Genetically engineered bacteria for preparing xylitol and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a genetically engineered bacterium for preparing xylitol and its application, wherein the genetically engineered bacterium is a genetically engineered bacterium that co-expresses a xylose reductase gene, an L-arabinitol-4-dehydrogenase gene, and an L-xylulose reductase gene, and can catalyze the production of xylitol using a hemicellulose hydrolyzate as a substrate. Compared with existing xylitol-producing engineering bacteria, the xylose reductase gene, the L-arabinitol-4-dehydrogenase gene, and the L-xylulose reductase gene in the genetically engineered bacteria of the present invention are widely available, the xylose conversion rate in the fermentation product can exceed 99%, and there is no generation of arabinitol, which greatly simplifies the subsequent xylitol separation and purification process.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a genetic engineering bacterium for preparing xylitol and an application thereof. Background Art

[0002] The molecular formula of xylitol is C5H 12 O5 is a five-carbon sugar alcohol, which is a white crystal or crystalline powder. It tastes cool, with a sweetness equivalent to sucrose and a calorie equivalent to glucose. Xylitol is easily soluble in water, can absorb a lot of calories when dissolved in water, and produces a pleasant cool feeling in the mouth when consumed. It is slightly soluble in ethanol and methanol. Xylitol has the characteristics of hygroscopicity, high sweetness, and good thermal stability. At present, xylitol has been widely used as a natural sweetener for high-end foods, and was rated as one of the 12 most promising basic chemicals from biomass by the US Department of Energy in 2004. Xylitol has attracted more and more attention. For example, in the field of pharmacy, it is used as an excipient of pharmaceuticals and a xylitol surfactant; in the field of medicine, it is used as a high-quality infusion carrier, and can be made into infusion with antibacterial drugs, cardiovascular and cerebrovascular drugs, digestive system drugs, anti-tumor drugs, etc. It can also be used for parenteral nutrition, treatment of diabetes, treatment of liver disease, prevention of dental caries, prevention of respiratory tract infections, inhibition of macrophage adhesion, treatment of chronic sinusitis, etc. The global xylitol market size has generally shown an upward trend year by year, rising from US$800 million in 2017 to US$1.11 billion in 2023, with great market potential.

[0003] The preparation methods of xylitol mainly include natural extraction, chemical synthesis and biotransformation. Due to the low content of xylitol in nature, it is difficult and costly to prepare by extraction, and there is no value for industrial production. Chemical synthesis first separates hemicellulose and hydrolyzes sugar to obtain pure xylose, and then reduces xylose to xylitol through high-pressure hydrogenation. This is a method commonly used in industry at present; due to its relatively complex production process and high cost, the price of xylitol has remained high for a long time, and compared with other commonly used sweeteners, it has no price advantage. The production of xylitol by biotransformation is a recently emerging preparation process. Compared with the chemical method, this process has many advantages: microbial fermentation reaction is carried out at room temperature and pressure, with the advantages of low energy consumption, simple equipment, and safe operation. Therefore, it has gradually attracted people's attention and is expected to become an alternative process for xylitol production. The biological production of xylitol is to reduce xylose to xylitol by enzyme catalysis or microbial fermentation, and then purify it to obtain xylitol.

[0004] Although the microbial method has these advantages, the specificity of xylose reductase in existing genetically engineered bacteria is relatively poor. When fermenting hemicellulose hydrolysate (mainly containing glucose, D-xylose (D-Xylose) and L-arabinose (L-Arabinose) and other sugars) as substrates, xylose reductase catalyzes the reduction of xylose to xylitol and also reduces arabinose to arabitol. Xylitol and arabitol are diastereomers with very similar physical and chemical properties. The generated arabitol brings difficulties to the separation and purification of xylitol. In recent years, researchers have continuously improved the specificity of xylose reductase in microorganisms to xylose through screening, optimization, genetic engineering and other means to increase the yield of xylitol, simplify the xylitol separation and purification process and reduce production costs, but the results are very limited and the effect is not good. Summary of the invention

[0005] In view of the problem in the prior art that the impurity arabitol exists in the product xylitol and is difficult to remove, the present invention provides a genetically engineered bacterium for preparing xylitol and its application, and the specific technical scheme is as follows:

[0006] The present invention first provides a genetically engineered bacterium, wherein the genetically engineered bacterium is a genetically engineered bacterium that co-expresses a xylose reductase gene, an L-arabinitol-4-dehydrogenase gene and an L-xylulose reductase gene.

