Genetic engineering modified vibrio natriticus and application thereof in production of xylitol

Abstract

The invention provides a genetic engineering modified vibrio natriureticus and an application of the genetic engineering modified vibrio natriureticus in production of xylitol. Specifically, a genetic engineering technology is utilized for the first time to introduce a heterologous gene for coding arabinose-1-dehydrogenase and a heterologous gene for coding NADPH oxidase into the vibrio natriticus, so that the modified vibrio natriticus can co-express the arabinose-1-dehydrogenase and the NADPH oxidase. Furthermore, the invention provides an application of the genetic engineering modified vibrio natriureticus disclosed by the invention in production of xylitol. L-arabinose in hemicellulose hydrolysate can be completely converted into L-arabinoic acid by utilizing the vibrio natriureticus modified by genetic engineering, so that the generation of a byproduct L-arabitol which is difficult to separate is avoided, the adverse effect of the L-arabitol is eliminated, and the production efficiency of xylitol is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a genetically engineered Vibrio natriureticus and its application in producing xylitol. Background Art

[0002] Xylitol (molecular formula C5H 12 Xylitol (X5) is a five-carbon sugar alcohol that appears as white crystals or crystalline powder. Xylitol is a sweetener with the sweetness equivalent to sucrose and the caloric value equivalent to glucose. In 2004, it was named one of the 12 most promising biomass-based chemicals by the U.S. Department of Energy. The global xylitol market has enormous potential, with its size showing an overall annual growth trend, rising from US$800 million in 2017 to US$993 million in 2021. It is projected to reach US$1.11 billion in 2023.

[0003] Currently, chemical methods are widely used in industry to produce xylitol. This involves separating pure xylose from hemicellulose hydrolysate through a complex separation process, followed by high-pressure hydrogenation to reduce the xylose to xylitol. Due to the relatively complex and costly production process, the price of xylitol has remained high for a long time. Biological methods for producing xylitol offer several advantages over chemical methods, including mild catalytic conditions, safe operation, and low energy consumption. Consequently, these methods have garnered industry attention in recent years and are expected to become alternatives to chemical methods. Biological methods utilize enzymes or microbial cells to directly convert xylose from hemicellulose hydrolysate into xylitol, which is then separated to produce the xylitol product.

[0004] Xylose reductase is a key enzyme in the existing bioprocess for producing xylitol. Wild-type xylose reductase typically has poor specificity. When bioprocessing hemicellulose hydrolysate, xylose reductase catalyzes the reduction of xylose to xylitol while also reducing arabinose in the hemicellulose hydrolysate to arabitol. However, arabitol's structure and physicochemical properties are very similar to those of xylitol, resulting in the byproduct arabitol affecting the separation and purification of xylitol. In recent years, numerous studies have sought to improve the specificity of xylose reductase by exploring novel xylose reductases and molecularly modifying them. While these studies have achieved some success, they still cannot completely eliminate the catalytic activity of xylose reductase towards arabinose during the production of xylitol from hemicellulose hydrolysate. In other words, existing technologies are still unable to completely eliminate the impact of the difficult-to-separate byproduct arabitol. Summary of the Invention

[0005] To address the problems of the prior art, the present invention provides a genetically engineered Vibrio natriuresis. Specifically, the present invention utilizes genetic engineering techniques for the first time to introduce heterologous genes encoding arabinose-1-dehydrogenase and NADPH oxidase into Vibrio natriuresis, enabling the modified Vibrio natriuresis to co-express arabinose-1-dehydrogenase and NADPH oxidase. The genetically engineered Vibrio natriuresis of the present invention can completely convert L-arabinose in hemicellulose hydrolysate into L-arabinonic acid, thereby avoiding the production of the difficult-to-separate byproduct L-arabinitol, eliminating the adverse effects of L-arabinitol, and improving the production efficiency of xylitol. Furthermore, the present invention further provides the use of the genetically engineered Vibrio natriuresis of the present invention in the production of xylitol.

[0006] In this regard, the present invention includes but is not limited to the following:

[0007] In one aspect, the present invention provides an engineered bacterium, which is obtained by introducing a heterologous gene encoding arabinose-1-dehydrogenase and a heterologous gene encoding NADPH oxidase into a host cell, so as to co-express arabinose-1-dehydrogenase and NADPH oxidase in the host cell, wherein the host cell is Vibrio natriuresis.

[0008] In one aspect, the GenBank accession number of the arabinose-1-dehydrogenase of the present invention is BAD95974.1, WP_004041122.1, NP_353239.1, WP_004083016.1, AAD35400.1, WP_004083078.1, AIS92476.1, AIT75903.1 or AIS92478.1; preferably, the arabinose-1-dehydrogenase The GenBank accession number of the enzyme is BAD95974.1, WP_004083016.1, AIT75903.1 or AIS92478.1; more preferably, the GenBank accession number of the arabinose-1-dehydrogenase is WP_004083016.1 or AIT75903.1; most preferably, the GenBank accession number of the arabinose-1-dehydrogenase is WP_004083016.1.

[0009] In one aspect, the GenBank accession number of the arabinose-1-dehydrogenase of the present invention is BAD95974.1, WP_004083016.1, AIT75903.1, or AIS92478.1. Preferably, in one aspect, the GenBank accession number of the arabinose-1-dehydrogenase of the present invention is WP_004083016.1 or AIT75903.1. More preferably, in one aspect, the GenBank accession number of the arabinose-1-dehydrogenase of the present invention is WP_004083016.1.

[0010] In one aspect, the GenBank accession number of the NADPH oxidase of the present invention is AXI93343.1, BAB19268.1, ABJ69145.1, BDR59011.1, QNN75144.1 or QFR22240.1; preferably, the GenBank accession number of the NADPH oxidase is BAB19268.1, QNN75144.1, AXI93343.1 or QFR22240.1; more preferably, the GenBank accession number of the NADPH oxidase is AXI93343.1 or QFR22240.1; most preferably, the GenBank accession number of the NADPH oxidase is QFR22240.1.

[0011] In one aspect, the GenBank accession number of the NADPH oxidase of the present invention is BAB19268.1, QNN75144.1, AXI93343.1, or QFR22240.1. Preferably, in one aspect, the GenBank accession number of the NADPH oxidase of the present invention is AXI93343.1 or QFR22240.1. More preferably, in one aspect, the GenBank accession number of the NADPH oxidase of the present invention is QFR22240.1.

[0012] In one aspect, the heterologous gene encoding arabinose-1-dehydrogenase and / or the heterologous gene encoding NADPH oxidase of the present invention is further codon-optimized according to the preference of the expression vector or host cell.

[0013] In one aspect, the heterologous gene sequence encoding arabinose-1-dehydrogenase of the present invention is shown as SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, and / or the heterologous gene sequence encoding NADPH oxidase is shown as SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

[0014] In one aspect, the heterologous gene sequence encoding arabinose-1-dehydrogenase of the present invention is shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13. Preferably, in one aspect, the heterologous gene sequence encoding arabinose-1-dehydrogenase of the present invention is shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 13; more preferably, in one aspect, the heterologous gene sequence encoding arabinose-1-dehydrogenase of the present invention is shown in SEQ ID NO: 1 or SEQ ID NO: 12.

[0015] In one aspect, the heterologous gene sequence encoding NADPH oxidase of the present invention is shown as SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18. Preferably, in one aspect, the heterologous gene sequence encoding NADPH oxidase of the present invention is shown as SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 18. More preferably, in one aspect, the heterologous gene sequence encoding NADPH oxidase of the present invention is shown as SEQ ID NO: 3 or SEQ ID NO: 14.

[0016] In one aspect, the amino acid sequence of the arabinose-1-dehydrogenase of the present invention is shown in SEQ ID NO: 2.

[0017] In one aspect, the amino acid sequence of the NADPH oxidase of the present invention is shown in SEQ ID NO:4.

[0018] In one aspect, the amino acid sequence of the arabinose-1-dehydrogenase of the present invention is shown as SEQ ID NO: 2, and / or the amino acid sequence of the NADPH oxidase of the present invention is shown as SEQ ID NO: 4.

[0019] In one aspect, the heterologous gene sequence encoding arabinose-1-dehydrogenase of the present invention is shown as SEQ ID NO: 1.

[0020] In one aspect, the heterologous gene sequence encoding NADPH oxidase of the present invention is shown as SEQ ID NO: 3.

[0021] In one aspect, the heterologous gene sequence encoding arabinose-1-dehydrogenase of the present invention is shown as SEQ ID NO: 1, and the heterologous gene sequence encoding NADPH oxidase is shown as SEQ ID NO: 3.

[0022] In one aspect, the heterologous gene encoding arabinose-1-dehydrogenase and / or the heterologous gene encoding NADPH oxidase of the present invention is expressed in a host cell in the form of a recombinant plasmid and / or inserted into the host cell genome in the form of an expression cassette for expression. Preferably, the recombinant plasmid is a recombinant pET-28a plasmid.

[0023] In one aspect, the heterologous gene encoding arabinose-1-dehydrogenase and / or the heterologous gene encoding NADPH oxidase of the present invention is expressed in a host cell in the form of a recombinant plasmid.

[0024] In one aspect, the heterologous gene encoding arabinose-1-dehydrogenase and / or the heterologous gene encoding NADPH oxidase of the present invention is expressed in a host cell in the form of a recombinant pET-28a plasmid.

[0025] In one aspect, the heterologous gene encoding arabinose-1-dehydrogenase and / or the heterologous gene encoding NADPH oxidase of the present invention is inserted into the host cell genome in the form of an expression cassette for expression.

[0026] In one aspect, the copy number of the heterologous gene encoding arabinose-1-dehydrogenase of the present invention is 2 or 3, and / or the copy number of the heterologous gene encoding NADPH oxidase is 10.

[0027] In one aspect, the heterologous gene sequence encoding arabinose-1-dehydrogenase of the present invention is as shown in SEQ ID NO: 1, and its copy number is 2 or 3. Preferably, in one aspect, the heterologous gene sequence encoding arabinose-1-dehydrogenase of the present invention is as shown in SEQ ID NO: 1, and its copy number is 2.

[0028] In one aspect, the heterologous gene sequence encoding NADPH oxidase of the present invention is shown in SEQ ID NO: 3, and its copy number is 10.

[0029] In one aspect, the heterologous gene sequence encoding arabinose-1-dehydrogenase of the present invention is shown as SEQ ID NO: 1, and its copy number is 2 or 3; the heterologous gene sequence encoding NADPH oxidase is shown as SEQ ID NO: 3, and its copy number is 10.

