| Mackerel is an important economic fish in China,but it is a safety concern because of its high biogenic amine content in fish due to its high susceptibility to spoilage.Studies on biogenic amines associated with the spoilage of mackerel have been limited to environmental factors affecting decarboxylase activity,such as p H,temperature,water activity and oxygen.However,the signalling pathways between these environmental factors and biogenic amine synthesis are poorly studied and the upstream regulators of the amino acid decarboxylase gene cluster are unclear.The c-di-GMP is an important bacterial second messenger that receives extracellular signals through the receptor domain of its metabolic enzymes and is involved in the regulation of various biological functions such as cell differentiation,bacterial membrane formation,virulence factors and bioluminescence.c-di-GMP needs to be further investigated to determine whether it is also involved in the regulation of biogenic amine synthesis.Therefore,in this study,the key amine-producing bacterium Klebsiella oxytoca was used to investigate the regulation mechanism of biogenic amine synthesis by c-di-GMP signaling molecules,to provide a theoretical basis and new ideas for the study of the regulatory network of biogenic amine synthesis in amine-producing bacteria.The main results of this study are as follows:(1)In this paper,a strain of Klebsiella oxytoca,a high biogenic amine-producing spoilage bacterium obtained from mackerel,was used as the target of this study.Through whole gene sequencing,structural domain alignment,overexpression strain construction,relevant phenotypic determination and prokaryotic transcriptomic analysis,the regulatory role of c-diGMP on biogenic amine production was determined.The overexpression of c-di-GMP synthase proteins Koxy＿2511 and Koxy＿5178 significantly increased intracellular c-di-GMP levels and enhanced cadaveric amine production in the strain.Koxy＿2511 and Koxy＿5178 reduced lysinebased protein synthesis,decreased lysine consumption other than cadaverine synthesis and promoted population sensing and ABC transporter family gene expression.Koxy＿2511 and Koxy＿5178 were also found to promote the expression of the inducible lysine decarboxylase gene cad A without affecting the transcript level of the lysine decarboxylase manipulator gene cad C,suggesting that c-di-GMP can co-regulate biogenic amine synthesis in multiple ways.(2)Modelling of the proteins Koxy＿2511 and Koxy＿5178 with Robetta.Using the modelled proteins as receptor targets,inhibitors were screened from a database of food components and the effect of the active substance on traits such as cadaverine production was determined,resulting in a biogenic amine inhibitor(-)-gallocatechin gallate with high scores for both proteins and low toxicity.400 μM and 800 μM of(-)-gallocatechin gallate could reduce intracellular c-di-GMP levels and cadaverine production without affecting the growth of the strain.Molecular docking analysis revealed that(-)-gallocatechin gallate bound to key active sites of two c-di-GMP metabolic proteins,thereby interfering with protein function.Analysis of molecular dynamics revealed that(-)-gallocatechin gallate was able to influence the structure and function of the proteins by forming hydrogen bonds.In this study,the correlation between c-di-GMP and cadaveric amine synthesis in Klebsiella oxytoca was initially determined,and two DGCs that could regulate cadaveric amine biosynthesis were identified,which provided genetic support for the subsequent in-depth study of the molecular mechanism of c-di-GMP signalling pathway regulating biogenic amine synthesis.At the same time,effective inhibitors of c-di-GMP and biogenic amines were obtained by computer-based screening,providing a new pathway for the control of biogenic amines. |
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