Xylose transport protein mutant with improved xylose transport capacity and gene and application thereof

Abstract

The invention relates to the technical field of agricultural biology, in particular to a xylose transporter mutant with improved xylose transport capacity as well as a gene and application of the xylose transporter mutant. Compared with a wild type, after overexpression of the xylose transporter Xyl, the utilization rate of xylose by the engineering strain of the mycelia thermophila reaches 29%; after xylose transporter mutants M365F, M365L, M365Y, M365S and M365V are over-expressed, the xylose utilization rates of the engineering strains of the destructor thermophila respectively reach 78%, 71%, 64%, 45% and 41%. The xylose transshipment of the xylose transshipment protein mutants M365F, M365L, M365Y, M365S and M365V, which are overexpressed, is improved by 163 percent, 140 percent, 119 percent, 53 percent and 38 percent under a mixed sugar system. The xylose transshipment of the xylose transshipment protein mutants M365F, M365L, M365Y, M365S and M365V is improved.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and in particular to a xylose transport protein mutant with improved xylose transport capacity, a gene thereof and an application thereof. Background Art

[0002] Currently, microbial efficiency in simultaneously utilizing glucose and xylose in straw hydrolysates is low, a bottleneck that severely restricts the industrial application of straw-based biomass. Research has found that the "glucose inhibition" phenomenon, caused by carbon source inhibition, is a key factor contributing to the low xylose utilization rate.

[0003] To achieve efficient utilization of mixed sugars by microorganisms, improving the efficiency of xylose transport is a key scientific issue that needs to be addressed urgently. Among them, the competitive inhibition effect caused by the nonspecific binding of glucose to xylose transporters is the main bottleneck restricting xylose transport efficiency. However, research on xylose transporters has been slow. To date, only about 30 sugar transporters from 9 different microorganisms have been confirmed to have the ability to transport xylose, but they generally show high sensitivity to glucose inhibition and inherent low transport efficiency. Therefore, there is an urgent need to further explore and develop highly active xylose transporters to relieve the inhibitory effect of glucose on xylose metabolism and provide a solution to break through the bottleneck of microbial mixed sugar utilization. Summary of the Invention

[0004] The purpose of the present invention is to provide a highly active xylose transporter mutant.

[0005] Another object of the present invention is to provide applications of the xylose transporter mutant.

[0006] The present invention obtains a mutant protein by protein engineering the xylose transporter Xyl. Compared with the wild type, the mutant has significantly improved xylose transport activity and can be widely used to construct microbial cell factories capable of synchronously co-utilizing mixed sugars.

[0007] According to the technical solution of the present invention, the amino acid sequence of the wild-type xylose transporter Xyl is shown in SEQ ID NO: 1, and the 365th amino acid position of the enzyme is substituted to obtain a mutant.

[0008] The amino acid sequence of the sugar transporter Xyl is shown in SEQ ID NO: 1.

[0009] SEQ ID NO: 1 .

[0010] In an embodiment of the present invention, the mutants include: M365F, M365L, M365Y, M365S and M365V.

[0011] The present invention also relates to a gene encoding the xylose transporter mutant.

[0012] The present invention also relates to a recombinant expression vector comprising the above gene.

[0013] The present invention also relates to a recombinant expression vector comprising the above gene.

[0014] In an embodiment of the present invention, the host cell is Myceliophthora thermophila.

[0015] The recombinant expression vector is transferred into a host cell of Myceliophthora thermophila for recombinant expression, thereby obtaining an engineered Myceliophthora thermophila strain with enhanced xylose utilization ability under a mixed sugar system.

[0016] The beneficial effects of the present invention are: The xylose transporter mutants M365F, M365L, M365Y, M365S, and M365V of the present invention significantly enhance the xylose transport capacity of Myceliophthora thermophila in a mixed sugar system. This invention provides key functional elements for constructing chassis cells that efficiently co-utilize glucose and xylose in straw hydrolyzate, and has important application value and technical significance for promoting the efficient bioconversion of lignocellulosic biomass. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Showing the xylose utilization of the thermophilic Myceliophthora engineered strain overexpressing a xylose transporter mutant under a mixed sugar system. DETAILED DESCRIPTION

[0018] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0019] 1. Strains Thermophila ATCC42464 is an existing industrial filamentous fungus used to produce cellulase and is stored in the American Type Culture Collection. alp 1 was used as the starting strain. Compared with Myceliophthora thermophila ATCC42464, the protein was more stable after expression and was not easily degraded by proteases.

