Engineering bacterium for producing D-glucosamine by fermentation method and application of engineering bacterium

Abstract

The invention discloses an engineering bacterium for producing D-glucosamine by a fermentation method and application of the engineering bacterium. The engineering bacteria comprise modification (a) and modification (e) for escherichia coli; the transformation (a) comprises the following transformation: knocking out a pfkA gene, knocking out a pfkB gene, knocking out a zwf gene, knocking out manXYZ, knocking out a nagE gene, knocking out a nagB gene, enhancing the expression of an araE gene, replacing a ptsG gene with a glk gene, and replacing a galR gene with a Zglf gene; the modification (e) comprises the following modification: introducing and expressing an EcGlmS gene and a BtGlmP gene. The engineering bacteria prepared by the invention can grow and accumulate biomass by using glycerol or grease as a carbon source, and meanwhile, D-glucose is further converted and synthesized into D-glucosamine by using the accumulated biomass. The engineering bacterium disclosed by the invention has a good application prospect in industrial production of D-glucosamine.

Description

Technical Field

[0001] The present invention relates to the fields of medicine, food and cosmetics, and specifically to an engineering bacterium for producing D-glucosamine by fermentation and its application, and particularly to an engineering bacterium for synthesizing D-glucosamine by using glycerol or oil and D-glucose and its application. Background Art

[0002] D-Glucosamine, commonly known as amino sugar, is a compound in which the 2nd hydroxyl group in the D-glucose molecule is replaced by an amino group. Its chemical formula is C6H 13 NO5. The structure of glucosamine is similar to that of glucose, but the hydroxyl group (-OH) on the second carbon atom is replaced by an amino group (-NH2). This replacement causes the chemical properties and biological activities of glucosamine to be different from those of glucose. Glucosamine is the main component of glycoproteins and proteoglycans and exists in almost all organisms. Due to its unique chemical properties and biological activities, glucosamine and its derivatives are widely used in medicine, food, cosmetics and other fields. Industrially produced glucosamine is divided into three types: D-glucosamine hydrochloride, D-glucosamine sulfate, and high-purity glucosamine sulfate.

[0003] Traditional methods for producing glucosamine mainly include biological extraction and biological fermentation. The biological extraction method refers to the extraction of chitin or chitosan from shrimp and crab shells and acid hydrolysis to produce glucosamine hydrochloride; the biological fermentation method uses glucose as raw material and produces glucosamine and other products through fermentation, extraction, purification, concentration, crystallization and drying. The hydrolysis method has relatively low technical barriers, but low production efficiency, so that the hydrolysis of chitin with concentrated acids such as hydrochloric acid also faces serious environmental pollution problems. At the same time, the source of raw materials is affected by the season, so the cost is relatively high.

[0004] The biosynthesis method uses natural raw materials that exist in large quantities in nature and uses biological enzymes as catalysts to prepare glucosamine. This production method is conducive to reducing the cost of industrial production and is in line with the current green and environmentally friendly production principles. Its production process is not limited by resources, has relatively little pollution to the environment, and the product does not contain animal-derived allergens. Escherichia coli is the most common glucosamine-producing bacterium. It mainly uses acetyl glucosamine as an amino donor and converts fructose-6-phosphate into glucosamine-6-phosphate through the catalysis of L-glutamine-D-fructose-6-phosphate aminotransferase. Limited by the feedback inhibition of glucosamine production by glucosamine-6-phosphate and the cytotoxicity produced by glucosamine, current glucosamine-producing bacteria have problems such as low bacterial fermentation density, low glucosamine production, and difficulty in extracellular accumulation. This has also become a key technical problem that limits the production of glucosamine by microbial fermentation.

[0005] China is a major producer of glucosamine raw materials, and the United States is a major consumer. According to public information, more than 95% of the world's glucosamine raw material manufacturers are in China, and Chinese glucosamine raw material manufacturers are mainly concentrated in coastal areas such as Jiangsu and Zhejiang. At present, the production methods of glucosamine are biological extraction and biological fermentation, each of which occupies a certain proportion. Among them, the fermentation method mostly uses glucose as a raw material to convert glucosamine into acetylglucosamine, and then uses deacetylase to convert acetylglucosamine into glucosamine. The production cycle is relatively long, and there is a problem of "competing with others for grain". The moisture, acid value and peroxide value of waste cooking oil seriously exceed the standard. As a non-edible oil, from the perspective of environmental protection, its hygiene and safety are extremely poor, and people are terrified. However, in my country, the catering industry produces about 20 million tons of waste cooking oil every year, and the total amount of animal and vegetable oils that are discarded or idle in the country is about 100 million tons each year. Calculated based on the extraction of about 800kg of oil from 1 ton of animal and vegetable oil, more than 80 million tons of oil can be generated, and a large amount of glycerol is generated as a by-product. If there is no correct way to recycle and utilize waste cooking oil, it is very easy to endanger human health or cause waste of resources. Therefore, it is urgent to efficiently and reasonably utilize waste cooking oil and reduce environmental pollution. Establishing a route that uses glycerol and oil as raw materials for microbial growth and glucose as raw material to synthesize D-glucosamine has great economic and social value. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to construct a recombinant Escherichia coli that can accumulate biomass using glycerol or oil as a carbon source and synthesize D-glucosamine using D-glucose as a raw material.

[0007] In order to solve the above technical problems, the present invention first provides a recombinant Escherichia coli.

[0008] The recombinant Escherichia coli provided by the present invention is obtained by subjecting Escherichia coli to multiple transformations;

[0009] The plurality of modifications include modification (a) and modification (e);

[0010] Modification (a) includes the following modifications (a1) to (a9):

[0011] Transformation (a1): knockout of the pfkA gene;

[0012] Transformation (a2): knockout of the pfkB gene;

[0013] Transformation (a3): knockout of zwf gene;

[0014] Transformation (a4): knockout of manXYZ gene;

[0015] Transformation (a5): knockout of nagE gene;

[0016] Transformation (a6): knockout of nagB gene;

[0017] Modification (a7): Enhanced expression of araE gene;

[0018] Transformation (a8): ptsG gene was replaced by glk gene;

[0019] Transformation (a9): galR gene was replaced by Zglf gene;

[0020] Transformation (e): Introduction and expression of EcGlmS gene and BtGlmP gene.

[0021] In order to solve the above technical problems, the present invention also provides a method for constructing a recombinant Escherichia coli that produces D-glucosamine.

[0022] The method for constructing a recombinant Escherichia coli producing D-glucosamine provided by the present invention comprises the following steps: performing multiple transformations on the Escherichia coli; the multiple transformations include transformation (a) and transformation (e);

[0023] Modification (a) includes the following modifications (a1) to (a9):

[0024] Transformation (a1): knockout of the pfkA gene;

[0025] Transformation (a2): knockout of the pfkB gene;

[0026] Transformation (a3): knockout of zwf gene;

[0027] Transformation (a4): knockout of manXYZ gene;

[0028] Transformation (a5): knockout of nagE gene;

[0029] Transformation (a6): knockout of nagB gene;

[0030] Modification (a7): Enhanced expression of araE gene;

[0031] Transformation (a8): ptsG gene was replaced by glk gene;

[0032] Transformation (a9): galR gene was replaced by Zglf gene;

[0033] Transformation (e): Introduction and expression of EcGlmS gene and BtGlmP gene.

[0034] In one embodiment of the present invention, the multiple modifications further include modification (b); the modification (b) includes the following modifications (b1)-(b9):

[0035] Transformation (b1): knockout of fadR gene;

[0036] Modification (b2): Enhanced expression of fadD gene;

[0037] Modification (b3): ​​Enhanced expression of fadL gene;

[0038] Modification (b4): Enhanced expression of sthA gene;

[0039] Modification (b5): Enhanced expression of the atoSC gene cluster;

[0040] Transformation (b6): knockout of the fabF gene;

[0041] Transformation (b7): knockout of the fabH gene;

[0042] Transformation (b8): knockout of the lpxM gene;

[0043] Modification (b9): Knockout of the iclR gene.

[0044] In one embodiment of the present invention, the multiple modifications further include modification (c); the modification (c) includes the following modifications (c1)-(c4):

[0045] Transformation (c1): knockout of alsE gene;

[0046] Transformation (c2): knockout of manA gene;

[0047] Transformation (c3): knockout of the mtlD gene;

[0048] Transformation (c4): Knockout of the srlD gene.

[0049] In one embodiment of the present invention, the multiple modifications further include modification (d); the modification (d) includes the following modifications (d1)-(d5):

[0050] Transformation (d1): knockout of glsA gene;

[0051] Transformation (d2): knockout of glsB gene;

[0052] Transformation (d3): knockout of glnB gene;

[0053] Transformation (d4): knockout of the glnE gene;

[0054] Modification (d5): ldhA gene was replaced with glutamine synthetase gene.

[0055] Any of the above-mentioned knockout of pfkA gene, knockout of pfkB gene, knockout of zwf gene, knockout of manXYZ gene, knockout of nagE gene, knockout of nagB gene, knockout of fadR gene, knockout of fabF gene, knockout of fabH gene, knockout of lpxM gene, knockout of iclR gene, knockout of alsE gene, knockout of manA gene, knockout of mtlD gene, knockout of srlD gene, knockout of glsA gene, knockout of glsB gene, knockout of glnB gene, and knockout of glnE gene is achieved by using targeting fragments, pCP20 plasmids, and pKD46 plasmids for knocking out the above-mentioned genes.

[0056] Among them, the targeting fragment for knocking out the pfkA gene is pfkAup-kan-pfkAdown, and the pfkAup-kan-pfkAdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers pfkA-1 / pfkA-2.

[0057] The targeting fragment for knocking out the pfkB gene is pfkBup-kan-pfkBdown, and the pfkBup-kan-pfkBdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers pfkB-1 / pfkB-2.

[0058] The target fragment for knocking out the zwf gene is zwfup-kan-zwfdown, and the zwfup-kan-zwfdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers zwf-1 / zwf-2.

[0059] The targeting fragment for knocking out the manXYZ gene is manXYZup-kan-manXYZdown, which can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers manXYZ-1 / manXYZ-2.

[0060] The targeting fragment for knocking out the nagE gene is nagEup-kan-nagEdown, and the nagEup-kan-nagEdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers nagE-1 / nagE-2.

[0061] The targeting fragment for knocking out the nagB gene is nagBup-kan-nagBdown, and the nagBup-kan-nagBdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers nagB-1 / nagB-2.

[0062] The target fragment for knocking out the fadR gene is fadRup-kan-fadRdown, and the fadRup-kan-fadRdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers fadR-1 / fadR-2.

[0063] The targeting fragment for knocking out the fabF gene is fabFup-kan-fabFdown, and the fabFup-kan-fabFdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers fabF-1 / fabF-2.

[0064] The target fragment for knocking out the fabH gene is fadHup-kan-fadHdown, and the fadHup-kan-fadHdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers fadH-1 / fadH-2.

