Thermophilic mycelia gene engineering strain with improved mixed sugar utilization capability and application thereof

Abstract

The invention relates to the technical field of agricultural biology, in particular to a myceliophthora thermophila genetic engineering strain with improved mixed sugar utilization capacity and application of the myceliophthora thermophila genetic engineering strain. The strain constitutively expresses the sugar transporter MtSTP of the mycelia thermophila, and the xylose utilization rate of the strain in a mixed sugar system is increased by 3.97 times compared with that of an original strain; the invention also relates to application of the strain with improved mixed sugar utilization capability in production of malic acid by fermentation of mixed sugar, and the malic acid yield of the strain in a mixed sugar system is improved by 0.63 times compared with that of an original strain. The method disclosed by the invention has important reference and guiding significance for construction of the chassis cells for efficiently utilizing the mixed sugar, and has important value for reducing the conversion cost of the lignocellulose biomass.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to a genetically engineered strain of Myceliophthora thermophila with improved mixed sugar utilization ability and application thereof. Background Art

[0002] Lignocellulosic biomass is an abundant renewable resource. Using it as a raw material and using microbial cell factories to synthesize high value-added products can not only reduce dependence on fossil resources, but also help achieve the carbon peak target, which has important environmental and economic significance. Among them, achieving efficient utilization of hydrolyzate mixed sugars by microorganisms is considered to be the key to reducing product costs. However, when most microorganisms ferment mixed sugars, they will preferentially utilize glucose in mixed sugars and delay the utilization of xylose due to the carbon source repression effect, which seriously restricts the conversion efficiency of lignocellulose. Therefore, the development of microbial cell factories that efficiently utilize mixed sugars is of great significance for the high-value utilization of lignocellulose.

[0003] In order to achieve efficient utilization of mixed sugars by microorganisms, the following two problems need to be solved: on the one hand, the problem of xylose transport, specifically the inhibition of xylose transport caused by the binding of glucose to xylose transporters; on the other hand, the problem of xylose metabolism, specifically the inhibition of transcriptional expression of genes related to xylose metabolism. In recent years, researchers have carried out a lot of research work on these two issues, but the research on xylose transporters has been slow. So far, only about 30 sugar transporters from 9 different microorganisms have been confirmed to have the ability to transport xylose, but the affinity of some sugar transporters for glucose is 10-100 times that of xylose, and the presence of glucose will strongly inhibit xylose transport. Therefore, finding and introducing high-affinity xylose transporters is crucial to improving the efficiency of microorganisms in utilizing mixed sugars.

[0004] Myceliophthora thermophila is a high-temperature filamentous fungus chassis strain with the ability to efficiently degrade biomass and secrete proteins. It has a high development and application value in the degradation and utilization of lignocellulose. However, in the process of mixed sugar utilization, it also has a carbon source repression effect and the problem of delayed utilization of xylose. At present, it is urgent to explore and introduce high-affinity xylose transporters to eliminate the inhibitory effect of glucose on xylose metabolism, improve the utilization efficiency of mixed sugars by Myceliophthora thermophila, and achieve low-cost production of target products of lignocellulosic biomass. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defect of low efficiency of mixed sugar utilization by microorganisms in the prior art, and the purpose of the present invention is to provide a genetically engineered strain of Myceliophthora thermophila with improved mixed sugar utilization ability.

[0006] Another object of the present invention is to provide a use of a genetically engineered strain of Myceliophthora thermophila with improved mixed sugar utilization ability in the production of malic acid by mixed sugar fermentation.

[0007] According to the genetically engineered Myceliophthora thermophila strain with improved mixed sugar utilization ability of the present invention, the genetically engineered Myceliophthora thermophila strain constitutively expresses a gene encoding a sugar transporter protein MtSTP, wherein the amino acid sequence of the sugar transporter protein MtSTP is shown in SEQ ID NO:3.

[0008] According to the genetically engineered Myceliophthora thermophila strain with improved mixed sugar utilization ability of the present invention, the genetically engineered Myceliophthora thermophila strain is an alkaline protease gene-deficient strain.

[0009] According to the genetically engineered strain of Myceliophthora thermophila with improved mixed sugar utilization ability of the present invention, the DNA sequence of the gene encoding the sugar transporter MtSTP is as shown in SEQ ID NO: 1, and the coding sequence of the sugar transporter MtSTP is as shown in SEQ ID NO: 2.

