Genetically engineered bacteria for producing xylitol using arabinose and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to genetically engineered bacteria for producing xylitol using arabinose and their applications. A genetically engineered bacterium is specifically provided, including a host cell and a target gene inserted into the host cell, wherein the target gene comprises an L-arabinose isomerase gene, a D-psicose-3-epimerase gene and an L-xylulose reductase gene; the protein coding sequence of the L-arabinose isomerase gene is shown in SEQ ID NO.1. The present invention successfully solves the problem of co-expression regulation of three enzymes in the xylitol production pathway of L-arabinose metabolism, and obtains genetically engineered bacteria with balanced expression of L-arabinose isomerase, D-psicose-3-epimerase and L-xylulose reductase by screening enzyme genes from different sources and promoter gene mutations, so that L-arabinose can be almost completely metabolized to produce xylitol.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to genetic engineering bacteria for producing xylitol by utilizing arabinose and application thereof. Background Art

[0002] Xylitol is a five-carbon sugar alcohol that exists naturally in animal and plant cells. The sweetness of xylitol is comparable to that of sucrose, and its metabolism is not regulated by insulin. It is completely metabolized in the human body, with a calorific value of 16.2 kJ / g, which is very low in calories. In the medical field, xylitol has the effects of preventing dental caries, preventing obesity, and anti-inflammatory. In the food field, xylitol, as a food additive, is a healthy and safe sugar substitute sweetener with important economic and social value.

[0003] At present, the industrial large-scale production of xylitol is mainly obtained by chemical catalytic hydrogenation after separation of hemicellulose hydrolyzed sugars and obtaining pure xylose (D-xylose). The process mainly includes 5 steps: hemicellulose hydrolysis, separation and purification of hydrolyzate, catalytic hydrogenation, xylitol purification, and xylitol crystallization. This method has the disadvantages of complex procedures, high energy consumption, large discharge of three wastes, and safety hazards of high-pressure hydrogenation. The biological method of producing xylitol is to reduce xylose to xylitol by enzyme catalysis or microbial fermentation, and then crystallize and separate xylitol. This process has the advantages of mild conversion conditions and green process, and is expected to replace the currently commonly used chemical hydrogenation method to produce xylitol. However, the current biological method only uses xylose in hemicellulose hydrolyzed sugars, and another pentose L-arabinose (L-Arabinose) cannot be used.

[0004] Gramineous hemicellulose is a polysaccharide with 1,4-β-D-pyranose as the main chain and 4-oxymethyl-pyranose glucuronic acid as the branch chain. It often contains L-furanose arabinose as the branch chain connected to the polyxylose main chain. The main components of the hemicellulose hydrolyzed sugar obtained after acid hydrolysis are D-xylose, D-glucose and L-arabinose. Although L-arabinose is classified in the list of "new food ingredients" by the National Health Commission of China, it inhibits sucrase, which will lead to more sucrose components in the intestines, which may cause bloating or diarrhea, which has led to a slow development of the application of L-arabinose. Therefore, the market demand for L-arabinose is not large, and it is necessary to develop new production processes and convert L-arabinose into xylitol to expand the application range of L-arabinose, thereby improving the utilization level of hemicellulose resources. Summary of the invention

[0005] In view of the technical demand for converting L-arabinose into xylitol in the prior art, the present invention provides a genetically engineered bacterium for producing xylitol using arabinose and its application. The specific technical scheme is as follows:

[0006] The present invention provides a genetically engineered bacterium, comprising a host cell and a target gene inserted into the host cell, wherein the target gene comprises an L-arabinose isomerase gene, a D-psicose-3-epimerase gene and an L-xylulose reductase gene; the L-arabinose isomerase gene is araA gene, whose protein coding sequence is shown in SEQ ID NO.1; the D-psicose-3-epimerase gene is dpe Gene, the dpe The gene is any one of NCBI accession numbers WP-138792167.1, WP-027674194.1, WP-313591370.1, WP-279658377.1, and AE008210; the L-xylulose reductase gene is lxr Gene, the lxr The gene is any one of NCBI accession numbers KAG7881166.1, KAF6014139.1, ANZ74972.1, ODV85849.1, and AJ583159.

[0007] L-arabinose is converted into xylitol through a metabolic pathway composed of three enzymes, with L-arabinose isomerase catalyzing the isomerization of L-arabinose into L-ribulose, L-psicose-3-epimerase catalyzing the epimerization of L-ribulose into L-xylulose, and L-xylulose reductase catalyzing the reduction of L-xylulose into xylitol.

[0008] Furthermore, the araA The 5' untranslated region of the gene is mutated, and the mutation is any one of the sequences shown in SEQ ID NO.8, SEQ ID NO.16, and SEQ ID NO.18.

[0009] wild type araA The gene has the defect of poor expression effect in Escherichia coli. araA The 5' untranslated region of the gene is mutated, and the 5' untranslated region of the gene is mutated. araA The gene was expressed in Escherichia coli, and the 5' non-translated region sequence with better expression effect in Escherichia coli was screened by measuring the enzyme activity.

[0010] Further, araA 5' untranslated region of the gene, dpe Genes and lxr The gene combinations are as follows:

[0011] (1) SEQ ID NO.8, WP-138792167.1, KAF6014139.1;

[0012] (2) SEQ ID NO.18, WP-138792167.1, AJ583159;

[0013] (3) SEQ ID NO.8, AE008210, KAF6014139.1;

[0014] (4) SEQ ID NO.18, AE008210, AJ583159;

[0015] (5) SEQ ID NO.8, WP-138792167.1, AJ583159;

[0016] (6) SEQ ID NO.8, AE008210, AJ583159.

[0017] Furthermore, the host cell is Escherichia coli.

[0018] Furthermore, the host cell is Escherichia coli E.coli W3110.

[0019] Furthermore, the E. coli genome araB , araD and lyxK One or more genes in the cell are knocked out or silenced.

[0020] Furthermore, the E. coli genome araB , araD and lyxK Genes are knocked out or silenced.

[0021] Furthermore, the araB The nucleotide sequence of the gene is shown in SEQ ID NO.19. araD The nucleotide sequence of the gene is shown in SEQ ID NO.20. lyxK The nucleotide sequence of the gene is shown in SEQ ID NO.21.

[0022] In order to improve the efficiency of engineering bacteria in metabolizing L-arabinose to produce xylitol, the present invention uses E.coli W3110△araBAD△lyxK was used as the host, and a genetically engineered bacterium was constructed to co-express L-arabinose isomerase with a 5' untranslated region (5'UTR) mutation, D-psicose-3-isomerase and L-xylulose reductase from different sources. The co-expression method was to construct a tandem expression plasmid pBAD18-araA-dpe-lxr and to express it in Escherichia coli. E.coli W3110△araBAD△lyxk was expressed, or the three gene co-expression cassettes were integrated into the genome to construct an integrated Escherichia coli E.coli W3110::(araA-dpe-lxr)△araBAD△lyxK was expressed.

