Study Of The Ubiquinone Biosynthesis In Pseudomonas Aeruginosa M18

Coenzyme Q（CoQ）is a kind of lipid soluble quinones,which plays a central role in the electron transport chain of aerobic respiration.Coenzyme Q is constituted of a conserved aromatic ring（2,3-dimethoxy-5-methyl-1,4-benzoquinone）and a polyisoprenoid side chain.The length of the side chain is different in different species.Phenazine-1-carboxylic acid（PCA）,a secondary metabolite produced by Pseudomonas aeruginosa M18,is the main component of the green pesticide Shenqinmycin.In this study,the types and biosynthesis mechanism of coenzyme Q in P.aeruginosa M18 were studied by means of bioinformatics and genetics,and the relationship between coenzyme Q biosynthetic pathway and PCA biosynthetic pathway was also explored.Firstly,coenzyme Q in P.aeruginosa M18 was extracted and purified.According to the identification of ultra-performance liquid chromatography-mass spectrometry（UPLC-MS）,CoQ₉is the predominant coenzyme Q synthesized by strain M18 with 9 isoprenoid units,and CoQ₇is produced as the minor coenzyme Q in the same strain.Through gene mining,we screened 15 genes that maybe involved in CoQ biosynthesis in strain M18.By gene knockout,point mutation and heterologous complementation,nds（PAM18＿4662）was confirmed to encode nonaprenyl diphosphate synthase（Nds）,and Met¹²³and Ile¹³⁵of Nds are the key residues for determining the number of isoprenoid units of coenzyme Q in M18.coq7^PA（PAM18＿0636）encodes C6-hydroxylase,which is involved in CoQ₉ biosynthesis;ubi C（PAM18＿5477）encodes chorismate lyase,which is responsible for the synthesis of 4-hydroxybenzoic acid,the precursor of CoQ aromatic ring;100C-terminal of UbiJ^PA（PAM18＿5179）,UbiB^PA（PAM18＿5180）and UbiK^PA（PAM18＿5410）form a complex to participate in the C5-hydroxylation of CoQ.Deletion of ubi J^PA and ubi B^PA,or overexpression of ubi C,ubi A and nds in CoQ biosynthetic pathway resulted in reduced PCA production in M18.Used the amino acid sequences of chorismate lyase,decarboxylase,C6-hydroxylase and UbiJ-like proteins in Escherichia coli as templates,the coenzyme Q biosynthetic gene clusters of 34 species of Gram-negative bacteria were divided into nine categories by amino acid sequence alignment,domain and phylogenetic tree analysis.This study revealed the genetic diversity of coenzyme Q biosynthetic genes in Gram-negative bacteria.In conclusion,this study further improved the biosynthetic mechanism of CoQ in bacteria and provided a theoretical basis for the study of Pseudomonas in biological control.