[0010] MtSTP genomic DNA sequence SEQ ID NO: 1

[0011] MtSTP coding sequence SEQ ID NO: 2

[0012] The amino acid sequence of MtSTP is shown in SEQ ID NO: 3 .

[0013] According to the genetically engineered strain of Myceliophthora with improved ability to produce malic acid using mixed sugars of the present invention, the genetically engineered strain of Myceliophthora thermophila constitutively expresses a gene encoding a sugar transporter MtSTP and expresses a gene encoding a heterologous malic acid transporter TrMae, wherein the amino acid sequence of the sugar transporter MtSTP is as shown in SEQ ID NO: 3, and the amino acid sequence of the heterologous malic acid transporter TrMae is as shown in SEQ ID NO: 5.

[0014] The nucleotide sequence of the gene encoding TrMae, SEQ ID NO: 4:

[0015] The amino acid sequence of TrMae, SEQ ID NO: 5: MKAAFPHAVDINDPNRPKLPIRQRLQHFTWAWYTLPMSTGGLSLLIYAQPYQFAGERVIGLVVYIINLIIFSLVTMAMMARFFLHAGEFVRSITHPREGFFVPTFFLSLATIITSTQRYAIPDDHPILEPAVKIAFWVYVALTAVLALGQYSYVFAAHNLSLKTFMPTLILPIFPIMLSGTIASVIAGTQ PEFDAMPILVAGLTCQGLGMSVAILMYAHMIGRLLQFGLPNREHRPGLFMCVGPPAFTALALVGMANGVPEGISELGIDKSVIQVVAILVAVFLWALSFWWFGIAVIAVVSSPPKYFHLGWWAMVFPNTGFTLATISIAKELLSPSLQWVTIGMSCCIFTIFIFVFVNHVRAVIIQDIMYPGRDENVEDH.

[0016] The method for improving the ability of Myceliophthora thermophila to produce malic acid by utilizing mixed sugars according to the present invention comprises the following steps: Constructing a recombinant expression vector comprising a gene encoding a sugar transporter protein MtSTP, wherein the gene encoding the sugar transporter protein MtSTP is regulated by a constitutive promoter in the recombinant expression vector, and the amino acid sequence of the sugar transporter protein MtSTP is shown in SEQ ID NO: 3; constructing a recombinant expression vector comprising a gene encoding a heterologous malate transporter TrMae; introducing the recombinant expression vector comprising the gene encoding the heterologous malate transporter TrMae into the thermophilic Myceliophthora; Transforming the constructed recombinant expression vector containing the gene encoding the sugar transporter MtSTP into Myceliophthora thermophila; The genetically engineered strain of Myceliophthora thermophila that was successfully transformed was screened.

[0017] According to the method for improving the ability of Myceliophthora thermophila to produce malic acid by utilizing mixed sugars, the amino acid sequence of the heterologous malic acid transporter TrMae is as shown in SEQ ID NO:5.

[0018] The present invention provides the use of the genetically engineered Myceliophthora thermophila strain with improved mixed sugar utilization ability for fermentation production of malic acid.

[0019] According to the genetically engineered Myceliophthora strain with improved malic acid production capability using mixed sugars of the present invention, the genetically engineered Myceliophthora thermophila strain is an alkaline protease gene-deficient strain.

[0020] According to the genetically engineered strain of Myceliophthora with improved ability to produce malic acid using mixed sugars of the present invention, the nucleotide sequence of the gene encoding the heterologous malic acid transporter TrMae is shown in SEQ ID NO:4.

[0021] The beneficial effects of the present invention are: Using the thermophilic Myceliophthora engineered strain with improved mixed sugar utilization ability of the present invention, its xylose utilization rate in the mixed sugar system is increased by 3.97 times compared with the starting strain; using the strain with improved mixed sugar utilization ability to produce malic acid by mixed sugar fermentation, its malic acid yield is increased by 0.63 times compared with the starting strain. The present invention has important reference and guiding significance for the construction of chassis cells for efficient utilization of mixed sugars, and has important value for reducing the cost of converting lignocellulosic biomass. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The mixed sugar system showed that the starting strain of Thermomycete alp 1 and genetically engineered strain Δ alp 1 -Mtstp Degradation rates of glucose and xylose; Figure 2 The mixed sugar system showed that the starting strain of Thermomycete alp 1- Trmae and genetically engineered strain Δ alp 1- Trmae-Mtstp Ability to synthesize malic acid; Figure 3 The mixed sugar system showed that the starting strain of Thermomycete alp 1- Trmae and genetically engineered strain Δ alp 1- Trmae-Mtstp Degradation rates of glucose and xylose during the synthesis of malic acid. DETAILED DESCRIPTION

[0023] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0024] 1. Strains and vectors Thermophilic Myceliophthora ATCC42464 is an existing industrial filamentous fungus used to produce cellulase and is stored in the American Type Culture Collection. alp 1 is used as the starting strain for genetic modification. Compared with the thermophilic Myceliophthora ATCC42464, the protein is more stable after expression and is not easily degraded by proteases.

