Study Of Culture Conditions For Ansamitocin Biosynthesis In Actinosynnema Mirum

Ansamitocins are a group of macrolide antibiotics, which include five components:AP-1, AP-2, AP-3, AP-3’and AP-4. Among these components, AP-3is an active and potent anti-tumor maytansinoid, which is usually produced by Actinosynnema pretiosum. Actinosynnema mirum, as a potential antibiotic producer, can produce nocardicin, penicillin, streptomycin and novobiocin. A. mirum is gaining more attention because of its high homology in phylogenetic tree with A. pretiosum. Based on the reported information on ansamitocin biosynthesis genns and the whole genome information of A. mirum, the BLAST results showed that A. mirum contains also the ansamitocin biosynthesis genes with only a slightly difference with that of A. pretiosum. In addition, LC-MS assay suggested that there was AP-3component in the fermtentation broth of A. mirum. This study focuses on the biosynthetic process of AP-3in A. mirum and the influence and mechanism of cultural conditions alteration, which will be the theoretical and experimental support for optimization of AP-3production by A. mirum.Different carbon sources (glucose, fructose, galactose, xylose and sucrose) were found to have large effects on the production of AP-3. Glucose is the best carbon source for the growth of A. mirum, and fructose is favorable for AP-3production. The intracellular metabolism was futher investigated for A. mirum cells grown on either glucose or fructose to explore the relationship between the different carbon sources metabolism and AP-3biosynthesis. We found that:(1) Experimental resultes showed that more succinate and acetate were excreted by glucose-growing A. mirum cells, and cells used the organic acid as carbon source after glucose was used up. It means organic acid degradation has negative influence on AP-3biosynthesis.(2) When gluose was used as the carbon source, the pentose phosphate pathway (PPP) and the tricarboxylic acid cycle (TCA) were more active, while the glycolytic pathway was mainly used in cells grown on fructose. So we deduced that flux through the glycolytic pathway was beneficial to AP-3biosythesis.(3) Transcriptional assay indicated that the regulatory gene asm29and the structural genes asm43, asm24, asm14were up-regulated in fructose-growing A. mirum cells. In addition, we found that cells cultivated on fructose might be in a higher reducing state in comparison to cells grown on glucose.The effects of addition of branched chain amino acids (BCAA) in medium were also studied. The yied and composition of Ansamitocins by A. mirum were significantly affected by different BCAAs. Experimental results showed the AP-3yield has been doubled with the addition of0.5g/L valine at48h of fermentation, while the supplementation of0.5g/L leucine or isoleucine exhibited significant inhibition on AP-3yield (by58.8%and83.3%respectively). Instead, the contents of AP-2and AP-4components were increased when leucine or isoleucine was added into the medium.