[0007] The genetically engineered bacteria provided by the present invention can ferment xylitol using hemicellulose hydrolysate as a substrate, and the hemicellulose hydrolysate mainly contains sugars such as glucose, D-xylose and L-arabinose. First, the xylose reductase expressed by the genetically engineered bacteria catalyzes the xylose in the hemicellulose hydrolysate to generate xylitol. At the same time, the L-arabinose in the hydrolysate is also reduced to L-arabinose by the xylose reductase, and then the L-arabinose is catalyzed by L-arabinose-4-dehydrogenase to generate L-xylulose, and finally the L-xylulose is further catalyzed by L-xylulose reductase to generate xylitol, so that the xylose and arabinose in the substrate are converted into xylitol, and no arabinose byproduct remains after fermentation, which greatly simplifies the process of xylitol separation and purification and reduces the production cost of xylitol.

[0008] Further, the xylose reductase gene is an XR gene, and the XR gene is one of NCBI accession numbers AF451326.3, KF752418.1, AF074484.1, AB002106.1, ALO17776.1, EAA34695.1, AAA99507.1, and Q9P8R5.1;

[0009] The L-xylulose reductase gene is an LXR gene, and the LXR gene is one of NCBI accession numbers KAG7881166.1, VEU21754.1, KAF6014139.1, ANZ74972.1, CCE79252.1, EIF45204.1, ODV85849.1, GCF00616.1, and AJ583159.

[0010] Furthermore, the combination of the XR gene and the LXR gene is as follows:

[0011] (1) AF451326.3, KAG7881166.1; (2) AF451326.3, GCF00616.1;

[0012] (3) KF752418.1, KAG7881166.1; (4) KF752418.1, GCF00616.1;

[0013] (5) AF074484.1, KAG7881166.1; (6) AF074484.1, GCF00616.1;

[0014] (7) AB002106.1, KAG7881166.1; (8) AB002106.1, GCF00616.1;

[0015] (9) ALO17776.1, KAG7881166.1; (10) ALO17776.1, GCF00616.1;

[0016] (11) EAA34695.1, KAG7881166.1; (12) EAA34695.1, GCF00616.1;

[0017] (13) AAA99507.1, KAG7881166.1; (14) AAA99507.1, GCF00616.1;

[0018] (15) Q9P8R5.1, KAG7881166.1; (16) Q9P8R5.1, GCF00616.1.

[0019] Further, the L-arabinitol-4-dehydrogenase gene is a LAD gene, and the LAD gene is one of NCBI accession numbers KX591955.1, KX591956.1, KX591957.1, AAL08944.1, EAA36547.1, BAC81768.1, EDP56335.1, XP_002569286.1, EDK37120.2, and CAH69383.1.

[0020] Furthermore, the starting strain of the genetically engineered bacteria is a wild-type Escherichia coli, or a strain that has a knockout or silenced ptsG Gene, ptsF Gene, xylA Gene, xylB coli after at least one gene in the gene.

[0021] Furthermore, the ptsG The nucleotide sequence of the gene is shown in SEQ ID NO.1. ptsF The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0022] Since the reaction catalyzed by xylose reductase requires the consumption of the coenzyme NADPH, in order to maximize the conversion of xylose into xylitol, the genetically engineered bacteria of the present invention have knocked out ptsG Genes can slow down the CCR effect, improve the co-metabolism efficiency of glucose and other sugars, and enable genetically engineered bacteria to metabolize glucose to produce coenzyme NADPH, thereby removing glucose from hemicellulose hydrolysate. ptsF Knockout of can reduce the transport of xylitol from the extracellular to the intracellular; xylA and xylB The knockout of can block the downstream decomposition pathway of xylose. Finally, a xylitol solution without other sugars or sugar alcohols can be obtained, and a high-purity xylitol product can be obtained after sterilization, protein removal and other refining processes.

[0023] In E. coli, xylA Genes and xylB Gene Two genes are linked together. xylA Genes and xylB Gene Two genes are collectively called xylAB Gene, when performing gene knockout or silencing, two genes are knocked out or silenced together, that is, xylAB Gene knockout or silencing.

[0024] Further, xylA Genes and xylB The genes are linked together, and the nucleotide sequence is shown in SEQ ID NO.3, that is, the xylABThe nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0025] Furthermore, the Escherichia coli is E.coli W3110.

[0026] The present invention further provides a method for preparing the above-mentioned genetically engineered bacteria, comprising: (1) integrating a xylose reductase gene, an L-arabinitol-4-dehydrogenase gene and an L-xylulose reductase gene into the genome of a starting strain; or, (2) transforming a plasmid containing the xylose reductase gene, the L-arabinitol-4-dehydrogenase gene and the L-xylulose reductase gene into the starting strain; and obtaining a genetically engineered bacteria co-expressing the xylose reductase gene, the L-arabinitol-4-dehydrogenase gene and the L-xylulose reductase gene.

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

[0028] In the genetically engineered bacteria of the present invention, the xylose reductase gene, L-arabinitol-4-dehydrogenase gene and L-xylulose reductase gene used can be of any source. When the expression products of the three genes in the starting strain have corresponding normal functions and activities, any combination of the xylose reductase gene, L-arabinitol-4-dehydrogenase gene and L-xylulose reductase gene of any source can achieve the function of catalyzing the production of xylitol using hemicellulose hydrolysate as a substrate.

