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

Abstract

The present application relates to the field of agricultural biotechnology, and in particular to a xylose transport protein mutant with improved xylose transport capacity, and genes and applications thereof. Compared with the wild type, after overexpression of xylose transport protein Xyl, the utilization rate of xylose by the engineered strain of Myceliopthora thermophila reached 29%; after overexpression of the xylose transport protein mutants M365F, M365L, M365Y, M365S and M365V, the utilization rate of xylose by the engineered strain of Myceliopthora thermophila reached 78%, 71%, 64%, 45% and 41% respectively. The engineered strain of Myceliopthora thermophila with overexpression of the xylose transport protein mutants M365F, M365L, M365Y, M365S and M365V had an increase of 163%, 140%, 119%, 53% and 38% in the transport of xylose under a mixed sugar system.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, in particular to a xylose transport protein mutant with improved xylose transport capacity, and genes and applications thereof. BACKGROUND

[0002] The current microorganism has low efficiency in simultaneous utilization of glucose and xylose in straw hydrolysate, and this bottleneck problem seriously restricts the industrial application process of straw biomass. Research has found that the "glucose inhibition" phenomenon caused by carbon source repression effect is the key factor causing low utilization rate of xylose.

[0003] To achieve efficient utilization of mixed sugar by microorganisms, improving the efficiency of xylose transport is a key scientific problem to be solved. Among them, the competitive inhibition effect caused by the non-specific binding of glucose and xylose transport protein is the main bottleneck restricting the efficiency of xylose transport. However, the research on xylose transport protein has been slow, and so far only about 30 sugar transport proteins from 9 different microorganisms have been confirmed to have xylose transport capacity, but they generally show high sensitivity to glucose inhibition and inherent low transport efficiency characteristics. Therefore, it is urgent to deeply explore and develop high-activity xylose transport proteins to relieve the inhibition effect of glucose on xylose metabolism, and to provide a solution to break through the bottleneck of mixed sugar utilization by microorganisms. SUMMARY

[0004] The purpose of the present application is to provide a high-activity xylose transport protein mutant.

[0005] Another purpose of the present application is to provide the use of the above-mentioned xylose transport protein mutant.

[0006] The present application obtains a mutant protein by protein engineering of xylose transport protein Xyl. Compared with the wild type, the xylose transport activity of the mutant is significantly improved, and it can be widely used in the construction of microbial cell factories with simultaneous co-utilization ability of mixed sugar.

[0007] According to the technical scheme of the present application, the amino acid sequence of the wild-type xylose transport protein Xyl is shown in SEQ ID NO: 1, and the mutant is obtained by substituting the amino acid at position 365 of the enzyme.

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

[0009] SEQ ID NO: 1

[0010] MGAGGGGDAGAFYDAALKKRQAMMGKSGPSALLKNFRVFRIAAFACIGGVLYGYNQGMFSGVLAMPSFNSHMGEYTTNQTKKGWLTAILELGAWLGTLLSSFLAEVLSRKYGVLVACAVFMLGVVIQTTAVSAGHNSILAGRFITGMGVGSLAMIIPIYNSEVAPPEVRGALVATQQLAICFGIMISFWIDYGTNYIGGTGEGQSDAAWLLPVCLQLAPAVILFVGMIFMPFSPRWLINHGREEEARKVLSELRGMPPDHELVEIEFLEIKAQSLFEKRSIAEMFPELSERTAWNIFKLQFVAIKKLFQTKAMFKRVIVATVTMFFQQWTGINAVLYYAPFIFQQLGLDLNTTSLLATGVVGIVMFIATIPSVLWVDRAGRKPVLTIGAIGMATCHIIIAVLVAKNINQWAEQRAAGWAAVCMVWLFVIHFGYSWGPCAWIIVAEIWPLSTRPYGVALGASSNWMNNFIVGQVTPDMLEGIPYGTYILFGLLTYLGAAFIWFLVPETKRLTLEEMDVLFGSEGTAAADYERMEEINNEIGLNQILRGEGRVTAPSTSDAEKPKGAEQMETV.

[0011] In an embodiment of the present application, the mutant comprises M365F, M365L, M365Y, M365S and M365V.

[0012] The present application also relates to a gene encoding the above-mentioned mutant of xylose transporter protein.

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

[0014] The present application also relates to a recombinant expression vector comprising the above-mentioned gene.

[0015] In an embodiment of the present application, the host cell is Myceliopthora thermophila.

[0016] The above-mentioned recombinant expression vector is transformed into a host cell of Myceliopthora thermophila for recombinant expression, so as to obtain an engineered strain of Myceliopthora thermophila with enhanced ability of utilizing xylose in a mixed sugar system.

