Mutant for relieving glucose inhibition effect

Abstract

The invention relates to a KmRgt1 protein mutant. Compared with a wild type KmRgt1 protein, the KmRgt1 protein mutant has amino acid mutations of S344A, S345A, S348A, S350A, S756A and T754A; preferably, the amino acid sequence of the KmRgt1 protein mutant is as shown in SEQ ID NO: 1. On the basis of the mutant, an engineering strain capable of relieving the glucose inhibition effect can be obtained, the obtained engineering strain basically keeps the original glucose utilization capacity, and meanwhile, the utilization of non-glucose carbon sources such as xylose, glycerol, cane sugar and lactose is not inhibited by 2-deoxyglucose any more; therefore, co-utilization of mixed sugars such as glucose and xylose can be realized, and the method has great potential of producing high-added-value bio-based products by utilizing biomass such as lignocellulose.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and discloses a yeast transcriptional repressor KmRgt1 mutant, and also discloses the application of the mutant in relieving the glucose repression effect. Background Art

[0002] Due to the current high dependence and unrestrained use of fossil fuels by humans, the finiteness and non-renewability of fossil fuels have made renewable biomass resources receive increasing attention [1]. Lignocellulosic biomass is inexpensive and abundant in reserves, and is one of the renewable resources with development potential [2]. Lignocellulosic biomass can be derived from wood, agricultural waste, marine organisms, etc., and is mainly composed of cellulose, hemicellulose and lignin. Its hydrolysate contains abundant hexoses and pentoses. Based on this, the production of high-value-added bio-based products not only reduces environmental pollution, but also has high economic feasibility and is more in line with the sustainable development goal. The comprehensive utilization of glucose and other carbon sources in other agricultural and pastoral wastes, such as glycerol, lactose, etc., is also important for sustainable economic development.

[0003] The glucose repression effect refers to the phenomenon that catabolite repression of the transcription of genes encoding certain inducible enzyme systems occurs by glucose or the catabolites of some easily utilizable carbon sources, also known as metabolite repression. In most microorganisms, it is manifested as preferentially using mixed carbon sources sequentially or selectively, resulting in an extended fermentation production cycle of microorganisms and a reduced economic feasibility. Therefore, the co-utilization of mixed carbon sources is crucial for the low-cost and high-efficiency fermentation of microorganisms [3]. Therefore, there is a great demand for strains with relieved glucose repression effect.

[0004] In Saccharomyces cerevisiae, when low-concentration glucose is present, the kinase Snf1 is phosphorylated and activated, indirectly phosphorylating the transcription factor Rgt1 and promoting the expression of the Mth1 protein. At this time, Rgt1 binds to Mth1 and recruits the general co-repressor complex Ssn6-Tup1 to inhibit the expression of the transporter HXT gene; Snf3 and Rgt2 sense the presence of environmental glucose and induce the expression of high-affinity glucose transporters Hxt2, Hxt4, Hxt7 and low-affinity glucose transporters (such as Hxt1). After Rgt2 and Snf3 are activated by glucose, they phosphorylate Mth1 and Std1 and are ubiquitinated or degraded by the proteasome. While Mth1 is being degraded, Mig1 inhibits the expression of Mth1. At this time, glucose-induced PKA exposes the phosphorylation site on Rgt1 and is over-phosphorylated and degraded. Therefore, the interaction between Snf1 and Rgt1 is at the core of Hxt gene expression and glucose repression. The inventors have conducted in-depth research on Rgt1 in Kluyveromyces marxianus and hope to construct mutants that can relieve the glucose repression effect through the modification of Rgt1.

[0005] As a microorganism that has obtained the "Qualified Safety Status" (QPS) and "Generally Recognized as Safe" (GRAS) certifications [4], Kluyveromyces marxianus has advantages that other yeasts do not have in engineering fermentation: 1. It is heat-resistant and can grow at temperatures as high as 52°C [5]; 2. It has a high growth rate of 0.86-0.99 h -1 , which will make the fermentation rate faster[6]; 3. It has a broad spectrum of substrates, including glucose, xylose, galactose, lactose, glycerol, etc.[7]; 4. It is highly economical. The ability of Kluyveromyces marxianus to grow at high temperatures makes simultaneous saccharification and fermentation (SSF) and pre-saccharification and simultaneous fermentation (PSSF) possible, reducing production and fermentation costs[8]. The optimal temperature for cellulase used in biomass saccharification is 45-50°C, so the use of heat-resistant Kluyveromyces marxianus is more advantageous and can reduce the risk of contamination[9]. Summary of the invention

[0006] To solve the above problems, the present invention uses Kluyveromyces marxianus as a platform to study its Rgt1 protein (KmRgt1), and the constructed KmRgt1 mutant successfully relieves the glucose inhibition effect of the strain. The engineered strain based on the KmRgt1 mutant KmRgt1-G6M can achieve the co-utilization of mixed sugars such as glucose and xylose, demonstrating broad application prospects for the production of high value-added bio-based products using biomass such as lignocellulose.

[0007] Specifically, the present invention focuses on the modification of key regulatory factors in the signal regulation pathway of glucose inhibition effect of Kluyveromyces marxianus, specifically constructing a mutant KmRgt1-G6M of the transcription factor KmRgt1, and detecting its application in relieving the glucose inhibition effect.

[0008] First, the present invention constructed a KmRGT1 knockout strain, expressed KmRgt1 fused with FLAG in the knockout strain, and then purified KmRgt1-FLAG under the culture condition with glucose as the carbon source, and performed mass spectrometry analysis to obtain its phosphorylation sites (serines at positions 344, 345, 348, 350, and 756 (S344, S345, S348, S350, S756)), and threonine at position 754 was phosphorylated (T754)) under the condition with glucose as the carbon source. Then, mutations were designed to change all phosphorylated site amino acids into alanines to obtain the KmRgt1 mutant KmRgt1-G6M. Subsequently, the mutant gene was transferred into the KmRGT1 knockout strain to obtain the engineered strain YΔKmRGT1-G6M. Through the drop plate experiment, the engineered strain YΔKmRGT1-G6M showed an ability to utilize glucose close to normal, and was no longer inhibited by 2-deoxyglucose when utilizing non-glucose carbon sources such as xylose, glycerol, sucrose, and lactose, and its ability to utilize glucose was not significantly affected, indicating that the mutant KmRgt1-G6M successfully relieved the glucose inhibition effect. Further RT-PCR analysis showed that in the presence of glucose, the expression levels of the xylose reductase gene (KmXYL1), xylitol dehydrogenase gene (KmXYL2), and xylulokinase gene (KmXYL3) related to xylose utilization were increased by 2.12-fold, 32.01-fold, and 8.52-fold, respectively, compared with the control strain, indicating that these genes were no longer inhibited by glucose. It was proved that this mutant could relieve the glucose inhibition effect of yeast strains. Further, expressing the mutant KmRgt1-G6M in the KmMth1 knockout strain could also achieve the relief of the glucose inhibition effect, proving that the relief of the glucose inhibition effect was independent of Mth1, which was different from the previous research task that the function of Rgt1 was dependent on Mth1.