[0029] The present invention also provides a method for producing xylitol, which specifically comprises: using hemicellulose hydrolyzate, glucose and an organic nitrogen source as substrates and using the above-mentioned genetically engineered bacteria to ferment and produce xylitol.

[0030] Furthermore, in the fermentation substrate, the organic nitrogen source is an organic nitrogen source for cell culture of animal or plant origin, preferably one or a combination of peptone, yeast powder or corn steep liquor powder.

[0031] Furthermore, in the fermentation substrate, the mass ratio of xylose to glucose is 5:1 to 2:1.

[0032] Furthermore, during the fermentation process, the dissolved oxygen is maintained between 0 and 50%, the pH is 6.0-8.0, and the temperature is 18-37°C, taking the dissolved oxygen under the initial fermentation conditions after inoculation as 100%.

[0033] 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.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The genetically engineered bacteria of the present invention co-express the XR gene, the LAD gene and the LXR gene, and can convert xylose and arabinose in the hemicellulose hydrolysate into xylitol. The conversion rate of xylose exceeds 99%, and no substrate miscellaneous sugars remain. No L-arabinitol is produced in the product, which greatly simplifies the process of biological preparation and separation and purification of xylitol and further reduces the production cost of xylitol.

[0036] (2) The XR gene, LAD gene and LXR gene in the genetically engineered bacteria of the present invention have a wide range of sources, and different combinations have good catalytic effects.

[0037] (3) The genetically engineered bacteria of the present invention convert the byproduct L-arabinitol in the original fermentation product into xylitol after fermentation, thereby further improving the yield of xylitol production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the reaction of xylose reductase (XR), L-arabinitol-4-dehydrogenase (LAD) and L-xylulose reductase (LXR) to prepare xylitol.

[0039] Figure 2 The integrated Escherichia coli genetically engineered bacteria W3110△ptsG△ptsF△xylAB-3-XR6-LAD5-LXR1 was used to produce xylitol by fed-batch fermentation of hemicellulose hydrolysate in industrial-grade culture medium. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] The gene names involved in the present invention are explained as follows:

[0045] ptsG Gene: Glucose phosphotransferase system enzyme II component gene;

[0046] ptsF Gene: Fructose phosphotransferase system enzyme II component gene;

[0047] xylA Gene: Xylose isomerase gene;

[0048] xylB Gene: Xylulose kinase gene;

[0049] XR: xylose reductase;

[0050] LAD: L-arabinose-4-dehydrogenase;

[0051] LXR: L-xylulose reductase.

[0052] ptsG The nucleotide sequence of the gene is shown in SEQ ID NO.1. ptsF The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0053] 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, xylAB The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0054] 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.

[0055] The metabolic pathways of xylose and arabinose of the engineered bacteria co-expressing XR, LAD and LXR genes obtained in the present invention are as follows: Figure 1 shown.

[0056] Example 1 Genetically engineered Escherichia coli E.coliConstruction of W3110△ptsG△ptsF△xylAB

[0057] According to the xylose metabolic pathway and its global regulation in Escherichia coli, the present invention is a E.coli A series of engineered bacteria were constructed based on W3110, such as ptsG , ptsF , xylA ,and xylB Knockout or inactivation of ptsG Knockout of α2+ attenuated the CCR effect. ptsF Knockout of can reduce the transport of xylitol from the extracellular to the intracellular xylA and xylB The knockout of can block the downstream decomposition pathway of xylose. Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl Environ Microbiol, 2015) ptsF , ptsG , xylA and xylB Knockout of genes.

[0058] The primer sequence information used in the following examples is shown in Table 1.

[0059] Table 1 Primer list

[0060]

[0061] (1) Knockout ptsG Gene strain E.coli Construction of W3110△ptsG

[0062] 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; then ptsG--uF and ptsG-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.

[0063] 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.

[0064] 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 Eppendorf 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 twice; d) Resuspend with 100 μL of 10% glycerol. Prepare E. coli W3110 competent cells using the above electroporation competent cell preparation method, electrotransform the pCas plasmid (electroporation conditions: 2.5 kV, 200 Ω, 25μF) into the competent cells, pick a single W3110 / pCas colony in a 5 mL LB test tube containing kanamycin and 10 mM arabinose, and culture at 37°C until OD 600 It is about 0.6, and then the above method is used to prepare W3110 / pCas electroporation competent state.

[0065] Electroporation: ptsG The repair template fragment and ptsG-N20 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.

[0066] 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 neither can grow, it indicates that both pCas and ptsG-N20 plasmids have been lost, and the plasmids are obtained. E.coli W310ΔptsG strain.

