Xylose isomerase mutants, nucleic acid molecules and expression vectors thereof and uses

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to a xylose isomerase mutant, a nucleic acid molecule and an expression vector thereof and application. The amino acid sequence of the xylose isomerase mutant is selected from at least one of SEQ ID NO. 2-12. More than ten xylose isomerase mutants with high expression activity in Zymomonas mobilis are obtained by using big data mining, semi-rational modification and heterologous recombinant expression and function screening, and the shortage of active xylose isomerase in Zymomonas mobilis is solved. Zymomonas mobilis is used as a host strain for expressing XI protein, and each mutant obtained is subjected to heterologous expression. The xylose isomerase mutant can endow the recombinant strain with high metabolic capacity for xylose, effectively improve the utilization rate of xylose and the yield of ethanol, and has important industrial application value and potential for constructing a recombinant microorganism with high conversion of xylose and obtaining a wood fiber fuel ethanol production strain with excellent properties.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a xylose isomerase mutant, a nucleic acid molecule and an expression vector thereof and application. BACKGROUND

[0002] Lignocellulose is widely present in various plants, especially in wood and crop straw such as corn straw and sugarcane residue. As the most abundant renewable biomass resource, efficient conversion and utilization of lignocellulose has become a research focus in the field of green biological manufacturing. The lignocellulose is converted into high-value products such as chemicals, materials and biofuels by microbial fermentation, and the production of fuel ethanol from lignocellulose is the main conversion and utilization way. This conversion method not only helps to reduce the dependence on fossil resources, but also effectively reduces the emission of carbon dioxide, thereby addressing the challenges brought by global climate change. Lignocellulose is mainly composed of cellulose, hemicellulose and lignin, and glucose and xylose can be released after pretreatment and enzymatic hydrolysis. Xylose accounts for 30%-40% of the hydrolysis products and is the second most abundant monosaccharide. Efficient xylose conversion is a key problem for fully utilizing lignocellulose resources and improving the utilization rate of hydrolysis products.

[0003] Zymomonas mobilis (Z. mobilis) is a gram-negative bacterium with a unique Entner-Doudoroff (ED) metabolic pathway and high sugar fermentation efficiency. It can convert glucose, fructose and sucrose into ethanol. Due to the efficient expression of pyruvate decarboxylase and ethanol dehydrogenase genes, the ethanol fermentation capacity is very outstanding. In addition, Z. mobilis has high tolerance to lignocellulose hydrolysate, and the related mechanism of tolerance to inhibitors in the hydrolysate is also relatively mature. Moreover, Z. mobilis has many industrial production advantages such as low fermentation cost, simple process, stable genome and suitability for large-scale long-term fermentation. In view of these unique physiological characteristics and excellent industrial production advantages of Z. mobilis, its application potential in the production platform of cellulosic ethanol is huge. However, the natural Z. 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 oxidation-reduction pathway catalyzed by xylose reductase (XR) and xylose dehydrogenase (XDH), and the other is the isomerization pathway catalyzed by xylose isomerase (XI). The oxidation-reduction pathway has the disadvantages of low efficiency and high energy consumption due to the difference in cofactor preference (NADPH and NADH) of XR and XDH. +), which leads to the imbalance of intracellular cofactors and the accumulation of toxic byproduct xylitol, limiting its application in industrial production. Wood sugar isomerase (XI) has a unique advantage in the bioconversion of lignocellulose because it can directly convert xylose to xylulose through a one-step isomerization reaction without the participation of cofactors. Although the XI pathway has many advantages, it still faces some challenges in practical application. For example, although the XI from bacteria can be normally expressed in Z. mobilis, its low enzyme activity in the low-temperature and weakly acidic environment in Z. mobilis hinders the utilization of lignocellulose raw materials by recombinant Z. mobilis. Therefore, it is of great significance to screen XI with high activity in Z. mobilis and optimize the xylose metabolic capacity to promote the efficient conversion and utilization of lignocellulose. SUMMARY

[0005] In order to solve the problems of lack of xylose isomerase suitable for Z. mobilis and low xylose metabolic capacity of Z. mobilis in the prior art, the present application provides a xylose isomerase mutant, a nucleic acid molecule containing the coding gene thereof and an expression vector, and further provides the application of the aforementioned xylose isomerase mutant, nucleic acid molecule and expression vector in enhancing the xylose conversion capacity of microorganisms or preparing products requiring the participation of xylose isomerase. The present application is realized by the following technical solutions:

[0006] The first aspect of the present application provides a xylose isomerase mutant, the amino acid sequence of which is selected from at least one of SEQ ID NO. 2-12.

[0007] Further, 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; and more further, at least one of SEQ ID NO. 9 and SEQ ID NO. 12.

[0008] The second aspect of the present application provides a nucleic acid molecule, which comprises a gene sequence encoding the xylose isomerase mutant as described above.

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

[0010] Further, the promoter is a constitutive strong promoter; and more further, the promoter is enolase promoter Peno.

[0011] The third aspect of the present application provides an expression vector, which comprises the nucleic acid molecule as described above.

