磷胁迫下草酸青霉有机酸代谢的分子机制

Penicillium oxalicum has high efficiency in phosphate solubilization by producing large amounts of organic acids under the stress of tricalcium phosphate and other insoluble inorganic phosphorus sources. Nevertheless, the types and amounts of the organic acids generated are significantly affected by the environmental factors, such as carbon and nitrogen sources, which cause a change of the microbial phosphate-solubilizing abilities to a large extent, and currently become a common and significant problem that seriously restricts the application of phosphate-solubilizing microorganisms. Therefore, research into the molecular mechanism of organic acids metabolism under phosphorus deficiency stress is urgently needed. .In our project, a unique pathway database of Penicillium oxalicum under tricalcium phosphate stress was constructed by means of KEGG, based on a subtractive cDNA library of Penicillium oxalicum under tricalcium phosphate stress constructed before. Then the pathway of the organic acids metabolism was found. Next, the metabolic networks of organic acids will be reconstructed in the light of the metabolic networks in database, in order to determine the key differentially expressed genes relating to accumulation of organic acids. All key genes above will be tracked in transcriptional level through Real-Time PCR, and simultaneously, the detection of related enzyme activity in biochemical level and qualitative and quantitative analysis of organic acids in level of metabolites will be conducted as supplements. Then the molecular mechanism of functional genes will be illuminated clearly, which are expressed under phosphorus deficiency stress during the metabolism of organic acids. The research aims to provide an important scientific basis for insight into the phosphate-solubilizing mechanism in molecular level and great significance on large-scale production and application of Penicillium oxalicum.

在磷酸钙等难溶性无机磷源胁迫条件下，草酸青霉以产生大量有机酸而实现高效溶磷能力，但由于环境中碳、氮等因素能显著影响产生的有机酸种类和数量，使得菌体溶磷能力发生较大变化，这也是严重制约溶磷微生物应用的共同瓶颈问题，探明磷胁迫下的有机酸代谢分子机制迫在眉睫。本项目基于已构建的磷酸钙胁迫下的草酸青霉cDNA消减文库，利用KEGG建立了该菌特有的磷胁迫代谢网络数据库，发现了有机酸代谢相关路径，拟进一步根据数据库中的物质代谢网络图重构有机酸代谢网络，从中确定与有机酸积累相关的关键差异表达基因，通过Real-time PCR对关键基因进行转录水平跟踪，辅之以生物化学水平上的相应酶活性检测和代谢产物水平上的有机酸定性定量分析，阐明磷胁迫下的功能基因表达响应在有机酸代谢过程中的分子作用机理，为深入了解草酸青霉的分子溶磷机制提供科学依据，对草酸青霉菌的规模化生产和应用具有重要意义。

构建了磷酸钙胁迫下草酸青霉正、反向cDNA消减文库；功能分类结果显示EST序列主要与物质和能量代谢、物质转运、细胞和机体防御、转录翻译表达调控、信号转导等相关；基于KEGG有机酸代谢网络数据库，确定磷胁迫响应的有机酸合成积累紧密相关的主要标志物有线粒体苹果酸脱氢酶、富马酸酶、磷酸烯醇丙酮酸羧激酶、乌头酸水合酶、己糖激酶。.利用荧光定量PCR技术在分子水平上研究了两个最关键基因线粒体苹果酸脱氢酶和富马酸酶基因对磷胁迫的相应机制，结果表明草酸青霉菌可先通过提高线粒体苹果酸脱氢酶的转录水平，直接增加草酰乙酸合成苹果酸的能力，富马酸酶基因的过量表达响应稍晚于苹果酸脱氢酶基因；对应1-7天的磷胁迫下两种关键酶活力的动态变化与各自的基因转录水平变化相一致，生化水平上验证了mMDH基因和富马酸酶基因的编码蛋白对草酸青霉菌响应磷胁迫具有重要作用；通过HPLC技术监测发酵液中有机酸含量动态变化情况发现，草酸青霉菌在正常生长状态下以分泌大量草酸为主要有机酸，但在低磷胁迫条件下却极大的减少分泌量；原本产生的少量乙酸，在磷胁迫下分泌完全受到抑制，而正常生长仅产生极少量的苹果酸却在低磷胁迫时表现出极大量的快速增加， 苹果酸的分泌量随时间变化趋势与关键基因的响应过程和对应的酶活性变化几乎吻合，在生理水平上清楚的说明草酸青霉菌在磷胁迫下，受体内苹果酸脱氢酶、富马酸酶等的基因表达调控，改变了原有的有机酸代谢路径，最终实现菌体对难溶磷的生物可利用性。.重构了利于苹果酸积累和分泌的代谢网络：草酸青霉由于某种原因关闭了乙醛酸代谢路径，减少的琥珀酸不利于三羧酸循环的推动。为了获得基本的生存能量，线粒体苹果酸脱氢酶和富马酸酶响应表达，催化草酰乙酸和富马酸合成苹果酸。因为三羧酸循环中间代谢产物苹果酸的双重作用（填补反应）是生成葡萄糖，过量的苹果酸在驱动TCA循环产能的同时，穿梭到线粒体膜外，在苹果酸脱氢酶的作用下合成草酰乙酸，为磷酸烯醇丙酮酸羧激酶提供催化底物进行糖异生，用于合成真菌细胞壁构成的多糖及其它物质转换，以抵抗逆境环境。同时，顺乌头酸酶协助柠檬酸向异柠檬酸转化，从而推动TCA循环。