CN112048446A - Yeast with relieved glucose inhibition effect and method for utilizing non-glucose carbon source by utilizing yeast - Google Patents

Abstract

The invention relates to Kluyveromyces marxianus CGMCC No.20304, wherein the glucose inhibition effect in the strain is relieved, the capability of non-glucose carbon sources such as xylose, glycerol, sucrose, raffinose, lactose and the like is not inhibited by 2-deoxyglucose any more, the utilization capability of glucose is kept, and particularly, the capability of the strain for utilizing xylose is higher.

Description

Yeast with relieved glucose inhibition effect and method for utilizing non-glucose carbon source by utilizing yeast

Technical Field

The invention belongs to the technical field of biology, and particularly relates to yeast with a relieved glucose inhibition effect and a method for utilizing a non-glucose carbon source by the yeast, in particular to a mutant strain of a heat-resistant yeast strain Kluyveromyces marxianus, wherein the glucose inhibition effect in the mutant strain is relieved, the utilization capacity of glucose is kept, and the capacity of utilizing the non-glucose carbon source such as xylose, glycerol, galactose, sucrose and raffinose, especially xylose, is high.

Background

Lignocellulosic biomass, such as corn stover, corn cobs, switchgrass, miscanthus, and the like, comprises major components including cellulose, hemicellulose, and lignin (Fatma et al, 2018), with the hydrolysis yielding major monosaccharides including glucose, xylose, arabinose, and fructose and lactose in minute amounts. And other waste liquids in the dairy industry, waste residues in the starch industry, waste residue treatment liquids and the like also contain saccharides other than glucose. The complete utilization of these sugars is very important for the economics of bio-based chemical products (Kim et al, 2012).

Glucose repression, also known as glucose repression or catabolism producing repression, refers to the phenomenon whereby glucose or certain readily available carbon sources whose catabolites repress the transcription of certain genes encoding inducible enzyme systems. Its presence in microorganisms severely inhibits the utilization of non-glucose carbon sources in biomass hydrolysates by the microorganisms and also significantly reduces the yield and rate of the target product, thereby reducing the production economy (Kim et al, 2012). In order to efficiently utilize lignocellulosic materials and various biomass-related wastes, it is necessary to achieve co-utilization of mixed sugars.

Therefore, there is a need for a microorganism that can utilize a non-glucose carbon source in a mixed sugar and has high ability to utilize a non-glucose carbon source, while eliminating the glucose inhibitory effect.

In order to counteract the glucose inhibitory effect, the applicant found in previous studies that the glucose effect could be eliminated by the knockout of the hexokinase gene in yeast (Hua et al, 2019), but this strategy would rapidly reduce the ability of the strain to utilize glucose, while losing the fructose utilization. Even though the glucose utilization efficiency is improved by overexpressing glucokinase gene therein, the strain still loses the fructose utilization ability and is difficult to be applied as a good platform strain.

SNF3 (sugar non-fermening 3, SNF3) is a cell membrane low glucose receptor, a high affinity receptor that induces hexose transport, and SNF3 is also sensitive to fructose and mannose, and has a homologous protein RGT 2. In s.cerevisiae, SNF3 is a glucose receptor highly homologous to sugar transporters, SNF3 and RGT2 sensing low and high concentrations of glucose, respectively (Ozcan et al, 1996).

If SNF3 and RGT2 in Saccharomyces cerevisiae are knocked out, the strain's remarkable ability to utilize glucose is reduced and growth becomes very slow with glucose (Ozcan, 2002). Knock-out of SNF3 in Saccharomyces cerevisiae alone results in loss of the ability of the strain to grow on sucrose and raffinose, as well as loss of the ability to utilize low concentrations of glucose (Neigeborn et al, 1986). Expression analysis of SUC2, a key gene for sucrose utilization, and beta-galactosidase, a key gene for galactose found that knock-out of SNF3 from saccharomyces cerevisiae did not alter the inhibition of sucrose and galactose utilization gene expression by glucose, wherein the loss of sucrose utilization was probably due to loss of sucrose absorption. Therefore, there has not yet been obtained a microorganism which has released the glucose inhibitory effect, not to mention a strain having a high ability to utilize a non-glucose carbon source based on this.

