Xylose isomerase mutant as well as nucleic acid molecule, expression vector and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a xylose isomerase mutant as well as a nucleic acid molecule, an expression vector and application thereof. The amino acid sequence of the xylose isomerase mutant is selected from at least one of SEQ ID NO.2-12. According to the invention, more than ten xylose isomerase mutants with high expression activity in Zymomonas mobilis are obtained through big data mining, semi-rational transformation, heterologous recombinant expression and function screening, and the awkward situation that the active xylose isomerase of Zymomonas mobilis is deficient is solved. Zymomonas mobilis is used as a host strain for XI protein expression, the obtained mutants are subjected to heterologous expression, and the xylose isomerase mutant can endow a recombinant strain with high metabolic capability on xylose, so that the utilization rate of xylose and the yield of ethanol are effectively improved; the method has important industrial application value and potential for constructing recombinant microorganisms capable of efficiently converting xylose and obtaining lignocellulose fuel ethanol production strains with excellent characters.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a xylose isomerase mutant, its nucleic acid molecule, expression vector and application. Background Art

[0002] Lignocellulose widely exists in various plants, especially in wood and crop straws such as corn stover and sugarcane bagasse. As the most abundant renewable biomass resource, its efficient conversion and utilization has become the research focus in the field of green biomanufacturing. Using microbial fermentation to convert lignocellulose into high-value products, such as chemicals, materials and biofuels, especially the production of fuel ethanol from lignocellulose is its main conversion and utilization approach. This conversion method not only helps to reduce the dependence on fossil resources, but also can effectively reduce carbon dioxide emissions, thus coping with the challenges brought by global climate change. Lignocellulose is mainly composed of cellulose, hemicellulose and lignin. After pretreatment and enzymatic hydrolysis, glucose and xylose can be released, among which xylose accounts for 30%-40% of the hydrolysis products and is the second most abundant monosaccharide. Efficient xylose conversion is the core issue for making full use of lignocellulose resources and improving the utilization rate of its hydrolysis products.

[0003] Zymomonas mobilis (Z. mobilis), a Gram-negative bacterium, has a unique Entner-Doudoroff (ED) metabolic pathway and a high sugar fermentation efficiency. It can convert glucose, fructose and sucrose into ethanol. Due to the high expression of its pyruvate decarboxylase and alcohol dehydrogenase genes, its ethanol fermentation ability is very prominent. In addition, Zymomonas mobilis has a high tolerance to lignocellulose hydrolysate, and the research on the tolerance mechanism to inhibitors in the hydrolysate is also relatively mature. Moreover, Zymomonas mobilis has many industrial production advantages such as low fermentation cost, simple process, stable genome and suitability for large-scale long-term fermentation. Given these unique physiological characteristics and excellent industrial production advantages of Zymomonas mobilis, it has great application potential as a cellulose ethanol production platform. However, natural Zymomonas mobilis lacks a xylose metabolic pathway, which greatly restricts its development and application in the high-value conversion of lignocellulose.

[0004] There are two main xylose metabolic pathways in nature: one is the redox pathway catalyzed by xylose reductase (XR) and xylitol dehydrogenase (XDH), and the other is the isomerization pathway catalyzed by xylose isomerase (XI). The redox pathway has different cofactor preferences for XR and XDH (NADPH and NAD +) It is likely to lead to intracellular cofactor imbalance and the accumulation of toxic byproduct xylitol, restricting its application in industrial production. Xylose isomerase (XI) has unique advantages in the bioconversion of lignocellulose because it can directly convert xylose into xylulose through a one-step isomerization reaction without the participation of cofactors. Despite the many advantages of the XI pathway, there are still some challenges in practical applications. For example, although XI from bacteria can be normally expressed in Zymomonas mobilis, its low enzyme activity in the low-temperature and weakly acidic environment within Zymomonas mobilis hinders the utilization of lignocellulosic raw materials by recombinant Zymomonas mobilis. Therefore, screening for XI that can be highly expressed in Zymomonas mobilis and optimizing xylose metabolism ability are of great significance for promoting the efficient conversion and utilization of lignocellulose. Summary of the Invention

[0005] Aiming at the problems of the lack of xylose isomerase applicable to Zymomonas mobilis and / or the low xylose metabolism ability of Zymomonas mobilis in the prior art, the present invention provides a xylose isomerase mutant, a nucleic acid molecule containing its coding gene, and an expression vector, and further provides the application of the foregoing xylose isomerase mutant, nucleic acid molecule, and expression vector in enhancing the xylose conversion ability of microorganisms or preparing products that require the participation of xylose isomerase. The present invention is achieved through the following technical solutions:

[0006] In the first aspect of the present invention, a xylose isomerase mutant is provided, and its amino acid sequence is selected from at least one of SEQ ID NO.2 - 12.

[0007] Furthermore, the amino acid sequence of the xylose isomerase mutant is selected from at least one of SEQ ID NO.2, SEQ ID NO.8 - 12; more preferably, from at least one of SEQ ID NO.9 and SEQ ID NO.12.

[0008] In the second aspect of the present invention, a nucleic acid molecule is provided, which includes a gene sequence encoding the xylose isomerase mutant as described above.

[0009] Furthermore, the nucleic acid molecule further includes a gene sequence encoding a promoter, and the xylose isomerase mutant is located downstream of the promoter.

[0010] Furthermore, the promoter is a constitutive strong promoter; more preferably, the promoter is the enolase promoter Peno.

[0011] In the third aspect of the present invention, an expression vector is provided, and the expression vector includes the nucleic acid molecule as described above.

[0012] Furthermore, the expression vector is a pEZ15a or pTZ28a vector.

[0013] The fourth aspect of the present invention provides the application of the xylose isomerase mutant, nucleic acid molecule or expression vector as described above in enhancing the xylose conversion ability of microorganisms.

[0014] Furthermore, the microorganism is Zymomonas mobilis.

[0015] The fifth aspect of the present invention provides the application of the xylose isomerase mutant, nucleic acid molecule or expression vector as described above in the preparation of products that require the participation of xylose isomerase.

