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Genetically engineered bacteria and application thereof in production of BT (D-1,2,4-butanetriol)

A genetically engineered bacteria and gene technology, applied to genetically engineered bacteria and its application in the production of D-1,2,4-butanetriol, can solve problems such as low efficiency

Active Publication Date: 2018-02-16
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problem of low efficiency still faced by existing BT biotransformation pathways, the present invention investigates xylonate dehydratases from different sources and 2-ketoacid decarboxylases from different sources

Method used

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  • Genetically engineered bacteria and application thereof in production of BT (D-1,2,4-butanetriol)
  • Genetically engineered bacteria and application thereof in production of BT (D-1,2,4-butanetriol)
  • Genetically engineered bacteria and application thereof in production of BT (D-1,2,4-butanetriol)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of genetically engineered bacteria BL21-02, BL21-08, BL21-09, BL21-10

[0037] 1) Construct clones expressing 2-ketoacid decarboxylase genes MdlC, KivD, KdcA, Aro10; xylose dehydrogenase gene XylB, D-xylose dehydratase YjhG and alcohol dehydrogenase gene YqhD; D-xylose dehydration Enzyme YjhG ( Escherichia coli ), GeneID: 946829; the xylose dehydrogenase gene XylB, Gene ID: 7329904; the alcohol dehydrogenase gene YqhD, GenBank: ADK47404.1; the α-keto acid decarboxylase ( Lactococcus lactis ), GenBank: AAS49166.1;

[0038] 2) The 2-ketoacid decarboxylase genes MdlC, KivD, KdcA, and Aro10 were respectively inserted into the plasmid pTRC99a (TransGen) Nco I and Sac Between the Ⅰ sites, the plasmids pTRC-MdlC, pTRC-KivD, pTRC-KdcA, pTRC-Aro10 were obtained, and the XylB gene was inserted into the plasmid pTRC99a Nco I and Bam Between the HI sites, pTRC99a-XylB was obtained. Then the XylB fragment with the TRC promoter was obtained by PCR, ...

Embodiment 2

[0041] The construction of embodiment 2 genetically engineered bacteria BL21-11, BL21-12, BL21-13

[0042] 1) Construct clones expressing benzoylformate decarboxylase gene MdlC and xylose dehydrogenase gene XylB, xylose dehydratase genes CcXylD, PsXylD, HvXylD and alcohol dehydrogenase gene YqhD.

[0043] 2) The MdlC gene was inserted into the plasmid pTRC99a Nco I and Sac Between the Ⅰ sites, the plasmid pTRC-MdlC was obtained, and the XylB gene was inserted into the plasmid pTRC99a Nco I and Bam Between the HI sites, pTRC99a-XylB was obtained. Then obtain the XylB fragment with the TRC promoter by PCR, insert the fragment into the plasmid pTRC-MdlC Sac I and Bam Between the H I sites, the plasmid pTRC-MdlC-TRC-XylB was obtained.

[0044] 3) The gene fragments CcXylD, PsXylD, HvXylD, and YqhD were respectively inserted into the pCWJ plasmids to obtain recombinant plasmids pCWJ-CcXylD, pCWJ-PsXylD, pCWJ-HvXylD, and pCWJ-YqhD. Using the pCWJ-YqhD plasmid as a templ...

Embodiment 3

[0046] Example 3 Fermentation of genetically engineered bacteria BL21-02, BL21-08, BL21-09, BL21-10 to produce D-1,2,4-butanetriol

[0047] Plate culture:

[0048] Take out the glycerin frozen bacteria stored at -80°C, and put them on the plate medium (peptone 10g / L, yeast powder 5g / L, NaCl10 g / L, ampicillin 100 mg / L, chloramphenicol 68 mg / L) Streak operation was performed on the plate, and then the plate was placed in a 37°C incubator for 12-16h.

[0049] Seed liquid culture:

[0050] Add 5 mL of seed medium (peptone 10g / L, yeast powder 5g / L, NaCl 10 g / L, ampicillin 100 mg / L, chloramphenicol 68 mg / L) into a 50 mL centrifuge tube, pick a small amount Glycerol-frozen bacteria were inoculated and cultured at 200 rpm and 37 °C for 10-12 h.

[0051] Shake flask fermentation culture:

[0052] 50 mL of fermentation medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 25 g / L D-xylose, 100 mg / L ampicillin, chloramphenicol 68 mg / L), the inoculum size was 1%, the rotation speed...

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Abstract

The invention discloses genetically engineered bacteria and an application thereof in production of BT (D-1,2,4-butanetriol). The genetically engineered bacteria are novel genetically engineered bacteria which are obtained as follows: a 2-keto acid decarboxylase gene MalC, an xylose dehydrogenase gene XylB, an xylonic acid dehydratase gene YjhG and an alcohol dehydrogenase gene YqhD are constructed, cloned and expressed, the genes are transferred into cells of host bacteria BL21(DE3), genetically engineered bacteria BL21-02 are obtained, and new xylonic acid dehydratase is screened on the basis of the genetically engineered bacteria; the genetically engineered bacteria are subjected to fermenting culture for production of BT. The capability of synthesizing BT from D-xylose can be improvedby screening the provided xylonic acid dehydratase gene CcXylD. The optimal xylonic acid dehydratase gene CcXylD and alpha-keto acid decarboxylase gene KdcA from lactococcus lactis are applied to theproduction process of BT, the optimal strain BL21-15 is obtained, and finally, the BT yield can reach 10.66 g / L.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a genetically engineered bacterium and its application in the production of D-1,2,4-butanetriol. Background technique [0002] D-1,2,4-Butanetriol (abbreviated as BT) is a four-carbon polyol with similar properties to glycerin, and is widely used in medicine, pesticide, cosmetics, papermaking, polymer materials, tobacco, military industry and other fields. BT and its related derivatives are important substrates in the synthesis of many natural products, and are also the synthetic precursors of many chiral compounds: in the military, due to the low impact sensitivity of BT’s nitro compounds, good thermal stability, low toxicity, moisture absorption It has good properties and can be used in combination with other energetic plasticizers to significantly improve the low-temperature mechanical properties of nitrocellulose-based gunpowder. Therefore, BT is often use...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/18C12R1/19
CPCC12N9/0006C12N9/88C12N15/70C12P7/18C12Y101/01C12Y101/01009C12Y401/01001C12Y402/01082
Inventor 陈可泉许娜娜王昕高倩胡社伟姜明均
Owner NANJING UNIV OF TECH
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