Desai Ruchir P, Rodriguez Eduardo, Galazzo Jorge L, Licari Peter
Kosan Biosciences, Inc., 3825 Bay Center Place, Hayward, California 94545, USA.
Biotechnol Prog. 2004 Nov-Dec;20(6):1660-5. doi: 10.1021/bp0497435.
The bioconversion of a 6-deoxyerythronolide B analogue to the corresponding erythromycin A analogue (R-EryA) by a Saccharopolyspora erythraea mutant lacking the ketosynthase in the first polyketide synthase module was significantly improved by changing fluxes at a key branch point affecting the erythromycin congener distribution. This was achieved by integrating an additional copy of the eryK gene into the chromosome under control of the eryAIp promoter. Real-time PCR analysis of RNA confirmed higher expression of eryK in the resulting strain, S. erythraea K301-105B, compared to its parent. In shake flasks, K301-105B produced less of the shunt product 15-fluoro-erythromycin B (15F-EryB), suggesting a shift in congener distribution toward the desired product, 15-fluoro-erythromycin A (15F-EryA). In bioreactor studies, K301-105B produced 1.3 g/L of 15F-EryA with 75-80% molar yield on fed precursor, compared with 0.9 g/L 15F-EryA with 50-55% molar yield on fed precursor by the parent strain. At higher precursor feed rates, K301-105B produced 3.5 g/L of 15F-EryA while maintaining 75-80% molar yield on fed precursor.
通过改变影响红霉素同系物分布的关键分支点处的通量，显著提高了缺少第一个聚酮合酶模块中酮合酶的糖多孢红霉菌突变体将6-脱氧红霉内酯B类似物生物转化为相应的红霉素A类似物（R-EryA）的效率。这是通过在eryAIp启动子的控制下将eryK基因的额外拷贝整合到染色体中来实现的。对RNA的实时PCR分析证实，与亲本相比，所得菌株糖多孢红霉菌K301-105B中eryK的表达更高。在摇瓶中，K301-105B产生的分流产物15-氟红霉素B（15F-EryB）较少，这表明同系物分布向所需产物15-氟红霉素A（15F-EryA）发生了转变。在生物反应器研究中，K301-105B以75-80%的摩尔产率产生了1.3 g/L的15F-EryA，而亲本菌株以50-55%的摩尔产率产生了0.9 g/L的15F-EryA。在更高的前体进料速率下，K301-105B产生了3.5 g/L的15F-EryA，同时保持了75-80%的摩尔产率。