[0020] 2. Culture medium and other reagents The culture medium used for culturing Myceliophthora thermophila was potato dextrose agar medium; Protoplast regeneration medium (100 mL): 2 mL 50 × Vogel's solution, 2 g sucrose, 18.2 g sorbitol, 0.75 g agarose, dilute to 100 mL; 50 × Vogel's solution (1 L): 130 g trisodium citrate dihydrate, 126 g potassium nitrate, 144 g ammonium dihydrogen phosphate, 80 g potassium dihydrogen phosphate, 10 g magnesium sulfate heptahydrate, 5 g calcium chloride dihydrate, 5 mL trace element solution, 2.5 mL 0.1 mg / mL biotin solution, 2 mL chloroform, dilute to 1000 mL; Trace element solution (100 mL): 5 g citric acid monohydrate, 5 g zinc sulfate heptahydrate, 1 g ferrous ammonium sulfate hexahydrate, 0.25 g copper sulfate pentahydrate, 0.05 g manganese sulfate monohydrate, 0.05 g boric acid, 0.05 g sodium molybdate dihydrate, dilute to 100 mL; Mixed sugar utilization function evaluation medium (100 mL): 2 mL 50 × Vogel's solution, 4 g glucose, 2 g xylose; Example 1 Site-directed mutagenesis of xylose transporter Xyl and preparation of recombinant expression vector of the mutant

[0021] Using the genomic DNA of the xylose transporter Xyl as a template, two fragments were separated at the mutation site for PCR amplification, and the fragments were electrophoresed on a 1% agarose gel and the target fragments were recovered from the gel. Subsequently, the two fragments were used as templates for PCR amplification again using primers Xyl-F and Xyl-R, and the fragments were electrophoresed on a 1% agarose gel. The target fragments were cut from the gel and then recovered and connected to the SESA-neo vector backbone by homologous recombination to obtain the recombinant expression vectors SESA-neo-Xyl-M365F, SESA-neo-Xyl-M365L, SESA-neo-Xyl-M365Y, SESA-neo-Xyl-M365S, SESA-neo-Xyl-M365V, SESA-neo-Xyl-M365V. The primers and sequences used for PCR amplification of the xylose transporter mutants are shown in Table 1 (the italicized parts indicate the homology arms).

