Recombinant strains, methods and applications of Escherichia coli

Abstract

The present application relates to the technical field of Escherichia coli, and specifically to a recombinant strain, method and application of Escherichia coli. The recombinant strain is a recombinant strain of Escherichia coli containing an operon carrying a xylose reductase gene and a xylose transferase gene. The recombinant strain has a xylose transport efficiency higher than that of its endogenous xylose transport system, and obtains a stable xylitol yield, and has broad application prospects in the synthesis of xylitol and related fields.

Description

Technical Field

[0001] The present application relates to the technical field of Escherichia coli, and in particular to recombinant strains, methods and applications of Escherichia coli. Background Art

[0002] Escherichia coli (E. coli) has the advantages of easy cultivation, fast cell growth, high yield, low cultivation cost, strong continuous fermentation ability, and easy separation and purification of expressed proteins from cells. It has become a widely used host for expressing exogenous proteins and is also often used as a cell factory. It is widely used in the field of industrial fermentation to produce various compounds, thereby exerting its important industrial value. Summary of the invention

[0003] The present application discloses a recombinant strain of Escherichia coli, a preparation method and an application thereof. The recombinant strain can have a higher xylose transport efficiency than its endogenous xylose transport system and obtain a stable xylitol yield, and has a wide range of application prospects in the synthesis of xylitol and related fields.

[0004] Based on this, the embodiments of the present application disclose at least the following technical solutions:

[0005] In a first aspect, the embodiment discloses a recombinant strain of Escherichia coli. The recombinant strain is an Escherichia coli containing an operon carrying a xylose reductase gene and a xylose transferase gene.

[0006] In the second aspect, the embodiment discloses a method for preparing a recombinant strain of Escherichia coli. The preparation method comprises: obtaining Escherichia coli; obtaining an operon carrying a xylose reductase gene and a xylose transferase gene; obtaining a recombinant plasmid containing the operon; and transferring the recombinant plasmid into the Escherichia coli.

[0007] In the third aspect, the embodiment discloses a method for preparing xylitol, which comprises: obtaining the recombinant strain described in the first aspect or the recombinant strain prepared by the preparation method described in the second aspect; fermenting the recombinant strain; and harvesting the xylitol from the fermentation product.

[0008] In a fourth aspect, the embodiments disclose applications of the recombinant strain described in the first aspect or the recombinant strain prepared by the preparation method described in the second aspect, wherein the application is selected from at least one of the following: synthesizing xylitol; preparing food additives; or preparing nutritional supplements. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The growth curves (A), xylitol production in the fermentation broth after 72 h (B), and xylose consumption after 72 h of fermentation (C) of the recombinant strain DH5α-xyrA and the recombinant strain BL21-xyrA provided in the examples.

[0010] Figure 2 The growth curves (A), xylitol concentration in the fermentation broth after 72 h (B), and xylose consumption after 72 h of fermentation (C) of the recombinant strain Trans110-xyrA and the recombinant strain BL21-xyrA provided in the examples.

[0011] Figure 3 The growth curve of the recombinant strain BL21-xyrA provided in the example in fermentation broth with different xylose contents (A), the xylitol production in the fermentation broth after 60 h (B), and the xylose consumption after 60 h of fermentation (C).

[0012] Figure 4 Growth curves (A), xylitol production in 121 h fermentation broth (B), and xylose consumption after 121 h of fermentation (C) of the recombinant strains BL21-xyrA, BL21-xyrA-glf-A18T, BL21-xyrA-glf-1028+T, BL21-xyrA-V275F, and BL21-xyrA-glf-2-RD5 provided in the examples.

[0013] Figure 5 Growth curves (A) and xylitol yield in 120 h fermentation broth (B) of the recombinant strains BL21-xyrA, BL21-xyrA-glf-L445I, and BL21-xyrA-sthA-glf-L445I provided in the examples.

