Burkholderia bidirectional ester synthase JG53625355, coding gene and application of Burkholderia bidirectional ester synthase JG53625355

A technology of Kholderia and Holderia, applied in the field of genetic engineering, can solve the problems of lack of research, unfavorable ester synthesis reaction, etc.

Active Publication Date: 2022-04-19
WULIANGYE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although ester-synthesizing microorganisms have been reported in the literature, relevant studies focusing on the key enzymes of ester synthesis are still lacking.
Previous studies have shown that organic phase systems such as n-hexane and n-heptane solvents are helpful for enzyme-catalyzed ester synthesis reactions. However, liquor is a solid-state fermentation system, and the water content of the fermented grains is between 53 and 58%. Considered as an aqueous system, it is not conducive to the classic ester synthesis reaction

Method used

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  • Burkholderia bidirectional ester synthase JG53625355, coding gene and application of Burkholderia bidirectional ester synthase JG53625355
  • Burkholderia bidirectional ester synthase JG53625355, coding gene and application of Burkholderia bidirectional ester synthase JG53625355
  • Burkholderia bidirectional ester synthase JG53625355, coding gene and application of Burkholderia bidirectional ester synthase JG53625355

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Cloning of the gene encoding the ester synthase JG536_25355 of embodiment 1

[0042] 1.1 Cultivation of Burkholderia and extraction of genomic DNA

[0043] Burkholderia BJQ0010 (CGMCC 24186) was inoculated into the fermentation medium, cultured on a shaker at 200±10r / min, 30±1°C for 24-72h. The fermentation medium is composed of soluble starch 10g / L, peptone 10g / L, NH 4 HSO 4 1g / L, K 2 HPO 4 1g / L, MgSO 4 0.8g / L, olive oil 10mL / L, natural pH, sterilized at 121°C for 15min.

[0044] The cultured bidirectional Burkholderia was collected by centrifugation, and the total DNA was extracted by BIOMIGA bacterial DNA extraction kit (BIOMIGA [Biomiga Shanghai Technical Service Center], model GD2411). The specific operation steps are carried out according to the conventional operation method of the kit.

[0045] 1.2 Specific amplification of the gene encoding ester synthase JG536_25355

[0046] The gene encoding Burkholderia-derived ester synthase JG536_25355 was amplifi...

Embodiment 2

[0054] Example 2 Construction of an E. coli engineering strain expressing ester synthase JG536_25355

[0055] 2.1 Linearization of pET-28a(+) vector

[0056] Linearization of the circular pET-28a(+) vector was performed using PCR. Primers were designed as follows:

[0057] Forward primer (SEQ ID NO.5): 5'-ATCGCACTCGAGCACCACC-3'

[0058] Reverse primer (SEQ ID NO.6): 5'-CATGGTATATCTCTCTTCTTA-3'

[0059] The PCR reaction system and amplification cycles are shown in Table 3 and Table 4, respectively.

[0060] Table 3 Example 2 PCR linearization reaction system of circular pET-28a(+)

[0061] Reagent Volume (μL) dd H 2 o

72.0 dNTP Mixture (2.5mM each) 8.0 10×Ex Taq Buffer 10.0 Forward primer (10μM) 4.0 Reverse primer (10μM) 4.0 pET-28a(+) vector 1.0 Q5 DNA Polymerase (5U / μl) 1.0 Total 100.0

[0062] Table 4 embodiment 2PCR amplification cycle

[0063]

[0064] 2.2 Plasmid construction

[0065] pass II...

Embodiment 3

[0071] Example 3 Ester synthase JG536_25355 crude enzyme liquid aqueous phase system catalyzes the synthesis of ethyl butyrate, ethyl valerate, ethyl hexanoate, ethyl caprylate and ethyl caprate

[0072] 3.1 Preparation of ester synthase JG536_25355

[0073] Use colony PCR to verify the cultured colonies, select the correct transformants and transfer them to SOC liquid test tubes (containing a final concentration of kanamycin sulfate 40-80mg / L), shaker at 150-250r / min, shake at 37±2°C for 10-15h, Then inoculate the Erlenmeyer flask containing the SOC medium with 1-5% (v / v), shake the shaker at 150-250r / min, shake and cultivate at 37±2°C until the cell density is 0.6-0.8 (OD 600nm ), and then add IPTG at a concentration of 0.01-0.10mM as an inducer, shake at 150-250r / min, and culture with shaking at 25±2°C for 15-24h. The cultured cells were collected by centrifugation at 8000 rpm for 5 min, washed with 0.05M Tris-HCl buffer solution (pH 7.5) twice and suspended. The suspende...

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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to bidirectional burkholderia derived ester synthase JG53625355, a coding gene and application of the bidirectional burkholderia derived ester synthase JG53625355. The amino acid sequence of the ester synthetase is as shown in SEQ ID NO. 1. The ester synthetase JG53625355 sourced from Burkholderia bidirectional BJQ0010 and the coding gene thereof are obtained through cloning and inducible expression, escherichia coli expression plasmids containing the coding gene of the ester synthetase are constructed, the plasmids are transferred into escherichia coli, and the enzyme is obtained through inducible expression. A catalytic system verifies that the ester synthase not only has the capability of catalytically synthesizing ethyl hexanoate and ethyl caprylate in a water phase system, but also can catalytically synthesize ethyl butyrate, ethyl valerate and ethyl caprate, so that the ester synthase can be used for catalytically synthesizing important flavor esters in the field of white spirit brewing.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a bidirectional Burkholderia-derived ester synthase JG536_25355, an encoding gene and application. Background technique [0002] Flavor analysis shows that the content of ethanol and water in Luzhou-flavor liquor accounts for about 98% of the total liquor body, and the remaining 2% is flavor substances, which are trace components in liquor. Although the content of flavor substances is low, it plays a decisive role in the quality of liquor. There are more than 2,400 kinds of trace components reported so far, among which esters are the most important type of flavor substances, with a total of 510 kinds reported. Due to the differences in the geographical distribution of raw materials, fermentation agents and brewing techniques, there are 12 major flavors of liquor. Among them, Luzhou-flavor liquor accounts for more than 70% of the liquor market share. It i...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/70C12P7/62C12G3/02
CPCC12N9/93C12N15/70C12G3/02C12P7/62C12Y601/02Y02A50/30
Inventor 赵东徐友强孙啸涛乔宗伟
Owner WULIANGYE
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