WO2017174038A1

WO2017174038A1 - Method for producing n-acetyl-d-glucosamine and/or d-glucosamine salt by microbial fermentation

Info

Publication number: WO2017174038A1
Application number: PCT/CN2017/080651
Authority: WO
Inventors: 孙镧
Original assignee: 孙镧
Priority date: 2016-04-05
Filing date: 2017-04-14
Publication date: 2017-10-12

Abstract

A method for producing N-acetyl-D-glucosamine and/or D-glucosamine salt by microbial fermentation: improving the effect of UDP-acetyl-D-glucosamine-2-isomerase in the microbe in order to produce N-acetyl-D-glucosamine and/or D-glucosamine salt more efficiently and with a higher yield.

Description

Method for producing N-acetyl-D-glucosamine and/or D-glucosamine salt by microbial fermentation

Technical field

The invention belongs to the field of microbial fermentation. In particular, the invention relates to a process for the production of N-acetyl-D-glucosamine by microbial fermentation and the further preparation of D-glucosamine salts.

Background technique

N-Acetyl-D-glucosamine (NAG or GlcNAc), also known as N-acetyl-glucosamine, N-acetylglucosamine, is the basic building block of many important polysaccharides in biological cells. It has important physiological functions in the living body. N-acetyl-D-glucosamine can be used clinically to: enhance the function of the human immune system; inhibit the growth of malignant tumors or fibroblasts; effectively treat various inflammations; as a low-calorie sweetener for diabetic patients and food additives for infants and young children, etc. . Hydrolyzed N-acetyl-D-glucosamine can be used to produce D-glucosamine hydrochloride, which can be used as a food supplement for anti-cancer, anti-cancer, hypolipidemic and blood pressure lowering. It is the third in the chitosan health food series. Generation of functional food additives. In addition, N-acetyl-D-glucosamine is the main raw material for synthesizing the anticancer drug chloramphenicol in the pharmaceutical industry; as a biochemical reagent, it can also be used as an immunoadjuvant for antibacterial infection and an activator for human anti-influenza virus.

In today's world, a large number of patients suffer from varying degrees of arthritis. In the United States alone, 33 million people suffer from osteoarthritis and joint pain. There are more than 150 million people in China. D-glucosamine products have been widely used in the treatment and health care of arthritis and joint pain, and have been widely used, and have become a very important drug substance variety in foreign markets.

N-acetyl-D-glucosamine is thought to have a similar effect as D-glucosamine, and it is known that uptake of N-acetyl-D-glucosamine can induce the production of new cartilage and prevent the onset of osteoarthritis, or in some cases Used to treat osteoarthritis. Since D-glucosamine has a bitter taste, and N-acetyl-D-glucosamine has a sweetness of 50% sucrose and is easily ingested, N-acetyl-D-glucosamine has been used as a substitute for D-glucosamine. attract attention.

At present, the source of glucosamine at home and abroad is mainly based on biological extraction. The biological extraction is mainly obtained by extracting chitin or chitosan from the shrimp and crab shell, and then preparing by hydrolysis with concentrated hydrochloric acid, or by extracting with acid and alkali using citric acid residue. The annual production is about 20,000 tons. However, when extracting with shrimp and crab shells, a large amount of waste residue and more than 100 tons of waste water will be produced for each ton of product obtained; when extracting with citric acid slag, 30-50 tons of waste acid slag will be produced for each ton of product obtained, which is a highly polluting process. It has been banned in many places. And, from the shell of aquatic products The extracted glucosamine is not suitable for many patients who are allergic to aquatic products. People who are allergic to aquatic products may cause severe allergic problems and even life-threatening problems. In addition, the biological extraction and purification process is complicated, and the product has a fishy smell and is unstable. In addition, due to environmental pollution, glucosamine extracted from shrimp and crab shells is inevitably contaminated by heavy metals.

Therefore, the bio-extraction method for producing glucosamine is difficult to meet people's needs in terms of quantity and quality, and a new alternative method must be opened up. If chemical synthesis is used to prepare, there are three disadvantages: high production cost; serious environmental pollution; and safety hazards. This method is currently not used at home and abroad. In comparison, the production of glucosamine by microbial fermentation is a good way. The microbial fermentation method uses glucose and inorganic salts as raw materials, selects excellent strains for liquid fermentation, and directly produces glucosamine by separation, concentration and purification. No harmful gases are produced during the production process. The glucosamine produced by the fermentation method has no fishy smell and the production is not limited by resources. Moreover, the use of metabolic engineering for strain improvement, high yield, industrialization and large production potential. Therefore, the production of glucosamine by microbial fermentation has undergone a major change in the technical process. Instead of the traditional biological extraction, it not only has an advantage in cost, but also has a certain environmental contribution in reducing the pollution of three wastes.

Conventional methods for the production of N-acetyl-D-glucosamine by microbial fermentation include a method involving the production of N-acetyl-D-glucosamine from chitin produced from shrimp shell material by an enzyme produced by a microorganism (for example, US 5998173, "Process for producing N-acetyl-D-glucosamine"); enzymatic hydrolysis by microorganisms (Trichoderma) or partial hydrolysis of acid to purify chitin from fungal residue (such as the slag of Aspergillus niger used in citric acid fermentation) to produce N- A method of acetyl-D-glucosamine (for example, US20030073666A1, "N-acetyl-D-glucosamine and process for producing N-acetyl-D-glucosamine"); direct use of glucose as a carbon source by Trichoderma does not require fungal residue Or a method for producing N-acetyl-D-glucosamine by fermentation of chitin and chitin oligosaccharide produced by shrimp shells (for example, US20110059489A1, "Method for fermentative production of N-acetyl-D-glucosamine by microorganism") Production of N-acetyl-D- by culturing Chlorella virus-infected Chlorella cells or recombinant E. coli introduced with a gene derived from Chlorella virus Glucosyl-based method (for example, JP2004283144A, "Method for producing glucosamine and N-acetylglucosamine"); fermentation-produced D-glucosamine or N-acetyl-D-glucosamine using genetically modified microorganisms, particularly genetically modified Escherichia coli The method (for example, US 6,372,457, "Process and materials for production of glucosamine"; WO2004/003175, "Process and materials for production of glucosamine and N-acetylglucosamine").

The use of enzymes produced by microorganisms or microorganisms to degrade chitin from crustaceans such as crabs and shrimps to produce N-acetyl-D-glucosamine is relatively traditional, and has low yield, high cost, and insufficient source of animals. problem. A method for producing N-acetyl-D-glucosamine by culturing Chlorella cells infected with Chlorella virus involves a step of crushing cells to obtain N-acetyl-D-glucosamine, which has problems such as complicated operation. A method for fermentative production of N-acetyl-D-glucosamine by directly using glucose as a carbon source, and having the advantage of not using a carbon source such as chitin or chitin oligosaccharide produced from crustacean shell or fungal residue, but Fungi such as Trichoderma has low fermentation temperature (27 ° C), long time (10 days), and low yield (15 mg/ml), which has the disadvantages of long production cycle, high cost, easy contamination of bacteria, and severely limits the method. Industrial application.

Obviously, the production of N-acetyl-D-glucosamine by genetically modified microorganisms is an important application method for large-scale industrial production in view of the growing market demand for glucosamine. New genetically modified microorganisms can be obtained in many ways, such as genetic recombination, gene transfer, gene mutation, gene deletion, gene overexpression, and metabolic pathway changes.

Methods and materials for the production of D-glucosamine by microbial fermentation are disclosed in U.S. Patent No. 6,372,457. The invention includes genetically modified microorganisms for use in the method of producing glucosamine of the invention, as well as recombinant nucleic acid molecules and proteins produced by the recombinant nucleic acid molecules. The genetically modified microorganism of the invention is primarily directed to genetic modifications that increase glucosamine-6-phosphate synthase activity, including multiple gene mutations or amino acid deletions and substitutions. However, this patent does not involve an increase or decrease in glucosamine-6-phosphate synthase activity by changes such as endogenous glucosamine-6-phosphate synthase gene promoter replacement or deletion. In addition, the patent mainly produces D-glucosamine by genetic modification of glucosamine-6-phosphate synthase, and does not involve N-acetyl-D-glucosamine production. Moreover, since D-glucosamine is very unstable in the fermentation broth, the degradation products may be toxic to microorganisms. The production of D-glucosamine by genetic modification has a low yield and has limitations in practical application.

Biosynthesis methods for the production of D-glucosamine and N-acetyl-D-glucosamine are disclosed in WO2004/003175. The method modifies the microorganism by fermentation to produce glucosamine and/or N-acetyl-D-glucosamine. The invention also discloses genetically modified microorganisms for the production of glucosamine and N-acetyl-D-glucosamine. Further, the invention also describes a method of recovering N-acetyl-D-glucosamine produced by a fermentation process, including a method of producing high-purity N-acetyl-D-glucosamine. The invention also discloses a process for producing D-glucosamine from N-acetyl-D-glucosamine. The genetically modified microorganism of the invention is primarily directed to a genetic modification that increases the activity of glucosamine-6-phosphate acetyltransferase. Yeast ammonia Expression of Glucosamine-6-phosphate Acetyltransferase Gene (GNA1) in E. coli can acetylate glucosamine-6-phosphate to acetylglucosamine-6-phosphate, which has also been reported and confirmed in previous literature (Mio T1, Yamada-Okabe T, Arisawa M, Yamada-Okabe H: Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis, J Biol Chem., 1999 Jan 1;274(1):424-9 .).

Summary of the invention

The present invention is directed to the metabolic pathway of N-acetyl-D-glucosamine, using a novel genetic modification method to transform microorganisms, and using the microorganism to produce N-acetyl-D-glucosamine (GlcNAc) and/or with higher efficiency and higher yield. Or D-glucosamine salt.

Specifically, the present invention enhances UDP in microorganisms by increasing the action of UDP-N-acetyl-D-glucosamine-2-epimerase (WecB) in microorganisms. -N-acetyl-D-glucosamine (UDP-GlcNAc) is converted to N-acetyl-D-mannosamine (ManNAc), thereby Microorganisms produce N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salts with higher efficiency and higher yield.

The present invention further relates to one or more of the following contents based on the above content:

1. By increasing the action of D-Glucosamine-6-phosphate deaminase (NagB) in microorganisms, it is preferred to simultaneously reduce Glucosamine-6-phosphate synthase (Glucosamine-6-phosphate synthase). , GlmS, also known as L-glutamine-6-phosphate fructose aminotransferase, enhances the glucose-6-phosphate (Glcose) in the microorganism. -6-P) is aminated to D-glucosamine-6-phosphate (GlcN-6-P). The reaction catalyzed by D-glucosamine-6-phosphate deaminase (NagB) is reversible, and the reaction catalyzed by glucosamine-6-phosphate synthase (GlmS) is irreversible, but has serious product inhibition problems. When the NagB-catalyzed reaction proceeds to the direction in which GlcN-6-P is produced by Glc-6-P, it has the same function as GlmS, and can replace GlmS without product inhibition problems. Increasing the effect of NagB, accelerating the NagB catalytic reaction to GlcN-6-P from Glc-6-P, preferably simultaneously reducing the effect of GlmS, attenuating the product inhibition problem of GlmS, and achieving the purpose of increasing GlcN-6-P, This allows the microorganism to produce N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt with higher efficiency and higher yield.

2. By increasing the glucosamine-6-phosphate synthase (GlmS, also known as L-glutamine-6-phosphate fructose aminotransferase, L-glutamine: D-fructose-6-phosphate The role of aminotransferase, and at the same time reduce the role of D-Glucosamine-6-phosphate deaminase (NagB), strengthen the glucose-6-phosphate (Glcose-6-phosphate, Glc) -6-P) is aminated to D-glucosamine-6-phosphate (GlcN-6-P). The reaction catalyzed by D-glucosamine-6-phosphate deaminase (NagB) is reversible. When the NagB-catalyzed reaction proceeds to the direction of Glc-6-P produced by GlcN-6-P, it is opposite to GlmS. Will offset the role of GlmS. Decreasing the effect of NagB, preventing the NagB catalytic reaction from proceeding to the Glc-6-P production by GlcN-6-P, and simultaneously overexpressing GlmS, accelerating GlmS-catalyzed Glc-6-P amination to GlcN-6-P, The purpose of GlcN-6-P is increased to allow the microorganism to produce N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt with higher efficiency and higher yield.

3. reducing the action of an enzyme or protein in the microorganism associated with the re-intake of the target product into the cell or the degradation of the beneficial intermediate, increasing the sugar conversion rate and the N-acetyl-D-glucosamine production in the microorganism, thereby allowing the microorganism to Higher efficiency and higher yields produce N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salts. Including but not limited to one or more of the following:

(1) Reduce the role of the mannose transporter EIIM, P/III ^man (Mannose transporter EIIM, P/III ^Man , ManXYZ) in the microorganism, and prevent the transport of hexose such as N-acetyl-D-glucosamine (GlcNAc) back into the cell. degradation.

(2) Reducing the action of N-acetylneuraminate lyase (NanA) in microorganisms to prevent degradation of N-acetyl-D-mannosamine (ManNAc) in microorganisms.

(3) Reducing the action of N-acetyl-glucoseamine-6-phosphate deacetylase (NagA) in microorganisms to prevent N-acetyl-D-glucosamine-6- in microorganisms Phosphoric acid (N-acetyl-D-glucosamine-6-phosphate, GlcNAc-6-P) was converted to D-glucosamine-6-phosphate (GlcN-6-P).

(4) reducing microbial acting N- acetyl -D- glucosamine-specific enzyme ^{II Nag (N-acetylglucosamine specific enzyme} II Nag, NagE) , preventing -D- N- acetyl glucosamine (GIcNAc) were transferred to microbial cells degradation.

(5) by increasing the phosphoglucosamine mutase in the microorganisms (phosphoglucosamine mutase, The role of GlmM) is to strengthen the conversion of D-glucosamine-6-phosphate (GlcN-6-P) into D-glucosamine-1-phosphate. GlcN-1-P).

(6) by increasing the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine transferase (bifunctional N-acetyl glucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyl transferase, GlmU) The role of strengthening D-glucosamine-1-phosphate (GlcN-1-P) in microorganisms to N-acetyl-D-glucosamine-1-phosphate (N-acetyl-D-glucosamine) 1-phosphate, GlcNAc-1-P), and further converted to UDP-N-acetyl-D-glucosamine (UDP-GlcNAc).

According to one embodiment of the invention, the invention relates to a method for the production of N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:

A) cultivating a microorganism in a fermentation medium comprising at least one genetic modification capable of increasing the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism;

B) Collection of N-acetyl-D-glucosamine (GlcNAc) produced from the cultivation step A).

Preferably, further comprising C) deacetylating from N-acetyl-D-glucosamine (GlcNAc) to give a D-glucosamine salt.

In the present invention, the genetic modification for increasing the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in microorganisms is selected from a) UDP-N-acetyl-D-glucosamine-2- in the microorganism The enzymatic activity of the isomerase (WecB) is increased; and/or b) UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is overexpressed in the microorganism.

It will be understood by those skilled in the art that in order to enhance the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in microorganisms, it can be screened and encoded by UDP-N-acetyl-D-glucosamine-2. - Gene mutants with increased enzymatic activity of the isomerase (WecB) are achieved. Screening for WecB gene mutants can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes. In order to enhance the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in microorganisms, it is also possible to increase the number of copies of the gene and replace the promoter with a higher expression level than the native promoter. This was achieved by overexpressing UDP-N-acetyl-D-glucosamine-2-isomerase (WecB). In a specific embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism.

In a preferred embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB).

In one aspect, the nucleic acid sequence encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) contains at least one increase in UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) Genetic modification of the enzyme activity). Preferably, the genetic modification comprises one or more of substitutions at positions corresponding to the amino acid sequence of SEQ ID NO: 17: the 34th cysteine is substituted with serine and the 145th histidine is Aspartic acid substitution, 226th cysteine substituted by phenylalanine and 245 valine substituted by glycine; more preferably, encoding said UDP-N-acetyl-D-glucosamine-2-isomer The nucleic acid sequence of the enzyme (WecB) is SEQ ID NO:26; the amino acid sequence of the UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is SEQ ID NO:27.

In another aspect, the UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) has at least about 30% identical, preferably at least about 50% identical to the amino acid sequence of SEQ ID NO:17, Further preferably at least about 70% identical, further preferably at least about 80% identical, still more preferably at least about 90% identical, most preferably at least about 95% identical amino acid sequence, wherein said UDP-N-acetyl-D-glucosamine -2-isomerase (WecB) has enzymatic activity.

In another aspect, the UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) has the amino acid sequence of SEQ ID NO:17.

In another aspect, the gene copy number encoding the UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the recombinant nucleic acid molecule is increased.

In another aspect, the recombinant nucleic acid molecule comprises an endogenous native promoter, a promoter having a higher expression level than the endogenous native promoter, an enhancer, a fusion sequence, and the like. Preferably, the recombinant nucleic acid molecule comprises a promoter having a higher expression level than the endogenous natural promoter, such as an HCE promoter, a gap promoter, a trc promoter, a T7 promoter, etc.; more preferably, the recombinant nucleic acid molecule comprises a trc promoter. child. The trc promoter is a split promoter of the trp promoter and the lac promoter, which has higher transcription efficiency than trp and strong promoter properties regulated by lacI repressor.

