Regulation of Manganese Peroxidase Gene Expression

金9723725白腐担子菌黄孢原毛平革菌的木质素降解系统可降解植物细胞壁聚合物、木质素和多种芳香污染物。这个非特异性氧化系统的主要胞外成分是两种含血红素的过氧化物酶，即锰过氧化物酶(MNP)和木质素过氧化物酶(LIP)。LDS在次生代谢期表达，次生代谢期由营养氮的消耗触发。锰离子、热休克和氧化应激也在基因转录水平上调节着MNP的表达。本项目的主要目的是在分子水平上阐明这些特定的MNP基因转录机制。新的MNP启动子-基因报告结构将用于研究氮、锰和热激对单个MNP基因的调节。利用推测的金属反应元件的定点突变、启动子缺失分析和合成启动子元件，将识别参与MNP调控的顺式作用启动子序列。可能的锰反应转录因子将被鉴定和纯化，编码锰反应和热休克转录因子的基因将被克隆。最后，将进一步研究MNP合成的转录后要求。木质素是第二丰富的天然聚合物，占木本植物细胞壁的15%-30%，并形成环绕木材中纤维素的基质。因此，木质素的降解是全球碳循环中的重要一步。此外，木质素是纤维素工业利用的主要障碍。白腐菌是唯一能将木质素完全降解为二氧化碳和水的生物。这些真菌中研究得最好的是黄孢原毛平革菌，它能有效地降解木质素和多种环境污染物。黄孢原毛霉和其他木质素降解真菌分泌锰过氧化物酶(MnP)。这种酶能氧化锰离子，而锰离子又能分解木质素。MNP的产生在基因水平上受到营养氮的消耗、锰的有效性以及温度和化学胁迫的调节。这项研究的目的是在分子水平上了解这些因素中的每一个控制MNP产生的机制。这是第一个在分子水平上研究锰对基因表达调控的例子。

Gold 9723725 The lignin degradative system (LDS) of the white-rot basidiomycetous fungus Phanerochaete chrysosporium degrades the plant cell wall polymer, lignin, and a wide range of aromatic pollutants. The major extracellular components of this nonspecific oxidative system are two heme-containing peroxidases, manganese peroxidase (MnP) and lignin peroxidase (LiP). The LDS is expressed during secondary metabolic growth, the onset of which is triggered by the depletion of nutrient nitrogen. MnP expression also is regulated at the level of gene transcription by Mn ion, by heat shock, and by oxidative stress. The primary goal of this project is to elucidate these specific mechanisms of mnp gene transcription at the molecular level. New mnp promoter-gene reporter constructs will be used to examine the regulation of individual mnp genes by nitrogen, Mn, and heat shock. The cis-acting promoter sequences involved in mnp regulation using site-directed mutagenesis of putative metal response elements, promoter deletion analysis, and synthetic promoter elements will be identified. The putative Mn-responsive transcription factor will be identified and purified, and genes encoding the Mn-responsive and heat shock transcription factors will be cloned. Finally, the post-transcriptional requirements for MnP synthesis will be examined further. Lignin is the second most abundant natural polymer, constituting 15-30% of woody plant cell walls, and forming a matrix that surrounds the cellulose in wood. Thus, the degradation of lignin is a significant step in the global carbon cycle. Furthermore, lignin presents a major obstacle to the industrial utilization of cellulose. White-rot fungi are the only organisms which can completely degrade lignin to carbon dioxide and water. The best studied of these fungi, Phanerochaete chrysosporium, efficiently degrades lignin and a wide range of environmental pollutants. P. chrysosporium and other lignin-degrading fungi secrete the enzyme manganese peroxidase (Mn P). This enzyme oxidizes manganese ion (Mn) which, in turn, depolymerizes lignin. The production of MnP is regulated at the level of the gene by the depletion of nutrient nitrogen, by the availability of Mn, and by temperature and chemical stresses. The goal of this research is to understand, at the molecular level, the mechanisms by which each of these factors control MnP production. This is the first example of Mn regulation of gene expression to be studied at the molecular level.