[0016] The advantages and positive effects of the present invention are as follows: By using big data mining, semi-rational modification, heterologous recombinant expression and functional screening, more than a dozen xylose isomerase mutants with high expression activity in Zymomonas mobilis are obtained, solving the dilemma of the lack of active xylose isomerase in Zymomonas mobilis. Using Zymomonas mobilis as the host strain for XI protein expression, heterologous expression of each obtained mutant is carried out. The xylose isomerase mutant can endow the recombinant strain with high metabolic ability for xylose, effectively improving the utilization rate of xylose and the yield of ethanol, and has important industrial application value and potential for constructing recombinant microorganisms with efficient xylose conversion and obtaining lignocellulosic fuel ethanol production strains with excellent traits. Brief Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 It is a fermentation performance test chart of recombinant Zymomonas mobilis transformed with xylose isomerase genes of different species in RMX5 and RMX10 media in the embodiments of the present invention; among them, Figures A - B are RMX5 and RMX10 media respectively;

[0019] Figure 2 It is a fermentation performance test chart of recombinant Zymomonas mobilis transformed with single-point mutants H20N, S114E, F145Y, T146S, V186I, V186L, V267I, V267L, S375E, K341R, Q415H, K420Q, R425H of PanXI in RMX5 medium in the embodiments of the present invention;

[0020] Figure 3Fermentation performance test chart of the recombinant strains transformed with the PanXI combined mutants F145Y-V267I, F145Y-V267L, F145Y-K341R, F145Y-S375E, and F145Y-K420Q in the RMX5 medium in the embodiments of the present invention;

[0021] Figure 4 Fermentation performance test chart of the recombinant strains transformed with the PanXI combined mutants V267I-F145Y, V267I-K341R, V267I-S375E, and V267I-K420Q in the RMX5 medium in the embodiments of the present invention;

[0022] Figure 5 Fermentation performance test chart of the recombinant strains transformed with the PanXI combined mutants V267L-F145Y, V267L-341R, V267L-S375E, and V267L-K420Q in the RMX5 medium in the embodiments of the present invention;

[0023] Figure 6 Test chart of the effect of temperature on the enzyme activity of wild-type PanXI and the mutants F145Y-V267L and F145Y-K420Q in the embodiments of the present invention;

[0024] Figure 7 Test chart of the effect of pH on the enzyme activity of wild-type PanXI and the mutants F145Y-V267L and F145Y-K420Q in the embodiments of the present invention;

[0025] Figure 8 Test chart of the effect of pH on the enzyme stability of wild-type PanXI and the mutants F145Y-V267L and F145Y-K420Q in the embodiments of the present invention;

[0026] Figure 9 Test chart of the specific enzyme activity of wild-type PanXI and the mutants F145Y-V267L and F145Y-K420Q under the conditions of temperature 30°C, pH 7.5, and pH 6 in the embodiments of the present invention. Detailed implementation manners

[0027] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] Based on the information contained in this application, various changes can be easily made to the precise description of the present invention by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, as these embodiments and other descriptions are merely for illustrative purposes of specific aspects of the present invention. In fact, all various changes that can be made by those skilled in the art or related fields to the embodiments of the present invention are covered within the scope of the appended claims.

[0029] For a better understanding of the present invention rather than limiting its scope, all numbers representing amounts, percentages, and other numerical values used in this application should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may vary according to the different desired properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by the conventional rounding method. Additionally, the meanings of terms such as "comprising", "including", "containing", "having", etc. are non-restrictive, that is, other steps and other components can be added without affecting the result.

[0030] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following provides a detailed description of specific embodiments of the present invention.

[0031] In the technology of cellulosic ethanol fermentation, the ability of microorganisms to utilize xylose and convert it into ethanol is the most crucial. Natural Zymomonas mobilis is an industrially commonly used ethanol-producing bacterium, but it lacks the xylose utilization pathway. Therefore, introducing the exogenous xylose isomerase (XI) pathway into Zymomonas mobilis to ferment lignocellulosic hydrolysates to produce ethanol has become a research hotspot. The core of this technology lies in the high-active expression of XI in Zymomonas mobilis to promote the conversion and utilization rate of xylose in lignocellulosic hydrolysates by Zymomonas mobilis. XI can directly convert xylose into xylulose, and xylulose enters the pentose phosphate pathway (PPP) through phosphorylation to generate pyruvate, which is ultimately fermented into ethanol.

[0032] Most of the XIs currently discovered have low expression activity in Zymomonas mobilis, especially in a low-temperature and weakly acidic environment, which severely limits the xylose conversion ability of recombinant Zymomonas mobilis. Therefore, the development and molecular modification of novel XIs are particularly important. In recent years, directed evolution has made remarkable progress in the modification of XIs. Some studies have conducted two rounds of random mutagenesis and screening on Thermotoga neapolitana xylose isomerase (TNXI) and obtained two mutants with increased activity under low-temperature and low-pH conditions: 3A2 (V185T / L282P) and 1F1 (V185T / L282P / F186S). Another study predicted the substrate channel flexibility of Thermoanaerobacterium saccharolyticum XI through molecular dynamics simulation and designed a single-point mutant W139F. The catalytic efficiency (K cat / K m ) of this mutant is 10 times higher than that of the wild type, and it has a longer half-life than the wild type. These studies demonstrate the potential of directed evolution and semi-rational design in improving XI activity.

[0033] Based on in-depth mining of online data in the NCBI database, the present invention obtained 6 candidate xylose isomerase genes from different sources. By constructing a heterologous expression system and performing functional characterization and screening in the Zymomonas mobilis xylose-utilizing strain 8b, a xylose isomerase (PanXI) gene with high expression activity was obtained, and its activity in catalyzing xylose conversion is significantly superior to existing xylose isomerases. To further improve the enzyme activity, based on natural PanXI, the present invention performed semi-rational modification based on consensus sequence analysis, obtained a series of mutants through a combination of site-directed mutagenesis and combinatorial mutagenesis, then ligated each mutant gene with the Peno promoter and inserted it into the pEZ15a expression vector, transformed it into the host strain 8b, obtained multiple recombinant xylose isomerase expression strains, compared the growth, xylose metabolism, and ethanol production capabilities of each group of recombinant strains with those of the wild type, and finally obtained the XI single mutants F145Y, V267I, V267L, K341R, S375E, and K420Q and double mutants F145Y-V267I, F145Y-V267L, F145Y-K341R, F145Y-S375E, and F145Y-K420Q with high activity expression in Zymomonas mobilis. The aforementioned mutants can effectively improve the xylose metabolism ability and ethanol yield of recombinant strains, providing important XI enzyme resources for the development of Zymomonas mobilis for cellulosic ethanol production.

