US20130266987A1 - Method and agents for producing n-acetylneuraminic acid (neunac) - Google Patents

Method and agents for producing n-acetylneuraminic acid (neunac) Download PDF

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US20130266987A1
US20130266987A1 US13/993,979 US201113993979A US2013266987A1 US 20130266987 A1 US20130266987 A1 US 20130266987A1 US 201113993979 A US201113993979 A US 201113993979A US 2013266987 A1 US2013266987 A1 US 2013266987A1
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neunac
nucleic acid
glcnac
epimerase
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Astrid Mach-Aigner
Robert Mach
Matthias G. Steiger
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Technische Universitaet Wien
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    • C12Y205/01056—N-acetylneuraminate synthase (2.5.1.56)
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    • C12Y501/03014—UDP-N-acetylglucosamine 2-epimerase (non-hydrolysing) (5.1.3.14)

Definitions

  • the present invention relates to methods and means for producing N-acetylneuraminic acid (NeuNAc).
  • N-acetylneuraminic acid pertains to the group of sialic acids.
  • sialic acids are usually present as a terminal residue of sugar conjugates on the cell surface. Due to their terminal position and the negative carboxylation function sialic acids play an important role in cellular recognition and adhesion processes.
  • NeuNAc Derivatives of NeuNAc are employed as neuraminidase inhibitors for the treatment of viral infections, such as influenza.
  • NeuNAc serves as a starting material for the manufacture of such medicaments, such as oseltamivir and zanamivir.
  • NeuNAc may either be extracted from corresponding raw materials, such as milk and eggs, or chemically synthesized.
  • NeuNAc is made exclusively from raw materials, such as N-acetylglucosamine, wherein a proportion of the known methods comprise enzymatically catalyzed steps.
  • Document EP 1 154 018 A1 describes an N-acetylglucosamine 2-epimerase having a specific amino acid sequence.
  • said epimerase can be recombinantly produced in various host cells, i. a. in yeasts, wherein expression vectors can be used which have a promoter operably linked to a nucleic acid molecule encoding N-acetylglucosamine 2-epimerase.
  • Document EP 1 484 406 A1 describes a method for producing N-acetylneuraminic acid.
  • a variety of inducible promoters are mentioned which are capable of controlling the expression of the enzymes required for the production of N-acetylneuraminic acid.
  • document EP 0 578 825 discloses a process for the production of NeuNAc in which N-acetylglucosamine and pyruvic acid are reacted with N-acetylneuraminic acid lyase.
  • the present invention relates to an isolated nucleic acid molecule comprising at least one promoter that is active in fungal cells of the genus Trichoderma and has a nucleic acid sequence encoding an N-acetylglucosamine 2-epimerase and/or an N-acetylneuraminic acid synthase operably linked thereto, wherein said at least one promoter that is active in fungal cells is a constitutive promoter.
  • NeuNAc can be produced in a simple and efficient manner in a fungal cell of the genus Trichoderma , provided said fungal cell is capable of constitutively expressing N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid synthase.
  • the fungal cell used herein should also be capable of providing a sufficient amount of N-acetyl-D-glucosamine in order to produce N-acetyl-D-mannosamine with the aid of the N-acetylglucosamine 2-epimerase, wherein NeuNAc is eventually synthesized by the reaction of N-acetyl-D-mannosamine and the N-acetylneuraminic acid synthase.
  • fungal cells which are not capable of providing N-acetyl-D-glucosamine. In such a case, N-acetyl-D-glucosamine would have to be added to the culture medium or organisms would have to be used that are capable of providing N-acetyl-D-glucosamine to the medium.
  • nucleic acid molecules comprising the corresponding nucleic acid sequences have to be introduced into the fungal cells used.
  • the nucleic acid molecule to be introduced may comprise the nucleic acid sequences of both enzymes.
  • the encoding nucleic acid sequences thereof are operably linked to a promoter that acts constitutively in fungal cells.