[0022] Table 1 Primer information for constructing xylose transporter mutants Primer Name Sequence Information (5'-3') Xyl-F(SEQ ID NO:2) ATGGGCGCTGGCGGAGGCGGCGACGC Xyl-R(SEQ ID NO:3) TTAGACAGTCTCCATCTGTTCCGC Xyl-M365F-F(SEQ ID NO:4) GTGGTCGGCATCGTCTTCTTCATCGCGACCATCCCGTCCGTC Xyl-M365F-R(SEQ ID NO:5) GGTCGCGATGAAGAAGACGATGCCGACCACGCCCGTGGCCAGGAGC Xyl-M365L-F(SEQ ID NO:6) TGGTCGGCATCGTCCTGTTCATCGCGACCATCCCGTCCGTCC Xyl-M365L-R(SEQ ID NO:7) GGGATGGTCGCGATGAACAGGACGATGCCGACCACGCCCGTG Xyl-M365Y-F(SEQ ID NO:8) TGGTCGGCATCGTCTACTTCATCGCGACCATCCCGTCCGTCC Xyl-M365Y-R(SEQ ID NO:9) GGGATGGTCGCGATGAAGTAGACGATGCCGACCACGCCCGTGG Xyl-M365S-F(SEQ ID NO:10) TGGTCGGCATCGTCTCCTTCATCGCGACCATCCCGTCCGTCC Xyl-M365S-R(SEQ ID NO:11) GGGATGGTCGCGATGAAGGAGACGATGCCGACCACGCCCGTG Xyl-M365V-F(SEQ ID NO:12) TGGTCGGCATCGTCGTGTTCATCGCGACCATCCCGTCCGTCC Xyl-M365V-R(SEQ ID NO:13) GGGATGGTCGCGATGAACACGACGATGCCGACCACGCCCGTGG Xyl-M365G-F(SEQ ID NO:14) TGGTCGGCATCGTCGGCTTCATCGCGACCATCCCGTCCGTCC Xyl-M365G-R(SEQ ID NO:15) GGATGGTCGCGATGAAGCCGACGATGCCGACCACGCCCGTG Xyl-M365C-F(SEQ ID NO:16) TGGTCGGCATCGTCTGCTTCATCGCGACCATCCCGTCCGTCC Xyl-M365C-R(SEQ ID NO:17) GGATGGTCGCGATGAAGCAGACGATGCCGACCACGCCCGTG Xyl-M365I-F(SEQ ID NO:18) TGGTCGGCATCGTCATCTTCATCGCGACCATCCCGTCCGTCC Xyl-M365I-R(SEQ ID NO:19) GGATGGTCGCGATGAAGATGACGATGCCGACCACGCCCGTG Xyl-M365D-F(SEQ ID NO:20) TGGTCGGCATCGTCGACTTCATCGCGACCATCCCGTCCGTCC Xyl-M365D-R(SEQ ID NO:21) GGATGGTCGCGATGAAGTCGACGATGCCGACCACGCCCGTG Xyl-M365H-F(SEQ ID NO:22) TGGTCGGCATCGTCCACTTCATCGCGACCATCCCGTCCGTCC Xyl-M365H-R(SEQ ID NO:23) GGATGGTCGCGATGAAGTGGACGATGCCGACCACGCCCGTG Xyl-M365N-F(SEQ ID NO:24) TGGTCGGCATCGTCAACTTCATCGCGACCATCCCGTCCGTCC Xyl-M365N-R(SEQ ID NO:25) GGATGGTCGCGATGAAGTTGACGATGCCGACCACGCCCGTG Xyl-M365P-F(SEQ ID NO:26) TGGTCGGCATCGTCCCGTTCATCGCGACCATCCCGTCCGTCC Xyl-M365P-R(SEQ ID NO:27) GGGATGGTCGCGATGAACGGGACGATGCCGACCACGCCCGTG Xyl-M365Q-F(SEQ ID NO:28) TGGTCGGCATCGTCCAGTTCATCGCGACCATCCCGTCCGTCC Xyl-M365Q-R(SEQ ID NO:29) GGATGGTCGCGATGAACTGGACGATGCCGACCACGCCCGTG Xyl-M365A-F(SEQ ID NO:30) TGGTCGGCATCGTCGCGTTCATCGCGACCATCCCGTCCGTCC Xyl-M365A-R(SEQ ID NO:31) GGATGGTCGCGATGAACGCGACGATGCCGACCACGCCCGTG Xyl-M365T-F(SEQ ID NO:32) TGGTCGGCATCGTCACCTTCATCGCGACCATCCCGTCCGTCC Xyl-M365T-R(SEQ ID NO:33) GGGATGGTCGCGATGAAGGTGACGATGCCGACCACGCCCGTG Xyl-M365R-F(SEQ ID NO:34) TGGTCGGCATCGTCCGGTTCATCGCGACCATCCCGTCCGTCC Xyl-M365R-R(SEQ ID NO:35) GGATGGTCGCGATGAACCGGACGATGCCGACCACGCCCGTG Xyl-M365W-F(SEQ ID NO:36) TGGTCGGCATCGTCTGGTTCATCGCGACCATCCCGTCCGTCC Xyl-M365W-R(SEQ ID NO:37) GGGATGGTCGCGATGAACCAGACGATGCCGACCACGCCCGTGG Xyl-M365K-F(SEQ ID NO:38) TGGTCGGCATCGTCAAGTTCATCGCGACCATCCCGTCCGTCC Xyl-M365K-R(SEQ ID NO:39) GGGATGGTCGCGATGAACTTGACGATGCCGACCACGCCCGTG Xyl-M365E-F(SEQ ID NO:40) TGGTCGGCATCGTCGAGTTCATCGCGACCATCCCGTCCGTCC Xyl-M365E-R(SEQ ID NO:41) GGATGGTCGCGATGAACTCGACGATGCCGACCACGCCCGTG Example 2 Construction of an engineered strain of Myceliophthora thermophila overexpressing a xylose transporter mutant

[0023] Protoplast preparation: Thermomycete alpThe spore suspension was spread on potato dextrose agar plates and cultured at 37°C for 16 h. The germinated mycelia were collected and placed in 20 mL of 10 mg / mL lysing enzyme solution and digested at 75 rpm at 28°C for 2 h. The filtrate was collected by filtration into a 50 mL centrifuge tube and centrifuged at 3000 rpm and 4°C for 10 minutes. Then, 10 mL of STC solution (1.2 M sorbitol, 50 mM calcium chloride, 35 mM sodium chloride, pH 7.0) was added to suspend the protoplasts and centrifuged at 3000 rpm and 4°C for 10 minutes. The supernatant was discarded and 200 μL of STC solution was added to resuspend the protoplasts. 50 μL of PEG solution and gene fragment were gently shaken and incubated on ice for 20 minutes. Then, 2 mL of PEG solution was added and gently shaken. The tube was incubated at room temperature for 5 minutes. 4 mL of STC solution was added and then mixed with protoplast regeneration medium supplemented with 100 μg / mL G418. The tube was cultured at 45°C for 3 days until transformants grew.