[0034] Based on this, an embodiment of the present invention provides a xylose isomerase mutant, and the xylose isomerase mutant has any one of the following amino acid site mutations (a)-(g) compared with the wild-type xylose isomerase (the amino acid sequence is shown in SEQ ID NO.1):

[0035] (a) Mutate the 145th amino acid of wild-type xylose isomerase from F to Y (F145Y);

[0036] (b) Mutate the 267th amino acid of wild-type xylose isomerase from V to I (V267I);

[0037] (c) Mutate the 267th amino acid of wild-type xylose isomerase from V to L (V267L);

[0038] (d) Mutate the 341st amino acid of wild-type xylose isomerase from K to R (K341R);

[0039] (e) Mutate the 375th amino acid of wild-type xylose isomerase from S to E (S375E);

[0040] (f) Mutate the 420th amino acid of wild-type xylose isomerase from K to Q (K420Q);

[0041] (g) Contain (a), and optionally contain one amino acid site mutation in (b)-(f).

[0042] The above respectively correspond to F145Y, V267I, V267L, K341R, S375E, K420Q, F145Y-V267I, F145Y-V267L, F145Y-K341R, F145Y-S375E, F145Y-K420Q mutants, and their amino acid sequences are shown in SEQ ID NO.2-12 respectively.

[0043] The present invention has obtained more than ten xylose isomerase mutants with high expression activity in Zymomonas mobilis by using big data mining, semi-rational modification, heterologous recombination expression and functional screening, solving the dilemma of the lack of active xylose isomerase in Zymomonas mobilis. Using Zymomonas mobilis as the host strain for XI protein expression, heterologous expression of each obtained mutant is carried out. The xylose isomerase mutant can endow the recombinant strain with high metabolic ability to xylose, effectively improve the utilization rate of xylose and the yield of ethanol, and has important industrial application value and potential for constructing recombinant microorganisms for efficient conversion of xylose and obtaining lignocellulosic fuel ethanol production strains with excellent traits.

[0044] By comprehensively comparing the growth, xylose metabolism, and ethanol production results of different mutants, it is preferred that the xylose isomerase mutant contains at least the F145Y site mutation, that is, it is selected from at least one of the F145Y, F145Y-V267I, F145Y-V267L, F145Y-K341R, F145Y-S375E, and F145Y-K420Q mutants, and their amino acid sequences are shown in SEQ ID NO.2, SEQ ID NO.8-12 respectively.

[0045] More preferably, the xylose isomerase mutant is selected from the F145Y-V267L and F145Y-K420Q mutants. Analysis of the enzymatic properties of the aforementioned mutants showed that: the specific enzyme activity of the mutant F145Y-K420Q was higher than that of the wild type under the conditions of 30°C - 60°C. The specific enzyme activity of the mutant F145Y-V267L was basically equivalent to that of the wild type XI at 30°C - 40°C, but at its optimal temperature of 50°C, the specific enzyme activity was significantly higher than that of the wild type. Under weakly acidic conditions (pH 4 - 6), the specific enzyme activities of the two mutants F145Y-K420Q and F145Y-V267L were both higher than that of the wild type, and they showed stronger acid resistance compared to the wild type, with less loss of enzyme activity during long-term incubation in an acidic solution environment and higher enzyme activity stability. In addition, the pure enzyme activity was measured. At a temperature of 30°C and a weakly alkaline (pH 7.5) environment, the specific activity of the F145Y-K420Q mutant was 2.39 U / mg, which was higher than the specific enzyme activity of the wild type PanXI of 1.91 U / mg. The enzyme activity of the F145Y-V267L mutant was basically equivalent to that of the wild type PanXI. At a temperature of 30°C and a weakly acidic (pH 6) condition, the specific enzyme activities of F145Y-V267L and F145Y-K420Q were 1.74 and 2.09 U / mg respectively, which were significantly higher than the specific activity of the wild type PanXI of 1.52 U / mg. These results show that these mutants have higher enzyme catalytic activity and better acid resistance in an acidic environment. When Zymomonas mobilis ferments xylose without artificially controlling the pH, the initial pH of the xylose fermentation medium is about 5.8 - 6.0, and it can drop to about 4 at the end. The mutant XI with enhanced acid resistance can better adapt to this application scenario, which is beneficial to improving the xylose metabolism performance in Zymomonas mobilis and enhancing the economy and yield of lignocellulosic ethanol. Therefore, in a more preferred embodiment of the present invention, the xylose isomerase mutant is selected from the F145Y-V267L and / or F145Y-K420Q mutants.

[0046] Another embodiment of the present invention provides a nucleic acid molecule, which includes a gene sequence encoding the xylose isomerase mutant as described above.

[0047] The advantages of the nucleic acid molecule over the prior art are the same as those of the xylose isomerase mutant over the prior art as described above, and will not be elaborated here.

[0048] The nucleic acid molecule includes a DNA molecule (such as genomic DNA or cDNA) and / or an RNA molecule (such as mRNA), and the molecule can be single-stranded or double-stranded. The sequence of the nucleic acid molecule can be obtained by conventional means such as codon coding rules based on the XI amino acid (AA) sequence. The full-length sequence or a fragment of the nucleic acid molecule can usually be obtained by PCR amplification, recombination, or artificial synthesis methods.

[0049] Optionally, the nucleic acid molecule further includes a gene sequence encoding a promoter, and the xylose isomerase mutant is located downstream of the promoter. The promoter is used to initiate the transcription and translation processes of XI and can be adaptively selected according to the receptor cell and the expression of the xylose isomerase mutant.

[0050] In a preferred embodiment, in order to achieve the high-level expression of the xylose isomerase mutant, the promoter is selected from constitutive strong promoters, such as the enolase promoter Peno, and its gene sequence is shown in SEQ ID NO.14.

[0051] Another embodiment of the present invention provides an expression vector, and the expression vector includes the nucleic acid molecule as described above.

[0052] The advantages of the expression vector over the prior art are the same as those of the xylose isomerase mutant over the prior art as described above, and will not be elaborated here.

[0053] Optionally, the expression vector includes a prokaryotic expression vector, a eukaryotic expression vector, or a viral expression vector (such as a lentivirus, an adenovirus). Correspondingly, the receptor cells transformed or transfected by the expression vector can be prokaryotic cells and eukaryotic cells, which are selected according to the type of the expression vector. For example, when it is a prokaryotic expression vector, the receptor cells are prokaryotic cells, and common examples of prokaryotic cells include Escherichia coli, Bacillus, Corynebacterium, Zymomonas mobilis, Streptomyces, etc.; when it is a eukaryotic expression vector, the receptor cells are eukaryotic cells, and common examples of eukaryotic cells include Saccharomyces cerevisiae, Saccharomyces pastorianus, filamentous fungi, etc.