[0023] Furthermore, the E. coli genome was inserted glpF Gene, glpK Gene, glpD At least one of the genes.

[0024] Furthermore, the E. coli genome was inserted glpF Gene, glpK Gene, glpD Gene.

[0025] Furthermore, the glpF The nucleotide sequence of the gene is shown in SEQ ID NO.22. glpK The nucleotide sequence of the gene is shown in SEQ ID NO.23. glpD The nucleotide sequence of the gene is shown in SEQ ID NO.24.

[0026] Since the L-xylulose reductase in the metabolic pathway needs to consume the coenzyme NADH when converting L-xylulose into xylitol, in order to ensure that the metabolic pathway can proceed in the forward direction, the present invention converts the glycerol transporter gene glpF and glycerol kinase gene glpK Co-expression cassette, glyceraldehyde-3-phosphate dehydrogenase gene glpD The expression cassette is integrated into the untranslated region of the E. coli genome; and by adding the metabolic co-substrate glycerol, more coenzyme NADH is produced, allowing L-xylulose to continuously produce xylitol.

[0027] The present invention provides the use of the genetically engineered bacteria in the biological preparation of xylitol.

[0028] The present invention also provides a method for producing xylitol by utilizing L-arabinose, comprising: inoculating the above-mentioned genetically engineered bacteria into a fermentation liquid containing L-arabinose and glycerol, and obtaining xylitol after fermentation.

[0029] Furthermore, the fermentation conditions are as follows: the genetically engineered bacteria are inoculated into a shake flask fermentation medium, cultured at 18-37° C. for 2-5 h, L-arabinose and glycerol are added to the medium, and fermented at 18-37° C. for 20-60 h.

[0030] Furthermore, the fermentation conditions are as follows: the genetically engineered bacteria are inoculated into a shake flask fermentation medium, cultured at 37°C for 2-5 h, 10 g / L of L-arabinose and 10 g / L of glycerol are added to the medium, and fermented at 30°C for 20-30 h.

[0031] Furthermore, the fermentation medium is composed of: yeast powder 5-10 g / L, Na2HPO4 1-5 g / L, KH2PO4 1-5 g / L, NH4Cl 1-3 g / L, NaCl 0.5-2 g / L, MgSO4 0.1-0.5 g / L, CaCl2 0.1-0.5 g / L.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The present invention uses Escherichia coli E.coli W3110 is the chassis cell, and a metabolic pathway of L-arabinose isomerase, D-psicose-3-epimerase and L-xylulose reductase genes is constructed and expressed in series in Escherichia coli W3110△araBAD△lyxK, thereby obtaining a genetically engineered bacterium that can convert L-arabinose into xylitol. araA , dpe and lxr The source combination is quite diverse.

[0034] (2) The present invention successfully solves the problem of co-expression regulation of three enzymes in the L-arabinose metabolism to xylitol production pathway. By screening enzyme genes from different sources and promoter gene mutations, a genetically engineered bacterium with balanced expression of L-arabinose isomerase, D-psicose-3-epimerase and L-xylulose reductase is obtained, so that L-arabinose can be basically completely metabolized to produce xylitol.

[0035] (3) The metabolic pathway and engineered bacteria constructed by the present invention can completely convert L-arabinose into xylitol, successfully solving the problem of L-arabinose utilization in hemicellulose hydrosaccharide and improving the utilization rate of hemicellulose resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Metabolic pathway for the conversion of L-arabinose to xylitol.

[0037] Figure 2 The results of shake flask fermentation before and after the enhancement of the glycerol metabolic pathway are shown; wherein G6 represents the multi-copy genome integrated engineered bacteria constructed in Example 9, and G7 represents the genetically engineered bacteria obtained after the glycerol pathway of the G6 strain constructed in Example 10 was enhanced. DETAILED DESCRIPTION

[0038] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0039] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0040] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0041] Detection of sugar alcohols: HPLC determination, the mobile phase is ultrapure water, Agilent Hi-Plex Ca chromatographic column (7.7 mm×300 mm), column temperature is 85℃; flow rate is 0.6 mL / min.

[0042] In the following examples, the final concentration of kanamycin in the culture medium is 50 μg / mL, and the final concentration of spectinomycin in the culture medium is 100 μg / mL.

[0043] The gene names involved in the present invention are explained as follows:

[0044] araA : L-arabinose isomerase gene;

[0045] dpe : D-psicose-3-epimerase gene;

[0046] lxr : L-xylulose reductase gene;

[0047] araB : ribulose kinase gene;

[0048] araD : L-ribulose-5-phosphate-4-epimerase gene;

[0049] lyxK : L-xylulose kinase gene;

[0050] glpF : Glycerol transporter gene;

[0051] glpK : Glycerol kinase gene;

[0052] glpD :Glyceryl triphosphate dehydrogenase gene.

[0053] The overall metabolic pathway of the strain of the present invention is as follows Figure 1 The primer information used in the following examples is shown in Table 1.

[0054] Table 1 Primer list

[0055]

[0056] Table 1 continued

[0057]

[0058] In the present invention, the araA The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0059] SEQ ID NO.1:

[0060]

[0061] Example 1 Knockout of ribulose kinase gene araB , L-ribulose-5-phosphate-4-epimerase gene araD and L-xylulose kinase gene lyxK Construction of engineered Escherichia coli

[0062] The present invention E.coli W3110 was used as the starting strain and the CRISPR / Cas9 gene editing method reported in the literature was used. Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl Environ Microbiol, 2015) araB , araD and lyxK Knockout of genes such as IL-61 can eliminate or alleviate the catabolism of L-ribulose and L-xylulose, the intermediate metabolites of the L-arabinose metabolic pathway.

[0063] (1) Knockout araB Gene strain E.coli Construction of W3110△araB

[0064] PCR amplification: Using ara-uF and ara-dR, ara-dF and ara-dR as primers, respectively, and W3110 genome as template, amplify fragments of 500 bp each, and recover them on gel; then using ara-uF and ara-dR as primers, the two recovered 500 bp fragments are used as templates to amplify a repair template fragment of about 1000 bp, and recover them on gel.

[0065] Construction of ara-target plasmid: Using ara-target-F and ara-target-R as primers and pTargetF-cadA as template, PCR amplified a fragment of about 2.1 kb, and after DpnI digestion, the ara-Target plasmid was constructed.

