CN118599751B

CN118599751B - Recombinant strains, methods and applications of Escherichia coli

Info

Publication number: CN118599751B
Application number: CN202410887652.5A
Authority: CN
Inventors: 杨世辉; 张蕾; 何桥宁; 杜军
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2024-07-03
Filing date: 2024-07-03
Publication date: 2025-02-28
Anticipated expiration: 2044-07-03
Also published as: CN118599751A

Abstract

The application relates to the technical field of escherichia coli, in particular to a recombinant strain of escherichia coli, a method and application thereof. The recombinant strain is an escherichia coli recombinant strain containing an operon carrying xylose reductase genes and xylose transferase genes. The recombinant strain has higher xylose transfer efficiency than an endogenous xylose transfer system, obtains stable xylitol yield, and has wide application prospects in the fields of xylitol synthesis and related fields.

Description

Recombinant strain of escherichia coli, method and application

Technical Field

The application relates to the technical field of escherichia coli, in particular to a recombinant strain of escherichia coli, a method and application thereof.

Background

Coli (e.coli) has advantages of easy culture, fast cell growth, high yield, low culture cost, strong continuous fermentation capability, easy separation and purification of expressed proteins from cells, and the like, becomes a host widely used for expressing exogenous proteins, is also often used as a cell factory, and is widely applied to the production of various compounds in the field of industrial fermentation, thereby exerting important industrial values thereof.

Disclosure of Invention

The application discloses a recombinant strain of escherichia coli, a preparation method and application thereof. The recombinant strain can have higher xylose transfer efficiency than an endogenous xylose transfer system, and stable xylitol yield is obtained, so that the recombinant strain has wide application prospects in the fields of xylitol synthesis and related fields.

Based on the above, the embodiment of the application at least discloses the following technical scheme:

in a first aspect, the examples disclose recombinant strains of E.coli. The recombinant strain is escherichia coli containing an operon carrying xylose reductase genes and xylose transferase genes.

In a second aspect, the examples disclose methods for preparing recombinant strains of E.coli. The preparation method comprises the steps of obtaining escherichia coli, obtaining an operon carrying xylose reductase genes and xylose transferase genes, obtaining a recombinant plasmid containing the operon, and transferring the recombinant plasmid into the escherichia coli.

In a third aspect, the examples disclose a method of preparing xylitol. The method comprises obtaining the recombinant strain according to the first aspect or the recombinant strain produced by the production method according to the second aspect, fermenting the recombinant strain, and harvesting the xylitol from the fermentation product.

In a fourth aspect, embodiments disclose the use of a recombinant strain according to the first aspect or a recombinant strain produced by the production method according to the second aspect, wherein the use is selected from at least one of the group consisting of the synthesis of xylitol, the production of a food additive, and the production of a nutritional supplement.

Drawings

FIG. 1 shows the growth curves (A) of recombinant strains DH 5. Alpha. -xyrA and BL21-xyrA, xylitol production (B) of 72h fermentation broth and xylose consumption (C) of 72h fermentation, provided in examples.

FIG. 2 shows the growth curves (A) of the recombinant strains Trans110-xyrA and BL21-xyrA, the xylitol concentration (B) of the 72h fermentation broth and the xylose consumption (C) of the 72h fermentation.

FIG. 3 shows the growth curves (A) of recombinant strain BL21-xyrA in different xylose fermentation broths, xylitol production (B) of the 60h fermentation broths and xylose consumption (C) of the 60h fermentation.

FIG. 4 shows the growth curves (A) of the recombinant strains BL21-xyrA, BL21-xyrA-glf-A18T, BL-xyrA-glf-1028+T, BL21-xyrA-V275F, BL-xyrA-glf-2-RD 5, the xylitol production (B) of the 121h broth and the xylose consumption (C) of the fermentation for 121h, provided in the examples.

FIG. 5 shows the growth curves (A) of the recombinant strains BL21-xyrA, BL21-xyrA-glf-L445I, BL-xyrA-sthA-glf-L445I and xylitol production (B) of the 120h fermentation broth provided in the examples.

Fig. 6 is a schematic diagram 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) according to an example embodiment.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The reagents which are not specifically described in the present application are conventional reagents and are commercially available, and the methods which are not specifically described in the present application are conventional experimental methods and are known from the prior art.