[0054] Typical vectors include plasmids (such as pUC series, pET series, pWB series, pGEX series, pDXW series, pBR322, pEZ15a, pTZ28a, pMA5, pPICZα, PIC9K, pSET152), viral vectors, phages (such as λgt4λB, λ-Charon, λΔz1, and M13), cosmids, and minichromosomes. Plasmids are the most commonly used vectors, and in the context of the present invention, unless otherwise specified, plasmids and vectors can be used interchangeably.

[0055] In a preferred embodiment, the expression vector is the pEZ15a or pTZ28a vector. The above nucleic acid molecule is inserted between the multiple cloning sites of the pEZ15a or pTZ28a vector to obtain a recombinant expression vector, which is then transformed into *Zymomonas mobilis* and cultured by conventional methods, and xylose isomerase can be expressed in the cells.

[0056] In a preferred embodiment of the present invention, the method for inserting the nucleic acid molecule into the vector includes: amplifying the above nucleic acid molecule respectively by primer pairs with vector homologous arms, and obtaining a linearized vector by amplifying the vector with primer pairs, and connecting the nucleic acid molecule with homologous arms to the linearized vector by the Gibson assembly method, and a recombinant expression vector can be obtained.

[0057] Another embodiment of the present invention provides the application of the above-mentioned xylose isomerase mutant, the nucleic acid molecule or expression vector containing the xylose isomerase encoding gene in enhancing the xylose conversion ability of microorganisms.

[0058] Optionally, the microorganism is *Zymomonas mobilis*, specifically it can be *Zymomonas mobilis* 8b or ZM4. The above-mentioned nucleic acid molecule or expression vector is transformed into *Zymomonas mobilis* to obtain a recombinant *Zymomonas mobilis* expressing xylose isomerase. This recombinant *Zymomonas mobilis* can efficiently utilize xylose, improve the xylose fermentation ability of the strain and contribute to the conversion of xylose into ethanol, enhancing the cellulose ethanol production ability.

[0059] The transformation of the nucleic acid molecule or expression vector into *Zymomonas mobilis* can be carried out by various methods known in the art, including: CaCl₂ transformation method, calcium phosphate-DNA co-precipitation, electroporation, gene gun bombardment, microinjection, conjugation transfer, liposome-mediated transfection, liposome fusion, etc.

[0060] The embodiments of the present invention also provide the application of the above-mentioned xylose isomerase mutant, the nucleic acid molecule or expression vector containing the xylose isomerase encoding gene in the preparation of products that require the participation of xylose isomerase.

[0061] The above-mentioned nucleic acid molecule or expression vector is transformed into *Zymomonas mobilis*, and xylose isomerase is obtained by induced expression, and then high-purity xylose isomerase can be obtained by conventional methods such as nickel column affinity purification.

[0062] The present invention will be further illustrated below with specific examples. The experimental methods without specific conditions noted in the following examples are usually carried out under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or according to the conditions recommended by the manufacturer.

[0063] The culture medium formulations in the following examples are as follows: (1) RMG5: 50 g / L glucose, 2 g / L KH2PO4, and 10 g / L yeast extract, sterilized at 108 °C for 30 min for standby; (2) RMX5 medium: 50 g / L xylose, 2 g / L KH2PO4, 10 g / L yeast extract. Xylose needs to be prepared as a stock solution and filtered through bacteria, and the remaining components are mixed and sterilized at 121 °C for 30 min for standby; (3) RMX10 medium: 100 g / L xylose, 2 g / L KH2PO4, and 10 g / L yeast extract. 15 g / L agar needs to be added to the solid medium of the aforementioned medium.

[0064] I. Screening of xylose isomerases from different sources

[0065] Through the analysis of the NCBI online database, 6 putative xylose isomerases and their encoding genes from different sources were mined, namely xylose isomerases of Pantoea, Enterobacter cancerogenus, Klebsiella pneumoniae, Lactiplantibacillus pentosus, Levilactobacillus brevis, and Klebsiella oxytoca, named PanXI (NCBI accession number: WP_161733287.1), EncXI (NCBI accession number: CAD5351441.1), KlpXI (NCBI accession number: WP_040169899.1), LapXI (NCBI accession number: P21938.1), LebXI (NCBI accession number: P29443.1), and KloXI (NCBI accession number: WP_004106797.1) in sequence. In order to screen for xylose isomerase genes highly expressed in Zymomonas mobilis, after connecting the above 6 xylose isomerase genes with the constitutive strong promoter - enolase promoter Peno, they were inserted into the multiple cloning site of the pEZ15a expression vector. Using Zymomonas mobilis 8b (the method for constructing the strain can be referred to the literature "Yang S, Franden MA, Wang X, et al. Transcriptomic Profiles of Zymomonas mobilis 8b to Furfural Acute and Long-Term Stress in Both Glucose and Xylose Conditions[J]. Front Microbiol, 2020, 11:13.") as the host strain for genetic transformation, after obtaining the recombinant xylose isomerase strain, xylose metabolism tests were carried out in RMX5 and RMX10 media. The gene and primer sequences for constructing the recombinant expression vector in this example are shown in Table 1, where F and R represent the upstream and downstream primers respectively.

[0066] Table 1 Genes and primer sequences for constructing recombinant expression vectors in the examples of the present invention

[0067]

[0068]

[0069] Construction of the pEZ15a recombinant expression vector: Using the primer pairs pEZ15a - PanXI - F / R, pEZ15a - EncXI - F / R, pEZ15a - KlpXI - F / R, pEZ15a - LapXI - F / R, pEZ15a - LebXI - F / R, pEZ15a - KloXI - F / R respectively, with the sequence synthesized by total gene synthesis as the template, the PanXI, EncXI, KlpXI, LapXI, LebXI, and KloXI genes were obtained by PCR amplification. At this time, homologous arm sequences homologous to the pEZ15a vector were carried at both ends of the genes. In addition, using the primer pair V - pEZ15a - F and V - pEZ15a - Peno - R, with the plasmid pEZ15a (the vector carries the Peno promoter) as the template, a linearized plasmid was amplified. The genes were ligated downstream of the Peno promoter of the linearized pEZ15a vector by the Gibson assembly method to form the recombinant pEZ15a - Peno expression vector. Specifically, it includes: Gel extraction of the above - amplified gene fragments and the linearized plasmid, mixing each gene fragment with the linearized plasmid at a molar concentration ratio of 3:1, adding 0.5 μL of 10×Buffer4 (purchased from NEB, catalog number M0633L) and 0.5 μL of exonuclease (T5 Exonuclease, purchased from NEB, catalog number M0663S), supplementing to 5 μL with appropriate ddH2O, and reacting on ice for 5 min. After the reaction, 20 μL of pre - thawed Escherichia coli DH5α competent cells were added to the reaction system, and left standing on ice for about 25 min. Then heat - shocked in a 42°C water bath for 45 s, added 500 μL of LB liquid medium after ice - bathing for about 2 min, and cultured and resuscitated in a shaker at 37°C and 250 rpm for about 1 h. Finally, the resuscitated culture was spread on a screening plate with spectinomycin, and after overnight culture, the positive recombinants on the plate were verified by colony PCR. After culturing the positive recombinants, plasmids were extracted for Sanger sequencing identification.

