A two-stage biological method for producing xylitol using hemicellulose hydrolysate

Abstract

The present invention provides a method for producing xylitol by a two-stage biological method using hemicellulose hydrolysate, the method comprising: using hemicellulose hydrolysate as a raw material, in a fermentation medium inoculated with fermentation engineering bacteria, performing a first-stage fermentation culture until there is no L-arabinose residue in the fermentation liquid, and obtaining a fermentation reaction liquid I; after adding glucose feed and an inducer to the fermentation reaction liquid I, continuing the second-stage fermentation culture to obtain a fermented product containing xylitol. The method of the present invention utilizes the pathway of xylitol production by L-arabinose metabolism constructed in the fermentation engineering bacteria, and the time node control of the fermentation process, so that both L-arabinose and D-xylose can be fully utilized and converted into xylitol, realizing a two-stage biological method for producing xylitol using hemicellulose hydrolysate as a raw material, significantly improving the yield of xylitol in the hemicellulose hydrolysate, and providing a new xylitol production process with better promotion for the xylitol production industry.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for producing xylitol by utilizing hemicellulose hydrolysate in a two-stage biological method. Background Art

[0002] Xylitol is a naturally occurring pentitol (molecular formula C5H 12 O5, molecular weight 152.15), white powdery crystalline substance, is an intermediate product of normal human metabolism. It is also found in many fruits, yeast, lichens, seaweed, mushrooms and vegetables, but the content is relatively low. At present, xylitol is widely used in food, medicine, cosmetics and health products. Its largest consumption is in the food industry as a natural sweetener. With the improvement of people's living standards, obesity has become a common problem that troubles the public. Consumers are increasingly favoring low-sugar and low-calorie foods. At the same time, xylitol also has the effect of preventing tooth decay. Therefore, it is used more and more in food additives and the demand is also increasing.

[0003] At present, the industrial production of xylitol mainly uses xylose from hemicellulose hydrolysate as raw material, which is produced by chemical catalytic hydrogenation under high temperature and high pressure conditions. The high production cost has pushed up the price of xylitol and has become the main factor restricting the wider application of xylitol. The biological production of xylitol is a new process for preparing xylitol. Its reaction conditions are mild and the production cost is relatively low. It is expected to replace the traditional chemical hydrogenation production process.

[0004] In recent years, it has received extensive attention at home and abroad. The biological production of xylitol mainly uses microorganisms such as bacteria, fungi, yeast or recombinant genetically engineered bacteria to ferment different biomass raw materials to obtain xylitol products. For example, the Chinese invention patent application with publication number CN104357339A discloses a tropical Candida, which ferments xylitol with high concentration xylose as the only carbon source, and the xylitol concentration can reach 20-65 g / L, and the residual sugar concentration is less than 5%. The Chinese invention patent with announcement number CN110982850B discloses a genetically engineered Aspergillus oryzae, which uses 50 g / L xylan as the only carbon source, produces xylitol at a concentration of 13.5 g / L, and its yield and productivity are 0.27 g / g and 0.16 g / L / h, respectively. A Chinese invention patent with Chinese publication number CN106661540B discloses a recombinant Pichia olmer yeast that uses 250 g / L glucose monohydrate as a raw material to produce 120 g / L of xylitol and 5 g / L of ribitol, with a xylitol yield and productivity of 0.48 g / g and 1.81 g / L / h, respectively.

[0005] Hemicellulose is the largest biomass raw material of pentose in nature, and the most economical solution is to prepare xylitol from hemicellulose hydrolysate. Patent applications with publication numbers CN108949839A and CN111440830A disclose the technology of fermenting corn cob hydrolysate (a hemicellulose hydrolysate) with genetically engineered Escherichia coli to prepare xylitol. However, since 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 a branch chain connected to the polyxylose main chain. Therefore, in addition to D-xylose (D-Xylose), hemicellulose hydrolysate also contains a variety of miscellaneous sugars, such as glucose (Glucose), L-arabinose (L-Arabinose), etc. In particular, L-arabinose will produce L-arabinitol after microbial metabolism and enter the subsequent process of xylitol production, thereby increasing the difficulty of refining xylitol products and increasing the manufacturing cost of xylitol products. Therefore, it is urgent to develop genetically engineered bacteria and their fermentation processes to accelerate the metabolic rate of miscellaneous sugars, avoid the production of impurity L-arabinitol, and increase the yield and production rate of xylitol. Summary of the invention

[0006] The present invention provides a method for producing xylitol by a two-stage biological method using hemicellulose hydrolysate. The method utilizes the pathway for producing xylitol by L-arabinose metabolism constructed in fermentation engineering bacteria and the time node control of the fermentation process, so that L-arabinose can be fully utilized and converted into xylitol, realizing a two-stage biological method for producing xylitol using hemicellulose hydrolysate as a raw material, significantly improving the yield of xylitol in the hemicellulose hydrolysate, and providing a new xylitol production process with better promotion for the xylitol production industry.

[0007] The specific technical solutions are as follows:

[0008] The present invention provides a two-stage biological method for producing xylitol using hemicellulose hydrolysate, comprising:

[0009] (1) using hemicellulose hydrolyzate as a raw material, performing a first stage fermentation culture in a fermentation medium inoculated with fermentation engineering bacteria until no L-arabinose remains in the fermentation liquid, thereby obtaining a fermentation reaction liquid I;

[0010] (2) After adding glucose feed and inducer to the fermentation reaction liquid I, the second stage fermentation culture is continued to obtain a fermentation product containing xylitol.

[0011] The method of the present invention utilizes the expression regulation of functional genes in fermentation engineering bacteria, as well as the control of glucose feeding and inducer addition time, to divide the fermentation stage of fermentation engineering bacteria fermenting hemicellulose hydrolyzate to produce xylitol into two stages; in the first stage, the cells rapidly proliferate and metabolize L-arabinose to produce xylitol; in the second stage, the cells are induced to produce xylose reductase, thereby co-metabolizing xylose and glucose to produce xylitol.

[0012] Furthermore, the fermentation engineering bacteria include: a host cell containing a xylose reductase XR gene and a target gene co-expressed in the host cell; the target gene includes an L-arabinose isomerase gene araA , D-psicose-3-epimerase gene dpe and L-xylulose reductase gene lxr .

[0013] The method of the present invention utilizes fermentation engineering bacteria to transform hemicellulose hydrolysate to produce xylitol, which is divided into two stages. In the first stage, under the action of L-arabinose isomerase, D-psicose-3-epimerase and L-xylulose reductase expressed by the fermentation engineering bacteria and NADH, all L-arabinose is converted into xylitol; in the second stage, under the action of an inducer, under the action of xylose reductase expressed by the fermentation engineering bacteria, unreacted xylose in the hemicellulose hydrolysate is completely converted into xylitol. After the two stages are completed, the conversion rate of the raw material is greater than 99%; there is no residual sugar in the fermentation product, and no L-arabinitol is produced. The method simplifies the refining process of xylitol and improves the production efficiency and product yield of xylitol produced from hemicellulose hydrolysate.