[0014] Figure 6 Schematic diagram of the structures of various operons (Pgap-xyrA, Pgap-xyrA-RBS2-glf, Pgap-xyrA-RBS2-glf-A18T, Pgap-xyrA-RBS2-glf-V275F, Pgap-xyrA-RBS2-glf-2-RD5, Pgap-xyrA-RBS2-glf-1028+T, Pgap-xyrA-RBS2-glf-L445I, Pgap-xyrA-RBS2-sthA-Pgap-glf-L445I) provided in the embodiments. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0016] Xylitol is a five-carbon sugar alcohol that can be used as a sweetener to replace sucrose. It is more helpful in preventing and treating dental caries. It is widely used in food processing, pharmaceutical manufacturing, chemical industry and other industries due to its unique physiological effects and excellent physical and chemical properties. In the prior art, chemical synthesis methods are often used to produce xylitol, but the environmental pollution problem is serious. Biosynthesis has gradually replaced chemical methods as a new method for producing xylitol. The synthesis of xylitol in microorganisms mainly follows the oxidoreductase pathway, also known as the xylose reductase (XR) and xylose dehydrogenase (XDH) pathways. Under the action of xylose reductase, xylose is directly converted into xylitol with reduced NAD(P)H as a cofactor. Subsequently, xylitol is catalytically oxidized to xylulose with NAD+ as a cofactor under the action of xylitol dehydrogenase. Xylulose is further converted into 5-phospho-xylulose by phosphorylation of xylulokinase, enters the pentose phosphate pathway, and finally enters the tricarboxylic acid cycle.

[0017] The term "recombinant strain" refers to a strain obtained by genetically modifying a wild-type strain, for example, by introducing a foreign gene into the strain.

[0018] However, the present application introduces an operon carrying the exogenous gene xylose reductase gene xyrA into Escherichia coli to construct a recombinant strain, which can obtain a xylose transport efficiency higher than that of its endogenous xylose transport system and obtain a stable xylitol yield, and has broad application prospects in the synthesis of xylitol and related fields.

[0019] In a first aspect, the embodiment discloses a recombinant strain of Escherichia coli. The recombinant strain is an Escherichia coli containing an operon carrying a xylose reductase gene and a xylose transferase gene.

[0020] In some embodiments, the xylose reductase gene is derived from Zymomonas mobilis ZM4 strain.

[0021] In some embodiments, the Escherichia coli is selected from Escherichia coli BL21, Escherichia coli DH5α, and Escherichia coli Trans110.

[0022] In the second aspect, the embodiment discloses a method for preparing a recombinant strain of Escherichia coli. The preparation method comprises: obtaining Escherichia coli; obtaining an operon containing a xylose reductase gene and a xylose transferase gene; obtaining a recombinant plasmid containing the operon; and transferring the recombinant plasmid into the Escherichia coli.

[0023] In some embodiments, the operon is formed by sequentially connecting a Pgap promoter (SEQ ID NO: 1), a xyrA gene (SEQ ID NO: 2), a RBS2 sequence (SEQ ID NO: 3) and a glf gene (SEQ ID NO: 4).

[0024] In some embodiments, the operon is formed by sequentially connecting the Pgap promoter, the xyrA gene, the RBS2 sequence and the glf-A18T gene (SEQ ID NO: 5).

[0025] In some embodiments, the operon is formed by sequentially connecting the Pgap promoter, the xyrA gene, the RBS2 sequence and the glf-V275F gene (SEQ ID NO: 6).

[0026] In some embodiments, the operon is formed by sequentially connecting the Pgap promoter, the xyrA gene, the RBS2 sequence and the glf-2-RD5 gene (SEQ ID NO: 7).

[0027] In some embodiments, the operon is formed by sequentially connecting the Pgap promoter, the xyrA gene, the RBS2 sequence and the glf-1028+T gene (SEQ ID NO: 8).

[0028] In some embodiments, the operon is formed by sequentially connecting the Pgap promoter, the xyrA gene, the RBS2 sequence and the glf-L445I gene (SEQ ID NO: 9).

[0029] In some embodiments, the operon is formed by sequentially connecting the Pgap promoter, the xyrA gene, the RBS2 sequence, the sthA gene (SEQ ID NO: 10), the Pgap promoter and the glf-L445I gene.

[0030] In some embodiments, the recombinant plasmid includes a pEZ15A plasmid and the operon inserted into the pEZ15A plasmid.

[0031] Among them, pEZ15A was obtained by referring to the method of "Yang S, Mohagheghi A, Franden MA, et al. Metabolic engineering of Zymomonas mobilis for 2,3-butanediol production from lignocellulosic biomass sugars [J]. Biotechnol Biofuels, 2016, 9 (1): 189." To obtain pEZ15A encoding different genes (such as resistance genes), different marker genes can be inserted into it by referring to "Construction and application of plasmid pUC19-CM-D [J]. Anhui Agricultural Science, 2010, Public Methods, Issue 19".