Xylitol is a five-carbon sugar alcohol, can be used as a sweetener for replacing sucrose, is more beneficial to preventing and treating dental caries, and has wide application in industries such as food processing, pharmaceutical manufacturing, chemical industry and the like due to unique physiological effects and excellent physical and chemical properties. In the prior art, xylitol is produced by a chemical synthesis method, but the problem of environmental pollution is serious. The biosynthesis process gradually replaces the chemical process to become a new xylitol production process. Xylitol synthesis in microorganisms mainly follows the oxidoreductase pathway, also known as Xylose Reductase (XR) and Xylose Dehydrogenase (XDH) pathways. Xylose is directly converted into xylitol by taking reduced NAD (P) H as a cofactor under the action of xylose reductase. Subsequently, xylitol is catalytically oxidized to xylulose under the action of xylitol dehydrogenase with nad+ as cofactor. Xylulose is further converted to 5-phospho-xylulose by phosphorylation of xylulokinase, enters the pentose phosphate pathway, and finally enters the tricarboxylic acid cycle.

The term "recombinant strain" refers to a strain obtained by genetically engineering a wild-type strain, for example, by introducing an exogenous gene into its cells.

However, the recombinant strain constructed by transferring the operon carrying the exogenous gene xylose reductase gene xyrA into the escherichia coli can obtain the xylose transfer efficiency higher than that of an endogenous xylose transfer system, and stable xylitol yield, and has wide application prospects in the fields of xylitol synthesis and related fields.

In some embodiments, the xylose reductase gene is derived from a zymomonas mobilis ZM4 strain.

In some embodiments, the E.coli is selected from the group consisting of E.coli BL21, E.coli DH 5. Alpha., E.coli Trans110.

In a second aspect, the examples disclose methods for preparing recombinant strains of E.coli. The preparation method comprises the steps of obtaining escherichia coli, obtaining an operon containing xylose reductase genes and xylose transferase genes, obtaining a recombinant plasmid containing the operon, and transferring the recombinant plasmid into the escherichia coli.

In some embodiments, the operon is formed by the sequential ligation of the Pgap promoter (SEQ ID NO: 1), the xyrA gene (SEQ ID NO: 2), the RBS2 sequence (SEQ ID NO: 3), and the glf gene (SEQ ID NO: 4).

In some embodiments, the operon is formed by the sequential ligation of the Pgap promoter, the xyrA gene, the RBS2 sequence, and the glf-A18T gene (SEQ ID NO: 5).

In some embodiments, the operon is formed by the sequential ligation of the Pgap promoter, the xyrA gene, the RBS2 sequence, and the glf-V275F gene (SEQ ID NO: 6).

In some embodiments, the operon is formed by the sequential ligation of the Pgap promoter, the xyrA gene, the RBS2 sequence, and the glf-2-RD5 gene (SEQ ID NO: 7).

In some embodiments, the operon is formed by the sequential ligation of the Pgap promoter, the xyrA gene, the RBS2 sequence, and the glf-1028+T gene (SEQ ID NO: 8).

In some embodiments, the operon is formed by the sequential ligation of the Pgap promoter, the xyrA gene, the RBS2 sequence, and the glf-L445I gene (SEQ ID NO: 9).

In some embodiments, the operon is formed by the sequential ligation of the Pgap promoter, the xyrA gene, the RBS2 sequence, the sthA gene (SEQ ID NO: 10), the Pgap promoter, and the glf-L445I gene.

In some embodiments, the recombinant plasmid comprises pEZ a plasmid and the operon inserted into the pEZ a plasmid.

Wherein pEZ a is obtained by reference to "Yang S,Mohagheghi A,Franden M A,et al.Metabolic engineering ofZymomonas mobilis for2,3-butanediolproduction from lignocellulosic biomass sugars[J].Biotechnol Biofuels,2016,9(1):189.". For pEZ a of different coding genes (e.g. resistance genes) reference can be made to the construction and application of the "plasmid pUC19-CM-D [ J ]. The method disclosed in the agricultural science of Anhui, 2010, stage 19" in which different marker genes are inserted.

Contains pEZ-Pgap-xyrA recombinant plasmid and BL21-xyrA recombinant strain

The examples provide recombinant plasmids (pEZ-Pgap-xyrA) of an operon (Pgap-xyrA) formed by the sequential ligation of the Pgap promoter (SEQ ID NO: 1) and the xyrA gene (SEQ ID NO: 2). And transferring the recombinant plasmid into escherichia coli BL21 (Baolaibo, BTN12-25 y) to obtain a recombinant strain.