[0066] Preparation of electroporated competent cells: a) Pick a single colony of E. coli and place it in a 5 mL LB test tube medium, and culture it at 37°C, 200 rpm until OD600 is about 0.6; b) Use sterilized 2 mL Eppendorf tubes to dispense 1.5 mL of the bacterial solution into each tube, centrifuge at 4°C, 4000 rpm for 10 min, and discard the supernatant; c) Add 1 mL of pre-cooled sterilized 10% glycerol, gently resuspend, centrifuge at 4°C, 4000 rpm for 10 min, and carefully discard the supernatant; repeat this step twice; d) Resuspend with 100 μL 10% glycerol. Escherichia coli W3110 competent cells were prepared using the above-mentioned electroporation competent cell preparation method, and the pCas plasmid was electroporated (electroporation conditions: 2.5 kV, 200 Ω, 25 μF) into the competent cells. A single W3110 / pCas colony was picked up in a 5 mL LB test tube containing kanamycin and 10 mM arabinose, and cultured at 37°C until OD600 was approximately 0.6, and then the above-mentioned method was used to prepare W3110 / pCas electroporation competent cells.

[0067] Electroporation: The ara repair template fragment and ara-Target plasmid were electroporated into W3110 / pCas competent cells, spread on LB plates containing spectinomycin and kanamycin, and cultured at 37°C overnight; single colonies were grown and verified by colony PCR using specific primers.

[0068] Plasmid loss: Pick a single colony that is positive after colony PCR verification, inoculate it into an LB test tube containing kanamycin, add rhamnose with a final concentration of 10 mM, and culture it at 37°C overnight; the next day, inoculate the bacterial solution in the test tube into an antibiotic-free LB test tube containing 10 g / L sucrose, and culture it at 37°C overnight; the next day, streak the bacterial solution in the test tube directly on an antibiotic-free LB plate, and culture it at 37°C overnight; the next day, pick a single colony and transfer it to an LB plate containing spectinomycin or kanamycin; if neither can grow, it indicates that both pCas and ara-Target plasmids have been lost, and the W3110△araB strain is obtained.

[0069] (2) Knockout araD Gene strain E.coli Construction of W3110△araBAD

[0070] PCR amplification: Using araD-uF and araD-dR, araD-dF and araD-dR as primers, respectively, and W3110 genome as template to amplify fragments of 500 bp each, and then recover on gel; then using araD-uF and araD-dR as primers, the two recovered 500 bp fragments were used as templates to amplify a repair template fragment of approximately 1000 bp, and then recover on gel.

[0071] Construction of araD-Target plasmid: Using araD-Target-F and araD-Target-R as primers and pTargetF-cadA as template, PCR amplified a fragment of about 2.1 kb, and after DpnI digestion, the araD-Target plasmid was constructed.

[0072] Prepared by the above electroporation competent preparation method E.coli W3110△araB competent cells, the pCas plasmid was electroporated into competent cells, a single colony of W3110△araB / pCas was picked up in a 5 mL LB test tube containing kanamycin and 10 mM arabinose, and cultured at 37°C until OD600 was about 0.6, and then the W3110△araB / pCas competent cells were prepared using the above method.

[0073] Electroporation: The araD repair template fragment and araD-Target plasmid were electroporated into W3110△araB / pCas competent cells, spread on LB plates containing spectinomycin and kanamycin, and cultured at 37°C overnight; single colonies were grown and verified by colony PCR using specific primers.

[0074] Plasmid loss: The pCas and ptsF-N20 plasmids were removed using the aforementioned method to obtain the W3110△araBAD strain.

[0075] (3) Knockout lyxK Gene strain E.coli Construction of W3110△araBAD△lyxK

[0076] PCR amplification: using lyxK-uF and lyxK-dR, lyxK-dF and lyxK-dR as primers, respectively, and W3110 genome as template to amplify fragments of 500 bp each, and then recover on gel; then using lyxK-uF and lyxK-dR as primers, the two recovered 500 bp fragments were used as templates to amplify a repair template fragment of about 1000 bp, and then recover on gel.

[0077] Construction of lyxK-Target plasmid: Using lyxK-target-F and lyxK-target-R as primers and pTargetF-cadA as template, PCR amplified a fragment of about 2.1 kb, and after DpnI digestion, the lyxk-Target plasmid was constructed.

[0078] The W3110△araBAD competent cells were prepared using the above-mentioned electroporation competent cell preparation method, the pCas plasmid was electroporated into the competent cells, a single W3110△araBAD / pCas colony was picked and placed in a 5 mL LB test tube containing kanamycin and 10 mM arabinose, and cultured at 37°C until the OD600 was approximately 0.6, and the W3110△araBAD / pCas competent cells were prepared using the above-mentioned method.

[0079] Electroporation: lyxK The repair template fragment and lyxK-Target plasmid were electroporated into W3110△BAD / pCas competent cells, spread on LB plates containing spectinomycin and kanamycin, and cultured at 37°C overnight; single colonies were grown and verified by colony PCR using specific primers.

[0080] Plasmid loss: The pCas and lyxK-Target plasmids were removed using the aforementioned method to obtain the W3110△araBAD△lyxK strain.

[0081] Example 2 5'UTR mutation araA Construction of gene expression cassette and characterization of enzyme activity

[0082] (1) 5'UTR mutation araA Construction of gene expression plasmid

[0083] a) First, the E. coli W3110 was used as a template and araA-XbaI-F and araA-SphI-R were used as primers to amplify the araA The gene was amplified by restriction endonucleases XbaI and SphI. araA The gene fragments were digested by enzymes and recovered by column.

[0084] b) Extract the pBAD18 plasmid using a plasmid extraction kit, and then digest the plasmid pBAD18 with restriction endonucleases XbaI and SphI, and recover the plasmid by gel extraction.

[0085] c) Use T4 ligase to cleave the XbaI and SphI digested araA The fragment was connected to the linearized pBAD18 plasmid, transformed into E. coli DH5α, coated with LB solid plates containing kanamycin, and cultured at 37°C overnight; single colonies were grown and verified by colony PCR using specific primers. The correct clone was pBAD18-araA17 plasmid.

[0086] d) Using pBAD18-araA17 plasmid as template, specific primers araA1-F / araA1-R, araA2-F / araA2-R, araA3-F / araA3-R, araA4-F / araA4-R, araA5-F / araA5-R, araA6-F / araA6-R, araA7-F / araA7-R, araA8-F / araA8-R, araA9-F / araA9-R, araA10-F / araA10-R, araA11-F / araA11-R, araA12-F / araA12-R, araA13-F / araA13-R, araA14-F / araA14-R, araA15-F / araA16-R -F / araA15-R and araA16-F / araA16-R were subjected to whole plasmid PCR and digested with DpnI to construct pBAD18-araA1, pBAD18-araA2, pBAD18-araA3, pBAD18-araA4, pBAD18-araA5, pBAD18-araA6, pBAD18-araA7, pBAD18-araA8, pBAD18-araA9, pBAD18-araA10, pBAD18-araA11, pBAD18-araA12, pBAD18-araA13, pBAD18-araA14, pBAD18-araA15 and pBAD18-araA16 plasmids, respectively.

