基于Fosmid文库筛选山羊瘤胃微生物源蛋白酶基因及其表达验证  

Screening and expression verification of rumen-derived protease genes of goat based on Fosmid library

作　　者：杨凯尧 刘功炜 李林芳 王智伟[1] 崔雯元 杨雨鑫[1] YANG Kaiyao;LIU Gongwei;LI Linfang;WANG Zhiwei;CUI Wenyuan;YANG Yuxin(College of Animal Science and Technology,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2024年第4期28-38,共11页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家重点研发计划项目(2022YFD1300201,2021YFD1600704);陕西省重点研发计划项目(2021ZDLNY05-02);国家绒毛用羊产业技术体系项目(CARS-39-12)。

摘　　要：【目的】利用Fosmid文库功能筛选法,获得山羊瘤胃微生物源蛋白酶基因并进行原核表达。【方法】利用功能底物筛选法从山羊瘤胃微生物源Fosmid文库中筛选具有蛋白酶活性的克隆子,对其进行Illumina二代测序,对预测到的基因进行功能注释,根据基因丰度结果确定后续研究的功能基因。利用大肠杆菌BL21(DE3)对功能基因进行诱导表达,对表达产物进行酶活性测定。通过CRISPR/Cas9基因编辑技术,将密码子优化后的功能基因分别敲入枯草芽孢杆菌C6基因组ctc位点并进行表达验证。【结果】从Fosmid文库1700个克隆子中筛选到1个具有蛋白酶活性的阳性克隆Pro4-C5。通过二代测序技术,鉴定出3个潜在的蛋白酶基因gene0833(内肽酶,EC编号:EC3.4.21.53)、gene0196(金属内肽酶,EC编号:EC3.4.24)和gene0585(羧肽酶,EC编号:EC3.4.17.14)以及1个L-天冬酰胺酶基因(gene0683,EC编号:EC3.5.1.1)。以pET-28a(+)为表达载体,通过大肠杆菌BL21(DE3)原核诱导表达发现,gene0585和gene0196未表达;gene0833表达的蛋白分子质量为87 ku,蛋白酶活性为10.45 U/mL;gene0683表达的蛋白分子质量为37 ku,L-天冬酰胺酶活性为88.52 U/mL,同时发现该蛋白也具有蛋白酶活性,酶活性为5.25 U/mL。将密码子优化后的gene0683和gene0833定点敲入枯草芽孢杆菌C6基因组中表达发现,gene0833未表达,gene0683表达的蛋白酶活性为109.72 U/mL,相比原始菌株(100.97 U/mL)显著提高了8.67%(P<0.01);L-天冬酰胺酶活性为31.63 U/mL,相比原始菌株(22.79 U/mL)显著提高了30.06%(P<0.01)。【结论】从山羊瘤胃微生物源Fosmid文库中筛选和鉴定出了2个具有蛋白酶活性的功能基因(gene0833和gene0683),均来源于微小杆菌属(Exiguobacterium),其中gene0683在大肠杆菌和枯草芽孢杆菌中的表达产物具有较高的蛋白酶和L-天冬酰胺酶酶活性。【Objective】This study identified goat rumen microbial protease genes and achieved their effective expressions via functional screening of the Fosmid library.【Method】Clones with protease activity were selected from the goat rumen microbial Fosmid library using the functional screening technique.The Illumina second generation was used to sequence the clones.The predicted genes were functionally annotated,and the functional genes were chosen based on gene abundance data.Escherichia coli BL21(DE3)was employed to induce the expression of functional genes and confirm their functionality.The codon-optimized functional genes were inserted into the ctc location of Bacillus subtilis C6 genome using CRISPR/Cas9 gene editing technology for expression and functional testing.【Result】Among 1700 clones in the Fosmid library,one Pro4-C5 clone exhibiting protease activity was found to be positive.Second-generation sequen-cing technology discovered three possible protease genes of gene0833(endopeptidase,EC3.4.21.53),gene0196(metalloendopeptidase,EC3.4.24)and gene0585(carboxypeptidase,EC3.4.17.14))as well as one L-asparaginase gene(gene0683,EC3.4.17.14).Genes 0585 and 0196 showed no expression when pET-28a(+)was used as the prokaryotic expression vector in Escherichia coli BL21(DE3).Gene0833 had a molecular weight of 87 ku and a protease activity of 10.45 U/mL.Gene0683 had a molecular weight of 37 ku,a L-asparaginase activity of 88.52 U/mL and a protease activity of 5.25 U/mL.After codon optimiza-tion,gene0683 and gene0833 from Bacillus subtilis C6 were cloned,and gene0683 activity was 109.72 U/mL,which was 8.67%(P<0.01)significantly higher than that of starting strain(100.97 U/mL).Compared to the original strain(22.79 U/mL),the L-asparaginase activity(31.63 U/mL)was considera-bly increased by 30.06%(P<0.01).【Conclusion】Two functional genes(gene0833 and gene0683)from Exiguobacterium were isolated and verified from goat rumen-derived Fosmid library and the expression products of gene0683 in Escherichia coli and Bacill

关 键 词：FOSMID文库 蛋白酶 L-天冬酰胺酶 原核表达 基因筛选 菌株生产 

分 类 号：S827.1[农业科学—畜牧学] Q786[农业科学—畜牧兽医]