Screening Of Strain TYF-FGY-BD17 And Study On Biosynthesis Mechanism Of Tetramethylpyrazine

Tetramethylpyrazine（TTMP）,also known as ligustrazine,is a bioactive nitrogen-containing heterocyclic compound,which has been widely used in medicine,food,chemical industry and other industries.The intermediates in the biosynthesis of tetramethylpyrazine are complex and difficult to detect,so there are still many difficulties in analyzing their metabolic pathways.In this study,a strain with high yield of tetramethylpyrazine was obtained by targeted screening,and the biosynthesis process of tetramethylpyrazine and its precursor acetoin was explored from its genome and transcriptomic information,providing important theoretical data for comprehensively analyzing the biosynthesis pathway of tetramethylpyrazine.In this project,48 strains that could produce acetoin were screened out from vinegar fermented grains in vinegar fermentation workshop,and then rescreened by HIGH performance liquid chromatography,it was found that a total of 20 strains could produce tetramethylpyrazine,and then monitored the fermentation process of these 20 strains.Bacillus subtilis and Bacillus Amyloliquefaciens strains TYF-FGY-BD17（Bacillus subtilis）and TYF-FGY-BM12（Bacillus amyloliquefaciens）with high yield of tetramethylpyrazine were selected to optimize fermentation conditions,and the optimal fermentation conditions of the two strains were determined.Strain TYF-FGY-BD17:Peptone 30 g/L,sucrose 50 g/L,yeast powder 15 g/L,diammonium hydrogen phosphate 30 g/L,p H value was maintained at the original p H value（7.2）,temperature 37℃,rotation speed 200 r/min,the yield of tetramethylpyrazine 2.80 g/L;Strain TYF-FGY-BM12:Peptone 30 g/L,sucrose 70 g/L,yeast powder 10 g/L,diammonium hydrogen phosphate 30 g/L,p H value was maintained at the original p H（7.2）,temperature45℃,rotation speed 200 r/min,the yield of tetramethylpyrazine 2.68 g/L;Based on the economy of fermentation raw materials,strain TYF-FGY-BD17 was selected for follow-up study,and the whole genome of the strain was sequed.Through the analysis of CAZy,COG,KEGG and other databases,it was found that the strain had the ability to degrade various carbon sources and exogenous proteins such as pentose,hexose,disaccharide and polysaccharide.Combined with the database and literature,The synthetic and metabolic pathways of TYF-FGY-BD17 acetoin were preliminarily constructed.In order to further clarify the biosynthesis process of acetoin and key genes of tetramethylpyrazine biosynthesis,the transcriptomic analysis of strain TYF-FGY-BD17 was carried out,and the characteristics of the acetoin biosynthesis pathway of strain TYF-FGY-BD17 were determined.The acetoin biosynthesis pathway of strain TYF-FGY-BD17 was not catalyzed byα-acetolactate decarboxylase.Instead,it is naturally decarboxylated to form diacetyl,which is then reduced to acetoin by diacetyl reductase.In addition,the operon acu ABC and aco ABCLR may be involved in the biodegradation of acetoin in the biosynthesis process.According to the formation process of tetramethylpyrazine and the changes in transcription levels of related genes,15 genes related to tetramethylpyrazine synthesis were found,including8 genes related to protein or peptide degradation,4 genes related to NH₃ production,and 3genes related to ammonia transfer.These genes had high expression levels and showed a synergistic trend in the rapid synthesis stage of tetramethylpyrazine.In order to verify the role of key genes in the tetramethylpyrazine biosynthesis pathway of strain TYF-FGY-BD17,q PCR was performed on the differentially expressed genes screened by transcriptomic data,and the results were consistent with the transcriptomic results.Through online prediction and analysis and prediction of transcription unit,won 11 and tetramethyl pyrazine biosynthesis related transcription unit,and has carried on the annotation to the function of each gene transcription unit and analysis,made clear its metabolic pathways,found that the strain b benzoin synthesis and biosynthesis of valine,leucine,associated with,the b benzoin biosynthetic pathway has significant characteristics.The biosynthesis pathway of TYF-FGY-BD17 tetramethylpyrazine was speculated as follows:Organic nitrogen sources decompose into-NH₄⁺under the action of biological enzymes,acetoin reacts with-NH₄⁺to generate 3-amino-2-butanone,or under the action of transaminase,the amino group of amino acids is transferred to acetoin to generate 3-amino-2-butanone,then 3-amino-2-butanone through condensation,oxidative dehydrogenation and other steps to eventually generate tetramethylpyrazine.