In the present invention, the recombinant nucleic acid molecule is transformed into a microorganism selected from the group consisting of a free form (i.e., a recombinant nucleic acid molecule is loaded into a plasmid) and an integrated type (i.e., a recombinant nucleic acid molecule is integrated into the genome of the microorganism). Preferably, the recombinant nucleic acid molecule is integrated into the genome of the microorganism.

In another preferred embodiment, the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB). Preferably, The endogenous native promoter of the gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is replaced by a promoter with a higher expression level, such as the HCE promoter, gap promoter, trc Promoter, T7 promoter, etc.; more preferably, the endogenous native promoter of the gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is replaced by the trc promoter.

According to a preferred embodiment of the invention, the microorganism further comprises one or more of the following genetic modifications:

(1) comprising at least one genetic modification capable of enhancing the action of D-glucosamine-6-phosphate deaminase (NagB) in a microorganism, preferably comprising at least one molecule capable of reducing glucosamine-6-phosphate synthase (GlmS) Genetic modification

(2) comprising at least one genetic modification capable of increasing the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism, and at the same time comprising at least one capable of reducing D-glucosamine-6-phosphate deaminase (NagB) Genetic modification.

In the above aspect (1), the genetic modification for enhancing the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism is selected from the group consisting of a) D-glucosamine-6-phosphate deaminase (NagB) in the microorganism. The enzyme activity is increased; and/or b) D-glucosamine-6-phosphate deaminase (NagB) is overexpressed in the microorganism.

It will be understood by those skilled in the art that in order to enhance the action of D-glucosamine-6-phosphate deaminase (NagB) in microorganisms, the enzyme activity encoding D-glucosamine-6-phosphate deaminase (NagB) can be screened. Increased gene mutants are achieved. Screening for NagB gene mutants can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes. In order to enhance the action of D-glucosamine-6-phosphate deaminase (NagB) in microorganisms, it is also possible to overexpress D-amino by increasing the number of copies of the gene and replacing a promoter with a higher expression level than the native promoter. Glucose-6-phosphate deaminase (NagB) is achieved. In a specific embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.

In a preferred embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding D-glucosamine-6-phosphate deaminase (NagB).

In one aspect, the nucleic acid sequence encoding D-glucosamine-6-phosphate deaminase (NagB) contains at least one genetic modification that increases the enzymatic activity of D-glucosamine-6-phosphate deaminase (NagB).

In another aspect, the gene copy number encoding D-glucosamine-6-phosphate deaminase (NagB) is increased in the recombinant nucleic acid molecule.

In another aspect, the recombinant nucleic acid molecule comprises an endogenous native promoter, a promoter having a higher expression level than the endogenous native promoter, an enhancer, a fusion sequence, and the like. Preferably, the recombinant nucleic acid molecule comprises a promoter having a higher expression level than the endogenous natural promoter, such as an HCE promoter, a gap promoter, a trc promoter, a T7 promoter, etc.; more preferably, the recombinant nucleic acid molecule comprises a trc promoter. child.

In another preferred embodiment, the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding D-glucosamine-6-phosphate deaminase (NagB). Preferably, the endogenous native promoter of the gene encoding D-glucosamine-6-phosphate deaminase (NagB) is replaced by a promoter with a higher expression level, such as the HCE promoter, the gap promoter, the trc promoter, The T7 promoter or the like; more preferably, the endogenous natural promoter of the gene encoding D-glucosamine-6-phosphate deaminase (NagB) is replaced by the trc promoter.

In the present invention, the genetic modification for reducing the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism is selected from a) a decrease in the enzymatic activity of glucosamine-6-phosphate synthase (GlmS) in the microorganism; and/or b) Reduced expression of glucosamine-6-phosphate synthase (GlmS) in microorganisms, including but not limited to: partial or complete deletion of the endogenous gene encoding glucosamine-6-phosphate synthase (GlmS) in the microorganism, or partial or Completely inactivated, and/or partially or completely deleted, or partially or completely inactivated, of an endogenous natural promoter encoding a glucosamine-6-phosphate synthase (GlmS) gene in a microorganism. Preferably, the genetic modification to reduce the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism is that the endogenous natural promoter encoding the glucosamine-6-phosphate synthase (GlmS) gene in the microorganism is completely deleted, ie, deleted.

In a specific embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism.

In the above aspect (2), the genetic modification for increasing the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism is selected from a) an increase in the enzymatic activity of glucosamine-6-phosphate synthase (GlmS) in the microorganism; And/or b) the glucosamine-6-phosphate synthase (GlmS) is overexpressed in the microorganism.

It will be understood by those skilled in the art that in order to enhance the action of glucosamine-6-phosphate synthase (GlmS) in microorganisms, it is possible to screen for a gene mutant encoding an increase in enzymatic activity of glucosamine-6-phosphate synthase (GlmS). achieve. Screening of GlmS gene mutants can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes. To increase glucosamine-6-phosphate synthase (GlmS) in microorganisms The effect can also be achieved by overexpressing glucosamine-6-phosphate synthase (GlmS) by increasing its gene copy number and replacing a promoter with a higher expression level than the native promoter. In a specific embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism.

In a preferred embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding glucosamine-6-phosphate synthase (GlmS).

In one aspect, the nucleic acid sequence encoding glucosamine-6-phosphate synthase (GlmS) contains at least one genetic modification that increases the enzymatic activity of glucosamine-6-phosphate synthase (GlmS).

In another aspect, the gene copy number encoding the glucosamine-6-phosphate synthase (GlmS) is increased in the recombinant nucleic acid molecule.

In another preferred embodiment, the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding glucosamine-6-phosphate synthase (GlmS). Preferably, the endogenous native promoter of the gene encoding glucosamine-6-phosphate synthase (GlmS) is replaced by a promoter with a higher expression level, such as the HCE promoter, the gap promoter, the trc promoter, the T7 promoter. More preferably, the endogenous native promoter of the gene encoding glucosamine-6-phosphate synthase (GlmS) is replaced by a trc promoter.

In the present invention, the genetic modification for reducing the action of D-glucosamine-6-phosphate deaminase (NagB) in microorganisms is selected from a) the reduction of the enzyme activity of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism. And/or b) reduced expression of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism, including but not limited to: encoding D-glucosamine-6-phosphate deaminase (NagB) in the microorganism Partial or complete deletion, or partial or complete inactivation of the gene, and/or partial or complete deletion of the endogenous natural promoter of the D-glucosamine-6-phosphate deaminase (NagB) gene encoding the microorganism, Partially or completely inactivated. Preferably, the genetic modification of reducing the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism is The endogenous natural promoter of the D-glucosamine-6-phosphate deaminase (NagB) gene in the code microorganism is completely deleted, ie, deleted.

In a specific embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.

(1) comprising at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III ^man (ManXYZ) in the microorganism;

(2) comprising at least one genetic modification capable of reducing the action of N-acetylneuraminic acid lyase (NanA) in the microorganism;

(3) comprising at least one genetic modification capable of reducing the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism;

(4) comprising at least one genetic modification capable of reducing the action of N-acetyl-D-glucosamine specific enzyme II ^Nag (NagE) in microorganisms;

(5) comprising at least one genetic modification capable of increasing the action of phosphoglucosamine mutase (GlmM) in the microorganism;

(6) comprising at least one genetic modification capable of enhancing the action of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in microorganisms.

In the above aspect (1), the genetic modification of reducing the action of the mannose transporter EIIM, P/III ^man (ManXYZ) in the microorganism includes, but is not limited to, encoding the mannose transporter EIIM, P/III ^man (ManXYZ) Partial or complete deletion, or partial or complete inactivation of the endogenous gene, and/or partial or complete encoding of the endogenous natural promoter of the mannose transporter EIIM, P/III ^man (ManXYZ) gene in the microorganism Missing, or partially or completely inactivated. Preferably, mannose transporter EIIM reducing microorganisms, genetic P / III ^man (ManXYZ) acting microorganism modified to encode the mannose transporter EIIM, the complete absence of P / III ^man (ManXYZ) an endogenous gene, is deleted . In a specific embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of the mannose transporter EIIM, P/III ^man (ManXYZ) in the microorganism.

In the above aspect (2), the genetic modification for reducing the action of N-acetylneuraminic acid lyase (NanA) in the microorganism includes, but is not limited to, N-acetylneuraminic acid lyase (NanA) in the encoded microorganism. Partial or complete deletion, or partial or complete inactivation of the endogenous gene, and/or partial or complete deletion of the endogenous natural promoter of the N-acetylneuraminic acid lyase (NanA) gene in the microorganism, or Partially or completely inactivated. Preferably, the genetic modification to reduce the action of N-acetylneuraminic acid lyase (NanA) in the microorganism is that the endogenous gene encoding the N-acetylneuraminic acid lyase (NanA) in the microorganism is completely deleted, ie, deleted. In a specific embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of N-acetylneuraminic acid lyase (NanA) in the microorganism.

In the above aspect (3), the genetic modification for reducing the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism includes, but is not limited to, encoding the N-acetyl-D-amino group in the microorganism. Partial or complete deletion, or partial or complete inactivation of the endogenous gene of glucose-6-phosphate deacetylase (NagA), and/or N-acetyl-D-glucosamine-6-phosphate deacetylase in the coding microorganism The endogenous natural promoter of the (NagA) gene is partially or completely deleted, or partially or completely inactivated. Preferably, the genetic modification to reduce the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism is to encode N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism. The endogenous gene is completely deleted and is deleted. In a specific embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism.

In the above aspect (4), the genetic modification for reducing the action of the N-acetyl-D-glucosamine specific enzyme II ^Nag (NagE) in the microorganism includes, but is not limited to, encoding the N-acetyl-D-glucosamine specific enzyme in the microorganism Partial or complete deletion, or partial or complete inactivation of the endogenous gene of II ^Nag (NagE), and/or endogenous natural coding of the N-acetyl-D-glucosamine specific enzyme II ^Nag (NagE) gene in the microorganism Partial or complete deletion of the promoter, or partial or complete inactivation. Preferably, the reducing microbial genetic -D- N- acetyl-glucosamine-specific enzyme II ^Nag (NagE) acting microorganism modified to encode the complete absence of N- acetyl -D- glucosamine-specific enzyme II ^Nag (NagE) an endogenous gene That is deleted. In a specific embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of the N-acetyl-D-glucosamine specific enzyme II ^Nag (NagE) in the microorganism.

In the above aspect (5), the genetic modification for increasing the action of the phosphoglucosamine mutase (GlmM) in the microorganism is selected from a) an increase in the enzymatic activity of the phosphoglucosamine mutase (GlmM) in the microorganism; and / Or b) the phosphoglucosamine mutase (GlmM) is overexpressed in the microorganism.

It will be understood by those skilled in the art that in order to enhance the action of phosphoglucosamine mutase (GlmM) in microorganisms, it is possible to screen for a gene encoding an increase in enzyme activity having a glucosamine glucosamine mutase (GlmM). Mutants are implemented. Screening for the GlmM gene mutant can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes. In order to enhance the action of phosphoglucosamine mutase (GlmM) in microorganisms, it is also possible to overexpress phosphoglucosamine mutase by increasing its gene copy number and replacing a promoter with a higher expression level than the native promoter. (GlmM) to achieve. In a specific embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of phosphoglucosamine mutase (GlmM) in the microorganism.

In a preferred embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a phosphoglucosamine mutase (GlmM).

In one aspect, the nucleic acid sequence encoding a phosphoglucosamine mutase (GlmM) contains at least one genetic modification that increases the enzymatic activity of a phosphoglucosamine mutase (GlmM).

In another aspect, the gene copy number encoding the phosphoglucosamine mutase (GlmM) is increased in the recombinant nucleic acid molecule.

In another preferred embodiment, the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding a phosphoglucosamine mutase (GlmM). Preferably, the endogenous native promoter encoding the gene for phosphoglucosamine mutase (GlmM) is replaced by a promoter with a higher expression level, such as the HCE promoter, gap promoter, trc promoter, T7 promoter, etc. More preferably, the endogenous native promoter of the gene encoding the phosphoglucosamine mutase (GlmM) is replaced by the trc promoter.

In the above aspect (6), the genetic modification for enhancing the action of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in the microorganism is selected from a) the bifunctional enzyme N in the microorganism - an increase in the enzymatic activity of acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU); and/or b) bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine transfer in microorganisms The enzyme (GlmU) is overexpressed.

It will be understood by those skilled in the art that in order to enhance the action of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in the microorganism, it can be screened and encoded by the bifunctional enzyme N-B. A gene mutant with increased enzymatic activity of acyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) is achieved. Screening for the GlmU gene mutant can be accomplished by error-producing PCR techniques to obtain high frequency mutant genes. In order to increase the role of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in microorganisms, it is also possible to increase the expression level of the gene by changing its gene copy number and replacing it with a higher expression level than the native promoter. The promoter is expressed by overexpressing the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU). In a specific embodiment, the microorganism comprises at least one genetically modified recombinant nucleic acid comprising at least one effect of enhancing the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in the microorganism Molecular transformation.

In a preferred embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU).

In one aspect, the nucleic acid sequence encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) contains at least one additional bifunctional enzyme N-acetyl-D-glucosamine-1- Genetic modification of the enzymatic activity of phosphouridine syltransferase (GlmU).

In another aspect, the gene copy number encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) is increased in the recombinant nucleic acid molecule.

In another preferred embodiment, the microorganism comprises at least one inheritance of an endogenous natural promoter of a gene encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) Modification. Preferably, the endogenous native promoter of the gene encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) is replaced by a promoter with a higher expression level, such as the HCE promoter. , a gap promoter, a trc promoter, a T7 promoter, etc.; more preferably, an endogenous natural promoter encoding a gene of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) Replaced by the trc promoter.

The invention further relates to the following preferred embodiments:

1. According to a preferred embodiment of the invention, the invention relates to a process for the production of N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the process comprising:

A) cultivating a microorganism in a fermentation medium comprising: at least one genetic modification capable of increasing the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism; and at least one Genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase (NagB) in microorganisms;

Preferably, the microorganism further comprises at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase (GlmS).

2. According to another preferred embodiment of the present invention, the present invention relates to a method for producing N-acetyl-D-glucosamine (GlcNAc) and/or D-glucosamine salt by microbial fermentation, the method comprising:

A) cultivating the microorganism in a fermentation medium comprising: at least one genetic modification capable of increasing the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism; at least one capable a genetic modification that enhances the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism; and at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase (NagB);

In a preferred embodiment of the above, further comprising C) deacetylating from N-acetyl-D-glucosamine (GlcNAc) to give a D-glucosamine salt.

In the above preferred embodiment, the microorganism further comprises: at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III ^man (ManXYZ) in the microorganism; at least one capable of reducing the N-acetyl nerve in the microorganism Genetic modification of the action of lyase (NanA); at least one genetic modification that reduces the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in microorganisms; and at least one that reduces microbial activity Genetic modification of the action of N-acetyl-D-glucosamine specific enzyme II ^Nag (NagE).

In one aspect of any of the above embodiments, the expression of any of the above recombinant nucleic acid molecules is inducible, including, but not limited to, induced by lactose, for example, lactose-induced expression can be achieved by the addition of lactose or the like to the culture broth.

Those skilled in the art will appreciate that various conventional fermentation media known in the art can be used in the present invention. In one aspect, the fermentation medium contains a source of carbon. In another aspect, the fermentation medium comprises a source of nitrogen. In another aspect, the fermentation medium comprises a source of carbon and a source of nitrogen. In another aspect, the fermentation medium comprises a carbon source, a nitrogen source, and an inorganic salt.

Those skilled in the art will appreciate that a variety of carbon sources known in the art can be used in the present invention, including organic carbon sources and/or inorganic carbon sources. Preferably, the carbon source is selected from one or more of the group consisting of glucose, fructose, sucrose, galactose, dextrin, glycerin, starch, syrup, and molasses. Preferably, the concentration of the carbon source is maintained from about 0.1% to about 5%. Those skilled in the art will appreciate that a variety of nitrogen sources known in the art can be used in the present invention, including organic nitrogen sources and/or inorganic nitrogen sources. Preferably, the nitrogen source is selected from the group consisting of ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, sodium nitrate, urea, yeast extract, meat extract, peptone, fish meal, soy flour, malt, corn syrup and cottonseed meal. One or more.

Preferably, the present invention employs a fed fermentation process. According to an aspect of the invention, the sugar-retaining liquid comprises glucose and ribose, preferably, the glucose concentration is 10%-85% (w/v), the ribose concentration is 0.5%-15% (w/v), further preferably, the glucose concentration 55%-75% (w/v), ribose concentration is 5%-7% (w/v); according to another aspect of the invention, the sugar-containing solution comprises glucose and gluconate, preferably, the glucose concentration is 10 %-85% (w/v), gluconate concentration 0.5%-15% (w/v), further preferably, glucose concentration 55%-75% (w/v), gluconate concentration 2% - 3% (w/v); According to another aspect of the invention, the sugar-comprising solution comprises glucose, ribose and gluconate, preferably having a glucose concentration of 10%-85% (w/v) and a ribose concentration of 0.5. %-15% (w/v), gluconate concentration 0.5%-15% (w/v), further preferably, glucose concentration 55%-75% (w/v), ribose concentration 5%-7 %(w/v), gluconate concentration is 2%-3% (w/v). Preferably, the gluconate is sodium gluconate.