[0065] The targeting fragment for knocking out the lpxM gene is lpxMup-kan-lpxMdown, and the lpxMup-kan-lpxMdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers lpxM-1 / lpxM-2.

[0066] The targeting fragment for knocking out the iclR gene is iclRup-kan-iclRdown, and the iclRup-kan-iclRdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers iclR-1 / iclR-2.

[0067] The target fragment for knocking out the alsE gene is alsEup-kan-alsEdown, which can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers alsE-1 / alsE-2.

[0068] The targeting fragment for knocking out the manA gene is manAup-kan-manAdown, and the manAup-kan-manAdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers manA-1 / manA-2.

[0069] The targeting fragment for knocking out the mtlD gene is mtlDup-kan-mtlDdown, which can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers mtlD-1 / mtlD-2.

[0070] The target fragment for knocking out the srlD gene is srlDup-kan-srlDdown, and the srlDup-kan-srlDdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers srlD-1 / srlD-2.

[0071] The target fragment for knocking out the glsA gene is glsAup-kan-glsAdown, and the glsAup-kan-glsAdown can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers glsA-1 / glsA-2.

[0072] The targeting fragment for knocking out the glsB gene is glsBup-kan-glsBdown, which can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers glsB-1 / glsB-2.

[0073] The targeting fragment for knocking out the glnB gene is glnBup-kan-glnBdown, which can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers glnB-1 / glnB-2.

[0074] The targeting fragment for knocking out the glnE gene is glnEup-kan-glnEdown, which can be obtained by PCR amplification using the genomic DNA of the Escherichia coli strain JW1176 as a template and primers glnE-1 / glnE-2.

[0075] Any of the above-mentioned methods for enhancing the expression of the araE gene is carried out by inserting the Escherichia coli constitutive promoter P upstream of the araE gene. CPA1Furthermore, the Escherichia coli constitutive promoter P is inserted upstream of the araE gene. CPA1 Through the targeting fragment araEup-kan-P CPA1 -araEdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment araEup-kan-P CPA1 -araEdown can be a gene fragment (5' to 3' is composed of araEup shown in SEQ ID No.5, frt-kan-frt screening marker fragment shown in SEQ ID No.1, P CPA1 The promoter fragment and araEdown shown in SEQ ID No.6 were used as templates and PCR amplification was performed using primers araE-1 / araE-2.

[0076] Any of the above-mentioned methods for enhancing the expression of the fadD gene is carried out by inserting an Escherichia coli constitutive promoter P upstream of the fadD gene. CPA1 Furthermore, the Escherichia coli constitutive promoter P is inserted upstream of the fadD gene. CPA1 By targeting the fadDup-kan-P CPA1 -fadDdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment fadDup-kan-P CPA1 -fadDdown can be a gene fragment (5' to 3' is sequentially composed of fadDup shown in SEQ ID No.13, frt-kan-frt screening marker fragment shown in SEQ ID No.1, P CPA1 The promoter fragment and fadDdown shown in SEQ ID No.14 were used as templates and PCR amplification was performed using primers fadD-1 / fadD-2.

[0077] Any of the above-mentioned methods for enhancing the expression of the fadL gene is carried out by inserting a constitutive promoter P of Escherichia coli upstream of the fadL gene. CPA1 Furthermore, the Escherichia coli constitutive promoter P is inserted upstream of the fadL gene. CPA1 By targeting the fadLup-kan-P CPA1 -fadLdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment fadLup-kan-P CPA1-fadLdown can be a gene fragment (5' to 3' is sequentially composed of fadLup shown in SEQ ID No.15, frt-kan-frt screening marker fragment shown in SEQ ID No.1, P CPA1 The promoter fragment and fadLdown shown in SEQ ID No.16 were used as templates and PCR amplification was performed using primers fadL-1 / fadL-2.

[0078] Any of the above-mentioned methods for enhancing the expression of the sthA gene is carried out by inserting a constitutive promoter P of Escherichia coli upstream of the sthA gene. CPA1 Furthermore, the Escherichia coli constitutive promoter P is inserted upstream of the sthA gene. CPA1 By targeting the fragment sthAup-kan-P CPA1 -sthAdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment sthAup-kan-P CPA1 -sthAdown can be a gene fragment (5' to 3' is composed of sthAup shown in SEQ ID No.17, frt-kan-frt screening marker fragment shown in SEQ ID No.1, P shown in SEQ ID No.3 CPA1 The promoter fragment and sthAdown shown in SEQ ID No. 18) were used as templates and PCR amplified using primers sthA-1 / sthA-2.

[0079] Any of the above-mentioned enhancements to the expression of the atoSC gene cluster are achieved by inserting an Escherichia coli constitutive promoter P upstream of the atoSC gene cluster. CPA1 Furthermore, the Escherichia coli constitutive promoter P is inserted upstream of the atoSC gene cluster. CPA1 By targeting the fragment atoSup-kan-P CPA1 -atoSdown, pCP20 plasmid and pKD46 plasmid. Further, the targeting fragment atoSup-kan-P CPA1 -atoSdown can be a gene fragment (5' to 3' is composed of atoSup shown in SEQ ID No.19, frt-kan-frt screening marker fragment shown in SEQ ID No.1, P CPA1 The promoter fragment and atoSdown shown in SEQ ID No.20 were used as templates and PCR amplified using primers atoS-1 / atoS-2.

[0080] Any of the above ptsG gene replacement with glk gene is ptsG gene replacement with constitutive promoter P 119 Furthermore, the ptsG gene is replaced by a constitutive promoter P 119 and glk genes by targeting the fragment ptsGup-kan-P 119 -glk-ptsGdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment ptsGup-kan-P 119 -glk-ptsGdown can be a gene fragment (5' to 3' is composed of ptsGup shown in SEQ ID No.7, frt-kan-frt screening marker fragment shown in SEQ ID No.1, P 119 The promoter fragment, glk gene, TrrnB terminator fragment shown in SEQ ID No.2, and ptsGdown shown in SEQ ID No.8 were used as templates and ptsG-1 / ptsG-2 were used as primers for PCR amplification.

[0081] Any of the above galR gene is replaced by Zglf gene or galR gene is replaced by constitutive promoter P CPA1 Furthermore, the galR gene is replaced by a constitutive promoter P CPA1 and glk gene by targeting fragment galRup-kan-P CPA1 -Zglf-galRdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment galRup-kan-P CPA1 -Zglf-galRdown can be a gene fragment (5' to 3' is composed of galRup shown in SEQ ID No.9, frt-kan-frt screening marker fragment shown in SEQ ID No.1, P CPA1 promoter, Zglf gene, TrrnB terminator fragment shown in SEQ ID No.2, and galRdown shown in SEQ ID No.10) as template, and galR-1 / galR-2 as primers were used to perform PCR amplification to obtain the obtained product.

[0082] Any of the above-mentioned glutamine synthetase genes may be a CgGS gene derived from Corynebacterium glutamicum, a ScGS gene derived from Saccharomyces cerevisiae, or a LaGS gene derived from Lactobacillus acidophilus. The ldhA gene may be replaced with a glutamine synthetase gene, or the ldhA gene may be replaced with a constitutive promoter P CPA1 and CgGS gene or ldhA gene replaced by constitutive promoter P CPA1 and ScGS gene or ldhA gene replaced by constitutive promoter P CPA1 and LaGS genes.

[0083] Furthermore, the ldhA gene is replaced by a constitutive promoter P CPA1 and CgGS gene by targeting fragment ldhAup-kan-P CPA1 -CgGS-ldhAdown, pCP20 plasmid and pKD46 plasmid were used to achieve this.

[0084] The ldhA gene was replaced with the constitutive promoter P CPA1 and ScGS gene by targeting fragment ldhAup-kan-P CPA1 -ScGS-ldhAdown, pCP20 plasmid and pKD46 plasmid were used to achieve this.

[0085] The ldhA gene was replaced with the constitutive promoter P CPA1 and LaGS gene by targeting fragment ldhAup-kan-P CPA1 -LaGS-ldhAdown, pCP20 plasmid and pKD46 plasmid were used to achieve this.

[0086] Furthermore, the targeting fragment ldhAup-kan-P CPA1 -CgGS-ldhAdown can be a gene fragment (5' to 3' is composed of ldhAup shown in SEQ ID No.11, frt-kan-frt screening marker fragment shown in SEQ ID No.1, P CPA1 The promoter fragment, CgGS gene, TrrnB terminator fragment shown in SEQ ID No.2 and ldhAdown shown in SEQ ID No.12 were used as templates and ldhA-1 / ldhA-2 were used as primers for PCR amplification.

[0087] The targeting fragment ldhAup-kan-P CPA1-ScGS-ldhAdown can be a gene fragment (5' to 3' is sequentially composed of ldhAup shown in SEQ ID No.11, frt-kan-frt screening marker fragment shown in SEQ ID No.1, P CPA1 The promoter fragment, ScGS gene, TrrnB terminator fragment shown in SEQ ID No.2 and ldhAdown shown in SEQ ID No.12 were used as templates and ldhA-1 / ldhA-2 were used as primers for PCR amplification.

[0088] The targeting fragment ldhAup-kan-P CPA1 -LaGS-ldhAdown can be a gene fragment (5' to 3' is sequentially composed of ldhAup shown in SEQ ID No.11, frt-kan-frt screening marker fragment shown in SEQ ID No.1, P CPA1 The promoter fragment, LaGS gene, TrrnB terminator fragment shown in SEQ ID No.2 and ldhAdown shown in SEQ ID No.12 were used as templates and ldhA-1 / ldhA-2 were used as primers for PCR amplification.

[0089] Any of the above-mentioned EcGlmS gene and BtGlmP gene is introduced through the recombinant expression vector pTrc99a-EcGlmS-BtGlmP. The recombinant expression vector pTrc99a-EcGlmS-BtGlmP can be obtained by connecting the EcGlmS-BtGlmP fragment and the pTrc fragment by the Gibson assembly method; the EcGlmS-BtGlmP fragment is composed of SEQ ID No.22 and SEQ ID No.24 in sequence; the pTrc fragment can be obtained by PCR amplification using plasmid pTrc99a as a template and primers pTrc99a-01F / pTrc99a-01R.

[0090] In one embodiment of the present invention, the Escherichia coli is an Escherichia coli containing the pfkA gene, the pfkB gene, the zwf gene, the manXYZ gene, the nagE gene, the nagB gene, the araE gene, the ptsG gene, the galR gene, the fadR gene, the fadD gene, the fadL gene, the sthA gene, the atoSC gene cluster, the fabF gene, the fabH gene, the lpxM gene, the iclR gene, the alsE gene, the manA gene, the mtlD gene, the srlD gene, the glsA gene, the glsB gene, the glnB gene, the glnE gene, and the ldhA gene.

[0091] In a specific embodiment of the present invention, the Escherichia coli is Escherichia coli BW25113 strain.

[0092] The sequences of any of the above primers are shown in Table 1 in the examples below.

[0093] The use of any of the above-mentioned recombinant Escherichia coli or methods in the preparation of D-glucosamine also falls within the protection scope of the present invention.