  • the term “operably linked to” means that the nucleotide sequence encoding the enzymes according to the present invention is bound to the regulatory sequence(s) such that the expression of the nucleotide sequence is possible and both sequences are linked together such that they fulfill the function that is predicted for and assigned to the sequence.
  • a nucleic acid is “operably linked” if it is brought into a functional relationship with another nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if it affects the transcription of the sequence. Binding is accomplished by means of ligation at suitable restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors are used according to conventional practice.
  • a “constitutive promoter” is a promoter that enables a gene or operon to be continuously expressed in a cell.
  • a “constitutive promoter” is transcriptionally active in most stages of growth.
  • the expression rate of genes or operons which are operably linked to “inducible promoters” can be specifically controlled, so that under certain conditions the transcription is completely down-regulated and is up-regulated under different, preferably extrinsic, conditions.
  • N-acetyl-D-glucosamine 2-epimerase and N-acetylglucosamine 2-epimerase (EC 5.1.3.8), respectively, catalyze the conversion of N-acetylglucosamine to form N-acetylmannosamine.
  • the coding nucleic acid sequence of this enzyme has been described, i. a., in mammals and bacteria, such as cyanobacteria, in which these enzymes are expressed.
  • the corresponding nucleic acid sequences encoding these enzymes may be used according to the present invention.
  • nucleic acid molecule according to the present invention In order to improve the expression in fungal cells it is possible to generate codon-optimized nucleic acid sequences from an epimerase amino acid sequence, which are finally used in the nucleic acid molecule according to the present invention. It is particularly preferred to provide the N-acetylglucosamine 2-epimerase from Anabaena sp. (GenBank ABG57042) in the nucleic acid molecule according to the present invention and to express it in fungal cells.
  • N-acetylneuraminic acid synthase (EC 2.5.1.56) catalyzes the reaction of N-acetylmannosamine to form NeuNAc. In addition, this reaction involves phosphoenolpyruvate and water as a co-substrate.
  • N-acetylneuraminic acid synthase is expressed in bacteria, such as E. coli, Campylobacter jejuni and Neisseria meningitidis . The corresponding nucleic and amino acid sequences are thus well known or identifiable to a sufficient extent. From the known sequences, it is possible to derive codon-optimized nucleic acid sequences which are transcribed and translated particularly well in fungal cells.
  • N-acetylneuraminic acid synthase from Campylobacter jejuni (e.g. C. jejuni NCTC11168) in the nucleic acid molecule according to the present invention and to express it in fungal cells.
  • Campylobacter jejuni e.g. C. jejuni NCTC11168
  • the N-acetyl-D-glucosamine 2-epimerase is encoded by the following nucleic acid sequence:
  • the N-acetylneuraminic acid synthase is encoded by the following nucleic acid sequence:
  • the fungal cells according to the present invention pertain to the genus Trichoderma.
  • Trichoderma are particularly useful in the biosynthesis of NeuNAc as the members of this genus are capable of providing a sufficient amount of N-acetyl-D-glucosamine.
  • the fungal cells are Trichoderma reesei cells.
  • the constitutive promoter is selected from the group consisting of promoters of the glycolysis genes, in particular pki, gpd or zwf1, tef1a, act, cox4, negl and sari.
  • a further aspect of the present invention relates to a vector comprising a nucleic acid molecule according to the present invention.
  • a still further aspect of the present invention relates to a fungal cell of the genus Trichoderma comprising a nucleic acid molecule or a vector according to the present invention.
  • nucleic acid molecules or vectors comprising nucleic acid sequences encoding N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid synthase can be introduced.
  • the regions encoding the enzymes are operably linked to a constitutive promoter.
  • nucleic acid molecule or the vector according to the present invention are introduced into the host cell using generally known methods.
  • the fungal cell according to the present invention pertains to the genus Trichoderma .
  • members of the genus Trichoderma are capable of providing N-acetylglucosamine in an amount that is sufficient for synthesizing a sufficient amount of NeuNAc with the aid of recombinantly expressed N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid synthase. Therefore, the use of fungal cells of this genus is particularly preferred according to the present invention.
