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Bacillus subtilis gene engineering bacterial producing Neu5Ac, construction method and application thereof

A technology of Bacillus subtilis and acetylneuraminic acid, which is applied in the field of bioengineering, can solve the problems of complex genetic modification and unclear metabolic background, and achieves the effect of simple production process and environmental friendliness.

Inactive Publication Date: 2014-07-16
武汉中科光谷绿色生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with Escherichia coli, the genetic modification of Bacillus subtilis is more complicated, and the metabolic background is not clear, and there is no report on the synthesis and production of N-acetylneuraminic acid after modification.

Method used

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  • Bacillus subtilis gene engineering bacterial producing Neu5Ac, construction method and application thereof
  • Bacillus subtilis gene engineering bacterial producing Neu5Ac, construction method and application thereof
  • Bacillus subtilis gene engineering bacterial producing Neu5Ac, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Construction of neuBC and glmS gene tandem expression vector

[0024] 1. Construction of neuBC gene expression vector pHT01-neuBC

[0025] 1) The neuBC gene was synthesized according to the neuBC gene sequence (GenBank No.AF400048), with BamHI and XbaI sites added at both ends. The specific sequence is shown below (SEQ ID NO.1).

[0026] GGATCCATGAAAGAAATAAAAATACAAAATATAATCATAAGTGAAGAAAAAGCACCCTTAGTCGTACCTGAAATAGGCATTAATCATAATGGCAGTTTAGAACTAGCTAAAATTATGGTAGATGCAGCCTTTAGCGCAGGTGCTAAGATTATAAAGCATCAAACTCATATTGTTGAAGATGAGATGAGTAAGGCCGCTAAAAAAGTAATTCCTGGTAATGCAAAAATAAGCATTTATGAGATTATGCAAAAATGTGCTTTGGATTATAAAGATGAGCTAGCACTTAAAGAATACACAGAAAAATTAGGTCTTGTTTATCTTAGCACACCTTTTTCTCGTGCAGGTGCGAACCGCTTAGAAGATATGGGAGTTAGTGCTTTTAAGATTGGTTCAGGTGAGTGTAATAATTATCCGCTTATTAAACACATAGCAGCCTTTAAAAAGCCTATGATAGTTAGCACAGGAATGAATAGTATTGAAAGTATAAAACCAACTGTAAAAATCTTATTAGACAATGAAATTCCTTTTGTTTTAATGCACACGACCAATCTTTACCCAACCCCGCATAATCTTGTAAGATTAAACGCTATGCTTGAGTTAAAAAAAGAATTTTCTTGTATGGTAGGCTTAAG...

Embodiment 2

[0036] Embodiment 2: Construction of N-acetylneuraminic acid Bacillus subtilis genetically engineered bacteria

[0037] 1) Cultivate the E. coli DH5α strain containing the vector pHT01-neuBC-glmS overnight in liquid LB medium, and extract the plasmid pHT01-neuBC-glmS.

[0038] 2) Cultivate Bacillus subtilis 164 and 168 strains respectively to prepare competent cells, and transform the plasmid vector pHT01-neuBC-glmS into it by electroporation to obtain genetically engineered strains 164 / pHT01-neuBC-glmS and 168 / pHT01-neuBC-glmS, namely N-acetylneuraminic acid-producing genetically engineered bacteria are available, named CASOV-3 and CASOV-4, which have been deposited in the Chinese Type Culture Collection on December 31, 2013 and February 18, 2014, respectively. Center, the deposit numbers are CCTCC NO:M2013731 and CCTCC NO:M2014038 respectively.

Embodiment 3

[0039] Example 3 Glucose is used as carbon source to ferment and produce N-acetylneuraminic acid

[0040] 1. Seeds and fermentation medium:

[0041] (NH4) 2 SO 4 4g / L; KH 2 PO 4 6g / L; K 2 HPO 4 ·3H 2 O8g / L; yeast extract (purchased from OXOID company) 5g / L; peptone 10g / L; glucose 5g / L.

[0042] Feed solution: 500g / L glucose.

[0043] 2. Fermentation process:

[0044] 1) Pick a single colony of CASOV-3 or CASOV-4 into a 4ml LB test tube, and incubate at 37°C for 8-12 hours.

[0045] 2) Inoculate 2ml of first-grade seeds into 200ml of seed medium and incubate at 37°C for 8-10 hours.

[0046] 3) The secondary seeds are inoculated in a fermenter with a liquid volume of 3.5L, at 37°C, with a stirring speed of 300-800 rpm, and the dissolved oxygen is kept above 30%, and the pH is controlled at 6.9 with ammonia water.

[0047] 4) After the glucose is used up, add glucose at a rate of 5g / L.h.

[0048] 5) OD of fermentation broth 600 Add IPTG (final concentration of IPTG i...

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Abstract

The present invention discloses Bacillus subtilis gene engineering bacterial producing Neu5Ac, a construction method and an application thereof. According to the present invention, according to the gene engineering bacterial, genes for coding UDP-N-acetyl glucosamine epimerase and Neu5Ac synthetase are introduced into Bacillus subtilis so as to be expressed, and expression of glucosamine 6-phosphate synthetase of the Bacillus subtilis is enhanced to construct the complete metabolic pathway from glucose or glycerol to Neu5Ac in the Bacillus subtilis so as to utilize glucose or glycerol as a substrate to carry out fermentation culture so as to produce Neu5Ac; and the adopted Bacillus subtilis is widely used in the food industry, is safe and harmless, and is suitable for production of Neu5Ac required by food and health products.

Description

technical field [0001] The invention relates to a Bacillus subtilis genetically engineered bacterium producing N-acetylneuraminic acid, a construction method and application thereof, and belongs to the technical field of bioengineering. Background technique [0002] Sialic acid, also known as bird's nest acid, is widely found in various organisms in nature. Sialic acid is a general term for a series of acidic amino sugars containing 9 carbon atoms and a pyranose structure. The most important component is N- Acetylneuraminic acid (Neu5Ac). [0003] The current application of N-acetylneuraminic acid mainly has two fields, one is used as a pharmaceutical intermediate for the synthesis of the anti-influenza drug zanamivir; the other is used as a food additive for infant milk powder to make it Close to breast milk, promote the development of the baby's nervous system. [0004] As a pharmaceutical intermediate, N-acetylneuraminic acid can be produced by biocatalysis and fermenta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/26C12R1/125
Inventor 柳鹏福孙立洁王纪袁丽霞刘洋吴金勇陈祥松史吉平姚建铭
Owner 武汉中科光谷绿色生物技术有限公司
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