[0024] The transformants were picked and cultured on potato dextrose agar plates for 3 days, and then the genomic DNA of the transformants was extracted and used as a template for PCR verification. alp 1 as the host, and the recombinant vectors SESA-neo-Xyl-M365F, SESA-neo-Xyl-M365L, SESA-neo-Xyl-M365Y, SESA-neo-Xyl-M365S, SESA-neo-Xyl-M365V, SESA-neo-Xyl-M365G, SESA-neo-Xyl-M365C, SESA-neo-Xyl-M365I, SESA-neo-Xyl-M365D, SESA-neo-Xyl-M365 H, SESA-neo-Xyl-M365N, SESA-neo-Xyl-M365P, SESA-neo-Xyl-M365Q, SESA-neo-Xyl-M365A, SESA-neo-Xyl-M365T, SESA-neo-Xyl-M365R, SESA-neo-Xyl-M365W, SESA-neo-Xyl-M365K, SESA-neo-Xyl-M365E gene fragments, to obtain the thermophilic Myceliophthora engineered strain Δ alp 1-OEXylM365F,Δ alp 1-OEXylM365L,Δ alp 1-OEXylM365Y,Δ alp 1-OEXylM365S,Δ alp 1-OEXylM365V,Δ alp1-OEXylM365G,Δ alp 1-OEXylM365C,Δ alp 1-OEXylM365I,Δ alp 1-OEXylM365D,Δ alp 1-OEXylM365H,Δ alp 1-OEXylM365N,Δ alp 1-OEXylM365P,Δ alp 1-OEXylM365Q,Δ alp 1-OEXylM365A,Δ alp 1-OEXylM365T,Δ alp 1-OEXylM365R,Δ alp 1-OEXylM365W,Δ alp 1-OEXylM365K,Δ alp 1-OEXylM365E. Example 3 Evaluation of xylose utilization function of an engineered strain of Myceliophthora thermophila overexpressing a xylose transporter mutant in a mixed sugar system

[0025] Thermophilic Myceliophthora starting strain Δ alp 1. The thermophilic Myceliophthora strains overexpressing xylose transporter and its mutants were inoculated on potato dextrose agar plates and cultured at 45°C for 7 days until spores were formed. 6 Conidia spores were inoculated into a mixed sugar utilization evaluation medium at a concentration of 10 spores / mL. The xylose transport activity of the xylose transporter was evaluated based on the amount of xylose remaining in the fermentation broth on day 4. Xylose content was determined by high-performance liquid chromatography using a Bio-Rad Aminex HPX-87H sugar analysis column at a column temperature of 45°C, a mobile phase of 5 mM sulfuric acid, a flow rate of 0.5 mL / min, and an injection volume of 10 μL.

[0026] The results are as follows Figure 1 As shown, the starting strain of Thermomycete alp1. Due to the carbon source repression effect, the strain is unable to utilize xylose. Overexpression of the xylose transporter Xyl increased the xylose utilization rate of the engineered Myceliophthora thermophila strain to 29%. Overexpression of the xylose transporter mutants M365F, M365L, M365Y, M365S, and M365V increased the xylose utilization rates of the engineered Myceliophthora thermophila strain to 78%, 71%, 64%, 45%, and 41%, respectively. Compared to the wild-type strain, the engineered Myceliophthora thermophila strains overexpressing the xylose transporter mutants M365F, M365L, M365Y, M365S, and M365V increased xylose transport by 163%, 140%, 119%, 53%, and 38% in a mixed sugar system. This result indicates that the xylose transporter mutants M365F, M365L, M365Y, M365S and M365V can significantly enhance the xylose transport ability of Myceliophthora thermophila in a mixed sugar system.

[0027] The above embodiments are only used to explain the technical solutions of the present application and do not limit the scope of protection of the present application.

Claims

1. A xylose transporter mutant having improved xylose transport capacity, characterized in that: The xylose transporter mutant has an amino acid sequence in which the amino acid at position 365 of the amino acid sequence shown in SEQ ID NO: 1 is substituted, wherein the amino acid at position 365 of the amino acid sequence shown in SEQ ID NO: 1 undergoes the following substitution: M365F, M365L, M365Y, M365S, or M365V.

2. A xylose transporter gene, characterized in that The xylose transporter gene encodes the xylose transporter mutant with improved xylose transport capacity according to claim 1.

3. A recombinant vector comprising the xylose transporter gene according to claim 2.

4. A recombinant cell comprising the xylose transporter gene according to claim 2.

5. The xylose transport protein mutant with improved xylose transport capacity according to claim 1 is used to improve the xylose transport efficiency of microorganisms.

6. The use according to claim 5, characterized in that The microorganism is an engineered strain of Myceliophthora thermophila.

7. The use according to claim 5, characterized in that The xylose transport protein mutant with improved xylose transport capacity is used for microbial degradation of straw.

8. A method for improving the xylose transport capacity of Myceliophthora thermophila, characterized in that: The method comprises the step of expressing the xylose transporter gene of claim 2 in Myceliophthora thermophila.

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