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

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

[0014] Further, the microorganism is Zymomonas mobilis.

[0015] The fifth aspect of the present application provides use of the xylose isomerase mutant, the nucleic acid molecule or the expression vector as described above in preparing a product requiring the participation of xylose isomerase.

[0016] The present application has the advantages and positive effects that: the present application obtains more than ten xylose isomerase mutants with high expression activity in Zymomonas mobilis by using big data mining, semi-rational modification and heterologous recombinant expression and function screening, and solves the predicament of the lack of active xylose isomerase in Zymomonas mobilis. The obtained mutants are expressed in Zymomonas mobilis as a host strain of XI protein, and the xylose isomerase mutants can endow the recombinant strain with high metabolic capacity for xylose, effectively improve the utilization rate of xylose and the yield of ethanol, and have important industrial application value and potential for constructing a recombinant microorganism with high conversion of xylose and obtaining a lignocellulosic fuel ethanol production strain with excellent traits. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0018] Figure 1 Figure is the fermentation performance test diagram of the recombinant Zymomonas mobilis transformed with different species xylose isomerase genes in the RMX5 and RMX10 culture media of the embodiments of the present application; wherein, figures A-B are the RMX5 and RMX10 culture media respectively;

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

[0020] Figure 3The fermentation performance test diagram of the recombinant strains transformed with PanXI combined mutants F145Y-V267I, F145Y-V267L, F145Y-K341R, F145Y-S375E, and F145Y-K420Q in RMX5 medium is shown in the embodiment of the present invention.

[0021] Figure 4 The image shows the fermentation performance test of recombinant strains transformed with PanXI combined mutants V267I-F145Y, V267I-K341R, V267I-S375E, and V267I-K420Q in RMX5 medium according to embodiments of the present invention.

[0022] Figure 5 The fermentation performance test diagram of the recombinant strains transformed with PanXI combined mutants V267L-F145Y, V267L-341R, V267L-S375E and V267L-K420Q in RMX5 medium is shown in the embodiment of the present invention.

[0023] Figure 6 This is a graph showing the effect of temperature on the enzyme activity of wild-type PanXI and F145Y-V267L and F145Y-K420Q mutants in an embodiment of the present invention.

[0024] Figure 7 This is a graph showing the effect of pH on the enzyme activity of wild-type PanXI and F145Y-V267L and F145Y-K420Q mutants in an embodiment of the present invention.

[0025] Figure 8 This is a graph showing the effect of pH on the enzyme stability of wild-type PanXI and F145Y-V267L and F145Y-K420Q mutants in an embodiment of the present invention.

[0026] Figure 9 The graph shows the enzyme activity test results of wild-type PanXI and F145Y-V267L, F145Y-K420Q mutants under the conditions of 30°C, pH 7.5 and pH 6 in the embodiments of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0028] Various modifications in the precise arrangement of parts, as well as in the details thereof, can be made by those skilled in the art without departing from the spirit and scope of the application as set forth in the claims. It is to be understood that the scope of the application is not to be interpreted as limited to the foregoing specific process, compositions, or components, and that all changes and modifications that come within the spirit and scope of the application are intended to be embraced by the claims.

[0029] For a better understanding of the present application, no limitation is intended to the scope of the application as embodied in the system or processes herein described and illustrated, it being intended that all such modifications, alterations, and permutations for completing the intended of the application here described are fully encompassed by the application. The application is intended to be limited only as specified in the claims.

[0030] For a better understanding of the present application, no limitation is intended to the scope of the application as embodied in the system or processes herein described and illustrated, it being intended that all such modifications, alterations, and permutations for completing the intended of the application here described are fully encompassed by the application. The application is intended to be limited only as specified in the claims.

[0031] In the cellulose ethanol fermentation technology, the ability of microorganism to utilize and convert xylose into ethanol is the most critical. Natural Zymomonas mobilis is an industrial commonly used ethanol production bacteria, but it lacks the xylose utilization pathway, therefore, by introducing the exogenous xylose isomerase (XI) pathway into Zymomonas mobilis to ferment lignocellulose hydrolysis sugars to produce ethanol becomes a research hotspot. The core of this technology is the high activity expression of XI in Zymomonas mobilis to promote the utilization rate of Zymomonas mobilis to convert and utilize xylose in lignocellulose hydrolysis liquid. XI can directly convert xylose into xylulose, and xylulose is phosphorylated into the pentose phosphate pathway (PPP) to generate pyruvic acid, and finally fermented into ethanol.

