Genetically engineered bacterium and application of genetically engineered bacterium in preparation of xylitol by whole-cell catalysis of xylose

Abstract

The invention belongs to the field of biological catalysis, and particularly relates to a genetically engineered bacterium and application of the genetically engineered bacterium in preparation of xylitol through whole-cell catalysis of xylose, the genetically engineered bacterium comprises a host cell and a target gene inserted into the host cell, and the host cell is vibrio natrieti; the target genes comprise a xylose reductase gene and a glucose dehydrogenase gene. According to the invention, heterologous xylose reductase and glucose dehydrogenase genes are introduced into vibrio natriticus, and the dosage ratio of the xylose reductase to the glucose dehydrogenase genes is adjusted in a genome integration form, so that the vibrio natriticus genetically engineered bacterium is constructed. Compared with escherichia coli, the whole-cell biocatalyst disclosed by the invention is short in preparation period, high in catalytic activity and relatively high in production efficiency.

Description

Technical Field

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

[0002] Xylitol (molecular formula C 5 H 12 O 5 ) is a five-carbon sugar alcohol, which is white crystals or crystalline powder. Xylitol 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 there is no need to purify the raw materials to obtain high-purity xylose. The green biomanufacturing of xylitol can be achieved by using the pretreated hemicellulose hydrolysate as the raw material. Among the technical processes for preparing xylitol by biological methods, the microbial fermentation method 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 synthesis of other metabolites. At the same time, there is also a mismatch between the coenzyme NADPH produced by the cells and the NADPH required in the synthesis of xylitol during the fermentation process, resulting in a high amount of glucose added, which is far below the theoretical value. The biocatalytic method is simple and efficient, and can effectively avoid these problems. Usually, 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 + In order to achieve the regeneration of NADPH, the reaction needs to be coupled with glucose dehydrogenase to oxidize glucose into gluconic acid. The substance with the highest content in hemicellulose hydrolysate is xylose, followed by glucose. Therefore, the biocatalytic method can use xylose and glucose to produce xylitol and gluconic acid.

[0004] Vibrio natriuresis Vibrio natriegens) is a Gram-negative bacterium. It is the fastest growing microorganism known, with a generation time of 7 to 10 minutes, which is half that of Escherichia coli. Studies have found that the number of ribosomes in a single cell of Vibrio natrii is as high as 115,000, while Escherichia coli has only about 70,000 to 90,000, which means that it has a faster biomass synthesis rate and stronger protein expression ability. Vibrio natrii has a central metabolic model similar to that of Escherichia coli, with a growth rate of 1.70 L / h and a conversion rate of 3.90g / g / h in glucose minimal medium, which is twice the metabolic rate of Escherichia coli. In addition, Vibrio natrii is not susceptible to phage contamination in large-scale production in factories. It can be seen that developing Vibrio natrii into a cell factory for protein recombinant expression can not only shorten a large amount of fermentation time, but also avoid phage contamination. Summary of the invention

[0005] In view of the need for using Vibrio natriuresis to produce xylitol in the prior art, the present invention provides a genetically engineered bacterium and its application in whole-cell catalysis of xylose to prepare xylitol. The specific technical scheme is as follows: In a first aspect, the present invention provides a genetically engineered bacterium, including a host cell and a target gene inserted into the host cell, wherein the host cell is Vibrio natriuresis; the target gene includes a xylose reductase gene and a glucose dehydrogenase gene.

[0006] Furthermore, a T7 RNA polymerase expression cassette is integrated into the genome of the Vibrio natriuresis.

[0007] Furthermore, the T7 RNA polymerase expression cassette is inserted into the genome of Vibrio natriuresis DNS Genetically, and DNS Gene inactivation.

[0008] Furthermore, the T7 RNA polymerase expression cassette is derived from Escherichia coli BL21 (DE3).

[0009] Furthermore, 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.

[0010] Furthermore, the accession number of the xylose reductase gene in the NCBI library is ALO17776.1.

[0011] Furthermore, the accession number of the glucose dehydrogenase gene in the NCBI library is WP_013055546.1, WP_012369122.1 or 8W0O_A.

[0012] 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 natrii had low enzyme activity, and only the xylose reductase with accession number ALO17776.1 showed activity that could be used for xylitol production.

[0013] Furthermore, in (B), the copy number of the xylose reductase gene is 4 or 5.

[0014] Furthermore, in (B), the copy number of the glucose dehydrogenase gene is 1 or 2.