[0067] (2) Knockout ptsF Gene strain E.coli Construction of W3110△ptsG△ptsF

[0068] PCR amplification: Using ptsF-uF and ptsF-dR, ptsF-dF and ptsF-dR as primers, and W3110 genome as template, amplify fragments of 500 bp each and recover on gel; then using ptsF--uF and ptsF-dR as primers, the two recovered 500 bp fragments were used as templates to amplify a repair template fragment of about 1000 bp and recover on gel.

[0069] 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.

[0070] The W3110△ptsG 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△ptsG / 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 then the W3110△ptsG / pCas competent cells were prepared using the above-mentioned method.

[0071] Electroporation: ptsF The repair template fragment and ptsF-N20 plasmid were electroporated into W3110△ptsG / 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.

[0072] Plasmid loss: Use the above method to remove the two plasmids pCas and ptsF-N20 to obtain E.coli W3110ΔptsGΔptsF strain.

[0073] (3) Knockout xylA and xylB Gene strain E.coli Construction of W3110△ptsG△ptsF△xylAB

[0074] PCR amplification: Using xylAB-uF and xylAB-dR, xylAB-dF and xylAB-dR as primers, respectively, and W3110 genome as template, amplify fragments of 500 bp each, and recover on gel; then using xylAB--uF and xylAB-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 on gel.

[0075] 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.

[0076] The W3110△ptsG△ptsF 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△ptsG△ptsF / 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 OD600 was approximately 0.6, and then the W3110△ptsG△ptsF / pCas competent cells were prepared using the above-mentioned method.

[0077] Electroporation: xylAB The repair template fragment and xylAB-N20 plasmid were electroporated into W3110△ptsG△ptsF / 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.

[0078] Plasmid loss: Use the above method to remove the two plasmids pCas and xylAB-N20 to obtain E.coli W3110ΔptsGΔptsFΔxylAB strain.

[0079] Example 2 Construction of genetically engineered bacteria expressing xylose reductase from different sources and shake flask fermentation

[0080] (1) Obtaining XR genes from different sources

[0081] According to the XR gene sequence published on NCBI, 11 wild-type or mutant XR genes from different sources were screened, and the specific gene sequences of wild-type or mutant XR from different sources were fully synthesized to obtain plasmids containing XR genes from different sources.

[0082] (2) Construction of genetically engineered bacteria containing wild-type XR genes from different sources

[0083] a) Prepare Escherichia coli according to the method in Example 1 E.coli W3110△ptsG△ptsF△xylAB electroporation competent state;

[0084] b) Place the prepared electroporation competent cells on ice for 5 min, add 2-3 μL of different XR expression plasmids for electroporation;

[0085] c) Finally, spread it evenly on an LB plate containing chloramphenicol and culture it in a 37°C incubator overnight to obtain genetically engineered bacteria containing XR plasmids from different sources: E.coli W3110△ptsG△ptsF△xylAB / XR1, W3110△ptsG△ptsF△xylAB / XR2, W3110△ptsG△ptsF△xylA B / XR3, W3110△ptsG△ptsF△xylAB / XR4, W3110△ptsG△ptsF△xylAB / XR5, W3110△ptsG△pts F△xylAB / XR6, W3110△ptsG△ptsF△xylAB / XR7, W3110△ptsG△ptsF△xylAB / XR8, W3110△pt sG△ptsF△xylAB / XR9, W3110△ptsG△ptsF△xylAB / XR10, W3110△ptsG△ptsF△xylAB / XR11.

[0086] (3) Shake flask fermentation experiments of wild-type xylose reductase from different sources

[0087] a) Pick out single clones of bacteria and place them in 5 mL LB-cm medium. Culture them at 37℃ with shaking at 220 rpm for 8 h to obtain seed solution.

[0088] b) Inoculate the culture medium into a shake flask at 2% inoculum and culture at 37°C until the OD reaches 0.6-0.8.

[0089] c) Add appropriate amounts of xylose, glucose and L-arabinose mother liquor to each shake flask fermentation medium, so that the final concentration of each substrate in the fermentation broth is: xylose 20 g / L, glucose 10 g / L, L-arabinose 2.5 g / L. After adding the substrate, culture at 30°C for 20-30 hours. After fermentation, detect the concentration of the fermentation product xylitol and L-arabinitol.

[0090] As can be seen from Table 2, the wild-type XR from different sources catalyze xylose and L-arabinose to produce xylitol and L-arabinitol with similar effects. Most XRs have poor catalytic specificity and can catalyze L-arabinose to L-arabinitol while catalyzing xylose. After 20-30 h of shake flask fermentation, some XRs (XR-1 / XR-4 / XR-6 / XR-11) completely converted xylose into xylitol, and almost all L-arabinose was converted into L-arabinitol. The sequence information and fermentation results of 11 wild-type XRs from different sources are shown in Table 2.