[0087] (2) 5'UTR mutation araA Construction of plasmid-expressing genetic engineering bacteria

[0088] a) Prepare E. coli W3110△araBAD△lyxK electroporation competent cells according to the method in Example 1.

[0089] b) Place the prepared electroporated competent cells in an ice bath for 5 min, and add 2-3 μL of different araA The expression plasmid was electroporated.

[0090] c) Finally, spread it evenly on the LB plate with kanamycin and culture it in a 37℃ incubator overnight to obtain the 5'UTR mutation. araAGenetically engineered bacteria expressing plasmids: W3110△araBAD△lyxK / pBAD18-araA1, W3110△araBAD△lyxK / pBAD18-araA2, W3110△araBAD△lyxK / pBAD18-araA3, W3110△araBAD△lyxK / pBAD18-araA4, W3110△araBAD△lyxK / pBAD18-araA5, W3110△araBAD△lyxK / pBAD18-araA6, W3110△araBAD△lyxK / pBAD18-araA7, W3110△araBAD△lyxK / pBAD18-araA8, W3110△araBAD△lyxK / pBAD18-araA9, W3110△araBAD△lyxK / pBAD18 -araA10, W3110△araBAD△lyxK / pBAD18-araA11, W3110△araBAD△lyxK / pBAD18-araA12, W3110△araBAD△lyxK / pBAD18-araA13, W3110△araBAD△lyxK / pBAD18-araA14, W3110△ara BAD△lyxK / pBAD18-araA15, W3110△araBAD△lyxK / pBAD18-araA16, W3110△araBAD△lyxK / pBAD18-araA17.

[0091] (3) 5'UTR mutation araA Determination of enzyme activity

[0092] a) 17 strains araA Single clones of the engineered bacteria were picked and inoculated into 5 mL of LB medium containing kanamycin, and the seed solution was obtained after culturing at 37°C with shaking at 220 rpm for 8 h.

[0093] b) Transfer the seed solution to 50 mL of fresh LB medium containing kanamycin at a 2% inoculum volume and collect the bacterial solution after culturing for 30-40 hours. The composition of the LB medium is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0094] c) Take 3-5 mL of the bacterial culture in step (b), centrifuge at 4000 rpm for 10 min at 4°C, collect the bacterial sludge, resuspend the bacteria in 0.2 M potassium phosphate buffer (pH 8.0), and ultrasonically disrupt them in an ice bath (each ultrasonication lasts 2 s, with an interval of 4 s between two ultrasonications, and the whole process takes 5 min) to obtain araA Crude enzyme solution.

[0095] d) Preparation of enzyme activity reaction system: Use 100 mM Tris-HCl (pH 7.5) as the buffer system to prepare 200 mM L-arabinose solution and 1 M MnCl2 solution respectively and store them at 4 ℃.

[0096] e) Enzyme catalyzed reaction: Add 500 μL crude enzyme solution, 500 μL 200 mM L-arabinose, and 1 μL 1 M MnCl2 to a 1.5 mL EP tube. React in a 30°C metal bath for 5 min, and then boil at 100°C for 10 min to terminate the reaction.

[0097] Enzyme activity definition: The consumption of L-arabinose during the reaction was determined by HPLC analysis. araA One unit (U) of activity was defined as the amount of enzyme required to consume 1 μmol of L-arabinose in 1 min.

[0098] 17 5'UTR mutations of L-arabinose isomerase ( araA ) are shown in SEQ ID NO.2~18 (Table 2).

[0099] Table 2. L-arabinose isomerase 5'UTR mutation library and enzyme activity assay results

[0100]

[0101] Example 3 D-psicose-3-epimerase from different sources ( dpe ) Gene acquisition and characterization

[0102] (1) Obtaining D-psicose-3-epimerase from different sources ( dpe ) Gene sequence

[0103] According to the D-psicose-3-epimerase ( dpe ) gene sequences, and screened out wild-type or mutant types from 5 different sources dpe Gene, wild type or mutant from different sources dpe The gene sequence was optimized by E. coli codon and synthesized to obtain dpe Plasmids containing the gene are pBAD18-dpe1, pBAD18-dpe2, pBAD18-dpe3, pBAD18-dpe4, and pBAD18-dpe5.

[0104] (2) Construction of D-psicose-3-epimerase containing different sources ( dpe ) Genetically engineered bacteria

[0105] E. coli W3110△araBAD△lyxK electroporation competent cells were prepared according to the method in Example 1 and electroporated dpe Recombinant expression plasmids containing different sources dpe Genetically engineered bacteria expressing plasmids: E.coli W3110△araBAD△lyxK / pBAD18-dpe1、 E.coli W3110△araBAD△lyxK / pBAD18-dpe2、 E.coli W3110△araBAD△lyxK / pBAD18-dpe3、 E.coli W3110△araBAD△lyxK / pBAD18-dpe4、 E.coli W3110△araBAD△lyxK / pBAD18-dpe5.

[0106] (3) Determination of D-psicose-3-isomerase activity from different sources

[0107] a) 5 plants dpe Single clones of the engineered bacteria were picked and inoculated into 5 mL of LB medium containing kanamycin, and the seed solution was obtained after culturing at 37°C with shaking at 220 rpm for 8 h.

[0108] b) Transfer the seed solution to 50 mL of fresh LB medium containing kanamycin at a 2% inoculum volume and collect the bacterial solution after culturing for 30-40 hours. The composition of the LB medium is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0109] c) Take 3-5 mL of the bacterial culture obtained in step (b), centrifuge at 4000 rpm for 10 min at 4°C, collect the bacterial sludge, resuspend the bacteria in 0.2 M potassium phosphate buffer (pH 8.0), and disrupt them by ultrasonication in an ice bath (each ultrasonication lasts for 2 s, with an interval of 4 s between two ultrasonications, and the whole process takes 5 min) to obtain crude enzyme solutions of D-psicose-3-isomerase from different sources.

[0110] d) Preparation of enzyme activity reaction system: Use 100 mM Tris-HCl (pH-8.0) as the buffer system, prepare 200 mM D-fructose solution and 1 M MnCl2 solution respectively, and store them at 4 ℃.