1. Preparation of recombinant plasmid pEZ-Pgap-xyrA

In some embodiments, the steps of preparing a pEZ-Pgap-xyrA recombinant plasmid comprise PCR amplifying ZM4 genomic sequence of Zymomonas mobilis using ZMOxyrA-Pgap-F and ZMOxyrA-Pgap-R as primer pairs to obtain xyrA sequence, PCR amplifying pEZ Asp (containing spectinomycin resistance gene) using pEZ-FK-F and Pgap-anti-R as primer pairs to obtain pEZ Asp antisense sequence containing Pgap, assembling the xyrA sequence with pEZ Asp antisense sequence containing Pgap to obtain a first junction sequence (containing operon Pgap-xyrA) by Gibbs, transferring the first junction sequence into E.coli DH5 alpha (Shanghai-Mei, LM-81024), screening positive clones, and extracting pEZ-Pgap-xyrA recombinant plasmid from the culture of positive clones.

The reaction system of Gibbsen assembly is shown in Table 1, and after the reaction system is placed on ice for 5min, competent E.coli BL21 is added for transformation. Positive colonies were screened using plates containing 100. Mu.g/mL spectinomycin and verified by colony PCR.

TABLE 1

Reagent(s)	Volume of
		DNAfragment	0.12pM
Vector	0.04pM
		10×Buffer4(Thermo)	0.5μL
T5Exonuclease	0.5U
		ddH₂O	To5μL

Wherein the colony PCR reaction system (10. Mu.L) was 1. Mu.L of water-soluble bacteria, 10. Mu.M of 15A-fwd (SEQ ID NO: 11)/15A-rev (SEQ ID NO: 12) primers each 0.4. Mu.L, 1×T5 Super PCR Mix 5. Mu.L, and double distilled sterile water was supplemented to 10. Mu.L. The colony PCR was performed for a total of 26 cycles with a pre-denaturation at 98℃for 3min, a denaturation at 98℃for 10s, an annealing at 55℃for 10s and an extension at 72℃for 50 s.

2. Preparation of BL21-xyrA recombinant Strain

In some embodiments, the step of transferring pEZ-Pgap-xyrA recombinant plasmid into escherichia coli BL21 to obtain BL21-xyrA recombinant strain comprises:

(1) Preparation of competent E.coli strains of interest

Coli BL21 was streaked on LB plates (10 g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract, 3g/L agar), and cultured for 1 day at 37℃with inversion. The activated single colonies were picked up and transferred to LB liquid medium (10 g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract) containing 10mL and shake cultured at 37℃and 250rpm to mid-log phase for use as seed liquid. Transferring the seed liquid into a 500mL triangular flask containing 100mL of LB liquid culture medium, controlling the initial OD to be between 0.025 and 0.03, performing shake culture at 18 ℃ and 250rpm for about 48 hours until OD=0.5 to 0.8, cooling on ice for 30 minutes, and centrifuging at 4 ℃ and 4000rpm for 10 minutes to collect thalli. Re-suspending the thallus with 4 deg.C TB buffer, standing on ice for 5min, shaking vigorously and standing, repeating for 3 times, centrifuging at 3000rpm for 10min at 4 deg.C, collecting thallus, re-suspending thallus with 4 deg.C TB buffer (10X OD 600), slowly adding 7% DMSO, packaging, quick freezing in liquid nitrogen, and storing at-80 deg.C.

(2) Transferring pEZ-Pgap-xyrA recombinant plasmid into competent cells of target escherichia coli

E.coli BL21 competent cells are taken on ice, 30 mu L of competent cells are taken after the competent cells are melted, the competent cells are added into a 1.5mL EP tube, 1-2 mu L of plasmid is added, the mixture is uniformly mixed, and the mixture is kept stand on ice for 30min. After standing, placing an EP tube into a 42 ℃ heat shock for 45 seconds, immediately placing the EP tube on ice for cooling for 2 minutes after the heat shock is finished, then adding 200 mu L of subpackaged LB liquid culture medium, resuscitating and culturing for 1 hour on a shaking table at 37 ℃ and 250rpm, coating a culture solution with a plate containing 100 mu g/mL of spectinomycin, placing the plate in a 37 ℃ incubator for inversion and culturing for 12 hours, and after positive colonies grow out, verifying that correct colonies are colonies of recombinant strains by adopting colony PCR. The obtained correct positive clone is subjected to glycerol sterilization after being activated in a liquid LB culture medium with resistance.

