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Fermentative production of n-acetylneuraminic acid

A kind of fermentation broth and microorganism technology, applied in the product field of N-acetylneuraminic acid

Pending Publication Date: 2020-08-18
CHR HANSEN HMO GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The disadvantages of the above method are: (i) only small-scale production is possible; and (ii) excess pyruvate is required to shift the reaction equilibrium towards Neu5Ac

Method used

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  • Fermentative production of n-acetylneuraminic acid
  • Fermentative production of n-acetylneuraminic acid
  • Fermentative production of n-acetylneuraminic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0201] Example 1: Metabolic engineering of Escherichia coli BL21 (DE3) strain producing N-acetylneuraminic acid

[0202] Metabolic engineering is achieved through mutagenesis and deletion of specific endogenous genes and genomic integration of heterologous genes. The genes lacZ and araA were inactivated by mutagenesis using mismatched oligonucleotides as described by Ellis et al. (Proc. Natl. Acad. Sci. USA 98:6742-6746 (2001)).

[0203] Genomic deletions were generated according to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). To prevent the degradation of N-acetylglucosamine, the following genes were deleted from the genome of E. coli strain BL21(DE3): N-acetylglucosamine-specific PTS enzyme II (nagE), N-acetylglucosamine-6 - Phosphate deacetylase (nagA) and glucosamine-6-phosphate deaminase (nagB). Also deleted are N-acetylmannosamine kinase (nanK), N-acetylmannosamine-6-phosphate epimerase (nanE), N-acetylneuraminic acid aldolase (na...

Embodiment 2

[0213] Example 2: Production of N-acetylneuraminic acid in a batch fermentation process

[0214] Escherichia coli BL21 (DE3) strain #NANA1 was cultured in a 3L fermenter (New Brunswick, Edison, USA) at 30°C, initially using 1000 mL of mineral salt medium containing 7 g L -1 NH 4 h 2 PO 4 , 7g·L - 1 K 2 HPO 4 , 2g·L -1 KOH, 0.3g·L -1 Citric acid, 2g·L -1 MgSO 4 x7·H 2 O, 5g·L -1 NH 4 Cl 2 and 0.015g·L - 1 CaCl 2 x6·H 2 O, add 1mL·L -1 Trace element solution (54.4g L -1 Ferric ammonium citrate, 9.8g·L -1 MnCl 2 ×4·H 2 O, 1.6g·L -1 CoCl 2 x6·H 2 O, 1g L -1 CuCl 2 x2·H 2 O, 1.9g·L -1 h 3 BO 3 , 9g·L -1 ZnSO 4 x7·H 2 O, 1.1g L -1 Na 2 MoO 4 x2·H 2 O, 1.5g·L -1 Na 2 SeO 3 , 1.5 L -1 NiSO 4 x6·H 2 O), and containing 2% (m / v) sucrose as carbon source and the antibiotic bleomycin (10 μg·mL -1 ). Cultures were initiated using a 2.5% (v / v) inoculum from a preculture grown in the same sucrose-containing medium. The end of the batch phase is c...

Embodiment 3

[0216] Example 3: Generation and cultivation of a single gene knockout mutant of E. coli strain #NANA1 exhibiting increased Neu5Ac production capacity

[0217] E. coli BL21(DE3) strain #NANA1 was further modified to generate deletion mutants that removed or disrupted the genes gltB, yjhC and ppC. Strains #NANA1, #NANA1△gltB, #NANA1△yjhC and #NANA1△ppc were grown in 96-well plates. Therefore, a single colony of the strain was transferred from the agar plate to a microtiter plate containing 200 μL of the minimal medium described in Example 2 and incubated at 30° C. for about 20 h with vigorous shaking. Subsequently, 50 μL of the culture solution was transferred to a deep-well 96-well plate (2.0 mL), each well containing 400 μL of minimal medium.

[0218] After further incubation for 48 hours, the culture was stopped, and the content of N-acetylneuraminic acid in the supernatant was determined by mass spectrometry using an LC triple quadrupole MS detection system in multiple rea...

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Abstract

Disclosed are non-naturally-occurring microorganisms for the production of N-acetylneuraminic acid, a method for the production of N-acetylneuraminic acid by fermentation of the non-naturally-occurring microorganisms, and nutritional compositions containing N-acetylneuraminic acid which has been produced by fermentation of the non-naturally-occurring microorganisms.

Description

[0001] The present invention relates to non-naturally occurring microorganisms capable of producing N-acetylneuraminic acid, methods for the fermentative production of N-acetylneuraminic acid using said non-naturally occurring microorganisms, N-acetylneuraminic acid produced by fermentation and products containing N-acetylneuraminic acid produced in this way. Background technique [0002] Sialic acid (Sia) is a family of negatively charged monosaccharides with a nine-carbon backbone. More than 50 forms of alpha-keto acids have been found in nature. The most abundant sialic acid appears to be N-acetylneuraminic acid (NANA, NeuNAc, Neu5Ac). [0003] Sialic acid is present as the terminal sugar of glycans present in glycoconjugates (glycoproteins and glycolipids) on the cell surface of vertebrates and higher invertebrates. Sialic acid is a component of lipopolysaccharide and capsular polysaccharide of pathogenic bacteria including Escherichia coli K1, Haemophilus influenzae, Ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/245C12P19/00C12N9/00C12N15/52A23L33/00
CPCC07K14/245C12N15/52A23L33/40C12P19/26C12N15/70A23L33/135C12P19/28
Inventor S·詹尼温D·瓦滕伯格
Owner CHR HANSEN HMO GMBH
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