[0025] 2. Culture medium and other reagents The 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 ammonium ferrous 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; Mixed sugar fermentation for malic acid production medium (1 L): 40 g glucose, 20 g xylose, 8 g peptone, 0.15 g potassium dihydrogen phosphate, 0.15 g potassium hydrogen phosphate, 0.10 g calcium chloride dihydrate, 0.10 g magnesium sulfate heptahydrate, 1 mL 0.5 g / L biotin, 1 mL trace element solution, 200 mL 40% calcium carbonate. Example 1 Preparation of recombinant expression vector of sugar transporter MtSTP

[0026] The genomic DNA of Myceliophthora thermophila was used as a template for PCR amplification. The target fragment was obtained by electrophoresis on a 1% agarose gel, and the fragment was recovered and connected to the SESA-neo vector backbone by homologous recombination to obtain the recombinant vector SESA-neo- Mtstp , where SESA is a commercial constitutive expression backbone. Mtstp Constitutive expression， The obtained recombinant product was transformed into the cloning competent cell Trans1-T1, and after culturing in a 37°C incubator for 12 hours, the transformants were picked for PCR verification and sequencing. The primers and primer sequences used for PCR amplification of the MtSTP coding gene are as follows (the italicized part is the homology arm): Mtstp -F:5' TCTACTACAATTATTAATTAAA ATGGGCGCTGGCGGAGGCGGCGACGC 3' (SEQ IDNO: 6), Mtstp -R: 5' ATCATAAGAAATTCGGATCCTCA TTAGACAGTCTCCATCTGTTCCGC 3' (SEQ ID NO: 7). Example 2 Construction of Myceliophthora thermophila strain constitutively expressing sugar transporter MtSTP

[0027] Protoplast preparation: Thermomycete alp 1 The spore suspension was spread on a potato dextrose agar plate and cultured at 37 °C for 16 h. The germinated mycelium was collected and placed in 20 mL of 10 mg / mL lysing enzyme solution and digested at 75 rpm at 28 °C for 2 h. Filter and collect the filtrate into a 50 mL centrifuge tube, centrifuge at 3000 rpm and 4 ℃ for 10 minutes; then add 10 mL STC solution (1.2 M sorbitol, 50 mM calcium chloride, 35 mM sodium chloride, pH 7.0) to suspend the protoplasts, centrifuge at 3000 rpm and 4 ℃ for 10 minutes; discard the supernatant, add 200 μL STC solution to resuspend the protoplasts, gently shake and mix the gene fragment with 50 μL PEG solution, and incubate on ice for 20 minutes; then add 2 mL PEG solution, shake and mix, incubate at room temperature for 5 minutes, add 4 mL STC solution, and then mix with protoplast regeneration medium containing antibiotics (100 μg / mL G418 or 100 μg / mL Zeocin), and culture at 45 ℃ for 3 days until transformants grow.

[0028] Transformants were selected and cultured on potato dextrose agar plates for 3 days, and then genomic DNA of the transformants was extracted and used as a template for PCR verification. alp 1 as the host, transform the recombinant vector SESA-neo- Mtstp The gene fragment of thermophilic Myceliophthora was obtained by constitutively expressing the sugar transport protein MtSTP. alp 1 -Mtstp .

[0029] Thermophilic Myceliophthora has an endogenous malic acid synthesis pathway, but multiple key genes in the malic acid synthesis pathway are not expressed or expressed weakly, and it does not have the ability to efficiently synthesize malic acid; previous studies have found that overexpression of the heterologous malic acid transporter TrMae can effectively increase the expression levels of multiple key genes in the malic acid synthesis pathway, thereby significantly improving the ability of Thermophilic Myceliophthora to synthesize malic acid.

