疏棉状嗜热丝孢菌脂肪酶LipA在毕赤酵母中的重组表达及产量优化  

Recombinant Expression and Yield Optimization of Lipase LipA from Thermomyces lanuginosus in Pichia pastoris

作　　者：郑毅恒 侯颖[1] 赵仕达 成莉凤[3] 李炳坤 李丁 ZHENG Yiheng;HOU Ying;ZHAO Shida;CHENG Lifeng;LI Bingkun;LI Ding(College of Food and Bioengineering,Henan University of Science and Technology,Luoyang 471023,China;Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Institute of Bast Fiber Crops,Chinese Academy of Agricultural Science,Changsha 410205,China;College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]河南科技大学食品与生物工程学院,洛阳471023 [2]江苏省农业科学院动物免疫工程研究所,南京210014 [3]中国农业科学院麻类研究所,长沙410205 [4]江西农业大学生物科学与工程学院,南昌330045

出　　处：《动物营养学报》2025年第2期1365-1375,共11页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：国家自然科学基金项目(32002316);湖南省自然科学基金项目(2023JJ50315)。

摘　　要：本研究将来源于疏棉状嗜热丝孢菌的脂肪酶LipA在毕赤酵母中进行高效表达,并通过多拷贝构建、共表达分子伴侣、高密度发酵等策略对其产量进行优化。首先,在毕赤酵母中对疏棉状嗜热丝孢菌脂肪酶LipA进行异源表达,并对表达产物进行纯化和酶学性质分析;接着,通过构建多拷贝酵母菌株提高目的基因的转录水平,通过共表达分子伴侣改善蛋白的折叠与运输,通过高密度发酵进一步挖掘酵母菌株的生产潜能。结果显示:LipA的最适pH为9,最适温度为50℃。LipA在温度和pH方面都具有良好的稳定性,非常适合作为饲料添加剂使用。含有2个拷贝菌株的2-α-LipA具有最高的LipA产量,酶活力可达2453.63 U/mL,与单拷贝菌株1-α-LipA的1753.69 U/mL相比提高了39.9%(P<0.05)。在此基础上,共表达了8种不同的分子伴侣,发现其中HAC1显著提高了LipA的产量(P<0.05),使酶活力进一步提高至3233.2 U/mL,增幅达47.6%。通过对优化后的菌株进行高密度发酵,其最高酶活力可达28932 U/mL。综上所述,本研究成功利用毕赤酵母对疏棉状嗜热丝孢菌脂肪酶LipA进行了异源表达,通过综合进行基因剂量优化、共表达分子伴侣和高密度发酵,LipA的最终产量相较于初始产量增加了16.49倍。The objective of this study was to efficiently express lipase LipA from Thermomyces lanuginosus in Pichia pastoris,and to optimize its yield by a combination of strategies including multi-copy integration,co-expression with molecular chaperones,and high-density fermentation.Initially,LipA was heterologously expressed in Pichia pastoris,followed by purification and analysis of the enzymatic properties.Subsequently,the transcriptional level of target gene was improved by constructing multi-copy yeast strains,the protein folding and transport was improved through co-expression of chaperones,and the production potential of yeast strains was further explored through high-density fermentation.The findings revealed that LipA displayed optimal activity at pH 9 and temperature of 50℃ LipA exhibited remarkable stability at various pH and temperatures,making it highly suitable as a feed additive.The 2-copy strain 2-α-LipA demonstrated the highest yield of LipA,reaching an enzyme activity of 2453.63 U/mL,a 39.9%increase compared with the single-copy1-α-LipA(1753.69 U/mL)(P<0.05).Additionally,the co-expression of eight different molecular chaperones was examined,among which HAC1 significantly enhanced LipA yield(P<0.05),resulting in an enzyme activity of 3233.2 U/mL,a 47.6% improvement.Finally,maximal enzyme activity of 28932 U/mL was achieved through high-density fermentation following strain optimization.In conclusion,the lipase LipA from Thermomyces lanuginosus is heterogenetically expressed by Pichia pastoris successfully in this study;by optimizing gene dosage,co-expressing molecular chaperones,and utilizing high-density fermentation,the final yield of LipA has increased 16.49-fold compared with the initial yield.

关 键 词：疏棉状嗜热丝孢菌 脂肪酶 基因剂量 分子伴侣 

分 类 号：Q814[生物学—生物工程]