[0032] Containing pEZ-Pgap-xyrA recombinant plasmid and BL21-xyrA recombinant strain

[0033] The embodiment provides a recombinant plasmid (pEZ-Pgap-xyrA) of an operon (Pgap-xyrA) formed by sequentially connecting the Pgap promoter (SEQ ID NO: 1) and the xyrA gene (SEQ ID NO: 2). The recombinant plasmid was transformed into Escherichia coli BL21 (Bio-Lab, BTN12-25y) to obtain a recombinant strain.

[0034] 1. Preparation of recombinant plasmid pEZ-Pgap-xyrA

[0035] In some embodiments, the step of preparing the pEZ-Pgap-xyrA recombinant plasmid includes: using ZMOxyrA-Pgap-F and ZMOxyrA-Pgap-R as a primer pair to PCR amplify the ZM4 genome sequence of Zymomonas mobilis to obtain the xyrA sequence; using pEZ-FK-F and Pgap-anti-R as a primer pair to PCR amplify pEZ15Asp (containing the spectinomycin resistance gene) to obtain a pEZ15Asp reverse-amplified sequence containing Pgap; the xyrA sequence and the pEZ15Asp reverse-amplified sequence containing Pgap are assembled by Gibson to obtain a first connection sequence (containing the operon Pgap-xyrA); the first connection sequence is transferred into Escherichia coli DH5α (Shanghai Lianmai Biotechnology, LM-81024); positive clones are screened; and the pEZ-Pgap-xyrA recombinant plasmid is extracted from the culture of the positive clones.

[0036] The reaction system assembled by Gibson is shown in Table 1. After the reaction system was placed on ice for 5 minutes, competent Escherichia coli BL21 was added for transformation. Positive colonies were screened using a plate containing 100 μg / mL spectinomycin, and the positive colonies were verified by colony PCR.

[0037] Table 1

[0038] Reagents volume DNA fragment 0.12pM Vector 0.04pM 10×Buffer4(Thermo) 0.5μL T5Exonuclease 0.5U <![CDATA[ddH2O]]> To5μL

[0039] The reaction system (10 μL) of the colony PCR was: 1 μL of water-soluble bacteria, 0.4 μL of 10 μM 15A-fwd (SEQ ID NO: 11) / 15A-rev (SEQ ID NO: 12) primers, 5 μL of 1×T5 Super PCR Mix, and double-distilled sterile water was added to 10 μL. The reaction program of the colony PCR was: 98°C pre-denaturation for 3 min; 98°C denaturation for 10 s, 55°C annealing for 10 s, 72°C extension for 50 s, for a total of 26 cycles.

[0040] 2. Preparation of BL21-xyrA recombinant strain

[0041] In some embodiments, the step of transforming the pEZ-Pgap-xyrA recombinant plasmid into Escherichia coli BL21 to obtain the BL21-xyrA recombinant strain comprises:

[0042] (1) Preparation of competent E. coli strain

[0043] Streak E. coli BL21 on LB plate (10g / L sodium chloride, 10g / L tryptone, 5g / L yeast extract, 3g / L agar) and invert at 37℃ for 1 day for activation. Pick the activated single colony and transfer it to 10mL LB liquid medium (10g / L sodium chloride, 10g / L tryptone, 5g / L yeast extract), and culture it at 37℃, 250rpm shaking incubator until the mid-logarithmic phase as seed liquid. Transfer the seed liquid to a 500mL Erlenmeyer flask containing 100mL LB liquid medium, control the initial OD between 0.025 and 0.03, and culture it at 18℃, 250rpm shaking incubator for about 48h until OD = 0.5-0.8, and cool it on ice for 30min; centrifuge at 4℃, 4000rpm / min for 10min to collect the bacteria. Resuspend the cells with 4°C TB buffer, let stand on ice for 5 min, shake vigorously and let stand, repeat 3 times, and finally collect the cells by centrifugation at 4°C and 3000 rpm for 10 min; resuspend the cells with 4°C TB buffer (10×OD600), slowly add DMSO with a final concentration of 7%, divide into portions, quickly freeze in liquid nitrogen, and store at -80°C.