[0017] The present application has the following beneficial effects:

[0018] Using the xylose transporter mutants M365F, M365L, M365Y, M365S and M365V of the present application can significantly enhance the xylose transport capacity of Myceliopthora thermophila under mixed sugar system. The present application provides key functional elements for constructing a chassis cell that can efficiently co-utilize glucose and xylose in straw hydrolysate, and has important application value and technical significance for promoting the efficient bioconversion of lignocellulosic biomass. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The utilization of xylose by the Myceliopthora thermophila engineering strain overexpressing the xylose transporter mutant under mixed sugar system is shown. DETAILED DESCRIPTION

[0020] The present application is further illustrated by the following examples without limiting the present application to the described examples. The experimental methods in the following examples, for which no specific conditions are indicated, are selected according to the conventional methods and conditions, or according to the instructions of the commercial products.

[0021] 1. Strains

[0022] Myceliopthora thermophila ATCC 42464 is an existing industrial filamentous fungus for producing cellulase, which is preserved in the American Type Culture Collection. In this example, the main alkaline protease gene-deficient strain Δ alp 1 is used as the starting strain, which is more stable after protein expression than Myceliopthora thermophila ATCC 42464 and is not easily degraded by protease.

[0023] 2. Culture medium and other reagents

[0024] The culture medium used for culturing Myceliopthora thermophila is potato glucose agar medium;

[0025] Protoplast regeneration medium (100 mL): 2 mL of 50 × Vogel's solution, 2 g of sucrose, 18.2 g of sorbitol, 0.75 g of agarose, and constant volume to 100 mL;

[0026] 50 × Vogel's solution (1 L): 130 g of trisodium citrate dihydrate, 126 g of potassium nitrate, 144 g of ammonium dihydrogen phosphate, 80 g of potassium dihydrogen phosphate, 10 g of magnesium sulfate heptahydrate, 5 g of calcium chloride dihydrate, 5 mL of trace element solution, 2.5 mL of 0.1 mg / mL biotin solution, 2 mL of chloroform, and constant volume to 1000 mL;

[0027] 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, make up to 100 mL;

[0028] Mixed sugar utilization function evaluation medium (100 mL): 2 mL 50 × Vogel's solution, 4 g glucose, 2 g xylose;

[0029] Example 1 Site-directed mutation of xylose transporter Xyl and preparation of recombinant expression vectors of mutants

[0030] The genomic DNA of xylose transporter Xyl was used as a template for PCR amplification of the left and right fragments at the mutation site. The target fragments were obtained by electrophoresis on a 1% agarose gel and gel recovery. Subsequently, the two fragments were used as templates for PCR amplification with primers Xyl-F and Xyl-R. The target fragments were obtained by electrophoresis on a 1% agarose gel and gel cutting. The fragments were 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-M365G, SESA-neo-Xyl-M365C, SESA-neo-Xyl-M365I, SESA-neo-Xyl-M365D, SESA-neo-Xyl-M365H, 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, and SESA-neo-Xyl-M365E. The primers used for PCR amplification of the xylose transporter mutants and the sequences are shown in Table 1 (the italicized part is the homologous arm).

[0031] Table 1 Primer information table for construction of xylose transporter mutants

[0032] 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) GGGATGGTCGCGATGAAGCCGACGATGCCGACCACGCCCGTG Xyl-M365C-R (SEQ ID NO: 17) GGATGGTCGCGATGAAGCAGACGATGCCGACCACGCCCGTG GGATGGTCGCGATGAAGCAGACGATGCCGACCACGCCCGTG Xyl-M365I-R (SEQ ID NO: 19) GGATGGTCGCGATGAAGATGACGATGCCGACCACGCCCGTG GGATGGTCGCGATGAAGATGACGATGCCGACCACGCCCGTG Xyl-M365D-R (SEQ ID NO: 21) GGATGGTCGCGATGAAGTCGACGATGCCGACCACGCCCGTG GGATGGTCGCGATGAAGTCGACGATGCCGACCACGCCCGTG Xyl-M365H-R (SEQ ID NO: 23) GGATGGTCGCGATGAAGTGGACGATGCCGACCACGCCCGTG GGATGGTCGCGATGAAGTGGACGATGCCGACCACGCCCGTG Xyl-M365N-R (SEQ ID NO: 25) GGATGGTCGCGATGAAGTTGACGATGCCGACCACGCCCGTG GGATGGTCGCGATGAAGTTGACGATGCCGACCACGCCCGTG Xyl-M365P-R (SEQ ID NO: 27) GGGATGGTCGCGATGAACGGGACGATGCCGACCACGCCCGTG GGGATGGTCGCGATGAACGGGACGATGCCGACCACGCCCGTG Xyl-M365Q-R (SEQ ID NO: 29) GGATGGTCGCGATGAACTGGACGATGCCGACCACGCCCGTG GGATGGTCGCGATGAACTGGACGATGCCGACCACGCCCGTG Xyl-M365A-R (SEQ ID NO: 31) GGATGGTCGCGATGAACGCGACGATGCCGACCACGCCCGTG GGATGGTCGCGATGAACGCGACGATGCCGACCACGCCCGTG ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Xyl-M365T-F (SEQ ID NO: 32) TGGTCGGCATCGTCCGGTTCATCGCGACCATCCCGTCCGTCC 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 alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp .

