Ziermann R, Betlach M C
KOSAN Biosciences, Burlingame, CA 94010, USA.
Biotechniques. 1999 Jan;26(1):106-10. doi: 10.2144/99261st05.
Efficient polyketide synthesis derived from plasmid-borne heterologous Streptomyces polyketide synthase (PKS) gene clusters necessitates a suitable host strain. Well-characterized laboratory strains such as Streptomyces coelicolor or Streptomyces lividans and their frequently used derivatives carry endogenous genes for the synthesis of actinorhodin (among other PKS genes), which might interfere with the efficient production of extrachromosomally encoded PKS proteins and the quantitative analysis of their secreted polyketide products. To circumvent this problem, a frequently used S. coelicolor derivative, designated CH999, was engineered to lack most of the actinorhodin gene cluster. However, this strain can only be transformed with methyl-free DNA. Additionally, unlike its otherwise isogenic parent CH1, CH999 exhibits low transformation efficiencies. Here, we report the construction of two S. lividans host strains, K4-114 and K4-155. With respect to the actinorhodin gene cluster, both are genotypically identical to CH999; however, both can be transformed at considerably higher frequencies and also with methylated DNA. Upon transformation with the appropriate expression vector, CH999, K4-114 and K4-155 all produce the erythromycin precursor 6-deoxyerythronolide B (6-dEB) equally well.
源自质粒携带的异源链霉菌聚酮合酶（PKS）基因簇的高效聚酮化合物合成需要合适的宿主菌株。特征明确的实验室菌株，如天蓝色链霉菌或变铅青链霉菌及其常用衍生物，携带用于合成放线紫红素的内源基因（以及其他PKS基因），这可能会干扰染色体外编码的PKS蛋白的高效产生及其分泌的聚酮化合物产物的定量分析。为了解决这个问题，一种常用的天蓝色链霉菌衍生物，命名为CH999，经过改造使其缺乏大部分放线紫红素基因簇。然而，该菌株只能用无甲基化的DNA进行转化。此外，与其他方面同基因的亲本CH1不同，CH999表现出较低的转化效率。在此，我们报告了两种变铅青链霉菌宿主菌株K4-114和K4-155的构建。就放线紫红素基因簇而言，两者在基因型上与CH999相同；然而，两者都能以相当高的频率进行转化，并且也能用甲基化的DNA进行转化。用合适的表达载体转化后，CH999、K4-114和K4-155均能同样良好地产生红霉素前体6-脱氧红霉内酯B（6-dEB）。