  • N-acetylglucosamine If chitin is used as a starting substance, it is reduced to form N-acetylglucosamine.
  • This monomer may be used as both a carbon and a nitrogen source for cell growth and also as a building block for the cell wall biosynthesis (a substantial component is chitin) as well as for the synthesis of N-acetyl neuraminic acid.
  • An inducible system will come into action in a selective manner and thus cause an overload with respect to the availability of N-acetylglucosamine.
  • a constitutive system will continuously withdraw N-acetylglucosamine and therefore enable a continuous product formation.
  • the fungal cell is Trichoderma reesei.
  • the fungal cell according to the present invention comprises at least one nucleic acid molecule whose nucleic acid sequence encodes an N-acetylglucosamine 2-epimerase and an N-acetylneuraminic acid synthase and is operably linked to a constitutive promoter that is active in fungal cells.
  • a further aspect of the present invention relates to a method for producing N-acetylneuraminic acid (NeuNAc), comprising the cultivation of fungal cells according to the present invention in the presence of an N-acetyl-D-glucosamine source.
  • NeuroNAc N-acetylneuraminic acid
  • N-acetyl-D-glucosamine is required as a substrate. Therefore, it is necessary to use fungal cells that are capable of providing this substrate.
  • the N-acetyl-D-glucosamine source is chitin.
  • FIG. 1 shows an overview of possible metabolic reaction pathways, from the polymer chitin as a starting substance to the formation of NeuNAc. Metabolic intermediate products are shown in rectangles and arrows represent enzyme-catalyzed reactions. Next to the arrow, the corresponding EC number of the enzymatic reaction is indicated. A circled plus denotes an enzymatic reaction which could be assigned to a gene in the genome of Trichoderma reesei . (http://genome.jgipsf.org/Trire2/Trire2.home.html). A circled minus indicates that no annotated gene could be found in the currently published genome.
  • FIG. 2 shows the formation of NeuNAc in an in vitro reaction with heterologously expressed T. reesei protein in the transgenic strain PEC/PSC1 using the substrates GlcNAc, ATP and PEP.
  • the chromatograms (1), (2) and (3) were obtained with the same samples as described in section (a), wherein chromatogram (2) is amplified 10-fold and chromatogram (3) is amplified 1.000-fold in relation to chromatogram (1).
  • (ad 1) includes the mass spectrum pertaining to chromatogram (1) at a retention time of 8.345 min.
  • (ad 2) shows the mass spectrum of chromatogram (2) at a retention time of 8.348 min.
  • FIG. 3 shows the in vivo production of NeuNAc in the transgenic T. reesei strain PEC/PSC1 after cultivation on GlcNAc for 66 h.
  • the chromatograms (1), (2) and (3) were obtained with the same samples as described in section (a), wherein chromatogram (2) is amplified 100-fold and chromatogram (3) is amplified 1.000-fold in relation to chromatogram (1).
  • (ad 1) includes the mass spectrum pertaining to chromatogram (1) at a retention time of 8.345 min.
  • (ad 2) shows the mass spectrum of chromatogram (2) at a retention time of 8.348 min.
  • Trichoderma reesei Hypocrea jecorina ) QM9414 (ATCC 26921) was used as the parent strain in this example and was cultured on malt extract agar.
  • Mycelia for the in vitro enzymatic reactions were obtained from cultures of the strains set up in 1,000 mL Erlenmeyer flasks with 200 mL of 3% (w/v) malt extract.
  • the flasks were inoculated with 10 ⁇ 8 conidia per liter and the cultivation was carried out at 30° C. and 250 rpm for 40 h.
  • the cultivation of T. reesei on colloidal chitin was performed in 1,000 mL Erlenmeyer flasks each containing 200 ml of Mandels-Andreotti medium including 1% (w/v) colloidal chitin and 0.1% (w/v) bacto peptone.
  • the inoculation was performed with 10 ⁇ 8 conidia per liter and the incubation was carried out at 30° C. and 250 rpm for 90 h.