[0030] Genetically engineered strain of Myceliophthora thermophila for producing malic acid alp 1- Trmae As the host, the gene fragment of the recombinant vector pPH-zeo-MtSTP was transformed, wherein pPH-zeo is a constitutive expression skeleton, and the genetic engineering strain of thermophilic Myceliophthora was obtained. alp 1- Trmae-Mtstp . Example 3 Evaluation of the mixed sugar utilization function of the genetically engineered strain of Myceliophthora thermophila

[0031] Thermophilic Myceliophthora starting strain Δ alp 1 and genetically engineered strain Δ alp 1 -Mtstp Inoculate on potato dextrose agar plates and culture at 45°C for 7 days until spores are produced. 6 The concentration of spores / mL was inoculated into the mixed sugar utilization function evaluation medium, and the biological function of the sugar transporter MtSTP was evaluated according to the residual amount of glucose and xylose in the fermentation broth on the sixth day. The glucose and xylose contents were detected by high performance liquid chromatography using a Bio-Rad 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 an injection volume of 10 μL.

[0032] The results are as follows Figure 1 As shown, the genetically engineered strain of Myceliophthora thermophila Δ alp 1 -Mtstp The utilization rate of xylose from the starting strain Δ alp 1 increased from 12.96% to 64.37%, an increase of 3.97 times; the genetically engineered strain of thermophilic myceliophthora Δ alp 1 - Mtstp The utilization rate of glucose is similar to that of the starting strain Δ alp 1, which were 83.27% and 80.11%, respectively. This result indicates that constitutive expression of the sugar transporter MtSTP significantly enhances the xylose utilization efficiency of Myceliophthora thermophila in a mixed sugar system; it also indicates that the sugar transporter MtSTP is a high-affinity xylose transporter that is less affected by the glucose repression effect. Example 4 Construction of the genetically engineered strain Δalp1-Trmae of Myceliophthora thermophila for malic acid production

[0033] PCR amplification was performed using Trichoderma reesei cDNA as a template. Electrophoresis was performed on a 1% agarose gel, and the gel was cut to obtain the target fragment. The fragment was recovered and connected to the SESA-neo vector backbone by homologous recombination to obtain the recombinant vector SESA-neo- Trmae The obtained recombinant product was transformed into the cloning competent cell Trans1-T1, and after culturing in a 37°C incubator for 12 hours, the transformants were picked for PCR verification and sequencing. The primers and primer sequences used for PCR amplification of the TrMae coding gene are as follows (the italicized part is the homology arm): Trmae -F: 5' TACTACAATTATTAATTAAA ATGAAAGCGGCATTCCCTCATGC 3' (SEQ ID NO: 8), Trmae -R: 5' ATAAGAAATTCGGATCCTCA TCAGTGATCCTCCACATTCTCATC 3' (SEQ ID NO: 9).

[0034] Referring to the above-mentioned thermophilic myceliophthora protoplast transformation method, the thermophilic myceliophthora genetic engineering strain Δ alp 1 as the host, transform the recombinant vector SESA-neo- Trmae The gene fragment of the thermophilic myceliophthora malic acid production genetic engineering strain Δ alp 1- Trmae . Example 5 Construction of a genetically engineered strain of Myceliophthora thermophila that constitutively expresses the sugar transporter MtSTP for malic acid production Δalp1-Trmae-Mtstp

[0035] The genomic DNA of Myceliophthora thermophila was used as a template for PCR amplification. The target fragment was obtained by electrophoresis on a 1% agarose gel, and the fragment was recovered and connected to the pPH-zeo vector backbone by homologous recombination to obtain the recombinant vector pPH-zeo- Mtstp The obtained recombinant product was transformed into the cloning competent cell Trans1-T1, and after culturing in a 37°C incubator for 12 hours, the transformants were picked for PCR verification and sequencing.

[0036] Referring to the above-mentioned thermophilic myceliophthora protoplast transformation method, the thermophilic myceliophthora malic acid production genetic engineering strain Δ alp 1- Trmae , transform the recombinant vector pPH-zeo- Mtstp The gene fragment of the thermophilic myceliophthora was obtained to obtain the malic acid production genetic engineering strain Δ alp 1- Trmae-Mtstp . Example 6 Effect of Genetically Engineered Thermophilic Myceliophthora Strain on Mixed Sugar Fermentation to Produce Malic Acid

[0037] The malic acid-producing strain of Myceliophthora thermophila Δ alp 1- Trmae and genetically engineered strain Δ alp 1 - Trmae-Mtstp Inoculate on potato dextrose agar plates and culture at 45°C for 7 days until spores are produced. 6 The concentration of spores / mL was inoculated into the mixed sugar fermentation malic acid production medium, and the biological function of the sugar transporter MtSTP was evaluated according to the remaining amount of glucose and xylose and the malic acid production in the fermentation broth on the second day. The contents of glucose, xylose and malic acid were detected by high performance liquid chromatography using a Bio-Rad Aminex HPX-87H sugar analysis column, with a column temperature of 65°C, a mobile phase of 5 mM sulfuric acid, a flow rate of 0.6 mL / min, and an injection volume of 10 μL.

