Comparative Genomics And Preliminary Study Of Metabolism Of Thermophilic Actinomycete

The Thermoactinomycetaceae are a dominant microorganism in the high-temperature environment represented by Moutai high-temperature Daqu,and studies in recent years have shown that this type of microorganism has great potential for development and application.However,due to the limitations of isolated methods,the exploration of Thermoactinomycetaceae resources is still insufficient.Therefore,it is necessary to comprehensively and deeply analyze the metabolic functions of Thermoactinomycetaceae and accelerate the development of their strain resources.In this paper,the Thermoactinomycetaceae in Moutai high-temperature Daqu were taken as the research object,in-depth genomic information resolution and metabolic functional potential was initially predicted through the acquisition of culturable strains.Meanwhile,combined with the two strains of Laceyella sacchari FBKL4.010 and Thermoflavimicrobium daqui FBKL4.011~Tisolated in the early stage of our research team,the comparative genomics and metabolomics studies between strains were carried out,and a preliminary attempt was made to initially interpret the potential functional characteristics of Thermoactinomycetaceae in the brewing environment of sauce-flavor liquor from the view of genes and metabolism.The major research contents of this paper are as follows:(1)A cultureable strain FBKL4.014 was screened from the Moutai high-temperature Daqu.Through the identification technology like morphology,physiological/biochemical characteristics and 16S r RNA phylogeny,it was determined that it probably belonged to Laceyella sacchari under the family Thermoactinomy-cetaceae and Laceyella.The genome sequencing of this strain was completed by combining the second and third generation high-throughput sequencing technology,and the total length of the genome sequence was determined to be 3.35Mb,the GC content was 48.67%,and a total of 3328 protein-coding genes were predicted,which were similar to the information of the known strain.The analysis of genomic information was also completed.The results preliminarily revealed that the strain had carbohydrates and a relatively complete amino acid metabolism pathway,and there was also a key enzyme-coding gene for tetramethylpyrazine synthesis in its genome,which suggested that this strain may be related to the synthesis of nitrogenous substances represented by pyrazine in soy sauce-flavor liquor.(2)The whole genome sequence of L.sacchari FBKL4.014 was compared with the whole genome sequences of L.sacchari FBKL4.010 and T.daqui FBKL4.011~Tisolated from Moutai high-temperature Daqu in our experimental group.The potential functional characteristics of the three Thermoactinomycetaceae were preliminarily analyzed from the view of genome.The results showed that the three strains had 240,967 and 169 unique gene families,and the strains L.sacchari FBKL4.010 and L.sacchari FBKL4.014 were closely related and had good genome similarity.Both of them were distantly related to the strain T.daqui FBKL4.011~T.GO and KEGG enrichment analysis of their unique genes showed that the genomes of all strains contained protein-coding genes formed to amino acid metabolism,nitrogenous compounds and sulfur-containing compounds,but there were more genes related to amino acid metabolism in the T.daqui FBKL4.011~T’s genome.(3)Furthermore,metabolomic analysis techniques were used to preliminarily evaluate and analyze the metabolic characteristics and differences of three strains in their simulated living environment.On the one hand,the metabolic characteristics of volatile compounds produced in the simulated habitat of L.sacchari FBKL4.010,T.daqui FBKL4.011~Tand L.sacchari FBKL4.014 were preliminarily evaluated and compared by GC-MS technology,the result shown that three strains were characterized to 25,19 and 21 typical volatile metabolites,respectively,which showed that there were some differences in the metabolism of volatile compounds in the three strains.Among them,strain T.daqui FBKL4.011~Thas its own characteristics in the production of volatile metabolites,and can metabolize and produce 5 unique volatile metabolites.Strain L.sacchari FBKL4.010 and L.sacchari FBKL4.014 were similar in the metabolism of volatile compounds,especially synthesizing a variety of pyrazine compounds.On the other hand,LC-MS was used to compare and analyze the metabolic characteristics of non-volatile substances of each strain,and a total of63 typical non-volatile metabolic differential products were determined.The analysis results showed that there were indeed some differences in the metabolic function of non-volatile products in the Thermoactinomycetaceae from Moutai high-temperature Daqu,but the overall difference between strain L.sacchari FBKL4.010 and L.sacchari FBKL4.014 was small,while that of strain T.daqui FBKL4.011~T.This is basically consistent with the analysis results of comparative genomics.The differential metabolites among strains mainly involved metabolic pathways including D-alanine,glycerolipid,peptidoglycan biosynthesis,folate biosynthesis,and glycerophospholipid,among which the metabolism of amino acids was more prominent.It was initially indicated that there were differences in amino acid metabolism among strains.