【作者】 李佳玮；

【导师】 徐威；

【作者基本信息】 沈阳药科大学 ， 微生物与生化药学， 2006， 硕士

【摘要】 阿维链霉菌是阿维菌素的产生菌，由于其发酵液较粘稠而导致溶氧较差，因此溶氧成为阿维菌素产量提高的限制因素之一。本研究旨在通过在阿维链霉菌中表达透明颤菌血红蛋白基因（vgb）来改善氧的传递，进而改善菌体生长，提高阿维菌素的产量。 对阿维链霉菌原生质体的制备和再生条件进行优化。首先考察了影响阿维链霉菌原生质体形成的因素，发现在R₂YE、YMB、SM、YEME几种链霉菌常用的液体培养基中，通过R₂YE培养基收获的菌体形成原生质体的效率最高。菌丝培养采用二级培养，即用R₂YE培养基28℃培养48h，转种到补加0.25％甘氨酸的R₂YE培养基中，28℃继续培养20～24h，溶菌酶作用浓度为2mg·mL^-1，30℃酶解30min，在此条件下，原生质体的制备量为6.3×10⁸个·mL^-1原生质体的再生率最高，达到1.98％。 应用双亲灭活原生质体融合技术选育阿维菌素高产菌株，得到了一株高产菌株Streptomyces avermitilis F32，阿维菌素总发酵单位达3904μg·mL^-1，其中B_1a组分产量较高，达1016μg·mL^-1，较出发菌株产抗单位提高了117.1％。 利用链霉菌质粒pIJ702和带vgb的大肠埃希菌质粒pUC19-vgb，构建大肠埃希菌-链霉菌穿梭质粒pSY702。将该质粒以PEG-介导的方式转入阿维链霉菌原生质体中，利用硫链丝菌素抗性筛选转化子。CO结合差光谱法检测在420nm处有吸收峰，表明血红蛋白已在阿维链霉菌中表达且具有生物活性。通过高效液相色谱（HPLC）对转化子摇瓶发酵产物进行分析表明，转化子仍然合成阿维菌素，而且转化子在供养不足时生产阿维菌素的能力比对照更强。说明含vgb的转化子比不含vgb的对照菌更能耐受贫氧环境，合成阿维菌素效率更高，在贫氧环境中表现出其优越性。 以筛选到的转化子S．avermitilis T26为实验菌株，对发酵条件进行优化。结果表明随着发酵时间的延长，阿维菌素产量不断提高，发酵10d阿维菌素的产量最高；选用18h的种龄、15％的转种量可使阿维菌素的产量提高。通过均匀设计优化发酵培养基配方，发酵培养基（％）为：玉米淀粉8.0，黄豆饼粉0.7，花生饼粉1.1，酵母粉0.875，酵母膏0.2，玉米浆0.35时，使得S．avermitilis T26的发酵效价提高了38％，B_1a发酵单位达到1206μg.mL^-1。更多还原

【Abstract】 Streptomyces avermitilis is a kind of microbe that can produce avermectin. Its fermentation broth is so mucous that it cannot dissolve too much oxygen, So DO （dissolved oxygen） becomes one of the restricted factor affecting the increase of avermectin yield. This study was carried out to improve the genetically engineered bacteria’s ability to transport and absorb oxygen, better its growth, as well as strengthen its synthesis of protein and secondary metabolites, by expression of Vitreoscilla hemoglobin gene in Streptomyces avermitilis .In this work, conditions for preparation and regeneration of protoplasts of Streptomyces avermitilis were optimized. Medium R₂YE appeared to be more suitable than YMB, SM and YEME for protoplast formation. The mycelia of Streptomyces avermitilis was cultivated in the form of secondary culture , namely cultivation at 28℃ for 48h in R₂YE culture, then transfered to medium R₂YE added 0.25% glycine, continues cultivation at 28℃ for 20～24h. The washed mycelia were incubated in P buffer containing 2mg·mL^-1 lysozyme at 30℃ for 30min. The final concentration and regeneration rate of protoplasts reached 6.3×10⁸·mL^-1 and 1.98%respectively.The inactivated parental strain protoplasts fusion method was used to select high avermectin-producing strain. The total titer of Streptomyces avermitilis F32 was up to 3 904μg·mL^-1 , and the titer of effective composition B_1a was 1 016μg·mL^-1, increased by 117.1% compared with that of S. avermitilis 620.An E.coli-streptomyces shuttle plasmid pSY702 was constructed and transformed into the protoplasts of Streptomyces avermitilis. Transformants were screened with the thiostrepton resistance. Expression of VHb protein in transformants and the bioactivity of the expressed VHb were verified by the analysis of a CO difference spectra experiments. It was shown by HPLC analysis that avermectin was produced by VHb-expressing transformants in flask fermentation. Under low dissolved oxygen conditions, the S. avermitilis T26 transformant produced higher yields of avermectin compared with the control strain in flask fermentation experiment. These results demonstrated that VHb expression in S. avermitilis can improve the productivity of avermectin even under the conditions that aeration was reduced.Several factors affecting avermectin production were studied. The highest avermectin更多还原