[0009] On the other hand, the present invention also relates to a method for constructing a strain YΔKmRGT1-G6M capable of expressing the KmRgt1 mutant, including: adopting the homologous recombination ligation method, designing a pair of primers KmRgt1-TB-F / R containing all target mutant codons, amplifying the original phosphorylated site fragment to obtain a target fragment containing the mutation site; using the rest of the plasmid as a vector, designing primers KmRgt1-HR-F / R, which contain a homologous arm of about 18 bp with the fragment primers, and linearizing the plasmid 2; treating and ligating the above two fragments with a homologous recombinase, and finally constructing a mutant with 6 phosphorylated sites mutated simultaneously under the glucose culture condition (designated as KmRgt1-G6M), that is, obtaining the KmRgt1-G6M mutant expression plasmid YEUKmTEF-KmRgt1-G6M; transforming the KmRGT1 knockout strain with the expression plasmid YEUKmTEF-KmRgt1-G6M to obtain the KmRgt1 mutant expression strain YΔKmRGT1-G6M.

[0010] On the other hand, the present invention also relates to an engineered strain, characterized in that the engineered strain is constructed based on the above-mentioned mutant, and is characterized in that the engineered strain is obtained by transforming a mutant expression plasmid into a KmRGT1-knockout Kluyveromyces marxianus.

[0011] Specifically, the present invention relates to the following technical solutions:

[0012] 1. A KmRgt1 protein mutant, which has amino acid mutations S344A, S345A, S348A, S350A, S756A and T754A relative to the wild-type KmRgt1 protein;

[0013] Preferably, the amino acid sequence of the KmRgt1 protein mutant is as shown in SEQ ID NO: 1.

[0014] 2. The KmRgt1 protein mutant according to item 1, wherein the wild-type KmRgt1 protein is derived from Kluyveromyces marxianus NBRC1777;

[0015] 3. A nucleic acid molecule encoding the KmRgt1 protein mutant according to item 1 or 2;

[0016] Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 2.

[0017] 4. An expression vector comprising the nucleic acid molecule according to item 3; preferably, the expression vector is an expression vector suitable for yeast bacteria.

[0018] 5. An engineered strain of Kluyveromyces marxianus comprising the nucleic acid molecule according to item 3 or the expression vector according to item 4; optionally, the KmRgt1 gene in the engineered strain is knocked out.

[0019] 6. Use of the engineered strain according to item 5 in relieving or reducing the glucose repression effect.

[0020] 7. A method for constructing an engineered strain with relieved or reduced glucose repression effect, which comprises introducing the expression vector according to item 4 into yeast;

[0021] Preferably, the yeast is Kluyveromyces marxianus;

[0022] Optionally, the Kluyveromyces marxianus is a strain with the KmRgt1 gene knocked out.

[0023] 8. The application according to item 6 or the construction method according to item 7, wherein the alleviation or reduction of the glucose repression effect enables the co-utilization of glucose and non-glucose as carbon sources.

[0024] In a specific embodiment of the present invention, the alleviation of the glucose repression effect means that when the engineered strain utilizes non-glucose carbon sources composed of xylose, glycerol, sucrose, lactose, galactose, and inulin, it is not inhibited by glucose such as 2-deoxyglucose.

[0025] 9. The application or construction method according to item 8, wherein the non-glucose includes xylose, glycerol, sucrose, lactose, galactose, and / or inulin.

[0026] 10. The application according to item 6 or the construction method according to item 7, wherein the alleviation or reduction of the glucose repression effect does not depend on the KmMth1 gene.

[0027] Advantages and positive effects

[0028] The present invention provides a yeast transcription factor KmRgt1 mutant, which successfully alleviates the glucose repression effect in Kluyveromyces marxianus without affecting the normal functions of the strain. It is expected that this mutant can be applied to other microorganisms, especially yeasts, to obtain microorganisms with similar characteristics, especially engineered yeasts.

[0029] The successful alleviation of the glucose repression effect by the KmRgt1 mutant in Kluyveromyces marxianus enables the KmRgt1 mutant to be used for constructing strains capable of co-utilizing mixed sugars such as glucose and xylose, and heat-resistant platform strains for the development and utilization of lignocellulosic biomass. Combining the characteristics of Kluyveromyces marxianus, this strain will have advantages such as a faster fermentation rate, lower cooling costs in the fermentation process, and a smaller risk of contamination. It can become the basis for subsequent engineering fermentation and has broad application potential in the efficient utilization of lignocellulosic biomass to produce high-value-added biobased products. Brief description of the drawings

[0030] Figure 1 . SDS-PAGE analysis of the purified KmRgt1-FLAG protein. Lane 1: DNA molecular weight standard, Lane 2: purified Rgt1 protein under xylose culture conditions, Lane 3: purified Rgt1 protein under glucose culture conditions. Ruby staining was used.

[0031] Figure 2. Analysis results of the drop plate experiment, which showed that strain YΔKmRGT1-G6M relieved the inhibition of glucose on the utilization of other carbon sources. Among them, YHJ010-URA3 was the wild-type control strain, YΔKmRGT1-G6M was the strain expressing the mutant KmRgt1-G6M, YKmTEF-KmRGT1 was the strain with overexpression of the KmRGT1 gene, and YΔKmRGT1 was the strain with the KmRGT1 gene knocked out.

[0032] Figure 3 . RT-PCR analysis of the expression of xylose utilization-related genes KmXYL1, KmXYL2, and KmXYL3 in strains YHJ010-URA3 and YΔKmRGT1-G6M under different carbon source conditions. Among them, A and B were the changes in the expression levels of xylose utilization genes in the control strains YHJ010-URA3 and YΔKmRGT1-G6M under different carbon source conditions, respectively; C was the relative expression intensity of the xylose utilization genes in the YΔKmRGT1-G6M strain compared with the control strain YHJ010-URA3.

[0033] Figure 4 . The drop plate experiment verified that the KmMTH1 gene knockout strain YΔKmMTH1-G6M could relieve the glucose inhibition effect by expressing KmRgt1-G6M. Among them, YΔKmMTH1 was the KmMTH1 gene knockout strain, YHJ010-URA3 was the control strain, YΔKmRGT1-G6M was the mutant expressing KmRgt1-G6M in the KmRGT1 knockout strain, and YΔKmMTH1-G6M was the strain expressing KmRgt1-G6M in the KmMTH1 knockout strain. The auxotrophic types of these strains were the same (both were deficient in tryptophan and leucine).