[0094] In order to solve the above technical problems, the present invention also provides a method for preparing D-glucosamine.

[0095] The preparation method of D-glucosamine provided by the present invention comprises the following steps: using D-glucose as a raw material and adopting the above-mentioned recombinant Escherichia coli to carry out biotransformation to obtain D-glucosamine.

[0096] In one embodiment of the present invention, the method further comprises the step of culturing the recombinant Escherichia coli using glycerol and / or oil as a carbon source.

[0097] In a preferred embodiment of the present invention, the oil comprises triglycerides (such as soybean oil, salad oil and corn oil, etc.) and fatty acids (such as palmitic acid, stearic acid, palmitic acid, etc.).

[0098] In a specific embodiment of the present invention, the glycerol is extracted from oils and fats.

[0099] In another specific embodiment of the present invention, the glycerol is produced by fermentation or synthesis (such as prepared from carbon dioxide or petrochemical raw materials).

[0100] In another specific embodiment of the present invention, the oil is extracted from waste cooking oil. Specifically, the extraction method includes the steps of recovery, pretreatment, esterification and distillation.

[0101] In another specific embodiment of the present invention, the oil is extracted from plant or animal tissues.

[0102] The nucleotide sequence of any of the above-mentioned pfkA genes is shown in Gene Bank as Gene ID: 948412, and the amino acid sequence of 6-phosphofructokinase 1 encoded by the gene is shown in NCBI as reference sequence number CAA26356.1.

[0103] The nucleotide sequence of any of the above-mentioned pfkB genes is shown in Gene Bank as Gene ID: 946230, and the amino acid sequence of 6-phosphofructokinase 2 encoded by the gene is shown in NCBI as reference sequence number NP_416237.

[0104] The nucleotide sequence of any of the above-mentioned zwf genes is shown in Gene ID: 94637 in Gene Bank, and the amino acid sequence of 6-phosphate glucose dehydrogenase encoded by the gene is shown in reference sequence number NP_416366 in NCBI.

[0105] Any of the above-mentioned manXYZ genes comprises a manX gene, a manY gene and a manZ gene, the nucleotide sequences of the manX gene, the manY gene and the manZ gene are respectively shown in Gene ID: 946334, Gene ID: 946332 and Gene ID: 946342 in Gene Bank, and the amino acid sequences of the manX protein, manY protein and manZ protein encoded by the manX gene, the manY gene and the manZ gene are respectively shown in reference sequence number NP_416331, reference sequence number NP_416332 and reference sequence number NP_416333 in NCBI.

[0106] The nucleotide sequence of any of the above-mentioned nagE genes is shown in Gene ID: 945292 in Gene Bank, and the amino acid sequence of the N-acetylglucosamine-specific PTS transporter encoded by the gene is shown in reference sequence number NP_415205 in NCBI.

[0107] The nucleotide sequence of any of the above-mentioned nagB genes is shown in Gene ID: 945290 in Gene Bank, and the amino acid sequence of the glucosamine-6-phosphate deaminase encoded by the gene is shown in reference sequence number NP_415204 in NCBI.

[0108] The nucleotide sequence of any of the above-mentioned araE genes is shown as 947341 in Gene Bank, and the amino acid sequence of the arabinose transporter encoded by the gene is shown as reference sequence number NP_417318 in NCBI.

[0109] The nucleotide sequence of any of the above-mentioned ptsG genes is shown in Gene ID: 947341 in Gene Bank, and the amino acid sequence of the phosphotransferase G subunit encoded by the gene is shown in reference sequence number NP_415619 in NCBI.

[0110] The nucleotide sequence of any of the above-mentioned glk genes is shown in Gene ID: 94734 in Gene Bank, and the amino acid sequence of the glucose kinase encoded by the gene is shown in reference sequence number NP_416889 in NCBI.

[0111] The nucleotide sequence of any of the above-mentioned galR genes is shown in Gene ID: 947314 in Gene Bank, and the amino acid sequence of the galactose repressor factor encoded by the gene is shown in reference sequence number NP_417314 in NCBI.

[0112] The nucleotide sequence of any of the above-mentioned Zglf genes is shown in the 185-1606 positions of the genome sequence of reference sequence number M60615.1 in NCBI, and the amino acid sequence of the glucose transport auxiliary factor encoded by the gene is shown in the reference sequence number AAA27691.1 in NCBI.

[0113] The nucleotide sequence of any of the above-mentioned EcGlmS genes is shown as SEQ ID No.22, and the amino acid sequence of the EcGlmS protein encoded by the gene is shown as SEQ ID No.21.

[0114] The nucleotide sequence of any of the above-mentioned BtGlmP genes is shown as SEQ ID No.24, and the amino acid sequence of the BtGlmP protein encoded by the gene is shown as SEQ ID No.23.

[0115] The nucleotide sequence of any of the above-mentioned fadR genes is shown in Gene ID: 94865 in Gene Bank, and the amino acid sequence of the fatty acid degradation transcription factor encoded by the gene is shown in reference sequence number NP_415705 in NCBI.

[0116] The nucleotide sequence of any of the above-mentioned fadD genes is shown in Gene ID: 94632 in Gene Bank, and the amino acid sequence of the fatty acyl-CoA synthetase encoded by the gene is shown in reference sequence number NP_416319 in NCBI.

[0117] The nucleotide sequence of any of the above-mentioned fadL genes is shown in Gene ID: 946820 in Gene Bank, and the amino acid sequence of the long-chain fatty acid transport protein encoded by the gene is shown in reference sequence number NP_416846 in NCBI.

[0118] The nucleotide sequence of any of the above-mentioned sthA genes is shown in Gene ID: 94846 in Gene Bank, and the amino acid sequence of the NAD(P) transhydrogenase encoded by the gene is shown in reference sequence number NP_418397 in NCBI.

[0119] Any of the above-mentioned atoSC gene clusters contains the atoS gene and the atoC gene, the nucleotide sequences of the atoS gene and the atoC gene are shown in Gene ID: 949011 and Gene ID: 947444 in Gene Bank, respectively, and the amino acid sequences of the atoS protein and the atoC protein encoded by the atoS gene and the atoC gene are shown in reference sequence number NP_416723 and reference sequence number NP_416724 in NCBI, respectively.

[0120] The nucleotide sequence of any of the above-mentioned fabF genes is shown in Gene ID: 946665 in Gene Bank, and the amino acid sequence of β-ketoacyl-ACP synthase II encoded by the gene is shown in reference sequence number NP_415613 in NCBI.

[0121] The nucleotide sequence of any of the above-mentioned fabH genes is shown in Gene ID: 946003 in Gene Bank, and the amino acid sequence of β-ketoacyl-ACP synthase III encoded by the gene is shown in reference sequence number NP_415609 in NCBI.

[0122] The nucleotide sequence of any of the above-mentioned lpxM genes is shown in Gene Bank as Gene ID: 945143, and the amino acid sequence of the lipid A biosynthesis myristoyltransferase encoded by the gene is shown in NCBI as reference sequence number NP_416369.

[0123] The nucleotide sequence of any of the above-mentioned iclR genes is shown in Gene ID: 948524 in Gene Bank, and the amino acid sequence of the isocitrate lyase inhibitor encoded by the gene is shown in reference sequence number NP_418442 in NCBI.

[0124] The nucleotide sequence of any of the above-mentioned alsE genes is shown in Gene ID: 948595 in Gene Bank, and the amino acid sequence of the D-psicose-6-phosphate epimerase encoded by the gene is shown in reference sequence number NP_418509 in NCBI.

[0125] The nucleotide sequence of any of the manA genes described above is shown in Gene Bank as Gene ID: 944840, and the amino acid sequence of the mannose-6-phosphate isomerase encoded by the gene is shown in NCBI as reference sequence number NP_416130.

[0126] The nucleotide sequence of any of the above mtlD genes is shown in Gene Bank as Gene ID: 948117, and the amino acid sequence of mannitol-1-phosphate 5-dehydrogenase encoded by the gene is shown in NCBI as reference sequence number NP_418057.

[0127] The nucleotide sequence of any of the above-mentioned srlD genes is shown in Gene Bank as Gene ID: 948937, and the amino acid sequence of the sorbitol-6-phosphate 2-dehydrogenase encoded by the gene is shown in NCBI as reference sequence number NP_417185.

[0128] The nucleotide sequence of any of the above-mentioned glsA genes is shown in Gene ID: 946187 in Gene Bank, and the amino acid sequence of glutaminase A encoded by the gene is shown in reference sequence number NP_415018 in NCBI.

[0129] The nucleotide sequence of any of the above-mentioned glsB genes is shown in Gene ID: 944973 in Gene Bank, and the amino acid sequence of glutaminase B encoded by the gene is shown in reference sequence number NP_416041 in NCBI.

[0130] The nucleotide sequence of any of the above-mentioned glnB genes is shown in Gene Bank as Gene ID: 947016, and the amino acid sequence of the nitrogen regulatory protein encoded by the gene is shown in NCBI as reference sequence number NP_417048.

[0131] The nucleotide sequence of any of the above-mentioned glnE genes is shown in Gene ID: 947552 in Gene Bank, and the amino acid sequence of the glutamine synthetase adenylase / adenylyltransferase encoded by the gene is shown in reference sequence number NP_417525 in NCBI.

[0132] The nucleotide sequence of any of the above-mentioned ldhA genes is shown as Gene ID: 946315 in Gene Bank, and the amino acid sequence of lactate dehydrogenase encoded by the gene is shown as reference sequence number NP_415898 in NCBI.

[0133] The nucleotide sequence of any of the above-mentioned CgGS genes is shown in the 2185037-2186470th position of the genome sequence of reference sequence number CP007724.1 in NCBI, and the amino acid sequence of glutamine synthetase encoded by the gene is shown in the reference sequence number AIK85642 in NCBI.

[0134] The nucleotide sequence of any of the above-mentioned ScGS genes is shown in Gene ID: 856147 in Gene Bank, and the amino acid sequence of the glutamine synthetase encoded by the gene is shown in reference sequence number NP_015360 in NCBI.

[0135] The nucleotide sequence of any of the above-mentioned LaGS genes is shown in the 1491778-1493115th position of the genome sequence of reference sequence number CP020620.1 in NCBI, and the amino acid sequence of glutamine synthetase encoded by the gene is shown in the reference sequence number AJP46821 in NCBI.

[0136] Any of the above constitutive promoters P CPA1 The nucleotide sequence is shown in SEQ ID No.3.

[0137] Any of the above constitutive promoters P 119 The nucleotide sequence is shown in SEQ ID No.4.

