Screening of Virginiamycin Inducing Factors in Antibiotic-Producing Atcinomycetes

1.通过对多种抗生素产生菌的筛选，筛选出11株能产生类VB诱导因子的菌株(链霉菌、结节链霉菌、抗生素链霉菌、金黄色葡萄球菌、黄色葡萄球菌、硬粒葡萄球菌、棒状葡萄球菌、灰链霉菌和短芽孢杆菌)。其VB活性分别为500、500、100、100、100、5、4、1、0.5和0.2U/ml。采用C&lt；18&gt；反相高效液相色谱法，在20%~80%CH3CN线性梯度洗脱下，天然诱导因子VB-A和VB-B在12分钟洗脱，VB-C在8分钟洗脱。在相同条件下，从上述11个菌株中分离出活性成分。其中，林肯链霉菌、棒状链球菌和短链球菌的活性仅由一种组分组成，结节链球菌、黑纹链霉菌和东京链球菌的活性由两种组分组成，长木链球菌、纤维黄链霉菌和硬粒链球菌的活性由f…组成。3个组分较多，抗生素和灰链霉菌主要由4个组分组成。为了证实在这些微生物中检测到的活性确实是由于类似VB的化合物所致，我们选择了抗生素链霉菌进行高产菌株的筛选，得到了高产菌株NF-18。通过对培养基碳源和氮源的优化，确定了产酶5000单位/毫升的条件。这一诱导量比野生型抗生素产生的诱导量高50倍。通过乙酸乙酯提取、活性炭柱层析和C&lt；18&gt；反相高效液相色谱分离，从5L发酵液中分离得到2.3 mg的纯诱导因子。经常规波谱分析(核磁共振、IR、MS和CD)鉴定为维吉尼亚丁内酯A，为以前从维吉尼亚中分离得到的化合物。化学合成了几个VB类似物，并比较了它们诱导维吉尼亚霉素产生的效果，从而建立了VB的基本部分结构，以发挥其诱导活性。较少

1. After screening many antibiotics-producing strains, 11 strains (Streptomyces longwoodensis, S. tubercidicus, S. antibioticus, S. phaeofaciens, S. toyocaensis, S. celluloflavus, S. sclerogranulatus, S. clavurigerus, S. griseus and Bacillus brevis) were found to produce VB-like inducing factors. The VB activity of them were 500 units/ml, 500 units/ml, 100 units/ml, 100 units/ml, 5 units/ml, 5 units/ml, 4 units/ml, 1 unit/ml, 0.5 unit/ml and 0.2 unit/ml, respectively.2. By C_<18> reverse phase HPLC using linear gradient elution from 20% to 80% CH_3CN, natural inducing factors VB-A and VB-B were eluted at 12 min and VB-C at 8 min. Under the same conditions, active compounds from the above 11 strains were separated. Of them, activity of S. lincolnensis, S. clavurigerus and B. brevis was found to be constituted from only one component, that of S. tubercidicus, S. phaeofaciens and S. toyokaensis was from 2 components, that of S. longwoodensis, S. celluloflavus and S. sclerogranulatus was f … More rom 3 components, and that of S. antibioticus and S. griseus was from 4 components.3. To confirm that the activity deected in those organisms was actually due to VB-like compounds, S. antibioticus was selected and screened for higher producing strains and high producer strain NF-18 was obtained. After optimization of carbon and nitrogen sources of cultivation medium, conditions to produce 5000 units/ml were found. This amount of inducing activity was 50-fold higher than that produced by the wild type S. antibioticus. From the 5-liter culture broth of S. antibioticus NF-18, 2.3mg of pure inducing factor was isolated by ethyl acetate extraction, active charcoal column chromatography and C_<18> reverse phase HPLC. By usual spectroscopic analyses (NMR, IR, MS and CD), the compound was identified as virginiae butanolide A which was previously isolated from S. virginiae.4. Several VB analogs were chemically synthesized and their effectiveness on inducing virginiamycin production were compared leading to the establishment of the essential partial-structure in VB to exert its inducing activity. Less