[0070] Preparation of competent cells of Zymomonas mobilis: Pick an activated single colony of Zymomonas mobilis into RMG liquid medium, culture overnight at 30°C, transfer an appropriate amount of the bacterial liquid in the logarithmic growth phase to 200 mL of RMG5 liquid medium, control the initial OD 600 to be between 0.025 - 0.05, culture at 30°C and 100 rpm for 4 h, and wait for the OD of the bacterial liquid600 When it reaches 0.3 - 0.5, centrifuge at room temperature at 400 rpm for 10 min to collect the bacteria, then wash once with sterile water and twice with 10% glycerol. Finally, resuspend the bacteria in 200 μL of 10% glycerol to obtain competent cells, and aliquot them into 50 μL portions for standby.

[0071] Electroporation transformation of Zymomonas mobilis 8b: Add 50 μL of Zymomonas mobilis 8b competent cells into an electroporation cuvette, then add 200 ng - 500 ng of the recombinant expression vector to be transformed. Use a Bio-road MicroPulser 165 - 2100 electroporator to perform electroporation according to the program of 1.6 kV / 1.8 kV, 25 μF, and 200 Ω. After electroporation, inoculate the transformed strain into RMG5 liquid medium and incubate at 30 °C for 4 - 6 h for resuscitation. Then spread the culture on a plate with the corresponding resistance, select single colonies for sequencing to verify the successful transformation of the recombinant expression vector, and obtain the target strain.

[0072] Xylose metabolism ability test: Inoculate the target strain into RMX medium containing xylose at an inoculum size with an initial OD 600 of 0.1, and conduct growth and fermentation tests at 30 °C and 100 rpm. Sample every 12 h to measure the OD 600 value of the bacterial liquid of the strain and the xylose concentration. Use high-performance liquid chromatography (HPLC) to measure the xylose and ethanol concentrations in the medium. Use a Shimadzu liquid analysis system equipped with a differential refractive index detector (RID-20A), and the chromatographic column is an organic acid chromatographic column (Bio-Rad Aminex HPX-87H, 300 mm × 7.8 mm); the detection cell temperature is 40 °C, the column oven temperature is 60 °C; the mobile phase is 5 mM sulfuric acid, the flow rate is 0.5 mL / min, and the injection volume is 20 μL. Use an ultraviolet spectrophotometer to measure OD 600 at a wavelength of 600 nm to monitor growth. Use the existing recombinant strains of xylose isomerase (EcXI and RsXI) in the laboratory as controls.

[0073] The relevant results are shown in Figure 1 , where Figure A is the fermentation performance test chart of the recombinant xylose isomerase strain in RMX5 medium, and Figure B is the fermentation performance test chart of the recombinant xylose isomerase strain in RMX10 medium. From left to right are the growth curve charts of the recombinant strains (the abscissa in the figure is time, and the ordinate is the strain concentration (OD 600 value)) and the xylose metabolism curve charts (the abscissa in the figure is time, and the ordinate is the xylose concentration). It can be seen that whether in RMX5 or RMX10 medium, the xylose isomerase PanXI shows stronger heterologous expression activity and higher xylose metabolism ability. Based on this, the following selects the xylose isomerase PanXI for further modification to improve its enzyme activity.

[0074] II. Screening of Xylose Isomerase PanXI Mutants

[0075] 2.1 Screening of Single-Point Mutants

[0076] Consensus sequence analysis was performed on the natural xylose isomerase PanXI (WT group) to determine the single-point mutants of xylose isomerase PanXI, namely H20N, S114E, F145Y, T146S, V267I / L, S375E, V186I / L, K341R, Q415H, K420Q, and R425H. Primers were designed respectively to perform single-point mutation on PanXI, and an expression vector with Peno as the promoter and pEZ15a as the plasmid backbone was constructed. After transforming it into the host strain 8b to obtain the recombinant xylose isomerase recombinant strain, xylose metabolism tests were carried out in RMX5 medium. The related operations are the same as above and will not be elaborated here. The mutation primer pairs for H20N, S114E, F145Y, T146S, V186I, V186L, V267I, V267L, K341R, S375E, Q415H, K420Q, and R425H are respectively: pEZ15a-PanXI H20N -F / R, pEZ15a-PanXI S114E -F / R, pEZ15a-PanXI F145Y -F / R, pEZ15a-PanXI T146S -F / R, pEZ15a-PanXI V186I -F / R, pEZ15a-PanXI V186L -F / R, pEZ15a-PanXI V267I -F / R, pEZ15a-PanXI V267L -F / R, pEZ15a-PanXI K341R -F / R, pEZ15a-PanXI S375E -F / R, pEZ15a-PanXI Q415H -F / R, pEZ15a-PanXI K420Q -F / R, pEZ15a-PanXI R425H -F / R. Using the plasmid pEZ15a-Peno-PanXI as the template, the PanXI mutant gene was obtained by PCR amplification. The primer sequences are shown in Table 2.

[0077] Figure 2The cell growth, xylose consumption, and ethanol production of the recombinant strains transformed with single-point mutants were shown at 24 h of fermentation. It can be seen that at 24 h of fermentation, the xylose consumption rates (sugar consumption rate = (initial sugar concentration - final sugar concentration) / time interval, unit: g / L / h) of the H20N, S114E, F145Y, T146S, V186I, V186L, V267I, V267L, K341R, S375E, Q415H, K420Q, and R425H mutants were 1.28, 1.32, 1.67, 1.41, 1.41, 1.23, 1.53, 1.54, 1.49, 1.49, 0.53, 1.52, and 1.44 g / L / h, respectively. The sugar consumption rates of WT and control (strain 8b) were 1.17 g / L / h and 0.26 g / L / h, respectively. In the present invention, mutants with a sugar consumption rate greater than 1.45 g / L / h were defined as excellent mutants (F145Y, V267I, V267L, K341R, S375E, and K420Q, respectively). At the same time, the ethanol production of these mutants was also increased compared with WT. The aforementioned mutants were selected for the next step of combined mutation research.

