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CN-121975706-A - Recombinant escherichia coli for high yield of N-acetylneuraminic acid and application thereof

CN121975706ACN 121975706 ACN121975706 ACN 121975706ACN-121975706-A

Abstract

A recombinant escherichia coli with high yield of N-acetylneuraminic acid and application thereof relate to the technical field of biological genetic engineering. The recombinant escherichia coli takes escherichia coli as a host, freely expresses an N-acetylmannosamine epimerase gene yihS derived from the thermophilic bacteria of the mansion gate (Thermophagus xiamenensis) or an N-acetylmannosamine epimerase gene ce_3 derived from the bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron) and an exogenous N-acetylneuraminic acid lyase gene nanA, and re-expresses N-acetylhexosamine 1-kinase nahK, UDP-N-acetylglucosamine pyrophosphorylase genes glmU and UDP-N-acetylglucosamine-2-epimerase gene neuC in another synthetic pathway of the ManNAc. The recombinant escherichia coli with high yield of N-acetylneuraminic acid can reach 23.08g/L under the shake flask fermentation condition, the yield of N-acetylneuraminic acid in a 5L fermentation tank reaches 71.25g/L by adopting a two-stage batch feeding strategy, the molar conversion rate of GlcNAc reaches 57.60%, and the recombinant escherichia coli has potential of industrial application.

Inventors

  • ZHANG HONGTAO
  • ZHANG ZIMENG
  • LIU DENGFENG
  • ZHAO JIANXIN
  • WU JIANRONG

Assignees

  • 江南大学

Dates

Publication Date
20260505
Application Date
20250508

Claims (10)

  1. 1. A recombinant E.coli with high yield of N-acetylneuraminic acid is characterized in that the E.coli is taken as a host, an N-acetylmannosamine epimerase gene yihS derived from the thermophilic bacteria of the mansion machine (Thermophagus xiamenensis) or an N-acetylmannosamine epimerase gene ce_3 derived from the bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron) and an exogenous N-acetylneuraminic acid lyase gene nanA are expressed in a free way, and N-acetylhexosamine 1-kinase nahK, UDP-N-acetylglucosamine pyrophosphorylase genes glmU and UDP-N-acetylglucosamine-2-epimerase gene neuC in another synthetic pathway of the Mannac are expressed in a recombinant way.
  2. 2. The recombinant escherichia coli with high yield of N-acetylneuraminic acid according to claim 1, wherein the nucleotide sequence of the N-acetylmannosamine epimerase gene yihS is shown in SEQ ID NO.1, the nucleotide sequence of the N-acetylmannosamine epimerase gene ce_3 is shown in SEQ ID NO.2, the nucleotide sequence of the N-acetylneuraminic acid lyase gene nanA is shown in SEQ ID NO.3, the nucleotide sequence of the N-acetylhexosamine 1-kinase nahK is shown in SEQ ID NO.4, the nucleotide sequence of the UDP-N-acetylglucosamine pyrophosphorylase gene glmU is shown in SEQ ID NO.5, and the nucleotide sequence of the UDP-N-acetylglucosamine 2-epimerase gene neuC is shown in SEQ ID NO. 6.
  3. 3. The recombinant E.coli with high N-acetylneuraminic acid yield according to claim 1 or 2, wherein the recombinant plasmid constructed by homologous recombination is transformed into competent cell E.coli JM109 (DE 3) to construct recombinant E.coli JM109 (DE 3)/pET Duet-1yihS-neuC+nanA-glmU-nahK by expressing genes of yihS, ce_3 and nanA by using pET Deut-1 vector, expressing genes of nahK, glmU and neuC by using pET 28a series vector, and connecting the two genes of yihS-neuC in one expression cassette and the three genes of nanA-glmU-nahK by using flexible protein linker (GGGS) 3.
  4. 4. The recombinant escherichia coli of claim 1, wherein the escherichia coli comprises, but is not limited to ESCHERICHIA COLI NISSLE1917 (DE 3), e.coli JM109 (DE 3).
  5. 5. The recombinant escherichia coli for high production of N-acetylneuraminic acid according to claim 4, wherein the escherichia coli EcN strain knocks out pMUT1 and pMUT2 plasmids in the genome.
  6. 6. A method for high-yield N-acetylneuraminic acid, which is characterized in that the recombinant escherichia coli whole cell catalysis method is adopted to synthesize the high-yield N-acetylneuraminic acid.
  7. 7. The method according to claim 6, wherein the method comprises the steps of using the recombinant escherichia coli, performing induction culture in a fermentation medium at 20-30 ℃ for at least 25 hours until OD600 is more than or equal to 0.8, stopping fermentation, and performing whole cell catalytic reaction for more than 20 hours at 20-30 ℃ and pH of 6.5-7.5 by using 100-400 mM GlcNAc as a substrate.
  8. 8. The method according to claim 7, wherein the method further comprises adding 100 to 400mM GlcNAc, 200 to 500mM pyruvic acid, ATP or UTP at a concentration of 10mM or less as a catalyst to promote the synthesis of N-acetylneuraminic acid.
  9. 9. The method according to claim 6, wherein the recombinant escherichia coli is used for supplementing a glucose solution in a fermentation medium by adopting a variable-speed feeding mode, the glucose concentration in the fermentation liquid is maintained at a constant level, the fermentation liquid is subjected to induction culture for at least 30 hours at 20-25 ℃, the feeding is stopped until the OD600 of the fermentation liquid is more than or equal to 105.4, and then the whole cell catalytic reaction is carried out for 26 hours at 30 ℃ and pH 7.5 by taking 400mM GlcNAc, 500mM pyruvic acid, 10mM UMP and 10mM ATP as catalytic substrates.
  10. 10. Use of a recombinant escherichia coli according to any one of claims 1 to 5 or a method according to any one of claims 6 to 9 for the production of a product comprising N-acetylneuraminic acid.

