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Ansamitocin P-3 (AP-3) is an antitumor agent produced by ^ÅíáåçëóååÉã~=éêÉíáçëìã with a market demand for large and cheap supply, but its productivity is yet low. This work investigated the effects of ammonium in medium on the productivity, enzyme activities, and gene transcription for AP-3 biosynthesis. As observed, AP-3 production was depressed by medium ammonium, although the dry cell weight and the consumption of total sugar and isobutanol were not influenced obviously. From the onset of AP-3 accumulation, isobutyrate accumulation showed a different behavior in medium with or without ammonium. Isobutanol dehydrogenase activity was enhanced during production phase in medium with ammonium, but valine dehydrogenase activity was not substantially changed. qRT-PCR analysis revealed that transcriptional levels of structure genes=~ëãNQI ~ëãOQI ~ëãQP, and ~ëãNV were down-regulated by medium ammonium. The transcription of regulatory gene ~ëãO,=~ëãOVI=and=~ëãPN were slightly up-regulated while that of ~ëãPV was down-regulated by ammonium. The results indicated that inhibition of AP-3 production by ammonium might be related to the AP-3 ester side chain supply and the repression of gene transcription responsible for 3-amino-5-hydroxybenzoic acid and methoxymalonyl-ACP biosynthesis. The information is useful for future AP-3 productivity enhancement. © KSBB hÉóïçêÇëW=^åë~ãáíçÅáå=mJPI=ãÉÇáìã=~ããçåáìã=ÉÑÑÉÅíI=ÖÉåÉ=íê~åëÅêáéíáçåI=ÉåòóãÉ=~Åíáîáíó= = = = =

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Our previous work showed that the biosynthesis of ansamitocin P-3 (AP-3), an anti-tumor agent, by Actinosynnema pretiosum was depressed by ammonium but enhanced by isobutanol in the medium. Here we show proteomics analyses on A. pretiosum in different fermentation conditions with and without ammonium or isobutanol using two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization, and linear ion trap quadrupole mass spectrometry. Pairwise comparison of repetitive 2-DE maps was performed to find differentially expressed spots, and eight proteins were identified as functionally annotated ones. Among these proteins, D-3-phosphoglycerate dehydrogenase (PGDH) and glyceraldehyde 3-phosphate dehydrogenase showed statistically significant up-regulation in ammonium vs. basic or isobutanol medium, while fatty acid synthetase, histidine-tRNA ligase, transposase, molecular chaperone GroEL, SAM-dependent methyltransferase, and Crp/Fnr family transcriptional regulator were overexpressed in ammonium vs. basic medium. Based on the 2-DE data, exogenous L-serine which could inhibit the PGDH activity was added to the cultures with isobutanol, and a lower AP-3 production was confirmed under 2.5 mM serine addition (24 or 48 h).