贝莱斯芽胞杆菌CS1.13生物合成TTMP相关基因分析及其发酵调控

Analysis of TTMP Biosynthesis Relation Genes in Bacillus velezensis CS1.13 and Its Fermentation Regulation

为解析芽胞杆菌四甲基吡嗪(TTMP)生物合成途径,对筛选自高盐稀态酱醪的贝莱斯芽胞杆菌CS1.13全基因组进行注释,结合固态发酵研究其TTMP的生物合成途径及调控特征,结果显示:COG、GO及碳水化合物活性酶注释发现CS1.13参与氨基酸代谢及转运、碳水化合物代谢及转运,编码糖苷水解酶基因丰富,KEGG注释发现CS1.13具有完整的TTMP关键底物——乙偶姻的代谢通路。固态发酵过程研究发现,发酵72 h还原糖及氨基酸态氮含量分别升高53.63,8.28 g/kg,TTMP积累159.86 mg/kg,证明CS1.13具有良好的碳氮代谢能力及TTMP合成潜力。根据TTMP合成通路,选择苏氨酸、醋酸、丙酮酸及2,3-丁二醇等关键物质研究TTMP生物合成的调控,分别添加上述物质2,0.2,0.2 mg/g和0.8 mg/g时,TTMP积累量达到最大值,较对照样分别提高了32.82%,10.30%,222.46%,160.93%。随着添加物含量的增加,TTMP积累量下降。除氨基供体苏氨酸外,碳架供体醋酸、丙酮酸或2,3-丁二醇在较低浓度时更利于3种吡嗪(TTMP、2,5-二甲基吡嗪及2,3,5-三甲基吡嗪)的积累。过多的碳架供体反而有利于丁二酮系化合物的积累,说明氨基供体和碳架供体对TTMP的合成具有调控作用。TTMP生物合成相关基因分析结合发酵调控,为贝莱斯芽胞杆菌CS1.13高效积累TTMP探索了1条有效途径,也为其在酱香食品中快速增香的应用奠定基础。

In order to characterize the biosynthetic pathway of 2,3,5,6-Tetramethylpyrazine(TTMP)in Bacillus spp.,the whole-genome information of Bacillus velezensis CS1.13 screened from high-salt liquid-state moromi was annotated,and its TTMP biosynthetic pathway and regulatory characteristics were studied in combination with solid-state fermentation.The results show that CS1.13 is rich in genes involved in amino acid metabolism and transport,carbohydrate metabolism and transport and coding glycoside hydrolase by COG,GO and carbohydrate active enzyme annotations.And KEGG annotation reveals that CS1.13 has a complete metabolic pathway of Acetoin,a key substrate of TTMP.The solid-state fermentation process revealed that the reducing sugar and amino acid nitrogen contents increased by 53.63 g/kg and 8.28 g/kg,respectively,and TTMP accumulated 159.86 mg/kg at 72 h of fermentation,which proved that CS1.13 has good carbon and nitrogen metabolism ability and TTMP synthesis potential.According to the TTMP synthesis pathway,the key substances such as threonine,acetic acid,pyruvic acid and 2,3-butanediol were selected to study the regulation of TTMP biosynthesis.The maximum accumulation of TTMP was achieved when threonine 2 mg/g,acetic acid 0.2 mg/g,pyruvic acid 0.2 mg/g and 2,3-butanediol 0.8 mg/g were added respectively,which increased 32.82%,10.30%,222.46%and 160.93%compared with the control sample.The accumulation of TTMP decreased as the content of the above additive increased.In addition to the amino donor threonine,the accumulation of the three pyrazines(TTMP,2,5-dimethylpyrazine and 2,3,5-trimethylpyrazine)was favored by the lower concentrations of carbon skeleton donors acetate,pyruvic acid or 2,3-butanediol,and the accumulation of butanedione family compounds was favored by excessive carbon skeleton donors instead,suggesting that the synthesis of TTMP was regulated by the amino donor and carbon skeleton donor.The analysis of TTMP biosynthesis relation genes combined with fermentation regulation explored a feasible and effective way for Bacillus velezensis CS1.13 to accumulate TTMP efficiently,which will also lay the foundation for its application in rapid aroma enhancement in Jiang-flavor foods.