地衣素合成酶关键模块 LchAD 蛋白的性质和功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-0996

摘要/Abstract

摘要：

【目的】 地衣素（lichenysin）作为一种脂肽类物质，对白酒风味形成有促进作用。为研究地衣素合成酶中硫酯酶模块LchAD蛋白的性质和功能，采用传统分离纯化方法从酿酒大曲样品中，筛选产地衣素的芽孢杆菌菌株。【方法】 采用质谱串联的方式检测发酵产物，通过形态学、生理生化实验和构建系统发育树，分析菌株的种属关系。以筛选出的菌株基因组DNA为模板克隆LchAD基因，在大肠杆菌中异源表达LchAD蛋白并用镍柱纯化。利用在线软件对LchAD蛋白进行生物信息学分析，探究LchAD 蛋白的性质和功能。【结果】 在大曲中筛选获得的菌株YC7为地衣芽孢杆菌（Bacillus licheniformis），其发酵产物中含有地衣素A（D或G）的同系物，表明菌株YC7为地衣素产生菌。LchAD蛋白的可溶性表达研究发现，在温度为20℃，IPTG终浓度为100 μg/mL条件下可诱导LchAD蛋白异源表达，并形成可溶性蛋白，其分子量约为27.62 kD。镍柱纯化结果显示，在咪唑浓度为250 mmol/L时，可获得较高纯度的LchAD蛋白。生物信息学分析结果表明，LchAD蛋白为稳定的亲水性蛋白，无信号肽，理论等电点（pI）为7.23，含有一个GrsT保守结构域。LchAD蛋白的二级结构包含43.5%的α-螺旋，13.82%的β-折叠，5.69%的β-转角，其三级结构与表面活性素合成酶的硫脂酶模块相似。【结论】 筛选得到的地衣芽孢杆菌YC7菌株可产地衣素，其LchAD基因编码的蛋白为27.62 kD的亲水性蛋白，异源表达获得可溶性LchAD蛋白，该蛋白可能与合成表面活性素的硫酯酶模块功能相似。

关键词: LchAD蛋白, 地衣素, 地衣芽孢杆菌, 质谱检测, 可溶性表达, 生物信息学

Abstract:

【Objective】 Lichenysin, as a kind of lipopeptide substance, can promote the formation of Baijiu flavor. To study the properties and functions of thioesterase module LchAD protein in lichenysin synthase, Bacillus strains were isolated from Daqu using traditional separation and purification methods.【Method】 The fermentation products were detected by primary and secondary mass spectrometry, and the species relationship of strains was analyzed by morphological, physiological and biochemical experiments and phylogenetic tree construction. LchAD gene was cloned using genomic DNA of a selected strain as template, and LchAD protein was heterologously expressed in Escherichia coli and purified by nickel column. Bioinformatics analysis was performed with online software to explore the properties and functions of LchAD protein.【Result】 The strain YC7 isolated from Daqu was Bacillus licheniformis, and its fermentation products contained the homologue of lichenysin A（D or G）, indicating that strain YC7 was a lichenysin producing bacterium. The study on the soluble expression of LchAD protein showed that LchAD protein was induced to form soluble protein at the temperature of 20℃ and the final concentration of IPTG was 100 μg/mL, with a molecular weight of 27.62 kD. The results of nickel column purification showed that the relatively pure LchAD protein was obtained when the imidazole concentration was 250 mmol/L. The results of bioinformatics analysis showed that LchAD protein was a stable hydrophilic protein without signal peptide, with a theoretical isoelectric point（pI）of 7.23 and a conserved GrsT domain. The secondary structure of LchAD protein consisted of 43.5% α-helix, 13.82% β-folding and 5.69% β-turn, and its tertiary structure had a high similarity with the thioesterase module of surfactant synthetase. 【Conclusion】 The isolated B. licheniformis strain YC7 can produce lichenysin, and the protein encoded by the LchAD gene of this strain is a hydrophilic protein of 27.62 kD. Soluble LchAD protein can be obtained by heterologous expression, which may be functionally similar to the thioesterase module of surfactant synthetase.

Key words: LchAD protein, lichenysin, Bacillus licheniformis, mass spectrometry detection, soluble expression, bioinformatics

杨伟杰, 杨周林, 朱浩东, 魏煜, 刘君, 刘训. 地衣素合成酶关键模块 LchAD 蛋白的性质和功能研究[J]. 生物技术通报, 2024, 40(3): 322-332.

YANG Wei-jie, YANG Zhou-lin, ZHU Hao-dong, WEI Yu, LIU Jun, LIU Xun. Study on the Properties and Functions of LchAD Protein, a Key Module of Lichenysin Synthase[J]. Biotechnology Bulletin, 2024, 40(3): 322-332.

