Study on the Properties and Functions of LchAD Protein, a Key Module of Lichenysin Synthase

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

Abstract

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

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.

Figures/Tables 9

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

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

Fig. 2 Primary mass spectrogram

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

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	+

Fig. 4 Phylogenetic tree of strain YC7

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

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

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

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