[0091] Table 2. Sequence information and fermentation results of wild-type xylose reductase from different sources

[0092]

[0093] Example 3 Acquisition and characterization of L-arabinitol-4-dehydrogenase genes from different sources

[0094] (1) Obtaining LAD genes from different sources

[0095] According to the LAD genes published on NCBI, 10 wild-type or mutant LAD genes from different sources were screened, and the specific gene sequences of the wild-type or mutant LAD from different sources were fully synthesized to obtain plasmids p18-LAD1, p18-LAD2, p18-LAD3, p18-LAD4, p18-LAD5, p18-LAD6, p18-LAD7, p18-LAD8, p18-LAD9, and p18-LAD10 containing LAD genes from different sources, respectively.

[0096] (2) Construction of genetically engineered bacteria containing wild-type or mutant LAD genes from different sources

[0097] Prepared according to the method in Example 1 E.coli W3110△ptsG△ptsF△xylAB was electroporated into competent cells and then electroporated into LAD expression plasmids to obtain genetically engineered bacteria containing LAD plasmids from different sources: E.coli W3110 △ptsG△ptsF△xylAB / LAD1, W3110△ptsG△ptsF△xylAB / p18-LAD2, W3110△ptsG△ptsF△xylAB / p18-LAD3, W3110△ptsG△ptsF△xylAB / p18-LAD4, W3110△ptsG△ptsF△xylAB / p18-LAD5, W3110 △ptsG△ptsF△xylAB / p18-LAD6, W3110△ptsG△ptsF△xylAB / p18-LAD7, W3110△ptsG△ptsF△xy lAB / p18-LAD8, W3110△ptsG△ptsF△xylAB / p18-LAD9, W3110△ptsG△ptsF△xylAB / p18-LAD10.

[0098] (3) Determination of LAD enzyme activity from different sources

[0099] a) Pick a single clone from each of the 10 groups of LAD engineered bacteria and inoculate it into 5 mL of LB medium containing kanamycin, and culture at 37°C with shaking at 220 rpm for 8 h to obtain the seed solution;

[0100] b) Transfer the seed solution to 50 mL of fresh LB medium containing kanamycin at a 2% inoculum volume and culture for 30-40 hours before collecting the bacterial solution. The composition of the LB medium is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl;

[0101] 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 0.2M 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 the crude LAD enzyme solution from different sources;

[0102] d) Enzyme activity reaction: Take 100 μL 1 M L-arabinitol solution, 50 μL 40 mM NAD + , 825 μL 0.1M Tris-HCl buffer in a 1.5 mL EP tube, mix well, and preheat in a 30°C metal bath for 10 min. Add 25 μL of the diluted LAD enzyme crude solution to the 1.5 mL EP tube containing the reaction solution, quickly invert it upside down several times, and transfer it to a 1 cm cuvette. Measure the OD within 1 minute (every 15 s). 340 The numerical value of .

[0103] Calculation of enzyme activity: The amount of enzyme that catalyzes the production of 1 μmol NADH per minute at 30°C is defined as 1 U; the calculation formula (Eq. 3-1) is as follows:

[0104] U⁄mL=(△A) / min×1 / ε×1 / d×Vt / Vs………………………… (Eq. 3-1)

[0105] The above formula can be simplified as (Eq. 3-2):

[0106] U⁄mL=△A×1 / 6.402×40×dilution factor of crude enzyme solution……………………(Eq. 3-2)

[0107] Among them, ΔA / min represents the change in absorbance per minute, Vt represents the total volume of the reaction solution (total reaction 1000 μL), Vs represents the sample volume (sample volume 250 μL), d represents the cuvette light path (1 cm), and ε represents the molar extinction coefficient. The sequence information and expression enzyme activity assay results of the wild-type and mutant LADs from 10 different sources are shown in Table 3.

[0108] Table 3. Sequence information and expression enzyme activity assay results of L-arabinitol-4-dehydrogenase from different sources

[0109]

[0110] Example 4 Acquisition and characterization of L-xylulose reductase genes from different sources

[0111] (1) Obtaining LXR genes from different sources

[0112] According to the LXR gene sequence published on NCBI, 11 wild-type or mutant LXR genes from different sources were screened, and the specific gene sequences of LXR from different sources were fully synthesized to obtain plasmids p18-LXR1, p18-LXR2, p18-LXR3, p18-LXR4, p18-LXR5, p18-LXR6, p18-LXR7, p18-LXR8, p18-LXR9, p18-LXR10, and p18-LXR11 containing LXR genes from different sources, respectively.

[0113] (2) Construction of genetically engineered bacteria containing LXR genes from different sources

[0114] Prepared according to the method in Example 1 E.coli W3110 △ptsG△ptsF△xylAB was electroporated into competent cells and then electroporated into LXR expression plasmids to obtain genetically engineered bacteria containing LXR plasmids from different sources: E.coli W3110△ptsG△ptsF△xylAB / p18-LXR1, W3110△ptsG△ptsF△xylAB / p18-LXR2, W3110△ptsG△ptsF△xylAB / p 18-LXR3, W3110△ptsG△ptsF△xylAB / p18-LXR4, W3110△ptsG△ptsF△xylAB / p18-LXR5, W3110△ptsG△ptsF△ xylAB / p18-LXR6, W3110△ptsG△ptsF△xylAB / p18-LXR7, W3110△ptsG△ptsF△xylAB / p18-LXR8, W3110△pts G△ptsF△xylAB / p18-LXR9, W3110△ptsG△ptsF△xylAB / p18-LXR10, W3110△ptsG△ptsF△xylAB / p18-LXR11.