In a preferred embodiment, the culturing step is carried out at a temperature of from about 20 ° C to about 45 ° C, and more preferably, the culturing step is carried out at a temperature of from about 33 ° C to about 37 ° C.

In a preferred embodiment, the culturing step is carried out at a pH of from about 4.5 to about pH 8.5. In one aspect, the culturing step is carried out at a pH of from about 6.7 to about pH 7.2.

Those skilled in the art will appreciate that N-acetyl-D-glucosamine (GlcNAc) can be collected in the present invention using various conventional methods known in the art. Preferably, N-acetyl-D-glucosamine can be collected from the extracellular product in the fermentation medium. Further preferably, the collecting step comprises the step of: (a) precipitating N-acetyl-D-glucosamine from the fermentation broth for removing microorganisms; (b) crystallizing N-acetyl-D- from the fermentation broth for removing microorganisms Glucosamine.

According to the invention, the collecting step further comprises the step of decolorizing the fermentation broth. The decolorization step may include, but is not limited to, performing prior to precipitation or crystallization of the fermentation broth, after one or more precipitation or crystallization re-dissolution of the fermentation broth, including decolorization including activated carbon treatment and/or chromatographic decolorization. The chromatographic decolorization comprises the step of contacting the fermentation broth with an ion exchange resin, including but not limited to an anion exchange resin and/or a cation exchange resin, for example, contacting the fermentation broth with a mixed bed of anion and cation exchange resin.

According to the present invention, D-glucosamine salts can be obtained by deacetylating N-acetyl-D-glucosamine, including but not limited to hydrochlorides, sulfates, sodium salts, phosphates, hydrogen sulfates and the like. For example, N-acetyl-D-glucosamine can be deacetylated under acidic and heated conditions to obtain a D-glucosamine salt, preferably in a 30% to 37% hydrochloric acid solution at 60 ° C to 90 ° C for deacetylation hydrolysis. N-acetyl-D-glucosamine gives D-glucosamine hydrochloride; it can also hydrolyze N-acetyl-D-glucosamine under the action of UDP-3-ON-acetylglucosamine deacetylase to obtain D-glucosamine. And further into salt.

According to another embodiment of the present invention, the present invention relates to a microorganism comprising at least one genetic modification capable of enhancing the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in a microorganism . This genetic modification has been described in detail above.

(2) comprising at least one genetic modification capable of increasing the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism, and at the same time comprising at least one capable of reducing D-glucosamine-6-phosphate deaminase (NagB) Genetic modification. These genetic modifications have been described in detail above.

(6) comprising at least one genetic modification capable of enhancing the action of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in microorganisms. These genetic modifications have been described in detail above.

The invention further relates to the following preferred embodiments:

1. According to a preferred embodiment of the invention, the invention relates to a microorganism comprising: at least one capable of enhancing the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in a microorganism Genetic modification; and at least one genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase (NagB) in the microorganism.

2. According to another preferred embodiment of the present invention, the present invention relates to a microorganism comprising: at least one capable of increasing UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in a microorganism Genetic modification of action; at least one genetic modification that increases the action of glucosamine-6-phosphate synthase (GlmS) in the microorganism; and at least one that reduces the action of D-glucosamine-6-phosphate deaminase (NagB) Genetic modification.

According to another embodiment of the present invention, the present invention relates to a UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) having a higher enzymatic activity, which has the SEQ ID NO: 27 Amino acid sequence. The invention further relates to a nucleic acid molecule encoding the above UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) having the nucleic acid sequence set forth in SEQ ID NO:26. The invention further relates to a vector comprising the above nucleic acid molecule. The invention further relates to a microorganism comprising the above vector. The invention further relates to a microorganism comprising the above nucleic acid molecule in the genome.

In the present invention, the microorganism may be any microorganism (for example, a bacterium, a protist, an alga, a fungus, or other microorganisms). In a preferred embodiment, the microorganism includes, but is not limited to, bacteria, yeast or fungi. Preferably, the microorganism is selected from the group consisting of bacteria or yeast. Further preferably, the bacterium includes, but is not limited to, a genus selected from the group consisting of Escherichia, Bacillus, Lactobacillus, Pseudomonas, or Streptomyces. More preferably, the bacteria include, but are not limited to, selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Lactobacillus brevis, Pseudomonas aeruginosa ( Pseudomonas aeruginosa) or a species of Streptomyces lividans. Further preferably, the yeast includes, but is not limited to, selected from the group consisting of Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, and gram. Yeast of Kluveromyces and Phaffia; more preferably, yeast includes, but is not limited to, Saccharomyce scerevisiae, Schizosaccharo mycespombe, Candida. Albicans), Hansenula polymorpha, Pichia pastoris, Pichia canadensis, Kluyveromyces marxianus or Phaffia rohodozyma. Preferably, the microorganism is a fungus; further preferably, the fungus includes, but is not limited to, selected from the group consisting of Aspergillus, Absidia, Rhizopus, Chrysosporium, and Neurospora a fungus belonging to the genus Neurospora or Trichoderma; more preferably, the fungus includes, but is not limited to, selected from the group consisting of Aspergillus niger, Aspergillus nidulans, and Absidia coerulea. ), Rhizopus oryzae, Chrysosporium lucknowense, Neurospora crassa, interstitial vein Neurospora intermedia or Trichoderma reesei. Particularly preferred E. coli strains include K-12, B and W, most preferably K-12. Although E. coli is a preferred microorganism and is used as an example of various embodiments of the present invention, it is understood that N-acetyl-D-glucosamine can be used in the method of the invention and can be genetically modified to increase N-acetyl-D. - any other microorganism that produces glucosamine. The microorganism used in the present invention may also be referred to as a production organism.

In the present invention, the term N-acetyl-D-glucosamine may be referred to as 2-acetamido-2-deoxy-D-glucose. The terms N-acetyl-D-glucosamine, N-acetyl-D-glucosamine-6-phosphate and N-acetyl-D-glucosamine-1-phosphate can be abbreviated as GlcNAc, GlcNAc-6-P and GlcNAc-1, respectively. -P. N-acetyl-D-glucosamine is also abbreviated as NAG. Similar to N-acetyl-D-glucosamine and derivatives, the terms D-glucosamine, D-glucosamine-6-phosphate and D-glucosamine-1-phosphate can be abbreviated as GlcN, GlcN-6-P and GlcN, respectively. -1-P. Similarly, the terms N-acetyl-D-aminomannose, N-acetyl-D-aminomannose-6-phosphate, glucose, glucose-6-phosphate, fructose-6-phosphate can be abbreviated as ManNAc, ManNAc-6, respectively. -P, Glc, Glc-6-P, Fru-6-P.

The term increasing the action of an enzyme in a microorganism means that the activity of the enzyme in the microorganism is increased and/or the enzyme is overexpressed, thereby increasing the amount of substrate-producing product catalyzed by the enzyme in the microorganism.

The term reducing the action of an enzyme in a microorganism means that the activity of the enzyme in the microorganism is reduced and/or the expression of the enzyme is reduced, thereby reducing the amount of substrate-producing product catalyzed by the enzyme in the microorganism.

The term increased enzyme activity refers to an increased ability of an enzyme to catalyze a certain chemical reaction. It covers an increase in the ability of the enzyme to self-catalyze a chemical reaction in the event that the enzyme is inhibited by the product and the enzyme has a constant affinity for the substrate, and/or because the enzyme is inhibited by product inhibition and/or the enzyme affinity for the substrate The ability to increase the enzyme-catalyzed chemical reactions is increased. The term enzyme reduced by product inhibition means that the activity of the enzyme catalyzing the reaction is reduced by the specific inhibition of its end product. The term enzyme increased affinity for a substrate refers to an increase in the affinity of the enzyme for the substrate being catalyzed.

Figure 1 illustrates, in the case of E. coli, the main aspects of the genetic modification of the amino sugar metabolic pathway disclosed in the present invention for the large-scale production of N-acetyl-D-glucosamine. With respect to Figure 1, bold arrows indicate that the present invention relates to genetically engineered production and/or increased metabolic flux. Figure 1 discloses several different methods for synthesizing N-acetyl-D-glucosamine, including modifications to WecB, which may further include modifications to NagB, GlmS, or a combination thereof, and may further include ManXYZ, NanA, NagA Modification of NagE, GlmM, GlmU or a combination thereof. Those skilled in the art will appreciate that other microorganisms have classes Similar sugar metabolism pathways, and genes and proteins have similar structures and functions in such pathways. Thus, the invention discussed herein is equally applicable to other microorganisms other than E. coli and other microorganisms are clearly included in the present invention.

Enzymes having the same biological activity are known in the art to have different names depending on which microorganism the enzyme is derived from. The following are alternative names for many of the enzymes referred to herein and specific gene names from some organisms encoding such enzymes. The names of these enzymes may be used interchangeably or, if appropriate, for a given sequence or organism, but the invention is intended to include enzymes from a given function of any organism.

For example, an enzyme generally referred to herein as "N-acetyl-D-aminomannose kinase" catalyzes the phosphorylation of N-acetyl-D-aminomannose to N-acetyl-D-aminomannose-6-P. N-acetyl-D-aminomannose kinase from E. coli is generally referred to as NanK. N-acetyl-D-aminomannose kinases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention.

The enzyme generally referred to herein as "N-acetyl-D-aminomannose-6-P isomerase" catalyzes the conversion of N-acetyl-D-aminomannose-6-P to N-acetyl-D-glucosamine- 6-P. The N-acetyl-D-aminomannose-6-P isomerase from E. coli is generally referred to as NanE. N-acetyl-D-aminomannose-6-P isomerases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention.

An enzyme generally referred to herein as "UDP-N-acetyl-D-glucosamine-2-isomerase" catalyzes the conversion of UDP-N-acetyl-D-glucosamine to N-acetyl-D-aminomannose. UDP-N-acetyl-D-glucosamine-2-isomerase from E. coli is generally referred to as WecB. UDP-N-acetyl-D-glucosamine-2-isomerases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention. For example, it is described herein that UDP-N-acetyl-D-glucosamine-2-isomerase from Escherichia coli has the amino acid sequence encoded by the nucleic acid sequence represented by SEQ ID NO: 16, and represented by SEQ ID NO: 17.

An enzyme generally referred to herein as "D-glucosamine-6-phosphate deaminase" catalyzes the reversible reaction of D-glucosamine-6-phosphate with water to form glucose-6-phosphate and ammonium. The enzyme is also known as D-glucosamine-6-phosphate isomerase, GlcN6P deaminase, D-glucosamine isomerase, D-glucosamine isomerase, D-glucosamine phosphate deaminase and 2-amino-2-deoxy-D-glucose-6-phosphate ethyl ketone alcohol isomerase (deamination). D-glucosamine-6-phosphate deaminase from various organisms is well known in the art and can be used in the genetic engineering strategies of the present invention. In E. coli and other bacteria, the enzyme is generally referred to as NagB.

An enzyme generally referred to herein as "D-glucosamine-6-phosphate synthase" catalyzes the formation of D-glucosamine-6-phosphate and glutamic acid from glucose-6-phosphate and glutamine. The enzyme is also called D-glucosamine-fructose-6-phosphate aminotransferase (isomerization), hexose phosphate aminotransferase, D-fructose-6-phosphate transamidase, D- Glucosamine-6-phosphate isomerase (formation of glutamine), L-glutamine-fructose-6-phosphate transamidase and GlcN6P synthase. D-glucosamine-6-phosphate synthase from various organisms is well known in the art and can be used in the genetic engineering strategies of the present invention. D-glucosamine-6-phosphate synthase from E. coli and other bacteria is generally referred to as GlmS.

An enzyme generally referred to herein as "N-acetyl-D-glucosamine-6-phosphate deacetylase" hydrolyzes N-acetyl-D-glucosamine-6-phosphate to D-glucosamine-6-phosphate and acetate . N-acetyl-D-glucosamine-6-phosphate deacetylases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention. For example, a method from E. coli called NagA is described herein.

An enzyme generally referred to herein as "N-acetylneuraminic lyase" catalyzes the degradation of N-acetyl-D-aminomannose to N-acetylneuraminic acid. N-acetylneuraminic lyases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention. For example, an N-acetylneuraminic lyase from E. coli is described herein as NanoA.

An enzyme generally referred to herein as "phosphoglucosamine mutase" catalyzes the conversion of D-glucosamine-6-phosphate to D-glucosamine-1-phosphate. Phospho D-glucosamine mutases from various organisms are well known in the art and can be used in the genetic engineering strategies of the present invention. The phosphoglucosamine mutase of this enzyme in Escherichia coli and other bacteria is generally referred to as GlmM.

An enzyme generally referred to herein as "D-glucosamine-1-phosphate N-acetyltransferase" converts D-glucosamine-1-phosphate and acetyl-CoA to N-acetyl-D-glucosamine-1-phosphate, and Release the CoA. As a bifunctional enzyme, it also functions as N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase, also known as UDP-N-acetyl-D-glucosamine pyrophosphorylase, UDP- N-acetyl-D-glucosamine diphosphatase further converts N-acetyl-D-glucosamine-1-phosphate to UDP-N-acetyl-D-glucosamine. D-glucosamine-1-phosphate N-acetyltransferase and N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase from various organisms are well known in the art and can be used in the inheritance of the present invention In the transformation strategy. This enzyme is called GlmU in E. coli and other bacteria.

The "Trc Promoter" has been subtly designed for prokaryotic expression, such as the E. coli expression system. Trc promoters are well known in the art and can be used in the genetic engineering strategies of the present invention. For example, the Trc promoter described herein has the nucleotide sequence represented by SEQ ID NO:28.

As disclosed in the WO2004/003175 invention, D-glucosamine is extremely unstable in the general pH range for E. coli growth. D-glucosamine and/or its degradation products have toxic effects on the strain. D-glucosamine at a concentration as low as 20g/L in culture medium (pH7.0) even before cell seeding Toxicity was also observed when pre-incubation for 3.5 hours. Toxicity is at least in part due to D-glucosamine degradation products in the medium with a starting pH of 7.0. GlcN is more stable at lower pH conditions and D-glucosamine does not degrade below pH 4.7. However, E. coli grows slowly at pH conditions below 6-7. Therefore, the scheme of performing D-glucosamine production in a fermentor at a relatively low pH is difficult to perform.

According to the present invention, Glc-6-P becomes D-glucosamine-6-P (GlcN-6-P) catalyzed by NagB and GlmS in cells, and further catalyzes the formation of UDP-N-acetyl group in GlmM and GlmU. -D-glucosamine (UDP-GlcNAc), converted to N-acetyl-D-aminomannose (ManNAc) under the catalysis of UDP-N-acetyl-glucosamine-2-isomerase (WecB), further converted to N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P) is phosphorylated by the action of phosphatase and is excreted into N-acetyl-D-glucosamine (GlcNAc). The method of the present invention avoids the formation of D-glucosamine, thereby avoiding the toxic effects of D-glucosamine and/or its degradation products on the strain.

Therefore, the present invention has the beneficial effects that the present invention proves that the completely natural N-acetyl-D-glucosamine can be directly produced by the microbial fermentation method; the new production method has no risk of heavy metal pollution, no antibiotics, drug residue risk, and production is not affected. Influence of raw material supply, long-term stable production, high yield and low cost; N-acetyl-D-glucosamine and D-glucosamine products produced are non-animal, chitin without shrimp shell, glucose, etc. Carbon source fermentation, is a vegetarian product, and an allergen of anhydrous products.

The entire disclosures and references cited or described herein are hereby incorporated by reference.

DRAWINGS

Figure 1 N-acetyl-D-glucosamine biosynthesis pathway and metabolic engineering strategy in Escherichia coli

detailed description

The invention will be further described in detail below with reference to specific embodiments. The following examples are merely illustrative of the invention and are not to be construed as limiting the scope of the invention. The technology implemented based on the present invention is intended to be within the scope of the present invention.

The starting materials and reagents used in the examples are commercially available unless otherwise stated.

The following is a list of various genetically modified microorganisms of the present invention relating to and/or described.

Example 1

This example describes the construction of an E. coli mutant that blocks the metabolic pathway associated with uptake of N-acetyl-D-glucosamine and degradation of beneficial intermediates.

The parent strain of the production strain was AT-001 (Escherichia coli ATCC 27325) belonging to the E. coli K-12 derivative from the American Type Culture Collection.

Blocking the N-acetyl-D-glucosamine uptake and degradation of intermediate metabolites can reduce the loss in the metabolic process and increase the accumulation of the target product (N-acetyl-D-glucosamine).

Construction of this mutant host strain can be made by completely or partially deleting the humanXYZ, nanA, nagA and nagE gene sequences on its chromosomal genome, thereby rendering N-acetyl-D-amino Glucose accumulation.

The deletion of the gene sequence on this chromosome can be done using Red recombination technology. Red recombination is a DNA homologous recombination technique mediated by the lambda phage Red operon and the Rac phage RecE/RecT system. By this technique, it is possible to easily and rapidly perform various modifications such as insertion, knockout, and mutation of any large DNA molecule. Red Recombination Technology simply states that the pKD46 plasmid carrying the recombinase gene is first transferred into the cells, and then the linear DNA fragment for targeting is prepared by electroporation, and the positive clones are screened. Finally, the resistance in the recombinant strain is determined. Gene elimination.