图/表 9

图1 地衣素生物合成示意图 A：地衣素产生和分泌（ACC：乙酰辅酶A羧化酶复合物；FabD：丙二酰辅酶A -ACP转酰基酶；FabH：3-酮酰基载体蛋白合成酶；FabF：3-氧酰基- ACP合成酶II；FabG：β-酮酰基ACP还原酶；FabI：酰基载体蛋白还原酶；FabZ：3-羟基酰基ACP脱水酶；IlvC：乙酰羟基酸异构酶；RocG：谷氨酸脱氢酶；YwaA：支链氨基酸转氨酶；IlvA：苏氨酸脱水酶；IlvBH*：工程乙酰羟基酸合成酶调控亚基；IlvD：二羟基酸脱水酶；FACS：脂肪酸酰基辅酶A连接酶）；B：地衣素的合成；地衣素合成酶由4个模块组成，其中与模块3相连的是TE I，即TE结构域；模块4是TE II，即LchAD

Fig. 1 Schematic diagram of lichenysin biosynthesis A: Production and secretion of lichenysin（ACC: acetyl CoA carboxylase complex; FabD: malonyl-CoA-ACP transacylase; FabH: 3-keto-acyl carrier protein synthase III; FabF: 3-oxoacyl-ACP-synthase II；FabG: β-ketoacyl-ACP reductase; FabI: enoyl-ACP reductases; FabZ: 3-hydroxyacyl-ACP dehydratase; IlvC: acetohydroxy-acid isomeroreductase; RocG: glutamic dehydrogenase; YwaA: branched chain amino acid transaminase; IlvA: L-threonine dehydratase; IlvBH*: regulatory subunits of engineering acetyl hydroxy acid synthase; IlvD: dihydroxyacid dehydratase; FACS: fatty acyl CoA ligases）. B: Synthesis of lichenysin. Lichenysin synthase, consists of four modules, with TE I, the TE domain, connected to module 3. Module 4 is TE II, i.e. LchAD

表1 菌落形态

Table 1 Colony morphology

菌株 Strain	菌落形态 Colony morphology
YC1	奶白色，菌落表面干燥，边缘规则，扁平
YC2	半透明、微黄色，菌落表面湿润，边缘不规则，扁平
YC7	微红色、菌落表面不光滑但湿润，边缘不规则，扁平

图2 一级质谱图

Fig. 2 Primary mass spectrogram

图3 m/z为1 007.4离子峰的二级质谱图（A）及该离子峰发生简单断裂（B）和发生双氢转移断裂（C）产生的特征离子碎片理论值

Fig. 3 Secondary mass spectrometry of ion peak with m/z of 1 007.4（A）and theoretical values of characteristic ion fragments generated by simple cleavage（B）and double hydrogen transfer cleavage（C）of this ion peak

表2 生理生化结果

Table 2 Physiological and biochemical results

实验项目Experiment item	实验结果Experimental result
革兰氏染色	+
芽孢染色	+
接触酶实验	+
厌氧生长实验	+
葡萄糖产酸实验	+
葡萄糖产气实验	-
硝酸盐还原实验	+
淀粉分解实验	+
V-P实验	+
明胶分解实验	+

图4 菌株YC7的系统进化树

Fig. 4 Phylogenetic tree of strain YC7

图5 重组质粒pET-28a-LchAD双酶切（A）和 LchAD蛋白表达（B）及纯化（C）结果（A）M：DNA maker；1、2、3是重组质粒双酶切验证；（B）M：蛋白maker；1：载体pET-28a的上清液；2：重组质粒在20℃未加IPTG诱导的上清液；3：重组质粒在20℃诱导表达的全细胞液；4：重组质粒在20℃诱导表达的上清液；5：重组质粒在20℃诱导表达的沉淀；（C）M：蛋白maker；1：流穿液；2-7：浓度分别为10、25、50、100、250、500 mmol/L的咪唑洗脱

Fig. 5 Results of double digestion of recombinant plasmid pET-28a-LchAD（A）and LchAD protein expression（B）and purification（C）（A）M: DNA maker; 1, 2 and 3 are double restriction endonuclease digestion validation of recombinant plasmids.（B）M: Protein maker. 1: The supernatant of carrier pET-28a. 2: Recombinant plasmid supernatant without IPTG induction at 20℃. 3: Recombinant plasmid induced expression in whole cell fluid at 20℃. 4: The supernatant of the recombinant plasmid induced expression at 20℃.5: Precipitation of recombinant plasmid induced expression at 20℃.（C）M: Protein maker. 1: Flow through fluid. 2-7: Imidazole elution with a concentration of 10, 25, 50, 100, 250 and 500 mmol/L, respectively

图6 LchAD蛋白的保守结构域（A）、亲疏水性（B）和信号肽（C）预测

Fig. 6 Prediction of conserved domain（A）, hydrophilicity and hydrophobicity（B）and signal peptide（C）of LchAD protein

图7 LchAD蛋白的二级结构（A）和三级结构（B）预测结果

Fig. 7 Predicted results of secondary structure（A）and tertiary structure（B）of LchAD protein

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