Background and overview[1][2]
Geranylpyrophosphate (GPP), also known as geranyl diphosphate (GDP), has a C10 skeleton and is one of the direct precursors of terpenes. It can be used in monoterpene synthase (TPS) Monoterpenes are generated under the action, and can also continue to catalyze the synthesis of carotenoids into farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPPS).
Geranyl ammonium pyrophosphate includes geranyl ammonium pyrophosphate, which is the biosynthetic farnesyl pyrophosphate, which is the HMG-CoA reductase of cholesterol, terpenes and terpenoids. Intermediates in the pathway; isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are condensed by geranyl pyrophosphate synthase (dimethylallyltransferase) to produce Geranyl pyrophosphate (GPP) and pyrophosphate.
The carbon backbones of DMAPP and IPP have been colored to indicate their position within GPP. The decomposition of carotenoids in the human body can form vitamin A. The content of carotenoids in plants has an important impact on stress resistance and growth and development. Geranyl ammonium pyrophosphate CAS number 763-10-0, chemical formula C10H20O7P2. Molecular weight 314.20900. Density 1.129g/cm3, boiling point 484.2ºC at 760 mmHg, flash point 20ºC, refractive index 1.51.
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Geranyl ammonium pyrophosphate is an intermediate in the HMG-CoA reductase pathway of cholesterol, terpenes and terpenoids, and can be used for the synthesis of cyclic lipopeptide antibiotics. Cyclic lipopeptides are a type of amphiphilic compound produced by microbial fermentation. One end of the peptide ring is composed of multiple amino acids to form a hydrophilic group, and the other end is composed of an aliphatic hydrocarbon chain to form a lipophilic group. They have unique chemical structures and physiological functions. Cyclic lipopeptide compounds are mainly produced by the metabolism of Gram-positive Bacillus and have biological activities such as antibacterial, anti-tumor, anti-inflammatory, anti-mycoplasma, and cholesterol-lowering.
Because cyclic lipopeptide compounds have broad-spectrum antibacterial activity and their antibacterial mechanism is different from any class of antibiotics currently on the market, they are clinically highly efficient, low-toxic, and less likely to develop drug resistance, making them valuable in the medical field. Good application prospects. However, since there are multiple cycloaliphatic peptides and their homochemical derivatives in the fermentation broth, and the content of each cycloaliphatic peptide is low, it is very difficult to obtain high-purity products in large quantities.
At present, the development of daptomycin, a cyclic lipopeptide compound derived only from actinomycetes, has been relatively successful. It was first launched in the United States in September 2003 for vancomycin-resistant Enterococcus faecalis and methicillin-resistant Enterococcus faecalis. Treatment of Gram-positive bacteria such as Staphylococcus aureus. Some studies have provided a method to increase the production of cyclic lipopeptide antibiotics from Bacillus amyloliquefaciens Q-426. Through the Fomc solid-phase synthesis method, a linear precursor peptide Pre-ComX P10 with 10 amino acid residues was artificially synthesized. After interacting with the ComQ protein, the signal molecule ComX P10 of the ComP/ComA two-component signal transduction system was obtained. .
The signaling molecule ComX P10 was co-cultured with Bacillus amyloliquefaciens Q-426 to artificially enhance the signal intensity of the ComP/ComA signal transduction pathway of the bacterium, thereby promoting the production of cyclic lipopeptide antibiotics. Including the following steps:
(1) Use Fmoc solid-phase method to synthesize Pre-ComX P10. The amino acid sequence of Pre-ComX P10 is YKWDGGFSIV; the specific process is: 2-chlorotrityl chloride resin substituted with Fomc protected tyrosine As the starting material, Fomc solid-phase synthesis method was used to connect 9 Fomc-protected amino acids in sequence, and a mixture of frozen trifluoroacetic acid, 1,2-ethanedithiol, and m-cresol was used as the lysis solution. Pre-ComX P10 is cut from 2-CTC resin; the amino acids are Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly- OH, Fmo vulcanizing agent c-Gly-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH and Fmoc-Val-OH; the trifluoroacetic acid, 1,2-ethanedi The volume ratio of thiol and m-cresol is 95:4:1.
(2) Preparation of signal molecule ComX P10; the specific process is to react Pre-ComX P10 with geranyl ammonium pyrophosphate and ComQ in a buffer system to generate ComX P10, more specifically, the reaction between Pre-ComX P10 and geranyl ammonium pyrophosphate is Ammonium pyrophosphate and ComQ react in a buffer system to generate ComX P10; the buffer system is N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid/sodium hydroxide (pH 7. 5, 5 μmol/L magnesium chloride, 10 μmol/L zinc chloride); the reaction temperature is 35-37°C; the reaction time is 3-7 hours. The reaction equation is as follows:
The preparation method of ComQ is as follows: use chemical transformation method to transfer the recombinant plasmid into recombinant Escherichia coli BL21 (E.coli BL21 (RIL)) competent cells, and culture it on LB solid medium containing ampicillin overnight. Culture, when the OD600 is between 0.6-1.2, add isopropylthiogalactopyranoside (IPTG) for induction, and collect the cells after culturing for 8 hours at 30°C. Suspend the collected cells in buffer and conduct ultrasonic disruption. Centrifuge at 13,000 rpm for 50 minutes to obtain the supernatant. The supernatant is subjected to metal ion chelation chromatography, ion exchange chromatography, and gel filtration chromatography in sequence to obtain the ComQ protein. (3) Fermentation by Bacillus amyloliquefaciens Q-426, adding signal molecule ComX P10 during the fermentation process.
Main reference materials
[1] Ma Yingrui, Gong Yifu, Zhou Jing, Liu Hao, Zhu Shuaiqi, & Wang Heyu. (2017). Bioinformatics and induced expression analysis of Dunaliella viridis geranyl pyrophosphate synthase gene (GPS). Journal of Nuclear Agriculture , 31(2), 248-254.
[2] CN201810320212.6 A method for improving the production of Bacillus amyloliquefaciens Q-426 cyclic lipopeptide antibiotics