[0034] Figure 5 Showed the map of plasmid YEUKmTEF. Specific implementation manner

[0035] The following content is a further description of the present invention in combination with embodiments, and the embodiments listed are only to illustrate the present invention. However, it should be understood that the content of the present invention is not limited to these embodiments.

[0036] The test methods in the following embodiments are all conventional or standard methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0037] Reagents and strains:

[0038] All reagents in the present invention are reagents above reagent grade purchased from the market.

[0039] (1)Culture medium

[0040] SD solid medium: 2% glucose, 0.67% yeast basic nitrogen source, 1.5% agar powder, tryptophan (uracil, leucine are supplemented according to experimental needs), mainly used for yeast transformation.

[0041] YPD liquid medium: 1% yeast extract, 2% peptone, 2% glucose (add 1.5% agar powder for solid medium), used for yeast pre-culture.

[0042] YPX liquid medium: 1% yeast extract, 2% peptone, 2% xylose (add 1.5% agar powder for solid medium), used for yeast culture.

[0043] YPDX liquid medium: 1% yeast extract, 2% peptone, 2% glucose and 2% xylose (add 1.5% agar powder for solid medium), used for yeast culture.

[0044] Among them, glucose, xylose, glycerol, sucrose, lactose, galactose, arabinose, uracil, tryptophan, and leucine are all from Sangon Biotech (Shanghai) Co., Ltd., and yeast extract and peptone are from Thermo Fisher Scientific.

[0045] (2)Materials and Reagents

[0046] The gel extraction kit and plasmid extraction kit are from Sangon Biotech (Shanghai) Co., Ltd. The KODplus high-fidelity PCR enzyme is purchased from Toyobo, and the DpnI restriction enzyme is from Thermo Fisher Scientific. The Escherichia coli competent cell DH5α is purchased from Dingxiu Biotech and used as the host bacterium for DNA operation. The Luria-Bertani (LB) medium containing 100 μg / ml ampicillin is used for culturing Escherichia coli.

[0047] The synthesis of common gene primers and the sequencing during plasmid construction are both completed by Sangon Biotech (Shanghai) Co., Ltd.

[0048] Table 1 Primer information involved in the present invention

[0049]

[0050]

[0051] Example 1. Construction of KmRgt1 gene knockout strain

[0052] In this embodiment, the KmRgt1 gene in Kluyveromyces marxianus was knocked out, wherein the construction method of the KmRgt1 gene knockout strain is a conventional operation method in the art, and those skilled in the art can design and construct the KmRgt1 gene knockout strain according to the conventional means of the prior art, and the specific method of this embodiment is only exemplary. Wherein, the purpose of knocking out the KmRgt1 gene is only to avoid the interference of the wild-type KmRgt1 gene for the subsequent experimental effect of relieving the glucose inhibition effect, and those skilled in the art can understand that for the strains in which the KmRgt1 gene is not knocked out, as long as the mutant KmRgt1-G6M of the KmRgt1 gene of the present invention is introduced or overexpressed, the effect of reducing or relieving the glucose inhibition effect can be achieved. In addition, for the convenience of subsequent screening of successfully transformed strains, the embodiments of the present invention are only exemplary with Kluyveromyces marxianus YHJ010 as the starting strain, and those skilled in the art can understand that the Kluyveromyces marxianus strains conventionally used in the prior art (as long as the mutant KmRgt1-G6M of the KmRgt1 gene of the present invention is introduced or overexpressed) can achieve the effect of the present invention.

[0053] First, the KmRgt1 gene knockout cassette was constructed. The genomic DNA of Kluyveromyces marxianus YHJ010 (which is a triple auxotrophic type of uracil, leucine and tryptophan constructed using Kluyveromyces marxianus NBRC1777 as the starting strain, and its construction method can be found in reference 10) was extracted, and the KmRgt1 expression cassette gene sequence was amplified from the Kluyveromyces marxianus genome (wherein the GenBank accession No. of the KmRgt1 gene is BAP73047.1), and inserted into the YEUKmTEF vector (its plasmid map can be found in Figure 5 , or a conventional vector suitable for Kluyveromyces marxianus in the prior art), and the obtained plasmid was named YEUKmTEF-KmRgt1-Knockout.

[0054] The specific operations are as follows:

[0055] (1) Extraction of the genome of Kluyveromyces marxianus YHJ010 by bead milling

[0056] a) Streak frozen Kluyveromyces marxianus YHJ010 and culture in a 37°C incubator for 24 hours.

[0057] b) Pick a single clone, inoculate it into 5 ml YPD liquid medium, and culture it overnight at 37°C with a shaker at 250 rpm.

[0058] c) Collect the cells, centrifuge at 12000 rpm for 1 minute, and discard the supernatant.

[0059] d) Resuspend the cells in 500 μl of sterile water, transfer to a 1.5 ml centrifuge tube, centrifuge at 12,000 rpm for 1 minute, discard the supernatant, and collect the cells.

[0060] e) Add 200 μl of lysis buffer (EDTA (1 mM), Triton X-100 (2% (w / v)), NaCl (100 mM), SDS (1% (w / v)), Tris-Cl (10 mM, pH 8.0)) to resuspend the cells.

[0061] f) Add 0.3 g of acid-washed glass beads and 200 μl of phenol-chloroform solution (25:24:1, pH 8.0), and vortex at high speed for 3 minutes.

[0062] g) Then add 200 μl of 1x TE buffer (1 mM EDTA, 10 mM Tris-Cl, pH 8.0), and gently shake for 30 seconds.

[0063] h) Centrifuge at 14,000 rpm at 4 °C for 15 minutes, transfer the upper aqueous phase to a new 1.5 ml centrifuge tube. At this time, the supernatant is the crudely extracted yeast genomic DNA, which can be used for PCR gene amplification reaction. If you want to further remove RNA, add 3 volumes of ice-cold absolute ethanol, invert and mix well, and let stand at -20 °C for 30 minutes.

[0064] i) Centrifuge at 14,000 rpm at 4 °C for 30 minutes, discard the supernatant, and resuspend the pellet in 400 μl of 1x TE buffer.

[0065] j) Add 2 μl of 2 mg / ml RNase, and incubate in a 37 °C water bath for 5 minutes.

[0066] k) Add 40 μl of 3 M sodium acetate solution and 3 volumes of pre-cooled absolute ethanol, invert and mix well, and let stand at -20 °C for 30 minutes.

[0067] l) Centrifuge at 14,000 rpm at 4 °C for 30 minutes, discard the supernatant, wash the pellet with 1 ml of pre-cooled 75% ethanol, centrifuge again to discard the supernatant, and centrifuge at 14,000 rpm at 4 °C for 10 minutes.