[0138] The transformation of Escherichia coli in the present invention includes the following (1)-(5):

[0139] (1) Enhancement of oil assimilation pathway: including enhancing the expression of fadD gene, enhancing the expression of fadL gene, enhancing the expression of sthA gene, enhancing the expression of atoSC gene cluster, knocking out lpxM gene, knocking out fadR gene, knocking out fabF gene, knocking out fabH gene, and knocking out iclR gene;

[0140] (2) Enhancement of the D-glucosamine biosynthesis pathway: including enhancing the expression of the araE gene, replacing the galR gene with the Zglf gene, replacing the ptsG gene with the glk gene, and introducing and expressing the EcGlmS gene and the BtGlmP gene;

[0141] (3) Weakening or blocking of the D-glucose and D-glucosamine consumption pathways: including knocking out the pfkA gene, knocking out the pfkB gene, knocking out the zwf gene, knocking out the manXYZ gene, knocking out the nagE gene, and knocking out the nagB gene;

[0142] (4) L-glutamine synthesis pathway is enhanced and consumption pathway is weakened or blocked: ldhA gene is replaced with glutamine synthetase gene, glsA gene is knocked out, glsB gene is knocked out, glnB gene is knocked out, glnE gene is knocked out;

[0143] (5) Weakening or blocking of byproduct-related genes: including knocking out the alsE gene, knocking out the manA gene, knocking out the mtlD gene, and knocking out the srlD gene.

[0144] The invention prepares a recombinant Escherichia coli which can utilize glycerol or oil as a carbon source to grow and accumulate biomass, and utilizes the accumulated biomass to further convert D-glucose into D-glucosamine. The recombinant Escherichia coli can be used for the industrial production of D-glucosamine. On the one hand, waste cooking oil is efficiently and reasonably utilized, which not only reduces production costs but also reduces environmental pollution. On the other hand, D-glucosamine is efficiently synthesized and the yield of D-glucosamine is increased, which has great economic value and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] Figure 1 Comparison of the production of D-glucosamine by fermentation of different recombinant Escherichia coli engineered strains BW25113, GN109, and GN110.

[0146] Figure 2 Comparison of the production of D-glucosamine by fermentation of different recombinant Escherichia coli engineered strains BW25113, GN209, and GN210.

[0147] FIG3 is a comparison of the yield of D-glucosamine produced by fermentation of different recombinant Escherichia coli engineered strains BW25113, GN305, GN408, GN409, and GN410. DETAILED DESCRIPTION

[0148] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0149] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples are repeated three times unless otherwise specified, and the results are averaged.

[0150] The liquid LB medium (pH 7.0) in the following examples contains 1 g / 100 mL NaCl, 1 g / 100 mL tryptone, 0.5 g / 100 mL yeast powder, and the balance is water. The solid medium is obtained by adding agarose to the liquid medium.

[0151] The E. coli BW25113 (CGSC#: 7636), pKD46 plasmid (CGSC#: 7739), and pCP20 plasmid (CGSC#: 7629) in the following examples are all products of the E. coli Genetic Collection Center (CGSC) of Yale University, USA. pKD46 plasmid: contains the temperature-sensitive replication origin oriR101, which can replicate normally when cultured at 30°C and will be automatically lost when cultured above 37°C; E. coli carrying the PKD46 plasmid is cultured at 30°C, and Gam, Exo, and Beta are efficiently expressed after the addition of arabinose induction, and once the exogenous dsDNA is electrotransferred into the cell, homologous recombination can occur with the genomic target sequence; it carries the ampicillin resistance gene as a selection marker. pCP20 plasmid: Escherichia coli carrying the pCP20 plasmid can express the flippase recombinase (FLP) gene when cultured at 30°C, eliminating a FRT site and the kanamycin resistance gene; Escherichia coli carrying the pCP20 plasmid will automatically lose the pCP20 plasmid when cultured at 42°C; it carries the ampicillin resistance gene as a screening marker.

[0152] The Escherichia coli JW1176 in the following examples is a product of the National Institute of Genetics (NIG, Japan), which is recorded in the document "Xiaoyu Piao, Lei Wang, Baixue Lin, Hao Chen, Weifeng Liu, Yong Tao. Metabolic engineering of Escherichia coli for production of L-aspartate and its derivative β-alanine with high stoichiometric yield. Metabolic Engineering. 2019 54: 244-254." The public can obtain the above-mentioned biological material from the applicant, and the obtained above-mentioned biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0153] The primer sequences involved in the following examples are shown in Table 1.

[0154] Table 1. Primer names and primer nucleotide sequences in the examples

[0155]

[0156]

[0157]

[0158]

[0159] Example 1. Construction of recombinant Escherichia coli engineered strain GN110

[0160] In this example, a basic bacterium GN110 was prepared that can efficiently utilize glycerol as a carbon source for bacterial growth and can convert D-glucose into D-glucosamine. The preparation method of the strain is as follows, and the primers used are shown in Table 1.

[0161] (1) Knockout of 6-phosphofructokinase 1 (hereinafter referred to as pfkA) gene

[0162] Starting from E. coli BW25113, the pfkA gene of E. coli BW25113 was knocked out to obtain recombinant E. coli GN101. The specific steps are as follows:

[0163] (1-a) Preparation of the targeting fragment pfkAup-kan-pfkAdown

[0164] The targeting fragment pfkAup-kan-pfkAdown comes from the Escherichia coli strain JW1176, which is a W3110 series strain containing the pfkA knockout trait, in which the gene pfkA encoding 6-phosphofructokinase 1 is replaced by a kanamycin resistance gene (about 1500 bp) with FRT sites at both ends, thereby knocking out the pfkA gene (Baba T, Ara T, et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2006; 2: 2006.0008.).

[0165] Using the genomic DNA of Escherichia coli strain JW1176 as a template, PCR amplification was performed with primers pfkA-1 / pfkA-2 to obtain the target fragment pfkAup-kan-pfkAdown containing 70 bp homology arms upstream and downstream of the pfkA gene site.

[0166] (1-b) Preparation of host bacteria containing pKD46 plasmid

[0167] The pKD46 plasmid was transformed into E. coli BW25113 by calcium chloride transformation method, and clones were selected after overnight culture at 30°C on LB plates containing ampicillin to obtain recombinant E. coli BW25113 / pKD46 containing plasmid pKD46. After arabinose induction, BW25113 / pKD46 expressed three recombinant proteins of λ phage, and the host bacteria had the ability of homologous recombination. Then, BW25113 / pKD46 competent cells were prepared by washing with 10% glycerol.

[0168] (1-c) Homologous recombination

[0169] The target fragment pfkAup-kan-pfkAdown prepared in step (1-a) was electroporated into the BW25113 / pKD46 competent cells prepared in step (1-b), and incubated overnight at 37°C on an LB plate containing kanamycin (at a concentration of 50 μg / ml). The clones were selected to extract genomic DNA, and PCR amplification was performed using pfkA-3 / Kan-R as primers. The amplified target band of about 1400 bp was positive, and the positive clones were selected and named GN101-kan. The sequencing analysis results showed that the genome of GN101-kan contained the pfkAup-kan-pfkAdown fragment.

[0170] GN101-kan was cultured at 42°C overnight to eliminate the temperature-sensitive plasmid pKD46.

[0171] (1-d) Elimination of resistance

[0172] The pCP20 plasmid was transformed into GN101-kan by the calcium chloride transformation method. The culture was carried out at 30°C overnight on an LB plate containing ampicillin, and the FLP recombinase on the pCP20 plasmid was used to eliminate kanamycin resistance. The culture was carried out at 42°C overnight to eliminate the temperature-sensitive plasmid pCP20. The monoclonal strain was amplified and identified by PCR using the pfkA-2 / pfkA-3 primers. The amplified target band of about 200 bp was positive, and the recombinant E. coli GN101 was obtained.

[0173] The nucleotide sequence of the pfkA gene is shown in Gene ID: 948412 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of 6-phosphofructokinase 1 encoded by the gene is shown in reference sequence number CAA26356.1 in NCBI (submission date: 2016.07.26).

[0174] (2) Construction of GN102, GN103, GN104, GN105, and GN106

[0175] Referring to the method in step (1), on the basis of GN101, the 6-phosphofructokinase 2 gene (hereinafter referred to as pfkB), the 6-phosphoglucose dehydrogenase gene (hereinafter referred to as zwf), the operon element manXYZ where the mannose-specific PTS transporter gene is located, the N-acetylglucosamine-specific PTS transporter gene (hereinafter referred to as nagE) and the glucosamine-6-phosphate deaminase gene (hereinafter referred to as nagB) are knocked out in sequence to obtain recombinant Escherichia coli GN102, GN103, GN104, GN105 and GN106. The only difference is that the primer combination pfkA-1 / pfkA-2 / pfkA-3 is replaced by the primer combination pfkB-1 / pfkB-2 / pfkB-3, the primer combination zwf-1 / zwf-2 / zwf-3, the primer combination manXYZ-1 / manXYZ-2 / manXYZ-3, the primer combination nagE-1 / nagE-2 / nagE-3, and the primer combination nagB-1 / nagB-2 / nagB-3. The transformed strain BW25113 is replaced by GN101, GN102, GN103, GN104, and GN105.

[0176] The nucleotide sequence of the pfkB gene is shown in Gene ID: 946230 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of 6-phosphofructokinase 2 encoded by the gene is shown in reference sequence number NP_416237 in NCBI (submission date: 2022.03.09).

[0177] The nucleotide sequence of the zwf gene is shown in Gene ID: 94637 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of 6-phosphate glucose dehydrogenase encoded by the gene is shown in reference sequence number NP_416366 in NCBI (submission date: 2022.03.09).

[0178] The operon manXYZ contains manX gene, manY gene and manZ gene, and the nucleotide sequences of manX gene, manY gene and manZ gene are shown in Gene ID: 946334 (submission date: 2024.05.02), GeneID: 946332 (submission date: 2024.05.02) and Gene ID: 946342 (submission date: 2024.05.02) in Gene Bank, and the amino acid sequences of manX protein, manY protein and manZ protein encoded by manX gene, manY gene and manZ gene are shown in reference sequence number NP_416331 (submission date: 2022.03.09), reference sequence number NP_416332 (submission date: 2022.03.09) and reference sequence number NP_416333 (submission date: 2022.03.09) in NCBI.

[0179] The nucleotide sequence of the nagE gene is shown in Gene ID: 945292 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of the N-acetylglucosamine-specific PTS transporter encoded by the gene is shown in reference sequence number NP_415205 in NCBI (submission date: 2022.03.09).

[0180] The nucleotide sequence of the nagB gene is shown in Gene ID: 945290 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of the glucosamine-6-phosphate deaminase encoded by the gene is shown in reference sequence number NP_415204 in NCBI (submission date: 2022.03.09).

[0181] (3) Enhanced expression of the arabinose transporter (araE) gene by inserting a promoter

[0182] Starting from the recombinant E. coli GN106, the E. coli constitutive promoter P was inserted upstream of the araE gene in this strain. CPA1 (SEQ ID No.3), and obtain recombinant Escherichia coli GN107, the specific steps are as follows:

[0183] (3-a) Targeting fragment araEup-kan-P CPA1 - Preparation of araEdown

[0184] GenScript was commissioned to synthesize the following DNA fragments: from 5' to 3', it contained the 70 bp homology arm fragment upstream of the araE gene (denoted as araEup, SEQ ID No.5), the frt-kan-frt screening marker fragment (SEQ ID No.1), the PCPA1 The promoter fragment (SEQ ID No.3) and the 70 bp fragment before the start codon of the araE gene (denoted as araEdown, SEQ ID No.6) were used as primers and the gene synthesized DNA fragment was used as a template for PCR amplification to obtain the target fragment araEup-kan-P CPA1 -araEdown.