  • the synthetic gene tbage was generated based on the protein sequence of Anabaena sp. CH1 GlcNAc 2-epimerase (GenBank: ABG57042) by translating the protein sequence into a DNA sequence using the software GeneOptimizer® (Geneart, Germany). In this process, the DNA sequence was optimized with respect to the codon usage of T. reesei (Table 1):
  • the synthetic gene tneub was generated which is based on the protein sequence of the NeuNAc synthase from Campylobacter jejuni NCTC11168 (http://old.genedb.org/genedb/cje juni/index.jsp, Cj1141) and whose DNA sequence was also adapted to the codon usage of T. reesei :
  • the synthetic genes tbage and tneub were cut from their production plasmid using XbaI/NsiI restriction digestion and were inserted into the plasmid pRLM ex30 (Mach, R. L. et al., 1994, Curr. Genet. 25:567-70), wherein the hph gene located between the XbaI and the NsiI restriction site was replaced by tbage and tneub, respectively.
  • the plasmid pGEX4T-2 (GE Healthcare, UK) was digested with EcoRI and XhoI.
  • tbage tneub were introduced into pGEX-MS via the restriction sites XbaI/NsiI, which resulted in the formation of the plasmids pGEX-epi and pGEX-syn.
  • T. reesei The protoplast transformation of T. reesei was carried out as mentioned in a previous article (Gruber, F. et al., 1990. Curr. Genet. 18, 71-6). A total amount of 10 ⁇ g of DNA was used per transformation.
  • pMS-PEC 4 ⁇ g
  • pMS-PSC 4 ⁇ g
  • pMS-PSC 4 ⁇ g
  • Recombinant strains were selected for hygromycin B resistance.
  • RNA extraction, reverse transcription and qPCR were performed as described in a previous article. Oligomer nucleotide sequences which were employed as primers are given in Table 1.
  • Sar1 was used as a reference gene for the normalization of the RT-qPCR.
  • the primers ManEfw and ManErev were used for the gene tbage in the qPCR at an optimal elongation temperature of 64° C. and with 2 mM MgCl 2 .
  • the primers NANAfw and NANArev were used for the gene tneub in the qPCR at an optimal elongation temperature of 64° C.
  • the primers pkifwR and pkirev were used in the qPCR at an optimal elongation temperature of 64° C. Data analysis was carried out using REST 2008.
  • Genomic DNA was isolated from the fungal mycelium, as described in a previous article. The hybridization and detection was carried out according to standard operating procedures using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Switzerland). The qPCR of genomic DNA was performed using about 50 ng of genomic template DNA. The same primers as in the RNA analysis were used for the genes tbage and tneub. pki served as a reference gene and was amplified with the primers pkifwD and pkirev at an elongation temperature of 64° C.
  • GST Glutathione S-Transferase
  • GST fusion proteins of GlcNAc 2-epimerase (GST: epi) and NeuNAc synthase (GST: syn) were produced by expression of the plasmids pGEX-epi and pGEX-syn in E. coli BL21 (DE3) cells. According to the standard operating protocol, the fusion proteins were purified with the aid of GSTrapTMFF columns having a column volume of 1 mL (GE Healthcare).
  • the enzymatic reaction was carried out in a similar manner as described by Vann et al. (Vann, W. F., et al., 1997, Glycobiology 7:697-701).
  • the reaction for detecting the activity of GlcNAc 2-epimerase involves 10 mM GlcNAc, 0.2 mM ATP, 100 mM bicine buffer (pH 8) and 10-40 ⁇ L of cell-free extract in a total volume of 100 ⁇ L.
  • the reaction for detecting the activity of NeuNAc synthase involves 10 mM ManNAc, 10 mM PEP, 12.5 mM MnCl 2 , 100 mM bicine buffer (pH 8) and 10-40 ⁇ L of cell-free extract in a total volume of 100 ⁇ L.
  • the combined reaction for detecting the activity of both GlcNAc 2-epimerase and NeuNAc synthase involves 10 mM GlcNAc, 10 mM PEP, 12.5 mM MnCl 2 , 100 mM bicine buffer (pH 8) and 40 ⁇ L of cell-free extract in a total volume of 100 ⁇ L.