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

[0033] Based on the online data of NCBI database, the present application obtains 6 candidate xylose isomerase genes from different sources. Through the construction of a heterologous expression system, functional characterization and screening are carried out in Z. mobilis xylose-utilizing strain 8b, and a high-expression-activity xylose isomerase (PanXI) gene is obtained, which has significantly better catalytic activity for xylose conversion than existing xylose isomerases. To further improve the enzyme activity, the present application carries out semi-rational modification based on consensus sequence analysis on the basis of natural PanXI, and obtains a series of mutants through the combination of site-directed mutagenesis and combinatorial mutation. Then the mutant genes are linked with the Peno promoter and inserted into the pEZ15a expression vector, which is transformed into the host strain 8b to obtain a plurality of recombinant xylose isomerase expression strains. By comparing the growth, xylose metabolism and ethanol production capacity of each group of recombinant strains with the wild type, the single mutants F145Y, V267I, V267L, K341R, S375E and K420Q and the double mutants F145Y-V267I, F145Y-V267L, F145Y-K341R, F145Y-S375E and F145Y-K420Q with high activity expression in Z. mobilis are finally obtained. The aforementioned mutants can effectively improve the xylose metabolism capacity and ethanol yield of the recombinant strain, and provide important XI enzyme resources for the development of cellulose ethanol production Z. mobilis.

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

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

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

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

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

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

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

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

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

[0043] The present application utilizes big data mining, semi-rational modification and heterologous recombinant expression, and functional screening to obtain more than ten xylose isomerase mutants with high expression activity in Zymomonas mobilis, solving the predicament of the lack of active xylose isomerase in Zymomonas mobilis. Using Zymomonas mobilis as the host strain for XI protein expression, the obtained mutants are heterologously expressed, and the xylose isomerase mutants can endow the recombinant strain with high metabolic capacity for xylose, effectively improving the utilization rate of xylose and the yield of ethanol, and have important industrial application value and potential for constructing a recombinant microorganism with high conversion of xylose and obtaining a lignocellulosic fuel ethanol production strain with excellent properties.

[0044] Preferably, the xylose isomerase mutant comprises at least a F145Y site mutation, i.e., at least one of F145Y, F145Y-V267I, F145Y-V267L, F145Y-K341R, F145Y-S375E, F145Y-K420Q mutant, the amino acid sequences of which are shown in SEQ ID NO. 2, SEQ ID NO. 8-12, respectively.

[0045] More preferably, the xylose isomerase mutant is selected from F145Y-V267L and F145Y-K420Q mutant. Analysis of the enzymatic properties of the aforementioned mutants shows that the specific enzyme activity of mutant F145Y-K420Q is higher than that of the wild type at a temperature of 30-60℃, and the specific enzyme activity of mutant F145Y-V267L is basically equivalent to that of wild type XI at 30-40℃, but is significantly higher than that of the wild type at the optimum temperature of 50℃. Under weak acidic conditions (pH 4-6), the specific enzyme activity of both mutants F145Y-K420Q and F145Y-V267L is higher than that of the wild type, and they exhibit stronger acid resistance than the wild type, with a lower loss of enzyme activity and higher enzyme activity stability under long-term incubation in an acidic solution environment. In addition, determination of the pure enzyme activity shows that the specific activity of F145Y-K420Q mutant is 2.39 U / mg under the conditions of temperature 30℃ and weak alkaline (pH 7.5) environment, which is slightly higher than the specific enzyme activity of 1.91 U / mg of the wild type PanXI, and the specific activity of F145Y-V267L mutant is basically equivalent to that of the wild type PanXI enzyme; and under the conditions of temperature 30℃ and weak acidic (pH 6), the specific enzyme activity of F145Y-V267L and F145Y-K420Q is 1.74 and 2.09 U / mg, respectively, which is significantly higher than the specific activity of 1.52 U / mg of the wild type PanXI. These results show that these mutants have higher enzyme catalytic activity and better acid resistance in acidic environment. In the fermentation of xylose by Zymomonas mobilis without artificial control of pH, the pH of the xylose fermentation medium is initially about 5.8-6.0, and can decrease to about 4 at the end, and the mutant XI with enhanced acid resistance can better adapt to this application scenario, thereby facilitating the improvement of the xylose metabolism performance of Zymomonas mobilis and the improvement of the economy and yield of lignocellulosic ethanol. Therefore, in a more preferred embodiment of the present application, the xylose isomerase mutant is selected from F145Y-V267L and / or F145Y-K420Q mutant.

[0046] In another embodiment of the present application, a nucleic acid molecule is provided, which comprises 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 repeated here.

[0048] The nucleic acid molecule includes DNA molecules (e.g. genomic DNA or cDNA) and / or RNA molecules (e.g. mRNA), which can be single-stranded or double-stranded. The sequence of the nucleic acid molecule can be deduced from the XI amino acid (AA) sequence by conventional means such as the codon-usage rules. The full-length sequence of the nucleic acid molecule or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis.

[0049] Optionally, 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 used to initiate the transcription and translation of XI, and can be adaptively selected according to the expression of the recipient cell and the xylose isomerase mutant.

[0050] In a preferred embodiment, in order to achieve efficient expression of the xylose isomerase mutant, the promoter is selected from a constitutively strong promoter, such as the enolase promoter Peno, the gene sequence of which is shown as SEQ ID NO. 14.

[0051] Another embodiment of the present application provides an expression vector comprising 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 repeated here.