[0115] (3) Determination of LXR enzyme activity from different sources

[0116] a) Pick a single clone from each of the 11 groups of LXR engineered bacteria and inoculate it into 5 mL of LB medium containing kanamycin, and culture at 37°C with shaking at 220 rpm for 8 h to obtain the seed solution;

[0117] b) Transfer the seed solution to 50 mL of fresh LB medium containing kanamycin at a 2% inoculum volume and culture for 30-40 hours before collecting the bacterial solution. The composition of the LB medium is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl;

[0118] 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 crude LXR enzyme solutions from different sources;

[0119] 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, stored at 4°C;

[0120] 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 carried out in a 30°C metal bath for 5 min and then terminated by boiling at 100°C for 10 min.

[0121] Definition of enzyme activity: The consumption of xylitol during the reaction was determined by HPLC analysis. One unit (U) of LXR activity was defined as the amount of enzyme required to consume 1 μmol of xylitol in 1 min.

[0122] The sequence information and expression enzyme activity assay results of 11 wild-type and mutant LXRs from different sources are shown in Table 4.

[0123] Table 4. Sequence information and expression enzyme activity determination results of L-xylulose reductase from different sources

[0124]

[0125] Example 5 Shake flask fermentation experiment of plasmid-based genetically engineered bacteria co-expressing XR, LAD and LXR genes from different sources

[0126] (1) Construction of recombinant plasmids for co-expressing LAD and LXR genes from different sources

[0127] LAD and LXR enzymes with 0 enzyme activity in the enzyme library were removed, and LAD5 (highest enzyme activity) and LAD8 (lowest enzyme activity) were selected from the LAD enzyme library and the LXR enzyme library, respectively, and LXR1 (highest enzyme activity) and LXR10 (lowest enzyme activity) were selected from the LXR enzyme library. Plasmid p18-LAD1 and plasmid p18-LAD8 were digested with BamHI and XbaI, respectively, and recombined into plasmids of p18-LXR1 and p18-LXR8 that had been digested with the same enzymes (BamHI and XbaI), to obtain recombinant plasmids p18-LAD5-LXR1, p18-LAD5-LXR10, p18-LAD8-LXR1 and p18-LAD8-LXR10.

[0128] (2) Construction of plasmid-based genetically engineered Escherichia coli DH5α-p18-LAD5-LXR1, DH5α-p18-LAD5-LXR10, DH5α-p18-LAD8-LXR1, and DH5α-p18-LAD8-LXR10

[0129] Take 100 μL of E. coli from -80℃ freezer E.coli DH5α competent cell suspension was thawed in an ice bath for 10 min; 2-10 μL of recombinant plasmids p18-LAD5-LXR1, p18-LAD5-LXR10, p18-LAD8-LXR1 and p18-LAD8-LXR10 were added respectively, mixed and placed on ice for 30 min; heat-shocked in a 42℃ water bath for 45 s and then quickly placed on ice for 1-2 min; 890 μL of liquid LB medium was added to the tube, mixed and revived at 37℃ for 1 h; the above bacterial solution was shaken and 100 μL was spread on an LB plate containing kanamycin and cultured at 37℃ overnight. Single clones on the plate were picked and cultured in 5 mL of LB medium with kanamycin for sequencing to obtain LAD and LXR recombinant plasmids of different sources with correct sequences.

[0130] (3) Construction of plasmid-based genetically engineered bacteria that co-express XR, LAD, and LXR from different sources

[0131] Prepared according to the method in Example 1 E.coli W3110△ptsG△ptsF△xylAB / (XR1-7,11) was electroporated into competent cells and LAD and LXR recombinant expression plasmids were electroporated to obtain plasmid-type genetically engineered bacteria that co-express any combination of XR, LAD and LXR from different sources.