[0111] e) Enzyme catalyzed reaction: Add 500 μL crude enzyme solution, 500 μL 200 mM D-fructose, and 1 μL 1 M MnCl2 to a 1.5 mL EP tube. React in a 30°C metal bath for 5 min, and then boil at 100°C for 10 min to terminate the reaction.

[0112] Enzyme activity definition: The consumption of D-fructose during the reaction was determined by HPLC analysis. The activity unit (U) of D-psicose-3-epimerase was defined as the amount of enzyme required to consume 1 μmol of D-fructose in 1 min.

[0113] The sequence information and expression enzyme activity assay results of D-psicose-3-epimerase from 5 different sources are shown in Table 3.

[0114] Table 3. Sequence information and expression enzyme activity assay results of D-psicose-3-epimerase from different sources

[0115]

[0116] Example 4 L-xylulose reductase from different sources ( lxr ) Gene acquisition and characterization

[0117] (1) Acquisition and characterization of L-xylulose reductase genes from different sources

[0118] According to the NCBI published lxr Gene sequencing, screening out wild-type or mutant types from multiple different sources lxr Gene, wild type or mutant from different sources lxr The gene sequence was optimized by E. coli codon and synthesized to obtain lxr Plasmids containing the gene are pBAD18-lxr1, pBAD18-lxr2, pBAD18-lxr3, pBAD18-lxr4, pBAD18-lxr5, pBAD18-lxr6, pBAD18-lxr7, pBAD18-lxr8, pBAD18-lxr9, pBAD18-lxr10, and pBAD18-lxr11.

[0119] (2) Construction of wild-type or mutant strains from different sources lxr Genetically engineered bacteria

[0120] Prepared according to the method in Example 1 E.coli W3110△araBAD△lyxK electroporation competent cell and electroporation lxr Expression plasmids containing different sources lxr Genetically engineered bacteria expressing plasmids: E.coli W3110△araBAD△lyxK / pBAD18-lxr1、 E.coli W3110△araBAD△lyxK / pBAD18-lxr2、 E.coli W3110△araBAD△lyxK / pBAD18-lxr3、 E.coli W3110△araBAD△lyxK / pBAD18-lxr4 、E.coli W3110△araBAD△lyxK / pBAD18-lxr5、 E.coli W3110△araBAD△lyxK / pBAD18-lxr6, E.coli W3110△araBAD△lyxK / pBAD18-lxr7、 E.coli W3110△araBAD△lyxK / pBAD18-lxr8、 E.coli W3110△araBAD△lyxK / pBAD18-lxr9、 E.coli W3110△araBAD△lyxK / pBAD18-lxr10、 E.coli W3110△araBAD△lyxK / pBAD18-lxr11.

[0121] (3) Determination of L-xylulose reductase activity

[0122] a) Single clones of the 11 engineered bacteria were selected and inoculated into 5 mL of LB medium containing kanamycin. The culture was shaken at 220 rpm at 37°C for 8 h, and then the seed solution was obtained.

[0123] b) Transfer the seed solution to 50 mL of fresh LB medium containing kanamycin at a 2% inoculum volume and collect the bacterial solution after culturing for 30-40 hours. The composition of the LB medium is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0124] c) Take 3-5 mL of the bacterial culture obtained in step (b), centrifuge at 4000 rpm for 10 min at 4°C, collect the bacterial sludge, resuspend the bacteria in 0.2 M potassium phosphate buffer (pH 8.0), and disrupt them by ultrasonication in an ice bath (each ultrasonication lasts for 2 s, with an interval of 4 s between two ultrasonications, and the whole process takes 5 min) to obtain crude L-xylulose reductase solutions from different sources.

[0125] d) Preparation of enzyme activity reaction system: Using 100 mM Tris-HCl (pH 9.0) as buffer system, prepare 250 mM xylitol solution, 20 mM NAD ＋ and 0.5 M MnCl2 solution and stored at 4°C.

[0126] e) Enzyme catalyzed reaction: Add 500 μL crude enzyme solution, 400 μL 250 mM xylitol, and 100 μL NAD into a 1.5 mL EP tube. ＋and 1 μL 0.5 M MnCl2. The reaction was allowed to proceed in a 30°C metal bath for 5 min, and then the reaction was terminated by boiling at 100°C for 10 min.

[0127] Definition of enzyme activity: The consumption of xylitol during the reaction was determined by HPLC analysis. One unit (U) of L-xylulose reductase activity was defined as the amount of enzyme required to consume 1 μmol of xylitol in 1 min.

[0128] The sequence information and expression enzyme activity assay results of 11 wild-type and mutant L-xylulose reductases from different sources are shown in Table 4.

[0129] Table 4. Sequence information and expression enzyme activity determination results of L-xylulose reductase from different sources

[0130]

[0131] Example 5 Co-expression dpe , araA6 and lxr4 Shake flask fermentation experiment of plasmid-type genetically engineered bacteria.

[0132] (1) Construction of co-expression of different sources dpe , araA6 and lxr4 of the recombinant plasmid.

[0133] In order to explore different sources dpe The effect of enzyme activity on the production of xylitol from L-arabinose metabolism was selected. araA6、 lxr4 With 5 different sources dpe Gene combinations are expressed in tandem.

[0134] a) The pBAD18-araA6 plasmid prepared in Example 2 was extracted using a plasmid extraction kit, and digested with restriction endonucleases XbaI and BamHI to obtain a linearized pBAD18-araA6 plasmid.

[0135] b) The pBAD18-lxr4 plasmid prepared in Example 4 was extracted using a plasmid extraction kit and digested with restriction endonucleases XbaI and BamHI to obtain lxr-4 Fragment.

[0136] c) Use T4 ligase to ligate the linearized pBAD18-araA6 plasmid and lxr4 The fragment was transformed into E. coli DH5α, coated with LB solid plates containing kanamycin, and cultured at 37°C overnight; single colonies were grown and verified by colony PCR using specific primers. The correct clone was pBAD18-araA6-lxr4 plasmid.

[0137] d) The pBAD18-araA6-lxr4 plasmid was digested with restriction endonucleases BamHI and EcoRI to obtain linearized pBAD18-araA6-lxr4 plasmid.

[0138] e) The plasmids pBAD18-dpe1, pBAD18-dpe2, pBAD18-dpe3, pBAD18-dpe4, and pBAD18-dpe5 were digested with restriction endonucleases BamHI and EcoRI, respectively, to obtain dpe1、dpe2、dpe3、dpe4、dpe5 Gene fragment.

