The Study Of 2,5-dimethylpyrazine Biosynthetic Pathway By Bacillus Subtilis And Construction Of A High-yield Strain

Alkylpyrazines are a class of nitrogen-containing heterocyclic compounds with alkyl groups,which are widely distributed in fermented foods as the important flavor contributors.Among alkylpyrazines,2,5-dimethylpyrazine?2,5-DMP?has the unique flavor contribution in fermented foods due to its strong baking odour.In addition,2,5-DMP can also be used as an important intermediate for the formation of medicine and fragrance.The production of2,5-DMP is mainly synthesized by chemical methods.Because biosynthetic pathway of2,5-DMP has not been elucidated,the research on the biological production of 2,5-DMP is lacking.In the preliminary work,a strain Bacillus subtilis XZ1124 was screened from the starters of a soy sauce aroma type Baijiu.The addition of L-threonine facilitates the synthesis ability of 2,5-DMP by Bacillus subtilis XZ1124,suggesting that 2,5-DMP can be produced by microorganisms fermentation.Based on this result,B.subtilis 168 was used as the study host for clarifying the biosynthetic pathway of 2,5-DMP.On this basis,it was speculated that the biosynthetic pathway of 2,5-DMP may be involved in the biosynthetic pathway of other alkylpyrazines containing a mono-methyl-semi-ring,which was verified with2,3,5-trimethylpyrazine?TMP?.Based on the elucidated biosynthetic pathway of 2,5-DMP,a metabolically engineered strain with the high yield of 2,5-DMP using L-threonine as the substrate was constructed.The efficient biotransformation of 2,5-DMP was realized for the first time.This study enriched the understanding of the biosynthetic pathway of alkylpyrazines and can improve the process control of important flavors for 2,5-DMP and other alkylpyrazines containing a mono-methyl-semi-ring in fermented foods.In addition,this study will provide a theoretical basis for the bioconversion or biocatalysis of 2,5-DMP,which can help promote the research on 2,5-DMP production by biological methods.The main results were as follows:?1?The study of 2,5-DMP biosynthetic pathway.It was confirmed that L-threonine can be used as the sole substrate and aminoacetone can be the intermediate in the biosynthetic pathway of 2,5-DMP via the experiments of substrate addition,stable isotope tracing and whole-cell catalysis.And the conversion from aminoacetone to 2,5-DMP is a pH-dependent non-enzymatic reaction.L-Threonine-3-dehydrogenase?TDH?derived from B.subtilis 168was determined to be the key enzyme in the biosynthetic pathway of 2,5-DMP by the experiments of TDH catalysis with L-threonine as the substrate and knockout and complementation of TDH-encoded gene tdh in B.subtilis 168.TDH catalyzes L-threonine to generate aminoacetone.The gene kbl encoding 2-amino-3-ketobutyrate CoA ligase?KBL?was knocked out and complemented,and the results showed that the kbl gene co-transcribed with the tdh gene plays an important role in the biosynthetic pathway of 2,5-DMP.The kbl-encoding enzyme KBL can significantly reduce the ability of 2,5-DMP biosynthesis using L-threonine as the substrate.?2?The preliminary study of TMP biosynthetic pathway.It was found that B.subtilis 168can simultaneously use L-threonine and D-glucose or alone use L-threonine to form TMP by the experiments of stable isotope tracing and whole-cell catalysis.In addition,the experiments of knockout and complementation of TDH-encoded gene tdh in B.subtilis 168 showed that TDH,the key enzyme in the 2,5-DMP biosynthetic pathway with L-threonine as the substrate,also plays a critical role in the biosynthetic pathway of TMP with L-threonine as a substrate.The biosynthetic pathway of 2,5-DMP with L-threonine as the substrate is involved in the biosynthesis of TMP,suggesting that the biosynthetic pathway of 2,5-DMP may be involved in the biosynthetic pathway of other alkylpyrazines containing a mono-methyl-semi-ring.?3?Construction of 2,5-DMP high-yield strain with L-threonine as the substrate.TDH derived from seven different microbial species has been exogenously expressed in B.subtilis168,respectively.It was found that the overexpression of TDH from Escherichia coli K-12 in B.subtilis 168 is more favorable to the formation of 2,5-DMP with L-threonine as the substrate.A NADH oxidase?NOX?was further exogenously expressed in TDH overexpression strain to promote cofactor regeneration.The results showed that on the basis of TDH overexpression in B.subtilis 168,the overexpression of NOX has positive effect on the production of 2,5-DMP.A genetic engineering strain B.subtilis 168/pMA0911-tdh?E.c?-nox with high yield of 2,5-DMP using L-threonine as the substrate was obtained.After 24 h of fermentation,the yield of 2,5-DMP was as high as 616.0 mg?L^-1 in LB with 5.8 g?L^-1 of L-threonine addition.Compared with the control strain?B.subtilis 168/pMA0911?,the yield was increased by 22.5 times.