Genetically engineered bacteria and their application in whole-cell catalysis of xylose to produce xylitol

Abstract

The present invention belongs to the field of biocatalysis, specifically relating to genetically engineered bacteria and their use in whole-cell catalysis of xylose to produce xylitol. The genetically engineered bacteria comprise a host cell, Vibrio natriuresis, and target genes inserted into the host cell. The host cell is Vibrio natriuresis; the target genes include xylose reductase and glucose dehydrogenase genes. The present invention introduces heterologous xylose reductase and glucose dehydrogenase genes into Vibrio natriuresis, and adjusts the gene dosage ratio of xylose reductase and glucose dehydrogenase through genomic integration to construct a genetically engineered Vibrio natriuresis. Compared to Escherichia coli, the whole-cell biocatalyst of the present invention has a shorter preparation cycle, higher catalytic activity, and higher production efficiency.

Description

Technical Field

[0001] The invention belongs to the field of biocatalysis, and particularly relates to genetically engineered bacteria and application thereof in whole-cell catalysis of xylose to prepare xylitol. Background Art

[0002] Xylitol (molecular formula C5H 12 Xylitol (Xylitol) is a five-carbon sugar alcohol that appears as white crystals or crystalline powder. It is a sweetener with the same sweetness as sucrose and the same calories as glucose. It does not affect blood sugar or insulin levels and can be used as a sugar substitute for diabetics.

[0003] The traditional xylitol production process is chemical hydrogenation. This catalytic reaction needs to be carried out under high temperature and high pressure, and the purity of the raw material xylose is required to be high, resulting in a high production cost of xylitol, which is about 10 times the price of sucrose. The biological method for preparing xylitol has mild conditions and does not require the raw materials to be refined and purified to obtain high-purity xylose. The green biomanufacturing of xylitol can be achieved using pretreated hemicellulose hydrolysate as raw material. Among the technical processes for the biological preparation of xylitol, microbial fermentation is the most studied. However, due to the complexity of the microbial metabolic network and the need for microbial cell growth, some raw materials will inevitably be used for cell growth or to synthesize other metabolites. At the same time, there is also a mismatch between the coenzyme NADPH produced by the cells and the NADPH required for the xylitol synthesis process during the fermentation process, resulting in a high amount of glucose added, far below the theoretical value. The biocatalytic method has a simple and efficient reaction and can effectively avoid these problems. Generally, the biocatalytic method uses xylose as the raw material and uses xylose reductase to reduce xylose to xylitol. This process consumes NADPH and produces NADP + To regenerate NADPH, the reaction needs to be coupled with glucose dehydrogenase to oxidize glucose to gluconic acid. The most abundant substance in hemicellulose hydrolysate is xylose, followed by glucose. Therefore, biocatalytic methods can utilize xylose and glucose to co-produce xylitol and gluconic acid.

[0004] Vibrio natriuresis ( Vibrio natriegens) is a Gram-negative bacterium and the fastest-growing microorganism known, with a generation time of 7 to 10 minutes, half that of Escherichia coli. Studies have found that a single cell of Vibrio natrii contains as many as 115,000 ribosomes, compared to approximately 70,000 to 90,000 ribosomes in E. coli. This indicates faster biomass synthesis and greater protein expression capacity. Vibrio natrii has a similar central metabolic model to E. coli, with a growth rate of 1.70 L / h and a conversion rate of 3.90 g / g / h in glucose minimal medium, twice the metabolic rate of E. coli. Furthermore, Vibrio natrii is less susceptible to phage contamination during large-scale factory production. Therefore, developing Vibrio natrii into a cell factory for recombinant protein expression can not only significantly shorten fermentation time but also prevent phage contamination. Summary of the Invention

[0005] In response to the existing demand for using Vibrio natriuresis to produce xylitol, the present invention provides a genetically engineered bacterium and its application in whole-cell catalysis of xylose to produce xylitol. The specific technical solution is as follows:

[0006] In a first aspect, the present invention provides a genetically engineered bacterium comprising a host cell and a target gene inserted into the host cell, wherein the host cell is Vibrio natriuresis; the target gene comprises a xylose reductase gene and a glucose dehydrogenase gene.