[0068] m) Dry the pellet in an oven at 60 °C for 30 minutes, and resuspend with isothermal ddH2O. This is the completely extracted genomic DNA of yeast YHJ010.

[0069] (2) Amplification of the gene KmRgt1 expression cassette:

[0070] Using KmRgt1-Knockout-F / R as primers and the genomic DNA of yeast YHJ010 obtained in step (1) above as a template, the target gene fragment was amplified using KOD plus PCR enzyme. The PCR reaction system is as follows:

[0071]

[0072] PCR reaction conditions:

[0073]

[0074] Thus, the PCR product amplified by PCR contains the fragment of the KmRGT1 gene expression cassette. In the same way, it was amplified with KmRgt1-URA3-line-F / R primers, and finally the linearized fragment of the YEUKmTEF vector was obtained.

[0075] (3) Ligation by fragment homologous recombination method:

[0076] The reaction enzyme was purchased from Shanghai Yeasen Biotechnology Co., Ltd. The homologous recombination reaction system is as follows:

[0077] Among them, the KmRgt1 fragment to be inserted is the fragment of the KmRGT1 gene expression cassette obtained by PCR in step (2) above, and the linearized fragment of the YEUKmTEF vector is the linearized fragment of the YEUKmTEF vector obtained in step (2) above.

[0078]

[0079] Mix the prepared reaction system evenly and incubate at 50 °C in a water bath for 25 minutes for ligation, which can be used for subsequent Escherichia coli transformation experiments.

[0080] (4) Escherichia coli transformation:

[0081] The ligation product obtained in step (3) above was transformed into Escherichia coli competent DH5α (purchased from Dingxiu Biology). After colonies grew out, single colonies were picked and subjected to Escherichia coli colony PCR experiment using YEUKmTEF-F / KmRgt1-Knockout-R as primers. After the PCR reaction, it was identified by agarose gel electrophoresis. The positive clones with the correct band size were picked and inoculated into a test tube containing fresh LB liquid medium and cultured overnight; the plasmid was extracted from the cultured overnight bacterial solution, and then the sequence information was confirmed by sequencing, and the plasmid YEUKmTEF-KmRgt1-Knockout containing the KmRGT1 gene expression cassette was obtained.

[0082] (5) Design of sgRNA corresponding to KmRGT1:

[0083] First, design sgRNAs for the CDS sequence of the KmRgt1 gene through the CRISPOR (tefor.net) website. Generally, select sgRNAs with high specificity and cleavage efficiency scores for subsequent experiments. The sequences are KmRgt1-sgRNA-R and KmRgt1-sgRNA-F in Table 1.

[0084] (6) Construction of a plasmid containing the KmRGT1 gene knockout cassette:

[0085] Truncate the sgRNA binding position (20 bp) in the CDS sequence of the KmRgt1 gene in the plasmid YEUkmTEF-KmRgt1-Knockout obtained in step (4) by inverse PCR, and insert the ScURA3 expression cassette into the truncated vector (the sequence from the 2061st base to the 3158th base of the complete ScURA3 expression cassette GenBank: AM697670.1), thereby obtaining a plasmid containing the gene knockout cassette, named plasmid YEUkmTEF-KmRgt1-Knockout-URA3.

[0086] Secondly, knockout the KmRgt1 gene of the Kluyveromyces marxianus strain YHJ010 as follows:

[0087] 1). Use the plasmid YEUkmTEF-KmRgt1-Knockout-URA3 containing the gene knockout cassette obtained in step (6) as the amplification template, and amplify the gene knockout cassette fragment with KmRgt1-Knockout-F and KmRgt1-Knockout-R as primers; use the PDCR4-Cas9 -sgRNA(cas9) plasmid (the construction method can refer to reference 11) as the template

[11] , and amplify fragment A by using primers SNR52-F / KmRgt1-sgRNA-R, fragment B by using primers KmRgt1-sgRNA-F / CYCT-R. Fragments A and B are subjected to overlap PCR with SNR52-F / CYCT-R as primers to obtain fragment C, that is, the KmRgt1 sgRNA expression cassette fragment, and use primers Cas9-PDCR4-F / R to obtain the Cas9 expression cassette fragment by PCR with the PDCR4-Cas9 -sgRNA(cas9) plasmid as the template. Then, concentrate and purify the amplified knockout cassette fragment and the two expression cassette fragments by ethanol precipitation.

[0088] Among them, the operation steps of the ethanol precipitation method are as follows:

[0089] a). Combine the DNA solutions obtained from the PCR reaction in a clean 1.5 ml centrifuge tube, add one-tenth volume of 3M sodium acetate solution, and mix well;

[0090] b). Add 3 volumes of pre-cooled absolute ethanol, invert thoroughly to mix, and let stand overnight at -20°C in the refrigerator.

[0091] c). Centrifuge at 4°C, 14000 rpm for 30 minutes, discard the supernatant, add 1 mL of pre-cooled 75% ethanol, invert the tube up and down to shake the precipitate off the tube wall, and then centrifuge at 4°C, 14000 rpm for 15 minutes.

[0092] d). After centrifugation, carefully discard the supernatant, then use a pipette to carefully aspirate the residual liquid, and place it open in a 60°C oven for 30 minutes to volatilize the remaining ethanol.

[0093] e). Add an appropriate amount of isothermal ddH2O to dissolve the obtained DNA precipitate, and then store it in a -20°C refrigerator.

[0094] 2). Transform the knockout cassette fragment and expression cassette fragment of the above gene into Kluyveromyces marxianus YHJ010 together by the lithium acetate transformation method, and screen through a synthetic medium plate supplemented with leucine and tryptophan. Only the strains that have successfully transferred the fragment and have the ability to synthesize uracil can survive on this plate.

[0095] Among them, the specific operation steps of the lithium acetate transformation method of the yeast are as follows:

[0096] a). Streak the Kluyveromyces marxianus strain YHJ010 on a YPD agar medium plate and culture it in a 37°C incubator for 24 hours.

[0097] b). Pick a single colony from the streaked plate and inoculate it into 5 ml of YPD liquid medium, and culture it overnight at 37°C on a shaker at 250 rpm.

[0098] c) Take 500 μl of the overnight cultured bacterial solution and inoculate it into 5 ml of fresh YPD liquid medium, and culture it at 37°C on a shaker at 250 rpm for 4 hours.

[0099] d). Centrifuge the bacterial solution after culturing for 4 h at 5000 rpm in batches for 1 minute, discard the supernatant, and collect the cells in a 1.5 mL centrifuge tube.

[0100] e). Prepare 1 ml of transformation buffer:

[0101] 800 μl of 50% PEG4000

[0102] 100 μl of 2M lithium acetate

[0103] 100 μl of 1M DTT

[0104] f). Resuspend the cells in 300 μl of transformation buffer and transfer them to a 1.5 ml centrifuge tube. Centrifuge at 5000 rpm for 1 minute and discard the supernatant.