  • FIG. 1 illustrates the presently known enzyme-catalyzed processes which lead to the formation of NeuNAc using the biopolymer chitin as a starting substance.
  • Trichoderma are enzymes which are required for catabolizing chitin. The first step from chitin to the monomer GlcNAc is catalyzed by chitinases (3.2.1.14).
  • hexokinase EC 2.7.1.1
  • GlcNAc 6-phosphate deacetylase EC 3.5.1.25
  • a glucosamine-6-phosphate deaminase EC 3.5.99.6
  • At least one potential enzyme in each case can be found in the annotated genome of T. reesei (Table 3).
  • genes can be found that are responsible for the biosynthesis of chitin, including a phosphoacetylglucosamine mutase (EC 5.4.2.3), an UDP-N-GlcNAc diphosphorylase (EC 2.7.7.23) and a plurality of chitin synthases (EC 2.4.1.16).
  • T. reesei that are responsible for the synthesis of ManNAc (EC 5.1.3.8 in bacteria, EC 5.1.3.4 in mammals) or for the synthesis of NeuNAc (EC 2.5.1.6. in bacteria, EC 2.7.1.60, EC 2.5.1.57, EC 3.1.3.29 in mammals).
  • estExt_GeneWisePlus. C — 140427, est-tExt_GeneWisePlus.C — 140421, estExt_Genewise1.C — 140432) could be found which encode a hexokinase, a GlcNAc-6-phosphate deacetylase and glucosamine-6-phosphate deaminase and are all located in close proximity to one another in the genome of T. reesei (location in the genome on “scaffold 14”: 714385-729984). Similar clusters are also present in other filamentous fungi, such as Neurospora crassa or Aspergillus nidulans , which is indicative of a conserved cluster for the catabolism of GlcNAc.
  • the hexokinase (Protein ID 79677) that is annotated in the genome of T. reesei can therefore be further specified as GlcNAc kinase (EC 2.7.1.59), analogous to the annotation and characterization in Candida albicans (39).
  • the gene (estExt_GeneWisePlus.C — 140419), which is located adjacent to the GlcNAc-6-phosphate deacetylase (estExt_GeneWisePlus.C — 140421), may also pertain to the cluster as a homologue of this gene in Neurospora crassa is annotated as ⁇ -N-acetylglucosaminidase ( N. crassa OR74A (NC10): Supercontig. 6: 560844-564980) is annotated.
  • a two-enzyme strategy was chosen for the production of NeuNAc in Trichoderma , wherein the first enzymatic step is catalyzed by a GlcNAc 2-epimerase (EC 5.1.3.8) and the second by a NeuNAc synthase (EC 2.5.1.99).
  • the protein sequence of the GlcNAc 2-epimerase from Anabaena sp. CH1 (GenBank: ABG57042) and, for the NeuNAc synthase, the protein sequence of Campylobacter jejuni NCTC11168 (Cj1141) were selected as candidates.
  • the protein sequences were translated into DNA sequences by means of the software GeneOptimizer® (Geneart) and the codon usage was adapted to that of T. reesei (Table 1).
  • the resulting synthetic genes were designated as tbage and tneub.
  • the coding sequences were inserted into the plasmid pRLMex30, wherein the coding sequence for the hph gene was substituted in this plasmid.
  • both genes were under the control of the constitutive pki promoter and the cbh2 terminator (plasmid pMS-PEC with tbage and plasmid pMS-PSC with tneub).
  • the pki promoter was replaced by the xyn1 promoter (plasmid pMS-XEX with tbage and plasmid pMS-XSC with tneub).
  • the parent strain QM9414 was transformed with various combinations of the plasmids pMS-PEC, pMS-PSC, pMS-XEX as well as pMS-XSC and pMS-Hylox2 (including the selection marker hph between two loxP sequences).
  • the plasmids containing the genes tbage and tneub were transformed both individually and in a combination of tneub/tbage.