[0053] Optionally, the expression vector comprises a prokaryotic expression vector, a eukaryotic expression vector or a viral expression vector (such as a lentivirus, an adenovirus). Correspondingly, the recipient cell transformed or transfected by the expression vector can be a prokaryotic cell and a eukaryotic cell, which is selected according to the type of the expression vector, for example, when the expression vector is a prokaryotic expression vector, the recipient cell is selected to be a prokaryotic cell, and examples of commonly used prokaryotic cells include Escherichia coli, Bacillus, Corynebacterium, Zymomonas mobilis and Streptomyces; when the expression vector is a eukaryotic expression vector, the recipient cell is selected to be a eukaryotic cell, and examples of commonly used eukaryotic cells include Saccharomyces cerevisiae, Saccharomyces pastorianus, filamentous fungi and the like.

[0054] Typical vectors include plasmids (such as pUC series, pET series, pWB series, pGEX series, pDXW series, pBR322, pEZ15a, pTZ28a, pMA5, pPICZ alpha, PIC9K, pSET152), viral vectors, bacteriophages (such as lambda gt4 lambda B, lambda-Charon, lambda Delta z1 and M13), cosmids and minichromosomes. Plasmids are the most commonly used vectors, and in the context of the present application, plasmids and vectors can be used interchangeably unless otherwise specified.

[0055] In a preferred embodiment, the expression vector is pEZ15a or pTZ28a vector, the nucleic acid molecule is inserted into the multiple cloning site of pEZ15a or pTZ28a vector to obtain a recombinant expression vector, which is transformed into Z. mobilis, and the xylose isomerase is expressed in the cell by conventional culture method.

[0056] In a preferred embodiment of the present application, the method for inserting the nucleic acid molecule into the vector comprises: amplifying the nucleic acid molecule by a primer pair with homologous arms, respectively, and amplifying the vector by a primer pair to obtain a linearized vector, and connecting the nucleic acid molecule with the homologous arms and the linearized vector by Gibson method to obtain the recombinant expression vector.

[0057] A further embodiment of the present application provides the use of the xylose isomerase mutant, the nucleic acid molecule or the expression vector containing the xylose isomerase coding gene in enhancing the xylose conversion ability of microorganisms.

[0058] Alternatively, the microorganism is Z. mobilis, in particular Z. mobilis 8b or ZM4. The nucleic acid molecule or the expression vector as described above is transformed into Z. mobilis to obtain a recombinant Z. mobilis expressing xylose isomerase, which can efficiently utilize xylose, improve the xylose fermentation ability of the strain, and help to convert xylose into ethanol, and enhance the cellulose ethanol production ability.

[0059] The transformation of the nucleic acid molecule or the expression vector into Z. mobilis can be achieved by various methods known in the art, including CaCl2 transformation method, calcium phosphate-DNA co-precipitation, electroporation, biolistic bombardment, microinjection, conjugative transfer, liposome-mediated transfection, liposome fusion, etc.

[0060] The present application further provides the use of the xylose isomerase mutant, the nucleic acid molecule or the expression vector containing the xylose isomerase coding gene in preparing products requiring the participation of xylose isomerase.

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

[0062] The present application is further described in conjunction with specific examples. The experimental methods in the following examples, for which no specific conditions are indicated, are generally performed according to 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 medium formula in the following examples is 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; (2) RMX5 medium: 50 g / L xylose, 2 g / L KH2PO4 and 10 g / L yeast extract, the xylose needs to be prepared as a mother liquor and filtered to sterilize, and the remaining components are mixed and sterilized at 121°C for 30 min; (3) RMX10 medium: 100 g / L xylose, 2 g / L KH2PO4 and 10 g / L yeast extract. The solid medium of the aforementioned medium needs to add agar 15 g / L.

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

[0065] Through analysis of the NCBI online database, six putative xylose isomerases from different sources and their encoding genes were mined, which were xylose isomerases from 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), KloXI (NCBI accession number: WP_004106797.1), respectively. In order to screen the xylose isomerase gene with high activity expressed in Z. mobilis, the above six xylose isomerase genes were connected with the constitutive strong promoter—enolase promoter Peno, and then inserted into the multiple cloning site of pEZ15a expression vector, and then genetic transformation was carried out with Z. mobilis 8b (the method for constructing the strain is described in 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, and then xylose metabolism test was carried out in RMX5 and RMX10 media. The genes and primer sequences for constructing the recombinant expression vector in this example are shown in Table 1, and F and R represent the upstream and downstream primers, respectively.