[0105] g). Resuspend the cells again in 150 μl of transformation buffer and add the ethanol-precipitated DNA to be transformed. Mix well and incubate at room temperature for 30 minutes on a rotary mixer at low speed.

[0106] h). Place the mixture in a 47°C water bath and heat shock for 15 minutes. During this time, prepare the glass rod for spreading in the laminar flow hood.

[0107] i). Spread the mixture evenly on two synthetic media plates for screening at a volume of approximately 100 μL. Seal with parafilm and invert in a 37°C incubator for 1 - 2 days. Take out when colonies are visible to the naked eye.

[0108] j) Number the monoclonal colonies on the plate and streak them on a new plate of the same type for secondary screening. Invert in a 37°C incubator and incubate overnight.

[0109] K). Verify the secondary screening clones by colony PCR. Pick the monoclonal colonies growing from the streak into a PCR strip tube containing 20 μL of 20 mM NaOH. Stir to dissolve the cells in the solution and place in a PCR instrument at 95°C for 25 minutes to lyse the cells. The lysed solution can be used as a template.

[0110] l). Prepare the colony PCR reaction system according to the following table. After vortexing and mixing, aliquot 16 μL of the reaction solution into a single tube of a PCR strip tube. Take 4 μL of the above lysed solution into the 16 μL reaction solution, mix well and perform PCR.

[0111]

[0112] m). Perform agarose gel electrophoresis. The length of the target gene amplified from the genome of the knockout strain is larger than that of the target gene amplified from the wild type by the size of a URA3 expression cassette gene sequence (1467 bp), indicating that the KmRgt1 gene has been successfully knocked out. Name it strain YΔKmRGT1.

[0113] Example 2. Purification of KmRgt1 Protein, Identification of Phosphorylation Sites and Construction of KmRgt1 Mutant Expression Plasmid

[0114] (1) Expression and Purification of KmRgt1 Protein

[0115] First, an expression strain of KmRgt1-FLAG was constructed:

[0116] Referring to the plasmid construction method described in Example 1, the CDS sequence of the KmRgt1 gene was amplified from the genomic DNA of YHJ010 using KmRgt1-F / R as primers. The YEUKmTEF linearized fragment was amplified using the KmRgt1-URA3-line-F / R primers. Through homologous recombination treatment, the YEUKmTEF-KmRgt1 plasmid was obtained. Then, using the YEUKmTEF-KmRgt1 plasmid as a template and KmRgt1-line-F / R as primers, the plasmid YEUKmTEF-KmRgt1 was linearized by inverse PCR at the stop codon of the KmRgt1 gene sequence (the conditions were the same as those in Example 1 except for the template and primers). At the same time, the reverse complementary primers KmRgt1-G4S-FLAG-HR-F / R were evenly mixed and denatured completely at 98°C, and after cooling and renaturation, the G4S-FLAG gene sequence with homologous arms at both ends was obtained. The above two fragments were treated with a homologous recombinase to construct an expression plasmid YEUKmTEF-KmRgt1-G4S-FLAG with G4S linked to a FLAG tag and transformed into the KmRgt1 knockout strain by the lithium acetate method to construct a strain YKmTEF-KmRGT1 of KmRgt1 fused with FLAG, which knocked out the KmRgt1 gene and was transformed with the plasmid YEUKmTEF-KmRgt1-G4S-FLAG.

[0117] Secondly, the strain YKmTEF-KmRGT1 was cultured on a large scale, and the obtained KmRgt1 protein was purified using the FLAG tag. The specific operation method includes the following steps a)-u):

[0118] a) Yeast cell large-scale culture: Take out the frozen strain of Kluyveromyces marxianus YHJ010 to be purified from the -80°C refrigerator, streak it on the YPD solid medium in a laminar flow hood, and culture it in a 37°C incubator for 24 hours. After growing a clone of appropriate size, take it out. Pick a single clone into 5 mL of YPD liquid medium and culture it in a 37°C, 250 rpm shaker for 12 hours. Inoculate the overnight culture broth into a YPD liquid medium of an appropriate volume according to the volume ratio and culture it in a 37°C, 250 rpm shaker for about 10 hours until the OD600 reaches about 8.

[0119] b) Centrifuge at 6000×g for 3 to 5 minutes at room temperature to collect the cells, discard the supernatant, quickly freeze the cells in liquid nitrogen, and then use a liquid nitrogen grinder to break the cells (liquid nitrogen grinder program: pre-cool for 5 minutes, single run time 5 minutes, run 5 times, run interval 2 minutes) until the cells completely become powdery, and store at -80°C.

[0120] c) Take out the broken yeast cells from the -80°C refrigerator, pour them into a clean beaker, place the beaker in an ice bath to maintain low temperature, and thaw for about 30 minutes. Stir the powder from time to time during this period to prevent the cells at the bottom from caking.

[0121] d) Add an appropriate volume of ultrapure water, extraction buffer (a buffer well-known in the prior art, the components of which include 0.25 mM Tris-Hcl, 0.1 M KoAC, 0.3 M AS, 2 mM EDTA, 35% Glycerol (v / v), 5 mM β-mercaptoethanol, 1% protease inhibitor mixture (v / v)), β-mercaptoethanol, and protease inhibitor mixture (used to improve the recovery efficiency of the target protein, purchased from Thermo Fisher Scientific). Stir with a magnetic stirrer for 1 hour to completely dissolve the protein into the extraction buffer.

[0122] e) Add protease inhibitor mixture, and centrifuge at 14000 rpm and 4 °C for 30 minutes.

[0123] f) After adding protease inhibitor mixture to the supernatant, aliquot it evenly into ultracentrifuge tubes.

[0124] g) Perform ultracentrifugation at 40000 rpm, 4 °C for 1.5 hours using rotor type 45Ti. After ultracentrifugation, the original supernatant will be divided into three layers. The bottom layer of turbid precipitate is chromatin, the middle layer is clear and transparent, which is the dissolved protein, and the top thin layer in flocculent form is lipid. During ultracentrifugation, weigh about 60 g of ammonium sulfate into a mortar and grind it into a fine powder.

[0125] h) Use a 5 mL pipette to aspirate the middle clear protein solution into a clean beaker, being careful not to aspirate the turbid part at the bottom. Measure the volume of the protein solution with a graduated cylinder, add 0.25% protease inhibitor mixture, weigh ammonium sulfate powder at a ratio of 28.6 g per 100 mL of protein solution, and then measure 1 M KOH into a 1.5 mL centrifuge tube at a ratio of 10 μL per gram of ammonium sulfate.

