Guangdong S, Jianlu D, Yiguang W
Institute of Medical Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tiantan, Beijing, PR China.
J Antibiot (Tokyo). 2001 Jan;54(1):66-73. doi: 10.7164/antibiotics.54.66.
Shengjimycin is a group of 4"-acylated spiramycins with 4"-isovalerylspiramycin as the major component, produced by recombinant S. spiramyceticus F21 harboring a 4"-O-acyltransferase gene from S. mycarofaciens 1748. A stable bioengineered strain of Streptomyces spiramyceticus WSJ-1 was constructed by integrating the 4"-O-acyltransferase gene (ist) by homologous recombination into the chromosome of the spiramycin-producing strain S. spiramyceticus F21. In this construction, a Streptomyces/E. coli shuttle plasmid pKC1139 (AmR) was used as the vector with the tsr gene used as selection marker for homologous recombination. The constructed strain, S. spiramyceticus WSJ-1,was genetically stable in production titer and proportion of components of shengjimycin as well as in maintaining the tsr selective marker when grown without selection. Southern hybridization confirmed the integrated status of the ist gene in the host genome. The production and the proportion of major component of 4"-isovalerylspiramycin of S. spiramyceticus WSJ-1 was also improved comparing with the strain harboring an autonomous plasmid -S. spiramyceticus F21/pIJ680(311) as shown by HPLC analysis. Physiological studies indicated that increase of the VDH ( valine dehydrogenase ) and LDH ( leucine dehydrogenase ) activities of WSJ-1 may be involved in this improvement.
生技霉素是一组4”-酰化螺旋霉素，以4”-异戊酰螺旋霉素为主要成分，由携带来自产朊假丝酵母1748的4”-O-酰基转移酶基因的重组螺旋链霉菌F21产生。通过同源重组将4”-O-酰基转移酶基因（ist）整合到螺旋霉素生产菌株螺旋链霉菌F21的染色体中，构建了稳定的基因工程菌株螺旋链霉菌WSJ-1。在该构建过程中，使用链霉菌/大肠杆菌穿梭质粒pKC1139（AmR）作为载体，tsr基因用作同源重组的选择标记。构建的菌株螺旋链霉菌WSJ-1在生技霉素的生产效价、成分比例以及在无选择条件下生长时维持tsr选择标记方面遗传稳定。Southern杂交证实了ist基因在宿主基因组中的整合状态。高效液相色谱分析表明，与携带自主质粒的菌株螺旋链霉菌F21/pIJ680(311)相比，螺旋链霉菌WSJ-1的4”-异戊酰螺旋霉素的产量和主要成分比例也有所提高。生理学研究表明，WSJ-1的缬氨酸脱氢酶（VDH）和亮氨酸脱氢酶（LDH）活性的增加可能与这种提高有关。