[0014] More preferably, the gene araA The nucleotide sequence of the gene is shown in SEQ ID NO.1; dpe The nucleotide sequence of the gene is shown in SEQ ID NO.4; lxr The nucleotide sequence is shown in SEQ ID NO.6.

[0015] More preferably, the gene araA The nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO.3.

[0016] In the present invention, the L-arabinose isomerase is selected from E.coli L-arabinose isomerase gene of W3110 araA The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; and the 5' untranslated region (5'UTR) thereof is modified to obtain a mutated 5'UTR sequence (abbreviated as araA17), the nucleotide sequence is shown in SEQ ID NO.3. The D-psicose-3-epimerase is selected from the group consisting of Rhizobium sp. . D-psicose-3-epimerase gene of RM dpe The nucleotide sequence is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.5. The L-xylulose reductase is selected from the original Brettanomyces cerevisiae Brettanomyces bruxellensis L-xylulose reductase gene of AWRI1499 lxr The nucleotide sequence is shown in SEQ ID NO.6, and the amino acid sequence is shown in SEQ ID NO.7.

[0017] Furthermore, the target gene is co-expressed in the host cell in one of the following ways:

[0018] (A) Co-expression in host cells in the form of a recombinant plasmid;

[0019] (B) Inserted into the host cell genome in the form of an expression cassette for co-expression.

[0020] Co-expressed genes araA ,Gene dpe and genes lxr The methods include plasmid co-expression and genome integration co-expression; further preferably, the recombinant plasmid is pBAD18.

[0021] Furthermore, in (B), the target gene is inserted into the non-coding region of the host cell genome; or araA ,Gene dpe and genes lxr Random Replacement ptsG Gene ,ptsF Gene, xylA and xylB Gene.

[0022] Genome-integrated co-expression is to integrate the genes related to glucose transport and metabolism in the genome of the engineered bacteria ptsG and ptsF , and genes related to xylose metabolism xylA and xylB Arabinose-inducible promoter pBAD as well as araA , dpe and lxr The gene co-expression cassette is then integrated into the genome of the engineered bacteria. In order to improve the co-expression efficiency, it can be integrated into the genome of the engineered bacteria in the form of multiple copies; in the present invention, integration into the genome of the engineered bacteria in the form of 3 copies is used as an example.

[0023] in, ptsGThe replacement of slows down the CCR effect and improves the co-metabolic efficiency of glucose and other sugars; the metabolism of glucose produces the cofactor NADPH, which ensures the supply of NADPH when xylose reductase catalyzes the reduction of xylose to xylitol. ptsF The replacement of can reduce the transport of xylitol from the extracellular to the intracellular; xylA and xylB The replacement of L - Arabinose isomerase gene ( araA17 )、D-psicose-3-epimerase gene( dpe1 ) and L-xylulose reductase gene ( lxr8 ) expression, strengthening the L - Efficiency of arabinose metabolism to xylitol.

[0024] Furthermore, the gene ptsG The nucleotide sequence is shown in SEQ ID NO.20, ptsF The nucleotide sequence is shown in SEQ ID NO.21.

[0025] Furthermore, the xylA and xylB The genes are linked together, and when replacing them, the two genes are replaced together. xylA Genes and xylB Gene Two genes are collectively called xylAB Gene, the xylAB The nucleotide sequence of the gene is shown as SEQ ID NO.22.

[0026] The host cell used in the method of the present invention can be a microorganism such as Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, etc.; further, the host cell provided by the present invention is Escherichia coli. Further, the host cell provided by the present invention is Escherichia coli. E.coli W3110.

[0027] There is more than one metabolic pathway for L-arabinose. In order to further enhance the yield of xylitol produced by metabolizing L-arabinose, the present invention further targets genes related to other pathways of L-arabinose metabolism. araB Gene, araD Genes and lyxK Gene knockout.

[0028] Therefore, further, at least one of the host cell genomes is knocked out araB Gene, araD Genes and lyxK One of the genes.

[0029] Furthermore, the gene araB The nucleotide sequence of the gene is shown in SEQ ID NO.12; araD The nucleotide sequence of the gene is shown in SEQ ID NO.13; lyxK The nucleotide sequence is shown in SEQ ID NO.14.

[0030] Since the xylitol production pathway of L-arabinose metabolism requires the consumption of the cofactor NADH, in order to improve the efficiency of xylitol production by L-arabinose metabolism, the present invention also adds the glycerol transporter gene glpF and glycerol kinase gene glpK Co-expression cassette, and glyceraldehyde-3-phosphate dehydrogenase gene glpD The expression cassette is integrated into the untranslated region of the engineered bacterial genome to enhance glycerol metabolism to produce the cofactor NADH.

[0031] Furthermore, at least a glycerol transporter gene is inserted into the non-coding region of the host cell genome. glpF , glycerol kinase gene glpK Glyceryl triphosphate dehydrogenase gene glpD One of them.

[0032] Furthermore, the gene glpF The nucleotide sequence of the gene is shown in SEQ ID NO.9; glpK The nucleotide sequence of the gene is shown in SEQ ID NO.10; glpD The nucleotide sequence is shown in SEQ ID NO.11.

[0033] Theoretically, the xylose reductase used in the present invention can be a xylose reductase gene from any source that can be functionally expressed in engineered bacteria and has corresponding catalytic activity.

[0034] Further preferably, the xylose reductase gene is derived from Candida boidinii Candida boidinii , Candida gracilis Candida tenuis CBS 4435 , Candida tropicalis Candida tropicalis IFO 0618, Slime Red Yeast Rhodotorula mucilaginosa Neurospora crassa Neurospora crassa , Kluyveromyces lactis Kluyveromyces lactis Aspergillus niger Aspergillus nigerThe xylose reductase genes are abbreviated as XR1 gene, XR4 gene, XR5 gene and XR6 gene in the present invention, and the NCBI accession numbers are AF451326.3, AF074484.1, AB002106.1, ALO17776.1, EAA34695.1, AAA99507.1, and Q9P8R5.1, respectively. The promoter used to express xylose reductase can be any inducible promoter except the arabinose promoter; further preferably, the inducible promoter is a rhamnose promoter, anhydrotetracycline promoter or lactose promoter.

[0035] Furthermore, the nucleotide sequence of the inducible promoter of the XR gene is one of the sequences shown in SEQ ID NOs. 15-19.

[0036] Furthermore, the hemicellulose hydrolyzate is obtained by hydrolyzing biomass rich in hemicellulose; the biomass rich in hemicellulose is corn cobs, papermaking short fibers, bagasse, straw or rice husks, etc.; the hemicellulose hydrolyzate contains at least L-arabinose, glucose and xylose.