[0033] Example 2 Construction of a thermophilic Myceliopthora thermophila engineering strain overexpressing xylose transporter mutants

[0034] Protoplast preparation: The starting strain of Myceliopthora thermophila Δ alp 1 spore suspension was spread on potato glucose agar medium plate, and after 16 hours of culture at 37 ℃, the germinated mycelium was collected and placed in 20 mL of 10 mg / mL lysing enzyme solution, and digested at 75 rpm and 28 ℃ for 2 hours. The filtrate was collected by filtration into a 50 mL centrifuge tube, and centrifuged at 3000 rpm and 4 ℃ 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 ℃ for 10 minutes; the supernatant was discarded, 200 μL of STC solution was added to resuspend the protoplasts, 50 μL of PEG solution was mixed gently with the gene fragment, and incubated on ice for 20 minutes; then 2 mL of PEG solution was added, mixed gently, and incubated at room temperature for 5 minutes, 4 mL of STC solution was added, and then mixed with the protoplast regeneration medium containing 100 μg / mL G418, and cultured at 45 ℃ for 3 days until the transformants grew.

[0035] The transformants were picked and cultured on potato glucose agar medium plate for 3 days, and then the transformant genomic DNA was extracted, which was used as a template for PCR verification. According to the above method, the recombinant vector 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-M365H, 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 fragment was transformed into the host Myceliopthora thermophila starting strain Δ alp 1, to obtain the xylose transporter overexpressing Myceliopthora thermophila engineering strain Δ alp 1-OEXylM365F, Δ Figure 1 1-OEXylM365L, Δ alp 1-OEXylM365Y, Δ alp 1-OEXylM365S, Δalp 1-OEXylM365V, Δ alp 1-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.

[0036] Example 3 Evaluation of the function of the overexpressing xylose transporter mutant of Myceliopthora thermophila engineering strain in mixed sugar system

[0037] The Myceliopthora thermophila starting strain Δ alp 1. The Myceliopthora thermophila engineering strain overexpressing xylose transporter and its mutants were inoculated on potato glucose agar culture plates, and incubated at 45°C for 7 days to produce spores, then inoculated in mixed sugar utilization function evaluation medium at a concentration of 10 6 spores / mL, and the transport activity of the xylose transporter was evaluated according to the residual amount of xylose in the fermentation broth on the fourth day. The content of xylose was detected by high performance liquid chromatography using a Borel Aminex HPX-87H sugar analysis column, with a column temperature of 45°C, a mobile phase of 5 mM sulfuric acid, a flow rate of 0.5 mL / min, and a sample injection amount of 10 μL.

[0038] The results are shown in alp Table 1. The Myceliopthora thermophila starting strain Δ alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp alp1The utilization of xylose was affected by carbon source repression effect. The utilization rate of xylose by the engineered strain of Myceliopthora thermophila reached 29% after overexpression of xylose transporter Xyl. The utilization rate of xylose by the engineered strain of Myceliopthora thermophila reached 78%, 71%, 64%, 45% and 41% after overexpression of xylose transporter mutants M365F, M365L, M365Y, M365S and M365V, respectively. Compared with the wild type, the transport of xylose by the engineered strain of Myceliopthora thermophila overexpressing xylose transporter mutants M365F, M365L, M365Y, M365S and M365V was increased by 163%, 140%, 119%, 53% and 38% in the mixed sugar system. This result showed that the xylose transporter mutants M365F, M365L, M365Y, M365S and M365V could significantly enhance the transport capacity of Myceliopthora thermophila for xylose in the mixed sugar system.

[0039] The above examples are only used to explain the technical solutions of the present application, and do not limit the protection scope of the present application.

Claims

1. A xylose transporter mutant with enhanced xylose transport capacity, characterized in that, The amino acid sequence of the xylose transporter mutant is the amino acid sequence obtained by substituting the 365th amino acid of the amino acid sequence shown in SEQ ID NO:1 with the following substitutions: M365F, M365L, M365Y, M365S or M365V.

2. A xylose transporter gene, characterized in that, The xylose transporter gene encodes a xylose transporter mutant with enhanced xylose transport capacity as described in claim 1.

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

4. Recombinant cells comprising the xylose transporter gene of claim 2.

5. The application of the xylose transporter mutant with enhanced xylose transport capacity as described in claim 1 for improving the xylose transport efficiency of microorganisms.

6. The application according to claim 5, characterized in that, The microorganism is an engineered strain of thermophilic filamentous fungus.

7. The application according to claim 5, characterized in that, The xylose transporter mutant with enhanced xylose transport capacity is used for microbial degradation of straw.

8. A method for improving the xylose transport capacity of *Thermophilus thymol*, characterized in that, The method includes the step of expressing the xylose transporter gene of claim 2 in *Thermophilus thymol*.

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