Recombinant gluconobacter oxydans engineering bacterium and use thereof in synthesis of miglitol intermediate

A technology of Gluconobacter oxydans and engineering bacteria, which is applied in the field of preparation of miglitol intermediates, can solve problems such as energy consumption, potential safety hazards, increase the pressure of fermentation tanks, etc. The effect of dehydrogenase activity enhancement

Inactive Publication Date: 2021-04-02
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Traditional methods such as increasing the stirring speed, introducing pure oxygen or in...

Method used

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  • Recombinant gluconobacter oxydans engineering bacterium and use thereof in synthesis of miglitol intermediate
  • Recombinant gluconobacter oxydans engineering bacterium and use thereof in synthesis of miglitol intermediate
  • Recombinant gluconobacter oxydans engineering bacterium and use thereof in synthesis of miglitol intermediate

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Construction of the recombinant Gluconobacter oxidans engineering strain G.oxydans / AB-VHb.

[0032] 1. Construction of recombinant co-expression plasmid pBBR1-gHp0169-sldAB-vhb:

[0033] The vhb gene sequence of artificially synthesized Vitreoscilla haemoglobin (GenBank number is JN418989.1), and the protein sequence accession number is AEO09414.1. The broad host vector pBBR1MCS-5 was purchased from Hangzhou Baosai Biotechnology Co., Ltd. and used to express the vhb gene. The promoter gHp0169 (SEQ ID NO.5) of Gluconobacter oxydans (G.oxydans) is used to regulate the expression of the vhb gene, because the gHp0169 promoter itself does not have an RBS sequence, so the target gene needs to carry its own RBS sequence. Plasmid pBBR1-gHp0169 with gHp0169 promoter and terminator (SEQ ID NO.6) ( figure 1 Middle A), and plasmid pBBR1-gHp0169-sldAB ( figure 1 The construction of B) refers to the patent CN110016455A.

[0034] (1) Construction of plasmid pBBR1-gHp016...

Embodiment 2

[0077] Example 2 Comparison of growth and sorbitol dehydrogenase activity of recombinant Gluconobacter oxydans genetically engineered bacteria G.oxydans / AB-VHb and G.oxydans / AB under shake flask culture conditions.

[0078] (1) Preparation of resting cells of recombinant Gluconobacter oxydans genetically engineered bacteria:

[0079] The recombinant bacteria G.oxydans / AB-VHb and G.oxydans / AB preserved in glycerol tubes were streaked and inoculated on the SY solid medium culture dish containing 50 μg / L gentamicin, and cultured at 30°C for 2 days to grow single Bacterial colonies; use an inoculation loop to inoculate a single colony into a 250-mL shake flask containing 40 mL of SY seed medium containing 50 μg / L of gentamicin antibiotic at a final concentration of 50 μg / L, and cultivate at 30 ° C, 150 rpm for 40 h to OD 600 5.0-5.5, to obtain seed liquid. The seed solution was inoculated into a 500-mL shake flask filled with 50 mL of fermentation medium SYK at an inoculum volume...

Embodiment 3

[0087] Example 3 Comparison of the growth and sorbitol dehydrogenase activity of the recombinant Gluconobacter oxidans genetic engineering strain G.oxydans / AB-VHb and G.oxydans / AB in a 5-L fermenter.

[0088] Bacterial culture conditions: Inoculate G.oxydans / AB-VHb and G.oxydans / AB single colonies in SY seed medium containing gentamicin at a final concentration of 50 μg / L, and culture at 30°C for 40 hours to OD 600 After reaching the plateau for 5.0-5.5, obtain the seed liquid; inoculate the seed liquid with the 5-L fermentor that contains the SYK fermentation medium of 4L final concentration 50 μ g / L gentamicin with the inoculation amount of volume concentration 5% (Sartorius, B, Germany), the ventilation rate was kept at 2vvm, the stirring speed was 400rpm, and the temperature was 30°C. After 18 hours, the tank was removed, and the resting cells of the recombinant bacteria were obtained by centrifuging the fermented liquid.

[0089] Sampling: sampling every 2h for OD 600...

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Abstract

The invention discloses a recombinant gluconobacter oxydans engineering bacterium and use thereof in a synthesis of a miglitol intermediate. The recombinant gluconobacter oxydans engineering bacteriumis constructed by jointly transferring a vitreoscilla hemoglobin gene vhb and a sorbitol dehydrogenase gene sldAB into a host bacterium. The host bacterium is gluconobacter oxydans. A growth rate ofthe recombinant gluconobacter oxydans and a final activity of a sorbitol dehydrogenase are both improved. Resting cells are used to produce 6-deoxy-6-amino (N-ethoxyl)-alpha-L-furan sorbose. The recombinant gluconobacter oxydans engineering bacterium shows a higher catalytic capacity, can catalytically synthesize 96.1 g/L of 6NSL in a single batch within 24 h, and improves yield of the 6NSL by 9.5% under the same condition compared with G.oxydans/pBBR-sldAB constructed by a patent CN110016455A.

Description

[0001] (1) Technical field [0002] The present invention relates to the preparation of a miglitol intermediate, in particular to a double-gene co-expressed recombinant Gluconobacter oxydans engineering bacterium and its application, especially to the production of miglitol intermediate 6 by catalyzing resting cells - Deoxy-6-amino(N-hydroxyethyl)-α-L-sorbofuranosose (6NSL). [0003] (2) Background technology [0004] Miglitol (Miglitol) is an N-hydroxyethyl derivative of 1-deoxynojirimycin, a new type of small intestinal α-glucosidase inhibitor, and one of the leading drugs for the treatment of type II diabetes. Its chemical name is 1-(2-hydroxyethyl)-2-(hydroxymethyl)-3,4,5-piperidine triol; boiling point: 246.0°C, melting point: -20°C, and its molecular structure is as follows: [0005] [0006] At present, miglitol is usually synthesized by a combination of biology and chemistry: N-hydroxyethylglucosamine (NHEG) is used as a substrate, and 6-deoxy-6-amino (NHEG) is bioc...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/53C12N15/31C12P19/26C12R1/01
CPCC12N9/0006C07K14/195C12N15/74C12P19/26C12Y101/01014
Inventor 胡忠策刘东柯霞郑裕国
Owner ZHEJIANG UNIV OF TECH
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