The specific operation process is described below:

1, delete the manXYZ gene sequence

The mannose transporter EIIM, P/III ^man (mannose transporter EIIM, P/III ^Man , ManXYZ) can be used as the second transporter of N-acetyl-D-glucosamine, which can be used for N-acetyl-D-glucosamine, etc. The hexose is transported into the cell, thereby transporting the extracellular and accumulated target product back into intracellular degradation. Deletion of the manXYZ gene sequence prevents extracellular N-acetyl-D-glucosamine from being transported back into the cell for degradation.

(1) Preparation of a linear DNA full-length PCR fragment for Red recombinant targeting

1) PCR amplification of fKanrf fragments

The fKanrf fragment, that is, the FRT-Kanr-FRT fragment, refers to a base sequence of the FRT site specifically recognized by the FLP recombinase at both ends of the kanamycin resistance gene (Kanr).

Primers were designed: forward primer (mfKanf-F) SEQ ID No. 1, reverse primer (mfKanf-R) SEQ ID No. 2.

Template: pPic9K.

PCR reaction conditions: first step: denaturation at 94 ° C for 1 min; second step: 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, 30 cycles; third step: 72 ° C for 10 min.

fKanrf size: 1.28kb. Its nucleotide sequence is SEQ ID No. 3.

The PCR product was separated and purified by 1% agarose gel electrophoresis.

2) PCR amplification of full-length linear DNA fragments for Red recombinant targeting

Designing a homology arm primer: Designing a homologous arm forward primer (manXYZKO-F) SEQ ID No. 5, reverse primer (manXYZKO-R) SEQ ID No. 6 to delete the manXYZ sequence according to the humanXYZ sequence SEQ ID No. 4. .

Template: amplified fKanrf PCR fragment.

Amplification product: homology arm + fKanrf + homology arm.

The PCR product was separated by agarose gel electrophoresis, purified and recovered, and 100 ng/μl of linear DNA full-length PCR fragment was obtained for Red recombinant targeting.

(2) Red reorganization operation

First, the pKD46 vector was transferred into the E. coli AT-001 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.

1) Transformation of pKD46 plasmid

The pKD46 vector is a plasmid carrying the gene for expression of the Red recombinase, and expresses the three gene segments of Exo, Bet and Gam. The three genes are placed under the arabinose promoter and can be expressed in a large amount by L-arabinose induction. In order to achieve the purpose of modifying the target gene on the chromosome by Red recombination, it is necessary to transform the pKD46 plasmid into E. coli.

1 Competency preparation: First, Escherichia coli ATCC 27325 stock solution stored at -20 ° C was inoculated in 10 ml of LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking for 2-3 hours. The culture solution was further added to a 10 ml centrifuge tube, 4000 g × 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl ₂ 5 ml for 5 min. Finally, it was centrifuged at 4000 g × 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl ₂ 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally. Among them, the preparation of 0.1M CaCl ₂ : using anhydrous CaCl ₂ with 1M CaCl ₂ , autoclaved with a vapor pressure of 15 lbf / in ₂ for 20 min, 1.5 ml of the mixture was stored at -20 ° C, and then melted 1:10 Proportionally diluted to 0.1 M CaCl ₂ .

2 Plasmid transformation: 250 μl of naturally-precipitated cells were taken, and 5 μl of pKD46 plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use.

2) Electroporation to prepare a linear DNA fragment for targeting, screening positive clones

1 Preparation of electrotransfer competent state: Inoculate pKD46Escherichia coli ATCC 27325 strain AT-001 in a test tube containing ampicillin (Amp) LB medium, shake overnight at 250 rpm, and inoculate 1% in the next day to Amp-containing In the LB medium, culture was carried out at 30 ° C. After the OD ₆₀₀ reached about 0.2, 0.2% of L-arabinose was added and induced at 30 ° C for 35 minutes until the OD ₆₀₀ reached about 0.4. Cool in the ice bath. Wash once with ultrapure water, wash twice with 10% glycerol, and finally resuspend with 10% glycerol. The amount of glycerin should be used to concentrate the cells at a final concentration of 500-1000 times.

2 Electric shock conversion: The 2 mm electric rotor was taken out from 70% ethanol, washed twice with sterile ultrapure water, and irradiated with ultraviolet light for 30 minutes. Pre-cool for 30 minutes at 4 °C. Take 90 μl of the finally resuspended cells, transfer to a pre-chilled centrifuge tube, add 5 μl (100 ng or more) of the full-length PCR fragment (linear DNA) obtained in step (1), mix gently with a gun, and ice bath for 30 minutes. . Electrical parameters: 2500V, 200Ω, 25μF.

3 Resuscitation and screening positive clones: 1 ml of LB liquid medium was added, 37 ° C, 100 rpm, 1 hour. Then, one carrageenin (Kan) plate was coated every 200 μl for a total of five. Evenly and dry. Incubate at 30 ° C for 24 hours. Clones grown under resistance to caramycin were picked for PCR identification and positive clones were screened.

The obtained strain number: AT-002-01 (AT-001, ΔmanXYZ::fKanrf).

(3) Elimination of resistance genes

For subsequent work, the resistance genes in the obtained strains (positive clones) can be eliminated. Elimination of resistance genes can be accomplished with the help of the pCP20 plasmid. pCP20 is a plasmid with ampicillin and chloramphenicol resistance gene, which can express FLP recombinase after heat induction. The enzyme can specifically recognize the FRT site, and the sequence between FRT sites can be deleted by recombination, leaving only one FRT site.

pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost. The antimony inoculum was plated on the antibiotic-free medium, and the grown monoclonal spot was picked onto the caramycin resistant plate, and the undeveloped clone in which the caramycin resistance gene had been deleted by the FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.

The obtained strain number: AT-002-02 (AT-001, ΔmanXYZ).

2, delete the nanA gene sequence

N-acetylneuraminate lyase (NanA) is capable of degrading N-acetyl-D-aminomannose (ManNAc) in microorganisms to N-acetyl-D-neuraminic acid (Neu5Ac). Deletion of the nanA gene sequence in the nanKETA operon prevents the degradation of N-acetyl-D-aminomannose (ManNAc) to N-acetyl-D-neuraminic acid (Neu5Ac).

Designing the homology arm primer: Designing the homologous arm primer for deletion of the nanA sequence according to the nanE sequence of nanE-nanK SEQ ID No. 7, forward primer (nanAKO-F) SEQ ID No. 8, reverse primer (nanAKO-R) SEQ ID No. 9.

Template: amplified fKanrf PCR fragment.

Amplification product: homology arm + fKanrf + homology arm.

(2) Red reorganization operation

First, the pKD46 vector was transferred into the E. coli AT-002-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.

1) Transformation of pKD46 plasmid

1 Competent preparation: First, Escherichia coli AT-002-02 (AT-001, △manXYZ) stock solution stored at -20 ° C, inoculated in 10 ml LB liquid medium at 1:50-100, 37 ° C, Incubate at 225 rpm for 2-3 hours with shaking. The culture solution was further added to a 10 ml centrifuge tube, 4000 g × 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl ₂ 5 ml for 5 min. Finally, it was centrifuged at 4000 g × 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl ₂ 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.

1 Preparation of electrotransfer competent state: pKD46Escherichia coli strain AT-002-02 was inoculated into a test tube containing ampicillin (Amp) LB medium, shaken at 250 rpm overnight, and inoculated to Amp-containing 1% in the next day. In the LB medium, culture was carried out at 30 ° C. After the OD ₆₀₀ reached about 0.2, 0.2% of L-arabinose was added and induced at 30 ° C for 35 minutes until the OD ₆₀₀ reached about 0.4. Cool in the ice bath. Wash once with ultrapure water, wash twice with 10% glycerol, and finally resuspend with 10% glycerol. The amount of glycerin should be used to concentrate the cells at a final concentration of 500-1000 times.

2 Electric shock conversion: The 2 mm electric rotor was taken out from 70% ethanol, washed twice with sterile ultrapure water, and irradiated with ultraviolet light for 30 minutes. Pre-cool for 30 minutes at 4 °C. Take 90 μl of the finally resuspended cells, transfer to a pre-chilled centrifuge tube, and add 5 μl (100 ng or more) of the full-length PCR fragment (linear DNA) obtained in step (1). Gently massage with a gun and mix for 30 minutes on ice. Electrical parameters: 2500V, 200Ω, 25μF.

The obtained strain number: AT-003-01 (AT-002-02, △nanA::fKanrf).

(3) Elimination of resistance genes

The obtained strain number: AT-003-02 (AT-002-02, △nanA).

3. Delete the nagA gene sequence

N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) can treat N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P) in microorganisms Conversion to D-glucosamine-6-phosphate (GlcN-6-P). Deletion of the nagA gene sequence in the nag operon (nagE-nagBACD) prevents the conversion of N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P) to D-glucosamine-6-phosphate (GlcN- 6-P).

Designing the homology arm primer: designing the homologous arm primer for deletion of the nagA sequence according to NCBI search NC_000913, Escherichia coli str. K-12N-acetyl-D-glucosamine-6-phosphate deacetylase gene nagA sequence SEQ ID No.10 : forward primer (nagAKO-F) SEQ ID No. 11, reverse primer (nagAKO-R) SEQ ID No. 12.

Template: amplified fKanrf PCR fragment.

Amplification product: homology arm + fKanf + homology arm.

The PCR product was separated by agarose gel electrophoresis, purified and recovered to obtain 100 ng/μl of linear DNA. Full length PCR fragments were used for Red recombination targeting.

(2) Red reorganization operation

First, the pKD46 vector was transferred into the E. coli AT-003-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.

1) Transformation of pKD46 plasmid

1 Competent preparation: First, the Escherichia coli AT-003-02 (AT-002-02, △nanA) stock solution stored at -20 °C was inoculated in 10 ml LB liquid medium at 1:50-100, 37 Incubate for 2-3 hours at 225 rpm with °C. The culture solution was further added to a 10 ml centrifuge tube, 4000 g × 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl ₂ 5 ml for 5 min. Finally, it was centrifuged at 4000 g × 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl ₂ 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.

1 Preparation of electrotransfer competent state: pKD46Escherichia coli strain AT-003-02 was inoculated into a test tube containing ampicillin (Amp) LB medium, shaken at 250 rpm overnight, and inoculated to Amp-containing 1% in the next day. In the LB medium, culture was carried out at 30 ° C. After the OD ₆₀₀ reached about 0.2, 0.2% of L-arabinose was added and induced at 30 ° C for 35 minutes until the OD ₆₀₀ reached about 0.4. Cool in the ice bath. Wash once with ultrapure water, wash twice with 10% glycerol, and finally resuspend with 10% glycerol. The amount of glycerin should be used to concentrate the cells at a final concentration of 500-1000 times.

The obtained strain number: AT-004-01 (AT-003-02, ΔnagA::fKanrf).

(3) Elimination of resistance genes

The obtained strain number: AT-004-02 (AT-003-02, ΔnagA).

4, delete the nagE gene sequence

The N- acetyl -D- glucosamine-specific enzyme ^{II Nag (N-acetyl-glucosamine} -specific enzyme II Nag, NagE) nagE gene sequence deleted, can prevent the degradation of extracellular GlcNAc is transported back to the cell.

Designing the homology arm primer: Designing the homologous arm forward primer (nagEKO-F1) SEQ ID No to delete the nagE gene sequence according to NCBI search NC_000913, Escherichia coli str. K-12nagB promoter and nagE gene sequence SEQ ID No. 13. .14, reverse primer (nagEKO-R1) SEQ ID No. 15.

Template: amplified fKanrf PCR fragment.

Amplification product: homology arm + fKanrf + homology arm.

(2) Red reorganization operation

First, the pKD46 vector was transferred into the E. coli AT-004-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.

1) Transformation of pKD46 plasmid

1 Competent preparation: First, the Escherichia coli AT-004-02 (AT-003-02, ΔnagA) stock solution stored at -20 ° C was inoculated in 10 ml LB liquid medium at 1:50-100, 37 Incubate for 2-3 hours at 225 rpm with °C. The culture solution was further added to a 10 ml centrifuge tube, 4000 g × 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl ₂ 5 ml for 5 min. Finally, it was centrifuged at 4000 g × 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl ₂ 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.

1 Preparation of electroporation competent state: pKD46Escherichia coli strain AT-004-02 was inoculated into a test tube containing ampicillin (Amp) LB medium, shaken at 250 rpm overnight, and inoculated to Amp-containing 1% in the next day. In the LB medium, culture was carried out at 30 ° C. After the OD ₆₀₀ reached about 0.2, 0.2% of L-arabinose was added and induced at 30 ° C for 35 minutes until the OD ₆₀₀ reached about 0.4. Cool in the ice bath. Wash once with ultrapure water, wash twice with 10% glycerol, and finally resuspend with 10% glycerol. The amount of glycerin should be used to concentrate the cells at a final concentration of 500-1000 times.

The obtained strain number: AT-005-01 (AT-004-02, ΔnagE::fKanrf).

(3) Elimination of resistance genes

The obtained strain number: AT-005-02 (AT-004-02, ΔnagE).

Example 2

This example describes the cloning of the gene UDPB of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) and the transformation of the wecB/pTrc99A plasmid in E. coli, and the ptrc-wecB gene cassette into the E. coli chromosome. Integration.

1. Cloning of wecB gene, transformation of wecB/pTrc99A plasmid in E. coli and its effect on the yield of N-acetyl-D-glucosamine

Escherichia coli UDP-N-acetyl-D-glucosamine-2-epimerase (WecB) gene wecB was placed under the control of Trc promoter to transform the strain to make it excessive Expression, can enhance UDP-GlcNAc (UDP-N-acetyl glucosamine, UDP-N-acetyl-D-glucosamine) into ManNAc (N-Acetyl-D-mannosamine, N-acetyl-D-aminomannose or N- Acetyl-D-mannosamine).

1) Cloning of the E. coli wecB gene

The nucleotide sequence of the E. coli wecB gene SEQ ID No. 16 was found according to NCBI, and its amino acid sequence is SEQ ID No. 17.

Primers were designed: forward primer (TrcwecB-F) SEQ ID No. 18, reverse primer (TrcwecB-R) SEQ ID No. 19.

Template: AT-001 (Escherichia coli ATCC 27325) genome.

Amplification product size: 1.13 kb.

The PCR product was separated and purified by 1% agarose gel electrophoresis.

The obtained PCR amplified fragment and the pUC57-T vector were ligated and sequenced to obtain wecB/pUC57.

2) Construction and transformation of the wecB gene under the control of the Trc promoter

1 Plasmid construction: The plasmid wecB/pUC57 was amplified, and the plasmid wecB/pUC57 and the vector pTrc99A were digested with Nco I and HindIII respectively. The wecB fragment and the pTrc99A fragment were separated and purified by agarose gel electrophoresis, and the T4 DNA ligase was used at 16 ° C. The mixture was ligated overnight and identified to obtain the wecB/pTrc99A plasmid.

2 Competent preparation: First, the AT-005-02 bacterial solution stored at -20 ° C was seeded in 10 ml of LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, and shaken for 2-3 hours. The culture solution was further added to a 10 ml centrifuge tube, 4000 g × 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl ₂ 5 ml for 5 min. Finally, it was centrifuged at 4000 g × 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl ₂ 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.

3 Plasmid transformation: 250 μl of naturally-precipitated cells were taken, and 5 μl of wecB/pTrc99A plasmid was added at -4 ° C for 30 min. Then, in a 42 ° C water bath for 1.5 min, 0.7 ml of SOC medium was added, and the mixture was shaken at 30 ° C for 2 hours. Take 0.2 ml of bacterial solution and apply penicillin plate. Incubate overnight (12-16 hours) at 30 °C. Monoclones were picked, cultured in 5 ml of LB liquid medium, and plasmids were identified. Preserve positive strains for use. Obtained recombinant strain wecB/pTrc99A (AT-005-02)

3) Effect of wecB/pTrc99A plasmid transformation on N-acetyl-D-glucosamine production

The recombinant strain wecB/pTrc99A (AT-005-02) and the control strain were subjected to a shake flask fermentation test. The monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. LB liquid medium composition: 5 g / l yeast powder, 10 g / l peptone, 10 g / l NaCl. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium). The initial OD _{600 was} about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours. At 24 hours and 48 hours, the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH. According to the sugar consumption of the fermentation broth, 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L. At the end of the fermentation, 1 ml of the fermentation broth was taken and centrifuged. The N-acetyl-D-glucosamine content was determined by HPLC.

Determination of 1N-acetyl-D-glucosamine by HPLC

Buffer: Add 3.5g of dipotassium hydrogen phosphate to a 1L volumetric flask, add enough water to dissolve, add 0.25mL of ammonia water, add water to dilute and mix, adjust the pH to 7.5 with phosphoric acid, and dilute to volume with water.

Mobile phase: acetonitrile: buffer (75:25).

Diluent: acetonitrile and water (50:50).

Standard solution: 1.0 mg/mL USP N-acetyl-D-glucosamine standard (RS) was dissolved in the diluent.

Sample solution: 1.0 mg/mL N-acetyl-D-glucosamine sample was dissolved in the diluent.