[0037] Furthermore, the mass ratio of L-arabinose, glucose and xylose is 5%~6%: 2%~5%: 35%~40%.

[0038] Furthermore, in step (1), the conditions for the first stage fermentation culture are: temperature of 18-37°C, pH of 6.0-8.0; the dissolved oxygen content under the initial fermentation conditions after inoculation is 100%, and the dissolved oxygen is controlled to be maintained between 0 and 50%.

[0039] Furthermore, in step (1), the fermentation medium includes a hemicellulose hydrolyzate containing L-arabinose, glucose and xylose, a nitrogen source (yeast powder or peptone), a carbon source (glycerol or glucose), an inorganic salt (such as phosphate), etc.

[0040] Furthermore, in step (2), based on the mass of xylose in the semi-hydrolyzate, the amount of glucose feed added is 10% to 50% of xylose; the inducer is one of lactose, rhamnose or anhydrotetracycline, and based on the initial fermentation volume, the amount of inducer added is 1 g / L to 10 g / L.

[0041] Preferably, the inducer is lactose.

[0042] Furthermore, in step (2), the fermentation culture conditions of the second stage are: temperature of 18-37°C, pH of 6.0-8.0; based on the dissolved oxygen under the initial fermentation culture conditions after inoculation as 100%, the dissolved oxygen is maintained between 0 and 50%.

[0043] Furthermore, in step (1), the OD of the fermentation engineering bacteria when inoculated into the fermentation medium is 600 It is 0.6~0.8. Based on the initial fermentation volume when loading the tank, the inoculation amount is 1~15%.

[0044] In the present invention, a 15 L fermenter is used as a fermentation container, and the fermentation engineering bacteria are inoculated into a fermentation medium containing hemicellulose hydrolyzate to perform two-stage fermentation;

[0045] Wherein, in step (1), the fermentation medium comprises at least a carbon source, a nitrogen source and an inorganic salt; based on the initial fermentation volume, the amount of the carbon source added is 10 to 50 g / L; the amount of the nitrogen source added is 5 to 30 g / L; preferably, the carbon source is glycerol; preferably, the amount of glycerol added is 0.6 times the mass of arabinose or more;

[0046] In step (2), based on the xylose content in the hydrolyzate, the amount of glucose feed added is 10% to 50% of the xylose in terms of mass percentage; the inducer is one of lactose, rhamnose or anhydrotetracycline; based on the initial fermentation volume, the amount of inducer added is 1 g / L to 10 g / L in terms of volume percentage.

[0047] Furthermore, in the first stage, the components and concentrations in the fermentation medium are: Na2HPO4 6-10 g / L, KH2PO4 3-5 g / L, glycerol 6-15 g / L, yeast powder 10-30 g / L, peptone 5-15 g / L; the final concentration of xylose in the hemicellulose hydrolysate is 100-150 g / L; the fermentation temperature is 18-37°C, and the time is 10-20 h;

[0048] In the second stage, there is no L-arabinose in the fermentation reaction liquid I, the concentration of the added inducer is 3-5 g / L, and the concentration of the added glucose is 10-75 g / L; the fermentation temperature is 18-37°C, and the time is 20-60 h.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] (1) The two-stage biofermentation method for producing xylitol of the present invention is simple and convenient. The xylose reductase gene expressed in the genetically engineered bacteria used is widely available and does not require mutation to modify its substrate selectivity.

[0051] (2) The genetically engineered bacteria of the present invention ferment the hemicellulose hydrolysate, and the conversion rate of L-arabinose and xylose exceeds 99%, and no miscellaneous sugars remain. There is no L-arabinitol in the product, which successfully solves the problems of complex process, difficult separation and high cost in the xylitol refining process.

[0052] (3) The genetically engineered bacteria of the present invention can completely convert the substrate L-arabinose in the hemicellulose hydrolysate into xylitol during fermentation, thereby making full use of the miscellaneous sugars in the hemicellulose hydrolysate and further improving the yield of xylitol. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The diagram is a schematic diagram of the metabolic pathway for producing xylitol using a two-stage biological method of hemicellulose hydrolysate in the present invention.

[0054] Figure 2 Schematic diagram of the metabolic pathway for the conversion of L-arabinose to xylitol.

[0055] Figure 3 This is a process curve diagram of producing xylitol by fermenting hemicellulose hydrolyzate with Escherichia coli fermentation engineering bacteria in Example 6.

[0056] Figure 4 This is the HPLC detection spectrum of the standard sample in Example 6; wherein, the peak at 11.6 min is D-glucose, the peak at 12.7 min is D-xylose, the peak at 14.4 min is L-arabinose, the peak at 18.1 min is arabitol, and the peak at 21.2 min is xylitol.

[0057] Figure 5 This is the HPLC detection spectrum of the hemicellulose hydrolyzate fermented at 0 h in Example 6; wherein, the peak at 11.6 min is D-glucose, the peak at 12.7 min is D-xylose, and the peak at 14.4 min is L-arabinose.

[0058] Figure 6 This is the HPLC detection spectrum of the hemicellulose hydrolyzate fermented for 48 hours in Example 6; wherein, the peak at 12.7 min is D-xylose, and the peak at 21.0 min is xylitol.

[0059] Figure 7 This is the HPLC detection spectrum of the hemicellulose hydrolyzate fermented for 60 hours in Example 6; wherein the peak at 20.7 min is xylitol. DETAILED DESCRIPTION

[0060] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0061] The experimental methods in the present invention are conventional methods unless otherwise specified. For details of gene cloning operations, please refer to the Molecular Cloning Experiment Guide compiled by J. Sambrook et al. The gene operation kit was purchased from TAKARA for characterization, and the reagents used: xylose, arabinose, glucose, xylitol, and arabitol were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0062] 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.

[0063] The gene names involved in the present invention are explained as follows:

[0064] ptsG : Glucose phosphotransferase system enzyme II component gene;

[0065] ptsF : Fructose phosphotransferase system enzyme II component gene;

[0066] xylA : Xylose isomerase gene;

[0067] xylB: Xylulokinase gene:

[0068] araA : L-arabinose isomerase gene;

[0069] dpe : D-psicose-3-epimerase gene;

[0070] lxr : L-xylulose reductase gene;

[0071] araB : ribulose kinase gene;

[0072] araD : L-ribulose-5-phosphate-4-epimerase gene;

[0073] lyxK : L-xylulose kinase gene;

[0074] glpF : Glycerol transporter gene;

[0075] glpK : Glycerol kinase gene;

[0076] glpD :Glycerol-3-phosphate dehydrogenase gene.

[0077] In E. coli, xylA Genes and xylB The two genes are linked together, and when performing gene knockout, the two genes are knocked out together. In the following examples, xylA Genes and xylB Gene Two genes are collectively called xylAB Gene.