[0007] Furthermore, a T7 RNA polymerase expression cassette is integrated into the genome of the Vibrio natriuresis.

[0008] Furthermore, the T7 RNA polymerase expression cassette is inserted into the genome of Vibrio natriuresis. dns Genetically, and dns Gene inactivation.

[0009] Furthermore, the T7 RNA polymerase expression cassette is derived from Escherichia coli BL21 (DE3).

[0010] Furthermore, the xylose reductase and glucose dehydrogenase genes are expressed in the host cell in one of the following ways:

[0011] (A) Expressed in host cells in the form of a recombinant plasmid;

[0012] (B) Inserted into the host cell genome in the form of an expression cassette for expression.

[0013] Furthermore, the accession number of the xylose reductase gene in the NCBI library is ALO17776.1.

[0014] Furthermore, the accession number of the glucose dehydrogenase gene in the NCBI library is WP_013055546.1, WP_012369122.1 or 8W0O_A.

[0015] In the present invention, different xylose reductases and glucose dehydrogenases were screened, and it was found that the expression products of most xylose reductase genes in Vibrio natriuresis had low enzyme activity, and only the xylose reductase with accession number ALO17776.1 showed activity that could be used for xylitol production.

[0016] Furthermore, in (B), the copy number of the xylose reductase gene is 4 or 5.

[0017] Furthermore, in (B), the copy number of the glucose dehydrogenase gene is 1 or 2.

[0018] Furthermore, in (B), the copy number of the xylose reductase gene is 5, and the copy number of the glucose dehydrogenase gene is 2.

[0019] The present invention found that when the xylose reductase gene and the glucose dehydrogenase gene were expressed in the genomic integration form in Vibrio natrii, the copy number of the xylose reductase gene significantly affected the enzymatic activity of the xylose reductase, and the copy number of the glucose dehydrogenase gene significantly affected the enzymatic activity of the glucose dehydrogenase. + The cycle of glucose dehydrogenase gene copies also affects the catalytic activity of xylose reductase in Vibrio natriuresis. In Vibrio natriuresis, glucose dehydrogenase activity is highest when the glucose dehydrogenase gene copy number is 2, and then decreases as the copy number increases. As the glucose dehydrogenase gene copy number gradually increases, the xylose reductase activity gradually decreases. Considering the efficiency of the entire production process, the xylose reductase gene copy number of 4 or 5 and the glucose dehydrogenase gene copy number of 1 or 2 were selected to construct genetically engineered Vibrio natriuresis.

[0020] In a second aspect, the present invention provides a method for preparing xylitol, using xylose and glucose as substrates and the whole cells of the above-mentioned genetically engineered bacteria as catalysts to form a catalytic reaction system to produce xylitol and gluconic acid.

[0021] Furthermore, the reaction temperature is 25-35°C.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention introduces heterologous xylose reductase and glucose dehydrogenase genes into Vibrio natriuresis and adjusts the gene dosage ratio of xylose reductase and glucose dehydrogenase through genomic integration to construct a genetically engineered strain of Vibrio natriuresis. Compared to Escherichia coli, the whole-cell biocatalyst of the present invention has a shorter preparation cycle, higher catalytic activity, and higher production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the reaction process diagram for the production of xylitol and gluconic acid catalyzed by whole cells of plasmid-type Vibrio natriuresis.

[0025] Figure 2 Schematic diagram of the relationship between the addition amount of integrated Vibrio natriuresis cells at different concentrations and the production of xylitol and gluconic acid; among them, A, B, C, and D represent the addition amount of Vibrio natriuresis cells of 1 g / L, 2 g / L, 3 g / L, and 4 g / L, respectively. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] In the following examples, HPLC was used to determine the contents of xylose, xylitol, glucose, and gluconic acid. The following conditions were used for the determination of xylose, xylitol, and glucose: 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. The following conditions were used for the determination of gluconic acid: a C18 column (250 mm × 4.6 mm, 5 μm), a detection wavelength of 210 nm, and a mobile phase of 10 mM K₂HPO₃-10 mM tetrabutylammonium hydrogen sulfate (pH 7.2): methanol (95:5 v / v), at a flow rate of 0.7 mL / min.