Liquid phase conditions:

Model: LC

Detector: UV 195nm

Column: 4.6-mm × 15-cm; 3-μm packing L8

Flow rate: 1.5ml/min

Column temperature: 35 ° C

Injection volume: 10μL

Preparation of 2M9 medium

First prepare 5×M9 medium: add about 64g of Na ₂ HPO ₄ ·7H ₂ O, 15g of KH ₂ PO ₄ , 2.5g of NaCl, 5.0g of NH ₄ Cl in about 800ml of double distilled water (ddH ₂ O), after dissolving Add water to 1000ml. Sterilize at 121 ° C for 30 minutes. 1 M MgSO ₄ , 1 M CaCl ₂ , 20% glucose were separately prepared and sterilized separately. Then, M9 culture solution was prepared according to Table 1, wherein 1000× trace element solution was prepared according to Table 2.

Table 1. Composition of M9 medium

Table 2. Composition of 1000× trace element solution

成分ingredient	用量(g/L)Dosage (g/L)
CoCl₂·6H₂OCoCl ₂ ·6H ₂ O	0.010.01
CuSO₄·5H₂OCuSO ₄ ·5H ₂ O	0.010.01
MnSO₄·H₂OMnSO ₄ ·H ₂ O	0.0330.033
Fe SO₄·7H₂OFe SO ₄ ·7H ₂ O	0.500.50
ZnSO₄·7H₂OZnSO ₄ ·7H ₂ O	0.380.38
H₃BO₃ H ₃ BO ₃	0.010.01
NaMoO₄·2H₂ONaMoO ₄ ·2H ₂ O	0.010.01
pHpH	33

Effect of 3wecB/pTrc99A plasmid transformation on the yield of N-acetyl-D-glucosamine in shake flask fermentation

The yield of shake flask fermentation is shown in Table 3. The results showed that the yield of the control strain AT-005-02 was very low and not detected, while the yield of the recombinant strain wecB/pTrc99A (AT-005-02) overexpressed by the wecB gene under the control of the Trc promoter was significantly increased.

Table 3. Fermentation yield of recombinant strain wecB/pTrc99A (AT-005-02) shake flask

2. Integration of the pTrc-wecB gene cassette into the E. coli chromosome

The nagE gene locus is the integration site of the pTrc-wecB gene cassette on the chromosome. In order to achieve integration of the pTrc-wecB gene cassette into the E. coli chromosome, first, the wecB fragment pTrc-wecB with the Trc promoter and the karamycin with the FLP recombinase recognition site (FRT site) on both sides were amplified. Resistance gene fragment: FRT-Kanr-FRT (fKanrf), and spliced. Then, the primers for deleting the homologous arm of the nagE gene sequence were designed again, and the full-length linear DNA fragment of Red recombinant targeting was amplified by using the fragment of pTrc-wecB and fKanrf splicing as a template.

The specific operation process is as follows:

(1) PCR amplification of pTrc-wecB fragment

Template: wecB/pTrc99A.

Primers were designed: forward primer (Trcff-F) SEQ ID No. 20, reverse primer (Trcff-R) SEQ ID No. 21.

Product size: 1.3 kb.

The PCR product was separated and purified by 1% agarose gel electrophoresis.

(2) PCR amplification of fKanrf fragments

Design primers: forward primer (mfKanf-F) SEQ ID No. 1, reverse primer (mfKanf-R) SEQ ID No. 2.

Template: pPic9K.

fKanrf size: 1.28kb. Its nucleotide sequence is SEQ ID No. 3.

The PCR product was separated and purified by 1% agarose gel electrophoresis.

(3) Amplify fKanrf docked with pTrc-wecB

Primers were designed: forward primer (fKanf-F) SEQ ID No. 22, reverse primer (fKanf-R) SEQ ID No. 23.

Template: fKanrf.

The size of the second amplified fKanrf was 1.3 kb.

The PCR product was separated and purified by 1% agarose gel electrophoresis.

(4) Preparation of a linear DNA full-length PCR fragment for Red recombinant targeting

Designing a homology arm primer: The forward primer (nagEKO-F2) SEQ ID No. 24, reverse primer (nagEKO-R2) SEQ ID No. 25, which deleted the homologous arm of the nagE gene sequence, was designed again.

Template: pTrc-wecB PCR fragment and secondary amplified fKanrf PCR fragment, 1:1 mixed.

Amplification product: homology arm + pTrc-wecB-fKanrf + homology arm.

(5) Red reorganization operation

1) Transformation of pKD46 plasmid

1 Competent preparation: First, the Escherichia coli AT-004-02 stock solution stored at -20 ° C was inoculated in 10 ml LB liquid medium at 1:50-100, and cultured at 37 ° C, 225 rpm, shaking for 2-3 hours. . The culture solution was further added to a 10 ml centrifuge tube, 4000 g × 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl ₂ 5 ml for 5 min. Finally, it was centrifuged at 4000 g × 5 min, the supernatant was discarded, and suspended in an ice bath of 0.1 M CaCl ₂ 5 ml. It was allowed to stand at -4 ° C for 12 hours and settled naturally.

2 Electric shock conversion: The 2 mm electric rotor was taken out from 70% ethanol, washed twice with sterile ultrapure water, and irradiated with ultraviolet light for 30 minutes. Pre-cool for 30 minutes at 4 °C. Take 90 μl of the finally resuspended cells, transfer to a pre-chilled centrifuge tube, add 5 μl (100 ng or more) of the full-length PCR fragment (linear DNA) obtained in step (4), mix gently with a gun, and ice bath for 30 minutes. . Electrical parameters: 2500V, 200Ω, 25μF.

The obtained strain number: AT-042-01 (AT-004-02, ΔnagE::pTrc-wecB-fKanrf).

(6) Elimination of resistance genes

The obtained strain number: AT-042-02 (AT-004-02, ΔnagE::pTrc-wecB).

3. Effect of pTrc-wecB gene cassette integration on N-acetyl-D-glucosamine production

The recombinant strain AT-042-02 and the control strain in which the pTrc-wecB gene cassette was integrated at the chromosome nagE gene locus were subjected to a shake flask fermentation test. The monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium). The initial OD _{600 was} about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours. At 24 hours and 48 hours, the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH. According to the sugar consumption of the fermentation broth, 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L. At the end of the fermentation, 1 ml of the fermentation broth was taken and centrifuged. The N-acetyl-D-glucosamine content was determined by HPLC.

The production of shake flask fermentation is shown in Table 4. The results showed that the yields of the control strains AT-001 and AT-005-02 were very low, but not detected, while the yield of the pTrc-wecB gene cassette integrated recombinant strain was significantly improved, and compared with the unintegrated recombinant strain wecB/pTrc99A (AT-005). -02) The output has also increased significantly.

Table 4. pTrc-wecB gene cassette integration recombinant strain shake flask fermentation yield

Example 3

This example describes the screening of a gene for the mutated UDP-N-acetylglucosamine-2-isomerase (WecB), which encodes a UDP-N-acetylglucosamine-2-isomerase with increased enzymatic activity.

In order to further increase the amount of N-acetyl-D-glucosamine synthesis in the production strain, a gene mutant encoding UDP-N-acetylglucosamine-2-isomerase having an increased enzyme activity was screened. In order to achieve this, the cloned gene is amplified by error-prone PCR technology, and the gene is amplified by a DNA polymerase for amplification under conditions that cause high frequency mismatches to obtain high frequency mutations in the PCR product. .

The specific operation process is as follows:

1. Error-prone PCR amplification of E. coli UDP-N-acetylglucosamine-2-isomerase gene wecB

Taq DNA polymerase does not have the 3'-5' proofreading property at high magnesium ion concentration (8mmol/L) and different concentrations of dNTP (where dATP and dGTP concentrations are 1.5mmol/L; dTTP and dCTP) The concentration was 3.0mmol/L) to control the frequency of random mutations, and random mutations were introduced into the target gene to construct a mutant library. The template concentration of A260 was 1000 ng/mL, the enzyme concentration was 5 U/μL, and the primer concentration was 100 μM.

Error-prone PCR reaction system (50 μl): 5 μl of PCR reaction buffer, 5 μl of dNTP (2.5 mM), 5 μl of MgCl ₂ (2.5 mM), 1 μl of forward primer (Trcwec B-F, SEQ ID No. 18), reversed Primer (TrcwecB-R, SEQ ID No. 19) 1 μl, DNA template (wecB/pUC57) 0.1 μl, Taq DNA polymerase 0.5 μl, ddH ₂ O 32.4 μl.

PCR procedure: pre-denaturation at 96 °C for 4 min; denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, extension at 75 °C for 2 min, 45 cycles; final extension at 75 °C for 15 min, recovery of PCR product by gel recovery method (product size: 1.13 kb); 5 μl of the product was examined by 1% agarose gel electrophoresis and stored at -20 ° C until use.

2. Construction of a gene mutant library of UDP-N-acetylglucosamine-2-isomerase

The above PCR product was digested with restriction endonucleases Nco I and Hind III, and ligated with the pTrc99A plasmid digested with Nco I and Hind III endonuclease, and then transformed into E. coli AT-005 with the ligation product mixture. -02, a large number of cloned transformants were obtained, and a transformed mutant library was constructed.

3. Screening for high enzyme activity mutants

From the transformed cell mutant library, 640 mutant clones were randomly picked and wild-type WecB/pTrc99A (AT-005-02) was used as control. The cells were inoculated into 5 ml LB medium containing 50 μg/mL penicillin (Amp). After incubation at 37 ° C, 150 rpm for 18 h, the cells were collected by centrifugation at 10,000 rpm at 5 mM. After discarding the supernatant, resuspend in 1 ml of PBS (pH 7.5, 10 mmol/L) at 4 ° C, select 300 V under ice bath conditions, ultrasonically disrupt it for 10 min after 6 s of ultrasonic, and centrifuge for supernatant. The enzyme activity was measured as a crude extract of the enzyme.

Activity detection of UDP-N-acetylglucosamine-2-isomerase: based on the conversion of UDP-N-acetyl-D-glucosamine to N-acetyl-D-aminomannose. That is, UDP-N-acetyl-D-glucosamine is reduced to the assay marker. Enzyme unit definition: The amount of enzyme required to reduce the equivalent of 1 μmol of UDP-N-acetyl-D-glucosamine per minute under enzymatic reaction conditions, defined as an enzyme activity unit (IU). Specific operation The procedure was as follows: a 20 ml reaction system was used as an enzyme activity assay system, which contained 45 mmol/L phosphate buffer (pH 7.5), 10 mM MgCl2 and 100 nCi of UDPGlcNAc, and 5 mg of crude enzyme solution. The enzyme was incubated for 30 min in a 37 ° C water bath. The reaction was terminated by the addition of ethanol. The radioactive compound was separated by chromatography on paper. The radioactivity was measured by a liquid scintillation counter. The solvent system used was n-propanol: 1 M sodium acetate, pH 5.0: water (7:1:2). The activity unit of UDP-N-acetylglucosamine-2-isomerase was calculated based on how much UDPGlcNAc was converted to ManNAc.

The results showed that the enzyme activity of the highest mutant strain was 653 IU/ml, and the enzyme activity of the control strain was 21.0 IU/ml. WecB was engineered by error-prone PCR to obtain a mutant strain with greatly improved enzyme activity. The mutant strain with the highest activity of the enzyme was selected and plasmid sequencing was performed. The results showed that the UDP-N-acetylglucosamine-2-isomerase mutant gene sequence is shown in SEQ ID No. 26, and the corresponding amino acid sequence is shown in SEQ ID No. 27. Compared with the wild-type UDP-N-acetylglucosamine-2-isomerase gene sequence, five base point mutations occurred: 101G/C, 433C/G, 677G/T, 734T/G, 1038T/ C; a missense mutation at amino acid 4, the mutation points are: C34S (the 34th cysteine to serine), H145D (the 145th histidine to aspartic acid), C226F (226 The position cysteine becomes phenylalanine), V245G (the 245th valine becomes glycine). The mutant gene was named wecBM.

4. The pTrc-wecBM gene cassette integrates into the nagE gene locus of E. coli

The nagE gene locus is the integration site of the pTrc-wecBM gene cassette on the chromosome. In order to achieve integration of the pTrc-wecBM gene cassette into the E. coli chromosome, first, the wecBM fragment pTrc-wecBM with the Trc promoter and the carrageenin with the FLP recombinase recognition site (FRT site) on both sides were amplified. Resistance gene fragment: FRT-Kanr-FRT (fKanrf), and spliced. Then, the primer for deleting the homologous arm of the nagE gene sequence was designed again, and the full-length linear DNA fragment of Red recombinant targeting was amplified by using the fragment fused with pTrc-wecBM and fKanrf as a template.

The specific operation process is as follows:

(1) PCR amplification of pTrc-wecBM fragments

Template: wecBM/pTrc99A.

Product size: 1.3 kb.

The PCR product was separated and purified by 1% agarose gel electrophoresis.

(2) PCR amplification of fKanrf fragments

Template: pPic9K.

fKanrf size: 1.28kb. Its nucleotide sequence is SEQ ID No. 3.

The PCR product was separated and purified by 1% agarose gel electrophoresis.

(3) Amplification of fKanrf docked with pTrc-wecBM

Template: fKanrf.

The size of the second amplified fKanrf was 1.3 kb.

The PCR product was separated and purified by 1% agarose gel electrophoresis.

Template: pTrc-wecBM PCR fragment and secondary amplified fKanrf PCR fragment, 1:1 mixed.

Amplification product: homology arm + pTrc-wecBM-fKanrf + homology arm.

(5) Red reorganization operation

1) Transformation of pKD46 plasmid

The obtained strain number: AT-043-01 (AT-004-02, ΔnagE::pTrc-wecBM-fKanrf).

(6) Elimination of resistance genes

pCP20 was transferred into the above-mentioned caramycin resistant clone, cultured at 30 ° C for 8 h, and then increased to 42 ° C overnight, heat-induced FLP recombinase expression, and the plasmid was gradually lost. Draw a plate of antimony-inoculated liquid on antibiotic-free medium, and pick up the monoclonal spot to the caramycin resistant plate. The ungrown caramycin resistant group A clone that has been deleted by FLP recombinase. Clones with the disappearance of resistance to caramycin were identified by PCR using the identified primers.

The obtained strain number: AT-043-02 (AT-004-02, ΔnagE::pTrc-wecBM).

5. Effect of pTrc-wecBM gene cassette integration on N-acetyl-D-glucosamine production

The recombinant strain AT-043-02 and the control strain in which the pTrc-wecBM gene cassette was integrated at the chromosome nagE gene locus were subjected to a shake flask fermentation test. The monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium). The initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours. At 24 hours and 48 hours, the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH. According to the sugar consumption of the fermentation broth, 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L. At the end of the fermentation, 1 ml of the fermentation broth was taken and centrifuged. The N-acetyl-D-glucosamine content was determined by HPLC.

The production of shake flask fermentation is shown in Table 5. The results showed that the yield of the control strain AT-005-02 was very low and was not detected. The yield of the recombinant pTrc-wecBM gene cassette integrated recombinant strain AT-043-02 was significantly improved, and compared with the unmutated control strain AT-042- 02 production has also increased significantly.

Table 5. pTrc-wecBM gene cassette integrated recombinant strain shake flask fermentation yield

The above results show that not only the overexpression of UDP-N-acetylglucosamine-2-isomerase can significantly increase the yield of N-acetyl-D-glucosamine, but also the screening of mutants by error-prone PCR technology can greatly improve N-acetyl-D. - Glucosamine yield, which is due to the increased enzyme activity of the isomerase mutant obtained.

Example 4

This example describes an E. coli strain integrated with a pTrc-wecBM cassette in which glucosamine-6- Phospho-synthase (GlmS) gene glmS and/or D-glucosamine-6-phosphate deaminase (NagB) gene nagB endogenous natural promoter replacement and / or deletion of N-acetyl-D-glucosamine The impact of production.

1. Replace the endogenous natural promoter of nagB gene with the Trc promoter, and further delete the endogenous natural promoter of glmS gene, and influence the production of N-acetyl-D-glucosamine in Escherichia coli strain integrated with pTrc-WecBM cassette. .

(1) Replace the endogenous natural promoter of nagB gene with the Trc promoter

The promoter of D-glucosamine-6-phosphate deaminase (NagB) gene in the nagE-nagBACD was deleted and replaced with the Trc promoter. The reaction catalyzed by D-Glucosamine-6-phosphate deaminase (NagB) is reversible, over-expressing nagB, accelerating the forward catalytic reaction of NagB, and increasing D-glucosamine- 6-phosphoric acid (GlcN-6-P) purpose.

First, the Trc promoter fragment and the fKanrf fragment were amplified and spliced. Then, a homology arm primer was designed to amplify a full-length linear DNA fragment for Red recombinant targeting.

1. Amplify the Trc promoter sequence

Based on the published information, the Trc promoter sequence was found: SEQ ID No. 28.

Primers were designed: forward primer (KanTrcRed-F) SEQ ID No. 29, reverse primer (KanTrcRed-R) SEQ ID No. 30.

Template: pTrc99A

Product size: 166 bp.

The PCR product was separated and purified by 1% agarose gel electrophoresis.

2. Amplify the caramycin resistance gene with FLP recombinase recognition site (FRT site) on both sides:

fKanrf

Template: pPic9K.

PCR reaction conditions: first step: denaturation at 94 ° C for 1 min; second step: 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 40 s, Cycle 30 times; third step: extend at 72 ° C for 10 min.

fKanrf size: 1.28kb. Its nucleotide sequence is SEQ ID No. 3.

The PCR product was separated and purified by 1% agarose gel electrophoresis.

