齿毛菌漆酶高效表达的分子机制研究

Laccases have great application potentials in food and many other industries; however, the applications of laccases are limited by low production yields. We have previously isolated a white rot fungus, namely Cerrena sp. strain HYB07, with high laccase yields. Copper ions and nonlimiting carbon and nitrogen sources are essential for its efficient laccase production. In order to further improve laccase productivity, the molecular mechanism underlying laccase transcription regulation by copper ions as well as carbon and nitrogen source levels in HYB07 will be elucidated. This project aims to identify potential transcription factors and cis-acting elements involved in laccase expression regulation with genomic and transcriptomic analysis, validate function of the candidate transcription factors with gene knockout or overexpression approaches, determine transcription factor binding sites, and reveal the molecular basis for differential regulation of laccase isozyme genes. The research findings will provide a theoretical foundation for genetic modification of laccase-producing strains and regulation of laccase expression to realize highly efficient laccase production and facilitate commercial utilization of laccases.

漆酶在食品及其他工业领域用途广泛，但是产量较低导致漆酶的应用受到限制。课题组前期获得了一株高产漆酶的白腐真菌——齿毛菌HYB07菌株，其漆酶合成不受碳氮营养阻遏，只需要铜离子和高碳氮源水平。为了进一步提高漆酶产量，本课题将解析铜离子和碳氮源水平对HYB07菌株漆酶转录调控的分子机制。通过基因组学和转录组学分析，筛选和漆酶表达调控相关的转录因子和顺式作用元件，利用基因敲除或过量表达手段验证转录因子功能，鉴定转录因子结合位点，揭示漆酶同工酶基因差异性调控的分子基础。课题研究成果可为漆酶生产菌株的改造和漆酶表达调控提供理论依据，从而最终实现漆酶的高效生产，促进漆酶的产业化应用。

本项目针对漆酶应用中的两个瓶颈（生产成本高和稳定性低）开展研究，具有较强的理论和应用意义。首先，通过测序得到了高产漆酶齿毛菌（Cerrena sp.）HYB07菌株的基因组序列和不同培养条件下的转录组序列。从转录组序列中获得了漆酶表达调控转录因子序列，RT-PCR验证了表达水平差异。建立了两种齿毛菌的遗传转化体系，利用过量表达、单核菌株解析等手段发现铜离子响应因子Cuf1对HYB07菌株的主要漆酶Lac7的表达起抑制作用。其次，构建了含HYB07 Lac7启动子的一色齿毛菌漆酶异源表达宿主。再次，探索了金属离子对HYB07漆酶的抑制效果和机理。制备不同的固定化漆酶、比较固定化漆酶的稳定性和应用效果。另外，得到了2株高产漆酶的一色齿毛菌（Cerrena unicolor）菌株，对其进行了转录组学分析。纯化得到其主要漆酶，表征酶学性质并与HYB07漆酶比较。将漆酶应用于兽药的生物降解和乙烯基单体的生物聚合。课题研究结果推动了对漆酶表达调控的理解，为提高漆酶产量和降低漆酶生产成本提供了理论基础和技术手段。新型漆酶资源的发现扩展了漆酶资源库，为漆酶的应用提供了更多优良候选漆酶。同时，拓宽了漆酶的应用，加深了对漆酶催化机理的认识，可促进漆酶的稳定工业化应用，降低应用成本。