[0126] i) Place the protein solution in an ice bath and stir slowly with a magnetic stirrer. Slowly add ammonium sulfate powder to the protein solution at a rate of 1 g per minute, and take 100 μL of 1 M KOH every ten minutes and add it to the protein solution.

[0127] j) After all the ammonium sulfate powder is added, add 0.25% protease inhibitor mixture and stir with a magnetic stirrer in the ice bath for 1 hour.

[0128] k) Add 0.25% (v / v) protease inhibitor mixture, centrifuge at 14000 rpm and 4 °C for 1 hour, discard the supernatant, and place the precipitate at -80 °C.

[0129] l) Take out the precipitate precipitated by ammonium sulfate from the -80 °C refrigerator the next morning and thaw it on ice for 20 minutes.

[0130] m). Equilibrate with Anti-FLAG magnetic beads: Place the Anti-FLAG magnetic beads in the column (gravity column), first add approximately 12 mL of Tris-buffered saline solution (components include Tris-HCl, NaCl, adjust pH) for rinsing; then rinse with 0.1 M, pH 3.5 glycine-HCl (add 1 mL each time for rinsing and complete within 20 min); next, equilibrate the column with approximately 5 column volumes of Tris-buffered saline solution; finally, equilibrate the column with the binding buffer (a buffer well-known in the prior art, including 25 mM Tris-Hcl, 0.2 M KoAc, 5 mM EDTA, 10% Glycerol (v / v), 5 mM β-mercaptoethanol, 0.01% NP-40 (v / v), 0.5% protease inhibitor mixture), without draining completely, and retain a part for later use (resuspend the magnetic beads).

[0131] n). Take the binding buffer into a centrifuge tube, add protease inhibitor mixture, 20% NP-40 (a non-ionic detergent), resuspend the above ammonium sulfate precipitate. After thorough resuspension, incubate on a rotating mixer at 4°C for 30 minutes.

[0132] o). Centrifuge at 14000 rpm at 4°C for 30 minutes, and transfer the supernatant to a new 50 mL centrifuge tube.

[0133] p). Add protease inhibitor mixture, resuspend the equilibrated anti-FLAG magnetic beads and aspirate and add them to the supernatant. Incubate on a rotating mixer at 4°C for 2 hours, and supplement protease inhibitor mixture during this period.

[0134] q). Let the supernatant incubated with the magnetic beads flow through the column by gravity. At this time, the protein is adsorbed on the magnetic beads and will not flow away.

[0135] r). Pipette the first wash buffer (a buffer well-known in the prior art, including 50 mM Tris-Hcl, 0.5 M KoAc, 1 mM EDTA, 10 μM ZnSO4, 10% Glycerol (v / v), 5 mM β-mercaptoethanol, 0.01% NP-40 (v / v), 0.5% protease inhibitor mixture), the second wash buffer (a buffer well-known in the prior art, including 50 mM Tris-Hcl, 50 mM AS, 1 mM EDTA, 10 μM ZnSO4, 10% Glycerol (v / v), 5 mM β-mercaptoethanol, 0.01% NP-40 (v / v), 0.5% protease inhibitor mixture) and the digestion buffer (a buffer well-known in the prior art, including 50 mM HEPES, 100 mM AS, 1 mM EDTA, 10 μM ZnSO4, 10% Glycerol (v / v), 5 mM β-mercaptoethanol, 0.01% NP-40 (v / v), 0.5% protease inhibitor mixture) into three centrifuge tubes. Add protease inhibitor mixture and 20% NP-40 to each tube. Flow through in the order of the first wash buffer - the second wash buffer - the digestion buffer. Finally, leave 500 μL of the digestion buffer unflowed and resuspend the magnetic beads.

[0136] s). Transfer to a new 1.5 mL centrifuge tube, add 3×FLAG peptide, and incubate on a rotary mixer at 4°C for 2 hours to competitively elute the protein.

[0137] t). Place the incubated centrifuge tube on a magnetic stand to allow the magnetic beads to adsorb the precipitate. Aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube, which is the E1 protein fraction. Pipette 500 μL of fresh digestion buffer through, which is the E2 fraction.

[0138] u). Decide how to aliquot into small tubes according to subsequent uses and store at -80°C in a refrigerator; Perform SDS-PAGE experiments on the KmRgt1 protein purified after culturing with xylose and glucose respectively above. Then stain the PAGE gel with Ruby (SYPRO™ Ruby Protein Gel Stain, purchased from Thermo Fisher Scientific), and image it under ultraviolet light using a gel imager. As Figure 1 shown, KmRgt1 is at the position of 140 KDa. Band 1 is the Marker, band 2 is the Rgt1 protein extracted from yeast cells cultured under xylose conditions, and band 3 is the Rgt1 protein extracted from yeast cells cultured under glucose conditions.

[0139] (2) Mass spectrometry determines that the phosphorylation sites of KmRgt1 are different under different culture conditions

[0140] Cut out the fragment with the target size in SDS-PAGE, and successively subject the sample to proteolysis into peptide segments, enrichment of phosphorylated peptide segments, and mass spectrometry analysis. Finally, verify the data and identify the sequences and modification sites of phosphorylated peptide segments. The results show that serine at position 187 (S187) is phosphorylated under the culture condition with xylose as the carbon source; under the glucose culture condition, serine at positions 344, 345, 348, 350, 756 (S344, S345, S348, S350, S756), and threonine at position 754 (T754) are phosphorylated.

[0141] (3) Construction of the KmRgt1 mutant expression plasmid

[0142] As can be seen from the above, the phosphorylation sites of the KmRgt1 protein obtained under the glucose culture condition are concentrated in two regions, namely, S344, S345, S348, S350 in one region, and S756, T754 in another region. The inventors mutated the sites in each region simultaneously. Using the expression plasmid YEUKmTEF-KmRgt1-G4S-FLAG obtained in Example 2 as a template, and using primers KmRgt1-TB-F / R (wherein, primer KmRgt1-TB-F corresponds to the mutated bases at positions S344, S345, S348, S350, and primer KmRgt1-TB-R corresponds to the mutated bases at positions S756, T754), amplify the target fragment containing the mutation sites; use the rest of the plasmid as a vector, and amplify the rest of the expression plasmid through primers KmRgt1-HR-F / R containing a homologous arm of about 18 bp with the fragment primers. Treat the above two fragments with homologous recombinase, and finally construct a recombinant plasmid containing the KmRgt1 mutant with all mutations at the phosphorylation sites under the glucose culture condition (that is, the amino acid sequence encoded by it has amino acid mutations S344A, S345A, S348A, S350A, S756A, and T754 relative to the original KmRgt1 protein, and name the mutant KmRgt1-G6M), that is, obtain the KmRgt1-G6M mutant expression plasmid YEUKmTEF-KmRgt1-G6M.