[0078] In the following examples, the final concentration of kanamycin in the culture medium is 50 μg / mL, the final concentration of spectinomycin in the culture medium is 100 μg / mL, and the final concentration of chloramphenicol in the culture medium is 25 μg / mL.

[0079] The following examples use a two-stage bioprocess to produce xylitol using hemicellulose hydrolysate, and the specific method is as follows: Figure 1 shown.

[0080] The primer sequence information used in the following examples is shown in Table 1.

[0081] Table 1. Primer list

[0082]

[0083] Example 1 Construction of a pathway for producing xylitol by metabolizing L-arabinose

[0084] 1. Construction of knockout genome araB , araD and lyxK Engineered bacteria

[0085] References (Multigene Editing in the Escherichia coli Genome via theCRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl Environ Microbiol, 2015) was used to perform Figure 2 As shown in 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.

[0086] (1) Knockout araB Gene strain E.coli Construction of W3110△araB

[0087] PCR amplification: ara-UF and ara-UR, ara-DF and ara-DR were used as primers, E.coli The W3110 genome was used as a template to amplify fragments of 500 bp each and recovered on gel; then ara-UF and ara-DR were used as primers, and the two recovered 500 bp fragments were used as templates to amplify a repair template fragment of about 1000 bp and recovered on gel.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] (2) Knockout araD Gene strain E.coli Construction of W3110△araBAD

[0094] PCR amplification: araD-UF and araD-UR, araD-DF and araD-DR were used as primers, E.coli The W3110 genome was used as a template to amplify fragments of 500 bp each and recovered on gel; then araD-UF and araD-DR were used as primers, and the two recovered 500 bp fragments were used as templates to amplify a repair template fragment of about 1000 bp and recovered on gel.

[0095] 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.

[0096] Prepared by the above electroporation competent preparation method E.coli W3110△araB competent cells, electroporate the pCas plasmid into competent cells, and pick E.coli A single colony of W3110△araB / pCas was cultured in a 5 mL LB tube containing kanamycin and 10 mM arabinose at 37 °C until the OD600 was approximately 0.6, and then prepared using the above method. E.coli W3110△araB / pCas competent state.

[0097] 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.

[0098] Plasmid loss: Use the above method to remove the two plasmids pCas and ptsF-N20 to obtain E.coli W3110ΔaraBAD strain.

[0099] (3) Knockout lyxK Gene strain E.coli Construction of W3110△araBAD△lyxK

[0100] PCR amplification: lyxK-UF and lyxK-UR, lyxK-DF and lyxK-DR were used as primers, E.coli The W3110 genome was used as a template to amplify fragments of 500 bp each and recovered on gel; then lyxK-UF and lyxK-DR were used as primers, and the two recovered 500 bp fragments were used as templates to amplify a repair template fragment of about 1000 bp and recovered on gel.

[0101] 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.

[0102] Prepared by the above electroporation competent preparation method E.coli W3110△araBAD competent cells, electroporate the pCas plasmid into competent cells, and pick E.coli A single colony of W3110△araBAD / pCas was cultured in a 5 mL LB tube containing kanamycin and 10 mM arabinose at 37°C until OD600 was approximately 0.6, and then prepared using the above method. E.coli W3110△araBAD / pCas competent state.

[0103] Electroporation: Electroporate the lyxK repair template fragment and lyxK-Target plasmid into E.coli W3110△BAD / pCas competent cells were 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.

[0104] Plasmid loss: Use the above method to discard the two plasmids pCas and lyxK-Target to obtain E.coli W3110ΔaraBADΔlyxK strain.

[0105] 2. Strengthening of the pathway for producing cofactor NADH by glycerol metabolism (insert glpF , glpK and glpD to the E. coli genome)

[0106] The engineered bacteria obtained in step (3) above E.coli W3110△araBAD△lyxK or engineered bacteria E.coli W3110 was used as the starting strain, and the glycerol transporter gene was inserted into the 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::(glpF-glpK)::glpD or W3110::(glpF-glpK)::glpD.

[0107] Example 2

[0108] one, araA , dpe , lxr Plasmid co-expression of genes

[0109] Plasmid pBAD18-kan (containing the arabinose-inducible promoter) was extracted using a plasmid extraction kit. Para , SEQID NO.8), pBAD18-dpe1 (containing gene dpe1 , SEQ ID NO.4), pBAD18-lxr8 (containing gene lxr8 , SEQ ID NO.6) and pBAD18-araA17 (containing gene araA and mutant 5'UTR, SEQ ID NO.1, SEQ ID NO.3), the plasmid pBAD18-kan was digested with restriction endonucleases EcoRI and SphI, the plasmid pBAD18-dpe1 was digested with EcoRI and BamHI, the plasmid pBAD18-lxr8 was digested with BamHI and XbaI, and the plasmid pBAD18-ara17 was digested with XbaI and SphI, and the four DNA fragments were recovered by nucleic acid electrophoresis. The four fragments were connected with T4 ligase to construct the plasmid pBAD18-dpe1-lxr8-araA17.

[0110] The plasmid was electroporated into the strain E.coli W3110, E.coli W3110△araBAD△lyxK, or E.coli W3110△araBAD△lyxK::(glpF-glpK)::glpD, obtained a strain capable of metabolizing L-arabinose to xylitol E.coli W3110 / pBAD18-dpe1-lxr8-araA17, or E.coli W3110△araBAD△lyxK / pBAD18-dpe1-lxr8-araA17, or E.coli W3110△araBAD△lyxK::(glpF-glpK)::glpD / pBAD18-dpe1-lxr8-araA17.

[0111] two, araA , dpe , lxr Genomic co-expression of (optimization of xylitol production pathway by co-metabolism of xylose and glucose)

[0112] According to the xylose metabolic pathway and its global regulation in Escherichia coli, in this example, E.coli W3110, E.coli W3110△araBAD△lyxK, or E.coliA series of engineered bacteria were constructed based on W3110△araBAD△lyxK::(glpF-glpK)::glpD: the genes related to glucose transport and metabolism were ptsG and ptsF Replace with dpe1 , lxr8 and araA17 Gene expression cassette; Genes related to xylose metabolism xylAB Also replaced by dpe1 , lxr8 and araA17 Gene expression cassette.

[0113] in, ptsG The replacement of slowed down the CCR effect and improved the co-metabolism efficiency of glucose and other sugars; ptsF The replacement of can reduce the transport of xylitol from the extracellular to the intracellular; xylA and xylB The replacement of L can block the downstream decomposition pathway of xylose. - Arabinose isomerase gene ( araA17 )、D-psicose-3-epimerase( dpe1 ) and L-xylulose reductase ( lxr8 ) expression, strengthening the L - The efficiency of xylitol production from arabinose metabolism in the Escherichia coli genome ptsF、ptsG、xylA and xylB The isogenic replacement operation was performed using the CRISPR / Cas9 gene editing method.

