Genomic Insights Into A Robust Gamma-Aminobutyric Acid-Producer Lactobacillus Brevis CD0817

Gamma-aminobutyric acid is the major inhibitory neurotransmitter in the mammalian central nervous system,has been extensively utilized in food and pharmaceutical industries as a bioactive factor,due to its important physiological functions,such as antidepressant,hypotensive and diuretic effects.Lactic acid bacteria are generally regarded as safe and closed related to human and animal life,which occupy an important status in food and pharmaceutical fields.The biosynthesis of gamma-aminobutyric acid by using lactic acid bacteria as bacterial cell factories exhibits the advantages of safety and efficiency,has become a focus of research in recent years.A robust gamma-aminobutyric acid-producer Lactobacillus brevis CD0817 was screened from the gut of a healthy adult that can produce the highest known amount of GABA(252 g/L)among lactic acid bacteria,and has been deposited in the China Center for Type Culture Collection(Wuhan,China)with the accession number of CCTCC NO: M 2018462.This manuscript was focused on the analysis of the complete genome sequence information of a robust gamma-aminobutyric acid-producer Lactobacillus brevis CD0817,including the complete genome sequencing of Lactobacillus brevis CD0817;the comparative genomics research on Lactobacillus brevis CD0817 and other Lactobacillus brevis strains;the analysis of GABA synthesizer(glutamate decarboxylase system)in Lactobacillus brevis CD0817 genome;and the development of a novel genome walking strategy.The main results are as follows.(1)The complete genome sequence of Lactobacillus brevis CD0817 was determined through next-generation sequencing technology.The Lactobacillus brevis CD0817 genome is comprised of one circular chromosome and four plasmids with total size of 3,096,874 bp and average GC content of 50.35%.The Lactobacillus brevis CD0817 complete genome sequence data has been deposited in GenBank under the accession numbers of CP032931.1(Chromosome),CP032932.1(Plasmid pCD0817-1),CP032933.1(Plasmid pCD0817-2),CP032934.1(Plasmid pCD0817-3),and CP032935.1(Plasmid pCD0817-4).A total of 2,990 protein-coding genes,51 tRNA genes,16 rRNA genes(four 16S-23S-5S operons and one 16S-23S-5S-5S operon)and 3 sRNAs;116 LTRs,68 DNA transposons,23 long interspered repeated sequences and 7 short interspersed repeated sequences;106 tandem repeats,80 minisatellite DNAs and 1 microsatellite DNAs;8 genomic islands,2 prophages,6 credible CRISPR loci and 1 bacteriocin synthesis gene cluster were predicted in Lactobacillus brevis CD0817 genome.1,290,1,556,1,407,2,563,1,030,2,459 and 79 of 2,990 protein-coding genes in Lactobacillus brevis CD0817 genome were classified into COG,GO,KEGG,NR,Swiss-Prot,TrEMBL and CAZy functional categories,respectively.(2)A comparative genomics research on Lactobacillus brevis CD0817 and other 15 completely sequenced Lactobacillus brevis genomes was performed through the analyses of core/pan gene,gene family,synteny and phyletic evolution.Lactobacillus brevis CD0817 is a specific strain and unique from other Lactobacillus brevis strains in genome structure,component and function.Lactobacillus brevis CD0817 might share a common ancestor with other Lactobacillus brevis strains,yet diverged in early stage of evolution and then formed a distinct branch from other Lactobacillus brevis strains.The gentic relationship between Lactobacillus brevis CD0817 and other Lactobacillus brevis strains was gradually becoming distant under the pressure of natural selection,and therefore Lactobacillus brevis CD0817 became a highly specific strain which exhibits a number of unique genome characteristics.(3)The gene distribution and sequence feature of glutamate decarboxylase system in Lactobacillus brevis CD0817 genome were analyzed and compared with other Lactobacillus strains in detail.In Lactobacillus brevis CD0817 genome,only one glutamate decarboxylase gene(gadA)was found,and formed a gadCA operon with its adjacent glutamate/gamma-aminobutyric acid antiporter(encoded by gadC).However,in other Lactobacillus brevis genomes,except gadCA operon,a separate glutamate decarboxylase gene(gadB)was also found located in its downstream of circa 1.7 Mb distance.Moreover,gadA in Lactobacillus brevis CD0817 exhibits obvious difference from those in other Lactobacillus brevis strains,as the DNA and protein identities are only 79% and 91%,respectively.Only one glutamate decarboxylase gene closed to gadA or gadB was found in other Lactobacillus genomes.The GadA protein from Lactobacillus brevis CD0817 encodes 481 amino acids and contains 7,497 atoms in total,its molecular formula,molecular weight,isoelectric point,estimated half-life,instability index,aliphatic index and grand average of hydropathicity are respectively C2409H3708N628O730S22,53.85 kDa,4.94,30 h,25.72,85.53 and-0.242,therefore,GadA was presumed as a stable hydrophilic protein.The tertiary structure prediction result shows that GadA protein from Lactobacillus brevis CD0817 is an asymmetric trimer formed by three polypeptide chains through alpha helix,beta sheet,beta turn and random coil.(4)A novel genome walking strategy termed stepwise partially overlapping primer-based PCR was developed for identification of neighboring unknown DNA sequences adjacent to known genomic regions.Compared to the conventional partially overlapping primer-based PCR,stepwise partially overlapping primer-based PCR has the merits of simplicity and efficacy due to requiring fewer primers and being suitable for making mix master.The stepwise partially overlapping primer-based PCR is an alternative of the existing genome walking methods and can be applied to gap closing in chromosome sequencing project for obtaining a complete genome sequence.