Kluyveromyces marxianus, a well-recognized safe microorganism (GRAS), is thermotolerant and has several advantages when used for fermentation: 1. safe and can be used in food industry; 2. high temperature fermentation, which generally results in faster fermentation rates and reduced cooling costs on the fermentation process, while reducing the risk of contamination (Zhang et al, 2016); 3. the substrate spectrum is broad and glucose, xylose, galactose, inulin, etc. can be used (Pentjuss et al, 2017); 4. it itself produces a variety of enzymes, if collagenase, beta-D-galactosidase and inulinase, etc., so that it can directly utilize a variety of raw materials (Fonseca et al, 2008). When chemicals are produced by taking lignocellulose biomass, waste liquid of dairy products and the like as substrates, the Kluyveromyces marxianus has more obvious advantages, so that the development of the Kluyveromyces marxianus into lactic acid production strains has very important application value.

But the kluyveromyces marxianus has the glucose effect, so that the utilization of other non-glucose carbon sources by the kluyveromyces marxianus under the condition of mixing sugar is inhibited, the production efficiency of chemicals is reduced, and the application of the kluyveromyces marxianus is limited.

Disclosure of Invention

The present inventors have focused on the transformation of Kluyveromyces marxianus to construct a platform strain with reduced or eliminated glucose response, and have conducted intensive studies.

Only one homologous gene KmSNNF 3(GenBank accession No.: BAP73238.1) of SNF3/RGT2 was present in the Kluyveromyces marxianus genome, annotated as SNF3, and no RGT2 gene was found. The present inventors knocked out gene KmSNF3 in Kluyveromyces marxianus and found that the obtained mutant strain shows many new characteristics which are not possessed by similar gene knockout in Saccharomyces cerevisiae: the mutant strain has no glucose inhibition effect, no decrease in glucose utilization capacity, and greatly improved xylose utilization capacity. Further, a mutant strain which has no glucose inhibitory effect and has high ability to utilize a non-glucose carbon source, and a method for utilizing a non-glucose carbon source are provided.

Specifically, the invention obtains a heat-resistant engineering yeast mutant strain with relieved glucose inhibition effect by knocking out a gene KmSNNF 3 in Kluyveromyces marxianus YHJ010 (triple auxotroph of uracil, leucine and tryptophan, Kluyveromyces marxianus NBRC1777 as an original strain by the inventor and storing the gene in the laboratory, wherein the construction method is shown in Hong et al, 2007): kluyveromyces marxianus CGMCC No.20304, (hereinafter sometimes referred to as Y.DELTA.SNF 3). Therefore, the invention provides Kluyveromyces marxianus CGMCC No.20304, wherein the gene KmSNF3 is non-dominant.

Y Δ SNF3 has the ability to utilize non-glucose carbon sources such as xylose, glycerol, sucrose, raffinose, lactose, etc., to relieve the glucose inhibitory effect. When cultured on solid media containing 2-deoxyglucose, the utilization of these carbon sources by Y Δ SNF3 was no longer inhibited by 2-deoxyglucose.

After the SNF3 gene knockout, Y delta SNF3 still maintains the capability of using glucose for growth, the growth speed is not obviously reduced, and the biomass is increased. Compared with a control strain YWD005 (the auxotrophy of uracil in the YHJ010 strain is complemented by expressing URA3, so that the auxotrophy is the same as that of Y delta SNF3, Wu et al, 2020), the final cell density of Y delta SNF3 is improved by 45.8%; and xylose consumption rose by 44.7% within 58 hours. The expression levels of xylose reductase, xylitol dehydrogenase and xylulokinase were increased by 10.98-fold, 15.11-fold and 2.51-fold, respectively. log2FC (Δ YSNF3/YWD005) was 3.71, 4.14, 1.94, respectively.

In one embodiment of the present invention, Kluyveromyces marxianus YHJ010 was used as a starting strain for SNF3 knock-out, but the starting strain is not limited thereto, and Pichia pastoris, yarrowia lipolytica, and Hansenula may be selected. Similarly, the plasmid used is not limited to pGEM-T easy vector, and includes: pMD18T, pUC57, pUC19 and the like.

In one embodiment of the present invention, the SNF3 gene is knocked out by using a plasmid having a knock-out frame, but other methods commonly used in genetic engineering, such as: crispr-cas9, Cre-LoxP and RNAi, wherein the SNF3 gene in the strain is non-dominant.

In one embodiment of the present invention, xylose, glycerol, sucrose, raffinose, and lactose are used as the non-glucose carbon source, but are not limited thereto, and examples thereof include: galactose, arabinose, etc.