[0114] (1) Replacement ptsG Gene

[0115] PCR amplification: ptsG-uF and ptsG-uR, ptsG-dF and ptsG-dR were used as primers, E.coli The W3110 genome was used as a template to amplify fragments of 500 bp each and recovered on gel; ATX-F and ATX-R were used as primers and plasmid / pBAD18-dpe1-lxr8-araA17 was used as a template to amplify a fragment of about 5.1 kb and recovered on gel; then ptsG-uF and ptsG-dR were used as primers and the three recovered DNA fragments were used as templates to amplify a repair template fragment of about 6.1 bp and recovered on gel.

[0116] Construction of ptsG-N20 plasmid: Using ptsG-N20-F and ptsG-N20-R as primers and pTargetF-cadA as template, PCR amplified a fragment of about 2.2 kb, and after DpnI digestion, the ptsG-N20 plasmid was constructed.

[0117] Preparation of electroporated competent cells: a) Pick a single colony of E. coli and place it in a 5 mL LB tube medium and culture at 37°C, 200 rpm until OD 600 About 0.6; b) Use a sterilized 2 mL EP tube, 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 2 more times; d) Resuspend with 100 μL of 10% glycerol. Prepare E. coli using the above electroporation competent preparation method E.coli W3110 competent state, E.coli W3110△araBAD△lyxK competent state, E.coli W3110△araBAD△lyxK::(glpF-glpK)::glpD competent cells, electroporate the pCas plasmid (electroporation conditions: 2.5 kV, 200 Ω, 25 μF) into competent cells, and pick E.coli W3110 / pCas single colony, E.coli W3110△araBAD△lyxK / pCas single colony, E.coli A single colony of W3110△araBAD△lyxK::(glpF-glpK)::glpD / pCas was cultured in a 5 mL LB tube containing kanamycin and 10 mM arabinose at 37°C until OD 600 About 0.6, and then prepare Escherichia coli using the above method E.coli W3110 / pCas electroporation competent state, E.coli W3110△araBAD△lyxK / pCas electroporation competent state, E.coli W3110△araBAD△lyxK::(glpF-glpK)::glpD / pCas electroporation competent state.

[0118] Electroporation: ptsG Repair template fragment and ptsG-N20 plasmid electroporation E.coli W3110△araBAD△lyxK::(glpF-glpK)::glpD / pCas competent cells were 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.

[0119] Plasmid loss: Pick a single colony that is positive after PCR verification of the colony, 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 both cannot grow, it indicates that both pCas and ptsG-N20 plasmids have been lost, and the plasmids are obtained. E.coli W3110△ptsG::(araA17-dpe1-lxr8) strain, E.coli W3110△araBAD△lyxK△ptsG::(araA17-dpe1-lxr8) strain, E.coli W3110ΔaraBADΔlyxKΔptsG::(araA17-dpe1-lxr8)::(glpF-glpK)::glpD strain.

[0120] (2) Replacement ptsF Gene

[0121] PCR amplification: Using ptsF-uF and ptsF-uR, ptsF-dF and ptsF-dR as primers, and W3110 genome as template to amplify fragments of 500 bp each, and then recover them on gel; using ATX-F and ATX-R as primers, and plasmid / pBAD18-dpe1-lxr8-araA17 as template to amplify a fragment of about 5.1 kb, and then recover them on gel; then using ptsF-uF and ptsF-dR as primers, the three recovered DNA fragments were used as templates to amplify a repair template fragment of about 6.1 bp, and then recover them on gel.

[0122] Construction of ptsF-N20 plasmid: Using ptsF-N20-F and ptsF-N20-R as primers and pTargetF-cadA as template, PCR amplified a fragment of about 2.2 kb, and after DpnI digestion, the ptsF-N20 plasmid was constructed.

[0123] Prepared by the above electroporation competent preparation method E.coli W3110△ptsG::(araA17-dpe1-lxr8) competent state, E.coli W3110△araBAD△lyxK△ptsG::(araA17-dpe1-lxr8) competent state, E.coliW3110△araBAD△lyxK△ptsG::(araA17-dpe1-lxr8)::(glpF-glpK)::glpD competent cells, electroporate the pCas plasmid into competent cells, and pick E.coli W3110△ptsG::(araA17-dpe1-lxr8) / pCas single colony, E.coli W3110△araBAD△lyxK△ptsG::(araA17-dpe1-lxr8) / pCas single colony, E.coli A single colony of W3110△araBAD△lyxK△ptsG::(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / pCas was cultured in a 5 mL LB tube containing kanamycin and 10 mM arabinose at 37°C until OD600 was approximately 0.6, and then prepared using the above method. E.coli W3110△ptsG::(araA17-dpe1-lxr8) / pCas competent state, E.coli W3110△araBAD△lyxK△ptsG::(araA17-dpe1-lxr8) / pCas competent state, E.coli W3110△araBAD△lyxK△ptsG::(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / pCas competent state.

[0124] Electroporation: Electroporate the ptsF repair template fragment and ptsF-N20 plasmid into E.coli W3110△ptsG::(araA17-dpe1-lxr8) / pCas、 E.coli W3110△araBAD△lyxK△ptsG::(araA17-dpe1-lxr8) / pCas、 E.coli W3110△araBAD△lyxK△ptsG::(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / pCas competent cells were 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.

[0125] Plasmid loss: Use the above method to remove the two plasmids pCas and ptsF-N20 to obtain E.coli W3110△ptsG△ptsF::2(araA17-dpe1-lxr8), E.coliW3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8), E.coli W3110ΔaraBADΔlyxKΔptsGΔptsF::2(araA17-dpe1-lxr8)::(glpF-glpK)::glpD strain.

[0126] (3) Replacement xylAB Gene

[0127] PCR amplification: Using xylAB-uF and xylAB-uR, xylAB-dF and xylAB-dR as primers, W3110 genome was used as a template to amplify fragments of 500 bp each, and then recovered on gel; using ATX-F and ATX-R as primers, plasmid / pBAD18-dpe1-lxr8-araA17 was used as a template to amplify a fragment of about 5.1 kb, and then recovered on gel; then using xylAB-uF and xylAB-dR as primers, the three recovered DNA fragments were used as templates to amplify a repair template fragment of about 6.1 bp, and then recovered on gel.

[0128] Construction of xylAB-N20 plasmid: Using xylAB-N20-F and xylAB-N20-R as primers and pTargetF-cadA as template, PCR amplified a fragment of about 2.2 kb, and after DpnI digestion, the xylAB-N20 plasmid was constructed.