[0044] (2) Transform the pEZ-Pgap-xyrA recombinant plasmid into the target Escherichia coli competent cells

[0045] Take the competent cells of E. coli BL21 on ice, wait for the competent cells to melt, take 30μL and add it to a 1.5mL EP tube, add 1-2μL plasmid, mix well, and let it stand on ice for 30min. After standing, put the EP tube into 42℃ heat shock for 45s, and immediately put it on ice to cool for 2min after the heat shock is completed; then add 200μL of LB liquid culture medium, and after 1h of recovery culture on a shaker at 37℃ and 250rpm, the culture liquid is coated on a plate containing 100μg / mL spectinomycin, and placed in a 37℃ incubator for inverted culture for 12h. After positive colonies grow out, use colony PCR to verify that the correct colony is the colony of the recombinant strain. After the correct positive clone is activated in the resistant liquid LB culture medium, the bacteria are preserved with glycerol.

[0046] The reaction system (10 μL) of the colony PCR was: 1 μL of water-soluble bacteria, 0.3 μL of 10 μM upstream primer 15A-fwd, 0.3 μL of 10 μM downstream primer 15A-rev, 5 μL of 2×T5 Super PCR Mix (Tsingke), and double-distilled sterile water was added to 10 μL. The reaction program of the colony PCR was: 98°C pre-denaturation for 3 min; 98°C denaturation for 10 s, 55°C annealing for 10 s, 72°C extension (set according to the fragment length at 10 s / kb), a total of 26 cycles; after the cycle reaction, keep 72°C for 2 min.

[0047] 3. Fermentation test of BL21-xyrA recombinant strain

[0048] The recombinant strain (BL21-xyrA) containing the pEZ-Pgap-xyrA recombinant plasmid was constructed for fermentation test. The test steps include: inoculating the glycerol bacteria of BL21-xyrA into a cryotube containing 1mL LB (with 2μL spectinomycin), culturing at 37°C, 250rpm shaking in a turbidity, culturing as a seed liquid at 37°C, 250rpm shaking in a 100mL Erlenmeyer flask to the middle and late logarithmic phase, inoculating into the culture medium LBX10 at 20% bottling volume and fermenting in the culture medium in a 100mL Erlenmeyer flask. The initial OD was controlled to be 0.1 at OD600. During the fermentation process, the optical density at OD value 600nm was measured with an ultraviolet spectrophotometer, the cell growth at different time points was determined, and the fermentation broth at the end of the fermentation was collected and then used in HPLC (high performance liquid chromatograph) to detect the content of xylose and xylitol.

[0049] HPLC detection of xylose content: Agilent 1100 series high performance liquid chromatograph (LC-20AD) of Shimadzu Trading Co., Ltd. was used; the detector was a differential refractive index detector (RID-10A); the chromatographic column was an organic acid chromatographic column (Bio-Rad Aminex HPX-87H, 300 mm×7.8 mm); the column oven temperature was 60°C; the mobile phase was 5 mM sulfuric acid, the flow rate was 0.5 mL / min, and the initial flow rate was set to 0.2 mL / min when the instrument was running. After the column pressure stabilized, the flow rate was gradually increased to 0.5 mL / min at a rate of 0.1 mL / min; the injection volume was 20 μL.

[0050] HPLC detection of xylitol content: Xylitol was separated using an aminex HPX-87H ion exchange column (Bio-Rad, CA, USA) and detected using a SPD-20A UV detector. The analysis was performed at 40°C with 5 mM sulfuric acid as the mobile phase at a flow rate of 0.5 mL / min. Each sample was analyzed three times.

[0051] The same method was used to transfer the recombinant plasmid pEZ-Pgap-xyrA into Escherichia coli DH5α and Escherichia coli Trans110 (Kelei Biotechnology, KL-J-0054), respectively, to obtain the recombinant strains DH5α-xyrA and Trans110-xyrA, respectively, and fermentation tests were performed on these strains.

[0052] Figure 1 The growth curves of the recombinant strain DH5α-xyrA and the recombinant strain BL21-xyrA, the xylitol production of the fermentation broth at 72h, and the xylose consumption at 72h (the difference between the xylose concentration in the fermentation broth at the end of fermentation (72h) and the start of fermentation (0h)) are shown. It can be seen that the recombinant strain BL21-xyrA has a larger cell mass at 72h, and the xylitol production can reach 64g / L, which is higher than the recombinant strain DH5α-xyrA.

