The First Part 4, 5 - Double Hydrogen - 7 - To Ammonia Formyl - 7 - Hydroxy - 19 - S - Methyl, De Toxin And De Toxin Biosynthesis, Pks Relations After Modification, The Second Part Will Spiramycin Biosynthesis Regulate Gene Acyb2 Heterologous Expression I

Geldanamycin (GDM) is a benzoquinone ansamycin. It is a specific inhibitor of heat shock protein90(Hsp90) and has some pharmacological properties, including antitumor and antiviral activities. However, GDM is not considered as a clinical candidate due to its hepatotoxicity and poor water-solubility. It is only used as a promising lead compound. Obtaining new geldanamycin analogues with good solubility and low hepatotoxicity is becoming a hot spot in recent years.In our searching for GDM analogue(s), we have studied on the secondary metabolism of the gdmN disrupted mutant of Streptomyces hygroscopicus17997and successfully obtained an expected novel GDM analogue, which was later proved to be4,5-dihydro-7-descarbomoyl-7-hydroxy-19-S-methylgeldanamycin.The fermentation supernatant of gdmN-mutant was extracted with ethylacetate, and then separated by silica gel TLC. The expected red compound of interest was analyzed by HPLC and HR-ESI(+)-MS. The red compound was further purified by HPLC for1H-and13C-NMR analysis, which confirmed its identity as4,5-dihydro-7-descarbomoyl-7-hydroxy-19-S-methylgeldanamycin.Bioconversion of the red compound was performed in a geldanamycin polyketide synthase gene disruption mutant of Streptomyces hygroscopicus17997. It can be bioconverted to4,5-dihydro-19-S-methylgeldanamycin.The discovery of4,5-dihydro-7-descarbomoy1-7-hydroxy-19-S-methylgeldanamycin, together with19-S-methylgeldanamycin and4,5-dihydro-19-S-methylgeldanamycin identified ealier in our laboratory, deepen our understanding of the relationship between the post-PKS tailoring process of geldanamycin biosynthesis and other modifications existing in Streptomyces hygroscopicus17997. In particular,19-S-methylation of4,5-dihydro-7-descarbomoyl-7-hydroxygeldanamycin does not interfere its carbamoylation, but blocks its C4,5-oxidation of the post-PKS tailoring process of geldanamycin biosynthesis. Bitespiramycin is a new antibiotic biosynthesized by a recombinant spiramycin producing strain, Streptomyces spiramyceticus WSJ-1(for short, WSJ-1) that harbored a4"-O-acyltransferase gene (ist, carE or acyBl) for the addition of an acyl group, mainly an isovaleryl group, at the4"-OH of spiramycin.Recent reports mainly focused on the three genes acyA and acyBl-B2with the function of acylation. The gene acyA has the function of acetylation. The gene acyBl-B2has the function to add an isovaleryl group. The gene acyBl encodes4"-O-acyltransferase. Arisawa guessed acyB2is the positive regulator of acyBl, but the DNA binding region of acyB2coding protein is not clear now. We want to find the binding region of acyB2coding protein by EMS A; find the direct evidence of the relationship between acyBl and acyB2. The data presented in this work set the stage for subsequent studies to delineate the complexity of secondary metabolism regulations of strep to mycete, as well as for designing strategies for the construction of strains with enhanced bitespiramycin production.We constructed a recombinant plasmid pMA01containing acyB2gene using pMD18-T Simple Vector. After DNA sequencing verification as without mutation, the acyB2gene was cut out by EcoRI-Xbal double-digestion, and then inserted into the pCold II Vector for expression in E. coli, which resulted in a recombinant plasmid pCA01. The plasmid pCA01was transformed into E. coli Competent Cell BL21. The acyB2gene was expressed in the form of inclusion body. Then we tested different E. coli Competent Cell BL21as host cells which contained recombinant plasmids for expressing chaperones. However, no soluble protein of acyB2was detected. So, the protein of acyB2was purified as inclusion body by us, and then refolded.The acyBl-acyB2genes are closely linked. There is a DNA sequence of about300bp linking the two genes, which we thought to contain the DNA binding region of acyB2protein for up-regulate the transcription and expression of acyBl gene. This300bp DNA sequence was PCR-amplified and labelled as probe for detecting the DNA binding site of the above purifed AcyB2protein by EMSA, but no positive result was observed.