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Recombinant bacteria synthesizing 2,5-dimethylpyrazine (2,5-DMP) by catalysis

A technology of dimethylpyrazine and recombinant bacteria, applied in the direction of microorganism-based methods, bacteria, oxidoreductase, etc., can solve the problems of flavor compound development obstacles, low yield, lack of biochemical pathways, etc.

Active Publication Date: 2019-08-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are microbial strains capable of synthesizing potentially valuable flavor compounds, their yields are often low; and the development of biotechnological production of flavor compounds is hampered by a lack of knowledge about biochemical pathways, enzymes, and metabolic regulation

Method used

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  • Recombinant bacteria synthesizing 2,5-dimethylpyrazine (2,5-DMP) by catalysis
  • Recombinant bacteria synthesizing 2,5-dimethylpyrazine (2,5-DMP) by catalysis
  • Recombinant bacteria synthesizing 2,5-dimethylpyrazine (2,5-DMP) by catalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Construction and fermentation of a 2,5-DMP-producing strain using L-threonine as a fermentation substrate

[0050] 1. Construction of TDH expression vectors from different sources

[0051] In this example, B. subtilis 168 was used as the starting strain. First, TDHs from different species were selected and expressed exogenously. By detecting the production of 2,5-DMP, TDHs with catalytic advantages were screened, and 2,5-DMP was obtained. -DMP-increasing strains.

[0052] By searching in NCBI, 7 TDH-encoding gene tdh (Table 1) from different species were identified: B. subtilis168 (NP_389581), B. licheniformis ATCC 14580 (WP_085959523), B. amyloliquefaciensDSM7 (WP_014470388), P. putida ( WP_064301272), E. coli K-12 (NP_418073), A. candidus (XP_024673913), A. uvarums (XP_025487049).

[0053] The entire genomes of B. subtilis 168, B. licheniformisATCC 14580, and B. amyloliquefaciens DSM7 were extracted, and used as templates to clone the TDH-encoding gene td...

Embodiment 2

[0067] Example 2: Construction of recombinant bacteria co-expressed with TDH and NOX

[0068] In Example 2, a genetically engineered strain B. subtilis168 / pMA0911-tdh (E.c) that can utilize L-threonine to high-yield 2,5-DMP was obtained. This example is further improved on the basis of B. subtilis 168 / pMA0911-tdh (E.c). This example realizes the co-expression of TDH and NOX, and realizes NAD through exogenous expression of NADH oxidase NOX + rapid regeneration.

[0069] 1. Construction of TDH and NOX co-expression vector

[0070] In this example, the NOX-encoding gene nox (Table 1) and the TDH-encoding gene tdh were transcribed and expressed in the same plasmid. First, the plasmid pMA0911-nox carrying the NOX transcription gene nox was obtained by total gene synthesis ( figure 1 B), and use this as a template to amplify the target gene nox( Figure 5 A), gene nox was constructed into restriction plasmid pMA0911-tdh(E.c)( Figure 5 A), the schematic diagram of the recombi...

Embodiment 3

[0083] Embodiment 3: Utilize UPLC to quantitatively detect 2,5-DMP in the fermented liquid

[0084] Quantitative analysis of 2,5-DMP: Quantitative detection of 2,5-DMP by ultra-high performance liquid chromatography (UPLC), the specific conditions are as follows: chromatographic column Waters BEH C18 (100mm×2.1mm, 1.7μmparticle) 2,5-DMP for liquid phase separation. Mobile phase A is 0.1% formic acid solution (unless otherwise specified, the percentages involved are volume fractions), and mobile phase B is chromatographic grade methanol. Mobile phase elution gradient: initial, 31% B; 0-3min, 31%-69%B; 3-10min, 31%B; running time is 10min. The flow rate is 0.20ml·min -1 , UV detection wavelength is 275nm. The loading volume is 1 μL.

[0085] Prepare 2,5-DMP standard solutions with different concentration gradients, use Origin software to fit the 2,5-DMP standard curve according to the changes in concentration and peak area, and then calculate the peak area according to the c...

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Abstract

The invention discloses recombinant bacteria synthesizing 2,5-dimethylpyrazine (2,5-DMP) by catalysis, and belongs to the technical field of biosynthesis. A strain with high yield of the 2,5-DMP of L-threonine can be used to perform recombinant expression of threonine dehydrogenase (TDH). Preferably, when recombinant bacillus subtilis co-expressing NOX and the E.coli-derived TDH is fermented for 24 h with 5.83 g / L L-threonine as a substrate, the yield can reach 616.04 mg / L, the production intensity can reach 25.67 mg / (L.h), and the conversion rate can reach 10.6%; compared with a wild-type strain, the strain is increased by 22.5 times in yield, the production intensity and conversion rate are greatly improved, and efficient biotransformation of the 2,5-DMP is realized.

Description

technical field [0001] The invention relates to a recombinant bacterium that catalyzes the synthesis of 2,5-dimethylpyrazine, and belongs to the technical field of biosynthesis. Background technique [0002] Alkylpyrazines are a kind of branched nitrogen-containing heterocyclic compounds with alkyl groups. As important flavor substances, they mainly contribute to the nutty, barbecue and toasty flavors in food. Due to the low threshold value, alkylpyrazine can show strong odor characteristics. It is a flavor substance allowed to be used in my country's GB2760-86 regulation. It is mainly used as a seasoning food additive and some spice intermediates in the food industry. [0003] In addition to its unique flavor value, alkylpyrazine also has important value in medicine and can be used as medicine or medicine intermediate. TTMP has been found to be used as a drug to treat some diseases, such as stroke, myocardial cell damage, knee osteoarthritis, etc. In addition, TTMP can al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P17/12C12R1/125
CPCC12N9/0006C12P17/12C12Y101/01
Inventor 徐岩张丽杰
Owner JIANGNAN UNIV
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