3. Amplify fKanrf docked with Trc promoter

Primers were designed: forward primer (fKanfRed-F1) SEQ ID No. 31, reverse primer (fKanfRed-R1) SEQ ID No. 32.

Template: fKanrf.

The size of the second amplified fKanrf was 1.3 kb.

The PCR product was separated and purified by 1% agarose gel electrophoresis.

4. Preparation of a linear DNA full-length PCR fragment for Red recombinant targeting

Designing the homology arm primer: Designing the forward primer (nagBKO-F1) SEQ ID of the homologous arm of the nagB promoter according to NCBI search NC_000913, Escherichia coli str.K-12nagB promoter sequence and nagE gene sequence SEQ ID No.13 No. 33, reverse primer (nagBKO-R1) SEQ ID No. 34.

Template: Trc promoter PCR fragment and secondary amplified fKanrf PCR fragment, 1:1 mixing.

Amplification product: homology arm + fKanrf + Trc promoter + homology arm.

5, Red reorganization operation

First, the pKD46 vector was transferred into the E. coli AT-043-02 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.

The obtained strain number: AT-044 (AT-043-02, ΔnagB promotor::Trc promoter).

(2) delete the glmS gene endogenous natural promoter

The Glucosamine-6-phosphate synthase (glmS) gene promoter sequence was deleted. Glucosamine-6-phosphate synthase (GlmS), also known as L-glutamine-6-phosphate aminotransferase, catalyzes glucose-6-phosphate (Glc-6) -P) amination to D-glucosamine-6-phosphate (GlcN-6-P), but severe The product inhibits the problem, deleting its promoter sequence, and losing the expression of the enzyme, which can inhibit the inhibition of GlcN-6-P product.

First, the fKanrf fragment was amplified, and then the homology arm primer was designed to amplify the full-length linear DNA fragment of Red recombinant targeting.

1) Amplification of the caramycin resistance gene with FLP recombinase recognition site (FRT site) on both sides: fKanrf

Template: pPic9K.

fKanrf size: 1.28kb. Its nucleotide sequence is SEQ ID No. 3.

The PCR product was separated and purified by 1% agarose gel electrophoresis.

2) Preparation of a linear DNA full-length PCR fragment for Red recombinant targeting

Design of homology arm primers: According to NCBI, find NC_000913, Escherichia coli str. K-12L-glutamine-6-phosphate fructose aminotransferase (GlmS) gene promoter sequence SEQ ID No. 35, design the glmS gene promoter sequence deletion Source arm forward primer (Proglms KO-F) SEQ ID No. 36, reverse primer (Proglms KO-R) SEQ ID No. 37.

Template: fKanrf PCR fragment.

Amplification product: homology arm + fKanf + homology arm.

3) Red reorganization operation

First, the pKD46 vector was transferred into E. coli AT-044 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.

The obtained strain number: AT-045 (AT-044, ΔglmS promotor).

(3) The effect of replacing the nagB promoter with a promoter with higher expression level and further deleting the glmS promoter on the yield of N-acetyl-D-glucosamine

For the strain in which the pTrc-wecBM cassette was integrated, the nagB promoter was replaced with a promoter of a higher expression level, and the recombinant strain in which the glmS promoter was further deleted was subjected to a shake flask fermentation test. The monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium). The initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours. At 24 hours and 48 hours, the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH. According to the sugar consumption of the fermentation broth, 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L. At the end of the fermentation, 1 ml of the fermentation broth was taken and centrifuged. The N-acetyl-D-glucosamine content was determined by HPLC.

The yield of shake flask fermentation is shown in Table 6. The results showed that the recombinant strain that replaced the nagB promoter with the Trc promoter significantly increased the yield of N-acetyl-D-glucosamine, and further reduced the yield of N-acetyl-D-glucosamine after removing the glmS promoter.

Table 6. Recombinant shake flask fermentation yields for replacement of the nagB promoter and further removal of the glmS promoter

2. The endogenous natural promoter of glmS gene was replaced by Trc promoter, and the endogenous natural promoter of nagB gene was further deleted, and the effect of N-acetyl-D-glucosamine production of Escherichia coli strain integrated with pTrc-WecBM cassette was obtained. .

(1) Replace the glmS gene endogenous natural promoter with the Trc promoter

The L-glutamine-6-phosphate aminotransferase gene promoter sequence was replaced with a Trc promoter sequence. L-Glutamine-6-phosphate fructose aminotransferase, also known as Glucosamine-6-phosphate synthase (GlmS), replaces its promoter sequence with the Trc promoter sequence, overexpressing glmS, accelerating GlmS catalyzes the function of increasing D-glucosamine-6-phosphate (GlcN-6-P).

First, the Trc promoter sequence fragment and the fKanrf fragment were amplified and spliced. Then, a homology arm primer was designed to amplify a full-length linear DNA fragment for Red recombinant targeting.

1) Amplification of full-length PCR fragments of linear DNA for red recombinant targeting

Designing the homology arm primer: designing the homologous arm forward primer (ProglmspTrc-F) SEQ ID No. 38, reverse primer (ProglmspTrc-R), which was replaced with the Trc promoter according to the glmS gene promoter sequence SEQ ID No. SEQ ID No. 39.

Amplification product: homology arm + fKanrf + Trc promoter + homology arm.

2) Red reorganization operation

The obtained strain number: AT-046 (AT-043-02, ΔglmS promotor::Trc promoter).

(2) Deletion of the NagB gene endogenous natural promoter

Deletion of the D-glucosamine-6-phosphate deaminase (NagB) gene promoter sequence in the nagE-nagBACD, which disables nagB and eliminates NagB reverse catalysis. , reducing GlcN-6-P to produce Glc-6-P.

First, the fKanrf fragment was amplified, and then the homology arm primer was designed to prepare a full-length linear DNA fragment for Red recombinant targeting.

1) Preparation of a linear DNA full-length PCR fragment for Red recombinant targeting

Designing the homology arm primer: Designing the homologous arm forward primer (NagBKO-F2) SEQ ID No. 40, reverse primer (NagBKO-) to delete the nagB promoter sequence according to the nagB promoter and the nagE gene sequence SEQ ID No. R2) SEQ ID No. 41.

Template: fKanrf PCR fragment

Amplification product: homology arm + fKanrf + homology arm.

2) Red reorganization operation

First, the pKD46 vector was transferred into the E. coli AT-046 strain. Then, a linear DNA fragment for targeting was prepared by electroporation, and positive clones were selected. Finally, the resistance gene is eliminated.

The obtained strain number: AT-047 (AT-046, ΔnagB promotor).

(3) The effect of replacing the glmS promoter with a promoter with a higher expression level and further deleting the nagB promoter on the production of N-acetylglucosamine

For the strain in which the pTrc-wecBM cassette was integrated, the glmS promoter was replaced with a promoter of a higher expression level, and the recombinant strain in which the nagB promoter was further deleted was subjected to a shake flask fermentation test. The monoclonal strain on the freshly cultured LB plate medium was inoculated into a 3 ml LB liquid medium test tube (13 x 150 mm), and cultured at 30 ° C, 225 rpm for about 8 hours. Then, the seed culture solution was taken, and 3% was inoculated into a 250 ml shake flask containing 50 ml of the fermentation broth (M9 medium). The initial OD600 was about 0.5, cultured at 225 rpm at 37 ° C, and the fermentation cycle was 72 hours. At 24 hours and 48 hours, the pH of the fermentation broth was adjusted to 7.0 with 10 M NaOH. According to the sugar consumption of the fermentation broth, 65% glucose solution was added in portions to maintain the glucose concentration at 20 g/L. At the end of the fermentation, 1 ml of the fermentation broth was taken and centrifuged. The N-acetyl-D-glucosamine content was determined by HPLC.

The yield of shake flask fermentation is shown in Table 7. The results showed that the recombinant strain that replaced the glmS promoter with the Trc promoter had no obvious effect on the increase of N-acetyl-D-glucosamine production, but the N-acetyl-D-glucosamine production after the removal of the nagB promoter was lower than that of the control strain. There is a significant improvement.

Table 7. Recombinant shake flask fermentation yields for replacement of the glmS promoter and further removal of the nagB promoter

Example 5

This example describes the production of N-acetyl-D-glucosamine fermentation in a 10 L fermentor.

The recombinant engineering strain AT-047 was used as the production strain, and the N-acetyl-D-glucosamine fermentation test was carried out in a 10 L fermentor.

1. Seed culture

(1) Primary seed culture: A monoclonal strain on freshly cultured LB plate medium was picked, inoculated into 8 ml of LB liquid medium, and cultured at 37 ° C, 225 rpm for 8 hours.

(2) Secondary seed culture: 6 ml of the first-stage seed culture solution was taken, inoculated into a 1000 ml shake flask containing 200 ml of M9 medium, and cultured at 37 ° C, 225 rpm for 16 hours. The OD ₆₀₀ value is 6.0-10, which is about the middle of logarithmic growth.

(3) The fermentation medium was prepared according to Table 8, wherein the trace element solution was prepared according to Table 9, and the multivitamin solution was prepared according to Table 10.

Table 8. Fermentation medium

成分ingredient	用量(/L)Dosage (/L)
K₂HPO₄ K ₂ HPO ₄	1.30g1.30g
KH₂PO₄ KH ₂ PO ₄	1.00g1.00g
MgSO₄.7H₂OMgSO ₄ .7H ₂ O	0.10g0.10g
NH₄ClNH ₄ Cl	0.02g0.02g
(NH₄)₂SO₄ (NH ₄ ) ₂ SO ₄	0.20g0.20g
NaH₂PO₄ NaH ₂ PO ₄	0.60g0.60g
聚醚类消泡剂Polyether defoamer	10ml10ml
微量元素溶液Trace element solution	4ml4ml
复合维生素溶液Multivitamin solution	4ml4ml
葡萄糖glucose	6.00g6.00g

Note:

1 The trace element solution is separately sterilized and added, and the vitamin solution is filtered and added;

2 glucose: concentration 65% (w / v), sterilized separately, added before inoculation, glucose addition: 6.0g / L;

After combining 3 or more, adjust the pH to 7.0 with 10M NH ₄ OH;

4 The fermentation medium is the basal medium before the addition of glucose, and the initial liquid volume of the basal medium (the initial volume of the medium accounts for the total volume of the fermenter): 50%.

Table 9. Trace element solution

成分ingredient	用量(g/L)Dosage (g/L)
CaCl₂·2H₂OCaCl ₂ ·2H ₂ O	1010
FeCl₃·6H₂OFeCl ₃ ·6H ₂ O	1010
MnSO₄·5H₂OMnSO ₄ ·5H ₂ O	2.52.5
AlCl₃·6H₂OAlCl ₃ ·6H ₂ O	2.52.5
CoCl₂·6H₂OCoCl ₂ ·6H ₂ O	1.751.75
ZnSO₄·2H₂OZnSO ₄ ·2H ₂ O	0.50.5
NaMoO₄·2H₂ONaMoO ₄ ·2H ₂ O	0.50.5
CuSO₄·5H₂OCuSO ₄ ·5H ₂ O	0.250.25
H₃BO₃ H ₃ BO ₃	0.1250.125
pHpH	3～43 to 4

Table 10. Multivitamin solution

2. Vaccination

The secondary seed solution was inoculated to the fermentor at 40 ml/L, inoculating amount: 2.5-5% by volume, and the initial OD ₆₀₀ was 0.3-0.5.

3, process parameters

The high-density fermentation was carried out by using a 10L self-controlled fermenter, and the data was collected by the machine belt software to realize online computer control. The control parameters are: air flow rate of 0.5-1vvm.; dissolved oxygen ≥ 20%, to increase the speed and ventilation adjustment; temperature 37 ° C; pH value of 7.0, automatic flow of saturated ammonia to maintain constant. The glucose is consumed in the basal medium, that is, the sugar is added when the dissolved oxygen rises. The sugar-filling rate is based on the control of the residual sugar concentration of 0.45 g/L or less. The sugar solution glucose was at a concentration of 65% (w/v) and was added with 2.5% sodium gluconate or 6% ribose. The fermentation was stopped at 60-72h. Total liquid volume: 75-80%.

4. Example (10L fermenter)

(1) Species number: AT-047. Batch number: 00204.

(2) Seed liquid concentration: OD ₆₀₀ was 2.6.

(3) Base material: 4L.

(4) The inoculum size was 200 mL.

(5) Sugar filling rate: The residual sugar concentration is controlled to be 0.45 g/L or less.

(6) Sugar solution: glucose is 65% (w/v) and 2.5% sodium gluconate is added.

(7) Tracking index: OD ₆₀₀ and residual sugar content (residual glucose in fermentation broth).

(8) Product: N-acetyl-D-glucosamine. Potency: 72 hours 135.0g / L.

Example 6

This example describes the separation and purification treatment process of N-acetyl-D-glucosamine and D-glucosamine hydrochloride.

1. N-acetyl-D-glucosamine refining

(1) Inactivation: Fermentation liquid 80 ° C, 30 min.

(2) Solid-liquid separation: Centrifugation at 4000-8000 rpm, discarding the slag and protein, and taking the fermentation broth. It can also be filtered with a ceramic membrane.

(3) Decolorization: Product: Water: Activated carbon = 1: (1.5-3): (0.01-0.1), stirred for 0.5-5 h.

(4) Desalting: Electrodialysis and salt removal. The initial salt concentration of the concentrated chamber tank into the fermentation liquid is 0.01-0.05 mol/L. The flow rate of the fermentation broth of the light room is 40-80 L/h, the flow rate of the fermentation broth of the concentrated chamber is 40-80 L/h, and the voltage of the single membrane pair is 0.5-1.4 V. Deionization can also be carried out using an anion-cation exchange resin.

(5) Concentration: After demineralization, the fermentation broth is heated at 50-80 ° C under vacuum conditions (0.095 MPa), and concentrated. 8-15h, to supersaturation, about 4-6 times.

(6) Crystallization: The concentrated fermentation broth is first cooled to 25-35 ° C at 25 ° C, and then cooled with 0 ° C water for 1-3 h to 0-10 ° C. Add absolute ethanol (5-20 times the weight of the product) and stir at 700-1500 rpm for 15 min-1 h.

(7) Washing: Add anhydrous ethanol (equal amount with the product) to stir 10-100 rpm, 0.5-2 h.

(8) Drying: 50-100 ° C, 3-10 h. Purity: 99.92%. The total yield was 91.1%.

2. D-glucosamine hydrochloride refining

(1) Inactivation: Fermentation liquid 80 ° C, 30 min.

(5) Concentration: After demineralization, the fermentation broth is heated at 50-80 ° C under vacuum conditions (0.095 MPa), concentrated for 8-15 h, to supersaturate, about 4-6 times.

(6) Hydrolysis: The concentrated fermentation broth is transferred to an enamel or glass container, concentrated hydrochloric acid (37%) is added to a final concentration of 12-16%, stirred well, and incubated at 70 ° C for 90 min. Hydrochloric acid can be recycled.

(7) Crystallization: firstly cool the water at 25 ° C to 25-35 ° C, and then use 0 ° C water to cool 1-3 h to 4 ° C.

(8) Washing: Add anhydrous ethanol (equal amount with the product) to stir 10-100 rpm, 0.5-2 h. Centrifugation at 700-1500 rpm for 15-60 min gave glucosamine hydrochloride with a conversion of 89.6%.

(9) Dissolution: The washed product is dissolved in water, and the amount of water is about the volume of the original fermentation broth.

(10) Decolorization: Add activated carbon (1%). Mix for 30 minutes. Centrifuge at 700-1500 rpm for 15-60 min. Or filtered to give a colorless filtrate.

(11) Recrystallization: The filtrate was vacuum evaporated to supersaturation at 50 ° C and 55 cm Hg vacuum. Add absolute ethanol (5-20 times the weight of the product) and stir at 700-1500 rpm for 15 min-1 h.

(12) Washing: Add anhydrous ethanol (equal amount of product) to stir 10-100 rpm, 0.5-2 h. Centrifuge at 700-1500 rpm for 15-60 min.

(13) Drying: 50-100 ° C, 3-10 h. Purity: 99.94%. The total yield was 84.2%.

Although the invention has been described in detail above with the aid of the general description and specific embodiments, However, it will be apparent to those skilled in the art that modifications or improvements can be made thereto based on the present invention. Therefore, such modifications or improvements made without departing from the spirit of the invention are intended to be within the scope of the invention.

Claims

A method for producing N-acetyl-D-glucosamine and/or D-glucosamine salt by microbial fermentation, the method comprising:

A) cultivating a microorganism in a fermentation medium comprising at least one genetic modification capable of increasing the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism;

B) Collecting N-acetyl-D-glucosamine produced from the cultivation step A).

Preferably, further comprising C) deacetylating from N-acetyl-D-glucosamine to give a D-glucosamine salt.

Further preferably, the salt is selected from the group consisting of a hydrochloride, a sulfate, a sodium salt, a phosphate, and a hydrogen sulfate.
The method according to claim 1, wherein the genetic modification for increasing the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism is selected from the group consisting of a) UDP-N-acetyl-D in the microorganism - an increase in the enzymatic activity of glucosamine-2-isomerase (WecB); and/or b) overexpression of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism.
The method of claim 2, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB).