[0129] Prepared by the above electroporation competent preparation method E.coli W3110△ptsG△ptsF::2(araA17-dpe1-lxr8) competent state, E.coli W3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8) competent state, E.coli W3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8)::(glpF-glpK)::glpD competent cells, electroporate the pCas plasmid into competent cells, and pick E.coli W3110△ptsG△ptsF::2(araA17-dpe1-lxr8) / pCas single colony, E.coli W3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8) / pCas single colony, E.coliA single colony of W3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / pCas was cultured in a 5 ml LB tube containing kanamycin and 10 mM arabinose at 37°C until OD600 was approximately 0.6, and then prepared using the above method. E.coli W3110△ptsG△ptsF::2(araA17-dpe1-lxr8) / pCas competent state, E.coli W3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8) / pCas competent state, E.coli W3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / pCas competent state.

[0130] Electroporation: xylAB Repair template fragment and xylAB-N20 plasmid electroporation E.coli W3110△ptsG△ptsF::2(araA17-dpe1-lxr8) / pCas, E.coli W3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8) / pCas, E.coli W3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8)::(glpF-glpK)::glpD competent cells were spread on LB plates containing spectinomycin and kanamycin and cultured at 37°C overnight; single colonies grew and were verified by colony PCR using specific primers.

[0131] Plasmid loss: Use the above method to remove the two plasmids pCas and xylAB-N20 to obtain E.coli W3110 ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8), or E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8), E.coli W3110ΔaraBADΔlyxKΔptsGΔptsFΔxylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD strain.

[0132] Example 3 Recombinant and efficient expression of xylose reductase

[0133] (1) Comparison of xylose reductase from different sources

[0134] Eleven wild-type XR genes from different sources were selected, codon optimized and fully synthesized by Nanjing GenScript Biotechnology Co., Ltd., and then constructed into plasmid pET-28a and introduced into E. coli. E.coli During the BL21 genetic engineering, shake flask fermentation and enzyme production were performed to obtain a xylose reductase enzyme library. The enzyme activities of xylose reductase with L-arabinose as substrate (the product is L-arabinitol) and D-xylose as substrate (the product is xylitol) were measured, and the results are shown in Table 2. References for shake flask fermentation and enzyme production: Evolution in reverse: engineering a D-xylose-specific xylose reductase. Chembiochem, 2008, 9(8): 1213-5.

[0135] Table 2. Sources of xylose reductase and enzyme activity assay results

[0136]

[0137] (2) Construction of a lactose-induced metabolic genetically engineered bacterium expressing recombinant xylose reductase

[0138] a) Eleven xylose reductase genes from different sources were reconstructed into plasmid pSU2718 and P lac The promoter was constructed upstream of the XR gene.

[0139] b) Prepare Escherichia coli according to the method in Example 2 E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD electroporation competent state;

[0140] c) Place the prepared electroporation competent cells on ice for 5 min, add 2-3 μL of xylose reductase expression plasmid for electroporation;

[0141] d) Finally, evenly spread it on an LB plate containing chloramphenicol and culture it in a 37°C incubator overnight to obtain a lactose-induced xylose reductase gene recombinant expression metabolic engineering bacterium:

[0142] E.coliW3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR1 (referred to as P1);

[0143] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR2 (referred to as P2);

[0144] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR3 (referred to as P3);

[0145] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR4 (referred to as P4);

[0146] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR5 (referred to as P5);

[0147] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR6 (referred to as P6);

[0148] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR7 (referred to as P7);

[0149] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR8 (referred to as P8);

[0150] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR9 (referred to as P9);

[0151] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR10 (referred to as P10);

[0152] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR11 (abbreviated as P11).

[0153] According to the above method, the above 11 P lac Promoter-induced XR plasmid transfer E.coli From the W3110△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8) strain, strains P12 to P22 were obtained.

[0154] According to the above method, the above 11 P lac Promoter-induced XR plasmid transfer E.coli From the W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8) strain, strains P23 to P33 were obtained.

[0155] (3) Shake flask fermentation experiment

[0156] a) Pick the 33 successfully constructed monoclonal engineered bacteria mentioned above and place them in 5 mL LB-cm medium. Culture them at 37°C with shaking at 220 rpm for 8-12 h to obtain the seed solution.

[0157] b) Inoculate the culture medium into a shake flask at 2% inoculum and culture at 37°C until the OD is 1-2.

[0158] c) Add appropriate amounts of L-arabinose, glycerol, xylose, and glucose mother liquor to each shake flask fermentation medium, so that the concentration of L-arabinose in the fermentation broth is 2.5 g / L, the concentration of glycerol is 2.5 g / L, the concentration of xylose is 20 g / L, and the concentration of glucose is 10 g / L; continue to culture at 37°C for 3-4 hours after the substrate is added until all L-arabinose is converted into xylitol. After fermentation, detect the concentration of the fermentation substrate L-arabinose and the concentration of the product xylitol, and calculate the conversion rate of L-arabinose.

[0159] d) When all L-arabinose is consumed, add an appropriate amount of lactose as an inducer to each shake flask, and make the final concentration of lactose in the fermentation broth 1 g / L. After the addition of the substrate, induce the culture at 30°C for 24-48 hours. After the fermentation culture, detect the concentration of the fermentation substrate xylose and the concentration of the products xylitol and L-arabinitol, and calculate the conversion rate of xylose.

[0160] 33 strains P lac The results of shake flask fermentation experiments of metabolically engineered bacteria containing xylose reductase genes from different sources and promoters are shown in Table 3.

[0161] As can be seen from Table 3, before induction, P1~P11 genetically engineered bacteria almost converted all L-arabinose into xylitol, among which the conversion rate of strain P5 was as high as 99.89%, but the conversion rate of xylose was extremely low; P23~P33 also achieved a good conversion rate of L-arabinose, but lower than P1~P11; P12~P22 had a low efficiency of converting L-arabinose into xylitol due to the presence of natural L-arabinose decomposition pathway. After L-arabinose was converted into xylitol, lactose further induced the expression of XR from different sources, thereby catalyzing xylose to produce xylitol. The efficiency of catalyzing xylose by the wild-type XR from 33 different sources was different, and most of the wild-type XR had a relatively high efficiency in catalyzing xylose. After induced fermentation at 30℃ for 24-48 h, P1~P11 strains also converted almost all xylose into xylitol, with a xylose conversion rate of up to 99.95%, and no L-arabinose was produced.

[0162] Table 3. P lac The results of shake flask fermentation of wild-type xylose reductase strains with promoter

[0163]

[0164] Example 4 Construction of engineered bacteria with different inducible promoters and shake flask fermentation

[0165] (1) Construction of different inducible promoter plasmids

[0166] The 33 strains constructed according to Example 3 were P lac The plasmids containing the promoter and the results of shake flask fermentation were selected to construct different inducible promoters (rhamnose promoter P rha , anhydrotetracycline promoter P tet , lactose promoter P tac or P trc ) Plasmids expressing XR-1, XR-5 and XR-6.