[0143] Example 3. Construction of strains YΔKmRGT1-G6M, YΔKmMTH1-G6M and related control strains

[0144] Using the above lithium acetate transformation method, the KmRgt1-G6M mutant expression plasmid YEUKmTEF-KmRgt1-G6M obtained in Example 2 was transformed into the strain YΔKmRGT1 with the KmRgt1 gene knocked out obtained in Example 1 to obtain a yeast strain expressing the mutant, which was named strain YΔKmRGT1-G6M (which knocked out the original KmRgt1 gene and introduced a plasmid containing the mutant KmRgt1-G6M gene). In addition, to verify the dependence of the glucose repression effect on the KmMth1 gene, the plasmid YEUKmTEF-KmRgt1-G6M was also transformed into the strain with the KmMTH1 gene knocked out

[12] (the construction method of this strain can be referred to the prior art document 12) to overexpress KmRGT1-G6M, and the YΔKmMTH1-G6M yeast strain was obtained (which knocked out the KmMTH1 gene and introduced a vector containing the mutant KmRgt1-G6M gene). In the strain with the KmRgt1 gene knocked out obtained in Example 1, the expression plasmid YEUKmTEF-KmRgt1-G4S-FLAG obtained in Example 2 was introduced to overexpress KmRgt1, and the YKmTEF-KmRGT1 strain was obtained (which knocked out the original KmRgt1 gene and introduced a vector containing the KmRgt1-G4S-FLAG gene).

[0145] Example 4. The KmRgt1 mutant relieves the glucose repression effect

[0146] To verify that the KmRgt1 mutant can relieve the glucose repression effect, the inventors detected the phenotypic function of the KmRgt1 mutant through the YΔKmRGT1-G6M strain constructed in Example 3 above. The results showed that the glucose repression on xylose utilization was relieved in the strain YΔKmRGT1-G6M expressing the mutant KmRgt1-G6M.

[0147] On the basis of maintaining the consistency of the genetic background and the credibility of the experiment, the strain YΔKmRGT1-G6M (expressing the mutant KmRgt1-G6M), YHJ010-URA3 (wild-type control, which was obtained by ligating the tag URA3 in the wild-type YHJ010, and ligating the URA3 tag in the wild-type yeast is a well-known conventional operation in the prior art), YKmTEF-KmRGT1 (overexpressing the wild-type KmRGT1) and YΔKmRGT1 (the strain with the KmRgt1 gene knocked out) were subjected to a spotting test on plates with and without 2-deoxyglucose (2-DG).

[0148] The specific operation of the spotting experiment is as follows:

[0149] (1) Streak the strains YΔKmRGT1-G6M, YHJ010-URA3, Y△KmRGT1, and YKmTEF-KmRGT1 onto YPD solid medium and incubate them overnight in a 37°C biochemical incubator. After colonies grow, take them out.

[0150] (2) The next day, pick single colonies from the plate and culture them in test tubes containing 5 ml of YPD liquid medium under the same culture conditions of 37°C and 250 rpm overnight.

[0151] (3) Measure the bacterial density OD600 of the overnight-cultured bacterial solution using an ultraviolet spectrophotometer, and control the concentration gradient of the samples for drop plating by calculation and dilution with sterile water: The first concentration gradient is OD600 = 1, and then sequential 10-fold dilutions are performed, i.e., OD600 = 10 -1 , 10 -2 , 10 -3 .

[0152] (4) Use a pipette to aspirate 1.5 μl of the diluted bacterial solutions with OD600 = 1, 10 -1 , 10 -2 and 10 -3 to be spotted, and drop them onto the corresponding positions on the plate. After the bacterial solutions dry, seal them with a sealing film and invert the plate and place it in a 37°C incubator for culture.

[0153] (5) After culturing for 12 hours, observe and photograph each plate under the Tanon 1600 full-automatic digital gel imaging analysis system.

[0154] The results showed that the strain YΔKmRGT1-G6M expressing the mutant KmRgt1-G6M relieved the glucose repression effect. As Figure 2 shown, on the medium with glucose as the carbon source, whether 2-deoxyglucose was present or not, there was little difference between the mutant and the wild-type control, and the mutant was even slightly weaker. However, on the medium with xylose as the carbon source, when 2-deoxyglucose was absent, the growth of all strains was similar. On the xylose medium with 2-deoxyglucose, the strain YΔKmRGT1-G6M expressing the mutant KmRgt1-G6M grew significantly better than other strains, indicating that expressing the mutant KmRgt1-G6M could relieve the inhibition of glucose on xylose utilization. On the media with glycerol, sucrose, and lactose, it was also visible that when 2-deoxyglucose was absent, the growth of all strains was similar; while when 2-DG was present, the growth of the strain YΔKmRGT1-G6M was significantly better than other strains, indicating that the glucose repression effect was relieved, and the strain YΔKmRGT1-G6M with the relieved glucose repression effect was successfully obtained.

[0155] Example 5. Quantitative RT-PCR further demonstrated that the KmRGT1-G6M mutant could relieve the glucose repression effect

[0156] The engineered strain YΔKmRGT1-G6M and the control strain YHJ010-URA3 were cultured at an initial OD600 of 0.3 in a medium containing 2% glucose (1% yeast extract, 2% peptone, and 2% glucose, named YPD medium), a medium containing 2% xylose (1% yeast extract, 2% peptone, and 2% xylose, named YPX medium), and a medium containing a mixture of both (1% yeast extract, 2% peptone, and 2% glucose and 2% xylose, named YPDX). They were cultured at 37°C until the logarithmic phase when glucose was not completely consumed. The cells were harvested by centrifugation at 8000 rpm and 4°C. Subsequently, RNA was extracted using a column-type total yeast RNA extraction and purification kit purchased from Sangon Biotech (Shanghai) Co., Ltd. according to the instructions. Secondly, the extracted RNA was reverse-transcribed into cDNA using the TOROBlue® All-in-One qRT Mix with dsDNase (RTQ-204) kit from TianShai (Shanghai) Technology Co., Ltd. Finally, using the TOROGreen® qPCRMaster Mix kit also purchased from TianShai (Shanghai) Technology Co., Ltd., the reference gene ACT was amplified with RT-ACT-F / R, and the genes KmXYL1, KmXYL2, and KmXYL3 were amplified with RT-XR-F / R, RT-XDH-F / R, and RT-XK-F / R, respectively. A Roche thermal gradient real-time PCR instrument was used for quantitative RT-PCR analysis of the expression levels of key xylose utilization enzyme genes.