[0078] 2.2 Screening of combined mutants

[0079] Using the three excellent single-point mutants (F145Y, V267I, V267L) with better effects screened above as templates, pairwise combinations and superpositions were carried out with other excellent single-point mutants on this basis to obtain 11 double-point mutants: F145Y-V267I, F145Y-V267L, F145Y-K341R, F145Y-S375E, F145Y-K420Q, V267I-K341R, V267I-S375E, V267I-K420Q, V267L-341R, V267L-S375E, and V267L-K420Q. Primer pairs were designed for combined mutation of PanXI respectively, an expression vector with Peno as the promoter and pEZ15a as the plasmid backbone was constructed, and after being transformed into the host strain 8b to obtain recombinant xylose isomerase recombinant strains, xylose metabolism tests were carried out in RMX5 medium. The mutation primers for F145Y-V267I, F145Y-V267L, F145Y-K341R, F145Y-S375E, and F145Y-K420Q were: pEZ15a-PanXI V267I -F / R, pEZ15a-PanXI V267L -F / R, pEZ15a-PanXI K341R -F / R, pEZ15a-PanXI S375E -F / R, pEZ15a-PanXI K420Q -F / R, with pEZ15a-Peno-PanXIF145Y as the template; the V267I-K341R, V267I-S375E, and V267I-K420Q mutant primers are pEZ15a-PanXI K341R -F / R, pEZ15a-PanXI S375E -F / R, pEZ15a-PanXI K420Q -F / R, with pEZ15a-Peno-PanXI V267I as the template; the V267L-341R, V267L-S375E, and V267L-K420Q mutant primer pairs are pEZ15a-PanXI K341R -F / R, pEZ15a-PanXI S375E -F / R, pEZ15a-PanXI K420Q -F / R, with pEZ15a-Peno-PanXI V267L as the template, and the PanXI combined mutant gene is obtained by PCR amplification. The primer sequences are shown in Table 2.

[0080] Figures 3 - 5The cell growth, xylose consumption, and ethanol production of the recombinant strain expressing the combinatorial mutant were shown at 24 h of fermentation. It can be seen that at 24 h of fermentation, the xylose consumption rates of the F145Y-V267I (or V267I-F145Y), F145Y-V267L (or V267L-F145Y), F145Y-K341R, F145Y-S375E, F145Y-K420Q, V267I-K341R, V267I-S375E, V267I-K420Q, V267L-K341R, V267L-S375E, and V267L-K420Q mutants were 1.57, 1.68, 1.41, 1.45, 1.60, 1.12, 1.29, 1.31, 1.29, 1.18, and 1.22 g / L / h, respectively. The xylose consumption rates of the F145Y, V267I, V267L mutants, and the WT were 1.54, 1.42, 1.53, and 1.12 g / L / h, respectively. It can be seen that the xylose metabolism and ethanol production of the double mutants based on the single mutant F145Y were better than those of the double mutants based on the single mutants V267I and V267L. Secondly, it was V267L, while the xylose metabolism of the double mutants based on V267I was weaker compared with the other two. In addition, by comprehensively comparing the growth, xylose metabolism, and ethanol production experimental results of the three groups, the F145Y-V267L and F145Y-K420Q with a xylose consumption rate ≥1.60 g / L / h were finally determined as the optimal mutants. Among them, the strain growth rates of the F145Y-V267L and F145Y-K420Q mutants were 0.163 and 0.155 / h, respectively, both higher than 0.148 / h of the wild type. At the same time, the xylose consumption amounts of the two double mutants at the mid-logarithmic growth phase (24 h) of fermentation were 40.32 and 38.4 g / L, respectively, both higher than 36.96 g / L of the single mutant F145Y and 26.88 g / L of the wild type WT. In addition, at 24 h of fermentation, the ethanol production amounts of the F145Y-V267L and F145Y-K420Q mutants were 17.72 and 16.94 g / L, respectively, higher than 16.16 g / L of the single mutant F145Y and 12.81 g / L of the wild type WT.

[0081] Table 2 Proteins and primer sequences involved in the newly screened XI mutants and the construction of their recombinant expression vectors in the examples of the present invention

[0082]

[0083]

[0084] Note: The amino acid mutation sites are shown in bold with shading, and the codon mutation sites are shown with shading and underlining. Additionally, Table 2 exemplarily gives the amino acid sequences of some mutants. The remaining mutants can be obtained by replacing the amino acids corresponding to the mutation sites in the wild-type XI with the mutated amino acids, so they are not shown repeatedly. In addition, the gene sequences of the mutants are obtained by replacing the amino acid codons at the mutation sites of the wild-type XI gene with the codons of the mutated amino acids. For example, when constructing the F145Y mutant, the TTC codon at position 145 of the gene sequence is replaced with TAT, and the coding gene of the F145Y mutant is obtained.

[0085] III. Heterologous Expression and Purification of Xylose Isomerase PanXI Mutants

[0086] Heterologous expression was performed on the single mutants and the combined mutants F145Y-V267L and F145Y-K420Q screened by xylose growth and metabolism tests. Using pTZ28a as the expression vector, the expression plasmids pTZ28a-PanXI, pTZ28a-PanXI F145Y-V267L , pTZ28a-PanXI F145Y-K420Q were constructed respectively, and were transferred into Zymomonas mobilis ZM4-T7 by electroporation. After sequencing verification, the recombinant strains were successfully constructed, and the recombinant Zymomonas mobilis ZM4-T7-PanXI, ZM4-T7-PanXI F145Y-V267L and ZM4-T7-PanXI F145Y-K420Q were obtained.

[0087] The ZM4-T7 strain is a recombinant strain obtained by transforming the T7 RNA polymerase (T7 RNAP) expression system in Zymomonas mobilis ZM4 (Z. mobilis ZM4, ATCC 31821). The T7 RNAP expression system includes a P BAD promoter, T7 RNAP, TetR operon, Ptet promoter, and araC operon from upstream to downstream. Among them, the expression of araC is regulated by Ptet, and the expression of T7 RNAP is regulated by P BAD , while the activity of the P BAD promoter is controlled by the expression product of araC. Therefore, the expression level of T7 RNAP is high only when both tetracycline and arabinose are present. The construction method of the ZM4-T7 strain can be found in the patent "CN114774453A Construction Method and Application of a Stringent Gene Expression Regulation System for Zymomonas mobilis (Publication Date: July 22, 2022)".