Preferably, the nucleic acid sequence encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) contains at least one UDP-N-acetyl-D-glucosamine-2-isomerase (WecB). Genetic modification of enzymatic activity; further preferably, the genetic modification comprises one or more of substitutions at positions corresponding to amino acid sequence SEQ ID NO: 17: substitution of cysteine at position 34 with serine, The 145th histidine is substituted by aspartic acid, the 226th cysteine is substituted by phenylalanine, and the 245th valine is substituted by glycine; more preferably, the UDP-N-acetyl-D- is encoded The nucleic acid sequence of glucosamine-2-isomerase (WecB) is SEQ ID NO:26.

Preferably, said UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) has at least about 30% identical to the amino acid sequence of SEQ ID NO: 17, preferably at least about 50% identical, further preferably at least About 70% identical, further preferably at least about 80% identical, still more preferably at least about 90% identical, most preferably at least about 95% identical amino acid sequence, wherein said UDP-N-acetyl-D-glucosamine-2- The isomerase (WecB) has enzymatic activity; further preferably, the UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) has the amino acid sequence of SEQ ID NO: 17.

Further preferably, the number of copies of the gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the recombinant nucleic acid molecule is increased.

Further preferably, the recombinant nucleic acid molecule comprises an endogenous natural promoter or a promoter having a higher expression level than the endogenous natural promoter; preferably, the promoter having a higher expression level than the endogenous natural promoter is selected from the group consisting of HCE promoters a promoter, a trc promoter, a T7 promoter; further preferably, the promoter having a higher expression level than the endogenous natural promoter is a trc promoter.
The method of claim 2, wherein the microorganism comprises at least one genetic modification of an endogenous natural promoter of a gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB); Preferably, the endogenous native promoter of the gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is replaced by a promoter having a higher expression level; further preferably, with a higher expression level The promoter is selected from the group consisting of the HCE promoter, the gap promoter, the trc promoter, and the T7 promoter; most preferably, the promoter having a higher expression level is the trc promoter.
The method of any one of claims 1 to 4, wherein the microorganism further comprises one or more of the following genetic modifications:

(1) comprising at least one genetic modification capable of enhancing the action of D-glucosamine-6-phosphate deaminase in a microorganism, preferably comprising at least one genetic modification capable of reducing the action of glucosamine-6-phosphate synthase;

(2) comprising at least one genetic modification capable of increasing the action of glucosamine-6-phosphate synthase in the microorganism, and at the same time comprising at least one genetic modification capable of reducing the action of D-glucosamine-6-phosphate deaminase.
The method according to claim 5, wherein the genetic modification for increasing the action of D-glucosamine-6-phosphate deaminase in the microorganism is selected from a) an increase in the enzyme activity of D-glucosamine-6-phosphate deaminase in the microorganism. And/or b) D-glucosamine-6-phosphate deaminase is overexpressed in the microorganism;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase in the microorganism.
The method of claim 6 wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding D-glucosamine-6-phosphate deaminase.

Preferably, the nucleic acid sequence encoding D-glucosamine-6-phosphate deaminase contains at least one genetic modification that increases the enzymatic activity of D-glucosamine-6-phosphate deaminase.

Further preferably, the gene copy number encoding the D-glucosamine-6-phosphate deaminase in the recombinant nucleic acid molecule is increased.

Further preferably, the recombinant nucleic acid molecule comprises an endogenous natural promoter or a promoter having a higher expression level than the endogenous natural promoter; preferably, the promoter having a higher expression level than the endogenous natural promoter is selected from the group consisting of HCE promoters a promoter, a trc promoter, a T7 promoter; further preferably, the promoter having a higher expression level than the endogenous natural promoter is a trc promoter.
The method of claim 6 wherein the microorganism comprises at least one genetic modification of an endogenous native promoter of a gene encoding D-glucosamine-6-phosphate deaminase. Preferably, the endogenous native promoter of the gene encoding D-glucosamine-6-phosphate deaminase is replaced by a promoter having a higher expression level; further preferably, the promoter having a higher expression level is selected from the HCE promoter , the gap promoter, the trc promoter, the T7 promoter; most preferably, the promoter with a higher expression level is the trc promoter.
The method according to claim 5, wherein the genetic modification for reducing the action of glucosamine-6-phosphate synthase in the microorganism is selected from a) a decrease in enzymatic activity of glucosamine-6-phosphate synthase in the microorganism; and/or b) Reduced expression of glucosamine-6-phosphate synthase in microorganisms;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase in the microorganism.

Further preferably, the genetic modification to reduce the action of glucosamine-6-phosphate synthase in the microorganism is selected from a partial or complete deletion, or partial or complete inactivation, of an endogenous gene encoding a glucosamine-6-phosphate synthase in the microorganism, and / or partial or complete deletion, or partial or complete inactivation of the endogenous natural promoter encoding the glucosamine-6-phosphate synthase gene in the microorganism; more preferably, reducing the action of glucosamine-6-phosphate synthase in the microorganism The endogenous natural promoter genetically modified to encode the glucosamine-6-phosphate synthase gene in the microorganism is completely deleted, ie, deleted.
The method of claim 5, wherein the synthesis of glucosamine-6-phosphate synthesis in the microorganism The genetic modification of the enzymatic action is selected from a) an increase in the enzymatic activity of the glucosamine-6-phosphate synthase in the microorganism; and/or b) an overexpression of the glucosamine-6-phosphate synthase in the microorganism;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of glucosamine-6-phosphate synthase in the microorganism.
The method of claim 10, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an glucosamine-6-phosphate synthase.

Preferably, the nucleic acid sequence encoding glucosamine-6-phosphate synthase contains at least one genetic modification that increases the enzymatic activity of glucosamine-6-phosphate synthase.

Further preferably, the gene copy number encoding the glucosamine-6-phosphate synthase in the recombinant nucleic acid molecule is increased.

Further preferably, the recombinant nucleic acid molecule comprises an endogenous natural promoter or a promoter having a higher expression level than the endogenous natural promoter; preferably, the promoter having a higher expression level than the endogenous natural promoter is selected from the group consisting of HCE promoters a promoter, a trc promoter, a T7 promoter; further preferably, the promoter having a higher expression level than the endogenous natural promoter is a trc promoter.
The method of claim 10, wherein the microorganism comprises at least one genetic modification of an endogenous natural promoter of a gene encoding glucosamine-6-phosphate synthase. Preferably, the endogenous natural promoter of the gene encoding glucosamine-6-phosphate synthase is replaced by a promoter having a higher expression level; further preferably, the promoter having a higher expression level is selected from the HCE promoter, gap promoter Promoter, trc promoter, T7 promoter; most preferably, the promoter with a higher expression level is the trc promoter.
The method according to claim 5, wherein the genetic modification for reducing the action of D-glucosamine-6-phosphate deaminase in the microorganism is selected from the group consisting of a) a decrease in the enzyme activity of D-glucosamine-6-phosphate deaminase in the microorganism And/or b) reduced expression of D-glucosamine-6-phosphate deaminase in the microorganism;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase in the microorganism.

Further preferably, the genetic modification to reduce the action of D-glucosamine-6-phosphate deaminase in the microorganism is selected from a partial or complete deletion, or a partial deletion, of an endogenous gene encoding D-glucosamine-6-phosphate deaminase in the microorganism. Or completely inactivated, and/or encode the endogenous source of the D-glucosamine-6-phosphate deaminase gene in the microorganism Partial or complete deletion, or partial or complete inactivation of the sex promoter; more preferably, genetic modification to reduce the action of D-glucosamine-6-phosphate deaminase in the microorganism to encode D-glucosamine-6-phosphate in the microorganism The endogenous natural promoter of the deaminase gene is completely deleted, ie deleted.
The method according to any one of claims 1 to 13, wherein the microorganism further comprises one or more of the following genetic modifications:

(1) comprising at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism;

(2) comprising at least one genetic modification capable of reducing the action of N-acetylneuraminic acid lyase (NanA) in the microorganism;

(3) comprising at least one genetic modification capable of reducing the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism;

(4) comprising at least one genetic modification capable of reducing the action of N-acetyl-D-glucosamine specific enzyme II Nag (NagE) in microorganisms;

(5) comprising at least one genetic modification capable of increasing the action of phosphoglucosamine mutase (GlmM) in the microorganism;

(6) comprising at least one genetic modification capable of enhancing the action of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in microorganisms.
The method according to claim 14, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism.

Preferably, the genetic modification to reduce the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism is selected from a partial or complete deletion of an endogenous gene encoding a mannose transporter EIIM, P/III man (ManXYZ) in a microorganism. , or partially or completely inactivated, and/or partially or completely deleted, or partially or completely inactivated, of the endogenous natural promoter of the mannose transporter EIIM, P/III man (ManXYZ) gene in the microorganism; more preferably reduced mannose transporter EIIM microorganism genetically P / III man (ManXYZ) acting microorganism modified to encode the mannose transporter EIIM, P / III man (ManXYZ ) complete deletion of an endogenous gene, is deleted.
The method of claim 14, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification capable of reducing the action of N-acetylneuraminic acid lyase (NanA) in the microorganism.

Preferably, the genetic modification to reduce the action of N-acetylneuraminic acid lyase (NanA) in the microorganism is selected from a partial or complete deletion of the endogenous gene encoding the N-acetylneuraminic acid lyase (NanA) in the microorganism, or a partial or Completely inactivated, and/or partially or completely deleted, or partially or completely inactivated, of an endogenous natural promoter of the N-acetylneuraminic acid lyase (NanA) gene in a microorganism; more preferably, reduced N- in a microorganism The genetic modification of the action of acetylneuraminic lyase (NanA) is the complete deletion of the endogenous gene encoding the N-acetylneuraminic lyase (NanA) in the microorganism, ie it is deleted.
The method according to claim 14, wherein the microorganism uses at least one recombinant nucleic acid molecule comprising at least one genetic modification capable of reducing the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism. Conversion.

Preferably, the genetic modification to reduce the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism is selected from the group consisting of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism. Partial or complete deletion, or partial or complete inactivation of an endogenous gene, and/or an endogenous natural promoter encoding the N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) gene in a microorganism Partially or completely absent, or partially or completely inactivated; more preferably, the genetic modification to reduce the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in microorganisms is to encode N-acetyl-D in the microorganism The endogenous gene of glucosamine-6-phosphate deacetylase (NagA) is completely deleted, ie deleted.
The method according to claim 14, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification capable of reducing the action of N-acetyl-D-glucosamine specific enzyme II Nag (NagE) in the microorganism.

Preferably, the genetic modification to reduce the action of the N-acetyl-D-glucosamine specific enzyme II Nag (NagE) in the microorganism is selected from the endogenous gene encoding the N-acetyl-D-glucosamine specific enzyme II Nag (NagE) in the microorganism. Partial or complete deletion, or partial or complete inactivation, and/or partial or complete deletion, or partial or partial, of the endogenous natural promoter of the N-acetyl-D-glucosamine specific enzyme II Nag (NagE) gene in the microorganism complete inactivation; more preferably, reduced microorganism microorganism encoding the genetically modified -D- N- acetyl-glucosamine-specific enzyme II Nag (NagE) acting -D- N- acetyl-glucosamine-specific enzyme II Nag (NagE) of The source gene is completely deleted, ie it is deleted.
The method according to claim 14, wherein the genetic modification for increasing the action of phosphoglucosamine mutase (GlmM) in the microorganism is selected from a) an increase in the enzymatic activity of the phosphoglucosamine mutase (GlmM) in the microorganism; / or b) the phosphoglucosamine mutase (GlmM) is overexpressed in the microorganism;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of phosphoglucosamine mutase (GlmM) in the microorganism.
The method of claim 19, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a phosphoglucosamine mutase (GlmM).

Preferably, the nucleic acid sequence encoding a phosphoglucosamine mutase (GlmM) contains at least one genetic modification that increases the enzymatic activity of the phosphoglucosamine mutase (GlmM).

Further preferably, the number of copies of the gene encoding the phosphoglucosamine mutase (GlmM) in the recombinant nucleic acid molecule is increased.

Further preferably, the recombinant nucleic acid molecule comprises an endogenous natural promoter or a promoter having a higher expression level than the endogenous natural promoter; preferably, the promoter having a higher expression level than the endogenous natural promoter is selected from the group consisting of HCE promoters a promoter, a trc promoter, a T7 promoter; further preferably, the promoter having a higher expression level than the endogenous natural promoter is a trc promoter.
The method according to claim 19, wherein the microorganism comprises at least one genetic modification of an endogenous natural promoter of a gene encoding a phosphoglucosamine mutase (GlmM); preferably, encoding a glucosamine phosphate The endogenous natural promoter of the gene of the enzyme (GlmM) is replaced by a promoter having a higher expression level; further preferably, the promoter having a higher expression level is selected from the group consisting of an HCE promoter, a gap promoter, a trc promoter, and a T7 Promoter; most preferably, the promoter with a higher expression level is the trc promoter.
The method according to claim 14, wherein the genetic modification for enhancing the action of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in the microorganism is selected from a) a bifunctional enzyme in the microorganism Increased enzymatic activity of N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU); and/or b) bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine in microorganism Transferase (GlmU) is overexpressed;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in the microorganism.
The method of claim 22, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU).

Preferably, the nucleic acid sequence encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) contains at least one additional bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate Genetic modification of the enzymatic activity of glycosyltransferase (GlmU).

Further preferably, the gene copy number encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) is increased in the recombinant nucleic acid molecule.

Further preferably, the recombinant nucleic acid molecule comprises an endogenous natural promoter or a promoter having a higher expression level than the endogenous natural promoter; preferably, the promoter having a higher expression level than the endogenous natural promoter is selected from the group consisting of HCE promoters a promoter, a trc promoter, a T7 promoter; further preferably, the promoter having a higher expression level than the endogenous natural promoter is a trc promoter.
The method according to claim 22, wherein the microorganism comprises at least one endogenous natural promoter for a gene encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) Genetic modification; preferably, the endogenous natural promoter of the gene encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) is replaced by a promoter with a higher expression level; Further preferably, the promoter having a higher expression level is selected from the group consisting of an HCE promoter, a gap promoter, a trc promoter, and a T7 promoter; most preferably, the promoter having a higher expression level is a trc promoter.
The method of any of claims 1 to 24, wherein the recombinant nucleic acid molecule is loaded into a plasmid or the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
The method of any of claims 1 to 25, wherein expression of the recombinant nucleic acid molecule is inducible; preferably, expression of the recombinant nucleic acid molecule can be induced by lactose.
The method according to any one of claims 1 to 26, wherein the culturing step A) is carried out at a temperature of from about 20 ° C to about 45 ° C; preferably, at a temperature of from about 33 ° C to about 37 ° C.

Preferably, said culturing step A) is carried out at a pH of from about 4.5 to about pH 8.5; preferably at a pH of from about 6.7 to about pH 7.2.

Preferably, said culturing step A) employs a fed fermentation process.

Further preferably, the sugar-retaining liquid contains glucose and ribose, preferably, the glucose concentration is 10%-85% (w/v), the ribose concentration is 0.5%-15% (w/v), and further preferably, the glucose concentration is 55%- 75% (w/v), ribose concentration 5%-7% (w/v);

Further preferably, the sugar-retaining liquid contains glucose and gluconate, preferably, the glucose concentration is 10%-85% (w/v), the gluconate concentration is 0.5%-15% (w/v), and further preferably, the glucose concentration 55%-75% (w / v), gluconate concentration of 2% -3% (w / v);

Further preferably, the sugar-retaining liquid comprises glucose, ribose and gluconate, preferably, the glucose concentration is 10%-85% (w/v), the ribose concentration is 0.5%-15% (w/v), and the gluconate concentration It is 0.5%-15% (w/v), further preferably, the glucose concentration is 55%-75% (w/v), the ribose concentration is 5%-7% (w/v), and the gluconate concentration is 2%. -3% (w/v).

More preferably, the gluconate is sodium gluconate.
The method according to any one of claims 1 to 27, wherein the collecting step B) comprises (a) precipitating N-acetyl-D-glucosamine from a fermentation broth for removing microorganisms; and/or (b) N-acetyl-D-glucosamine is crystallized from the fermentation broth for removing microorganisms.

Preferably, the collecting step B) further comprises the step of decolorizing the fermentation broth; further preferably, the decolorizing step is performed after the fermentation broth is precipitated or crystallized, after one or more precipitation or crystallization re-dissolution of the fermentation broth. More preferably, the decolorizing step comprises activated carbon treatment and/or chromatographic decolorization.
The method according to any one of claims 1 to 28, wherein the step C) is carried out under acidic and heating conditions or under enzymatic catalysis.

Preferably, the N-acetyl-D-glucosamine is deacetylated in a 30% to 37% hydrochloric acid solution at 60 ° C to 90 ° C to obtain D-glucosamine hydrochloride.

Preferably, N-acetyl-D-glucosamine is hydrolyzed by UDP-3-O-N-acetylglucosamine deacetylase to obtain D-glucosamine, and further salt is formed.
A microorganism comprising at least one genetic modification that enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in a microorganism.
The microorganism according to claim 30, wherein the genetic modification for enhancing the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism is selected from the group consisting of a) UDP-N-acetyl-D in the microorganism - an increase in the enzymatic activity of glucosamine-2-isomerase (WecB); and/or b) overexpression of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the microorganism.
The microorganism according to claim 31, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding N-acetyl-D-aminomannose-6-phosphate isomerase.