[0038] The results are as follows Figure 2 and Figure 3 As shown, in the mixed sugar fermentation system for producing malic acid, the genetically engineered strain of Thermomycetes Δ alp 1 -Trmae-Mtstp The utilization rate (i.e., degradation rate) of xylose was different from that of the starting strain Δ alp 1 -Trmae The 22.99% of the total strain was increased to 61.16%, an increase of 1.66 times; the genetically engineered strain of Myceliophthora thermophila Δ alp 1 -Trmae-Mtstp The utilization rate of glucose is similar to that of the starting strain Δ alp 1 -Trmae There was no significant difference, which were 92.75% and 90.18% respectively; the genetically engineered strain of thermophilic Myceliophthora Δ alp 1 -Trmae-Mtstp The yield of synthesized malic acid from the starting strain Δ alp 1 -Trmae The results showed that the constitutive expression of the sugar transporter MtSTP solved the problem of carbon source repression and delayed xylose utilization in the mixed sugar utilization process to a certain extent, and significantly enhanced the ability of Myceliophthora thermophila to produce malic acid in the mixed sugar system.

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

Claims

1. A genetically engineered strain of Myceliophthora thermophila with improved mixed sugar utilization ability, characterized in that: The genetically engineered strain of Myceliophthora thermophila constitutively expresses a gene encoding a sugar transporter protein MtSTP, wherein the amino acid sequence of the sugar transporter protein MtSTP is shown in SEQ ID NO:

3.

2. The genetically engineered strain of Myceliophthora thermophila with improved mixed sugar utilization ability according to claim 1, characterized in that The genetically engineered thermophilic Myceliophthora strain is an alkaline protease gene-deficient strain.

3. The genetically engineered strain of Myceliophthora thermophila with improved mixed sugar utilization ability according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the sugar transporter MtSTP is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

4. A genetically engineered strain of Myceliophthora thermophila with improved ability to produce malic acid using mixed sugars, characterized in that: The genetically engineered strain of Myceliophthora thermophila constitutively expresses a gene encoding a sugar transporter MtSTP and a gene encoding a heterologous malic acid transporter TrMae, wherein the amino acid sequence of the sugar transporter MtSTP is shown in SEQ ID NO: 3, and the amino acid sequence of the heterologous malic acid transporter TrMae is shown in SEQ ID NO:

5.

5. The genetically engineered strain of Myceliophthora thermophila with improved ability to produce malic acid using mixed sugars according to claim 4, characterized in that: The genetically engineered thermophilic Myceliophthora strain is an alkaline protease gene-deficient strain.

6. The genetically engineered strain of Myceliophthora thermophila with improved ability to produce malic acid using mixed sugars according to claim 4, characterized in that: The nucleotide sequence of the gene encoding the heterologous malate transporter TrMae is shown in SEQ ID NO:

4.

7. A method for improving the ability of Myceliophthora thermophila to produce malic acid using mixed sugars, characterized in that: The method comprises the following steps: Constructing a recombinant expression vector comprising a gene encoding a sugar transporter protein MtSTP, wherein the gene encoding the sugar transporter protein MtSTP is regulated by a constitutive promoter in the recombinant expression vector, and the amino acid sequence of the sugar transporter protein MtSTP is shown in SEQ ID NO: 3; constructing a recombinant expression vector comprising a gene encoding a heterologous malate transporter TrMae; introducing the recombinant expression vector comprising the gene encoding the heterologous malate transporter TrMae into the thermophilic Myceliophthora; Transforming the constructed recombinant expression vector containing the gene encoding the sugar transporter MtSTP into Myceliophthora thermophila; The genetically engineered strain of Myceliophthora thermophila that was successfully transformed was screened.

8. The method for improving the ability of Myceliophthora thermophila to produce malic acid using mixed sugars according to claim 7, characterized in that: The amino acid sequence of the heterologous malate transporter TrMae is shown in SEQ ID NO:

5.

9. Use of the genetically engineered Myceliophthora thermophila strain with improved mixed sugar utilization ability according to claim 1 for fermentation production of malic acid.

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