INBIOTEC, Instituto de Biotecnología de León, Leon, Spain.
Appl Environ Microbiol. 2011 Aug 15;77(16):5688-96. doi: 10.1128/AEM.00059-11. Epub 2011 Jul 1.
Filamentous fungi produce an impressive variety of secondary metabolites; many of them have important biological activities. The biosynthesis of these secondary metabolites is frequently induced by plant-derived external elicitors and appears to also be regulated by internal inducers, which may work in a way similar to that of bacterial autoinducers. The biosynthesis of penicillin in Penicillium chrysogenum is an excellent model for studying the molecular mechanisms of control of gene expression due to a good knowledge of the biochemistry and molecular genetics of β-lactam antibiotics and to the availability of its genome sequence and proteome. In this work, we first developed a plate bioassay that allows direct testing of inducers of penicillin biosynthesis using single colonies of P. chrysogenum. Using this bioassay, we have found an inducer substance in the conditioned culture broths of P. chrysogenum and Acremonium chrysogenum. No inducing effect was exerted by γ-butyrolactones, jasmonic acid, or the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine. The conditioned broth induced penicillin biosynthesis and transcription of the pcbAB, pcbC, and penDE genes when added at inoculation time, but its effect was smaller if added at 12 h and it had no effect when added at 24 h, as shown by Northern analysis and lacZ reporter studies. The inducer molecule was purified and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as 1,3-diaminopropane. Addition of pure 1,3-diaminopropane stimulated the production of penicillin by about 100% compared to results for the control cultures. Genes for the biosynthesis of 1,3-diaminopropane have been identified in the P. chrysogenum genome.
丝状真菌产生了令人印象深刻的各种次生代谢产物；其中许多具有重要的生物活性。这些次生代谢产物的生物合成通常是由植物来源的外部诱导子诱导的，而且似乎也受到内部诱导子的调节，这些诱导子的作用方式可能类似于细菌的自动诱导子。青霉素在产黄青霉中的生物合成是研究基因表达控制的分子机制的一个极好模型，因为我们对β-内酰胺抗生素的生物化学和分子遗传学有很好的了解，并且可以获得其基因组序列和蛋白质组。在这项工作中，我们首先开发了一种平板生物测定法，该方法允许使用产黄青霉的单个菌落直接测试青霉素生物合成的诱导子。使用该生物测定法，我们在产黄青霉和棘孢曲霉的条件培养肉汤中发现了一种诱导物质。γ-丁内酯、茉莉酸或青霉素前体 δ-(L-α-氨基己酰基)-L-半胱氨酸-D-缬氨酸没有诱导作用。当在接种时添加条件肉汤时，它会诱导青霉素生物合成和 pcbAB、pcbC 和 penDE 基因的转录，但如果在 12 小时添加，其效果较小，如果在 24 小时添加，则没有效果，如 Northern 分析和 lacZ 报告基因研究所示。通过质谱（MS）和核磁共振（NMR）分析，该诱导分子被鉴定为 1,3-二氨基丙烷。与对照培养物相比，添加纯 1,3-二氨基丙烷可使青霉素的产量提高约 100%。1,3-二氨基丙烷生物合成的基因已在产黄青霉基因组中鉴定出来。