Preferably, the nucleic acid sequence encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) contains at least one UDP-N-acetyl-D-glucosamine-2-isomerase (WecB). Genetic modification of enzymatic activity; further preferably, the genetic modification comprises one or more of substitutions at positions corresponding to amino acid sequence SEQ ID NO: 17: substitution of cysteine at position 34 with serine, The 145th histidine is substituted by aspartic acid, the 226th cysteine is substituted by phenylalanine, and the 245th valine is substituted by glycine; more preferably, the UDP-N-acetyl-D- is encoded The nucleic acid sequence of glucosamine-2-isomerase (WecB) is SEQ ID NO:26.

Preferably, said UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) has at least about 30% identical to the amino acid sequence of SEQ ID NO: 17, preferably at least about 50% identical, further preferably at least About 70% identical, further preferably at least about 80% identical, still more preferably at least about 90% identical, most preferably at least about 95% identical amino acid sequence, wherein said UDP-N-acetyl-D-glucosamine-2- The isomerase (WecB) has enzymatic activity; further preferably, the UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) has the amino acid sequence of SEQ ID NO: 17.

Further preferably, the number of copies of the gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) in the recombinant nucleic acid molecule is increased.

Further preferably, the recombinant nucleic acid molecule comprises an endogenous natural promoter or a promoter having a higher expression level than the endogenous natural promoter; preferably, has a higher expression level than the endogenous natural promoter The promoter is selected from the group consisting of an HCE promoter, a gap promoter, a trc promoter, and a T7 promoter; further preferably, the promoter having a higher expression level than the endogenous natural promoter is a trc promoter.
The microorganism according to claim 31, wherein the microorganism comprises at least one genetic modification of an endogenous natural promoter of a gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB); Preferably, the endogenous native promoter of the gene encoding UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) is replaced by a promoter having a higher expression level; further preferably, with a higher expression level The promoter is selected from the group consisting of the HCE promoter, the gap promoter, the trc promoter, and the T7 promoter; most preferably, the promoter having a higher expression level is the trc promoter.
The microorganism according to any one of claims 30 to 33, wherein the microorganism further comprises one or more of the following genetic modifications:

(1) comprising at least one genetic modification capable of enhancing the action of D-glucosamine-6-phosphate deaminase in a microorganism, preferably comprising at least one genetic modification capable of reducing the action of glucosamine-6-phosphate synthase;

(2) comprising at least one genetic modification capable of increasing the action of glucosamine-6-phosphate synthase in the microorganism, and at the same time comprising at least one genetic modification capable of reducing the action of D-glucosamine-6-phosphate deaminase.
The microorganism according to claim 34, wherein the genetic modification for increasing the action of D-glucosamine-6-phosphate deaminase in the microorganism is selected from a) an increase in the enzyme activity of D-glucosamine-6-phosphate deaminase in the microorganism. And/or b) D-glucosamine-6-phosphate deaminase is overexpressed in the microorganism;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of D-glucosamine-6-phosphate deaminase in the microorganism.
The microorganism according to claim 35, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding D-glucosamine-6-phosphate deaminase.

Preferably, the nucleic acid sequence encoding D-glucosamine-6-phosphate deaminase contains at least one genetic modification that increases the enzymatic activity of D-glucosamine-6-phosphate deaminase.

Further preferably, the gene copy number encoding the D-glucosamine-6-phosphate deaminase in the recombinant nucleic acid molecule is increased.

Further preferably, the recombinant nucleic acid molecule comprises an endogenous natural promoter or a promoter having a higher expression level than the endogenous natural promoter; preferably, the promoter having a higher expression level than the endogenous natural promoter is selected from the group consisting of HCE promoters a promoter, a trc promoter, a T7 promoter; further preferably, the promoter having a higher expression level than the endogenous natural promoter is a trc promoter.
The microorganism according to claim 35, wherein the microorganism comprises at least one genetic modification of an endogenous natural promoter of a gene encoding D-glucosamine-6-phosphate deaminase. Preferably, the endogenous native promoter of the gene encoding D-glucosamine-6-phosphate deaminase is replaced by a promoter having a higher expression level; further preferably, the promoter having a higher expression level is selected from the HCE promoter , the gap promoter, the trc promoter, the T7 promoter; most preferably, the promoter with a higher expression level is the trc promoter.
The microorganism according to claim 34, wherein the genetic modification for reducing the action of glucosamine-6-phosphate synthase in the microorganism is selected from a) a decrease in enzymatic activity of glucosamine-6-phosphate synthase in the microorganism; and/or b) Reduced expression of glucosamine-6-phosphate synthase in microorganisms;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of glucosamine-6-phosphate synthase in the microorganism.

Further preferably, the genetic modification to reduce the action of glucosamine-6-phosphate synthase in the microorganism is selected from a partial or complete deletion, or partial or complete inactivation, of an endogenous gene encoding a glucosamine-6-phosphate synthase in the microorganism, and / or partial or complete deletion, or partial or complete inactivation of the endogenous natural promoter encoding the glucosamine-6-phosphate synthase gene in the microorganism; more preferably, reducing the action of glucosamine-6-phosphate synthase in the microorganism The endogenous natural promoter genetically modified to encode the glucosamine-6-phosphate synthase gene in the microorganism is completely deleted, ie, deleted.
The microorganism according to claim 34, wherein the genetic modification for increasing the action of glucosamine-6-phosphate synthase in the microorganism is selected from a) an increase in the enzymatic activity of glucosamine-6-phosphate synthase in the microorganism; and/or b) Glucosamine-6-phosphate synthase is overexpressed in the microorganism;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of glucosamine-6-phosphate synthase in the microorganism.
The microorganism according to claim 39, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an glucosamine-6-phosphate synthase.

Preferably, the nucleic acid sequence encoding glucosamine-6-phosphate synthase contains at least one genetic modification that increases the enzymatic activity of glucosamine-6-phosphate synthase.

Further preferably, the gene copy number encoding the glucosamine-6-phosphate synthase in the recombinant nucleic acid molecule is increased.

Further preferably, the recombinant nucleic acid molecule comprises an endogenous natural promoter or a promoter having a higher expression level than the endogenous natural promoter; preferably, the promoter having a higher expression level than the endogenous natural promoter is selected from the group consisting of HCE promoters a promoter, a trc promoter, a T7 promoter; further preferably, the promoter having a higher expression level than the endogenous natural promoter is a trc promoter.
The microorganism according to claim 39, wherein the microorganism comprises at least one genetic modification of an endogenous natural promoter of a gene encoding glucosamine-6-phosphate synthase. Preferably, the endogenous natural promoter of the gene encoding glucosamine-6-phosphate synthase is replaced by a promoter having a higher expression level; further preferably, the promoter having a higher expression level is selected from the HCE promoter, gap promoter Promoter, trc promoter, T7 promoter; most preferably, the promoter with a higher expression level is the trc promoter.
The microorganism according to claim 34, wherein the genetic modification for reducing the action of D-glucosamine-6-phosphate deaminase in the microorganism is selected from a) a decrease in the enzyme activity of D-glucosamine-6-phosphate deaminase in the microorganism. And/or b) reduced expression of D-glucosamine-6-phosphate deaminase in the microorganism;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that reduces the action of D-glucosamine-6-phosphate deaminase in the microorganism.

Further preferably, the genetic modification to reduce the action of D-glucosamine-6-phosphate deaminase in the microorganism is selected from a partial or complete deletion, or a partial deletion, of an endogenous gene encoding D-glucosamine-6-phosphate deaminase in the microorganism. Or completely inactivated, and/or partially or completely deleted, or partially or completely inactivated, of the endogenous natural promoter of the D-glucosamine-6-phosphate deaminase gene in the microorganism; more preferably, reduced D in the microorganism The genetic modification of the action of glucosamine-6-phosphate deaminase is the complete deletion, ie deletion, of the endogenous natural promoter encoding the D-glucosamine-6-phosphate deaminase gene in the microorganism.
The microorganism according to any one of claims 30 to 42, wherein the microorganism further comprises One or more of the following genetic modifications:

(1) comprising at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism;

(2) comprising at least one genetic modification capable of reducing the action of N-acetylneuraminic acid lyase (NanA) in the microorganism;

(3) comprising at least one genetic modification capable of reducing the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism;

(4) comprising at least one genetic modification capable of reducing the action of N-acetyl-D-glucosamine specific enzyme II Nag (NagE) in microorganisms;

(5) comprising at least one genetic modification capable of increasing the action of phosphoglucosamine mutase (GlmM) in the microorganism;

(6) comprising at least one genetic modification capable of enhancing the action of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in microorganisms.
The microorganism according to claim 43, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification capable of reducing the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism.

Preferably, the genetic modification to reduce the action of the mannose transporter EIIM, P/III man (ManXYZ) in the microorganism is selected from a partial or complete deletion of an endogenous gene encoding a mannose transporter EIIM, P/III man (ManXYZ) in a microorganism. , or partially or completely inactivated, and/or partially or completely deleted, or partially or completely inactivated, of the endogenous natural promoter of the mannose transporter EIIM, P/III man (ManXYZ) gene in the microorganism; more preferably reduced mannose transporter EIIM microorganism genetically P / III man (ManXYZ) acting microorganism modified to encode the mannose transporter EIIM, P / III man (ManXYZ ) complete deletion of an endogenous gene, is deleted.
The microorganism according to claim 43, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification capable of reducing the action of N-acetylneuraminic acid lyase (NanA) in the microorganism.

Preferably, the genetic modification to reduce the action of N-acetylneuraminic acid lyase (NanA) in the microorganism is selected from a partial or complete deletion of an endogenous gene encoding a N-acetylneuraminic acid lyase (NanA) in the microorganism, Partially or completely inactivated, and/or partially or completely deleted, or partially or completely inactivated, of an endogenous natural promoter of the N-acetylneuraminic acid lyase (NanA) gene in a microorganism; more preferably, reduced microorganisms The genetic modification of the action of N-acetylneuraminic acid lyase (NanA) is that the endogenous gene encoding the N-acetylneuraminic acid lyase (NanA) in the microorganism is completely deleted, ie, deleted.
The microorganism according to claim 43, wherein the microorganism uses at least one recombinant nucleic acid molecule comprising at least one genetic modification capable of reducing the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism. Conversion.

Preferably, the genetic modification to reduce the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism is selected from the group consisting of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in the microorganism. Partial or complete deletion, or partial or complete inactivation of an endogenous gene, and/or an endogenous natural promoter encoding the N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) gene in a microorganism Partially or completely absent, or partially or completely inactivated; more preferably, the genetic modification to reduce the action of N-acetyl-D-glucosamine-6-phosphate deacetylase (NagA) in microorganisms is to encode N-acetyl-D in the microorganism The endogenous gene of glucosamine-6-phosphate deacetylase (NagA) is completely deleted, ie deleted.
The microorganism according to claim 43, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification capable of reducing the action of N-acetyl-D-glucosamine specific enzyme II Nag (NagE) in the microorganism.

Preferably, the genetic modification to reduce the action of the N-acetyl-D-glucosamine specific enzyme II Nag (NagE) in the microorganism is selected from the endogenous gene encoding the N-acetyl-D-glucosamine specific enzyme II Nag (NagE) in the microorganism. Partial or complete deletion, or partial or complete inactivation, and/or partial or complete deletion, or partial or partial, of the endogenous natural promoter of the N-acetyl-D-glucosamine specific enzyme II Nag (NagE) gene in the microorganism complete inactivation; more preferably, reduced microorganism microorganism encoding the genetically modified -D- N- acetyl-glucosamine-specific enzyme II Nag (NagE) acting -D- N- acetyl-glucosamine-specific enzyme II Nag (NagE) of The source gene is completely deleted, ie it is deleted.
The microorganism according to claim 43, wherein the genetic modification for increasing the action of the phosphoglucosamine mutase (GlmM) in the microorganism is selected from a) an increase in the enzymatic activity of the phosphoglucosamine mutase (GlmM) in the microorganism; / or b) the phosphoglucosamine mutase (GlmM) is overexpressed in the microorganism;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of phosphoglucosamine mutase (GlmM) in the microorganism.
The microorganism according to claim 48, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a phosphoglucosamine mutase (GlmM).

Preferably, the nucleic acid sequence encoding a phosphoglucosamine mutase (GlmM) contains at least one genetic modification that increases the enzymatic activity of the phosphoglucosamine mutase (GlmM).

Further preferably, the number of copies of the gene encoding the phosphoglucosamine mutase (GlmM) in the recombinant nucleic acid molecule is increased.

Further preferably, the recombinant nucleic acid molecule comprises an endogenous natural promoter or a promoter having a higher expression level than the endogenous natural promoter; preferably, the promoter having a higher expression level than the endogenous natural promoter is selected from the group consisting of HCE promoters a promoter, a trc promoter, a T7 promoter; further preferably, the promoter having a higher expression level than the endogenous natural promoter is a trc promoter.
The microorganism according to claim 48, wherein the microorganism comprises at least one genetic modification of an endogenous natural promoter of a gene encoding a phosphoglucosamine mutase (GlmM); preferably, encoding a glucosamine phosphate The endogenous natural promoter of the gene of the enzyme (GlmM) is replaced by a promoter having a higher expression level; further preferably, the promoter having a higher expression level is selected from the group consisting of an HCE promoter, a gap promoter, a trc promoter, and a T7 Promoter; most preferably, the promoter with a higher expression level is the trc promoter.
The microorganism according to claim 43, wherein the genetic modification for enhancing the action of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in the microorganism is selected from a) a bifunctional enzyme in the microorganism Increased enzymatic activity of N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU); and/or b) bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine in microorganism Transferase (GlmU) is overexpressed;

Preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that enhances the action of the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) in the microorganism.
The microorganism according to claim 51, wherein the microorganism contains at least one coded duplex Transformation of a recombinant nucleic acid molecule capable of catalyzing the nucleic acid sequence of N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU).

Preferably, the nucleic acid sequence encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) contains at least one additional bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate Genetic modification of the enzymatic activity of glycosyltransferase (GlmU).

Further preferably, the gene copy number encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) is increased in the recombinant nucleic acid molecule.

Further preferably, the recombinant nucleic acid molecule comprises an endogenous natural promoter or a promoter having a higher expression level than the endogenous natural promoter; preferably, the promoter having a higher expression level than the endogenous natural promoter is selected from the group consisting of HCE promoters a promoter, a trc promoter, a T7 promoter; further preferably, the promoter having a higher expression level than the endogenous natural promoter is a trc promoter.
The microorganism according to claim 51, wherein the microorganism comprises at least one endogenous natural promoter for a gene encoding a bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridyltransferase (GlmU) Genetic modification; preferably, the endogenous natural promoter of the gene encoding the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate uridine acyltransferase (GlmU) is replaced by a promoter with a higher expression level; Further preferably, the promoter having a higher expression level is selected from the group consisting of an HCE promoter, a gap promoter, a trc promoter, and a T7 promoter; most preferably, the promoter having a higher expression level is a trc promoter.
The microorganism according to any one of claims 30 to 53, wherein the recombinant nucleic acid molecule is loaded into a plasmid or the recombinant nucleic acid molecule is integrated into the genome of the microorganism.
The microorganism according to any one of claims 30 to 54, wherein expression of the recombinant nucleic acid molecule is inducible; preferably, expression of the recombinant nucleic acid molecule can be induced by lactose.
The method according to any one of claims 1 to 29, or the microorganism according to any one of claims 30 to 551, wherein the microorganism microorganism is a bacterium, a yeast or a fungus.

Preferably, the microorganism is selected from the group consisting of bacteria or yeast.

Further preferably, the bacterium is selected from the genus Escherichia, Bacillus, Lactobacillus, Pseudomonas or Streptomyces. Bacteria; further preferably, the bacteria are selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Lactobacillus brevis, Pseudomonas aeruginosa or A bacterium of the species Streptomyces lividans; more preferably, the bacterium is Escherichia coli; further preferably, the Escherichia coli is selected from the K-12, B and W strains; most preferably the Escherichia coli is a K-12 strain.

Further preferably, the yeast is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, and Kluyveromyces. (Kluveromyces) and yeast of Phaffia; more preferably, the yeast includes, but is not limited to, selected from the group consisting of Saccharomyce scerevisiae, Schizosaccharo mycespombe, Candida albicans, and more Hansenula polymorpha, Pichia pastoris, Pichia canadensis, Kluyveromyces marxianus or Phaffia rohodozyma.

Preferably, the microorganism is a fungus; further preferably, the fungus is selected from the group consisting of Aspergillus, Absidia, Rhizopus, Chrysosporium, Neurospora. Or a fungus belonging to the genus Trichoderma; more preferably, the fungus is selected from the group consisting of Aspergillus niger, Aspergillus nidulans, Absidia coerulea, Rhizopus oryzae ), Chrysosporium lucknowense, Neurospora crassa, Neurospora intermedia or Trichoderma reesei.
A UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) having a higher enzymatic activity, the enzyme having the amino acid sequence shown in SEQ ID NO:27.
A nucleic acid molecule encoding the UDP-N-acetyl-D-glucosamine-2-isomerase (WecB) according to claim 57, which has the nucleic acid sequence of SEQ ID NO: 26.
A vector comprising the nucleic acid molecule of claim 58.
A microorganism comprising the vector of claim 59.
A microorganism comprising a nucleic acid molecule according to claim 58 in a genome.