[0066] Table 1 Gene and primer sequences for constructing recombinant expression vector of the embodiment of the present application

[0067]

[0068]

[0069] Construction of pEZ15a recombinant expression vector: PanXI, EncXI, KlpXI, LapXI, LebXI, KloXI genes were amplified by PCR 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 full gene synthesized sequence as the template, at this time the gene carried homologous arm sequences homologous to the pEZ15a vector at both ends. In addition, the linearized plasmid was amplified using the V-pEZ15a-F and V-pEZ15a-Peno-R primer pairs, with the plasmid pEZ15a (the vector carrying the Peno promoter) as the template. The Gibbson assembly method was used to connect each gene downstream of the Peno promoter of the linearized pEZ15a vector to form a recombinant pEZ15a-Peno expression vector. Specifically, the above-mentioned amplified gene fragments and linearized plasmid were gel recovered, the gene fragments and linearized plasmid were mixed in a molar ratio of 3:1, and 0.5 μL of 10x Buffer4 (purchased from NEB, catalog number M0633L) and 0.5 μL of exonuclease (T5 Exonuclease, purchased from NEB, catalog number M0663S) were added, and the appropriate amount of ddH2O was added to 5 μL, and the reaction was carried out on ice for 5 min. After the reaction was completed, 20 μL of E. coli DH5α competent cells were added to the reaction system, and incubated on ice for about 25 min. Then heat shock at 42°C water bath for 45 s, ice bath for about 2 min, then add 500 μL of LB liquid medium, incubate at 37°C, 250 rpm shaker for about 1 h. Finally, the recovered culture was spread on a selection plate with spectinomycin, and after overnight incubation, the positive recombinants on the plate were verified by colony PCR, and the positive recombinants were cultured to extract plasmids for Sanger sequencing identification.

[0070] Preparation of Z. mobilis competent cells: a single colony of activated Z. mobilis was picked into RMG liquid medium, incubated overnight at 30°C, and an appropriate amount of bacterial solution in the logarithmic growth phase was transferred to 200 mL of RMG5 liquid medium, and the initial OD 600 was controlled at 0.025-0.05, and incubated at 30°C, 100 rpm for 4 h, and the OD600 When the bacterial concentration reaches 0.3-0.5, centrifuge at 400 rpm for 10 min at room temperature to collect the bacterial cells. Then wash once with sterile water and twice with 10% glycerol. Finally, resuspend the bacterial cells in 200 μL of 10% glycerol to obtain competent cells, and dispense into 50 μL portions for use.

[0071] Electroporation of *Fermentomonas motilityis* 8b: 50 μL of competent *Fermentomonas motilityis* 8b cells were added to an electroporation vessel, followed by 200-500 ng of the recombinant expression vector to be transformed. Electroporation was performed using a Bio-road MicroPulser 165-2100 electroporator at a program of 1.6 kV / 1.8 kV, 25 μF, and 200 Ω. After electroporation, the transformed strain was inoculated into RMG5 liquid medium and incubated at 30°C for 4-6 h. The culture was then plated on plates with the appropriate antibiotic resistance. Single colonies were selected for sequencing to verify successful transformation of the recombinant expression vector, yielding the target strain.

[0072] Xylose metabolism capacity test: The target strain was divided according to its initial OD... 600 The inoculum was 0.1 g / L in RMX medium containing xylose, and growth and fermentation tests were conducted at 30°C and 100 rpm. OD values ​​of the bacterial culture were collected every 12 hours. 600 Xylose and xylose concentrations were determined using high-performance liquid chromatography (HPLC) in the culture medium. A Shimadzu HPLC system with a differential detector (RID-20A) was used. The chromatographic column was an organic acid column (Bio-Rad Aminex HPX-87H, 300 mm × 7.8 mm). The detection cell temperature was 40 °C, and 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 injection volume was 20 μL. OD values ​​were measured at 600 nm using a UV spectrophotometer. 600 To monitor growth. Recombinant xylose isomerase strains (EcXI and RsXI) currently available in the laboratory were used as controls.

[0073] See relevant results Figure 1 Figure A shows the fermentation performance of the xylose isomerase recombinant strain in RMX5 medium, and Figure B shows the fermentation performance of the xylose isomerase recombinant strain in RMX10 medium. From left to right, the figures are the growth curves of the recombinant strains (the horizontal axis represents time, and the vertical axis represents the strain concentration (OD)). 600 The graphs of xylose metabolism (where the horizontal axis represents time and the vertical axis represents xylose concentration) show that the xylose isomerase PanXI exhibits stronger heterologous expression activity and higher xylose metabolism capacity in both RMX5 and RMX10 media. Based on this, the xylose isomerase PanXI was further modified to enhance its enzyme activity.

[0074] II. Screening of xylose isomerase PanXI mutants

[0075] 2.1 Screening of single-point mutants

[0076] The single-point mutants of xylose isomerase PanXI were determined by performing consensus sequence analysis on the native xylose isomerase PanXI (WT group), which were H20N, S114E, F145Y, T146S, V267I / L, S375E, V186I / L, K341R, Q415H, K420Q, and R425H, respectively. The primers were designed for the single-point mutation of PanXI, and the expression vector with Peno as the promoter and pEZ15a as the plasmid backbone was constructed. The recombinant xylose isomerase recombinant strain was obtained by transforming the expression vector into the host strain 8b, and the xylose metabolism test was performed in the RMX5 medium. The related operations were the same as above and will not be described here. The primer pairs for the mutations of H20N, S114E, F145Y, T146S, V186I, V186L, V267I, V267L, K341R, S375E, Q415H, K420Q, and R425H were as follows: 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, and the PanXI mutant gene was obtained by PCR amplification with the plasmid pEZ15a-Peno-PanXI as the template. The primer sequences are shown in Table 2.