[0185] (3-b) The subsequent operations refer to steps (1-b) to (1-d) in the above (1), and the Escherichia coli constitutive promoter P is inserted upstream of the araE gene. CPA1 coli GN107. In step (1-c), the 1400bp positive clone identified and amplified using primers pfkA-3 / Kan-R is changed to the 1700bp positive clone identified and amplified using primers araE-3 / Kan-R. In step (1-d), the 200bp positive clone identified and amplified using primers pfkA-2 / pfkA-3 is changed to the 500bp positive clone identified and amplified using primers araE-2 / araE-3.

[0186] The nucleotide sequence of the araE gene is shown in Gene ID: 947341 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of the arabinose transporter encoded by the gene is shown in reference sequence number NP_417318 in NCBI (submission date: 2022.03.09).

[0187] (4) Replace the phosphotransferase G subunit (ptsG) gene with the glucokinase (glk) gene

[0188] Starting from the recombinant Escherichia coli GN107, the ptsG gene in the strain was replaced by the constitutive promoter P 119 and glk gene to obtain recombinant Escherichia coli GN108. The specific steps are as follows:

[0189] (4-a) Targeting fragment ptsGup-kan-P 119 Preparation of -glk-ptsGdown

[0190] GenScript was commissioned to synthesize the following DNA fragments: from 5' to 3', it contained the 70 bp homology arm fragment upstream of the ptsG gene (denoted as ptsGup, SEQ ID No.7), the frt-kan-frt screening marker fragment (SEQ ID No.1), the P 119The promoter fragment (SEQ ID No. 4), glk gene, TrrnB terminator fragment (SEQ ID No. 2), and ptsG gene downstream 70 bp fragment (denoted as ptsGdown, SEQ ID No. 8). PCR amplification was performed using ptsG-1 / ptsG-2 as primers and the gene synthesized DNA fragment as a template to obtain the target fragment ptsGup-kan-P 119 -glk-ptsGdown.

[0191] (4-b) The subsequent operation is the same as step (3-b) in the above (3), and the constitutive promoter P is obtained. 119 The recombinant E. coli GN108 in which the ptsG gene is replaced by the glk gene is different in that the primer combination araE-1 / araE-2 / araE-3 is replaced by the primer combination ptsG-1 / ptsG-2 / ptsG-3.

[0192] The nucleotide sequence of the ptsG gene is shown in Gene ID: 947341 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of the phosphotransferase G subunit encoded by the gene is shown in reference sequence number NP_415619 in NCBI (submission date: 2022.03.09).

[0193] The nucleotide sequence of the glk gene is shown in Gene ID: 94734 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of glucose kinase encoded by the gene is shown in reference sequence number NP_416889 in NCBI (submission date: 2022.03.09).

[0194] (5) Replace the galactose repressor gene (hereinafter referred to as galR) with the glucose transport cofactor gene (derived from Zymomonas mobilis, hereinafter referred to as Zglf)

[0195] Starting from the recombinant Escherichia coli GN108, the galR gene in the strain was replaced by the constitutive promoter P CPA1 and Zglf genes to obtain recombinant Escherichia coli GN109. The specific steps are as follows:

[0196] (5-a) GenScript was commissioned to synthesize the following DNA fragments: from 5' to 3', it contained the 70 bp homology arm fragment upstream of the galR gene (denoted as galRup, SEQ ID No. 9), the frt-kan-frt screening marker fragment (SEQ ID No. 1), the P CPA1Promoter fragment (SEQ ID No.3), Zglf gene, TrrnB terminator fragment (SEQ ID No.2), 70bp fragment downstream of galR gene (denoted as galRdown, SEQ ID No.10). PCR amplification was performed using galR-1 / galR-2 as primers and the gene synthesized DNA fragment as template to obtain the target fragment galRup-kan-P CPA1 -Zglf-galRdown.

[0197] (5-b) The subsequent operation is the same as step (3-b) in the above (3), and the constitutive promoter P is obtained. CPA1 The recombinant E. coli GN109 in which the galR gene is replaced by the Zglf gene is different in that the primer combination araE-1 / araE-2 / araE-3 is replaced by the primer combination galR-1 / galR-2 / galR-3.

[0198] The nucleotide sequence of the galR gene is shown in Gene ID: 947314 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of the galactose repressor factor encoded by the gene is shown in reference sequence number NP_417314 in NCBI (submission date: 2022.03.09).

[0199] The nucleotide sequence of the Zglf gene is shown in the 185-1606 positions of the genome sequence in the NCBI reference sequence number M60615.1 (submission date: 1993.04.26), and the amino acid sequence of the glucose transport auxiliary factor encoded by the gene is shown in the NCBI reference sequence number AAA27691.1 (submission date: 1993.04.26).

[0200] (6) Introducing the L-glutamine-D-fructose-6-phosphate aminotransferase (EcGlmS, derived from Escherichia coli) gene and the glucosamine-6-phosphate phosphorylase (BtGlmP, derived from Bacteroides thetaiotaomicron) gene into Escherichia coli

[0201] Starting from the recombinant E. coli GN109, the expression plasmid pTrc99a-EcGlmS-BtGlmP containing the EcGlmS gene and the BtGlmP gene was introduced into GN109 to obtain the recombinant E. coli GN110. The specific steps are as follows:

[0202] (6-a) Preparation of pTrc99a-EcGlmS-BtGlmP plasmid

[0203] The nucleotide sequence of the EcGlmS gene is shown in SEQ ID No. 22, and the nucleotide sequence of the BtGlmP gene is shown in SEQ ID No. 24. The sequence containing the EcGlmS and BtGlmP genes and related expression elements was synthesized by Qingke Biotechnology. The synthesized sequence was used as a template, GlmS / P-01F and GlmS / P-01R were used as primers, and the gene fragment EcGlmS-BtGlmP composed of the EcGlmS gene and the BtGlmP gene was PCR amplified using high-fidelity TransStartFastPfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., product catalog AP221), and the target fragment was recovered by agarose gel electrophoresis. Then, the primers pTrc99a-01F / pTrc99a-01R were used, and the plasmid pTrc99a (Beijing Zhuangmeng International Biogene Technology Co., Ltd., product number ZK1610) was used as a template to PCR amplify the pTrc fragment and recover the large vector fragment pTrc. The EcGlmS-BtGlmP fragment was ligated with the pTrc fragment using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. methods. 2009; 6 (5): 343-345) to obtain the recombinant expression vector pTrc99a-EcGlmS-BtGlmP. Escherichia coli DH5α competent cells (Beijing Quanshijin Biotechnology Co., Ltd., product catalog CD201) were transformed using the CaCl2 method. They were evenly spread on an LB plate containing ampicillin and cultured at 37°C overnight. Clones were selected, and the clones that could amplify the target fragment were identified and sequenced using primers RV-M / pTrcHis-R. Positive clones were selected to extract plasmids, and the obtained positive plasmids were named pTrc99a-EcGlmS-BtGlmP.

[0204] (6-b) Plasmid transformation

[0205] The pTrc99a-EcGlmS-BtGlmP plasmid was transformed into recombinant Escherichia coli GN109 by the calcium chloride transformation method. After overnight culture at 37°C on an LB plate containing ampicillin, clones were selected, and positive clones were identified using primers RV-M / pTrcHis-R. They were named Escherichia coli GN110 and stored at -80°C.

[0206] Escherichia coli GN110 contains the EcGlmS gene sequence shown in SEQ ID No.22 and the BtGlmP gene sequence shown in SEQ ID No.24, and can express L-glutamine-D-fructose-6-phosphate aminotransferase with an amino acid sequence as shown in SEQ ID No.21 and glucosamine-6-phosphate phosphorylase with an amino acid sequence as shown in SEQ ID No.23.

[0207] Example 2: Production of D-glucosamine using recombinant Escherichia coli engineered strain GN110

[0208] In this example, the recombinant Escherichia coli engineered strain GN110 is used as the production strain, glycerol is used as the carbon source to complete the strain culture and amplification, IPTG is used to induce the expression of key enzymes, and finally D-glucose is converted into D-glucosamine, which specifically includes the following steps:

[0209] (1) Culture medium components

[0210] (1-a) The components and final concentrations of the glycerol growth medium are as follows (the solvent is water):

[0211] Na2HPO4: 25mM;

[0212] KH2PO4: 25mM;

[0213] NH4Cl: 50mM;

[0214] Na2SO4: 5mM;

[0215] MgSO4: 2mM;

[0216] Glycerol: 3% (g / 100mL);

[0217] Yeast powder: 0.5% (g / 100mL);

[0218] Trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM CoCl2, 2μM NiCl2, 2μM Na2MO4, 2μM Na2SeO3 and 2μM H3BO3.

[0219] (1-b) The transformation medium components and final concentrations are as follows (the solvent is water):

[0220] Na2HPO4: 100mM;

[0221] KH2PO4: 100mM;

[0222] MgCl2: 5mM;

[0223] D-glucose: 20 g / 100 mL (20%).

[0224] (2) Bacterial culture and enzyme induction

[0225] The wild-type strain BW25113, recombinant E. coli GN109 and GN110 cultured overnight were inoculated into a shake flask containing 200 mL of glycerol growth medium (GN110 growth medium contained ampicillin at a final concentration of 100 mg / L) at a 1% inoculum, and cultured at 30°C for 3-4 h until the OD 600nm When the concentration of IPTG reached 0.6-0.8, add IPTG to a final concentration of 0.5 mM, continue culturing at 30°C for 12 h, and collect the bacteria by centrifugation at 8000 rpm for 10 min.

[0226] (3) Whole-cell catalysis of D-glucose

[0227] The collected bacteria were resuspended in a conical flask containing 10 mL of transformation medium. The reaction process was monitored regularly using pH test paper, and 0.1 M NaOH and HCl solutions were used to adjust the pH to 7.0. After reacting at 37°C for 48 hours, the supernatant was centrifuged and filtered. The content of D-glucosamine was detected by HPLC.

[0228] HPLC used Hi-Plex Ca column (300 mm × 7.7 mm, 8 μm); mobile phase was ultrapure water, flow rate was 0.6 mL / min; column temperature was 70°C; detector was differential refractive index detector; temperature was 40°C. D-Glucosamine hydrochloride standard was purchased from the National Drug Standard Substance Ordering Platform (Cat. No.: 140649).

[0229] The results showed that the production of D-glucosamine by wild-type strain BW25113 and recombinant E. coli GN109 was 0, and the average production of D-glucosamine by engineered strain GN110 was 70 g / L. Figure 1 Therefore, D-glucose can be converted into D-glucosamine using the engineered strain GN110 with a conversion rate of 35%.