Description

Recombinant escherichia coli for high yield of N-acetylneuraminic acid and application thereof Technical Field The invention relates to the technical field of biological genetic engineering, in particular to recombinant escherichia coli with high yield of N-acetylneuraminic acid, and a construction method and application thereof. Background N-acetylneuraminic acid (Neu 5 Ac) is a nine-carbon sugar with an N-acetyl group at the C-5 position. Neu5Ac is present in humans and can be incorporated as a precursor into the nervous system of infants to form gangliosides, thereby promoting nervous system development. The supplement of Neu5Ac in daily diet is helpful for promoting bone growth and brain development of infants, enhancing memory, and has positive effect on maintaining brain function of the elderly population. Notably, neu5Ac officially obtained european food additive approval in 2017. Of the natural substances, bird's nest has the highest Neu5Ac content, which can be up to about 10%, and in the Chinese dietary system, bird's nest is regarded as a very valuable and popular nutritional supplement, but its cost is relatively high. Neu5Ac is a key precursor for synthesizing N-acetylneuraminic acid Human Milk Oligosaccharides (HMOs), and about 25% of HMOs are modified by N-acetylneuraminic acid, which plays an important role in infant development. Currently, N-acetylneuraminic acid (Neu 5 Ac) can be obtained by artificial synthesis. Biosynthesis is the most widely used method at present and can be further divided into enzyme-catalyzed production, whole-cell catalysis and de-novo synthesis. Among them, whole cell catalytic production of N-acetylneuraminic acid is generally performed by overexpressing exogenous N-acetylglucosamine-2-isomerase AGE to synthesize the key precursor N-acetylmannosamine (ManNAc). However, certain thermodynamic bottlenecks exist in the AGE catalytic reaction process, and the metabolic flux strength cannot meet the requirement of large-scale industrial production at the present stage. CN116555147A discloses a construction method and application of recombinant escherichia coli with high yield of N-acetylneuraminic acid, and the recombinant escherichia coli with high yield of N-acetylneuraminic acid is obtained by taking escherichia coli as an initial strain, knocking out genes nanA, nanT, nanK and nagB, expressing exogenous genes neuC and neuB freely, and overexpressing UDP-GlcNAc synthetic pathway genes glmM, glmU and GlmS mutant genes glmS. The recombinant escherichia coli has complex construction steps and low yield of N-acetylneuraminic acid. N-acetylmannosamine (ManNAc) can be used as a substrate for the one-step synthesis of Neu5Ac, and this process has reported two methods, ① ManNAc with pyruvic acid to produce Neu5Ac under the catalysis of N-acetylneuraminic acid lyase (Comb and Roseman,1960; aug et al, 1984; rodri I guez-Apariio et al, 1995; mahmoudian et al, 1997); ② ManNAc and phosphoenolpyruvic acid produce Neu5Ac under the catalysis of N-acetylneuraminic acid synthetase (Blacklow and Warren,1962; vann et al, 1997; tabata et al, 2002), wherein two pathways exist for synthesizing ManNAc with GlcNAc as a substrate, ① GlcNAc can be directly isomerized into ManNAc by isomerase (Rodr I guez-Apariio et al, 1995; vann et al, 1997; tabata et al, 2002; sola-Carvajal et al, 2012), ② GlcNAc produces GlcNAc-1P by N-acetylhexosamine 1-kinase catalysis (Cai et al, 2009), UDP-N-acetylglucosamine pyrophosphorylase produces UDP-GlcNAc (Hu et al, 2021), UDP-N-acetylglucosamine 2-epimerase catalyzes the production of ManNAc (Lundgren and 352007). Therefore, finding a suitable N-acetylneuraminic acid synthesis pathway of E.coli and optimizing the enzyme expression level of the key pathway for the synthesis of ManNAc using N-acetylglucosamine (GlcNAc) as a substrate are effective solutions for improving the yield of the N-acetylneuraminic acid whole cell catalytic method. Disclosure of Invention Aiming at the problem that the currently reported synthetic approach can not meet the industrial production requirement of N-acetylneuraminic acid, the invention provides recombinant escherichia coli with high yield of N-acetylneuraminic acid and a construction method of the recombinant escherichia coli, and the invention also optimizes a method for preparing the N-acetylneuraminic acid by using the whole-cell catalysis method of the recombinant escherichia coli. The technical scheme adopted for solving the technical problems is as follows: A recombinant E.coli with high yield of N-acetylneuraminic acid is prepared by taking E.coli as a host, freely expressing N-acetylmannosamine epimerase gene yihS from Xiamen thermophilic bacteria (Thermophagus xiamenensis) or N-acetylmannosamine epimerase gene ce_3 from Bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron) and exogenous N-acetylneuraminic acid lyase gene nanA, and recombining and expressing N-acetylhexosamine 1-kinase nahK, UDP-N-acetylglucosami