[0077] Figure 2The recombinant strains of the transformed single-point mutants were fermented to 24 h, and the cell growth, xylose consumption and ethanol production were shown. It can be seen that 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, and the sugar consumption rates of the WT and control (8b strain) were 1.17 g / L / h and 0.26 g / L / h, respectively. The 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), and the ethanol production of these mutants was also increased compared with the WT. The aforementioned mutants were selected for further combination mutation study.

[0078] 2.2 Screening of combination mutants

[0079] The three excellent single-point mutants (F145Y, V267I, and V267L) screened above were used as templates, and were combined with other excellent single-point mutants 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 combination mutation of PanXI, and expression vectors with Peno as the promoter and pEZ15a as the plasmid backbone were constructed. The recombinant xylose isomerase strains were obtained by transforming the expression vectors into the host strain 8b, and xylose metabolism tests were performed in RMX5 medium. The F145Y-V267I, F145Y-V267L, F145Y-K341R, F145Y-S375E, and F145Y-K420Q mutant primers were as follows: V267I pEZ15a-PanXI V267L -F / R, pEZ15a-PanXI K341R -F / R, pEZ15a-PanXI S375E -F / R, pEZ15a-PanXI K420Q -F / R, pEZ15a-PanXIF145Y PanXI-F / R, pEZ15a-PanXI K341R PanXI-F / R, pEZ15a-PanXI S375E PanXI-F / R, pEZ15a-PanXI K420Q PanXI-F / R, pEZ15a-Peno-PanXI V267I PanXI-F / R, pEZ15a-PanXI K341R PanXI-F / R, pEZ15a-PanXI S375E PanXI-F / R, pEZ15a-PanXI K420Q PanXI-F / R, pEZ15a-Peno-PanXI V267L PanXI-F / R, pEZ15a-PanXI

[0080] Figures 3-5The recombinant strain of the conversion combination mutant is fermented to 24 h, and the cell growth, xylose consumption and ethanol production are shown. It can be seen that the xylose consumption rates of 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, V267L-K420Q mutants are 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, and the xylose consumption rates of F145Y, V267I, V267L mutants and WT are 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-point mutant based on the single-point mutant F145Y are better than those of the double-point mutants based on the single-point mutants V267I and V267L, followed by V267L, and the xylose metabolism of the double-point mutant based on V267I is weaker than the other two. In addition, by comparing the growth, xylose metabolism and ethanol production of the three groups, it is finally determined that F145Y-V267L and F145Y-K420Q with xylose consumption rate ≥1.60 g / L / h are the optimal mutants. Among them, the strain growth rates of F145Y-V267L and F145Y-K420Q mutants are 0.163 and 0.155 / h respectively, which are higher than that of the wild type 0.148 / h; at the same time, the xylose consumption amounts of the two double-point mutants at the logarithmic growth medium (24 h) are 40.32 and 38.4 g / L respectively, which are higher than that of the single-point mutant F145Y 36.96 g / L and the wild type WT 26.88 g / L; in addition, the ethanol production of F145Y-V267L and F145Y-K420Q mutants at 24 h is 17.72 and 16.94 g / L respectively, which is higher than that of the single-point mutant F145Y 16.16 g / L and the wild type WT 12.81 g / L.

[0081] Table 2 XI mutants newly screened in the embodiments of the application and protein and primer sequences involved in construction of recombinant expression vectors thereof

[0082]

[0083]

[0084] Note: the amino acid mutation site is indicated by shading and bold, and the codon mutation site is indicated by shading and underlining. In addition, Table 2 exemplarily shows the amino acid sequences of some mutants. The amino acid sequences of 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, and thus are not repeated. In addition, the gene sequence of the mutant is obtained by replacing the amino acid codon of the mutation site in the wild-type XI gene with the codon of the mutated amino acid. For example, when constructing the F145Y mutant, the TTC codon at position 145 of the gene sequence is replaced with TAT, i.e., the coding gene of the F145Y mutant is obtained.

[0085] III. Heterologous expression and purification of xylose isomerase PanXI mutants

[0086] The single-point mutants and the combined mutants F145Y-V267L and F145Y-K420Q selected by the xylose growth and metabolism test were heterologously expressed. The expression plasmids pTZ28a-PanXI, pTZ28a-PanXI F145Y-V267L , and pTZ28a-PanXI F145Y-K420Q were constructed using pTZ28a as the expression vector, and were transformed into Z. mobilis ZM4-T7 by electroporation. The recombinant strains were verified by sequencing, and the recombinant Z. mobilis ZM4-T7-PanXI, ZM4-T7-PanXI F145Y-V267L , and ZM4-T7-PanXI F145Y-K420Q were successfully constructed.