[0053] Figure 2 The growth curves of the recombinant strain Trans110-xyrA and the recombinant strain BL21-xyrA, the xylitol concentration of the fermentation broth at 72h, and the xylose consumption at 72h of fermentation are shown. It can be seen that the recombinant strain BL21-xyrA has a larger cell mass at 72h and a higher xylitol production than the recombinant strain Trans110-xyrA.

[0054] Optimizing the substrate xylose concentration

[0055] The embodiment also discloses a method for preparing xylitol, which comprises: fermenting the recombinant strain BL21-xyrA; and harvesting the xylitol from the fermentation product.

[0056] Among them, the BL21-xyrA strain was fermented in fermentation medium (LBX2, LBX4, LBX6, LBX8, LBX10) containing different xylose concentrations for 60 h, and the bacterial liquid was taken during the fermentation to measure the OD 600 A growth curve was drawn and the fermentation broth at the end point of the fermentation was taken for HPLC detection.

[0057] Among them, LBX2 medium contains 10g / L sodium chloride, 10g / L tryptone, 5g / L yeast extract and 20g / L xylose. LBX4 medium contains 10g / L sodium chloride, 10g / L tryptone, 5g / L yeast extract and 40g / L xylose. LBX6 medium contains 10g / L sodium chloride, 10g / L tryptone, 5g / L yeast extract and 60g / L xylose. LBX8 medium contains 10g / L sodium chloride, 10g / L tryptone, 5g / L yeast extract and 80g / L xylose. LBX10 medium contains 10g / L sodium chloride, 10g / L tryptone, 5g / L yeast extract and 100g / L xylose.

[0058] Results of the fermentation experiment Figure 3 The results showed that the biomass of E. coli tended to increase with the increase of xylose concentration, indicating that high concentration of xylose promoted the growth of E. coli, which may be related to the endogenous xylose metabolic pathway in E. coli. Higher concentration of xylose enables E. coli to better utilize xylose for metabolism. Figure 3 The results show that as the xylose concentration in the culture medium increases, the xylitol production of the recombinant strain increases and the xylose consumption gradually increases. This indicates that the production of xylitol by the recombinant expression strain BL21-xyrA is positively correlated with the xylose concentration in the culture medium. A culture medium with a high xylose concentration can be used for fermentation to obtain a higher xylitol yield.

[0059] Recombinant plasmid and recombinant strain containing glf gene and its mutant

[0060] Substrate transport is a key step in the entire metabolic system that converts substrate raw materials into target chemicals or fuels. Improving the efficiency of xylose transport is very important for the high yield of xylitol in the heterologous xylitol synthesis pathway. There are two main mechanisms for xylose to enter the cell of Escherichia coli: one is the high-affinity D-xylose ABC transporter XylFGH; the other mechanism is through the low-affinity xylose-H+ proton symporter XylE. The glucose transporter glf from Zymomonas mobilis is currently the only known bacterial glucose promoter that catalyzes the diffusion of monosaccharides as a monosaccharide transporter and belongs to the major promoter superfamily (MFS). Due to the passive transport mechanism of glf that can transport xylose without additional energy input, it is an energy-saving alternative to other costly xylose transport systems, and mutants A18T, V275F, 2-RD5, 1028+T, and L445I have been shown to improve the xylose transport efficiency, indicating that the xylose transport kinetics of glf can be enhanced by mutagenesis.

[0061] The embodiment provides a recombinant plasmid of an operon consisting of Pgap, xyrA and glf genes, and a recombinant plasmid of an operon consisting of Pgap, xyrA and a glf mutant. These recombinant plasmids were respectively transformed into Escherichia coli BL21 to obtain a variety of recombinant strains capable of efficiently producing xylitol.

[0062] 1. pEZ-Pgap-xyrA-glf recombinant plasmid

[0063] The preparation of pEZ-Pgap-xyrA-glf operon includes: using xyrA-RBS2-R primer (SEQ ID NO: 13) and PEZ-XYRA-ZT-F primer (SEQ ID NO: 14) to amplify pEZ-Pgap-xyrA recombinant plasmid to obtain the reverse amplification sequence of pEZ-Pgap-xyrA; using glf-V275F-pgap-R primer (SEQ ID NO: 15) and glf-V275F-F primer (SEQ ID NO: 16) to amplify pEZ-Pgap-xyrA recombinant plasmid to obtain the reverse amplification sequence of pEZ-Pgap-xyrA; NO:16) amplify the ZM4 genome to obtain the glf sequence; obtain the second connection sequence (containing the operon Pgap-xyrA-RBS2-glf) by Gibson assembly of the reverse amplification sequence of pEZ-Pgap-xyrA and the glf sequence; transfer the second connection sequence into Escherichia coli DH5α; screen positive clones; extract the pEZ-Pgap-xyrA-glf recombinant plasmid from the culture of the positive clones.