[0030] In one aspect, the heterologous gene sequence encoding arabinose-1-dehydrogenase of the present invention is shown as SEQ ID NO: 1, and its copy number is 2; the heterologous gene sequence encoding NADPH oxidase is shown as SEQ ID NO: 3, and its copy number is 10.

[0031] In one aspect, the heterologous gene encoding arabinose-1-dehydrogenase and / or the heterologous gene encoding NADPH oxidase of the present invention is expressed under the control of a T7 promoter.

[0032] In one aspect, the present invention integrates a T7 RNA polymerase expression cassette into the genome of the host cell; preferably, the T7 RNA polymerase expression cassette is integrated into the dns gene locus of the host cell. In one aspect, the present invention integrates a T7 RNA polymerase expression cassette into the dns gene locus of the host cell.

[0033] In another aspect, the present invention provides a two-stage biological method for producing xylitol using hemicellulose hydrolysate, comprising:

[0034] (1) using a hemicellulose hydrolyzate containing xylose and L-arabinose as a raw material, and performing a first stage fermentation culture in a fermentation medium inoculated with the engineered bacteria according to claim 1, until no L-arabinose remains in the fermentation liquid, thereby obtaining a fermentation reaction liquid I containing L-arabinonic acid;

[0035] (2) After adding the engineered bacteria co-expressing xylose reductase and glucose dehydrogenase to the fermentation reaction liquid I, the second stage of fermentation culture is continued to obtain a fermentation product containing xylitol.

[0036] In one aspect, the engineered bacteria co-expressing xylose reductase and glucose dehydrogenase described in the present invention are natriuretic Vibrio co-expressing xylose reductase and glucose dehydrogenase; preferably, it is the natriuretic Vibrio inserted with the xylose reductase (XR) gene and the glucose dehydrogenase (GDH) gene as described in patent application CN2025105923567; more preferably, it is the natriuretic Vibrio integrated with 5 copies of XR gene and 2 copies of GDH gene in the genome as described in patent application CN2025105923567, wherein the accession number of XR gene in the NCBI library is ALO17776.1 and the accession number of GDH gene in the NCBI library is WP_013055546.1, WP_012369122.1 or 8W0O_A.

[0037] In one aspect, the engineered bacteria co-expressing xylose reductase and glucose dehydrogenase according to the present invention are Vibrio natriuresis in which 5 copies of the XR gene and 2 copies of the GDH gene are integrated into the genome, wherein the accession number of the XR gene in the NCBI library is ALO17776.1 and the accession number of the GDH gene in the NCBI library is WP_013055546.1, WP_012369122.1 or 8W0O_A.

[0038] In one aspect, the engineered bacteria co-expressing xylose reductase and glucose dehydrogenase of the present invention is Vibrio natriuresis with 5 copies of XR gene and 2 copies of GDH gene integrated into the genome, wherein the accession number of XR gene in the NCBI library is ALO17776.1 and the accession number of GDH gene in the NCBI library is WP_012369122.1.

[0039] In one aspect, in the method of the present invention, the hemicellulose hydrolysate further contains glucose.

[0040] In one aspect, in the method of the present invention, the hemicellulose hydrolyzate contains xylose with a mass fraction of 28%-35%, L-arabinose with a mass fraction of 3.7-4.7%, and glucose with a mass fraction of 3%-4%.

[0041] In one aspect, in the method of the present invention, in addition to the glucose in the hemicellulose hydrolysate, glucose is additionally added, and the added amount is 31%-40%.

[0042] In one aspect, in the method of the present invention, the reaction system is 200-500 g of hemicellulose hydrolyzate, the pH is adjusted to 6.0-8.0 with ammonia water, the reaction temperature is 25-35°C, the first stage reaction time is 1.5-2.5 h, and the second stage reaction time is 3.5-5.5 h.

[0043] In one aspect, in the method of the present invention, the amount of the first-stage whole-cell catalyst added is 1-5 g / L of cell wet weight, and the amount of the second-stage whole-cell catalyst added is 1-5 g / L of cell wet weight.

[0044] In one aspect, in the methods of the invention, NADP + The addition amount is 0.04%-0.10%.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The two-stage whole-cell catalytic production of xylitol using the genetically engineered Vibrio natriuresis described in the present invention can efficiently convert xylose in the hemicellulose hydrolysate into xylitol, and efficiently convert glucose and arabinose into easily separable byproducts gluconic acid and arabinonic acid, thereby avoiding the production of the difficult-to-separate byproduct arabinitol.

[0047] (2) The present invention is the first to apply arabinose-1-dehydrogenase and NADPH oxidase to the preparation of xylitol. Arabinose-1-dehydrogenase and NADPH oxidase from different sources have good catalytic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is shown that plasmid-type Vibrio natriuresis whole cells catalyze the conversion of L-arabinose to L-arabinonic acid.

[0049] Figure 2 A schematic diagram of the two-stage whole-cell catalytic production of xylitol is shown.

[0050] Figure 3 A two-stage whole-cell catalytic production of xylitol is shown. DETAILED DESCRIPTION

[0051] In order to make those skilled in the art better understand the present invention, the technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is only a part of the embodiments of the present invention, rather than all embodiments.

[0052] 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.

[0053] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled 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 detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0054] Detection of xylitol, glucose, arabinose, arabitol, and xylose: HPLC determination with ultrapure water as the mobile phase, an Agilent Hi-Plex Ca column (7.7 mm × 300 mm), a column temperature of 85°C, and a flow rate of 0.6 mL / min.

[0055] Detection of gluconic acid and arabinonic acid: HPLC determination using a C18 column (250 mm × 4.6 mm 5 pm) at a detection wavelength of 210 nm; the mobile phase was 10 mM K2HPO3-10 mM tetrabutylammonium hydrogen sulfate (pH 7.2): methanol (95:5 v / v); the injection volume was 20 μL; and the flow rate was 0.7 mL / min.

[0056] In the following examples, the activity of arabinose-1-dehydrogenase was determined as follows: 1 mL of the reaction system included: 100 μL of 1M L-arabinose stock solution (final concentration 100 mM), 0.4 M NADP + Prepare 50 μL of stock solution (final concentration 20 mM), 750 μL of PBS buffer, and 100 μL of cell lysis buffer. Preheat the reaction mixture and cell lysis buffer in a metal bath at 30°C for 10 minutes, then rapidly mix them and monitor the absorbance change at 340 nm using a spectrophotometer. Enzyme activity units are defined as the amount of enzyme required to generate 1 μM NADPH per minute under standard reaction conditions. AraDH activity is calculated using the following formula: Enzyme activity (U / mL) = (ΔA / min) × (1 / ε) × (1 / d) × (Vt / Vs) × X, where ΔA / min represents the absorbance change per minute; ε represents the molar extinction coefficient, which is 6.402 mL / (μmol*cm); d represents the cuvette optical path length, which is 1 cm; Vt represents the total reaction volume, which is 1000 μL; Vs represents the cell lysis buffer volume, which is 100 μL; and X represents the dilution factor.

[0057] In the following examples, NADPH oxidase (NOX) activity was determined as follows: a 1 mL reaction system included: 50 μL of 0.4 M NADPH stock solution (final concentration 20 mM), 850 μL of PBS buffer, and 100 μL of cell lysis solution. The reaction solution and cell lysis solution were preheated in a metal bath at 30°C for 10 minutes, then rapidly mixed and the absorbance at 340 nm was monitored by a spectrophotometer. Activity unit definition: 1 μM NADP is generated per minute under standard reaction conditions. +The required amount of enzyme. NOX enzyme activity is calculated using the following formula: enzyme activity (U / mL) = (△A / min) × (1 / ε) × (1 / d) × (Vt / Vs) × X, where △A / min represents the change in absorbance per minute; ε represents the molar extinction coefficient, which is 6.402 mL / (μmol*cm); d represents the cuvette optical path length, which is 1 cm; Vt represents the total reaction volume, which is 1000 μL; Vs represents the volume of the cell lysis solution, which is 100 μL; and X represents the dilution factor.

[0058] In the following examples, the final concentration of kanamycin in the culture medium was 200 μg / mL.

[0059] Example 1 Expression of arabinose-1-dehydrogenase in Vibrio natriuresis

[0060] The present invention screened 10 arabinose-1-dehydrogenase (AraDH) enzymes from different sources (see Table 1). These enzyme gene sequences were codon-optimized and fully synthesized in E. coli (see the sequence listing for the gene sequences). These enzymes were then constructed on the pET-28a plasmid (pET-28a plasmid was purchased from Shanghai Qincheng Biotechnology Co., Ltd., catalog number QCP0023; primers required for the construction process are shown in Table 2). Ten plasmids were constructed. VnDX is a strain of Vibrio natriuresis with a T7 RNA polymerase expression cassette integrated into the dns locus. The 10 plasmids were electroporated into VnDX respectively by electroporation to obtain 10 strains of Vibrio natriuresis carrying AraDH expression plasmids: VnDX / pET-28a-AraDH1, VnDX / pET-28a-AraDH2, VnDX / pET-28a-AraDH3, VnDX / pET-28a-AraDH4, VnDX / pET-28a-AraDH5, VnDX / pET-28a-AraDH6, VnDX / pET-28a-AraDH7, VnDX / pET-28a-AraDH8, VnDX / pET-28a-AraDH9 and VnDX / pET-28a-AraDH10.

[0061] Activate 10 strains, including VnDX / pET-28a-AraDH1, by streaking on solid plates. Pick a single colony and inoculate it into a 5 ml tube of LB medium containing kanamycin and culture at 30°C and 220 rpm for 6-8 hours. Take 0.5 mL of the overnight bacterial culture and inoculate it into 50 mL of fermentation medium containing kanamycin (12 g / L peptone, 24 g / L yeast powder, 15 g / L NaCl, 0.5% (v / v) glycerol, 2.31 g / L KH2PO4, 12.5 g / L K2HPO4), and culture it on a shaker at 30°C and 220 rpm for 2 hours. Then, add IPTG to a final concentration of 0.3 mM for induction, cool to 28°C, and culture it on a shaker at 220 rpm for 9 hours.