Wherein the colony PCR reaction system (10. Mu.L) was 1. Mu.L of water-soluble bacteria, 0.3. Mu.L of 10. Mu.M of upstream primer 15A-fwd, 0.3. Mu.L of 10. Mu.M of downstream primer 15A-rev, 5. Mu.L of 2 XT 5 Super PCRMix (Tsingke), and double distilled sterile water was supplemented to 10. Mu.L. The colony PCR was performed by pre-denaturing at 98℃for 3min, denaturing at 98℃for 10s, annealing at 55℃for 10s, and extending at 72℃for 26 cycles (set at 10s/kb depending on the fragment length), and maintaining at 72℃for 2min after the end of the cycling reaction.

3. Fermentation test of BL21-xyrA recombinant strain

A recombinant strain (BL 21-xyrA) containing pEZ-Pgap-xyrA recombinant plasmid was constructed for fermentation testing. The test procedure comprises inoculating BL21-xyrA glycerol bacteria into a freezing tube containing 1mL LB (2. Mu.L spectinomycin) and shaking at 37 ℃ and 250rpm until turbid, shaking at 37 ℃ and 250rpm until late logarithmic phase as seed solution, inoculating to culture medium LBX10, and fermenting in 100mL triangular flask culture medium with 20% bottling amount. The initial OD was controlled to 0.1 at OD 600. In the fermentation process, the optical density at the OD value of 600nm is measured by an ultraviolet spectrophotometer, the cell growth at different time points is measured, and fermentation liquor at the fermentation end point is collected and then used for detecting the content of xylose and xylitol in HPLC (high performance liquid chromatograph).

The HPLC is used for detecting xylose content, namely, an Agilent 1100 series high performance liquid chromatograph (LC-20 AD) of Shimadzu commercial company, inc., a differential refractive detector (RID-10A) is adopted, the chromatographic column is an organic acid chromatographic column (Bio-RadAminex HPX-87H,300mM multiplied by 7.8 mM), the temperature of a column temperature box is 60 ℃, the flow rate of sulfuric acid of 5mM is 0.5mL/min, the initial flow rate is set to 0.2mL/min when the instrument is operated, the flow rate gradually increases to 0.5mL/min after the column pressure is stabilized, and the sample injection amount is 20 mu L.

HPLC detection of xylitol content xylitol was separated using aminexHPX-87H ion exchange column (Bio-Rad, CA, USA) and detected using SPD-20A UV detector. Performed at 40℃with 5mM sulfuric acid as the mobile phase and a flow rate of 0.5mL/min. Three replicates were performed for each sample.

By the same method, recombinant plasmids pEZ-Pgap-xyrA are respectively transferred into escherichia coli DH5 alpha and escherichia coli Trans110 (Ke Lei organism, KL-J-0054) to respectively obtain recombinant strains DH5 alpha-xyrA and Trans110-xyrA. And fermentation tests were performed on these strains, respectively.

FIG. 1 shows the growth curves of recombinant strains DH 5. Alpha. -xyrA and BL21-xyrA, the xylitol production of the 72h fermentation broth, and the xylose consumption (the difference between the fermentation end (72 h) and the xylose concentration in the fermentation broth at the fermentation start (0 h)) for fermentation for 72 h. As can be seen, the recombinant strain BL21-xyrA has more 72h thallus quantity, the xylitol yield can reach 64g/L, and the xylitol yield is higher than that of the recombinant strain DH5 alpha-xyrA.

FIG. 2 shows the growth curves of recombinant strains Trans110-xyrA and BL21-xyrA, the xylitol concentration of the 72h fermentation broth, and the xylose consumption of the fermentation for 72 h. As can be seen, the recombinant strain BL21-xyrA has more 72h cell mass and higher xylitol yield than the recombinant strain Trans110-xyrA.

Optimizing substrate xylose concentration

The example also discloses a method for preparing xylitol. The method comprises fermenting recombinant strain BL21-xyrA, and harvesting xylitol from the fermentation product.

Wherein BL21-xyrA is fermented for 60 hours in fermentation culture mediums (LBX 2, LBX4, LBX6, LBX8 and LBX 10) containing different xylose concentrations respectively, bacterial liquid is taken during fermentation to measure OD ₆₀₀ as a growth curve, and fermentation liquid at the fermentation end point is taken to carry out HPLC detection.

Wherein the LBX2 medium contains 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract and 20g/L xylose. The LBX4 medium contained 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract and 40g/L xylose. The LBX6 medium contained 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract and 60g/L xylose. The LBX8 medium contained 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract and 80g/L xylose. The LBX10 medium contained 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract and 100g/L xylose.