[0015] Furthermore, in (B), the copy number of the xylose reductase gene is 5, and the copy number of the glucose dehydrogenase gene is 2.

[0016] When the xylose reductase gene and the glucose dehydrogenase gene are expressed in the form of genome integration in Vibrio natrii, it is found that the copy number of the xylose reductase gene significantly affects the enzymatic activity of the xylose reductase, and the copy number of the glucose dehydrogenase gene significantly affects the enzymatic activity of the glucose dehydrogenase. + The cycle of glucose dehydrogenase gene copies also affects the catalytic ability of xylose reductase in Vibrio natrii. In Vibrio natrii, the enzymatic activity of glucose dehydrogenase is the highest when the copy number of glucose dehydrogenase gene is 2, and then decreases with the increase of copy number. As the copy number of glucose dehydrogenase gene gradually increases, the enzymatic activity of xylose reductase gradually decreases. Considering the efficiency of the entire production process, the copy number of xylose reductase gene is 4 or 5, and the copy number of glucose dehydrogenase gene is 1 or 2, and the genetically engineered bacteria of Vibrio natrii are constructed.

[0017] 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.

[0018] Furthermore, the reaction temperature is 25-35°C.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The invention introduces heterologous xylose reductase and glucose dehydrogenase genes into Vibrio natrii, adjusts the dosage ratio of xylose reductase and glucose dehydrogenase genes by genome integration, and constructs a genetically engineered Vibrio natrii. Compared with Escherichia coli, the whole-cell biocatalyst of the invention has a short preparation cycle, high catalytic activity, and high production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is the reaction process diagram of the preparation of xylitol and gluconic acid catalyzed by whole cells of plasmid-type Vibrio natriuresis.

[0021] 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

[0022] 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.

[0023] 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.

[0024] 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.

[0025] In the following examples, HPLC was used to detect the contents of xylose, xylitol, glucose and gluconic acid. The detection conditions for xylose, xylitol and glucose were as follows: mobile phase was ultrapure water, Agilent Hi-Plex Ca chromatographic column (7.7 mm × 300 mm), column temperature was 85°C, and flow rate was 0.6 mL / min. The detection conditions for gluconic acid were as follows: C18 chromatographic column (250 mm × 4.6 mm5pm), detection wavelength was 210 nm, and mobile phase was 10 mM K 2 HPO 3 -10 mM tetrabutylammonium hydrogen sulfate (pH 7.2): methanol (95:5 V / V); flow rate: 0.7 mL / min.

[0026] In the following examples, the xylose reductase activity determination method is as follows: the determination is carried out using a 1 mL reaction system, which includes: 100 μL of 1 M xylose mother solution (final concentration 100 mM), 50 μL of 0.4 M NADPH mother solution (final concentration 20 mM), 750 μL of PBS buffer, and 100 μL of cell lysis solution. After preheating the reaction solution and cell lysis solution at 30 °C in a metal bath for 10 minutes, the two are quickly mixed and the absorbance change at 340 nm is monitored by a spectrophotometer. Enzyme activity unit definition: Under standard reaction conditions, 1 μM NADP is generated per minute. + The amount of enzyme required. The formula for calculating the activity of xylose reductase 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 light path of the cuvette, which is 1 cm; Vt represents the total reaction volume, which is 1000 μL; Vs represents the volume of the cell lysate, which is 100 μL; and X represents the dilution multiple.

[0027] In the following examples, the glucose dehydrogenase activity was determined by a 1 mL reaction system, which included: 100 μL of 1 M glucose mother solution (final concentration 100 mM), 0.4 M NADP + 50 μL of mother solution (final concentration 20 mM), 750 μL of PBS buffer, and 100 μL of cell lysis solution. After preheating the reaction solution and cell lysis solution in a metal bath at 30 °C for 10 minutes, the two were quickly mixed and the absorbance change at 340 nm was monitored by a spectrophotometer. Definition of enzyme activity unit: the amount of enzyme required to generate 1 μM NADPH per minute under standard reaction conditions. Glucose dehydrogenase activity calculation formula: 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 light path of the cuvette, which is 1 cm; Vt represents the total reaction volume, which is 1000 μL; Vs represents the volume of cell lysis solution, which is 100 μL; X represents the dilution multiple.

[0028] Example 1 Expression of xylose reductase in Vibrio natriuresis 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 pET-28a plasmid to obtain 11 plasmids. DNSThe 11 plasmids were electroporated into VnDX to obtain 11 strains of Vibrio natriuresis carrying XR expression plasmids: 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.