[0157] In the control strain YHJ010-URA3, when glucose (YPD) or glucose + xylose (YPDX) was used as the carbon source, the expression of the genes for xylose reductase (KmXYL1), xylitol dehydrogenase (KmXYL2), and xylulokinase (KmXYL3) was inhibited by glucose ( Figure 3 A); when xylose (YPX) was used as the carbon source, the expression of these genes was significantly upregulated. A similar phenomenon was also observed in YΔKmRGT1-G6M, but the magnitude of the upregulation of the expression of these three genes induced by xylose was much smaller than that in the strain YHJ010-URA3 ( Figure 3 B). The expression levels of the genes KmXYL1, KmXYL2, and KmXYL3 under the three conditions (YPD, YPDX, and YPX) were compared between the strain YΔKmRGT1-G6M and the control strain. The results showed that in the strain YΔKmRGT1-G6M, even in the presence of glucose, the expression levels of the above three genes were much higher than those in the control strain. Especially for the KmXYL2 gene, it was more than 20 times higher than that in the control strain when glucose was present, while when xylose alone was used as the carbon source, the difference was not significant. Figure 3C), which indicates that the expression of the mutant KmRGT1-G6M has led to the release of the inhibition of the expression of xylose utilization genes in the presence of glucose, resulting in a high background expression. Therefore, when induced by xylose alone, the up-regulation amplitude is reduced.

[0158] Example 6. The KmRgt1-G6M mutant relieves glucose repression independently of KmMth1

[0159] Since Mth1 is essential for relieving glucose repression in Saccharomyces cerevisiae, we introduced the expression plasmid YEUKmTEF-KmRgt1-G6M of the KmRgt1-G6M mutant into the strain with the KmMTH1 gene knocked out (the construction method of this strain can be referred to the prior art document 13), thereby obtaining the strain YΔKmMTH1-G6M overexpressing the mutant KmRGT1-G6M. A drop plate experiment was carried out together with YΔKmMTH1, YHJ010-URA3, and YΔKmRGT1-G6M. The results showed that the growth of these four strains was similar on glucose, xylose, glycerol, sucrose, and lactose media. However, after adding 2-deoxyglucose to these media, the growth state of the strain YΔKmMTH1-G6M was close to that of the strain YΔKmRGT1-G6M and was much better than that of other strains. This indicates that the strain YΔKmMTH1-G6M also relieves glucose repression and is independent of the KmMth1 gene ( Figure 4 ).

[0160] Sequence:

[0161] SEQ ID NO: 1, Amino acid sequence of the KmRgt1 mutant

[0162]

[0163] SEQ ID NO: 2, Nucleotide sequence of the KmRgt1 mutant

[0164]

[0165] References:

[0166] 1. Rai, A.K., et al., Recent Developments in LignocellulosicBiofuels, a Renewable Source of Bioenergy. Fermentation, 2022. 8(4): p. 161.

[0167] 2. Ojo, A.O., An Overview of Lignocellulose and Its BiotechnologicalImportance in High-Value Product Production. Fermentation, 2023. 9(11): p.990.

[0168] 3. Wu, Y., et al., Metabolic Engineering Strategies for Co-Utilization of Carbon Sources in Microbes. Bioengineering, 2016. 3(1): p. 10.

[0169] 4. Karim, A., N. Gerliani, and M. Aïder, Kluyveromyces marxianus: Anemerging yeast cell factory for applications in food and biotechnology.International Journal of Food Microbiology, 2020. 333: p. 108818.

[0170] 5. Zhang, B., et al., Identification of a xylose reductase gene inthe xylose metabolic pathway of Kluyveromyces marxianus NBRC1777. Journal ofIndustrial Microbiology and Biotechnology, 2011. 38(12): p. 2001-2010.

[0171] 6. Zhang, B., et al., Engineered Kluyveromyces marxianus for pyruvateproduction at elevated temperature with simultaneous consumption of xyloseand glucose. Bioresource Technology, 2017. 224: p. 553-562.

[0172] 7. Pentjuss, A., et al., Model-based biotechnological potentialanalysis of Kluyveromyces marxianus central metabolism. Journal of IndustrialMicrobiology and Biotechnology, 2017. 44(8): p. 1177-1190.

[0173] 8. de Souza, C.J.A., et al., The influence of presaccharification,fermentation temperature and yeast strain on ethanol production fromsugarcane bagasse. Bioresource Technology, 2012. 109: p. 63-69.

[0174] 9. Alvira, P., et al., Pretreatment technologies for an efficientbioethanol production process based on enzymatic hydrolysis: A review.Bioresource Technology, 2010. 101(13): p. 4851-4861.

[0175] 10. Hong, J., et al., Construction of thermotolerant yeast expressing thermomostable cellulase genes. Journal of Biotechnology, 2007. 130(2): p. 114-123.

[0176] 11. Wang Jichao, Study on the inhibitory mechanism of glucose on xylose utilization in Kluyveromyces marx. 2017, University of Science and Technology of China. Postdoctoral report (National Library online view link: http: / / find.nlc.cn / search / showDocDetails?docId=-2224397139300533093&dataSource=ucs01&query.)

[0177] 12.Wang, L., et al., The novel properties of Kluyveromyces marxianusglucose sensor / receptor repressor pathway and the construction of glucoserepression-released strains. Microbial Cell Factories, 2023. 22(1): p. 123.

Claims

1. A KmRgt1 protein mutant having amino acid mutations S344A, S345A, S348A, S350A, S756A and T754A relative to the wild-type KmRgt1 protein; Preferably, the amino acid sequence of the KmRgt1 protein mutant is as shown in SEQ ID NO:

1.

2. The KmRgt1 protein mutant according to claim 1, wherein, The wild-type KmRgt1 protein is derived from Kluyveromyces marxianus NBRC1777.

3. A nucleic acid molecule encoding the KmRgt1 protein mutant according to claim 1 or 2; Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:

2.

4. An expression vector comprising the nucleic acid molecule according to claim 3; preferably, the expression vector is an expression vector suitable for yeast bacteria.

5. An engineered strain of Kluyveromyces marxianus comprising the nucleic acid molecule according to claim 3 or the expression vector according to claim 4; optionally, the KmRgt1 gene in the engineered strain is knocked out.

6. Use of the engineered strain according to claim 5 in relieving or reducing the glucose repression effect.

7. A method for constructing an engineered strain with a relieved or reduced glucose repression effect, which comprises introducing the expression vector according to claim 4 into yeast; Preferably, the yeast is Kluyveromyces marxianus; Optionally, the Kluyveromyces marxianus is a strain with the KmRgt1 gene knocked out.

8. The use according to claim 6 or the construction method according to claim 7, wherein the relieving or reducing the glucose repression effect is the ability to co-utilize glucose and non-glucose as carbon sources.

9. The application or construction method according to claim 8, wherein The non-glucose includes xylose, glycerol, sucrose, lactose, galactose and / or inulin.

10. The use according to claim 6 or the construction method according to claim 7, wherein the relieving or reducing the glucose repression effect is independent of the KmMth1 gene.

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