[0088] The construction process of the pTZ28a recombinant expression vector includes: using the V-pTZ28a-F / R primer pair, with plasmid pTZ28a as the template, to amplify the linearized pTZ28a plasmid. Using the pTZ28a-PanXI-F / R primer pair, respectively with plasmids pEZ15a-PanXI, pEZ15a-PanXI F145Y-V267L , pEZ15a-PanXI F145Y-K420Q as templates, to amplify PanXI, PanXI F145Y -V267L and PanXI F145Y-K420Q genes. Through the Gibson assembly method, each gene is ligated with the linearized pTZ28a vector to form the recombinant pTZ28a-PanXI, pTZ28a-PanXI F145Y-V267L , pTZ28a-PanXI F145Y-K420Q expression vectors. The related primers are shown in Table 2.

[0089] Inoculate the recombinant Zymomonas mobilis seed liquid into the RMG5 medium supplemented with Kana antibiotic, control the initial OD 600 to be 0.1, and at the same time add appropriate amounts of the inducers 0.8 μg / mL tetracycline and 3 mg / mL arabinose, and then place it in a shaker at 30 °C and 100 rpm for 24 h; collect the bacterial liquid and centrifuge it at 4 °C and 4000 rpm for 10 min, and wash the bacterial cell precipitate once with 50 mM Tris-HCl buffer and resuspend it; then ultrasonically disrupt the bacterial cell suspension until it is colorless and transparent, indicating complete disruption. Centrifuge the disrupted bacterial liquid at 4 °C and 15000 rpm at high speed for 30 min; filter the supernatant after centrifugation and load it into a chromatography column pre-equilibrated with nickel column packing (purchased from Cytiva, product number 17531806) and incubate it at 4 °C for 1 h; after incubation, collect the flow-through liquid, and sequentially perform gradient elution with 50 mM Tris-HCl buffer with different imidazole concentrations (imidazole concentration is 10 mM - 250 mM), collect the target protein elution liquid, pipette 10 μL of the protein elution liquid and mix it with 100 μL of the G250 solution. Observe the color change from blue to brown, indicating that the protein has been eluted cleanly at this concentration, and start the elution with the next imidazole concentration; after elution, take 40 μL of the elution liquid under different imidazole concentration conditions and mix it with 10 μL of 5×loading buffer, and heat it at 100 °C for 10 min for subsequent SDS-PAGE detection; determine the elution liquid corresponding to the target protein with the appropriate imidazole concentration by SDS-PAGE detection, and concentrate and change the liquid of it with an ultrafiltration tube of appropriate size. The concentrated protein is used for subsequent enzymatic experiment characterization.

[0090] IV. Enzymatic property analysis of the xylose isomerase PanXI mutant

[0091] The in vitro enzyme activity assay system for xylose isomerase (the concentration of each reagent is the final concentration in the system) includes: 66 mM xylose, 10 mM MgCl2, 50 mM Tris-HCl and an appropriate amount of enzyme solution. The reaction system is placed in a 30 °C water bath for 30 min. After inactivation, it is cooled to room temperature and the amount of xylulose produced is detected by HPLC. One unit of xylose isomerase activity is defined as the amount of enzyme required to produce 1 μM xylulose per minute under the assay conditions.

[0092] The content of xylulose was determined by HPLC. The method includes: using a Shimadzu liquid phase analysis system equipped with a differential refractive index detector (RID-20A), and the chromatographic column is an organic acid chromatographic column (Bio-Rad Aminex HPX-87H, 300 mm × 7.8 mm); the temperature of the detection cell is 40 °C, and the temperature of the column oven is 60 °C; the mobile phase is 5 mM sulfuric acid, the flow rate is 0.5 mL / min, and the injection volume is 20 μL.

[0093] The enzyme sample used for the following analysis of enzymatic properties is a crude enzyme, which is obtained by ultrasonic disruption of the bacterial cell suspension and centrifugation to collect the supernatant.

[0094] 4.1 Effect of temperature on enzyme activity

[0095] Temperature is an important indicator affecting the progress of enzyme reactions. When an enzyme is expressed in a microorganism, a low-temperature enzyme is often desired. However, when an enzyme is applied in industrial production, a high-temperature-resistant enzyme is more desired. Therefore, studying the optimal temperature of an enzyme is crucial for its application scenario. The prepared enzyme activity assay system was reacted at 30, 40, 50, 60, 70, 80 and 90 °C under the condition of pH 7.5 for 30 min, and the enzyme activity was measured.

[0096] The experimental results are as Figure 6 shown. The abscissa is the temperature and the ordinate is the specific enzyme activity. It can be seen that the relative activity trends of the wild type and the mutants are relatively consistent at different temperatures. However, the difference is that the optimal temperature of the mutant F145Y-V267L is 10 °C lower than that of the wild type and F145Y-K420Q, which is 50 °C, indicating that this mutant has greater potential under low-temperature conditions. In addition, the specific enzyme activity of the mutant F145Y-K420Q is higher than that of the wild type at temperatures from 30 °C to 60 °C. When the temperature is 30 °C, the growth temperature of Zymomonas mobilis, the specific enzyme activity of the mutant F145Y-K420Q is the highest, which is 1.52 U / mg, while the specific enzyme activity of the mutant F145Y-V267L is slightly lower than that of the WT. And in the case of 70 °C - 90 °C, the specific enzyme activity of the wild type is higher.

[0097] 4.2 Effect of pH on enzyme activity

[0098] The acidity and alkalinity of the system during the enzymatic reaction process are also important factors affecting enzyme activity. Extreme deviation of the reaction system pH (too acidic or too alkaline) may lead to irreversible denaturation of the enzyme structure, causing it to lose its catalytic ability; while moderate pH fluctuations may temporarily inhibit enzyme activity through reversible conformational changes. Set the pH change range as citrate-disodium hydrogen phosphate buffer (4, 5, 6, 7), Tris-hydrochloric acid buffer (7, 8, 9) and glycine-sodium hydroxide buffer (9, 10) to explore the catalytic activities of this xylose isomerase and its mutants under different pH conditions; measure the enzyme activities of the prepared enzyme activity assay systems at pH 4, 5, 6, 7, 8, 9 and 10 at 30 °C respectively.