[0087] The ZM4-T7 strain is a recombinant strain of Z. mobilis ZM4 (Z. mobilis ZM4, ATCC 31821) transformed with a T7 RNA polymerase (T7 RNAP) expression system. The T7 RNAP expression system includes, from upstream to downstream, a P BAD promoter, a T7 RNAP, a TetR operon, a Ptet promoter, and an araC operon. The expression of araC is regulated by the Ptet, the expression of T7 RNAP is regulated by the P BAD , and the activity of the P BAD promoter is controlled by the expression product of araC. Therefore, the expression amount of T7 RNAP is high only when both tetracycline and arabinose are present. The construction method of the ZM4-T7 strain is described in the patent “CN114774453A A method for constructing a strict regulation system for gene expression in Z. mobilis and its application (publication (announcement) date: 2022-07-22)”.

[0088] The construction process of the pTZ28a recombinant expression vector includes: using the V-pTZ28a-F / R primer pair, with plasmid pTZ28a as a template, to amplify the linearized pTZ28a plasmid. Then, using the pTZ28a-PanXI-F / R primer pair, with plasmids pEZ15a-PanXI and pEZ15a-PanXI, respectively... F145Y-V267L pEZ15a-PanXI F145Y-K420Q Using this as a template, PanXI and PanXI were amplified. F145Y -V267L and PanXI F145Y-K420Q Genes. Each gene was ligated to a linearized pTZ28a vector using the Gibson assembly method to form recombinant pTZ28a-PanXI and pTZ28a-PanXI. F145Y-V267L pTZ28a-PanXI F145Y-K420Q The expression vector and related primers are shown in Table 2.

[0089] Recombinant *Saccharomyces cerevisiae* seed culture was inoculated into RMG5 medium supplemented with Kana antibiotic, and the initial OD was controlled. 600 The concentration was 0.1, and appropriate amounts of inducing agents 0.8 μg / mL tetracycline and 3 mg / mL arabinose were added. The mixture was then incubated in a shaker at 30°C and 100 rpm for 24 h. The bacterial suspension was collected and centrifuged at 4°C and 4000 rpm for 10 min. The bacterial pellet was washed once with 50 mM Tris-HCl buffer and resuspended. The bacterial suspension was then sonicated until it was colorless and transparent, indicating complete disruption. The disrupted bacterial culture was centrifuged at 15,000 rpm for 30 min at 4 °C. The supernatant was filtered and loaded into a pre-equilibrated chromatography column packed with nickel (Cytiva, catalog number 17531806) and incubated at 4 °C for 1 h. After incubation, the flow-through was collected, and gradient elution was performed sequentially with different imidazole concentrations of 50 mM Tris-HCl buffer (imidazole concentrations of 10 mM-250 mM). The target protein eluent was collected, and 10 μL of the protein eluent was mixed with 100 μL of G250 solution. The color change from blue to brown indicated that the protein at that concentration had been completely eluted, and the next imidazole concentration elution was started. After elution, 40 μL of the eluent under different imidazole concentrations was mixed with 10 μL of 5× loading buffer. The mixture was mixed with buffer and heated at 100°C for 10 min for subsequent SDS-PAGE analysis. The target protein was determined by SDS-PAGE analysis to be eluted with the corresponding imidazole concentration. The protein was then concentrated and the solution was changed using an ultrafiltration tube of appropriate size. The concentrated protein was used for subsequent enzymatic characterization experiments.

[0090] IV. Enzymatic Properties Analysis of Xylose Isomerase PanXI Mutant

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

[0092] The HPLC method is used to determine the content of xylulose, which includes using a Shimadzu liquid chromatography system with a differential detector (RID-20A), and an organic acid chromatographic column (Bio-Rad Aminex HPX-87H, 300 mm x 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.

[0093] The enzyme sample used in the following enzymatic property analysis 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 that affects the progress of enzyme reactions. When enzymes are used for in vivo expression of microorganisms, it is often desirable to obtain low-temperature enzymes, but when enzymes are used for industrial production, it is more desirable to obtain high-temperature resistant enzymes. Therefore, studying the optimum temperature of the enzyme is crucial for its application scenarios. The prepared enzyme activity assay system was reacted at 30, 40, 50, 60, 70, 80, and 90°C at pH 7.5 for 30 min, and the enzyme activity was determined.

[0096] The experimental results are shown in Figure 6 The horizontal axis is temperature, and the vertical axis is specific enzyme activity. It can be seen that the relative activity trends of the wild type and the mutants at different temperatures are consistent, but the difference is that the optimum 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 the mutant has greater potential at low temperatures. In addition, the specific enzyme activity of mutant F145Y-K420Q is higher than that of the wild type at temperatures of 30-60°C. When the temperature is 30°C, the growth temperature of Zymomonas mobilis, the specific enzyme activity of mutant F145Y-K420Q is the highest, reaching 1.52 U / mg, while the specific enzyme activity of mutant F145Y-V267L is slightly lower than that of WT. At 70-90°C, the specific enzyme activity of the wild type is higher.

[0097] 4.2 Effect of pH on enzyme activity

[0098] The pH of the reaction system is also an important factor affecting enzyme activity. Extreme deviation of the pH of the reaction system (over-acid or over-alkali) can cause irreversible denaturation of the enzyme structure, resulting in loss of catalytic ability; and moderate pH fluctuations can temporarily inhibit enzyme activity through reversible conformational changes. The pH variation range was set as citric acid-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 activity of the xylose isomerase and its mutants under different pH conditions; the prepared enzyme activity determination system was measured for enzyme activity at pH 4, 5, 6, 7, 8, 9 and 10 at 30°C.