[0230] Example 3: Construction of recombinant Escherichia coli engineered strain GN210

[0231] In this example, a basic bacterium GN210 was prepared that can use fatty acids as carbon sources for bacterial growth and convert D-glucose into D-glucosamine. The preparation method of the strain is as follows, and the primers used are shown in Table 1.

[0232] (1) Knockout of the fatty acid degradation transcription factor (fadR) gene

[0233] Starting from recombinant E. coli GN109, the fadR gene of the GN109 strain was knocked out to obtain recombinant E. coli GN201. The specific steps were referred to steps (1-a) to (1-d) in Example 1. The difference was that the primer combination pfkA-1 / pfkA-2 / pfkA-3 was replaced by the primer combination fadR-1 / fadR-2 / fadR-3.

[0234] The nucleotide sequence of the fadR gene is shown in Gene ID: 94865 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of the fatty acid degradation transcription factor encoded by the gene is shown in reference sequence number NP_415705 in NCBI (submission date: 2022.03.09).

[0235] (2) Enhance the expression of fatty acyl-CoA synthetase (fadD) gene by inserting a promoter

[0236] Starting from the recombinant E. coli GN201, the E. coli constitutive promoter P was inserted upstream of the fadD gene in this strain. CPA1 (SEQ ID No. 3) to obtain recombinant E. coli GN202. The specific steps are as in step (3) of Example 1. The only difference is that the targeting fragment fadDup-kan-P CPA1 -fadDdown, and use the primer combination fadD-1 / fadD-2 / fadD-3 to replace the primer combination araE-1 / araE-2 / araE-3. The specific steps are as follows:

[0237] (2-a) Targeting fragment fadDup-kan-P CPA1 Preparation of -fadDdown

[0238] GenScript was commissioned to synthesize the following DNA fragments: from 5' to 3', it contained a 70 bp homology arm fragment upstream of the fadD gene (denoted as fadDup, SEQ ID No. 13), a frt-kan-frt screening marker fragment (SEQ ID No. 1), a P CPA1 The promoter fragment (SEQ ID No. 3) and the 70 bp fragment before the start codon of the fadD gene (recorded as sequence fadDdown, SEQ ID No. 14) were used as primers and the gene synthesized DNA fragment was used as a template for PCR amplification to obtain the target fragment fadDup-kan-P CPA1 -fadDdown.

[0239] (2-b) The subsequent operations were carried out in accordance with steps (1-b) to (1-d) in Example 1 (1), and the Escherichia coli constitutive promoter P was inserted upstream of the fadD gene. CPA1 coli GN202. In step (1-c), the 1400bp positive clone identified and amplified using primers pfkA-3 / Kan-R is changed to the 1400bp positive clone identified and amplified using primers fadD-3 / Kan-R. In step (1-d), the 200bp positive clone identified and amplified using primers pfkA-2 / pfkA-3 is changed to the 300bp positive clone identified and amplified using primers fadD-2 / fadD-3.

[0240] The nucleotide sequence of the fadD gene is shown in Gene ID: 94632 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of the fatty acyl-CoA synthetase encoded by the gene is shown in reference sequence number NP_416319 in NCBI (submission date: 2022.03.09).

[0241] (3) Enhance the expression of the long-chain fatty acid transporter (fadL) gene by inserting a promoter

[0242] Following the same method, starting from the recombinant E. coli GN202, a constitutive promoter P was inserted upstream of the fadL gene in the GN202 strain. CPA1 The operation process is similar to step (3) in Example 1, except that the targeting fragment fadLup-kan-P is obtained. CPA1 -fadLdown, and use the primer combination fadL-1 / fadL-2 / fadL-3 to replace the primer combination araE-1 / araE-2 / araE-3. The specific steps are as follows:

[0243] (3-a) Targeting fragment fadLup-kan-P CPA1 Preparation of -fadLdown

[0244] GenScript was commissioned to synthesize the following DNA fragments: from 5' to 3', it contained the 70 bp homology arm fragment upstream of the fadL gene (denoted as fadLup, SEQ ID No.15), the frt-kan-frt screening marker fragment (SEQ ID No.1), the P CPA1 The promoter fragment (SEQ ID No. 3) and the 70 bp fragment before the start codon of the fadL gene (recorded as sequence fadLdown, SEQ ID No. 16) were used as primers and the gene synthesized DNA fragment was used as a template for PCR amplification to obtain the target fragment fadLup-kan-PCPA1 -fadLdown.

[0245] (3-b) The subsequent operations refer to steps (1-b) to (1-d) in Example 1 (1) to obtain a constitutive promoter P inserted upstream of the fadL gene. CPA1 coli GN203. In step (1-c), the 1400bp positive clone identified and amplified using primers pfkA-3 / Kan-R is changed to the 1400bp positive clone identified and amplified using primers fadL-3 / Kan-R. In step (1-d), the 200bp positive clone identified and amplified using primers pfkA-2 / pfkA-3 is changed to the 300bp positive clone identified and amplified using primers fadL-2 / fadL-3.

[0246] The nucleotide sequence of the fadL gene is shown in Gene ID: 946820 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of the long-chain fatty acid transport protein encoded by the gene is shown in reference sequence number NP_416846 in NCBI (submission date: 2022.03.09).

[0247] (4) Enhanced expression of NAD(P) transhydrogenase (sthA) gene by inserting a promoter

[0248] Following the same method, starting from the recombinant E. coli GN203, a constitutive promoter P was inserted upstream of the sthA gene in the strain. CPA1 The operation process is similar to step (3) in Example 1, except that: the targeting fragment sthAup-kan-P is obtained. CPA1 -sthAdown, and use the primer combination sthA-1 / sthA-2 / sthA-3 to replace the primer combination araE-1 / araE-2 / araE-3. The specific steps are as follows:

[0249] (4-a) Targeting fragment sthAup-kan-P CPA1 Preparation of -sthAdown

[0250] GenScript was commissioned to synthesize the following DNA fragments: from 5' to 3', it contained the 70 bp homology arm fragment upstream of the sthA gene (denoted as sthAup, SEQ ID No. 17), the frt-kan-frt screening marker fragment (SEQ ID No. 1), the P CPA1The promoter fragment (SEQ ID No. 3) and the 70 bp fragment before the start codon of the sthA gene (recorded as the sequence sthAdown, SEQ ID No. 18) were used as primers and the gene synthesized DNA fragment was used as a template for PCR amplification to obtain the target fragment sthAup-kan-P CPA1 -sthAdown.

[0251] (4-b) The subsequent operations refer to steps (1-b) to (1-d) in Example 1 (1), and a constitutive promoter P is inserted upstream of the sthA gene. CPA1 coli GN204. In step (1-c), the 1400bp positive clone identified and amplified using primers pfkA-3 / Kan-R is changed to the 1400bp positive clone identified and amplified using primers sthA-3 / Kan-R. In step (1-d), the 200bp positive clone identified and amplified using primers pfkA-2 / pfkA-3 is changed to the 300bp positive clone identified and amplified using primers sthA-2 / sthA-3.

[0252] The nucleotide sequence of the sthA gene is shown in Gene ID: 94846 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of the NAD(P) transhydrogenase encoded by the gene is shown in reference sequence number NP_418397 in NCBI (submission date: 2022.03.09).

[0253] (5) Enhance the expression of the short-chain fatty acid degradation regulatory gene cluster atoS and atoC by inserting a promoter

[0254] Following the same method, starting from the recombinant E. coli GN204, a constitutive promoter P was inserted upstream of the atoSC gene cluster in this strain. CPA1 , and obtain recombinant E. coli GN205. The operation process is the same as that in Example 1 (3), except that: the targeting fragment atoSup-kan-P CPA1 -atoSdown, and use the primer combination atoS-1 / atoS-2 / atoS-3 to replace the primers araE-1 / araE-2 / araE-3. The specific steps are as follows:

[0255] (5-a) Targeting fragment atoSup-kan-P CPA1 - Preparation of atoSdown

[0256] GenScript was commissioned to synthesize the following DNA fragments: from 5' to 3', it contained the 70 bp homology arm fragment upstream of the atoS gene (denoted as atoSup, SEQ ID No.19), the frt-kan-frt screening marker fragment (SEQ ID No.1), the P CPA1 The promoter fragment (SEQ ID No.3) and the 70 bp fragment before the start codon of the atoS gene (recorded as sequence atoSdown, SEQ ID No.20) were used as primers and the gene synthesized DNA fragment was used as a template for PCR amplification to obtain the target fragment atoSup-kan-P CPA1 -atoSdown.

[0257] (5-b) The subsequent operations refer to steps (1-b) to (1-d) in Example 1 (1) to obtain a constitutive promoter P inserted upstream of the atoSC gene cluster. CPA1 coli GN205. In step (1-c), the 1400bp positive clone identified and amplified using primers pfkA-3 / Kan-R is changed to the 1400bp positive clone identified and amplified using primers atoS-3 / Kan-R. In step (1-d), the 200bp positive clone identified and amplified using primers pfkA-2 / pfkA-3 is changed to the 300bp positive clone identified and amplified using primers atoS-2 / atoS-3.

[0258] Among them, the atoSC gene cluster contains the atoS gene and the atoC gene. The nucleotide sequences of the atoS gene and the atoC gene are shown in Gene ID: 949011 (submission date: 2024.05.02) and Gene ID: 947444 (submission date: 2024.05.02) in Gene Bank, respectively, and the amino acid sequences of the atos protein and atoC protein encoded by the atoS gene and the atoC gene are shown in reference sequence number NP_416723 (submission date: 2022.03.09) and reference sequence number NP_416724 (submission date: 2022.03.09) in NCBI, respectively.

[0259] (6) Using GN205 as the starting strain, the β-ketoacyl-ACP synthase II (hereinafter referred to as fabF) gene, the β-ketoacyl-ACP synthase III (hereinafter referred to as fabH) gene, the lipid A biosynthesis myristoyltransferase (hereinafter referred to as lpxM) gene and the isocitrate lyase inhibitor (hereinafter referred to as iclR) gene were knocked out in sequence to obtain recombinant Escherichia coli GN206, GN207, GN208 and GN209. The specific operation steps refer to steps (1-a) to (1-d) in Example 1 (1). The only difference is that the primer combination pfkA-1 / pfkA-2 / pfkA-3 is replaced by the primer combination fabF-1 / fabF-2 / fabF-3, the primer combination fabH-1 / fabH-2 / fabH-3, the primer combination lpxM-1 / lpxM-2 / lpxM-3, and the primer combination iclR-1 / iclR-2 / iclR-3.

[0260] The nucleotide sequence of the fabF gene is shown in Gene ID: 946665 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of β-ketoacyl-ACP synthase II encoded by the gene is shown in reference sequence number NP_415613 in NCBI (submission date: 2022.03.09).

[0261] The nucleotide sequence of the fabH gene is shown in Gene ID: 946003 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of β-ketoacyl-ACP synthase III encoded by the gene is shown in reference sequence number NP_415609 in NCBI (submission date: 2022.03.09).