[0167] (2) Construction of genetically engineered bacteria with different inducible promoters

[0168] a) Prepare Escherichia coli according to the method in Example 2 E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD electroporation competent state;

[0169] b) Place the prepared electroporation competent cells on ice for 5 min, add 2-3 μL of XR expression plasmid for electroporation;

[0170] c) Finally, spread evenly on LB plate containing chloramphenicol and culture in a 37°C incubator overnight to obtain genetically engineered bacteria expressing xylose reductase genes from different sources with different inducible promoters:

[0171] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / prha-XR1 (referred to as P34);

[0172] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / prha-XR5 (referred to as P35);

[0173] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / prha-XR6 (referred to as P36);

[0174] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / ptet-XR1 (referred to as P37);

[0175] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / ptet-XR5 (referred to as P38);

[0176] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / ptet-XR6 (referred to as P39);

[0177] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / ptac-XR1 (referred to as P40);

[0178] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / ptac-XR5 (referred to as P41);

[0179] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / ptac-XR6 (referred to as P42);

[0180] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / ptrc-XR1 (referred to as P43);

[0181] E.coliW3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / ptrc-XR5 (referred to as P44);

[0182] E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / ptrc-XR6 (abbreviated as P45).

[0183] (3) Shake flask fermentation experiment of genetically engineered bacteria with different inducible promoters

[0184] a) Pick out P1, P5, P6, P34, P35, P36, P37, P38, P39, P40, P41, P42, P43, P44 and P45 monoclonal bacteria respectively in 5 mL LB-cm medium, culture at 37℃ with shaking at 220 rpm for 8-12 h, and then obtain the seed solution.

[0185] b) Inoculate the culture medium into a shake flask at 2% inoculum and culture at 37°C until the OD is 1-2.

[0186] c) Add appropriate amounts of L-arabinose, glycerol, xylose and glucose mother liquor to each shake flask fermentation medium, so that the concentration of L-arabinose in the fermentation broth is 2.5 g / L, the concentration of glycerol is 2.5 g / L, the concentration of xylose is 20 g / L, and the concentration of glucose is 10 g / L. After adding the substrate, continue to culture at 37°C for 3-4 hours until all L-arabinose is converted into xylitol.

[0187] d) When all L-arabinose is consumed, add appropriate amounts of the corresponding inducers to each shake flask so that the final concentration of lactose inducer in the fermentation broth is 1 g / L, the final concentration of rhamnose inducer is 10 mM, and the final concentration of anhydrotetracycline inducer is 1 g / L. After the addition of the substrate, induce the culture at 30°C for 24-48 h. After the fermentation culture, detect the concentrations of the fermentation substrates xylose and L-arabinose and the concentrations of the products xylitol and L-arabinitol, and calculate the conversion rates of xylose and arabinose.

[0188] The results of shake flask fermentation experiments of 15 genetically engineered bacteria expressing wild-type xylose reductase genes from different sources with different inducible promoters are shown in Table 4.

[0189] As can be seen from Table 4, after adding the substrate L-arabinose for cultivation (before induction), the 15 strains of genetically engineered bacteria were also able to convert all L-arabinose into xylitol, among which the L-arabinose conversion rate of strain P39 was as high as 99.38%; after L-arabinose was converted into xylitol, the wild-type XR from different sources was further induced to express by adding the corresponding inducer, thereby catalyzing xylose to produce xylitol. The efficiency of catalyzing xylose by different inducers inducing the expression of XR1, XR5 and XR6 was different. Among them, the efficiency of catalyzing xylose by the xylose reductase induced by the lactose inducible promoter was higher than that by the rhamnose and anhydrotetracycline inducible promoters. P lac The engineered strain P6 with the promoter as the highest xylose conversion rate reached 99.98%, while the efficiency of XR catalyzed by rhamnose and anhydrotetracycline was low, among which the strain P15 with anhydrotetracycline as the promoter had a xylose conversion rate of 78.29%. In the shake flask fermentation experiment, no L-arabinitol was detected.

[0190] Table 4. Shake flask fermentation results of engineered bacteria with different inducible promoters

[0191]

[0192] Example 5 Optimization of the glycerol addition ratio in the culture medium

[0193] According to the results of shake flask fermentation in Example 3, P6 ( E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR6) was used as the fermentation strain for shake flask fermentation. The fermentation experimental conditions were the same as those in Example 3, wherein the variable of each shake flask fermentation experiment was the amount of glycerol added, and the amount of glycerol added was 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, and 2.5 g / L, respectively. The amount of other sugars added was fixed, wherein L-arabinose was 2.5 g / L, and glycerol / L-arabinose (m / m) was 0.2, 0.4, 0.6, 0.8, and 1.0, respectively. The shake flask fermentation results are shown in Table 5.

[0194] The conversion rate of L-arabinose increases with the increase of the glycerol addition ratio; when the glycerol addition ratio increases to 0.6, the conversion rate of L-arabinose exceeds 99%; with the further increase of the glycerol addition ratio, the conversion rate of L-arabinose will no longer increase, indicating that when the mass concentration ratio of glycerol to L-arabinose in the fermentation system is 0.6, the coenzyme NADH provided by the fermentation bacteria through glycerol metabolism is sufficient for the metabolic consumption of L-arabinose.

[0195] Table 5. Optimization experimental results of glycerol addition

[0196]

[0197] Example 6 Preparation of xylitol by fermentation of hemicellulose hydrolysate with genetically engineered bacteria

[0198] With P6 ( E.coli W3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD / plac-XR6) was used as the fermentation strain, and corn cob hydrolysate was used as the raw material to ferment and produce xylitol.

[0199] The specific steps of fermentation are:

[0200] a) The hemicellulose hydrolysate was concentrated to a certain concentration and added to the fermentation basal medium, so that the total concentration of xylose was about 130 g / L, the total concentration of glucose was about 17 g / L, and the total concentration of L-arabinose was about 27 g / L; the volume of the medium was 10 L (15 L fermenter).

[0201] The other components and their concentrations in the fermentation basal medium are as follows: Na2HPO4 10 g / L, KH2PO4 3 g / L, glycerol 15 g / L, yeast powder 10 g / L, and peptone 5 g / L.

[0202] In addition, prepare 1 L of fermentation feed medium: glucose 500 g / L.

[0203] b) Inoculate the P6 seed solution (LB medium) into the sterilized basal medium at an inoculation rate of 10-15%, and culture for about 12 hours at 37°C, with an aeration ratio of 0.8 vvm and a pH of 7.0 (controlled by feeding NH3•H2O), until all L-arabinose in the hemicellulose hydrolysate is consumed. During the fermentation process, the speed is adjusted to keep the dissolved oxygen at 20%-40%.

[0204] c) After L-arabinose is completely consumed, the fermentation temperature is lowered to 30°C and the culture is continued; at the same time, lactose inducer with a final concentration of 5 g / L is added to induce the expression of xylose reductase, and feed medium is continuously added.

