基于蛋白质折叠调控的链霉菌谷氨酰胺转胺酶高效分泌表达研究

The efficient production of transglutaminase (TGase) is always a hot spot of research both at home and abroad. It was found that high folding rate of the TGase may be unfavorable for its secretion vis Sec pathway while its pro-peptide could inhibit the protein folding. In this study, the methods from molecular biology, bioinformatics, and etc. will be adopted to systematically analyze the intracellular interactions between the pro-peptide and mature enzyme; and, the amino acid positions in the pro-peptide and mature enzyme, which is critical for the folding rate of the TGase, will be determined; then, the pro-peptide mutants with higher inhibitory efficiency to the folding of TGase and the TGase with slow folding rate will be identified; the high efficient secretion of active TGase by the typical G- bacterium (E. coli) and G+ bacterium (Streptomyces lividans) can be achieved through co-expressing those TGase mutants with the pro-peptide mutants or singly expressing the TGase mutants with slow folding rate; finally, the kinetic relationship between the folding rate and secretion rate of the TGase will also be found. The results obtained here will reveal the intimate relationship between the protein folding and secretion rate of many proteins.

谷氨酰胺转胺酶(TGase)的高效生产一直以来是国内外研究的热点。大量研究表明，较高的折叠速率可能是链霉菌TGase难以经由细菌Sec途径高效分泌的重要原因，而其酶原区对酶折叠有抑制作用。本课题拟综合运用分子生物学、生物信息学等技术，基于前期构建的共表达系统，解析酶原区与TGase成熟酶在胞内相互作用的区域及动态变化，并确定两者序列中影响TGase折叠速率的关键氨基酸位点；在此基础上，筛选对TGase折叠有高抑制活性的酶原区突变体和低折叠速率的TGase突变体，通过共表达上述两类突变体或仅表达TGase低速折叠突变体，阐明TGase折叠速率与分泌速率的动力学关系，最终实现活性TGase在典型革兰氏阴性菌(Escherichia coli)和革兰氏阳性菌(Streptomyces lividans)中高效分泌。研究结论将有助于进一步揭示蛋白质折叠与其分泌性能的内在联系。

谷氨酰胺转氨酶（EC 2.3.2.13, TGase）是一种酰基转移酶，能催化蛋白质分子间的交联反应，在食品、制药和纺织等领域具有广泛的应用前景。因此，TGase的高效生产一直以来是国内外研究的热点。大量研究表明，较高的折叠速率可能是链霉菌TGase难以经由细菌Sec途径高效分泌的重要原因，而其酶原区对酶折叠有抑制作用。为提高链霉菌TGase表达效率，分析了TGase前导肽中影响其折叠的关键氨基酸位点，研究TGase在大肠杆菌、茂原链霉菌（Streptomyces mobaraensis）及解脂耶氏酵母(Yarrowia lipolytica)等宿主表达中的调控策略。在大肠杆菌中，通过部分缺失及定点突变确定Streptomyces hygroscopicus TGase前导肽中影响其在大肠杆菌中正确折叠的氨基酸位点为Tyr12; 基于酶原区替换、融合蛋白质表达标签、共表达蛋白酶及分子伴侣、分泌信号肽序列优化等策略，使S. mobaraensis TGase在大肠杆菌中胞外产量达到9.51 U/mL。在S. mobaraensis 中，基于ARTP诱变、流式细胞分选、微孔板培养及检测技术开发高产TGase高通量选育平台，获得了高产TGase 的突变株smY2019；通过增加基因拷贝数构建得到smY2019-3C，其胞外TGase酶活达到40.7 U/mL，是目前报道的最高水平。在Yarrowia lipolytica中，通过酶原区替换及插入Kex2 酶识别位点等策略实现了活性S. mobaraensis TGase 的高效表达，胞外酶活达到5.62 U/mL。研究结果，初步解析了TGase折叠调控的影响因素，同时为TGase工业化生产提供了高效菌株。研究过程中，开发的基因操作方法和表达调控策略也半为其它工业酶的微生物合成提供参考。