Construction and physiological studies on a stable bioengineered strain of shengjimycin.
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.
Guangdong
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Institute of Medical Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tiantan, Beijing, PR China.
Jianlu
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Yiguang
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D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D015139:Blotting, Southern; D002455:Cell Division / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D005285:Fermentation; D005818:Genetic Engineering / Q000379:methods*; D050803:Leucine Dehydrogenase; D011995:Recombination, Genetic; D015572:Spiramycin / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*; D013302:Streptomyces / Q000502:physiology*; D013997:Time Factors; D050802:Valine Dehydrogenase (NADP+)
D000900:Anti-Bacterial Agents; D015572:Spiramycin; D000594:Amino Acid Oxidoreductases; D050802:Valine Dehydrogenase (NADP+); D050803:Leucine Dehydrogenase; C423662:carrimycin