[0132] (4) Shake flask fermentation experiment of genetically engineered bacteria co-expressing XR, LAD and LXR

[0133] a) Pick out the monoclonal bacteria with good growth and place them in 5 mL of LB medium with kanamycin and chloramphenicol, and culture them at 37℃ for 12 h to obtain the seed solution;

[0134] b) Inoculate the cultured seed liquid into the fermentation medium at an inoculation rate of 2%, and culture at 37°C until the OD is 0.6-0.8. Add appropriate amounts of xylose, glucose and L-arabinose mother liquor to each shake flask fermentation medium, so that the final concentration of each substrate in the fermentation broth is: xylose 20 g / L, glucose 10 g / L, L-arabinose 2.5 g / L. After adding the substrate, culture at 30°C for 96 h. After fermentation, detect the concentration of the fermentation product xylitol and L-arabinitol, and calculate the conversion rate of xylose and L-arabinitol;

[0135] As can be seen from Table 5, the efficiency of fermentation to produce xylitol and L-arabinitol is different when different XR, LAD and LXR combinations are expressed. A portion of L-arabinitol in each XR, LAD and LXR combination is converted into xylitol, which increases the yield of xylitol. Among them, the efficiency of xylitol production by genetically engineered bacteria expressed in the combination of XR-6, LAD-5 and LXR-1 is the highest. After 30 hours of fermentation, the conversion rate of xylose is 98.88%, and the conversion rate of L-arabinitol is 91.96%. With the extension of fermentation time, after 96 hours, the conversion rates of xylose and L-arabinitol exceeded 99.9%, indicating that the xylose reductase gene, L-arabinitol-4-dehydrogenase gene and L-xylulose reductase gene can be derived from any source as long as they can be functionally expressed in the host cell, without affecting the final conversion rate of xylose and L-arabinitol.

[0136] Table 5 Plasmid-based genetically engineered bacteria E.coli W3110△ptsG△ptsF△xylAB / XR / LAD-LXR shake flask fermentation results

[0137]

[0138] Example 6 Construction of multi-copy genome-integrated engineered bacteria co-expressing XR, LAD and LXR genes and shake flask fermentation

[0139] (1) Construction of the integration system of three copies of the target genes XR, LAD and LXR in the Escherichia coli genome

[0140] The CRISPR / Cas9 gene editing tool in Example 1 was used to integrate the XR, LAD and LXR genes from different sources into Escherichia coli. E.coli In the genome of W3110△ptsG△ptsF△xylAB, an integrated genetic engineering bacterium with 3 copies of XR, LAD and LXR genes was obtained. E.coliW3110△ptsG△ptsF△xylAB::3(XR-LAD-LXR).

[0141] (2) Shake flask fermentation of genetically engineered bacteria co-expressing XR, LAD and LXR

[0142] The shake flask fermentation culture experiment was the same as in Example 5. The results of the shake flask fermentation experiment of the integrated genetically engineered bacteria are shown in Table 6. As can be seen from Table 6, the efficiency of the fermentation and conversion of xylose and L-arabinitol by the integrated genetically engineered bacteria with different combinations of 3 copies of XR, LAD and LXR genes is different. Comparing the results of the shake flask fermentation of the plasmid-type expressed genetically engineered bacteria, it was found that the effect of the shake flask fermentation of the integrated genetically engineered bacteria was significantly better than that of the plasmid-type genetically engineered bacteria, and the conversion rate of xylose and L-arabinitol by the integrated genetically engineered bacteria was higher. Among them, the integrated genetically engineered bacteria expressed in combination of XR-6, LAD-5 and LXR-1 had the best effect in producing xylitol. After 30 hours of fermentation, the conversion rates of xylose and L-arabinitol exceeded 99.9%, which shows that the form of co-expressing XR, LAD and LXR genes can also be the integration of related genes into the genome of the host bacteria for co-expression.

[0143] Table 6. Integrated genetically engineered bacteria E.coli W3110△ptsG△ptsF△xylAB::3(XR-LAD-LXR) shake flask fermentation results

[0144]

[0145] Example 7 Fermentation in a fermenter with multi-copy genome-integrated engineered bacteria that co-express XR, LAD and LXR genes

[0146] According to the results of shake flask fermentation in Example 5 and Example 6, a genetically engineered bacterium G1 ( E.coli W3110△ptsG△ptsF△xylAB::3(XR6-LAD5-LXR1)) is the fermentation strain. The specific fermentation steps are:

[0147] The integrated strain was inoculated into a 15 L fermenter at 30°C for 72 h with an inoculation rate of 10%. The initial liquid volume was 6 L, the rotation speed was 400 rpm, the aeration ratio was 0.8 vvm, and the pH was 7.0 (controlled by NH3·H2O addition). During the fermentation process, the dissolved oxygen was maintained at 30% by adjusting the rotation speed. When the OD600 reached about 15 (about 8 h), an appropriate amount of concentrated hemicellulose hydrolysate was added to make the final concentration of xylose about 40 g / L. At the same time, an organic nitrogen source with a final concentration of 10 g / L was added. At about 20 h and 40 h of fermentation, 1 L of hemicellulose hydrolysate was added to make the final concentration of xylose about 60 g / L, and glucose with a final concentration of 40 g / L and an organic nitrogen source with a final concentration of 5 g / L were added. Excluding the dilution effect, the total concentration of xylose was about 160 g / L, and the total culture volume in the fermenter was 12 L. During the fermentation process, samples were taken every 8 h 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 L-arabinitol.