[0030] In the following examples, the xylose reductase activity was determined using a 1 mL reaction system consisting of 100 μL of a 1 M xylose stock solution (final concentration 100 mM), 50 μL of a 0.4 M NADPH stock solution (final concentration 20 mM), 750 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 using a spectrophotometer. Enzyme activity unit definition: 1 μM NADP is generated per minute under standard reaction conditions. + The required amount of enzyme. The formula for calculating xylose reductase activity is: Enzyme activity (U / mL) = (△A / min) × (1 / ɛ) × (1 / d) × (Vt / Vs) × X, where △A / min represents the change in absorbance per minute; ɛ 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 lysed cell solution, which is 100 μL; and X represents the dilution factor.

[0031] In the following examples, the glucose dehydrogenase activity was determined using a 1 mL reaction system containing 100 μL of 1 M glucose solution (final concentration 100 mM), 0.4 M NADP-HCl, and 0.5 M HCl. + 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 absorbance changes at 340 nm using a spectrophotometer. Enzyme activity is defined as the amount of enzyme required to generate 1 μM NADPH per minute under standard reaction conditions. Glucose dehydrogenase activity is calculated using the following formula: Enzyme activity (U / mL) = (ΔA / min) × (1 / ɛ) × (1 / d) × (Vt / Vs) × X, where ΔA / min represents the absorbance change per minute; ɛ 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.

[0032] Example 1 Expression of xylose reductase in Vibrio natriuresis

[0033] The sequences of 11 xylose reductase XR genes from different sources (as shown in Table 1) were codon optimized and fully synthesized in E. coli, and then constructed on the pET-28a plasmid to obtain 11 plasmids. dnsThe 11 plasmids were electroporated into VnDX to obtain 11 strains of VnDX carrying the XR expression plasmid: VnDX / pET-28a-XR1, VnDX / pET-28a-XR2, VnDX / pET-28a-XR3, VnDX / pET-28a-XR4, VnDX / pET-28a-XR5, VnDX / pET-28a-XR6, VnDX / pET-28a-XR7, VnDX / pET-28a-XR8, VnDX / pET-28a-XR9, VnDX / pET-28a-XR10, and VnDX / pET-28a-XR11.

[0034] Eleven strains, including VnDX / pET-28a-XR1, were streaked onto solid plates. Single colonies were selected and inoculated into 5 mL tubes of LB medium supplemented with 200 μg / mL kanamycin and cultured at 30°C and 220 rpm for 6-8 hours. 0.5 mL of each overnight bacterial culture was inoculated into 50 mL of fermentation medium (12 g / L peptone, 24 g / L yeast extract, 15 g / L NaCl, 0.5% (v / v) glycerol, 2.31 g / L KH2PO4, 12.5 g / L K2HPO4) supplemented with 200 μg / mL kanamycin. The culture was shaken at 30°C and 220 rpm for 2 hours. IPTG was then added for induction to a final concentration of 0.3 mM. The tubes were cooled to 28°C and shaken at 220 rpm for 9 hours.

[0035] 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. The enzyme activity of XR was determined by UV spectrophotometry (Table 1). The results in Table 1 show that the enzyme activities of different XR in Vibrio natriuresis vary greatly, among which the enzyme from Rhodotorula mucilaginosa The enzyme activity of XR5 unit fermentation broth was the highest, reaching 49.21 U / mL.

[0036] Table 1 Enzyme activity assay results of recombinant XR from different sources expressed in Vibrio natriuresis

[0037]

[0038] Example 2 Expression of glucose dehydrogenase in Vibrio natriuresis

[0039] The sequences of four glucose dehydrogenase (GDH) genes (shown in Table 2) were codon-optimized and fully synthesized in E. coli. These genes were then constructed on the pET-28a plasmid, resulting in four plasmids. These plasmids were then electroporated into VnDX to generate four strains of V. natriuresis carrying the GDH expression plasmids: VnDX / pET-28a-GDH1, VnDX / pET-28a-GDH2, VnDX / pET-28a-GDH3, and VnDX / pET-28a-GDH4.