[0064] 2. pEZ-Pgap-xyrA-glf-A18T recombinant plasmid

[0065] The preparation of pEZ-Pgap-xyrA-glf-A18T operon includes:

[0066] The pEZ-Pgap-xyrA-glf plasmid was amplified by PCR using A18T-F (SEQ ID NO: 17) and Pgap-anti-R (SEQ ID NO: 18) as a primer pair and ZMOxyrA-Pgap-F (SEQ ID NO: 19) and A18T-R (SEQ ID NO: 20) as a primer pair, and the two obtained DNA fragments were assembled by Gibson to obtain a third connection sequence (containing the operon Pgap-xyrA-RBS2-glf-A18T); the third connection sequence was transformed into Escherichia coli DH5α; positive clones were screened; and the pEZ-Pgap-xyrA-glf-A18T recombinant plasmid was extracted from the culture of the positive clones.

[0067] 3. pEZ-Pgap-xyrA-glf-V275F recombinant plasmid

[0068] The preparation of pEZ-Pgap-xyrA-glf-V275F operon includes:

[0069] The pEZ-Pgap-xyrA-glf plasmid was amplified by PCR using V275F-F (SEQ ID NO: 21) and Pgap-anti-R as a primer pair and V275F-R (SEQID NO: 22) and ZMOxyrA-Pgap-F as a primer pair, and the two obtained DNA fragments were assembled by Gibson to obtain a fourth connecting sequence (containing the operon Pgap-xyrA-RBS2-glf-V275F); the fourth connecting sequence was transferred into Escherichia coli DH5α; positive clones were screened; and the pEZ-Pgap-xyrA-glf-V275F recombinant plasmid was extracted from the culture of the positive clones.

[0070] 4. pEZ-Pgap-xyrA-glf-2-RD5 recombinant plasmid

[0071] The preparation of the pEZ-Pgap-xyrA-glf-2-RD5 operon includes: amplifying the pEZ-Pgap-xyrA recombinant plasmid using the xyrA-RBS2-R and PEZ-XYRA-ZT-F primer pairs to obtain the reverse amplification sequence of pEZ-Pgap-xyrA; synthesizing the glf-2-RD5 sequence (SEQ ID NO: 7); obtaining the fifth connection sequence (containing the operon Pgap-xyrA-RBS2-glf-2-RD5) by Gibson assembly of the reverse amplification sequence of pEZ-Pgap-xyrA and the glf-2-RD5 sequence; transferring the fifth connection sequence into Escherichia coli DH5α; screening positive clones; and extracting from the culture of the positive clones to obtain the pEZ-Pgap-xyrA-glf-2-RD5 recombinant plasmid.

[0072] 5. pEZ-Pgap-xyrA-glf-1028+T recombinant plasmid

[0073] The preparation of pEZ-Pgap-xyrA-glf-1028+T operon includes:

[0074] The pEZ-Pgap-xyrA-glf plasmid was amplified by PCR using glf-itmut-F (SEQ ID NO: 23) and Pgap-anti-R as a primer pair and glf-itmut-R (SEQ ID NO: 24) and ZMOxyrA-Pgap-F as a primer pair, and the two obtained DNA fragments were assembled by Gibson to obtain the sixth connecting sequence (containing the operon Pgap-xyrA-RBS2-glf-1028+T); the sixth connecting sequence was transformed into Escherichia coli DH5α; positive clones were screened; and the pEZ-Pgap-xyrA-glf-1028+T recombinant plasmid was extracted from the culture of the positive clones.