[0148] The other components of the fermentation medium in the fermenter are as follows: Na2HPO4 6-10 g / L, KH2PO4 3-5 g / L, NH4Cl 1-5 g / L, NaCl 0.5-2 g / L, MgSO4 0.1-0.5 g / L, and CaCl2 0.1-0.5 g / L.

[0149] Depend on Figure 2 It can be seen that after 64 h of fermentation in a 15 L fermentor, the conversion rate of the substrate xylose in the hemicellulose hydrolysate exceeded 99.9%, and no other substrates (glucose, L-arabinose) remained, no L-arabinitol was produced in the product, and the conversion rate of L-arabinitol to xylitol exceeded 99.9%, with a xylitol yield of up to 169.48 g / L.

[0150] Comparative Example 1

[0151] According to the method of Example 5, a genetically engineered bacterium co-expressing L-arabinitol-4-dehydrogenase and L-xylulose reductase genes was constructed. E.coli W3110 ΔptsGΔptsFΔxylAB / p18-LAD5-LXR1.

[0152] The shake flask fermentation experiment of genetically engineered bacteria was also carried out according to Example 5. After the fermentation culture, the concentrations of xylitol and L-arabitol of the fermentation products were detected, and the conversion rates of xylose and L-arabitol were calculated.

[0153] After 48 h of fermentation, the xylose conversion rate was 1.09% and no L-arabinitol was detected.

Claims

1. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria are genetically engineered bacteria that co-express a xylose reductase gene, an L-arabinitol-4-dehydrogenase gene, and an L-xylulose reductase gene; The xylose reductase gene is an XR gene, and the XR gene is one of NCBI accession numbers AF451326.3, KF752418.1, AF074484.1, AB002106.1, ALO17776.1, EAA34695.1, AAA99507.1, and Q9P8R5.1; The L-xylulose reductase gene is an LXR gene, and the LXR gene is one of NCBI accession numbers KAG7881166.1 and GCF00616.1; The L-arabinitol-4-dehydrogenase gene is a LAD gene, and the LAD gene is one of NCBI accession numbers EAA36547.1 and XP_002569286.1; The starting strain of the genetically engineered bacteria is Escherichia coli in which the ptsG gene, ptsF gene, xylA gene and xylB gene are knocked out or silenced; The Escherichia coli is E. coliW3110.

2. The genetically engineered bacterium according to claim 1, characterized in that: The combination of the XR gene and the LXR gene is as follows: (1)AF451326.3, KAG7881166.1; (2)AF451326.3, GCF00616.1; (3)KF752418.1, KAG7881166.1; (4)KF752418.1, GCF00616.1; (5)AF074484.1, KAG7881166.1; (6)AF074484.1, GCF00616.1; (7)AB002106.1, KAG7881166.1; (8)AB002106.1, GCF00616.1; (9)ALO17776.1, KAG7881166.1; (10)ALO17776.1, GCF00616.1; (11)EAA34695.1, KAG7881166.1; (12)EAA34695.1, GCF00616.1; (13)AAA99507.1, KAG7881166.1; (14)AAA99507.1, GCF00616.1; (15)Q9P8R5.1, KAG7881166.1; (16) Q9P8R5.1, GCF00616.

1.

3. Use of the genetically engineered bacteria according to any one of claims 1 to 2 in the biological preparation of xylitol.

4. The method for preparing a genetically engineered bacterium according to any one of claims 1 to 2, characterized in that: (1) integrating the xylose reductase gene, L-arabinitol-4-dehydrogenase gene and L-xylulose reductase gene into the genome of the starting strain; or, (2) transforming a plasmid containing a xylose reductase gene, an L-arabinitol-4-dehydrogenase gene, and an L-xylulose reductase gene into a starting strain; A genetically engineered bacterium co-expressing a xylose reductase gene, an L-arabinitol-4-dehydrogenase gene and an L-xylulose reductase gene is obtained.

5. A method for preparing xylitol, characterized in that: Hemicellulose hydrolysate, glucose and an organic nitrogen source are used as substrates, and the genetically engineered bacteria described in any one of claims 1 to 2 are used to ferment and produce xylitol; the hemicellulose hydrolysate includes xylose and L-arabinose.

6. The method according to claim 5, characterized in that: In the fermentation substrate, the organic nitrogen source is an organic nitrogen source for cell culture of animal or plant origin.

7. The method according to claim 5, characterized in that: The organic nitrogen source is one or more of peptone, yeast powder or corn steep liquor powder.

8. The method according to claim 5, characterized in that: In the fermentation substrate, the mass ratio of xylose to glucose is 5:1 to 2:

1.

9. The method according to claim 5, characterized in that: During the fermentation process, the dissolved oxygen is maintained between 0 and 50%, the pH is between 6.0 and 8.0, and the temperature is between 18 and 37°C, taking the dissolved oxygen under the initial fermentation condition after inoculation as 100%.

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