[0062] 4 mL of bacterial liquid was taken from each shake flask of the above fermentation, 4 mL of phosphate buffer (pH 7.5) was added, and the bacteria were lysed with an ultrasonic cell disruptor (operation 3s, pause 7s, 30 times) to obtain a crude enzyme solution. The enzyme activity of AraDH was determined by ultraviolet spectrophotometry (Table 1). As can be seen from the results in Table 1, the enzyme activities of different AraDHs in Vibrio natrii varied greatly, among which the AraDH5 unit fermentation liquid from Thermotogamaritima had the highest enzyme activity, reaching 23.78 U / ml, followed by AraDH9. Moreover, the 10 AraDHs excavated by the present invention have no catalytic activity for the isomer substrate xylose, and will not convert xylose in the hemicellulose hydrolyzate into xylonic acid. Therefore, AraDH5 was selected for further study.

[0063] Table 1 Results of enzyme activity assays of AraDH from different sources recombinantly expressed in Vibrio natriuresis

[0064]

[0065]

[0066] Table 2 Primers required for constructing AraDH expression plasmid

[0067] Primer Name Sequence (5’-3’) SEQ ID No. pET-28a-F ctcgagcaccaccaccacca SEQ ID NO:19 pET-28a-R ccatggtatatctccttcttaaagttaaacaaaattatttc SEQ ID NO:20 AraDH1-F ggagatataccatggatgagtgatcaggtttcgctgg SEQ ID NO:21 AraDH1-R gtggtggtgctcgagtcagcggccgaacgcgtcgg SEQ ID NO:22 AraDH2-F ggagatataccatggATGGCCCGCATCGCAGTGAC SEQ ID NO:23 AraDH2-R gtggtggtgctcgagTTACTCCACAACGGTAGCAGAGT SEQ ID NO:24 AraDH3-F ggagatataccatggatgaaggaaatcggtcatttcattaatgg SEQ ID NO:25 AraDH3-R gtggtggtgctcgagttatttcatcgtcggcatgacgaat SEQ ID NO:26 AraDH4-F ggagatataccatggATGAAAGAGGTGTTCGACCTGC SEQ ID NO:27 AraDH4-R gtggtggtgctcgagTTAGTTGGCGGTCCAACCGC SEQ ID NO:28 AraDH5-F [[ID=۳۷]]ggagatataccatggATGAATATCCTGGAAAAACTGTTCTCTCTG SEQ ID NO:29 AraDH5-R gtggtggtgctcgagTTACCATACCGTGTAACCACCAT SEQ ID NO:30 AraDH6-F ggagatataccatggATGAAACCACTGCGTAAAATTCGTC SEQ ID NO:31 AraDH6-R gtggtggtgctcgagTTACAGCAGGGAAGAAACAAAAACTTTG SEQ ID NO:32 AraDH7-F ggagatataccatggATGAATTTTCAGGGTAAAGTAGTACTGATCAC SEQ ID NO:33 AraDH7-R gtggtggtgctcgagTTAGACCGCGGTGGAGCCGC SEQ ID NO:34 AraDH8-F ggagatataccatggATGTCTGCTTTGAGAGTTGCTATTGT SEQ ID NO:35 AraDH8-R gtggtggtgctcgagTCAATCTTCGAATGGTTCAACAAAATTTCTTC SEQ ID NO:36 AraDH9-F ggagatataccatggATGTCTCCAATTAACTTGGCTATTGTTG SEQ ID NO:37 AraDH9-R gtggtggtgctcgagTCAATCATGGAAAGCATCAACAAATTTTCT SEQ ID NO:38 AraDH10-F ggagatataccatggATGCAACCTATTAGATTGGGTTTGGT SEQ ID NO:39 AraDH10-R gtggtggtgctcgagTCAATCATAAAAAGCTTCAACAGAAACTCTT SEQ ID NO:40

[0068] Example 2 Expression of NADPH Oxidase in Vibrio natriuresis

[0069] The present invention screened out six NADPH oxidases (NOX) from different sources (see Table 3). These enzyme gene sequences were codon-optimized and fully synthesized in Escherichia coli (see the sequence listing for the gene sequences). These enzymes were then constructed on the pET-28a plasmid (primers required for the construction process are shown in Table 4), resulting in six plasmids. VnDX is a strain of Vibrio natriuresis with a T7 RNA polymerase expression cassette integrated into the dns locus. The six plasmids were electroporated into VnDX to obtain six strains of Vibrio natriuresis carrying the NOX expression plasmid: VnDX / pET-28a-NOX1, VnDX / pET-28a-NOX2, VnDX / pET-28a-NOX3, VnDX / pET-28a-NOX4, VnDX / pET-28a-NOX5, and VnDX / pET-28a-NOX6.

[0070] Solid plate streaking activated the aforementioned six strains, including VnDX / pET-28a-NOX1. Pick a single colony and inoculate it into a 5mL test tube of LB medium containing kanamycin, and culture it at 30°C and 220rpm for 6-8h. Take 0.5mL of the overnight bacterial solution and inoculate it into 50mL fermentation medium containing kanamycin (12g / L peptone, 24g / L yeast powder, 15g / L NaCl, 0.5% (v / v) glycerol, 2.31g / L KH2PO4, 12.5g / L K2HPO4), and culture it at 30°C and 220rpm for 2 hours. Then add IPTG to a final concentration of 0.3mM for induction, cool it to 28°C, and culture it at 220rpm for 9 hours.

[0071] 4 mL of bacterial culture was taken from each shake flask of the above fermentation, 4 mL of phosphate buffer (pH 7.5) was added, and the cells were lysed using an ultrasonic cell disruptor (operation 3s, pause 7s, 30 times) to obtain a crude enzyme solution. NOX enzyme activity was measured using a UV spectrophotometer (Table 3). As shown in Table 3, the enzyme activity of different NOX species in Vibrio natriuresis was three orders of magnitude lower than that of AraDH, likely due to the lack of oxygen during the activity measurement. Among the six NOX species, NOX6 had the highest enzyme activity, reaching 98.8 U / L. Therefore, NOX6 was selected for further study.

[0072] Table 3 Results of enzyme activity assays of NOX from different sources expressed by recombinant Vibrio natriuresis

[0073] Source GenBank Enzyme activity U / L NOX1 Lacticaseibacillus rhamnosus GG AXI93343.1 78.56 NOX2 [[ID=..]] ​ 44.88 ​ ​ ​ 9.71 ​ ​ ​ 20.77 ​ ​ ​ 45.32 ​ ​ ​ 98.80

[0074] Table 4 Primers required for constructing NOX expression plasmids

[0075] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ SEQ ID NO:46 NOX3-F ggagatataccatggATGAAAATTCTGGTCATTGGCGC SEQ ID NO:47 NOX3-R gtggtggtgctcgagTTAAGCCACCGGGGTCTTCT SEQ ID NO:48 NOX4-F ggagatataccatggATGAAAGTTGCAGTGATTGGTTGC SEQ ID NO:49 NOX4-R gtggtggtgctcgagTTATTTGCTAGAAGACTTTGCGCAT SEQ ID NO:50 NOX5-F ggagatataccatggATGAAAGTCGCAGTTATCGGTTG SEQ ID NO:51 NOX5-R gtggtggtgctcgagTTACTCCGCTGCAGCAGCCAT SEQ ID NO:52 NOX6-F ggagatataccatggATGAAAATCATCGTTATTGGCGCAAC SEQ ID NO:53 NOX6-R gtggtggtgctcgagTTAAATTGCCGGTTCCGCCT SEQ ID NO:54

[0076] Example 3 Co-expression of AraDH5 and NOX6 in Vibrio natriuresis

[0077] AraDH5 and NOX6 were cloned into two multiple cloning sites of the pETDuet-1 plasmid (purchased from Shanghai Zeye Biotechnology Co., Ltd., product number ZY1237) to construct the pETDuet-AraDH5-NOX6 plasmid (primers required for construction are shown in Table 5), which was then electroporated into Vibrio natriuresis VnDX to obtain the strain VnDX / pETDuet-AraDH5-NOX6. The AraDH5 enzyme activity was determined according to the method of Example 1, and the NOX6 enzyme activity was determined according to the method of Example 2. The results are shown in Table 6. When the two genes were co-expressed in Vibrio natriuresis, the enzyme activity did not decrease compared to when expressed alone. However, the enzyme activities of AraDH and NOX differed greatly. Therefore, the next step was to narrow the gap between the two enzyme activities by changing the copy number of the genes.

[0078] Table 5 Primers required for constructing pETDuet-AraDH5-NOX6

[0079]

[0080]

[0081] Table 6 Enzyme activities of Vibrio natriuresis co-expressing AraDH5 and NOX6

[0082] Name Enzyme activity AraDH5 20.11 U / mL NOX6 103.86 U / L

[0083] Example 4 Plasmid-type Vibrio natriuresis whole cells catalyze the conversion of L-arabinose into L-arabinonic acid

[0084] The strain VnDX / pETDuet-AraDH5-NOX6 was cultured in a shake flask according to the method of Example 1 and the cells were collected by centrifugation. A 200 mL whole-cell catalytic reaction system was prepared, wherein the initial concentration of L-arabinose was 150 mM, NADP + The initial concentration was 0.6 mM, and the genetically engineered bacterial cells were 5 g. The whole-cell catalytic reaction was carried out at 30°C under oxygen conditions. The pH of the reaction system was controlled to maintain at 7.5 with ammonia water. Samples were taken every 1 hour to detect the content of L-arabinose and L-arabinonic acid. The experimental results are as follows: Figure 1 shown.

[0085] Depend on Figure 1 It can be seen that when the reaction was carried out for 8 hours, 150mM L-arabinose was not completely converted into L-arabinonic acid. The final L-arabinose concentration was 91mM, the L-arabinonic acid concentration was 58mM, and the L-arabinose conversion rate was only 39.33%. This shows that the NOX enzyme activity in this system is much lower than that of AraDH, resulting in the conversion of NADP +The supply of NOx is insufficient, so the conversion rate is low. This result further suggests that it is necessary to improve NOx activity.

[0086] Example 5 Multi-copy integration of NOX and AraDH into the genome

[0087] Because NOX enzyme activity is much lower than that of AraDH, the present invention utilizes the NT-CRISPR method disclosed in the literature (see Daniel Stukenberg et al, NT-CRISPR, combining natural transformation and CRISPR-Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens, Commun Biol, (2022) 5:265, DOI:10.1038 / s42003-022-03150-0) to integrate NOX into the genome in multiple copies. Table 7 shows the primers required for genomic multi-copy integration. Vibrio natriegens with 1-10 copies of NOX integrated were obtained using NT-CRISPR. The strains were named S1 to S10, and enzyme activity was tested according to the method in Example 1. The results are shown in Table 8. As shown in Table 8, NOX enzyme activity continued to increase with increasing NOX copy number. The enzyme activity of 10 copies of NOX reached 289 U / L, which is 2.8 times that of the plasmid type.