[0029] Activate 11 strains including VnDX / pET-28a-XR1 by streaking on solid plates. Pick a single clone and inoculate it into a 5 mL test tube containing LB medium with 200 μg / mL kanamycin, and culture it at 30°C and 220 rpm for 6-8 h. Take 0.5 mL of overnight bacterial culture and inoculate it into 50 mL fermentation medium containing 200 μg / mL kanamycin (peptone 12 g / L, yeast powder 24 g / L, NaCl 15 g / L, glycerol 0.5% (v / v), KH 2 PO 4 2.31 g / L, K 2 HPO 4 The cells were incubated in a shaking incubator at 30°C and 220 rpm for 2 h, and then IPTG was added to a final concentration of 0.3 mM for induction. The cells were cooled to 28°C and incubated at 220 rpm for 9 h.

[0030] Take 4 mL of bacterial liquid from each shake flask of the above fermentation, add 4 mL of phosphate buffer (pH 7.5), and use an ultrasonic cell disruptor (operate 3s, pause 7s, 30 times) to lyse the bacteria to obtain a crude enzyme solution. The enzyme activity of XR was determined by UV spectrophotometer (Table 1). As shown in Table 1, the enzyme activities of different XR in Vibrio natrii vary greatly, among which the enzymes from Rhodotorula mucilaginosa The XR5 unit fermentation broth had the highest enzyme activity, reaching 49.21 U / mL.

[0031] Table 1 Results of enzyme activity assay of recombinant XR from different sources expressed in Vibrio natriuresis

[0032] Example 2 Expression of glucose dehydrogenase in Vibrio natriuresis The sequences of four glucose dehydrogenase GDH genes (as shown in Table 2) were codon optimized and fully synthesized in E. coli, and then constructed on pET-28a plasmid to obtain four plasmids. The four plasmids were electrotransformed into VnDX by electroporation to obtain four strains of Vibrio natrii with GDH expression plasmids: VnDX / pET-28a-GDH1, VnDX / pET-28a-GDH2, VnDX / pET-28a-GDH3, and VnDX / pET-28a-GDH4.

[0033] Activate four strains including VnDX / pET-28a-GDH1 by streaking on solid plates. Pick a single clone and inoculate it into a 5 mL test tube containing LB medium with 200 μg / mL kanamycin, and culture it at 30°C and 220 rpm for 6-8h. Take 0.5 mL of overnight bacterial culture and inoculate it into 50 mL fermentation medium containing 200 μg / mL kanamycin (peptone 12 g / L, yeast powder 24 g / L, NaCl 15 g / L, glycerol 0.5% (v / v), KH 2 PO 4 2.31 g / L, K 2 HPO 4 The cells were incubated in a shaking incubator at 30°C and 220 rpm for 2 h, and then IPTG was added to a final concentration of 0.3 mM for induction. The cells were cooled to 28°C and incubated at 220 rpm for 9 h.

[0034] Take 4 mL of bacterial solution from each shake flask of the above fermentation, add 4 mL of phosphate buffer (pH 7.5), and use an ultrasonic cell disruptor (operate 3 s, pause 7 s, 30 times) to lyse the bacteria to obtain a crude enzyme solution. The enzyme activity of XR was determined by UV spectrophotometer (Table 2). As shown in Table 2, the enzyme activities of different GDH in Vibrio natrii vary greatly, among which the enzyme activity of Exiguobacterium artemiae The GDH2 unit fermentation broth had the highest enzyme activity, reaching 68.32 U / mL.

[0035] Table 2 Results of enzyme activity assay of GDH from different sources expressed by recombinant Vibrio natriuresis

[0036] Example 3 Co-expression of xylose reductase and glucose dehydrogenase in Vibrio natriuresis The pETDuet-1 plasmid has two multiple cloning sites. The present invention clones the XR5 open reading frame and the GDH1, GDH2, and GDH4 open reading frames into the two multiple cloning sites of the pETDuet-1 plasmid, respectively, to construct pETDuet-XR5-GDH1, pETDuet-XR5-GDH2, and pETDuet-XR5-GDH4 plasmids, which are then electrotransduced into the natriuretic Vibrio vnDX to obtain strains VnDX / pETDuet-XR5-GDH1, VnDX / pETDuet-XR5-GDH2, and VnDX / pETDuet-XR5-GDH4. Microbial culture and cell collection are performed according to the shake flask fermentation method 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 the enzyme activity of XR5 being 28.56 U / mL and the enzyme activity of GDH being 61.32 U / mL. When the two enzymes were co-expressed, the enzyme activities decreased to varying degrees compared to their individual expression.