[0139] f) T4 ligase was used to ligate the linearized pBAD18-araA6-lxr4 plasmid into dpe1、dpe2、dpe3、dpe4、 dpe5 The gene fragments were connected and transformed into Escherichia coli DH5α, coated with LB solid plates containing kanamycin, and cultured at 37°C overnight; single colonies were grown and verified by colony PCR using specific primers. Five recombinant expression plasmids were obtained: pBAD18-dpe1-lxr4-araA6, pBAD18-dpe2-lxr4-araA6, pBAD18-dpe3-lxr4-araA6, pBAD18-dpe4-lxr4-araA6 and pBAD18-dpe5-lxr4-araA6.

[0140] (2) Construction of co-expression of different sources dpe , araA6 and lx4 Plasmid-based genetically engineered bacteria

[0141] E. coli W3110△araBAD△lyxK electroporation competent cells were prepared according to the method in Example 1 and electroporated dpe , araA6 and lxr4 Recombinant expression plasmids were used to co-express different sources of dpe , ara A 6 and lxr4 Plasmid-based genetically engineered bacteria E.coli W3110△araBAD△lyxK / pBAD18-dpe1-lxr4-araA6, E.coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr4-araA6、 E.coli W3110△araBAD△lyxK / pBAD18-dpe3-lxr4-araA6、 E.coli W3110△araBAD△lyxK / pBAD18-dpe4-lxr4-araA6 and E.coli W3110△araBAD△lyxK / pBAD18-dpe5-lxr4-araA6, named strains P1, P2, P3, P4, and P5, respectively.

[0142] (3) Co-expression dpe , araA6 and lxr4 Shake flask fermentation experiment of plasmid-type genetically engineered bacteria

[0143] a) Pick out five monoclonal bacterial strains with good growth and place them in 5 mL of LB medium supplemented with kanamycin and chloramphenicol. Culture them at 37°C for 12 h to prepare the seed solution.

[0144] b) The cultured seed solution was inoculated into the fermentation medium at an inoculation rate of 2%, and cultured at 37°C until OD600 was 0.6-0.8. Appropriate amounts of L-arabinose and glycerol mother solution were added to each shake flask fermentation medium, so that the final concentration of each substrate in the fermentation broth was: L-arabinose 10 g / L, glycerol 10 g / L, and cultured at 30°C for 20-30h after the substrate was added. After fermentation, the concentration of the fermentation product xylitol was detected. The results of the shake flask fermentation experiment are shown in Table 5.

[0145] Other components of the shake flask fermentation medium were: yeast powder 10 g / L, Na2HPO4 5 g / L, KH2PO4 1 g / L, NH4Cl 1 g / L, NaCl 1 g / L, MgSO4 0.2 g / L, and CaCl2 0.1 g / L.

[0146] As can be seen from Table 5, dpe and araA6 (expressed enzyme activity 271.83 U / L) and lxr - 4 (Expression enzyme activity 158.04 U / L) When the combination is expressed and fermented to produce xylitol, dpe The enzyme activity is not the limiting factor of the metabolic pathway. Its high or low catalytic activity does not affect the xylitol yield. The xylitol yield is about 6 g / L. dpe2 (Expressed enzyme activity 1562.61 U / L) Next step lxr Effect of enzyme activity on metabolic pathways for xylitol production.

[0147] Table 5. Results of shake flask fermentation of plasmid-based genetically engineered bacteria

[0148]

[0149] Example 6 Co-expression of different sources lxr , dpe2 and araA6Shake flask fermentation experiment of plasmid-type genetically engineered bacteria.

[0150] (1) Construction of co-expression of different sources lxr , dpe2 and araA6 Recombinant plasmid of gene.

[0151] In order to explore different sources lxr The effect of the enzyme activity of the gene on the metabolic pathway of L-arabinose to produce xylitol in the present invention is selected dpe2 , araA6 Genes from 11 different sources lxr Gene combinations are expressed in tandem.

[0152] a) Use a plasmid extraction kit to extract the pBAD18-dpe2-lxr4-araA6 plasmid, and digest it with restriction endonucleases BamHI and XBaI to obtain the linearized plasmid pBAD18-dpe2-araA6.

[0153] b) Use a plasmid extraction kit to extract plasmids pBAD18-lxr1, pBAD18-lxr2, pBAD18-lxr3, pBAD18-lxr5, pBAD18-lxr6, pBAD18-lxr7, pBAD18-lxr8, pBAD18-lxr9, pBAD18-lxr10, and pBAD18-lxr11. Digest with restriction endonucleases BamHI and XBaI to obtain lxr Snippet lxr1、lxr2、lxr3、lxr5、lxr6、 lxr7、lxr8、lxr9、lxr10、lxr11 .

[0154] c) T4 ligase was used to ligate the linearized plasmid pBAD18-dpe2-araA6 into lxr Snippet lxr1、lxr2、lxr3、 lxr5、lxr6、lxr7、lxr8、lxr9、lxr10、lxr11 The clones were connected and transformed into Escherichia coli DH5α, respectively, and coated on LB solid plates containing kanamycin and cultured at 37°C overnight; single colonies were grown and verified by colony PCR using specific primers. Ten recombinant expression plasmids were obtained: pBAD18-dpe2-lxr1-araA6, pBAD18-dpe2-lxr2-araA6, pBAD18-dpe2- lxr3-araA6, pBAD18-dpe2-lxr5-araA6, pBAD18-dpe2-lxr6-araA6, pBAD18-dpe2-lxr7-araA6, pBAD18-dpe2-lxr8-araA6, pBAD18-dpe2-lxr9-araA6, pBAD18-dpe2-lxr10-araA6, and pBAD18-dpe2-lxr11-araA6.

[0155] (2) Construction of co-expression of different sources lxr , dpe2 and araA6 Plasmid-based genetically engineered bacteria

[0156] According to the method in Example 1, Escherichia coli W3110△araBAD△lyxK electroporation competent cells were prepared and electroporated into different sources. lxr , dpe 2 and araA 6 recombinant expression plasmids to obtain co-expression of different sources lxr , dpe2 and araA6 Plasmid-based genetically engineered bacteria E.coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA6、 E.coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr2-araA6、 E.coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr3-araA6, E.coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr5-araA6、 E.coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr6-araA6、 E.coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr7-araA6、 E.coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr8-araA6、 E.coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr9-araA6、 E.coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr10-araA6 and E.coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr11-araA6, named P6, P7, P8, P9, P10, P11, P12, P13, P14, and P15, respectively.

[0157] (3) Co-expression of different sources lxr , dpe2 and araA6 Shake flask fermentation experiment of plasmid-type genetically engineered bacteria.

[0158] According to the method of Example 5, a shake flask fermentation experiment of genetically engineered bacteria was carried out on the strains P6 to P15 constructed and the strain P2 constructed in Example 5. The results of the shake flask fermentation experiment are shown in Table 6.