[0088] Using S10 as the starting strain, strains S11, S12, and S13 were constructed by integrating 1 to 3 copies of AraDH5 into the S10 genome using the same NT-CRISPR method. A 200 mL catalytic conversion experiment was performed using the reaction system described in Example 4. The L-arabinose conversion rates are shown in Table 9. As shown in Table 9, under oxygen flow, the strain with 10 integrated copies of NOX and co-expressed with 2 copies of AraDH (S12 strain) was able to completely convert 150 mM L-arabinose.

[0089] Table 7 Primers required for multi-copy integration of NOX and AraDH genome

[0090]

[0091]

[0092] Table 8 Integrated NOX enzyme activity

[0093]

[0094]

[0095] Table 9 Enzyme activities of integrated AraDH5 and NOX6

[0096]

[0097] Example 6 Two-stage whole-cell catalytic preparation of xylitol

[0098] Strain S12 is a natriuretic vibrio with 10 copies of the NADPH oxidase (NOX6) gene (sequence as shown in SEQ ID NO: 1) and 2 copies of the AraDH gene (AraDH5, sequence as shown in SEQ ID NO: 2) integrated into the genome, which can convert L-arabinose into L-arabinonic acid. Strain VnXY-10 is a natriuretic vibrio with 5 copies of the XR5 gene integrated into the genome and 2 copies of the GDH2 gene integrated into the genome (reference patent application 2025105923567), which can efficiently convert xylose and glucose into xylitol and gluconic acid. Therefore, the present invention constructs a two-stage whole-cell catalytic method ( Figure 2 ), strain S12 was used as the whole-cell catalyst in the first stage, and strain VnXY-10 was used as the whole-cell catalyst in the second stage. The whole-cell catalytic system was as follows: 300g corncob hydrolyzate (containing 31% xylose, 3.47% glucose, 4.3% L-arabinose, g / g), 105g glucose, NADP + 0.24g, adjust the pH to 7.5 with ammonia water, the reaction temperature is 30℃, and oxygen is passed. According to the shake flask culture method in Example 1, strains S12 and VnXY-10 are cultured separately, and whole cell catalysts are obtained by centrifugation. In the first stage, 1.6g (4g / L) of wet weight of S12 cells are added. After the arabinose in the hydrolyzate is completely consumed, the second stage of catalysis is entered, and 0.8g (2g / L) of wet weight of VnXY-10 cells are added. The experimental results are shown in the figure. Figure 3 As shown, the first stage is from 0 to 2 hours of the reaction, during which L-arabinose is completely converted into L-arabinonic acid with a mass fraction of 5.1%; the second stage is from 3 to 8 hours of the reaction, during which xylose is reduced to xylitol and glucose is oxidized to gluconic acid, ultimately obtaining 31% xylitol and 41% gluconic acid.

[0099] Sequence Listing

[0100] SEQ ID NO:1 AraDH5 gene sequence:

[0101] ATGAATATTCTGGAAAAACTGTTTAGCCTGAAACGTAAAGTGGCGCTGGTTACCGGCG

[0102] GCGGCCAGGGCATCGGCAAAGCGATCGCGCAGGCCCTGGCGGCGGCGGGCGCGGCCG

[0103] TGCTGATTATGGATATCAATGAAGAAACGGCCCGTCGTACCGTGGAAGAAATCAAAG

[0104] AAAAAGGCGGTGAGGCGGATTTTTATGTGGGCGATGTGACCAAAGAAGAAGATTGCT

[0105] TTGGCGCGGTGAAAAAAGCCCTGGATCGCTGGGGCAAACTGGATATTGGCGTGAACA

[0106] ATGCCGGCATTGGCGATTGGTGCGAAGCGGAAAACTATCCGGTGGAAAAGTGGAAAA

[0107] AAGTGATTGATGTGAACCTGGTGGGCGTGTTTCTGAGCGCGAAAGCGGAATTTCATGC

[0108] GATGAAAGAACGCAAATATGGCAAAATTATTAATATTGCGAGCATGAGCGGCCATAT

[0109] TGTGAATAAACCGCAGAAACAGACCGCGTATAATGCCAGCAAAGCGGGCGTGATCCA

[0110] TCTGACCCGTAGCCTGGCGGCCGAATGGGCGCCGTATGGCATTCGTGTGAATTCTATT

[0111] AGCCCGGGCTATATTCGTACCCCGCTGATTGAAAGTCCGAATGTGAAAGATTTAGTGC

[0112] CGCTGTGGCTGGATATGATTCCGCTGGGCCGCCTGGGCGAAGTGGATGATCTGATCGG

[0113] CGCGGCGATTTTCCTGGCGAGCCCGGCGAGCGATTACATGACCGGCCACGATCTGGTG

[0114] ATTGATGGCGGTTATACCGTGTGGTAA

[0115] SEQ ID NO:2 Amino acid sequence of AraDH5:

[0116] MNILEKLFSLKRKVALVTGGGQGIGKAIAQALAAAGAAVLIMDINEETARRTVEEIKEKGG

[0117] EADFYVGDVTKEEDCFGAVKKALDRWGKLDIGVNNAGIGDWCEAENYPVEKWKKVIDV

[0118] NLVGVFLSAKAEFHAMKERKYGKIINIASMSGHIVNKPQKQTAYNASKAGVIHLTRSLAAE

[0119] WAPYGIRVNSISPGYIRTPLIESPNVKDLVPLWLDMIPLGRLGEVDDLIGAAIFLASPASDYMTGHDLVIDGGYTVW*

[0120] SEQ ID NO:3 NOX6 gene sequence:

[0121] ATGAAAATTATTGTGATTGGTGCCACCCACGCGGGCACCTTTGCGACCCAGCAGATTC

[0122] TGACCGAACATCCGGATTATGCGGTGACTGTGTACGAACGTAACGATAACCTGAGCTT

[0123] CCTGAGCTGCGGCATTGCCCTGTGGGTGGGCAATAACGTGAGCGACCCGAACAAAAT

[0124] GTTCTATAGCAGCCCTGATGCACTGGCCCAGCTGGGCGCGACCATGCGTATGGAACAC

[0125] AACGTCACCGCGATTGATCCGGATGCCAAAACCATTGCGGCGACCAATTTAGTGACCG

[0126] GTGAACAGGTCACCGATCACTACGATAAACTGGTGTTCACCCCGGGCAGCGCCCCGGT

[0127] TATTCCGCCGATTCCGGGCATTCAGTCGGAACGTGTGTTCCTGTGCAAAAACTGGACC

[0128] GATGCAAAAAAACTGCACGATAATGCCCAGAGCATTAACAGTGTGGTGGTGATTGGC

[0129] GCCGGCTATATTGGCGCGGAACTGGCGGAACAGTACGCGCTGAGCGGCAAAAAAGTG

[0130] ACCCTGGTGGATGCCTTTGATCGCGTGCTGGCGAAAAATTTTGACCGCGATATTACCG

[0131] ATCGCATTGAAGCCCTGTACACCAAGCATGGCACCGCGCTGGCCCTGGGCCAGAAAG

[0132] TGACTAGCTTTACCACCGCGGCCGATCAGATTACCGTGACCACCGATCAGGCGGCGTA

[0133] CACCGCGGATATTGCGGTGCTGGCCGTGGGCTTTCGTCCGCGTACCGAACTGCTGCAG

[0134] AATCAGGTGGATATGCTGGCGAACGGCGCGGTTATTACCGATGCGTATATGCAGAGC

[0135] AGCCGCCCGGAAATTTTTGCGGCGGGCGATAGCGCCGCCGTGCATTATAATCCGACCG

[0136] GCCAGGATGATTACATTCCGCTGGCAACCAACGCGGTTCGTCAGGGCATCCTGATTGG

[0137] CAAAAATATTGCGGCCCCGACCGAACGTTACCTGGGCACCCAGGCGAGCAGCGCAGT

[0138] GGCCCTGTTCGACATGACCCTGGCGGCAAGCGGCCTGACCGCCGCAGGCGCGGCCGC

[0139] CCGTGGCATTACCGCGAACAGCGTTACCATTGAACAGAATTATCGTCCGGAATTTATG

[0140] CTGACCACCACCCCGGTGGCAGCGAGCCTGACTTGGGATCCGACCACGCACCAGGTG

[0141] CTGGGCGGCGCGTTTTACAGCGCGCATGACGTGAGCATGAGCGCGAATATGATTAGC

[0142] ATGGCGATTCAGACCAAAATGACCATTGAAACCCTGGCGATGGTGGATACCTTCTTCC

[0143] AGCCGAATTTTGATCAGCCGATTAATTGGGTGAATGCGGTTGCGATGGCCGCGGTGGA

[0144] AAAAGCGAAACAGGCGGAACCGGCGATTTAA

[0145] SEQ ID NO:4 NOX6 Amino Acid Sequence:

[0146] MKIIVIGATHAGTFATQQILTEHPDYAVTVYERNDNLSFLSCGIALWVGNNVSDPNKMFYSS

[0147] PDALAQLGATMRMEHNVTAIDPDAKTIAATNLVTGEQVTDHYDKLVFTPGSAPVIPPIPGIQ

[0148] SERVFLCKNWTDAKKLHDNAQSINSVVVIGAGYIGAELAEQYALSGKKVTLVDAFDRVLA

[0149] KNFDRDITDRIEALYTKHGTALALGQKVTSFTTAADQITVTTDQAAYTADIAVLAVGFRPRT

[0150] ELLQNQVDMLANGAVITDAYMQSSRPEIFAAGDSAAVHYNPTGQDDYIPLATNAVRQGILI

[0151] GKNIAAPTERYLGTQASSAVALFDMTLAASGLTAAGAAARGITANSVTIEQNYRPEFMLTT

[0152] TPVAASLTWDPTTHQVLGGAFYSAHDVSMSANMISMAIQTKMTIETLAMVDTFFQPNFDQPINWVNAVAMAAVEKAKQAEPAI*

[0153] SEQ ID NO:5 Arabidopsis thaliana DH1 gene sequence:

[0154] ATGAGCGATCAAGTTAGCCTGGGTGTTGTTGGTATCGGTAAAATTGCACGCGACCAGC

[0155] ACCTGCCGGCAATCGACGCCGAGCCGGGCTTCAAACTGACGGCGTGCGCATCTCGTCA

[0156] TGCGGAAGTGACGGGCGTTCGTAACTATCGTGACCTGCGCGCGCTGCTGGCAGCCGA

[0157] GCGTGAGCTGGATGCTGTGTCCCTGTGTGCTCCGCCGCAGGTCCGTTACGCACAGGCC

[0158] CGTGCTGCGCTGGAAGCAGGCAAGCACGTCATGCTGGAGAAGCCACCGGGTGCCACT

[0159] CTGGGCGAAGTAGCTGTGCTGGAAGCTCTGGCGCGTGAACGTGGTCTGACTCTGTTCG

[0160] CAACCTGGCACTCTCGTTGCGCTTCTGCGGTTGAACCGGCACGTGAATGGCTGGCCAC

[0161] CCGTGCAATTCGCGCCGTCCAAGTACGCTGGAAGGAAGACGTTCGTCGTTGGCACCCG

[0162] GGCCAACAGTGGATCTGGGAACCAGGTGGTCTGGGCGTATTCGACCCGGGTATTAAT

[0163] GCGCTGAGCATCGTGACTCGTATTCTGCCGCGTGAACTGGTTCTGCGTGAGGCTACCC

[0164] TGATCGTCCCGAGCGACGTGCAAACCCCGATCGCTGCAGAACTGGACTGCGCAGACA

[0165] CCGATGGCGTTCCGGTGCGTGCAGAATTCGATTGGCGCCACGGCCCGGTCGAACAGTG

[0166] GGAGATCGCGGTTGATACCGCTGACGGTGTACTGGCGATCAGCCGTGGTGGTGCACA

[0167] GCTGAGCATCGCGGGTGAACCGGTTGAACTGGGTCCGGAACGTGAATACCCGGCGCT

[0168] GTACGCGCATTTCCACGCACTGATCGCACGTGGCGAGTCCGATGTAGACGTTCGTCCA

[0169] CTGCGCCTGGTTGCTGACGCGTTCCTGTTCGGCCGTCGTGTTCAGACGGACGCATTCG

[0170] GTCGTTAA

[0171] SEQ ID NO:6 AraDH2 gene sequence:

[0172] ATGGCTCGTATTGCAGTAACCGGTGCGGCAGGTAACGTTGGTCGCGTGACGGTTGAAG

[0173] CACTGGCTAGCGATCACGATGTTACCCCGATCACGCACCGTGAACGCGAAGGTCTGG

[0174] ACTCCGTAATCCTGGATGTTCGCGACGAAGATGCGCTGACCGAAGCATTCGAAGGTCA

[0175] CGACATCGTTGTTCACCTGGCGGCTAACCCAAACCCGGATGCGGCGTGGGACTCCGTT

[0176] TATGAAGTGAATATCGGCGGCACCTACAACGTTTACGAGGCAGCTCTGGCAGCCGAT

[0177] ATCGATCGTCTGGTGTTCGCGAGCACCAACCATGTGCACCAGATGTATAACATCGCCG

[0178] ACGCTACCCGTCCGGAAACTCTGGCAGCGGATGCTGAGGCTGTAGGCGTTTCTGATCC

[0179] GCCGCGTCCTGACTCTTATTATGGCGTCTCTAAAGTGTTTGGTGAAGCACTGGGCAAC

[0180] TACTATGCAGATCGCCACGGTCTGGAGGTTCTGAACCTGCGTATCGGTTGGCTGCTGA

[0181] CCGCGGACGAAGTACGTGAGAAAATGGATGAAGAGGAGTCCGTTGCTCGTTACGTAC

[0182] GTGCAATGTGGCTGTCTCCGGGTGATTGCGAACAAGGTATGCGCCGTGCCGTAGAAGC

[0183] GTCTCTGCCAGATTCTCCGCTGGCAGTTAATCTGATCAGCGCGAACGACGACCGTTAC

[0184] CTGAGCCTGACTGAGACCATGCGTGCGATCGGCTACCGTCCTCGCGACAACTCCGCAA

[0185] CCGTGGTTGAGTAA

[0186] SEQ ID NO:7 AraDH3 gene sequence:

[0187] ATGCTGTGCACCAACTCTTCCGCAATGCTGCACCCATCTAAAAACCGTGGCGGTCTGC

[0188] CGATGAAGGAAATCGGCCACTTCATCAATGGCAAACACGTTCCGGGTGCGTCTGGTCG

[0189] TACCAGCAACGTGTACAACCCGGCGACTGGTGAAGTACAAGCAACTGTGGCCCTGGC

[0190] TAGCGACGCTGAACTGCGTGCGGCAGTTGAGAGCGCTAAAGCTGCGCAGCCGAAATG

[0191] GGCAGCTACCAACCCTCAGCGTCGCGCGCGTGTGTTCTTCAAGTTTGTGGAACTGCTG

[0192] AACCGCGACCTGAACGAACTGGCCGTTCTGCTGTCCAGCGAACACGGTAAAACCGTC

[0193] GAGGACAGCAAGGGTGATATCATCCGTGGTCTGGAAGTATGCGAATTCGTTTGCGGC

[0194] ATCCCGCACCTGCAGAAAGGCGAATTTACCGAAGGCGCCGGTCCGGCGATCGACATG

[0195] TATAGCATCCGTCAGCCGGTAGGTATCGGTGCAGGCATCACCCCGTTTAACTTCCCGG

[0196] GCATGATTCCGATGTGGATGTTTGCACCGGCGATCGCGTGTGGTAACGCTTTCATTCT

[0197] GAAACCGTCCGAGCGCGATCCGTCCCTGCCGATCCGCCTGGCGGAACTGATGATTGAG

[0198] GCGGGCCTGCCGGCTGGCATGCTGAACGTTGTTAACGGTGATAAGGGTGCCGTTGACG

[0199] CTATCCTGACTGACCCGGACATCGGTGCAGTTTCTTTCGTTGGCAGCACCCCAATCGC

[0200] ACGTTATGTGTATGGCACTGCTGCGATGAACGGCAAGCGTGCGCAGTGTTTTGGCGGT

[0201] GCGAAAAACCACATGATCATTATGCCTGACGCTGATCTGGACCAGGCGGTAAACGCC

[0202] CTGATGGGTGCAGGCTATGGTTCCGCCGGTGAGCGTTGCATGGCGGTTAGCGTGGCTG

[0203] TGCCGGTGGGTGAGGATACCGCAAACCGTCTGGTGGAGAAACTGATCCCGCAGATCG

[0204] AGTCCCTGCGTATCGGCCCGTATACCGACGATAAAGCAGACATGGGCCCGCTGGTTAC

[0205] TAAAGAGGCGCAGACCCGTGTGCGTGGCCTGATCGATAGCGGCGTGGAACAGGGTGC

[0206] AAAACTGCTGGTTGACGGCCGCGATTTTAAGCTGCAGGGCTATGAAGACGGTTATTTC

[0207] GTTGGTGGTTGCCTGTTTGACCACGTTACTCCGGACATGGATATCTACAAAACCGAGA

[0208] TCTTCGGTCCGGTGCTGAGCGTTGTTCGTGCCAAAAACTACGAAGATGCACTGGAACT

[0209] GCCGATGAAACATGAATACGGTAACGGCGTAGCGATTTACACCCGCGATGGTGACGC

[0210] AGCTCGTGATTTCGCGAGCCGCATCAACATTGGCATGATCGGTATCAATGTGCCGATC

[0211] CCGGTTCCGCTGGCGTACCACAGCTTCGGTGGTTGGAAAGCCTCTTCTTTCGGTGATCT

[0212] GAACCAGCACGGCACCGACAGCATTAAGTTCTGGACCAAGACCAAGACCATCACCTC

[0213] TCGTTGGCCGTCCGGTATTAAATCCGGTGCCGAATTTGTAATGCCGACGATGAAATAASEQ ID NO:8 AraDH4 gene sequence:

[0214] ATGAAGGAAGTTTTCGATCTGCGCGGCCGTGTTGCCCTGGTTACCGGTGGCTCTCGTG

[0215] GCCTGGGCTTCGGCATTGCTCAGGGTCTGGCTGAGGCCGGTTGCTCTGTTGTTGTAGC

[0216] ATCTCGCAACCTGGAGGAAGCAAGCGAAGCGGCGCAGAAGCTGACTGAGAAATACG

[0217] GCGTCGAAACCATGGCATTCCGTTGCGACGTGTCCAACTATGAAGAGGTCAAGAAAC

[0218] TGCTGGAAGCCGTTAAAGAGAAATTCGGCAAACTGGATACCGTGGTTAACGCTGCCG

[0219] GCATCAACCGTCGTCATCCTGCTGAAGAATTTCCGCTGGACGAATTCCGCCAAGTAAT

[0220] CGAGGTAAACCTGTTTGGTACCTACTACGTTTGCCGCGAAGCGTTCAGCCTGCTGCGT

[0221] GAAAGCGATAACCCGTCCATCATTAACATCGGCTCTCTGACCGTTGAGGAGGTCACGA

[0222] TGCCGAACATTTCTGCGTATGCTGCAAGCAAAGGCGGCGTGGCAAGCCTGACCAAGG

[0223] CTCTGGCCAAAGAATGGGGTCGTTATGGTATCCGTGTAAACGTAATCGCTCCAGGTTG

[0224] GTACCGCACGAAAATGACCGAAGCCGTCTTCTCTGACCCTGAGAAACTGGACTACATG

[0225] CTGAAACGTATCCCGCTGGGTCGTACCGGTGTTCCGGAAGATCTGAAAGGTGTAGCGG

[0226] TCTTCCTGGCAAGCGAGGAGGCCAAATATGTCACTGGCCAGATCATCTTTGTTGATGG

[0227] TGGCTGGACCGCAAACTAA

[0228] SEQ ID NO:9 AraDH6 gene sequence:

[0229] ATGAAACCTCTGCGCAAAATTCGTCTGGGCATCGTTGGCTGCGGTATTGCGGCTCGTG

[0230] AACTGCACCTGCCAGCCCTGAAAAATCTGTCCCATCTGTTCGAAATCACCGCTGTGAC

[0231] CTCTCGTACGCGTTCTCACGCGGAAGAATTCGCGAAAATGGTCGGTAACCCGGCGGTT

[0232] TTCGACAGCTACGAAGAGCTGCTGGAATCCGGCCTGGTTGATGCAGTGGATCTGACTC

[0233] TGCCGGTTGAACTGAACCTGCCTTTTATTGAAAAAGCGCTGCGCAAAGGCGTACACGT

[0234] TATCTGTGAAAAGCCAATCTCCACCGACGTTGAGACTGGCAAAAAAGTGGTGGAACT

[0235] GTCTGAAAAAAGCGAAAAAACTGTGTACATCGCGGAAAACTTTCGTCACGTTCCGGC

[0236] CTTCTGGAAGGCAAAGGAACTGGTTGAATCCGGTGCGATCGGTGACCCGGTGTTTATG

[0237] AACTGGCAGATCTGGGTCGGCATGGACGAAAACAACAAATACGTCCACACCGACTGG

[0238] CGCAAAAAACCAAAACACGTGGGCGGTTTCCTGTCTGATGGTGGCGTCCACCATGCA

[0239] GCGGCTATGCGTCTGATCCTGGGTGAGATTGAATGGATTTCCGCTGTTGCTAAGGACC

[0240] TGTCTCCGCTGCTGGGTGGCATGGACTTCCTGAGCTCTATCTTTGAGTTCGAAAACGG

[0241] CACCGTAGGCAACTACACCATCTCCTATAGCCTGAAGGGCAACGAGCGCTTCGAGATT

[0242] ACGGGTACCAAAGGCAAAATTTCCATCAGCTGGGATAAGATCGTCCTGAACGAGGAG

[0243] GAAATGAAAGTACCGCAGGAGAATAGCTACCAGAAAGAATTCGAGGATTTCTACCAG

[0244] GTTGTGGCGGAGGGTAAACCAAACGACCTGGGCTCTCCGGTTCAGGCCCTGAAAGAT

[0245] CTGGCTTTCATCGAAGCATGTGTGCGCAGCGCAGGTAACAAAGTTTTCGTTTCCTCCCT

[0246] GCTGTAA

[0247] SEQ ID NO:10 AraDH7 gene sequence:

[0248] ATGAATTTTCAAGGCAAAGTTGTGCTGATTACTGGCGCTGGTTCTGGTATCGGTAAAA

[0249] AGGCCGCAGTAATGTTCGCAGAGCGCGGTGCAAAAGTGGCCATCAACGACATCAGCG

[0250] AAGAAAAGGGCAAAGAAACCGTGGAGCTGATCAAAAGCATGGGTGGTGAAGCCGCA

[0251] TTCATTTTCGGTGACGTGGCTAAAGACGCTGAACAGATTGTTAAGAAAACGGTTGAAA

[0252] CCTTTGGCCGTCTGGACATCCTGGTCAACAACGCGGGTATTGTTCCGTATGGCAACAT

[0253] CGAAGAAACCTCTGAGGAAGACTTCGATAAAACCATGGCGGTTAACGTTAAGGGCCC

[0254] GTTCCTGCTGTCTAAATACGCAGTGGAGCAAATGAAAAAGCAAGGTGGCGGCGTTAT

[0255] TGTTAACGTATCCAGCGAAGCTGGTCTGATCGGTATCCCGCGTCGTTGCGTATACTCC

[0256] GTGTCCAAAGCTGCTCTGCTGGGTCTGACCCGTTCTCTGGCGGTAGATTATGTTGATTA

[0257] CGGCATCCGTGTCAACGCCGTTTGTCCGGGCACCACCCAGAGCGAGGGTCTGATGGCG

[0258] CGTGTTAAAGCTTCTCCAAACCCGGAGGAACTGCTGAAAAAGATGACCAGCCGCATT

[0259] CCGATGAAACGTCTGGGCAAGGAAGAGGAAATCGCATTCGCGATCCTGTTCGCAGCA

[0260] TGTGATGAAGCAGGTTTCATGACCGGCTCCATTATCAACATCGATGGCGGTAGCACGG

[0261] CTGTGTAA

[0262] SEQ ID NO:11 Nucleotide sequence of AraDH8 gene:

[0263] ATGTCCGCGCTGCGCGTTGCAATTGTTGGCTTTGGTAAAATCGCACGTGACCAGCATG

[0264] TGCCGGCAATCGCGGCTACCGAAGGTGTAACTCTGACGGCTGTCGCGTCCCGTAATGC

[0265] TTCCCTGCCAGGTCTGCCTCACTTCGGTAGCATTGAAGAACTGCTGCGCGACGGCCCG

[0266] GAAATCGATGCCGTTGCGCTGTGTACTCCGCCGCAGGTTCGTCGTGCTCAAGCTCAGG

[0267] CAGCGCTGGAAGCGGGTAAACACGTTATGCTGGAAAAACCGCCGGGCGCTGCGGTAA

[0268] CCGAACTGGACCCGCTGATTGCGCTGGCGAACGCACGTCAGCGTACCCTGTTTGCGAC

[0269] CTGGCACTCTCGTTACGCTCCGGCTGTAGAACCGGCTCGCACCTGGCTGTCTAGCCGC

[0270] CAGATCCGTTCCGTCCGTATCGATTGGAAAGAGGACGTTCGTGTCTGGCACCCGGGTC

[0271] AGGCGTGGATTTGGGAACCGGGTGGCCTGGGTGTGTTCGATCCGGGCATCAACGCGCT

[0272] GTCCATTCTGACGCGCATTCTGCCAGAACCGGTATTCCTGACCGAAGCGGAGCTGTCC

[0273] TTCCCTAGCAATCGCCAAGCTCCAATTGCTGCAAACCTGTCTCTGACGACCGCCTCTG

[0274] ACCTGCCGATCGCCGCTGAATTCGACTTCCGTCAGACCGGTCCGCAGTCCTGGGATAT

[0275] CCGCATCGAAACCGATGCGGGTCAGCTGAAACTGTCTCTGGGCGGCGCGCGTATGTCT

[0276] CGTGATGATCAGGTCATGATCGATGAGAAAGAACGTGAGTACCCGGGCCTGTATCGT

[0277] CGCTTTGTTGCCCTGACGAAAACTGGTGCGAGCGATGTCGATCTGGCACCGCTGCGTC

[0278] TGGTTGCGGATGCTTTCCTGCTGGGCCGTCGCAACTTCGTGGAACCGTTCGAGGATTA

[0279] A

[0280] SEQ ID NO:12 Arabidopsis thaliana DH9 gene sequence:

[0281] ATGAGCCCGATTAACCTGGCAATTGTTGGCGTCGGTAAAATTGTACGTGACCAGCATC

[0282] TGCCGTCTATCGCGAAGAATGACGATTTCAAACTGGTGGCTACCGCTTCTCGTCACGG

[0283] TACCGTTGAGGGTATTAACTCTTACACCACCATCGAAGCAATGCTGGATGCTGAACCG

[0284] TCCATTGATGCTGTCTCTCTGTGTATGCCTCCGCAGTACCGTTATGAGGCGGCGTATAA

[0285] AGCGCTGGTTGCAGGTAAGCACGTTTTCCTGGAAAAGCCACCGGGTGCGACGCTGTCT

[0286] GAAGTAGCAGACCTGGAGGCCCTGGCAGCAAAGCAGGGCGCTTCTCTGTTTGCTAGCT <00�00749>GGCACTCTCGCTATGCTCCAGCGGTAGAGGCGGCAAAGGCCTTTCTGGCATCTACCAC

[0288] CATCAAGTCCGTGCACGTGATCTGGAAAGAGGATGTCCGTCACTGGCATCCAAACCA

[0289] GGATTGGATCTGGCAGGCCGGCGGTCTGGGTGTATTCGACCCGGGTATTAACGCCCTG

[0290] AGCATCGTGACTCATATCCTGCCGCGTCCGATCTTCATCACCGAAGCCGTTCTGGAAT

[0291] TCCCGGAAAACCGCGATGCTCCTATTGCATCCGACATCCGCTTTCGCGATGCTGATGG

[0292] TCTGCCGGTTCACGCGGAGTTCGATTGGCGTCAAACTGGTAAACAGAGCTGGGACATT

[0293] GTGGCTGAAACCGCTGCTGGTCAGATGGTCCTGGCTGAGGGTGGCGCAAAACTGTCTA

[0294] TTGATGGTACCCTGACTTTTGCAGAGCCGGAACAGGAATACCCGTCTCTGTACCGTCG

[0295] CTTCGCTGAAATCATCAAAGCAGGTACCAGCGATGTAGACCTGGCGCCGCTGCGTCAT

[0296] GTTGCAGACGCGTTTATGCTGGGCCGTCGTAAATTCGTTGACGCATTCCACGACTAASEQ ID NO:1'sequence of AraDH10 gene:

[0297] ATGCAGCCTATCCGTCTGGGCCTGGTAGGTTACGGTAAAATCGCTCAGGACCAGCACG

[0298] TGCCGGCCATCAACGCTAACCCGGCTTTCACCCTGGTGTCCGTAGCAACCCAGGGCAA

[0299] It should be noted that there may be some inaccuracies in the translation due to the complexity and potential ambiguity of the original text which seems to be a nucleotide sequence. The above translation is an attempt to maintain the integrity of the original text as much as possible.ACCGTGCCCGGGCGTAGAAAACTTCCAGTCCCTGGGTGAACTGCTGGAGAACGGTCC

[0300] GCCGGTGGACGCGATTGCGTTCTGCACTCCGCCGCAGGGCCGTTTCGCACTGGTTCAG

[0301] CAAGCACTGGCTGCTGGCAAACACGTGCTGGTTGAAAAACCGCCGTGCGCCACCCTG

[0302] GGCAAAGCGGCTCTGTGGATCAAACGCGAACAGGCATCTGCTCCATGCTCTCCGTGCA

[0303] TCGCCTATGCGCCGGCTATCGCAGCAGCTCGCGACTGGCTGGCTACTCGTACCCTGCA

[0304] GAGCGTGCAGATCGACTGGAAAGAGGACGTTCGCAAATGGCACCCGGGCCAGGCGTG

[0305] GATTTGGCAACCGGGTCTGGGTGTGTTCGACCCGGGTATCAACGCGCTGTCCATTGTC

[0306] ACCCATCTGCTGCCGCTGCCGCTGTTCGTAGAATCTGCGGAGCTGCGCGTGCCAAGCA

[0307] ATTGTCAGTCCCCGATCGCCGCCTCTATCAAGATGTCTGATCCGCGTCTGCTGGATGTG

[0308] CGTGCCGAATTCGACTTTGATCACGGTCACGACGAACTGTGGTCCATCCAGATCCGTT

[0309] GCGCGGAAGGCACCCTGCGTCTGGATAATGGTGGTGCCCTGCTGTCCATCGACGGCGT

[0310] TCGTCAGACCGTTGCAGAGGAAGGTGAGTACGCGGCTGTGTACCGTCATTTCCAGCAG

[0311] CTGATCGGCGATAAGACGTCCGATGTTGATGTACAGCCGCTGCGTCTGGTAGCCGATT

[0312] CTTTTTTCGTGGGCTCTCGTGTTTCCGTTGAGGCATTCTACGATTAA

[0313] SEQ ID NO:14 NOX1 gene sequence:

[0314] ATGAAAATTCTGGTAATTGGTGCAACCCATGCAGGTACTTTCGCAACCCAGCAGATTC

[0315] TGACTGACCACCCGGACGCTGAGGTTACTGTCTATGAACGTAACAACAACCTGAGCTT

[0316] CCTGTCCTGTGGTATCGCGCTGTGGGTAGGTGACCATGTAAGCGACCCGGACAAGATG

[0317] TTCTACAGCAGCCCTGAGGCCCTGGCGAAACTGGGTGCCAATATGCAGATGGAACAC

[0318] GACGTTCTGAATATCGATCCGGCGACTAAAACCGTCGAGGTCAAAGATCTGAAAACG

[0319] GGTACCGTAACCACCGACACCTATGACAAACTGGTGTATACCACCGGCTCCACCCCGA

[0320] TTATTCCGAACATTCCGGGTATTCATGATTCTAACGTCTATCTGTGTAAGAACTGGAGC

[0321] GATGCTAAAACTCTGAAGGATCTGGCTCCGTCCATCAAAAGCGCCATTGTCATTGGCG

[0322] CCGGTTATATTGGCGCGGAACTGGCTGAACAATTTGCCCTGACCGACAAGGAAGTGA

[0323] CTCTGATCGACGGTCTGCCGCGTGTCCTGGCAAAGAACTTCGATGCTACCATTACTGA

[0324] CCGTGTTGAGAAACTGTATACCGATCACGGCGTCCACCTGGCACTGAATGAAATGGTT

[0325] ACTGAATTCGCGCAGGCAGACCAGGGCATCAAAGTCACCACCAACAAAGGCGATTAC

[0326] ACTGCCGATATTGCTATCCTGTGCACCGGTTTCCGTCCTAACACTGACCTGCTGAAGG

[0327] ACCACCTGGACACCCTGCCGAACGGTGCGGTTATCACTAACGCGTACATGCAGACTAG

[0328] CGATCCGGACATTTTCGCTGCAGGTGATACCGCGACCGTCCACTATAACCCGACTGGT

[0329] AAGAACGACTACATCCCACTGGCTACCAATGCGGTTCGTCAGGGTATCCTGGTGGGTA

[0330] AAAACATTATGACGCCGACCGAGAAATACCTGGGTACGCAGTCCAGCAGCGCAGTGG

[0331] AACTGTTTGATCACGCTATCGCTGCGTCTGGCCTGACCGTAGAAGGTGCACATACTCG

[0332] TGGCCTGGAACTGGACTCCGTAACCATCGAACAGGATTACCGTCCAGATTTCATGCTG

[0333] ACCACGACCCCGGTACTGTGTTCTCTGACTTGGGACCCGAAAACCCACGAAGTGAAA

[0334] GGTGGTGCGTTTTTCTCCAAACACGACATCTCTCAAAGCGCGAACGTGATCAGCCTGG

[0335] CTATCCAGACGCATATGACTATCGAGACCCTGGCGATGGTTGACATGCTGTTTCAGCC

[0336] GAACTTCGATCAGCCAATCAACTGGGTAAACGCTGTGGCTATGGCAGCTGTGGACAA

[0337] GGCCAAAAAAAAACCGACTACCCCGGTAGCGTAA

[0338] SEQ ID NO:15 NOX2 gene sequence:

[0339] ATGAAAGTTATCGTAGTTGGTTGTACTCATGCTGGTACTTTTGCTGTTAAACAAACGAT

[0340] TGCTGACCACCCGGATGCAGATGTGACCGCTTACGAAATGAACGACAACATCTCTTTT

[0341] CTGAGCTGCGGCATCGCCCTGTACCTGGGCAAGGAAATCAAAAACAATGATCCGCGT

[0342] GGTCTGTTCTACTCTTCTCCGGAAGAACTGTCCAACCTGGGTGCTAACGTGCAGATGC <OPTIONAL>[[ID=OPTIONAL]]

[0343] GTCACCAAGTCACGAACGTAGACCCGGAGACCAAAACCATCAAAGTGAAGGACCTGA <OPTIONAL>[[ID=OPTIONAL]]

[0344] TCACCAACGAAGAAAAAACCGAAGCGTACGACAAACTGATCATGACCACTGGTTCTA <OPTIONAL>[[ID=OPTIONAL]]

[0345] AACCGACCGTTCCACCAATCCCAGGTATTGATTCTTCTCGCGTCTACCTGTGTAAGAA <OPTIONAL>[[ID=OPTIONAL]]

[0346] CTATAACGACGCTAAGAAACTGTTCGAGGAGGCGCCAAAAGCGAAGACCATCACCAT

[0347] CATCGGCAGCGGTTACATCGGTGCTGAGCTGGCAGAAGCTTACTCCAACCAGAATTAC

[0348] AACGTTAACCTGATCGACGGCCACGAGCGCGTGCTGTATAAGTATTTCGACAAAGAGT

[0349] TTACCGATATCCTGGCCAAAGACTATGAAGCTCACGGCGTAAACCTGGTACTGGGCTC

[0350] TAAAGTTGCTGCTTTCGAAGAGGTTGATGACGAGATCATCACTAAGACCCTGGATGGC

[0351] AAAGAAATTAAAAGCGATATCGCTATTCTGTGCATTGGCTTCCGTCCGAACACCGAAC

[0352] TGCTGAAAGGCAAAGTTGCGATGCTGGACAACGGCGCGATTATCACCGATGAATACA

[0353] TGCACTCTAGCAACCGCGACATCTTCGCGGCGGGTGATTCTGCTGCGGTTCACTACAA

[0354] CCCGACCAACTCTAACGCGTACATTCCGCTGGCGACCAACGCAGTGCGTCAGGGCCGT

[0355] CTGGTGGGCCTGAACCTGACTGAAGACAAAGTTAAAGACATGGGCACGCAGAGCTCT

[0356] TCTGGTCTGAAACTGTACGGTCGCACTTACGTGTCCACTGGTATCAACACTGCACTGG

[0357] CAAAAGCTAACAACCTGAAAGTATCTGAAGTAATTATCGCTGACAACTACCGCCCGG

[0358] AATTCATGCTGTCCACTGATGAGGTACTGATGTCCCTGGTTTACGACCCGAAAACCCG

[0359] TGTTATCCTGGGTGGTGCTCTGAGCTCCATGCACGATGTGTCTCAGTCCGCGAACGTTC

[0360] TGAGCGTATGCATCCAGAACAAGAACACGATTGACGATCTGGCGATGGTAGATATGC

[0361] TGTTCCAACCGCAGTTCGATCGTCCGTTCAATTACCTGAATATCCTGGGTCAAGCAGC

[0362] GCAAGCTCAAGCGGACAAAGCTCATAAATAA

[0363] SEQ ID NO:16 NOX3 gene sequence:

[0364] ATGAAGATTCTGGTAATCGGCGCGACCCATGCAGGTACTTTTGCGACTCAGCAGATCC

[0365] TGACGGAACACCCGGATTACGACGTAACCGTTTACGAACGTAACCATAACCTGTCTTT

[0366] CCTGTCTTGCGGCATCGCTCTGTGGGTTGGTGATCATGTATCCGATCCGGAGAAAATG

[0367] TTTTACAGCTCCCCGGAAGCTCTGGCGGCACTGGGCGCGAACATGCAGATGGAGCAC

[0368] GATGTGCTGGCTATCGACCCGGTTGCGAAAACCGTAGAGGTCAAGGACCTGAAAACC

[0369] GGTAACGTAACTACGGACACCTACGACAAACTGGTTTACACCACTGGTTCCACGCCGA

[0370] TCGTCCCAAACATCCCGGGTATTCATGACACTGATGTTCACCTGTGCAAAAACTGGCA

[0371] CGACGCCAAAGCACTGAAAGCTCTGGCACCGACTATCAAGTCTGCAATTGTGATTGGC

[0372] GCAGGTTATATTGGTGCGGAACTGGCGGAACAGTACGCTCTGACTGACAAACAGGTC

[0373] ACCCTGATTGATGGCCTGCCGCGTGTACTGGCGAAAAATTTCGACGCCAACATTACTG

[0374] ACCGCGTGGAAAAACTGTACACGGACCATGGTGTAAATCTGGCACTGGGCGAAATGG

[0375] TGACCGGCTTCACTCAAGGCACCGACGGTGAAATGACCGTTACTACCGACAAGGGTA

[0376] GCTACACTGCTGATATCGCTGTCCTGTGCACCGGCTTCCGTCCGAATACGGAACTGCT

[0377] GAAAGACCACCTGGAAACTCTGCCAAATGGCGCTGTCATCACCGACGCGTACATGCA

[0378] AACCTCTGATCCGGCAATCTTCGCAGCTGGTGATACTGCTACCGTGCACTATAACCCG

[0379] ACCGGCAAAAACGATTACATTCCGCTGGCAACTAACGCGGTTCGTCAGGGTATCCTGG

[0380] TGGGTAAAAACATCGAAAAACCGACCGAGAAGTATCTGGGTACTCAGTCTTCTTCTGC

[0381] GGTCGAACTGTTCGAACACGCCATTGCTGCCTCTGGTATGACCACTGAAGGTGCGAAA

[0382] GCTCGTGGCATCGAAGTTGCGTCTGTTACCATCGAACAGGATTATCGCCCGGACTTTA

[0383] TGCTGACCACCACTCCGGTACTGTGTTCTCTGACCTGGGATCCGAAAACCCATGAAGT

[0384] TAAAGGTGGCGCCTTCTTCAGCAAACATGACATTTCCCAGAGCGCGAACGTCATCTCT

[0385] CTGGCGATCCAGACCCACATGACCATTGAAACGCTGGCAATGGTAGACATGCTGTTTC

[0386] AGCCGAATTTCGATCAGCCGATTAACTGGGTCAATGCCGTTGCTATGGCAGCCGTGGC

[0387] CAAAGCTCAGGAAATGGAAAAAACCCCAGTTGCTTAA

[0388] SEQ ID NO:17 NOX4 gene sequence:

[0389] ATGAAAGTAGCTGTTATCGGTTGTACGCACGCTGGTACCTTTTCTGCCCAGGAAATCC

[0390] TGACTCAGCACCCGGATGCCCAGGTGACTGTGTATGAACGTAACGATAACCTGAGCTT

[0391] CCTGTCCTGTGGTATCGCTCTGTGGGTTGGCAACAATGTGTCCGACCCGAAAAAAATG

[0392] TTCTACAGCTCCCCTGAAGCACTGACTAAACTGGGTGCTACCATGAAAATGGAACATG

[0393] ATGTGACCGACGTAGACCTGGATCAGAAAACTGTCACCGCGACCAACCTGAAGACCG

[0394] GTGAAACTAAAACCGAAGCTTTCGATAAAATCGTGATCACTACCGGTTCCAAACCAGT

[0395] TGTACCGAATCTGCCGGGTATCCATGGCGATAAAGTATACCAGTGCAAGAACTGGAA

[0396] CGATGCTAACCGCATTAAGGAAGCCTCTGAGAAAATTTCCAGCGCTGTGGTGATCGGC

[0397] GCAGGTTACATCGGTGCGGAAATCGCCGAGCAGCTGAGCCTGATCAATAAAGAGGTA

[0398] ACGCTGATCGACGGTCTGGATCGTGTGCTGGCCAACAACTTCGATCAGAAAGTCACCG

[0399] ACCGCATTGAAGATGAATACCGCAACCACGGCGTAAAACTGGCCCTGGGTGAAATGG

[0400] TGAAATCTTTCAACGAAGAAAATGACCAGGTAACCGTGGAGACCGACAAAGGCTCTT

[0401] ATACGGCAGACATCGCTGTTCTGGCAATCGGTTTCCTGCCGCGTACCGACCTGTTCAC

[0402] CGATAAACTGGATATGCTGCCGAACGGTGCCATCATCACCAACGAATACATGGAAAC

[0403] CTCCAAAAAGGACGTTTTTGCCGCCGGTGATGCTTCTTCTGTATTTTACAACCCGACCG

[0404] GTAAGGCAGACTACATTCCTCTGGCGACTAACGCTGTGCGTCAAGGTATTCTGGTTGG

[0405] TAACAACATCGAAAAACCGACGGTTAAATACCTGGGTACCCAGGCTACCAGCGCAGT

[0406] AGAACTGTACGGCTACGCACTGGCTGCGTCCGGTCTGAACGCGATGGGTGCTAAAAA

[0407] ACGTGGCGTTGAAATTGAGGAAGTGAGCATTGAACCGGACTATCGTCCGGACTTTATG

[0408] CCTACCACCACCAAAGTTCTGTGCACCCTGGTTTGGGATCCGACCAACCGTCAGGTAC

[0409] TGGGCGGCTCTTTTATGGCCAAACACGATATCTCCCAGGCTGCGAACGTTATTTCTCTG

[0410] GCGATCCAGTCTAAAATGACGATCGATGACCTGGCCATGGTGGACTTCTTTTTCCAGC

[0411] CGAACTTCGACCAGCCGATCAACTATATTGGTGCCGTGGCAAGCGCTGCGTGTGCGAA

[0412] ATCTAGCTCTAAGTAA

[0413] SEQ ID NO:18 NOX5 gene sequence:

[0414] ATGAAAGTTGCGGTAATCGGTTGCACCCACGCGGGCATTTTTAGCTCTCGTGAAATCC

[0415] TGAAAAATAACCCGGATGCCGAAATCACGGTCTTCGAGCGTAACGATACCGTGTCCTT

[0416] TCTGTCCTGCGGCATTGCGCTGTGGGTTGGTAATCACGTATCCTCTAGCGAAAAGATG

[0417] TTCTACGATAGCGTGGCGGCCATGCAGGCAGATGGCATCCAGATGAAAATGCAGCAC

[0418] GACGTTACCGCTGTTGACCTGGCTACCAAAACCCTGACTGCGGTTGACCTGAAAACCA

[0419] ATGAAAGCAAAACTGAAACCTTCGATAAAATCGTTATTACCACCGGTAGCAAACCGG

[0420] TAATGCCGCCAATTCCGGGCATCGACGGTGCGAACGTCTATAAATGCAAAAACTGGG

[0421] ACGACGCCAAGGCTATCAAGGAAGCGGCGAAAAGCGCGAAATCCGCAATTGTAATTG

[0422] GTGCGGGTTATATCGGTGCTGAACTGGCGGAACAGTTCTCTGTTAACAATATCAAAAC

[0423] CACGCTGATCGATGGTCTGGACCGCGTCCTGGCAAAAAACTTCGACAAAGACATCAC

[0424] GGATGAGGTTGAAGCTCAGTACCAAGCTCATGGCGTTACCCTGGCACTGGGTCAGATG

[0425] GTGAAGTCTTTCGAGGAGACTGAGACTGGCGTCAAAGTCACCACCGACAAGGGTGTG

[0426] TACGAGGCCGATATTGCTGTCCTGGGTATTGGCTTTCTGCCTCGCACGGACCTGTTCAC

[0427] CGGCCAGGTAGACATGATCAAAAATGGTGCAATCATCGTCGACAAGTACATGCAGAC

[0428] TAGCGTTAAAGATGTATACGCTGCAGGTGACTCTGCGACTGTTTTTTACAACCCGACC

[0429] CAGAAAGACGATTACATCCCGCTGGCCACTAACGCGATTCGTCAAGGTATCCTGGTGG

[0430] GCAAGAATATTAATACCCCGCAGGTTGCATATCTGGGTACCCAGTCCACCTCCGCGGT

[0431] CGAACTGTACGGTTACGCTATGGCTGCGTCTGGCCTGAACCAACAACTGGCGGAAGCT

[0432] CGCGGCATTACTGGTATCAAAGAGATCACCATCGAACAGGACTACCGTCCGGATTTTA

[0433] TGCTGACTACTACTCCGGTTCGCGCCACCCTGACCTGGGACGAGAAAACTCGTCAGGT

[0434] TCTGGGCGGCTCCTTTTATAGCAAGCACGACATTAGCCAGACCGCGAACGCTCTGTCC

[0435] CTGGCAATTCAGAACAAAATGACCATTGATGACCTGGCTATGTCCGATTTCCTGTTCC

[0436] AGCCGAATTTTTCCCAACCGATCAACTTCCTGGGTGCCGTTGCAATGGCTGCAGCTGC

[0437] AGAGTAA

Claims

1. An engineered bacterium, characterized in that: The engineered bacteria are obtained by introducing a heterologous gene encoding arabinose-1-dehydrogenase and a heterologous gene encoding NADPH oxidase into a host cell, so as to co-express arabinose-1-dehydrogenase and NADPH oxidase in the host cell, wherein the host cell is Vibrio natriuresis.

2. The engineered bacterium according to claim 1, wherein the GenBank accession number of the arabinose-1-dehydrogenase is BAD95974.1, WP_004041122.1, NP_353239.1, WP_004083016.1, AAD35400.1, WP_004083078.1, AIS92476.1, AIT75903.1 or AIS92478.1; preferably, the arabinose- The GenBank accession number of the arabinose-1-dehydrogenase is BAD95974.1, WP_004083016.1, AIT75903.1 or AIS92478.1; more preferably, the GenBank accession number of the arabinose-1-dehydrogenase is WP_004083016.1 or AIT75903.1; most preferably, the GenBank accession number of the arabinose-1-dehydrogenase is WP_004083016.

1.

3. The engineered bacterium according to claim 1, wherein the GenBank accession number of the NADPH oxidase is AXI93343.1, BAB19268.1, ABJ69145.1, BDR59011.1, QNN75144.1 or QFR22240.1; preferably, the GenBank accession number of the NADPH oxidase is BAB19268.1, QNN75144.1, AXI93343.1 or QFR22240.1; more preferably, the GenBank accession number of the NADPH oxidase is AXI93343.1 or QFR22240.1; most preferably, the GenBank accession number of the NADPH oxidase is QFR22240.

1.

4. The engineered bacteria according to claim 1, characterized in that The heterologous gene encoding arabinose-1-dehydrogenase and / or the heterologous gene encoding NADPH oxidase is further codon-optimized according to the preference of the expression vector or host cell.

5. The engineered bacteria according to claim 4, characterized in that The heterologous gene sequence encoding arabinose-1-dehydrogenase is shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, and / or the heterologous gene sequence encoding NADPH oxidase is shown in SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO:

18.

6. The engineered bacteria according to any one of claims 1 to 5, characterized in that The heterologous gene encoding arabinose-1-dehydrogenase and / or the heterologous gene encoding NADPH oxidase is expressed in the host cell in the form of a recombinant plasmid and / or inserted into the host cell genome in the form of an expression cassette for expression. Preferably, the recombinant plasmid is a recombinant pET-28a plasmid.

7. The engineered bacteria according to claims 1-5, characterized in that The copy number of the heterologous gene encoding arabinose-1-dehydrogenase is 2 or 3, and / or the copy number of the heterologous gene encoding NADPH oxidase is 10.

8. The engineered bacteria according to claims 1-5, characterized in that The heterologous gene encoding arabinose-1-dehydrogenase and / or the heterologous gene encoding NADPH oxidase is expressed under the control of a T7 promoter.

9. The engineered bacteria according to claim 8, characterized in that A T7 RNA polymerase expression cassette is integrated into the genome of the host cell; preferably, the T7 RNA polymerase expression cassette is integrated into the dns gene site of the host cell.

10. A two-stage biological method for producing xylitol using hemicellulose hydrolysate, characterized in that: include: (1) using a hemicellulose hydrolyzate containing xylose and L-arabinose as a raw material, and performing a first stage fermentation culture in a fermentation medium inoculated with the engineered bacteria according to claim 1, until no L-arabinose remains in the fermentation liquid, thereby obtaining a fermentation reaction liquid I containing L-arabinonic acid; (2) After adding the engineered bacteria co-expressing xylose reductase and glucose dehydrogenase to the fermentation reaction liquid I, the second stage of fermentation culture is continued to obtain a fermentation product containing xylitol.

11. The method according to claim 10, characterized in that The engineered bacteria co-expressing xylose reductase and glucose dehydrogenase is Vibrio natriuresis co-expressing xylose reductase and glucose dehydrogenase.

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