[0099] The experimental results are as Figure 7 shown. The abscissa is pH and the ordinate is specific enzyme activity. It can be seen that the optimal pH of both the wild type and the mutants is pH 7.5, and this enzyme has stronger adaptability under alkaline pH conditions and can maintain relatively high enzyme activity. Under acidic conditions (pH 4 - 5), the enzyme activities of PanXI and its optimal mutants decreased significantly. Especially at pH 4, the specific enzyme activities of the wild type (WT) and mutants F145Y-V267L and F145Y-K420Q were 0.11, 0.20 and 0.14 U / mg respectively, only 9.7%, 17.6% and 11.9% of the enzyme activity of mutant F145Y-K420Q under the optimal pH 7.5 condition (subsequent ratios are all based on this). At pH 6, the enzyme activities of the wild type and mutants could be maintained at about 80%, and the specific enzyme activities were 0.76, 0.86 and 0.93 U / mg respectively. At the optimal pH 7.5, the specific enzyme activities of the three xylose isomerases were 1.03, 0.97 and 1.20 U / mg respectively. It is worth noting that although the specific enzyme activity of mutant F145Y-V267L was slightly lower than that of the wild type under the optimal pH condition, under weak acidic conditions (pH 4 - 6), the enzyme activities of the two mutants were higher than that of the wild type, and the order of their enzyme activities was F145Y-K420Q > F145Y-V267L > WT. Through measurement, it was found that the initial pH of the xylose fermentation medium RMX5 of Zymomonas mobilis after sterilization was about 5.8 - 6.0 without artificially controlling the pH. Along with the continuous acid production during fermentation, the pH of the medium could drop to about 4 in the later stage. Therefore, it can be inferred that the mutated xylose isomerase has stronger acid resistance than the wild type, which may be one of the reasons for the improvement of its xylose metabolism performance in Zymomonas mobilis.

[0100] 4.3 Effect of pH on enzyme stability

[0101] Since fermentation is a relatively long reaction process that requires a long reaction time under certain pH conditions, the pH stability of the enzyme is also an important index to be considered. To investigate the enzyme activity stability of PanXI and its optimal mutants, the enzymes were incubated on ice for 6 h in different buffers with pH values ranging from 4 to 10, and the enzyme activity was measured at pH 7.5 and 60 °C after incubation. The enzyme activity of F145Y-K420Q without pH buffer incubation was defined as 100%.

[0102] The experimental results are as Figure 8 shown. The abscissa is pH and the ordinate is relative enzyme activity. At pH 4, the enzyme activity was significantly lost, and the relative enzyme activities of WT, F145Y-V267L, and F145Y-K420Q were only 22.3%, 23.5%, and 28.1% respectively. At pH values from 5 to 10, most of the relative enzyme activities remained above 70%, indicating that PanXI and its mutants had good stability under weakly acidic to moderately alkaline conditions. Among them, at pH values from 4 to 6, the relative enzyme activities of the two mutants were higher than that of the wild type, showing better tolerance to weak acidity.

[0103] V. In vitro enzyme activity test of xylose isomerase PanXI mutants

[0104] Since the in vitro enzyme activity tests of most xylose isomerases are carried out under the optimal conditions (pH 7.5) of the enzyme, while the actual application environment pH in this study is about 6. In addition, through the study of enzyme properties, it was found that the two mutants had stronger tolerance to low temperature and weak acid pH conditions compared with the wild type. Therefore, the in vitro enzyme activities of xylose isomerase and its mutants were tested at pH 7.5, pH 6, and temperature 30 °C in this invention, and the enzyme samples used in this experiment were pure enzymes purified by nickel column.

[0105] The experimental results are as Figure 9 shown. The specific enzyme activities of wild-type XI, F145Y-V267L, and F145Y-K420Q mutants at pH 7.5 were 1.91, 1.83, and 2.39 U / mg respectively. The specific activity of the mutant F145Y-K420Q increased by 25.1% compared with the wild type, while the specific activity of F145Y-V267L was slightly lower than that of the wild type. At pH 6, the specific enzyme activities of wild-type XI, F145Y-V267L, and F145Y-K420Q mutants were 1.52, 1.74, and 2.09 U / mg respectively. Among them, the enzyme activity of F145Y-V267L increased by 14.4% compared with the wild type, and the enzyme activity of F145Y-K420Q increased by 37.5% compared with the wild type.

[0106] In summary, the enzyme activities of mutants F145Y-V267L and F145Y-K420Q are improved compared to the wild type under low temperature and weak acid conditions, and they have higher stability in a weakly acidic environment. The improved performance after mutation is more compatible with the application scenario of producing ethanol by weak acid fermentation of Zymomonas mobilis, which may be one of the reasons for the improved xylose metabolism performance of the mutants.

[0107] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A xylose isomerase mutant, characterized in that, The amino acid sequence of the xylose isomerase mutant is selected from at least one of SEQ ID NO.2-12.

2. The xylose isomerase mutant according to claim 1, wherein The amino acid sequence of the xylose isomerase mutant is selected from at least one of SEQ ID NO.2, SEQ ID NO.8-12.

3. The xylose isomerase mutant according to claim 2, characterized in that, The amino acid sequence of the xylose isomerase mutant is selected from at least one of SEQ ID NO.9 and SEQ ID NO.

12.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a gene sequence encoding the xylose isomerase mutant according to any one of claims 1-3.

5. The nucleic acid molecule according to claim 4, wherein The nucleic acid molecule further comprises a gene sequence encoding a promoter, and the xylose isomerase mutant is located downstream of the promoter; the promoter is an enolase promoter.

6. An expression vector, characterized in that, Comprising the nucleic acid molecule according to any one of claims 4-5.

7. The expression vector according to claim 6, wherein The expression vector is a pEZ15a or pTZ28a vector.

8. Use of the xylose isomerase mutant according to any one of claims 1-3, the nucleic acid molecule according to any one of claims 4-5, or the expression vector according to any one of claims 6-7 in enhancing the xylose conversion ability of microorganisms.

9. Use of the xylose isomerase mutant, nucleic acid molecule or expression vector according to claim 8 in enhancing the xylose conversion ability of microorganisms, characterized in that, The microorganism is Zymomonas mobilis.

10. Use of the xylose isomerase mutant according to any one of claims 1-3, the nucleic acid molecule according to any one of claims 4-5, or the expression vector according to any one of claims 6-7 in the preparation of a product that requires the participation of xylose isomerase.

Images