The results of fermentation experiments are shown in FIG. 3, which shows that the biomass of the escherichia coli tends to rise along with the increase of the xylose concentration, and shows that high-concentration xylose has a promoting effect on the growth of the escherichia coli, and probably is related to an endogenous xylose metabolic pathway contained in the escherichia coli, and higher-concentration xylose enables the escherichia coli to better utilize xylose for metabolism in the body. From the results of FIG. 3, it can be seen that the xylitol production of the recombinant strain gradually increases with increasing xylose concentration in the medium, indicating that the yield of xylitol produced by the recombinant expression strain BL21-xyrA is positively correlated with the xylose concentration in the medium, and that a medium with a high xylose concentration can be used for fermentation to obtain a higher xylitol yield.

Recombinant plasmid and recombinant strain containing glf gene and mutant thereof

Substrate transport is a key step in the overall metabolic system for conversion of substrate raw materials to target chemicals or fuels, and improving xylose transport efficiency is important for high xylitol yield of heterologous xylitol synthesis pathways. The intracellular mechanism of xylose entry in E.coli is mainly two, namely a D-xylose ABC transporter XylFGH with high affinity and a xylose-H+ proton cotransporter XylE with low affinity. 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). Since glf is a passive transport mechanism capable of transporting xylose without additional energy input, it is an energy-efficient alternative to other costly xylose transport systems, and mutants a18T, V275F, 2-RD5, 1028+t, L445I proved to increase xylose transport efficiency, suggesting that the transport kinetics of glf's xylose can be enhanced by mutagenesis.

Examples provide recombinant plasmids of operons consisting of the Pgap, xyrA and glf genes, and recombinant plasmids of operons consisting of Pgap, xyrA and glf mutants. And the recombinant plasmids are respectively transferred into escherichia coli BL21 to obtain a plurality of recombinant strains capable of efficiently producing xylitol.

1. PEZ-Pgap-xyrA-glf recombinant plasmid

PEZ-Pgap-xyrA-glf operon preparation comprises amplifying pEZ-Pgap-xyrA recombinant plasmid with xyrA-RBS2-R primer (SEQ ID NO: 13), PEZ-XYRA-ZT-F primer (SEQ ID NO: 14) to obtain a pEZ-Pgap-xyrA reverse amplification sequence, amplifying ZM4 genome with glf-V275F-Pgap-R primer (SEQ ID NO: 15), glf-V275F-F primer (SEQ ID NO: 16) to obtain a glf sequence, assembling pEZ-Pgap-xyrA reverse amplification sequence and glf sequence by Gibbsen to obtain a second ligation sequence (containing operon Pgap-xyrA 2-glf), transferring the second ligation sequence into E.coli DH 5. Alpha., screening positive clones, and extracting pEZ-Pgap-xyrA recombinant plasmid from the positive clones.

2. PEZ-Pgap-xyrA-glf-A18T recombinant plasmid

Preparation of pEZ-Pgap-xyrA-glf-A18T operon included:

PCR amplification of pEZ-Pgap-xyrA-glf plasmid with A18T-F (SEQ ID NO: 17) and Pgap-anti-R (SEQ ID NO: 18) as primer pairs and ZMOxyrA-Pgap-F (SEQ ID NO: 19) and A18T-R (SEQ ID NO: 20) as primer pairs, respectively, to obtain two DNA fragments, gibbsen assembly to obtain the third connecting sequence (containing the operon Pgap-xyrA-RBS 2-glf-A18T), transferring the third connecting sequence into E.coli DH 5. Alpha. Screening positive clones, and extracting pEZ-Pgap-xyrA-glf-A18T recombinant plasmid from the culture of the positive clones.

3. PEZ-Pgap-xyrA-glf-V275F recombinant plasmid

Preparation of pEZ-Pgap-xyrA-glf-V275F operon included:

The pEZ-Pgap-xyrA-glf plasmid is amplified by PCR by using V275F-F (SEQ ID NO: 21) and Pgap-anti-R as primer pairs and V275F-R (SEQ ID NO: 22) and ZMOxyrA-Pgap-F as primer pairs, respectively, the obtained two DNA fragments are assembled by Gibbon to obtain a fourth connecting sequence (containing an operon Pgap-xyrA-RBS 2-glf-V275F), the fourth connecting sequence is transferred into E.coli DH5 alpha, positive clones are screened, and pEZ-Pgap-xyrA-glf-V275F recombinant plasmids are extracted from cultures of the positive clones.