[0040] Four strains, including VnDX / pET-28a-GDH1, were streaked onto solid plates. Single colonies were selected and inoculated into 5 mL tubes of LB medium supplemented with 200 μg / mL kanamycin and cultured at 30°C, 220 rpm, for 6-8 hours. 0.5 mL of each overnight bacterial culture was inoculated into 50 mL of fermentation medium (12 g / L peptone, 24 g / L yeast extract, 15 g / L NaCl, 0.5% (v / v) glycerol, 2.31 g / L KH2PO4, 12.5 g / L K2HPO4) supplemented with 200 μg / mL kanamycin. Cultures were shaken at 30°C, 220 rpm, for 2 hours. IPTG was then added for induction to a final concentration of 0.3 mM. The culture was cooled to 28°C and shaken at 220 rpm for 9 hours.

[0041] 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 3 s, pause 7 s, 30 times) to obtain a crude enzyme solution. The enzyme activity of XR was determined by UV spectrophotometry (Table 2). The results in Table 2 show that the enzyme activities of different GDHs in Vibrio natriuresis vary greatly, among which the GDH from Exiguobacterium artemiae The GDH2 unit fermentation broth had the highest enzyme activity, reaching 68.32 U / mL.

[0042] Table 2 Results of enzyme activity assays of GDH from different sources recombinantly expressed in Vibrio natriuresis

[0043]

[0044] Example 3 Co-expression of xylose reductase and glucose dehydrogenase in Vibrio natriuresis

[0045] The pETDuet-1 plasmid has two multiple cloning sites. In the present invention, the XR5 open reading frame and the GDH1, GDH2, and GDH4 open reading frames were cloned into the two multiple cloning sites of the pETDuet-1 plasmid to construct the pETDuet-XR5-GDH1, pETDuet-XR5-GDH2, and pETDuet-XR5-GDH4 plasmids, which were then electroporated into Vibrio natriuresis VnDX to obtain strains VnDX / pETDuet-XR5-GDH1, VnDX / pETDuet-XR5-GDH2, and VnDX / pETDuet-XR5-GDH4. Microbial culture and cell collection were performed according to the shake flask fermentation method described in Example 1. The XR enzyme activity was determined according to the method of Example 1, and the GDH enzyme activity was determined according to the method of Example 2. The results are shown in Table 3. Combination 2 had the best effect, with an XR5 enzyme activity of 28.56 U / mL and a GDH enzyme activity of 61.32 U / mL. When the two enzymes were co-expressed, the enzyme activities decreased to varying degrees compared to those expressed alone.

[0046] Table 3 Enzyme activities of xylose reductase XR5 and glucose dehydrogenase GDH co-expressed in Vibrio natriuresis

[0047]

[0048] Example 4 Catalytic Production of Xylitol and Gluconic Acid by Whole Cells of Plasmid-Type Vibrio natriuresis

[0049] The strain VnDX / pETDuet-XR5-GDH2 was cultured according to the shake flask fermentation method in Example 1 and the cells were collected by centrifugation. A 200 mL whole-cell catalytic reaction system was prepared, wherein the initial concentration of xylose was 1.5 M, the initial concentration of glucose was 1.8 M, and the NADP + The initial concentration was 0.6 mM, and the genetically engineered bacterial cells were 1 g. The whole-cell catalytic reaction was carried out at 30°C. 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 xylose, xylitol, glucose, and gluconic acid. The experimental results are as follows: Figure 1 shown.

[0050] Depend on Figure 1 As can be seen, after 8 hours of reaction, 1.8M glucose was completely converted to gluconic acid, with a final gluconic acid concentration of 1.74M. However, the conversion rate of 1.5M initial xylose was only 56%, and the final xylitol yield was 0.84M. This shows that although the glucose conversion rate of this system is high, the xylose conversion rate is low, resulting in a low xylitol yield, so there is still considerable room for optimization. The reason for this phenomenon is speculated to be the inconsistent enzymatic activities of XR and GDH due to the co-expression of two enzymes in the plasmid.