[0262] The nucleotide sequence of the lpxM gene is shown in Gene ID: 945143 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of the lipid A biosynthesis myristoyltransferase encoded by the gene is shown in reference sequence number NP_416369 in NCBI (submission date: 2022.03.09).

[0263] The nucleotide sequence of the iclR gene is shown in Gene ID: 948524 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of the isocitrate lyase inhibitor encoded by the gene is shown in reference sequence number NP_418442 in NCBI (submission date: 2022.03.09).

[0264] (7) Starting from the recombinant Escherichia coli GN209, the expression plasmid pTrc99a-EcGlmS-BtGlmP containing the EcGlmS and BtGlmP genes was introduced into GN209 to obtain the recombinant Escherichia coli GN210. The specific steps are as follows: step (6) in Example 1.

[0265] Example 4: Production of D-glucosamine using recombinant Escherichia coli engineered strain GN210

[0266] In this example, the recombinant E. coli engineered strain GN210 is used as the production strain, fatty acids are used as the carbon source to complete the strain culture and amplification, IPTG is used to induce the expression of key enzymes, and finally D-glucose is converted into D-glucosamine, which specifically includes the following steps:

[0267] (1) Culture medium components

[0268] (1-a) The components and final concentrations of fatty acid growth medium are as follows:

[0269] Medium A: Medium A is a sterile culture medium composed of solutes and solvents, the solvent is water, and the solutes and their concentrations are: 25mM NaHPO4, 25mM KH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4, 0.5% glycerol by volume, 0.5% yeast powder by mass, 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM CoCl2, 2μM NiCl2, 2μM Na2MO4, 2μM Na2SeO3 and 2μM H3BO3.

[0270] Medium B: Medium B is a sterile culture medium obtained by adding palmitic acid and polyoxyethylene ether Brij58 emulsifier (Merck, Inc., Product Number: P5884-100G) to medium A, wherein the mass percentage concentration of palmitic acid is 0.5%, and the mass percentage concentration of polyoxyethylene ether Brij58 emulsifier is 0.2%.

[0271] (1-b) The transformation medium components and final concentrations are as follows (the solvent is water):

[0272] Na2HPO4: 100mM;

[0273] KH2PO4: 100mM;

[0274] MgCl2: 5 mM;

[0275] D-glucose: 20 g / 100 mL (20%).

[0276] (2) Bacterial culture and enzyme induction

[0277] The wild-type strain BW25113, recombinant E. coli GN209 and GN210 cultured overnight were inoculated at 1% in a shake flask containing 200 mL of fatty acid growth medium B (wherein the GN210 growth medium contained ampicillin at a final concentration of 100 mg / L) and cultured at 30°C for 3-4 h until the OD reached 0. 600nm When the concentration of IPTG reached 0.6-0.8, add IPTG to a final concentration of 0.5 mM, continue culturing at 30°C for 12 h, and collect the bacteria by centrifugation at 8000 rpm for 10 min.

[0278] (3) Whole-cell catalysis of D-glucose

[0279] The collected cells were resuspended in a triangular flask containing 10 mL of transformation medium, and the pH was kept at 7.0 during the reaction. After reacting at 37°C for 48 hours, the supernatant was centrifuged and filtered. The content of D-glucosamine was detected by HPLC. The content of D-glucosamine was detected by referring to the method described in Example 2.

[0280] The results showed that the production of D-glucosamine by wild-type strain BW25113 and recombinant E. coli GN209 was 0, and the average production of D-glucosamine by recombinant E. coli GN210 was 68 g / L. Figure 2 Therefore, D-glucose can be converted into D-glucosamine using the engineered strain GN210 with a conversion rate of 34%.

[0281] Example 5: Construction of recombinant Escherichia coli engineered strain GN305

[0282] In this example, a recombinant E. coli engineered strain GN305 with weakened by-product production was constructed based on GN209. The construction method of the strain is as follows, and the primers used are shown in Table 1.

[0283] (1) Knockout of the D-psicose-6-phosphate isomerase (alsE) gene

[0284] Starting from recombinant E. coli GN209, the alsE gene in the GN209 strain was knocked out to obtain recombinant E. coli GN301. The specific steps are referred to steps (1-a) to (1-d) in Example 1. The only difference is that the primer combination pfkA-1 / pfkA-2 / pfkA-3 is replaced by the primer combination alsE-1 / alsE-2 / alsE-3.

[0285] The nucleotide sequence of the alsE gene is shown in Gene ID: 948595 (submission date: 2024.05.02) in Gene Bank, and the amino acid sequence of the D-psicose-6-phosphate isomerase encoded by the gene is shown in reference sequence number NP_418509 in NCBI (submission date: 2024.05.02).

[0286] (2) Using GN301 as the starting strain, the mannose-6-phosphate isomerase (hereinafter referred to as manA) gene, the mannitol-1-phosphate 5-dehydrogenase (hereinafter referred to as mtlD) gene and the sorbitol-6-phosphate 2-dehydrogenase (hereinafter referred to as srlD) gene were knocked out in sequence to obtain recombinant Escherichia coli GN302, GN303 and GN304, respectively. The specific operation steps refer to steps (1-a) to (1-d) in Example 1 (1). The only difference is that the primer pfkA-1 / pfkA-2 / pfkA-3 combination is replaced by the primer manA-1 / manA-2 / manA-3 combination, the primer mtlD-1 / mtlD-2 / mtlD-3 combination, and the primer srlD-1 / srlD-2 / srlD-3 combination.

[0287] The nucleotide sequence of the manA gene is shown in Gene ID: 944840 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of mannose-6-phosphate isomerase encoded by the gene is shown in reference sequence number NP_416130 in NCBI (submission date: 2022.03.09).

[0288] The nucleotide sequence of the mtlD gene is shown in Gene ID: 948117 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of mannitol-1-phosphate 5-dehydrogenase encoded by the gene is shown in reference sequence number NP_418057 in NCBI (submission date: 2022.03.09).

[0289] The nucleotide sequence of the srlD gene is shown in Gene ID: 948937 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of sorbitol-6-phosphate 2-dehydrogenase encoded by the gene is shown in reference sequence number NP_417185 in NCBI (submission date: 2022.03.09).

[0290] (3) Starting from the recombinant Escherichia coli GN304, the expression plasmid pTrc99a-EcGlmS-BtGlmP containing the EcGlmS and BtGlmP genes was introduced into GN304 to obtain GN305. The specific steps are as follows: step (6) in Example 1.

[0291] Example 6: Construction of recombinant Escherichia coli engineered strains GN408, GN409 and GN410

[0292] In this example, based on GN304, recombinant E. coli engineered bacteria GN408, GN409 and GN410 were constructed to enhance the synthesis pathway of glutamine and weaken the metabolic pathway of glutamine consumption, so as to further increase the production of D-glucosamine. The construction method of the strain is as follows, and the primers used are shown in Table 1.

[0293] (1) Knockout of the glutaminase A (glsA) gene

[0294] Starting from recombinant E. coli GN304, the glsA gene in the GN304 strain was knocked out to obtain recombinant E. coli GN401. The specific steps were referred to steps (1-a) to (1-d) in Example 1. The only difference was that the primer combination pfkA-1 / pfkA-2 / pfkA-3 was replaced by the primer combination glsA-1 / glsA-2 / glsA-3.

[0295] The nucleotide sequence of the glsA gene is shown in Gene ID: 946187 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of glutaminase A encoded by the gene is shown in reference sequence number NP_415018 in NCBI (submission date: 2022.03.09).

[0296] (2) Using GN401 as the starting strain, the glutaminase B (hereinafter referred to as glsB) gene, nitrogen regulatory protein (hereinafter referred to as glnB) gene and glutamine synthetase adenylase / adenylyltransferase (hereinafter referred to as glnE) gene were knocked out in sequence to obtain recombinant Escherichia coli GN402, GN403 and GN404, respectively. The specific operation steps refer to steps (1-a) to (1-d) in Example 1 (1). The only difference is that the primer pfkA-1 / pfkA-2 / pfkA-3 combination is replaced by the primer glsB-1 / glsB-2 / glsB-3 combination, the primer glnB-1 / glnB-2 / glnB-3h combination and the primer glnE-1 / glnE-2 / glnE-3 combination in sequence.

[0297] The nucleotide sequence of the glsB gene is shown in Gene ID: 944973 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of glutaminase B encoded by the gene is shown in reference sequence number NP_416041 in NCBI (submission date: 2022.03.09).

[0298] The nucleotide sequence of the glnB gene is shown in Gene ID: 947016 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of the nitrogen-regulating protein encoded by the gene is shown in reference sequence number NP_417048 in NCBI (submission date: 2022.03.09).

[0299] The nucleotide sequence of the glnE gene is shown in Gene ID: 947552 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of glutamine synthetase adenylase / adenylyltransferase encoded by the gene is shown in reference sequence number NP_417525 in NCBI (submission date: 2022.03.09).

[0300] (3) Replacing the lactate dehydrogenase (ldhA) gene with the glutamine synthetase (derived from Corynebacterium glutamicu, CgGS) gene

[0301] Starting from the recombinant Escherichia coli GN404, the ldhA gene in the strain was replaced by the constitutive promoter P CPA1 and CgGS gene to obtain recombinant E. coli GN405. The specific steps are as follows:

[0302] (3-a) Targeting fragment ldhAup-kan-P CPA1 Preparation of -CgGS-ldhAdown

[0303] GenScript was commissioned to synthesize the following DNA fragments: from 5' to 3', it contained the 70 bp homology arm fragment upstream of the ldhA gene (denoted as ldhAup, SEQ ID No.11), the frt-kan-frt screening marker fragment (SEQ ID No.1), the P CPA1 The promoter fragment (SEQ ID No. 3), CgGS gene, TrrnB terminator fragment (SEQ ID No. 2), 1dhA gene downstream 70bp fragment (denoted as ldhAdown, SEQ ID No. 12). PCR amplification was performed using ldhA-1 / ldhA-2 as primers and the gene synthesized DNA fragment as a template to obtain the target fragment ldhAup-kan-P CPA1 -CgGS-ldhAdown.

[0304] (3-b) The subsequent operation is the same as step (3-b) in Example 1 to obtain a constitutive promoter P CPA1The recombinant E. coli GN405 in which the ldhA gene is replaced by the CgGS gene is only different in that the primer combination araE-1 / araE-2 / araE-3 is replaced by the primer combination ldhA-1 / ldhA-2 / ldhA-3.

[0305] The nucleotide sequence of the ldhA gene is shown in Gene ID: 946315 in Gene Bank (submission date: 2024.05.02), and the amino acid sequence of lactate dehydrogenase encoded by the gene is shown in reference sequence number NP_415898 in NCBI (submission date: 2022.03.09).

[0306] The nucleotide sequence of the CgGS gene is shown in the 2185037-2186470 position of the genome sequence of the reference sequence number CP007724.1 (submission date: 2014.08.21) in NCBI, and the amino acid sequence of the glutamine synthetase encoded by the gene is shown in the reference sequence number AIK85642 (submission date: 2014.08.21) in NCBI.

