Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (3): 322-332.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0996
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YANG Wei-jie1(), YANG Zhou-lin1, ZHU Hao-dong1, WEI Yu1, LIU Jun1,2(), LIU Xun1()
Received:
2023-10-23
Online:
2024-03-26
Published:
2024-04-08
Contact:
LIU Jun, LIU Xun
E-mail:18783502843@163.com;xunliu0123@suse.edu.cn;liujunbio@suse.edu.cn
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.
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
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 |
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. 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
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 | + |
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. 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
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