固体培养条件下水活度对米曲霉hacA^i基因表达的影响

The Influence of Water Activity in the Expression of hacA^i Under Solid-state Cultivation

米曲霉在食品工业及相关行业中应用广泛,具有高效分泌蛋白质的能力。在利用重组丝状真菌生产异源蛋白时,液体培养方式的异源蛋白产量相当低,固体培养方式却能高产异源蛋白。这可能是由于蛋白分泌途径的调控机制在不同培养条件下有所不同,然而目前蛋白分泌途径的机制尚不十分清楚。内质网中蛋白质的未折叠或错误折叠是蛋白分泌途径中的主要瓶颈,因而对蛋白分泌途径的研究热点集中于未折叠蛋白反应(UPR)。近年来,黑曲霉,李氏木霉和构巢曲霉等丝状真菌液体培养时的UPR机制已经得到研究,但固体培养时的UPR机制尚未见到文献报道。通过荧光定量PCR技术对比分析米曲霉在固体培养和液体培养时UPR关键转录因子hacAmRNA剪切成熟体hacA^i的表达情况,并进一步分析固体培养时水活度对hacA表达水平的影响。结果表明:hacA^i在固体培养条件下的表达水平明显高于液体培养,并且随着水活度的降低,hacA^i表达量逐渐上升。

Aspergillus oryzae has received increasing attention as a source for homologous and heterologous protein production due to its ability to secrete large amounts of protein.The amount of enzymes secreted by Aspergillus oryzae in the solid-state culture greatly exceeds that secreted in the submerged culture.However,the mechanisms involved in protein production remain unknown.Studies have focused on the unfolded protein response in the endoplasmic reticulum(ER) because this step in protein maturation represents the most probable bottleneck in the protein secretory pathway.As of the present time,the UPR pathway has been identified in A.nidulans,T.reesei and A.niger,but UPR regulation under solid-state culture is still unknown.The expressional level of the hacA' between solid-state and submerged cultures was compared through real-time PCR,the differences in the UPR between the two culture conditions were discussed.The results show that the expressional level of the hacA^i under solid-state cultivation of A.oryzae is higher than under submerged cultivation and increased with low water activity.