响应面法优化必特螺旋霉素发酵培养基

目的利用响应面法对新一代必特螺旋霉素基因工程菌(Streptomyces spiramyceticus,WSJ-2),即含有整合型双拷贝4″位异戊酰基转移酶基因(4″-isovaleryltrasferase gene,ist)工程菌发酵培养基进行优化。方法以发酵效价和异戊酰螺旋霉素组分含量为指标,通过部分因子析因设计实验,筛选出影响较大的主要影响因子。其次,进行最陡爬坡实验,最后,通过中心组合设计,利用DX7trail 7.0软件进行回归分析,确定主要影响因子的最佳浓度,得到优化发酵培养基。结果综合培养基组成对发酵效价和组分含量的影响获得2个主要影响因子,即KH2PO4和NaCl,通过最陡爬坡实验和响应面法,确定了优化发酵培养基组成为(g.L-1):淀粉60、碳酸钙7.0、MgSO42.0、NH4NO36.0、酵母粉3.0、鱼粉15、MnCl20.10、NaCl 12.5、KH2PO40.645。验证实验表明在优化培养基中发酵相对效价为142%与预测值139%比较接近,效价比原配方提高了42%,而其总异戊酰基螺旋霉素组分的含量与原配方基本一致。结论对新构建的必特螺旋霉素基因工程菌采用响应面中心组合设计,可以较有效地进行发酵培养基的优化。

Optimization of fermentation medium for bitespiramycin production by response surface methodology

Objective To optimize the fermentation medium components for bitespiramycin production in a new gene-engineered bitespiramycin-producing strain with integrated double copy of isovaleryltransferase (ist) gene. Methods Response surface methodology (RSM) was adopted，two level fractional factorial design (FFD) was first used to evaluate the influence of six related factors. The path of steepest ascent was used to approach the optimal region of the fermentation conditions subsequently. The concentrations of those two main factors were further optimized by using central composite design (CCD). Results Two factors were determined to play important roles in the medium, which were KH₂PO₄ and NaCl. By solving the quadratic regression model equation, the optimal concentrations of the variables were determined as KH₂PO₄ 0.645 g·L^-1, NaCl 12.5 g·L^-1. An optimized fermentation medium was obtained for this newly developed strain, which was composed of (g·L^-1): starch 60, CaCO₃ 7.0, MgSO₄ 2.0, NH₄NO₃ 6.0, yeast powder 3.0, fish meal 15, MnCl₂ 0.10, NaCl 12.5, KH₂PO₄ 0.645. Under the optimum conditions, the relative bitespiramycin titer of 139％ as predicted in theory and 142% in the experiment were obtained with an increase of 42% compared to the initial medium. Meanwhile the relative proportion of total isovalerylspiramycin was no significant changes. Conclusions Response surface methodology as a statistical tool is efficient to improve the productivity of a new gene-engineered strain of antibiotic producer.