[0159] As can be seen from Table 6, lxr and araA6 (enzyme activity 271.83 U / L) and dpe2 (enzyme activity 1562.49 U / L) When the combination was expressed and fermented to produce xylitol, lxr The enzyme activity will have a certain degree of influence on the metabolic pathway for xylitol production, namely lxr If the enzyme activity is too low (enzyme activity < 100 U / L), the production of xylitol will be limited. When the enzyme activity is > 100 U / L, the production of xylitol will not change significantly, so lxr1 Next step: exploring genes araA Effect of enzyme activity on xylitol production.

[0160] Table 6. Results of shake flask fermentation of plasmid-based genetically engineered bacteria

[0161]

[0162] Example 7 Co-expression of 5'UTR mutations araA , dpe2 and lxr1 Shake flask fermentation experiment of plasmid-type genetically engineered bacteria.

[0163] (1) Construction of co-expression of 5'UTR mutations araA , dpe2 and lxr1 Recombinant plasmid

[0164] In order to explore the effect of araA gene expression on the metabolic pathway of xylitol production from L-arabinose in the present invention, dpe 2. lxr 1 gene with 17 different 5'UTR mutations araA Gene combinations are expressed in tandem.

[0165] a) The pBAD18-dpe2-lxr1-araA6 plasmid was extracted using a plasmid extraction kit and digested with restriction endonucleases XBaI and SphI to obtain the linearized plasmid pBAD18-dpe2-lxr1.

[0166] b) The 16 pBAD18-araA plasmids (except pBAD18-araA6) in Example 1 were extracted using a plasmid extraction kit. The plasmids were digested with restriction endonucleases XBaI and SphI to obtain araA Snippet araA1、araA2、araA3、 araA4、araA5、araA7、araA8、araA9、araA10、araA11、araA12、araA13、araA14、araA15、 araA16、araA17 .

[0167] c) T4 ligase was used to ligate the linearized plasmid pBAD18-dpe2-lxr1 to araA Snippet araA 1. araA2、 araA3, araA4, araA5, araA7, araA8, araA9, araA10, araA11, araA12, araA13, araA14, araA15, araA16, araA17 The cells were connected and transformed into Escherichia coli DH5α, coated with LB solid plates containing kanamycin, and cultured at 37°C overnight; single colonies were grown and verified by colony PCR using specific primers. 16 recombinant expression plasmids were obtained: pBAD18-dpe2-lxr1-araA1, pBAD18-dpe2-lxr2-araA2, pBAD18-dpe2-lxr3-araA3, pBAD18-dpe2-lxr5-araA4, pBAD18-dpe2-lxr6-araA5, pBAD18-dpe2-lxr7-araA7, pBAD18-dpe2-lxr8-araA8, pBAD18-dpe2-lxr9-araA9, pBAD18-dpe2-lxr10-araA11, pBAD18-dpe2-lxr11-araA12, pBAD18-dpe2-lxr12-araA13, pBAD18-dpe2-lxr13-araA14, pBAD18-dpe2-lxr14-araA15, pBAD18-dpe2-lxr15-araA16, pBAD18-dpe2-lxr16-araA17, pBAD18-dpe2-lxr17-araA18 AD18-dpe2-lxr10-araA10, pBAD18-dpe2-lxr11-araA11, pBAD18-dpe2-lxr11-araA12, pBAD18-dpe2-lxr11-araA13, p BAD18-dpe2-lxr11-araA14, pBAD18-dpe2-lxr11-araA15, pBAD18-dpe2-lxr11-araA16, pBAD18-dpe2-lxr11-araA17.

[0168] (2) Construction of co-expression of 5'UTR mutations araA, dpe2 and lxr1 Plasmid-based genetically engineered bacteria

[0169] According to the method in Example 1, the E. coli W3110△araBAD△lyxK electroporation competent cells were prepared and the above recombinant expression plasmids were electroporated to obtain the co-expression of different 5'UTR mutations. araA, dpe2 and lxr1 Plasmid-based genetically engineered bacteria E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA1、 E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA2, E.coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA3, E. coliW3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA4、 E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA5、 E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA7、 E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA8、 E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA9, E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA10, E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA11, E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA12, E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA13, E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA14, E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA15, E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA16 and E. coli W3110△araBAD△lyxK / pBAD18-dpe2-lxr1-araA17 were named P16, P17, P18, P19, P20, P21, P22, P23, P24, P25, P26, P27, P28, P29, P30, and P31, respectively.

[0170] (3) Co-expression of 5'UTR mutations araA, dpe2 and lxr1 Shake flask fermentation experiment of plasmid-type genetically engineered bacteria.

[0171] According to the method of Example 5, a shake flask fermentation experiment was performed on the above 16 genetically engineered bacteria and the strain P6 constructed in Example 6 (a total of 17 genetically engineered bacteria). The results of the shake flask fermentation experiment are shown in Table 7.

[0172] As can be seen from Table 7, the 5'UTR mutation araAGene and lxr1 (expressed enzyme activity 429.82 U / L) and dpe2 (expression enzyme activity 1562.49 U / L) When the combination was expressed and fermented to produce xylitol, it was found araA The enzyme activity is the key factor affecting the metabolic pathway for xylitol production. araA The expression of genes plays a decisive role. araA When the enzyme activity is low (enzyme activity < 50 U / L), no xylitol is produced; araA With the further improvement of enzyme activity, the production of xylitol also gradually increased. When the enzyme activity approached 400 U / L, the production of xylitol tended to be stable, and almost all L-arabinose (10 g / L) was converted into xylitol (the mass ratio of L-arabinose to xylitol was 1 g / 0.98 g, that is, 1 g L-arabinose produced 0.98 g xylitol).

[0173] Table 7. Results of shake flask fermentation of plasmid-based genetically engineered bacteria

[0174]

[0175] Example 8 Co-expression of different combinations araA, dpe and lxr Shake flask fermentation experiment of plasmid-type genetic engineering bacteria

[0176] (1) Constructing different co-expression combinations araA, dpe and lxr Recombinant plasmid

[0177] In order to further verify the effect of enzyme activities of three genes in the metabolic pathway on xylitol fermentation, DPE Select from gene bank dpe1 (expressed enzyme activity 142.76 U / L) and dpe5 (expressed enzyme activity 1238.57U / L), from lxr Select from gene bank lxr3, lxr8 and lxr11 , the different sources DPE , lxr Respectively araA1, araA4 , araA6, araA7, araA15, araA17 According to the method of enzyme digestion and ligation in the above embodiment, 24 araA, dpe and lxr Co-expression recombinant plasmid.