  • the strain PEC/PSC1 exhibits a transcription of the tbage gene that is approximately 2-fold higher than that of the strain PEC/PSC10.
  • the strain PEC/PSC10 exhibits an about 2-fold higher transcription of the gene family tneub than the strain PEC/PSC1.
  • the cell-free extract was tested for the presence of GlcNAc 2-epimerase and NeuNAc synthase.
  • the conversion of the substrates PEP and GlcNAc to form ManNAc and NeuNAc was measured subsequently to the addition of the cell-free enzyme-containing extract.
  • the conversion reaction was analyzed by HPLC-MS and the corresponding chromatograms are shown in FIG. 2 .
  • GST fusion proteins of GlcNAc 2-epimerase (tbage) and NeuNAc synthase (tneub), which are produced by expression in E. coli were used as a positive control.
  • ManNAc and NeuNAc shows indicates that the two synthetic genes tbage and tneub are functionally expressed in Trichoderma ( FIG. 2 a 2 and FIG. 2 b 2 ). Also, the positive control with the GST fusion proteins shows the formation of ManNAc and NeuNAc ( FIG. 2 a 1 and FIG. 2 b 1 ). Neither ManNAc nor NeuNAc are formed in the enzymatic reaction when an extract of the original strain QM9414 is used. This result shows that no significant GlcNAc 2-epimerase activity is present in the parent strain. Furthermore, it was also exclusively tested for NeuNAc synthase activity in strain QM9414, wherein ManNAc and PEP were used as a substrate in the enzymatic reaction. Neither in this case any activity in the parent strain could be observed, which suggests that there is neither NeuNAc synthase activity nor GlcNAc 2-epimerase activity in natural isolates of Trichoderma reesei.
  • the T. reesei strain PEC/PSC1 was cultured on colloidal chitin as a carbon source. During the cultivation the increase in chitinase activity was monitored. After 90 h of cultivation time the chitinase activity reached its maximum and the supernatant was tested for the ability to hydrolyze chitin. The results are presented in Table 6. Colloidal chitin from crab shells yields ten times more GlcNAc than untreated chitin from crab shells. The GlcNAc thus released may be used as a starting substance for the production of NeuNAc with the strain PEC/PSC1.
  • the recombinant strain PEC/PSC1 produces ManNAc ( FIG. 3 a 2 ) and NeuNAc ( FIG. 3 b 2 , 10 ⁇ g per g of dry biomass). This result indicates that NeuNAc can be produced in T. reesei by co-expression of two bacterial enzymes.
  • the parental strain QM9414 shows neither a formation of NeuNAc nor of ManNAc ( FIG. 3 a 3 and FIG. 3 b 3 ).
  • reesei produces a plurality of chitinases (Table 3) and is capable of effectively degrading the polymer chitin to yield its monomer GlcNAc.
  • the specific biosynthesis of NeuNAc starts with intermediates of the chitin metabolic pathway (GlcNAc or UDP-GlcNAc) (see FIG. 1 ) which are available in T. reesei .
  • GlcNAc or UDP-GlcNAc intermediates of the chitin metabolic pathway
  • no genes can be found in this organism that are similar to genes encoding an UDP-GlcNAc 2-epimerase, a ManNAc kinase, a NeuNAc 9-phosphate synthase or a NeuNAc 9-phosphatase.
  • the second enzyme a NeuNAc synthase
  • the codon usage of both genes was optimized with respect to the codon usage of Trichoderma reesei in order to improve the expression of the bacterial genes in the fungal host.
  • the constitutive promoter of the pki gene on the one hand and the well-controllable promoter of the xyn1 gene on the other hand were chosen for the expression of the genes. Under the control of the xyn1 promoter no successful expression of the two genes could be achieved. Although it was shown that the genes are transcribed, no enzymatic activity could be detected.
  • the two heterologously expressed genes can not only be transcribed, but the corresponding enzymatic activity could also be detected.
  • the formation of NeuNAc could also be demonstrated in vivo.
