| Maytansinoids are extraordinary antitumor macrolactam agents in phase II clinical trials in America. Ansamitocins are classified as maytansinoids, and biosynthesized by Actinosynnema proetiosum, therein ansamitocin P-3 (AP-3) has an excellent activity.Due to the important physiological activities, the interest in ansamitocins has increased around the world in recent years. Many studies have focused on strain improvement, medium optimization, and pathway regulation to enhance AP-3 production. However, its commercial application is still limited resulted from the low productivity and high price of AP-3. Until now there is no information related to environmental factor on AP-3 production based on the fermentation process and the molecular mechanism for regulation of AP-3 biosynthesis, albeit those knowledge is significant for increasing antibiotics productivity.In this study, we first adopted the reported medium to produce AP-3 in the fermentation of Actinosynnem pretiosum spp. auranticum 31565, however, only an amount of 1-2 mgL-1 was observed. Thus, the Plackett-Burman and fold-over Plackett-Burman designs were applied to select fermentation medium, and the maximum AP-3 production was 12.9 mgL-1. Furthermore, one-time-one-factor method was used to simplify the medium compositions, and found that ammonium and isobutanol exerted negative and positive effects on AP-3 production respectively. The center composition design as well as response surface method were then employed to optimize the medium concentrations, the final fermentation medium were (gL-1):glucose 5, yeast extract 10, glycerol 40, corn filtrate 20, CaCO35, K2HPO40.5, FeSO4·7H2O 0.002, and isobutanol 3, the maximum AP-3 production was 68.2 mgL-1 with an inoculua of 1.64×109 CFUL-1 cultivated for 96 h in 60 mL/250 mL flask at 220 rpm, the productivity (17.1 mgL-1d-1) was higher among the published references. Fermentation based on flask initial KLa was carried out in 3-L bioreactor successfully, and the AP-3 production achieved 52 mgL-1 at 96 h.It was observed that removal of ammonium from the fermentation medium was positive to AP-3 production. However, ammonium is an intermediate of nitrogen-contained compounds which always accumulated during antibiotics fermentation, information on ammonium effect from the viewpoint of biosynthetic gene transcription and proteomics has not yet reported, albeit it is beneficial to medium selection and pathway reconstruction. Hence, inhibition of AP-3 production was studied with (37 mM) and without ammonium based on fermentation process. Fermentation profiles showed that mycelium growth rate was repressed by ammonium, but the absorption rate of extracellular isobutyrate was facilitated. Assay of enzymatic activities showed that the activity of valine dehydrogenase, the first enzyme in valine metabolism pathway that always inhibited by ammonium in other systems, was induced during the whole process. Transcriptional levels of genes in AP-3 biosynthetic pathway were down-regulated by ammonium, which was in agreement with the production reduction. About 1600 protein spots by two-dimensional electrophoresis with pH3-5.6 and PAGE concentration of 13.5% were obtained; therein spots above 1.5-fold differential expression determined by mass determination were D-3-phosphoglycerate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, and fatty acid synthase etc. The results showed that ammonium influenced the biosynthesis of fatty acid and serine, and regulated the transcription of AP-3 biosynthetic genes.Previous work implied that isobutanol might act as a precursor of isobutyryl-CoA (side chain of AP-3), and the enhancement of AP-3 production by isobutanol addition was also found during the process of medium optimization. We first optimize the addition conditions, and found that isobutanol added at 0 h with 36 mM was optimal, resulting in 3.4-fold improvement of AP-3 and 4-fold decrease of AP-2 production at 96 h. Accumulation profiles of intracellular CoAs showed that acetyl-CoA and malonyl-CoA concentration increased in isobutanol supplemented culture, suggested isobutanol also influence pools of extender units of AP-3. In isobutanol supplemented culture activities of both isobutanol dehydrogenase and valine dehydrogenase were enhanced for about 2 folds in the growth phase and production phase, respectively; further combined addition of valine at 36 h based on isobutanol resulted in 35% improvement of AP-3 yield (82.3 mgL"1). Transcriptional response of genes in AP-3 biosynthetic and isobutyryl-CoA catabolic pathways showed a similar way as the increase of AP-3 production, therein the side-chain incorporating gene asm19 and regulatory gene asm40 in the former pathway were up-regulated by isobutanol significantly, and genes in the latter pathway were also induced, suggested a shunt of isobutyryl-CoA metabolism by isobutanol. Furthermore, protein expression of catalase and LysR family regulator related to the state of cell redox was up-regulated by isobutanol resulted from proteomics study. In addition, to testify the responses of asm19 and asm40 to isobutanol, their engineering strains were cultivated, and it was observed that overexpression of asm19 resulted in more tolerance of isobutanol than wild type, while deletion of asm40 induced AP-3 production for 20% by isobutanol, suggested an indirect role of asm40 in response to isobutanol. The information indicated that isobutanol disturbed the pools of precursor CoAs for AP-3 biosynthesis, and influenced the expression of redox-related genes and proteins in cells.Collectively, this work revealed that ammonium could activate the biosynthetic pathways of fatty acid and serine, as well as down-regulated of the transcription of AP-3 biosynthetic genes; meanwhile, the positive role of isobutanol was caused by changes of precursor supply, and influence of expression of genes and proteins related to the redox state of cells. The information is helpful to development of fermentation medium and reconstitution of AP-3 biosynthetic pathway, and also taken as a reference to other antibiotics fermentation. |
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