[0037] Table 3 Enzyme activity of xylose reductase XR5 and glucose dehydrogenase GDH co-expressed in Vibrio natriuresis

[0038] Example 4 Preparation of xylitol and gluconic acid catalyzed by whole cells of plasmid-type Vibrio natriuresis 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, 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, and 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.

[0039] Depend on Figure 1 It can be seen that when the reaction was carried out for 8 hours, 1.8M glucose was completely converted into gluconic acid, and the final gluconic acid concentration was 1.74M. However, the initial xylose conversion rate of 1.5M was only 56%, and the final xylitol yield was 0.84M. It can be seen 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 a lot of room for optimization. The reason for this phenomenon is speculated to be that the plasmid type co-expresses two enzymes, and the enzyme activities of XR and GDH are inconsistent.

[0040] Example 5 Xylose reductase and glucose dehydrogenase genomic integration and copy number optimization In order to optimize the ratio of xylose reductase XR5 and glucose dehydrogenase GDH2 and ensure the consistency of the enzyme activities of XR and GDH per unit cell, the present invention intends to optimize the gene dosage to improve the catalytic efficiency of the whole cell. The genome editing method of Vibrio natrii was carried out according to the NT-CRISPR disclosed in the literature (DOI: 10.1038 / s42003-022-03150-0). Vibrio natrii with 1-8 copies of XR5 integrated were obtained by NT-CRISPR, and the strains were named VnXY-1 to VnXY-8, respectively. The enzyme activity was detected according to the method in Example 1, and the results are shown in Table 4. It can be seen from the results that as the copy number increases, the enzyme activity of XR5 first increases and then decreases, among which the enzyme activity of 5 copies of XR5 is as high as 67.52 U / mL, which is higher than the plasmid type.

[0041] Taking VnXY-5 as the starting strain, 1-5 copies of GDH2 were integrated into the VnXY-5 genome by the NT-CRISPR method to construct strains VnXY-9 to VnXY-13. The XR5 enzyme activity was determined according to the method in Example 1, and the GDH2 enzyme activity was determined according to the method in Example 2. The results are shown in Table 5. As can be seen from the results in Table 5, by integrating XR and GDH into the genome and optimizing the gene dosage by adjusting the copy number, the enzyme activities of GDH2 and XR5 in the final strain VnXY-10 matched, the XR5 enzyme activity was higher than that of single plasmid expression and plasmid co-expression, and the GDH enzyme activity basically reached the effect of single plasmid expression. In this strain, GDH2 is 2 copies and XR5 is 5 copies.

[0042] Table 4 Enzyme activity of integrated XR5

[0043] Table 5 Enzyme activities of integrated XR5 and GDH2

[0044] Example 6 Preparation of xylitol and gluconic acid catalyzed by integrated Vibrio natriuresis whole cells According to the shake flask fermentation method in Example 1, the VnXY-10 strain was cultured and the cells were collected by centrifugation. The 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, 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, the pH of the reaction system was controlled to maintain 7.5 with ammonia water, and samples were taken every 1 hour to detect the content of xylose, xylitol, glucose, and gluconic acid. The experimental results are shown in Figure 2 As shown. Figure 2 It can be seen that with the increase of cell addition, the efficiency of catalytic production of xylitol and gluconic acid increased. Finally, under the condition of adding 4 g / L WCW, 1.5 M xylose can be completely converted into 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.

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 1, characterized in that: The accession number of the xylose reductase gene in the NCBI database is ALO17776.

1.

5. The genetically engineered bacterium according to claim 1, characterized in that: The accession number of the glucose dehydrogenase gene in the NCBI library is WP_013055546.1, WP_012369122.1 or 8W0O_A.

6. The genetically engineered bacterium according to claim 1, characterized in that: In (B), the copy number of the xylose reductase gene is 4 or 5.

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

8. A method for preparing xylitol, characterized in that: Xylose and glucose are used as substrates, and the whole cells of the genetically engineered bacteria described in any one of claims 1 to 7 are used as catalysts to form a catalytic reaction system to produce xylitol and gluconic acid.

9. The genetically engineered bacterium according to claim 8, characterized in that: The reaction temperature is 25-35°C.

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