草酸青霉纤维素酶基因的新关键调控基因POX01167的功能鉴定

Biorefineries from sugarcane bagasse can improve economic performance of cane sugar industry, but it hasn’t been industrized because of low cellulase production from microorganisms. Penicillium oxalicum HP7-1 can efficiently degrade sugarcane baggase cellulose. A mutant ΔPOX01167 derived from P. oxalicum HP7-1 produces negligible cellulase in medium containing crystalline cellulose Avicel as the carbon source, while normally produces cellulase to the wild-type level in medium containing wheat bran and Avicel. In this proposed project, the regulon of the key regulatory gene POX01167 will be identified and the candidate regulatory genes acting downstream of POX01167 will be screened by differential transcriptomic analysis of ΔPOX01167 and background strain ΔPoxKu70 cultivated in media containing Avicel, and wheat bran plus Avicel as the carbon source. The screened candidate regulatory genes will be respectively knocked-out in ΔPoxKu70 and over-expressed in ΔPOX01167. The cellulase activity of the constructed mutants will be measured. The regulation of the found genes by POX01167 will be verified by electrophoretic mobility shift assay and reporter gene assay in vivo. Key regulatory genes acting downstream of POX01167 will be thus identified. Furthermore, key amino acid residues in the DNA-binding domain of POX01167, and bound DNA sequences and key bases by POX01167 will be characterized. .The expected outcome of this suggested project is of great theoretical significane in understanding regulation mechanism of cellulase gene expression in fungi, and thus provide a solid basis for rational genetic modification of fungal strains to reduce the cost of biorefinery from sugarcane bagasse.

甘蔗渣生物炼制可提高甘蔗糖业的经济效益，但微生物的纤维素酶产量低使其不能实现产业化。草酸青霉HP7-1能高效降解甘蔗渣纤维素，其突变体ΔPOX01167在含微晶纤维素Avicel培养基中几乎不产生纤维素酶，但在含麦麸加Avicel培养基中其正常产生纤维素酶。本项目拟通过比较分析ΔPOX01167和出发菌株ΔPoxKu70分别在含Avicel、和麦麸加Avicel培养基中的差异转录组，鉴定POX01167的调控元，筛选POX01167下游候选调控基因，通过在ΔPoxKu70中敲除和在ΔPOX01167中过量表达候选基因、突变体的酶活测定、凝胶迁移实验和体内报告基因检测，鉴定出下游关键调控基因。鉴定POX01167的DNA结合功能域的关键氨基酸残基和POX01167结合的DNA序列及关键碱基。对认识真菌纤维素酶基因的表达调控机理具有重要意义，为理性改造真菌、降低甘蔗渣生物炼制成本提供理论依据。

甘蔗渣生物炼制可提高甘蔗糖业的综合经济效益，但真菌的纤维素酶产量低、成本高，导致其不能实现产业化。草酸青霉（Penicillium oxalicum）菌株HP7-1能高效水解甘蔗渣纤维素，其突变体ΔPOX01167在Avicel培养基中几乎不产纤维素酶，但在含Avicel加麦麸培养基中正常产纤维素酶。然而，基因POX01167的分子调控机理不清楚。.本项目主要是鉴定POX01167的调控元及其下游新的关键调控基因。鉴定POX01167的DNA结合功能域及关键氨基酸残基，鉴定POX01167结合的DNA序列及序列上的关键碱基。.本项目鉴定了草酸青霉POX01167的调控元，包括4211个基因，鉴定了POX01167下游调控草酸青霉纤维素酶和木聚糖酶产量的8个关键调控基因以及1个相关基因，其中PoxCxrC、PoxCbh、PoxMK1和PoxPLA2 4个基因是第一次报道的。.发现POX01167的DNA结合功能域为POX01167（17-58），关键氨基酸残基为18(R), 19(R), 27(Q), 30(K), 32(K) 和38(P)，以及其在纤维二糖水解酶基因cbh1和调控基因PoxClrB启动子区结合的DNA序列分别为5’-ATCAGATCCTCAAAGA-3’和5’-GCTGAGTCCTT-3’，其中5’-TCCT-3’是关键碱基。.另外，发现在草酸青霉中同时敲除基因PoxAtf1和PoxCxrC导致其纤维素酶产量提高2.4-29.1倍，木聚糖酶产量提高78.9%-130.8%；所产粗酶液水解预处理甘蔗渣96小时，水解产物葡萄糖和木糖产量增加10.6%-13.5%。.上述研究成果对认识真菌纤维素酶基因和木聚糖酶基因的表达调控机理具有重要的科学意义，为理性分子设计、定向改造真菌菌株以提高酶产量提供理论依据和可借鉴的案例，具有应用潜力。