[0178] (2) Co-expression of different combinations araA, dpe and lxr Construction of plasmid-type genetic engineering bacteria

[0179] According to the method in Example 1, the E. coli W3110△araBAD△lyxK electroporation competent cells were prepared and the above 24 recombinant expression plasmids were electroporated respectively to obtain co-expression of different combinations of araA , DPE and lxr The plasmid-type genetically engineered bacteria containing the genes were named P32, P33, P34, P35, P36, P37, P38, P39, P40, P41, P42, P43, P44, P45, P46, P47, P48, P49, P50, P51, P52, P53, P54, and P55. The specific gene combinations are shown in Table 8.

[0180] (3) Co-expression of different combinations araA , DPE and lxr Shake flask fermentation experiment of plasmid-type genetically engineered bacteria

[0181] The shake flask fermentation experiment of genetically engineered bacteria was carried out according to the method of Example 5. The results of the shake flask fermentation experiment are shown in Table 8.

[0182] It can be seen from Table 8 that different combinations DPE , lxr and araA There is no interaction between the effects of xylitol production, that is, the main limiting factor of the xylitol production metabolic pathway of L-arabinose in the present invention is the gene araA The expression lxr Genes have a certain influence on metabolic pathways. DPE The effects of genes on metabolic pathways were independent of their source.

[0183] Table 8. Results of shake flask fermentation of plasmid-based genetically engineered bacteria

[0184]

[0185] Example 9 Co-expression araA , DPE and lxr Construction of multi-copy genome-integrated engineered bacteria and shake flask fermentation

[0186] (1) Integration of three copies of the genome araA , DPE and lxr E. coli construction of the co-expression cassette

[0187] According to the results of shake flask fermentation in Example 8, araA7, dpe1 and dpe5 and lxr3, lxr8 and lxr11 Combined co-expression, using the CRISPR / Cas9 gene editing tool in Example 1, the combined expressed genes were edited for three rounds respectively. araA7, dpe1 and dpe5 and lxr3, lxr8 and lxr11 Three copies were integrated into the genome of Escherichia coli W3110△araBAD△lyxK, resulting in six strains with three copies of integration. araA, dpe and lxr The integrated genetically engineered bacteria of the expression cassettes were named G1, G2, G3, G4, G5, and G6, respectively. The specific gene combinations are shown in Table 9.

[0188] (2) Shake flask fermentation culture of integrated genetically engineered bacteria

[0189] The above 6 strains of integrated genetically engineered bacteria were fermented in shake flasks, and the culture method was the same as in Example 5. The results of the shake flask fermentation experiment of integrated genetically engineered bacteria are shown in Table 9. It can be seen from Table 9 that different combinations of 3 copies araA , DPE and lxr The effect of xylitol production by gene-integrated genetically engineered bacteria was similar to that of xylitol production by the same combination of plasmid-type genetically engineered bacteria. Using 10 g / L L-arabinose as the substrate, all L-arabinose was converted into xylitol after 20 to 30 hours of fermentation.

[0190] Table 9. Results of shake flask fermentation of integrated genetically engineered bacteria

[0191]

[0192] Example 10 Construction of genetically engineered bacteria for enhancing the glycerol pathway and shake flask fermentation

[0193] (1) Construction of engineered bacteria with enhanced glycerol pathway

[0194] The G6 strain obtained in Example 9 was used as the starting strain, and the glycerol transporter gene was inserted into the glycerol transporter gene using the CRISPR / Cas9 method in Example 1. glpF and glycerol kinase gene glpK Co-expression cassette, glyceraldehyde-3-phosphate dehydrogenase gene glpD The expression cassette was integrated into the untranslated region of the E. coli genome to construct W3110△araBAD△lyxK::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD, named G7.

[0195] (2) Shake flask fermentation of engineered bacteria with enhanced glycerol pathway

[0196] The G7 and G6 engineered bacteria constructed above were subjected to shake flask fermentation, and the culture method was the same as in Example 5. The results of the shake flask fermentation experiment of the engineered bacteria are shown in FIG. Picture 2 As shown. Picture 2It can be seen that the engineered bacteria after the glycerol pathway is strengthened have a higher coenzyme NADH yield, which can convert L-arabinose into xylitol more quickly, that is, the xylitol yield of the engineered bacteria after the glycerol pathway is strengthened is 40% faster than that before the glycerol pathway is strengthened. The xylitol yield is the same before and after the glycerol pathway is strengthened, that is, both can completely convert L-arabinose into xylitol. Therefore, strengthening the glycerol metabolic pathway can save a lot of fermentation time.

Claims

1. A genetically engineered bacterium comprising a host cell and a target gene inserted into the host cell, It is characterized in that The target genes include L-arabinose isomerase gene, D-psicose-3-epimerase gene and L-xylulose reductase gene; The L-arabinose isomerase gene is araA Gene, the nucleotide sequence of which is shown in SEQ ID NO.1; The D-psicose-3-epimerase gene is dpe Gene, dpe The gene is any one of the NCBI accession numbers WP-138792167.1 and AE008210; The L-xylulose reductase gene is lxr Gene, lxr The gene is any one of the NCBI accession numbers KAF6014139.1 and AJ583159; Said araA The 5' untranslated region of the gene is mutated, and the mutation is any one of the sequences shown in SEQ ID NO.8, SEQ ID NO.16, and SEQ ID NO.18; The host cell is Escherichia coli; The host cell is Escherichia coli E. coli W3110; Escherichia coli genome araB Gene, araD Genes and lyx Genes are knocked out or silenced.

2. The genetically engineered bacteria according to claim 1, It is characterized in that araA 5' untranslated region of the gene, dpe Genes and lxr The gene combinations are as follows: (1) SEQ ID NO.8, WP-138792167.1, KAF6014139.1; (2) SEQ ID NO.18, WP-138792167.1, AJ583159; (3) SEQ ID NO.8, AE008210, KAF6014139.1; (4) SEQ ID NO.18, AE008210, AJ583159; (5) SEQ ID NO.8, WP-138792167.1, AJ583159; (6) SEQ ID NO.8, AE008210, AJ583159.

3. The genetically engineered bacterium according to claim 1, It is characterized in that Insertion into the E. coli genome gf Gene, gL Gene, gL At least one of the genes.

4. Use of the genetically engineered bacteria as claimed in claims 1 to 3 in the biological preparation of xylitol.

5. A method for producing xylitol using arabinose, It is characterized in that The genetically engineered bacteria according to any one of claims 1 to 3 are inoculated into a fermentation liquid containing L-arabinose and glycerol, and xylitol is obtained after fermentation.

6. The method for producing xylitol using arabinose according to claim 5, It is characterized in that The fermentation conditions are as follows: the genetically engineered bacteria are inoculated into a shake flask fermentation medium, cultured at 18-37° C. for 2-5 hours, L-arabinose and glycerol are added to the medium, and fermented at 18-37° C. for 20-60 hours.

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