Functional Gene Screening Of The Bacteria-degrading Feed Flavor Agent Raw Thiothiazole And Construction Of Engineering Bacteria

Thiothiazole is an organic compound with the chemical name 4-methyl-5-（β-hydroxyethyl）-thiazole.As one of the raw materials for the production of feed additives,in recent years,with the strengthening of national environmental awareness,the requirements for the emission of volatile organic compounds in feed flavor additive production plants have become increasingly strict.In order to solve the environmental pollution caused by thiothiazole production process and use process,currently using microbial treatment method for degradation is one of the methods to solve the environmental pollution.The use of microbial degradation can eliminate the adverse effects on the environment caused by the substance that overflows due to volatility during production and use.This study is based on the second generation of high-throughput sequencing and metagenomic sequencing to screen and mine bacteria and their functional genes that degrade thithiazole.Then construct the engineering bacteria according to the functional genes.In this study,high-throughput sequencing detected a total of 27 phylum,37 classes,72orders,128 families,and 207 genera in environmentally-friendly equipment that degrades fishy incense.A total of 888 OTUs were generated from the 10 samples collected in the two phases.Among them,the number of OTUs contained in the first stage sample（YX1）in the water tank of the environmental protection equipment is 614,the number of OTUs contained in the second stage sample（YX2）is 765,the number of jointly owned OTUs is 491,and the number of OTUs unique to YX1 and YX2 are respectively For 123 and 274.After two stages,the diversity of bacterial communities in the water tank of the release equipment was more abundant.The second stage increased 151 OTUs over the first stage,indicating that the second stage had more unique bacterial species than the first stage.The Chao1 and ACE indexes of the YX1 group samples were extremely significant（p<0.01）smaller than those of the YX2 group samples;the Simpson index of the YX1 group samples was significantly smaller（p<0.05）than the YX2 group samples;the Shannon index of the YX1 group samples was extremely significant（p<0.001）Less than YX2 group samples.It is shown that the bacterial abundance and bacterial community diversity of the YX2 sample are higher than that of the YX1 sample.Over time,the bacterial abundance and bacterial community structure diversity in the water tank of environmental protection equipment increase.By analyzing the level distribution of each bacterial community phylum and genera,it was found that there were ten dominant species at the phylum level,including Proteobacteria（66.40%）,Bacteroides（9.42%）,Actinomyces（3.15%）,Unidentified bacteria（17.61%）accounted for 96%of the total number of bacteria in each group of samples,and were the dominant bacteria in the samples.There are thirty dominant species at the genus level,among which Brevimonas（26.36%）,Azospirillum（4.00%）,Chrysanthemum（3.46%）,and unidentified bacteria（17.22%）The dominant bacteria accounted for 51.04%of the total sample bacteria.Through metagenomic sequencing,the KEGG function database was used to find the metabolic pathway map of thithiazole.The primary metabolism is divided into six categories,including:cellular processes,environmental information processing,genetic information processing,human diseases,metabolism,and biological systems.Under the metabolism classification,the thiamine metabolic pathway map00 780 was screened.In the metabolic pathway,there are 14 enzymes involved in metabolism in the sample.Among them,thiothiazole can be transformed by hydroxyethylthiazole kinase through the metabolic pathway and eventually participate in the synthesis of vitamin B₁.The hydroxyethylthiazole kinase was identified as thiM,and the thiM gene sequence was searched in the NCBI database.The ID number was 938 519.The source was Bacillus subtilis subsp.Subtilis str.168 and the size was 819 bp.The plasmid T-Vector pMD19 was used as a vector and transferred into E.coli DH5αCompetent Cells to construct engineering bacteria.Finally,it was determined that the engineered bacteria successfully degraded thiothiazole 77.48%and Bacillus subtilis 62.83%.