  • the fungus was cultured on the biopolymer chitin, which resulted in the release of the monomer GlcNAc.
  • the production of NeuNAc could be detected in the mycelium ( FIG. 3 b 2 ).

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Publication number Priority date Publication date Assignee Title
US10378034B2 (en) 2014-05-27 2019-08-13 Universitetet I Tromsø—Norges Arktiske Universitet Use of a N-acetylneuraminate lyase derived from the bacterium Aliivibrio salmonicida in the production of neuraminic acid and derivatives thereof
CN112741821A (zh) * 2021-01-15 2021-05-04 厦门诺康得生物科技有限公司 一种唾液酸纳米颗粒及其制备方法和应用
WO2024036508A1 (zh) * 2022-08-17 2024-02-22 深圳大学 融合蛋白酶、融合表达载体和工程菌以及n-乙酰神经氨酸的生产方法

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AT517831B1 (de) * 2015-12-30 2017-05-15 Technische Universität Wien Verfahren zur Herstellung von Sekundärmetaboliten
CA3048521A1 (en) * 2016-12-27 2018-07-05 Inbiose N.V. In vivo synthesis of sialylated compounds
EP3473644A1 (de) 2017-10-17 2019-04-24 Jennewein Biotechnologie GmbH Fermentative herstellung von n-acetylneuraminsäure
EP3486326A1 (de) 2017-11-21 2019-05-22 Jennewein Biotechnologie GmbH Verfahren zur reinigung von n-acetylneuraminsäure aus einer fermentationsbrühe

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5610034A (en) * 1987-04-29 1997-03-11 Alko Group Ltd. Immunoglobulin production by trichoderma
US20040265953A1 (en) * 2000-08-11 2004-12-30 Harman Gary E. Production and use of inducible enzymes from trichoderma and bacteria for control of plant pests and for industrial processes

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3131655B2 (ja) 1992-02-03 2001-02-05 マルキン忠勇株式会社 Ｎ−アセチルノイラミン酸の製造法
GB9311873D0 (en) 1993-06-09 1993-07-28 Glaxo Group Ltd Process
AU2459300A (en) * 1999-02-09 2000-08-29 Kyowa Hakko Kogyo Co. Ltd. N-acetylglucosamine 2-epimerase and dna encoding the enzyme
US6846656B1 (en) * 1999-08-30 2005-01-25 Kyowa Hakko Kogyo Co., Ltd. Process for producing N-acetylneuraminic acid
US7863020B2 (en) * 2000-06-28 2011-01-04 Glycofi, Inc. Production of sialylated N-glycans in lower eukaryotes
US20050260729A1 (en) * 2004-03-17 2005-11-24 Hamilton Stephen R Method of engineering a cytidine monophosphate-sialic acid synthetic pathway in fungi and yeast
FI120310B (fi) * 2001-02-13 2009-09-15 Valtion Teknillinen Parannettu menetelmä erittyvien proteiinien tuottamiseksi sienissä
US7329514B2 (en) * 2002-02-28 2008-02-12 Kyowa Hakko Kogyo Co., Ltd. Process for producing n-acetylneuraminic acid

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5610034A (en) * 1987-04-29 1997-03-11 Alko Group Ltd. Immunoglobulin production by trichoderma
US20040265953A1 (en) * 2000-08-11 2004-12-30 Harman Gary E. Production and use of inducible enzymes from trichoderma and bacteria for control of plant pests and for industrial processes

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10378034B2 (en) 2014-05-27 2019-08-13 Universitetet I Tromsø—Norges Arktiske Universitet Use of a N-acetylneuraminate lyase derived from the bacterium Aliivibrio salmonicida in the production of neuraminic acid and derivatives thereof
CN112741821A (zh) * 2021-01-15 2021-05-04 厦门诺康得生物科技有限公司 一种唾液酸纳米颗粒及其制备方法和应用
WO2024036508A1 (zh) * 2022-08-17 2024-02-22 深圳大学 融合蛋白酶、融合表达载体和工程菌以及n-乙酰神经氨酸的生产方法

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