chemical studies on lincomycin.1. The structure of lincomycin. J Am Chem Soc 86:4223-4224
[8]邬行彦,熊宗贵,胡章助.抗生素生产工艺学.北京：化学工业出版社,1989年,402-403
[9]李丽燕.微生物药品化学[M].北京：中国医药科技出版社,1990,125-126
[10]王镜岩.生物化学(第三版)[M]. 北京：教育出版社.2002
[11]陈梅芳.代谢工程在芳香化合物生物合成研究中的应用.氨基酸和生物资源[J],2003,25(2)：37—40
[12]杨蕴刘,焦瑞身.林肯链霉菌合成林可霉素代谢调节的研究.生物工程学报[J],1998,14(1)： 101-105
[13]焦瑞身等.林肯链霉菌谷氨酰胺合成酶活力调节的研究.微生物学报[J],2001,41(4)：481-488
[14]杨蕴刘,焦瑞身.林肯链霉菌合成林可霉素代谢调节的研究.生物工程学报,1998,1(14),101-105
[15]李小兵.林可霉素发酵过程中磷的调节.中国医药工业杂志,1999年,卷号：30(6),246-248
[16]杨蕴刘,焦瑞身.林肯链霉菌合成林可霉素代谢调节的研究.生物工程学报,1998,1(14),101-105
[17]Madigan M T, Martinko J M Parker, J.微生物生物学[M].北京：科学出版社。2001.
[18]顾觉奋,王鲁燕,倪盂样.抗生素[M].上海：上海科学技术出版社,2001.
[19]白兰芳.西罗莫司产生菌Streptomyces hygroscopicus WY-93的诱变育种与代谢研究[J].中国抗生素杂志,2001,26(1)：35—38.
[20]胡永兰等.梧宁霉素产生菌诱变选育的研究[J].微生物学杂志,1995,15(2)：40—-44.
[21]贾红华,周华,韦萍.微波诱变育种研究及应用进展[J].工业微生物,2003,33(2)：46—50.
[22]周建琴,高荣梅.康乐霉素C产生菌的微波诱变.生物学杂志[J],2002,19(4)： 12—13.
[23]施巧琴,吴松刚.工业微生物育种学[M].北京：科学出版社,2003.
[24]刘连碧.离子注入诱变育种技术在柔红霉素高产菌选育中的应用[J].中国医药工业杂志,2001,32(7)：297—299.
[25]王纪.离子注入麦角甾醇酵母选育及发酵工艺[J].微生物学报,1998,18(4)：25—28.
[26]龚加顺.单宁酸酶产生菌氮离子注入的诱变效应研究[J].食品与发酵工业,2000,26(5)：9—13.
[27]杨素红.氮氖激光辐照诱发棉病囊霉突变产生核黄素[J].应用激光,1992,12(6)：265—268.
[28]陈有为等.激光诱变微生物的遗传和刺激效应机理及育种研究[J].激光生物学,1996,5(1)：800—803.
[29]赵炎生.脱落酸高产荫的激光诱变效应研究[J].激光生物学报。1999,8(3)：205—208
[30]李金国等.空间条件对棘孢小单孢菌的诱变效应[J].航天医学与医学工程。1995,8(2)：113—116.
[31]贾士芳.空间条件对芽孢杆菌的蒯氧化物歧化酶和其他性质的影响[J].航天医学与医学工程,1995,8(1)：12—14.
[32]王璋、刘新征等.“神舟”4号空间飞行对搭载的转谷氨酰胺酶链霉菌选育的影响[J].航天医学与医学工程。2004,17(4)：275—281.
[33]周希贵.粘杆菌素高产菌株的选育[J].微生物学通报,2001,28(5)：49—51.
[34]顾正华等.L-组氨酸产生菌的选育[J].无锡轻工大学学报,2002,21(5)：533—535.
[35]盂庆繁.透明质酸产生菌的化学诱变及发酵条件探索[J].吉林大学学报,2004,42(1)：121—125.
[36]王世梅,等.阿扎霉素B产生菌吸水链霉菌NND-52的诱变筛选[J].微生物学通报,2001,28(1)：64—67.
[37]程世清.产色素菌T17-2-39的诱变育种试验.江苏食品与发酵[J],2000,101(2)：9—12.
[38]王弋博等.用紫外诱变及紫外+硫酸二乙酯复合诱变方法选育高产异淀粉酶菌株.青海大学学报[J],2003,21(4)：7—10.
[39]张怡轩等.氨甲酰一妥布霉素产生菌的高产菌株筛选.沈阳药科大学学报[J],1999,16(1)：53—57.
[40]刘长云.肌苷高产菌株的选育.中国药科大学学报[J],1994,25(1)：56—60.
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