[0205] d) During the fermentation process, samples were taken at regular intervals to detect the concentrations of the remaining substrates glucose, xylose and L-arabinose in the fermentation broth, as well as the concentrations of the products xylitol and arabinitol.

[0206] The concentration changes of various substances during the fermentation process are as follows Figure 3 As shown, the HPLC detection spectra of various standard samples are as follows Figure 4As shown in the figure, the HPLC detection spectrum of hemicellulose hydrolysate at 0 h of fermentation is as follows Figure 5 As shown in the figure, the HPLC detection spectrum of hemicellulose hydrolysate after 48 h of fermentation is as follows Figure 6 As shown in the figure, the HPLC detection spectrum of hemicellulose hydrolysate after 60 h of fermentation is as follows Figure 7 As shown. Figures 3 - 7 It can be seen that in the first stage of fermentation (fermentation 0-12 h), L-arabinose in the hemicellulose hydrolysate was continuously consumed and gradually converted into xylitol. After 12 h of fermentation, all L-arabinose in the hemicellulose hydrolysate had been converted into xylitol. In the second stage of fermentation, with the addition of lactose inducer, xylose reductase was expressed and catalyzed the reduction of xylose in the hemicellulose hydrolysate to xylitol. After 60 h of fermentation, all xylose in the hemicellulose hydrolysate had been converted into xylitol, and the xylose conversion rate was close to 100%, and there was basically no residual miscellaneous sugars (glucose and L-arabinose), and the production of L-arabinitol was not detected. Finally, 11.5 L of fermentation broth was obtained, and the concentration of xylitol reached 136.5 g / L. Since L-arabinose was also converted into xylitol, the yield of xylitol to xylose reached 1.2 g / g, and the xylitol yield reached 2.3 g / L / h.

Claims

1. A two-stage biological method for producing xylitol using hemicellulose hydrolysate, characterized in that: include: (1) using hemicellulose hydrolyzate as a raw material, performing a first stage fermentation culture in a fermentation medium inoculated with fermentation engineering bacteria until no L-arabinose remains in the fermentation liquid, thereby obtaining a fermentation reaction liquid I; (2) After adding glucose feed and inducer to the fermentation reaction liquid I, the second stage of fermentation culture is continued to obtain a fermentation product containing xylitol; The fermentation engineering bacteria include: a host cell containing a xylose reductase XR gene and a target gene co-expressed in the host cell; The target gene includes L-arabinose isomerase gene araA , D-psicose-3-epimerase gene dpe and L-xylulose reductase gene lxr ; The host cell is Escherichia coli; At least one of the genes in the genome of the host cell is knocked out araB Gene, araD Genes and lyx One of the genes; The promoter that drives the expression of the XR gene is an inducible promoter; the inducible promoter is a non-arabinose inducible promoter.

2. The method for producing xylitol using a two-stage biological method using hemicellulose hydrolysate as claimed in claim 1, characterized in that: The gene araA The nucleotide sequence of the gene is shown in SEQ ID NO.1; dpe The nucleotide sequence of the gene is shown in SEQ ID NO.4; lxr The nucleotide sequence is shown in SEQ ID NO.

6.

3. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 1, characterized in that: The target gene is co-expressed in the host cell in one of the following ways: (A) Co-expression in host cells in the form of a recombinant plasmid; (B) Inserted into the host cell genome in the form of an expression cassette for co-expression.

4. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 3, characterized in that: In (A), the original expression vector of the recombinant plasmid is pBAD18; (B) The target gene is inserted into the non-coding region of the host cell genome; or araA ,Gene dpe and genes lxr Random Replacement ptsG Gene ,ptsF Gene, xyA and xyB Gene.

5. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to any one of claim 1, characterized in that: The host cell is Escherichia coli E. coli W3110.

6. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 5, characterized in that: At least a glycerol transporter gene is inserted into the non-coding region of the genome of the host cell gf , glycerol kinase gene gL Glyceryl triphosphate dehydrogenase gene gL One of them.

7. The method for producing xylitol using a two-stage biological method using hemicellulose hydrolysate as claimed in claim 6, characterized in that: The gene gf The nucleotide sequence of the gene is shown in SEQ ID NO.9; gL The nucleotide sequence of the gene is shown in SEQ ID NO.10; gL The nucleotide sequence is shown in SEQ ID NO.

11.

8. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 1, characterized in that: The NCBI accession number of the XR gene is one of AF451326.3, AF074484.1, AB002106.1, ALO17776.1, EAA34695.1, AAA99507.1 and Q9P8R5.

1.

9. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 1, characterized in that: The inducible promoter of the XR gene is a rhamnose promoter, anhydrotetracycline promoter or a lactose promoter.

10. The method for producing xylitol using a two-stage biological method using hemicellulose hydrolysate as claimed in claim 9, characterized in that: The nucleotide sequence of the inducible promoter of the XR gene is one of the sequences shown in SEQ ID NO.15-19.

11. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 1, characterized in that: The hemicellulose hydrolyzate is obtained by hydrolyzing hemicellulose-containing biomass; the hemicellulose-containing biomass is corn cobs, papermaking short fibers, bagasse, straw or rice husks; the hemicellulose hydrolyzate contains at least L-arabinose, glucose and xylose.

12. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 1, characterized in that: In step (1), the conditions of the first stage fermentation culture are: temperature of 18-37°C, pH of 6.0-8.0; based on the dissolved oxygen under the initial fermentation conditions after inoculation as 100%, the dissolved oxygen is maintained between 0 and 50%.

13. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 1, characterized in that: In step (1), the OD of the fermentation engineering bacteria when inoculated into the fermentation medium is 600 It is 0.6~0.

8. Based on the initial fermentation volume when loading the tank, the inoculation amount is 1~15%.

14. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate as claimed in claim 1, characterized in that: In step (1), the fermentation medium comprises at least a carbon source, a nitrogen source and an inorganic salt; based on the initial fermentation volume, the amount of the carbon source added is 10 to 50 g / L; the amount of the nitrogen source added is 5 to 30 g / L.

15. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 14, characterized in that: The carbon source is glycerol.

16. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 15, characterized in that: The added amount of glycerol is more than 0.6 times the mass of arabinose.

17. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 11, characterized in that: In step (2), based on the xylose content in the hydrolyzate, the amount of glucose feed added is 10% to 50% of the xylose in terms of mass percentage; the inducer is one of lactose, rhamnose or anhydrotetracycline; based on the initial fermentation volume, the amount of inducer added is 1 g / L to 10 g / L in terms of volume percentage.

18. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 1, characterized in that: In step (2), the fermentation culture conditions of the second stage are: temperature of 18-37°C, pH of 6.0-8.0; based on the dissolved oxygen under the initial fermentation conditions after inoculation as 100%, the dissolved oxygen is maintained between 0 and 50%.

Images