[0307] (4) Replace the lactate dehydrogenase (ldhA) gene with the glutamine synthetase (derived from Saccharomyces cerevisiae, ScGS) gene

[0308] Starting from the recombinant Escherichia coli GN404, the ldhA gene in the strain was replaced by the constitutive promoter P CPA1 and ScGS gene to obtain recombinant E. coli GN406. The specific steps are as follows:

[0309] (4-a) Targeting fragment ldhAup-kan-P CPA1 Preparation of -ScGS-ldhAdown

[0310] GenScript was commissioned to synthesize the following DNA fragments: from 5' to 3', it contained the 70 bp homology arm fragment upstream of the ldhA gene (denoted as ldhAup, SEQ ID No.11), the frt-kan-frt screening marker fragment (SEQ ID No.1), the P CPA1 The promoter fragment (SEQ ID No. 3), ScGS gene, TrrnB terminator fragment (SEQ ID No. 2), 1dhA gene downstream 70bp fragment (denoted as ldhAdown, SEQ ID No. 12). PCR amplification was performed using ldhA-1 / ldhA-2 as primers and the gene synthesized DNA fragment as a template to obtain the target fragment ldhAup-kan-P CPA1 -ScGS-ldhAdown.

[0311] (4-b) The subsequent operation refers to step (3-b) in Example 1 to obtain a constitutive promoter P CPA1 The recombinant E. coli GN406 in which the ldhA gene is replaced by the ScGS gene is only different in that the primer combination araE-1 / araE-2 / araE-3 is replaced by the primer combination ldhA-1 / ldhA-2 / ldhA-3.

[0312] The nucleotide sequence of the ScGS gene is shown in Gene ID: 856147 in Gene Bank (submission date: 2024.03.17), and the amino acid sequence of the glutamine synthetase encoded by the gene is shown in reference sequence number NP_015360 in NCBI (submission date: 2024.04.12).

[0313] (5) Replace the lactate dehydrogenase (ldhA) gene with the glutamine synthetase (from Lactobacillus acidophilus, LaGS) gene

[0314] Starting from the recombinant Escherichia coli GN404, the ldhA gene in the strain was replaced by the constitutive promoter P CPA1 and LaGS gene to obtain recombinant Escherichia coli GN407. The specific steps are as follows:

[0315] (5-a) Targeting fragment ldhAup-kan-P CPA1 Preparation of -LaGS-ldhAdown

[0316] GenScript was commissioned to synthesize the following DNA fragments: from 5' to 3', it contained the 70 bp homology arm fragment upstream of the ldhA gene (denoted as ldhAup, SEQ ID No.11), the frt-kan-frt screening marker fragment (SEQ ID No.1), the P CPA1 The promoter fragment (SEQ ID No. 3), LaGS gene (SEQ ID No. 2), TrrnB terminator fragment (SEQ ID No. 2), 70 bp fragment downstream of ldhA gene (denoted as ldhAdown, SEQ ID No. 12) were amplified by PCR using ldhA-1 / ldhA-2 as primers and the gene synthesized DNA fragment as a template to obtain the target fragment ldhAup-kan-P CPA1 -LaGS-ldhAdown.

[0317] (5-b) The subsequent operation is as in step (3-b) of Example 1, and the constitutive promoter P is obtained. CPA1The recombinant E. coli GN407 in which the ldhA gene is replaced by the LaGS gene is only different in that the primer combination araE-1 / araE-2 / araE-3 is replaced by the primer combination ldhA-1 / ldhA-2 / ldhA-3.

[0318] The nucleotide sequence of the LaGS gene is shown in the 1491778-1493115th position of the genome sequence of the reference sequence number CP020620.1 (submission date: 2018.04.10) in NCBI, and the amino acid sequence of the glutamine synthetase encoded by the gene is shown in the reference sequence number AJP46821 (submission date: 2015.04.15) in NCBI.

[0319] (6) Starting from recombinant Escherichia coli GN405, GN406 and GN407, the expression plasmid pTrc99a-EcGlmS-BtGlmP containing the EcGlmS and BtGlmP genes was introduced into GN405, GN406 and GN407 respectively to obtain recombinant Escherichia coli GN408, GN409 and GN410. The specific steps refer to step (6) in Example 1.

[0320] Example 7: Production of D-glucosamine using recombinant Escherichia coli engineered strains GN305, GN408, GN409 and GN410

[0321] In this example, recombinant E. coli engineered strains GN305, GN408, GN409 and GN410 are used as production strains, mixed carbon sources are used as raw materials to complete strain culture and amplification, IPTG is used to induce the expression of key enzymes, and finally D-glucose is converted into D-glucosamine, which specifically includes the following steps:

[0322] (1) Culture medium components

[0323] (1-a) The components and final concentrations of the mixed carbon source growth medium are as follows:

[0324] Medium A: Medium A is a sterile culture medium composed of solutes and solvents, the solvent is water, and the solutes and their concentrations are: 25mM NaHPO4, 25mM KH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4, 0.5% glycerol by volume, 0.5% yeast powder by mass, 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM CoCl2, 2μM NiCl2, 2μM Na2MO4, 2μM Na2SeO3 and 2μM H3BO3.

[0325] A1 medium: Based on the ingredients of A medium, it also contains 0.5% palmitic acid and 0.2% polyoxyethylene ether Brij58 emulsifier.

[0326] A2 medium: Based on the composition of A medium, it also contains 3% glycerol.

[0327] A3 medium: Based on the ingredients of A medium, it also contains 1% glucose and 1.5% glycerol.

[0328] A4 medium: based on the composition of A medium, it also contains 1% soybean oil and 15 mg / L lipase (Merck Co., Ltd., catalog number: V900486).

[0329] (1-b) The transformation medium components are as follows:

[0330] Na2HPO4: 100mM;

[0331] NaH2PO4: 100mM;

[0332] MgCl2: 5 mM;

[0333] D-Glucose: 20%.

[0334] (2) Bacterial culture and enzyme induction

[0335] The wild-type strain BW25113 and the recombinant E. coli engineered strains GN305, GN408, GN409 and GN410 were inoculated into shake flasks containing 200 mL of A1-A4 growth medium at 1% inoculation rate and cultured at 30°C for 3-4 h until the OD 600nm When the pH value is 0.6-0.8 (determined after extracting the oil components of A1 and A4 medium with n-hexane), add IPTG at a final concentration of 0.5 mM and continue culturing at 30°C for 12 hours. Collect the cells.

[0336] (3) Whole-cell catalysis of D-glucose

[0337] The collected cells were resuspended in a beaker containing 10 mL of transformation medium. The pH was kept at 7.0 during the reaction. After reacting at 37°C for 24 hours, the supernatant was centrifuged and filtered. The content of D-glucosamine was detected by HPLC. The content of D-glucosamine was detected by referring to the method described in Example 2.

[0338] The results showed that after the recombinant E. coli engineered strains were grown on different carbon sources and then subjected to whole-cell transformation, the yield of D-glucosamine was 82-126 g / L (Table 2, Figure 3), and the conversion rate was 41-63%.

[0339] Table 2. Comparison of the yield of D-glucosamine prepared by GN305, GN408, GN409 and GN410

[0340]

[0341] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Recombinant E. coli is obtained by performing multiple modifications on E. coli; The plurality of modifications include modification (a) and modification (e); Modification (a) includes the following modifications (a1) to (a9): Transformation (a1): knockout of the pfkA gene; Transformation (a2): knockout of the pfkB gene; Transformation (a3): knockout of zwf gene; Transformation (a4): knockout of manXYZ gene; Transformation (a5): knockout of nagE gene; Transformation (a6): knockout of nagB gene; Modification (a7): Enhanced expression of araE gene; Transformation (a8): ptsG gene was replaced by glk gene; Transformation (a9): galR gene was replaced by Zglf gene; Transformation (e): Introduction and expression of EcGlmS gene and BtGlmP gene.

2. A method for constructing a recombinant Escherichia coli that produces D-glucosamine, comprising the following steps: performing multiple transformations on the Escherichia coli; the multiple transformations include transformation (a) and transformation (e); Modification (a) includes the following modifications (a1) to (a9): Transformation (a1): knockout of the pfkA gene; Transformation (a2): knockout of the pfkB gene; Transformation (a3): knockout of zwf gene; Transformation (a4): knockout of manXYZ gene; Transformation (a5): knockout of nagE gene; Transformation (a6): knockout of nagB gene; Modification (a7): Enhanced expression of araE gene; Transformation (a8): ptsG gene was replaced by glk gene; Transformation (a9): galR gene was replaced by Zglf gene; Transformation (e): Introduction and expression of EcGlmS gene and BtGlmP gene.

3. The recombinant Escherichia coli according to claim 1 or the method according to claim 2, characterized in that: The plurality of modifications also includes modification (b); The transformation (b) includes the following transformations (b1) to (b9): Transformation (b1): knockout of fadR gene; Modification (b2): Enhanced expression of fadD gene; Modification (b3): ​​Enhanced expression of fadL gene; Modification (b4): Enhanced expression of sthA gene; Modification (b5): Enhanced expression of the atoSC gene cluster; Transformation (b6): knockout of the fabF gene; Transformation (b7): knockout of the fabH gene; Transformation (b8): knockout of the lpxM gene; Modification (b9): Knockout of the iclR gene.

4. The recombinant Escherichia coli or method according to any one of claims 1 to 3, characterized in that: The plurality of modifications also includes modification (c); The transformation (c) includes the following transformations (c1) to (c4): Transformation (c1): knockout of alsE gene; Transformation (c2): knockout of manA gene; Transformation (c3): knockout of the mtlD gene; Transformation (c4): Knockout of the srlD gene.

5. The recombinant Escherichia coli or method according to any one of claims 1 to 4, characterized in that: The plurality of modifications also includes modification (d); The transformation (d) includes the following transformations (d1)-(d5): Transformation (d1): knockout of glsA gene; Transformation (d2): knockout of glsB gene; Transformation (d3): knockout of glnB gene; Transformation (d4): knockout of the glnE gene; Modification (d5): ldhA gene was replaced with glutamine synthetase gene.

6. The recombinant Escherichia coli or method according to any one of claims 1 to 5, characterized in that: The glutamine synthetase gene is a CgGS gene derived from Corynebacterium glutamicu, a ScGS gene derived from Saccharomyces cerevisiae, or a LaGS gene derived from Lactobacillus acidophilus.

7. Use of the recombinant Escherichia coli or the method according to any one of claims 1 to 6 in the preparation of D-glucosamine.

8. A method for preparing D-glucosamine, comprising the following steps: using D-glucose as a raw material and using the recombinant Escherichia coli according to any one of claims 1 to 6 to carry out biotransformation to obtain D-glucosamine.

9. The method according to claim 8, characterized in that: The method further comprises the step of culturing the recombinant Escherichia coli using glycerol and / or oil as a carbon source.

10. The method according to claim 8 or 9, characterized in that: The oil includes triglycerides and fatty acids.

Images