[0051] Example 5 Xylose reductase and glucose dehydrogenase genomic integration and copy number optimization

[0052] To optimize the ratio of xylose reductase XR5 and glucose dehydrogenase GDH2 and ensure consistent XR and GDH activities per cell, the present invention proposes optimizing gene dosage to improve whole-cell catalytic efficiency. The genome editing method for Vibrio natriuresis was performed according to the published NT-CRISPR method (DOI: 10.1038 / s42003-022-03150-0). Vibrio natriuresis strains with 1 to 8 copies of XR5 were generated using NT-CRISPR, designated VnXY-1 to VnXY-8. Enzyme activity was assayed according to the method described in Example 1, and the results are shown in Table 4. The results show that XR5 enzyme activity initially increases and then decreases with increasing copy number. The activity of the strain with 5 copies of XR5 reached 67.52 U / mL, significantly higher than that of the plasmid-derived strain.

[0053] Using VnXY-5 as the starting strain, strains VnXY-9 to VnXY-13 were constructed by integrating 1 to 5 copies of GDH2 into the VnXY-5 genome using the same NT-CRISPR method. XR5 enzyme activity was determined according to the methods in Example 1, and GDH2 enzyme activity was determined according to the methods in Example 2. The results are shown in Table 5. As shown in Table 5, by integrating XR and GDH in the genome and optimizing gene dosage by adjusting copy number, the GDH2 and XR5 enzyme activities in the final strain VnXY-10 matched each other. XR5 enzyme activity was higher than that observed with both plasmid expression alone and plasmid co-expression, while GDH enzyme activity essentially matched that observed with plasmid expression alone. This strain contained two copies of GDH2 and five copies of XR5.

[0054] Table 4 Enzyme activity of integrated XR5

[0055]

[0056] Table 5 Enzyme activities of integrated XR5 and GDH2

[0057]

[0058] Example 6 Catalytic Production of Xylitol and Gluconic Acid by Integrated Vibrio natriuresis Whole Cells

[0059] The VnXY-10 strain was cultured according to the shake flask fermentation method in Example 1 and the cells were collected by centrifugation. Whole-cell catalysis was carried out according to the method in Example 4. A 200 mL whole-cell catalytic reaction system was prepared, wherein the initial concentration of xylose was 1.5 M, the initial concentration of glucose was 1.8 M, and the NADP +The initial concentration was 0.6 mM, and the amount of genetically engineered bacteria wet cells added was 0.2 g (1 g / L WCW) (WCW refers to wet cell weight) or 0.4 g (2 g / L WCW) or 0.6 g (3 g / L WCW) or 0.8 g (4 g / L WCW). The whole-cell catalytic reaction was carried out at 30°C, and 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 xylose, xylitol, glucose, and gluconic acid. The experimental results are shown in the figure. Figure 2 As shown. Figure 2 As shown, the efficiency of catalytic production of xylitol and gluconic acid increased with increasing cell addition. Ultimately, with the addition of 4 g / L WCW, 1.5 M xylose was completely converted to 1.5 M xylitol within 5 hours, with a space-time yield of 0.3 mol / L / h.

Claims

1. A genetically engineered bacterium comprising a host cell and a target gene inserted into the host cell, characterized in that: The host cell is Vibrio natriuresis; the target genes include xylose reductase gene and glucose dehydrogenase gene; The xylose reductase gene has an accession number of ALO17776.1 in the NCBI database; The accession number of the glucose dehydrogenase gene in the NCBI library is WP_013055546.1, WP_012369122.1 or 8W0O_A.

2. The genetically engineered bacterium according to claim 1, characterized in that A T7 RNA polymerase expression cassette is integrated into the genome of the Vibrio natriuresis.

3. The genetically engineered bacterium according to claim 1, characterized in that The xylose reductase and glucose dehydrogenase genes are expressed in the host cell in one of the following ways: (A) Expressed in host cells in the form of a recombinant plasmid; (B) Inserted into the host cell genome in the form of an expression cassette for expression.

4. The genetically engineered bacterium according to claim 3, characterized in that In (B), the copy number of the xylose reductase gene is 4 or 5.

5. The genetically engineered bacterium according to claim 3, characterized in that In (B), the copy number of the glucose dehydrogenase gene is 1 or 2.

6. A method for preparing xylitol, characterized in that: Xylose and glucose are used as substrates, and the whole cells of the genetically engineered bacteria according to any one of claims 1 to 5 are used as catalysts to form a catalytic reaction system to produce xylitol and gluconic acid.

7. The method according to claim 6, characterized in that The reaction temperature is 25-35°C.

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