Discovery Of New Natrul Products From Micromonospra Sp. A1304Guided By Bioinformatics

In recent years, a new method for natural products discovery, named "genome mining", has been widely used. Discovery of new natural products using this method is oriented by bioinformatics analysis. This method of discovery of new chemical entities from the source is much more challenging, and may form the basis for new drug leads.Micromonospora sp. A1304was discovered by the Japanese scientist Tamotsu Furumai in2002. And Kosinostatin, an anthracycline antibiotics with anti-tumor activity, was isolated from it. Recently, we have found the key genes, AHBA synthase, oxidoreductase and phosphatase, involved in ansamycin biosynthesis in the genome of Micromonospora sp. A1304based on bioinformatics analysis. These genes are always conversed and play an important role in the biosynthesis of3-amino-5-hydroxy benzoic acid (AHBA), the key start unit of ansamycin biosynthesis. Thus, we speculated that the bacteria may be able to produce ansamycin.To explore the new ansamycin, the fosmid genomic library of Micromonospora sp. A1304with high efficiency was constructed with pCC1FOS as the vector. And part of the biosynthetic gene cluster has been cloned through a variety of strategies. The HPLC analysis conditions of the fermentation product were explored at the same time. Gene replacement and in frame deletion mutants was constructed for preliminary study of its biosynthetic pathway. Also, we tried to isolate the target compounds, and initial results have been achieved.In addition, we constructed the gene disruption mutant strain mKOSD3to investigate the function of kstD3gene, encoding a sugar3,5-epimerase involved in the KST biosynthetic gene cluster. A novel compound, deoxy-isoquinocycline B was discovered and isolated from this mutant strain and its structure was characterized. This compound could be considered as an analogue of isoquinocycline B and a byproduct of the KST biosynthetic pathway. Compared with isoquinocycline B, its anti-tumor activity decreased significantly. We also found that the glycosyltransferase KstD5is more substrate-flexible than anticipated, which lay the foundation for further using the characteristics of this enzyme to obtain more analogues.In conclusion, we have used the method of geneome mining to explore new natural products from Micromonospora sp. A1304. In the follow-up work, we hope to clone the complete biosynthetic gene cluster, and determine the molecular structure of the target compounds by the means of combination of genetic information analysis and compounds characterization.