[0075] 6. pEZ-Pgap-xyrA-glf-L445I recombinant plasmid

[0076] The preparation of pEZ-Pgap-xyrA-glf-L445I operon includes:

[0077] pEZ-Pgap-xyrA-glf plasmid was amplified by PCR using glf-L445I-F (SEQ ID NO: 25) and Pgap-anti-R as primer pairs and glf-D445I-R (SEQ ID NO: 26) and ZMOxyrA-Pgap-F as primer pairs, and the obtained two DNA fragments were assembled by Gibson to obtain the seventh connecting sequence (containing the operon Pgap-xyrA-RBS2-glf-L445I); the seventh connecting sequence was transformed into Escherichia coli DH5α; positive clones were screened; and the pEZ-Pgap-xyrA-glf-L445I recombinant plasmid was extracted from the culture of the positive clones.

[0078] 7. pEZ-Pgap-xyrA-sthA-Pgap-glf-L445I recombinant plasmid

[0079] The preparation of the pEZ-Pgap-xyrA-glf-L445I operon includes: using the xyrA-RBS2-R and PEZ-XYRA-ZT-F primers to amplify the pEZ-Pgap-xyrA recombinant plasmid to obtain the reverse amplification sequence of pEZ-Pgap-xyrA; using the xyrA-RBS-sthA-F primer (SEQ ID NO: 29) and sthA-nonh-R primer (SEQ ID NO: 30) to amplify the BL21 genome to obtain the sthA sequence; using the sthA-Pgap-F primer (SEQ ID NO: 31) and pgap-glf-R primer (SEQ ID NO: 32) to amplify the pEZ-Pgap-xyrA recombinant plasmid to obtain the Pgap sequence; using the primers glf-V275F-F (SEQ ID NO: 27) and glf-V275F-pgap-R (SEQ ID NO: 33) to amplify the pEZ-Pgap-xyrA recombinant plasmid to obtain the Pgap sequence; using the primers glf-V275F-F (SEQ ID NO: 27) and glf-V275F-pgap-R (SEQ ID NO: 34) to amplify the pEZ-Pgap-xyrA recombinant plasmid to obtain the Pgap sequence; using the primers glf-V275F- NO:28), using plasmid pEZ-Ptet-spe-glf-L445I as a template for PCR amplification to obtain the glf-L445I sequence; the eighth connecting sequence (containing the operon Pgap-xyrA-RBS2-sthA-Pgap-glf-L445I) was obtained by Gibson assembly of the reverse amplification sequence of pEZ-Pgap-xyrA, the sthA sequence, the Pgap sequence, and the glf-L445I sequence; the eighth connecting sequence was transferred into Escherichia coli DH5α; positive clones were screened; and the pEZ-Pgap-xyrA-sthA-Pgap-glf-L445I recombinant plasmid was extracted from the culture of the positive clones.

[0080] 8. Recombinant strains

[0081] The same method as in the above example was used to recombinant plasmids pEZ-Pgap-xyrA-glf, pEZ-Pgap-xyrA-glf-A18T, pEZ-Pgap-xyrA-glf-V275F, pEZ-Pgap-xyrA-glf-2-RD5, pEZ-Pgap-xyrA-glf-1028+T, pEZ-Pgap-xyrA-glf-L445I and pEZ-Pgap-xyrA-sthA -Pgap-glf-L445 were respectively transformed into Escherichia coli BL21 to obtain recombinant strains BL21-xyrA-glf, BL21-xyrA-glf-A18T, BL21-xyrA-glf-V275F, BL21-xyrA-glf-2-RD5, BL21-xyrA-glf-1028+T, BL21-xyrA-glf-L445I and BL21-xyrA-sthA-Pgap-glf-L445.

[0082] 9. Fermentation test

[0083] LBX10 was used as the fermentation medium, the initial OD600 was controlled to be 0.1, and the fermentation was carried out at 37°C and 250rpm. Figure 4 , Figure 5 As shown, the strain BL21-xyrA-glf-L445I with the highest yield was obtained.

[0084] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A recombinant strain of Escherichia coli, wherein the recombinant strain is an Escherichia coli containing an operon carrying a xylose reductase gene and a xylose transferase gene, wherein the operon is formed by sequentially connecting a Pgap promoter, a xyrA gene, a RBS2 sequence and a glf-L445I gene, wherein: The Pgap promoter is shown as SEQ ID NO:1, the xyrA gene is shown as SEQ ID NO:2, the RBS2 sequence is shown as SEQ ID NO:3, and the glf-L445I gene is shown as SEQ ID NO:

9.

2. A method for preparing xylitol, comprising: Obtaining the recombinant strain according to claim 1; fermenting the recombinant strain; as well as The xylitol is harvested from the fermentation product.

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