[0099] The experimental results are shown in Figure 7 As shown in the table, the abscissa is pH, and the ordinate is specific enzyme activity. It can be seen that the optimal pH of the wild type and the mutants is pH 7.5, and the enzyme has stronger adaptability under alkaline pH conditions and can maintain higher enzyme activity. Under acidic conditions (pH 4-5), the enzyme activity of PanXI and its optimal mutants decreases significantly, especially at pH 4, the specific enzyme activities of the wild type (WT) and the mutants F145Y-V267L and F145Y-K420Q are 0.11, 0.20 and 0.14 U / mg, respectively, which are only 9.7%, 17.6% and 11.9% of the enzyme activity of the mutant F145Y-K420Q under the optimal pH 7.5 conditions (the subsequent ratios are based on this). At pH 6, the enzyme activity of the wild type and the mutants can be maintained at about 80%, and the specific enzyme activities are 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 are 1.03, 0.97 and 1.20 U / mg, respectively. It is worth noting that although the specific enzyme activity of the mutant F145Y-V267L is slightly lower than that of the wild type under the optimal pH conditions, the enzyme activity of the two mutants is higher than that of the wild type under weak acidic conditions (pH 4-6), and the enzyme activity is in the order of F145Y-K420Q>F145Y-V267L>WT. It is found by measurement that the initial pH of the xylose fermentation medium RMX5 of Zymomonas mobilis after sterilization is about 5.8-6.0 without artificial pH control. With the continuous production of acid during fermentation, the pH of the medium can be reduced 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 improved performance of xylose metabolism in Zymomonas mobilis.

[0100] 4.3 Effect of pH on enzyme stability

[0101] Since fermentation is a long reaction process, it needs to be carried out under certain pH conditions for a long time. Therefore, the pH stability of the enzyme is also an important index to be referred to. In order to explore the enzyme activity stability of PanXI and the optimal mutant, the enzyme was placed in different buffers with pH of 4-10 on ice for 6h, and then the enzyme activity was measured under the condition of pH 7.5 and 60℃. The enzyme activity of F145Y-K420Q without pH buffer incubation treatment was defined as 100%.

[0102] The experimental results are shown in Figure 8 The horizontal coordinate is pH, and the vertical coordinate is relative enzyme activity. Under the condition of pH 4, the enzyme activity was significantly lost, and the relative enzyme activity of WT, F145Y-V267L and F145Y-K420Q was only 22.3%, 23.5% and 28.1% respectively. Under the condition of pH 5-10, the relative enzyme activity was mostly maintained above 70%, indicating that PanXI and its mutants had good stability under weakly acidic to moderately strong alkaline conditions. Among them, under the condition of pH 4-6, the relative enzyme activity of the two mutants was higher than that of the wild type, showing better resistance to weak acid.

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

[0104] Since the in vitro enzyme activity test of most xylose isomerases is carried out under the optimal conditions of the enzyme (pH 7.5), and the actual application environment of this study is about pH 6. In addition, through the study of enzyme properties, it was found that the two mutants had stronger resistance to low temperature and weak acid pH conditions compared with the wild type. Therefore, the in vitro enzyme activity of xylose isomerase and its mutants was tested under the conditions of pH 7.5 and 6 and temperature 30℃. The enzyme sample used in this experiment was the pure enzyme obtained by nickel column purification.

[0105] The experimental results are shown in Figure 9 The specific enzyme activity of wild type XI and F145Y-V267L, F145Y-K420Q mutants under the condition of pH 7.5 was 1.91, 1.83 and 2.39 U / mg respectively. The specific activity of 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. The specific enzyme activity of wild type XI and F145Y-V267L, F145Y-K420Q mutants under the condition of pH 6 was 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 mutant F145Y-V267L and F145Y-K420Q have higher enzyme activity and stability in low temperature and weak acid conditions than the wild type. The performance improvement of these mutants is more consistent with the application scenario of Zymomonas mobilis for weak acid fermentation for ethanol production, which may be one of the reasons for the improved performance of the mutant xylose metabolism.

[0107] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A xylose isomerase mutant, characterized in that, The amino acid sequence of the xylose isomerase mutant is shown as SEQ ID NO.

9.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a gene sequence encoding the xylose isomerase mutant as claimed in claim 1.

3. The nucleic acid molecule of claim 2, 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.

4. An expression vector, characterized by, The nucleic acid molecule comprises a gene sequence encoding the xylose isomerase mutant as claimed in claim 1.

5. The expression vector of claim 4, wherein, The expression vector is a pEZ15a or pTZ28a vector.

6. Use of a xylose isomerase mutant according to claim 1, a nucleic acid molecule according to any of claims 2-3 or an expression vector according to any of claims 4-5 for enhancing the ability of a microorganism to convert xylose, characterized in that, The microorganism is Zymomonas mobilis.

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