4. PEZ-Pgap-xyrA-glf-2-RD5 recombinant plasmid

PEZ-Pgap-xyrA-glf-2-RD5 operon preparation comprises amplifying pEZ-Pgap-xyrA recombinant plasmid with xyrA-RBS2-R and PEZ-XYRA-ZT-F primer pair to obtain pEZ-Pgap-xyrA reverse-amplified sequence, synthesizing glf-2-RD5 sequence (SEQ ID NO: 7), assembling pEZ-Pgap-xyrA reverse-amplified sequence and glf-2-RD5 sequence by Gibbsen to obtain fifth connecting sequence (containing operon Pgap-xyrA-RBS2-glf-2-RD 5), transferring the fifth connecting sequence into E.coli DH5 alpha, screening positive clones, and extracting pEZ-Pgap-xyrA-glf-2-RD5 recombinant plasmid from the culture of positive clones.

5. PEZ-Pgap-xyrA-glf-1028+T recombinant plasmid

Preparation of pEZ-Pgap-xyrA-glf-1028+T operon included:

The pEZ-Pgap-xyrA-glf plasmid is amplified by PCR using glf-itmut-F (SEQ ID NO: 23) and Pgap-anti-R as primer pairs and glf-itmut-R (SEQ ID NO: 24) and ZMOxyrA-Pgap-F as primer pairs, respectively, the two obtained DNA fragments are assembled by Gibbon to obtain a sixth connecting sequence (containing an operon Pgap-xyrA-RBS 2-glf-1028+T), the sixth connecting sequence is transferred into E.coli DH5 alpha, positive clones are selected, and pEZ-Pgap-xyrA-glf-1028+T recombinant plasmids are extracted from cultures of the positive clones.

6. PEZ-Pgap-xyrA-glf-L445I recombinant plasmid

Preparation of pEZ-Pgap-xyrA-glf-L445I operon included:

The 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, respectively, and the two obtained DNA fragments were assembled by Gibbon to obtain the seventh junction sequence (containing the operon Pgap-xyrA-RBS 2-glf-L445I), the seventh junction sequence was transferred into E.coli DH 5. Alpha. And positive clones were selected, and the pEZ-Pgap-xyrA-glf-L445I recombinant plasmid was extracted from the culture of the positive clones.

7. PEZ-Pgap-xyrA-sthA-Pgap-glf-L445I recombinant plasmid

The pEZ-Pgap-xyrA-glf-L445I operon is prepared by amplifying pEZ-Pgap-xyrA recombinant plasmid by using xyrA-RBS2-R and PEZ-XYRA-ZT-F primer pairs to obtain pEZ-Pgap-xyrA reverse-amplified sequence; the BL21 genome was amplified using the xyrA-RBS-sthA-F primer (SEQ ID NO: 29), sthA-nonh-R primer (SEQ ID NO: 30) to give sthA sequences; the pEZ-Pgap-xyrA recombinant plasmid was amplified using sthA-Pgap-F primer (SEQ ID NO: 31), pgap-glf-R primer (SEQ ID NO: 32) to obtain Pgap sequence, primer glf-V275F-F (SEQ ID NO: 27), primer glf-V275F-Pgap-R (SEQ ID NO: 28), glf-L445I sequence was obtained by PCR amplification using plasmid pEZ-Ptet-spe-glf-L445I as a template, the reverse-amplified sequence, sthA sequence, pgap sequence, glf-L445I sequence of pEZ-Pgap-xyrA were obtained by Gibbsen assembly (comprising operon Pgap-xyrA-RBS 2-sthA-Pgap-glf-L445I), the eighth ligation sequence was transferred into E.coli strain 5α, DH positive clones were selected, and the pEZ-Pgap-xyrA recombinant plasmid was obtained from the cultures of positive clones.

8. Recombinant strains

By the same method as in the above example, these 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 transformed into E.coli BL21, respectively, to give 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.

9. Fermentation test

LBX10 was used as a fermentation medium, the initial OD600 was controlled to 0.1, and fermentation was performed at 37℃and 250 rpm. The results after fermentation are shown in FIG. 4 and FIG. 5, and the strain BL21-xyrA-glf-L445I with the highest yield is obtained.

The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the 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 in SEQ ID NO: 1, the xyrA gene is shown in SEQ ID NO: 2, the RBS2 sequence is shown in SEQ ID NO: 3, and the glf-L445I gene is shown in SEQ ID NO: 9.

2. A method for preparing xylitol, comprising:

Obtaining the recombinant strain according to claim 1;

fermenting the recombinant strain; and

The xylitol is harvested from the fermentation product.