In one embodiment of the present invention, the knockout cassette for SNF3 gene knockout contains the ScURA3 expression cassette, and the ScURA3 complete expression cassette (sequence from 2061 to 3158 nucleotides in GenBank: AM 697670.1) is used as the expression cassette. Among them, a nucleotide sequence having a function equivalent to that of a protein encoded by substituting, deleting or adding one or several nucleotides to the sequence, or a homologous gene or a fragment of a homologous gene having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more similarity to the SNF3 gene of kluyveromyces marxianus in other microorganisms, or a nucleotide sequence optimized depending on the kind of microorganism may be used.

In one embodiment of the present invention, lithium acetate transformation is used as the transformation method, and other commonly used yeast transformation methods, such as electric transformation, chemical transformation such as lithium chloride transformation, PEG100 transformation, protoplast transformation, etc., can also be used.

Specifically, the present invention comprises the following:

1. kluyveromyces marxianus YHJ010 mutant strain in which gene KmSNF3 was knocked out, released glucose inhibitory effect, maintained glucose-utilizing ability, and had high xylose-utilizing ability, wherein KmSNF3 GenBank access No.: BAP 73238.1.

2. The mutant strain according to item 1, which is Kluyveromyces marxianus (Kluyveromyces. marxianus) CGMCC No. 20304.

3. A method for constructing a mutant strain having relieved glucose suppressive effect, which comprises making the gene KmSNNF 3 of Kluyveromyces marxianus, preferably a strain derived from Kluyveromyces marxianus NBRC1777, more preferably Kluyveromyces marxianus YHJ010, non-dominant, preferably by knock-out.

4. The method according to item 3, wherein the knockout is performed by constructing a vector having a knockout box containing a ScURA3 expression box, preferably a ScURA3 complete expression box GenBank: the sequence from base 2061 to base 3158 of AM697670.1,

preferably, the vector with the knockout frame is plasmid pKMSNF 3-U.

5. The vector used in the construction method described in item 3 or 4 is preferably a plasmid pKMSNF3 or pKMSNF 3-U.

6. Use of the vector of item 5 for constructing a strain having a relieved glucose inhibitory effect.

7. Use of the mutant strain of item 1 or 2 for the utilization or degradation of a non-glucose carbon source.

8. A method for utilizing a non-glucose carbon source, which comprises culturing the mutant strain described in item 1 or 2 or treating a solution containing a non-glucose carbon source with the strain constructed by the method described in item 3 or 4,

the non-glucose carbon source is preferably xylose, glycerol, galactose, sucrose, raffinose, more preferably xylose, and the solution is preferably biomass hydrolysate or a bio-based chemical product, or a waste residue treatment solution thereof.

Advantages and positive effects

Kluyveromyces marxianus (Kluyveromyces marxianus) YHJ010 mutant strain Y delta SNF3 is the first heat-resistant strain which can realize the effect of relieving glucose inhibition without losing other sugars, so the strain can be used as an important starting strain for constructing engineering bacteria utilizing lignocellulose. The mutant strain Y delta SNF3 of the invention relieves the inhibition of glucose on the utilization of carbon sources such as xylose, lactose, galactose, glycerol, sucrose, raffinose and the like, maintains the growth capability of utilizing glucose, and improves the utilization of xylose, thereby being used as the basis for the construction of a platform strain for the co-utilization of carbon sources such as glucose, xylose and the like, and being applied to the utilization of biomass hydrolysate, bio-based chemical products, waste residue treatment liquid thereof and the like. And has wide application prospect in the aspect of constructing engineering bacteria for efficiently utilizing the lignocellulose biomass.

Meanwhile, the invention provides a method for constructing a microorganism for removing the glucose suppression effect by knocking out the homologous gene of the gene SNF3, the Kluyveromyces marxianus capable of removing the glucose suppression effect without losing other sugars is obtained by the method, and the method is applied to other microorganisms, particularly yeast strains, so that the microorganism, particularly yeast mutant engineering strains with similar characteristics can be expected to be obtained.

Deposit description

The mutant strain Kluyveromyces marxianus (Kluyveromyces marxianus) Y delta SNF3 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 7 months and 7 days in 2020 (CGMCC, the institute of microbiology of China academy of sciences, No.3 of the national institute of sciences, Nacio 1, Nacio, Chaozhou, Beijing, China), and the preservation number is CGMCC No. 20304.

Drawings

FIG. 1 PCR validation of knock-out of KmSNNF 3 gene in Y.DELTA.SNF 3.

FIG. 2. results of measurement of glucose inhibitory effect of Y.DELTA.SNF 3 using glucose analog 2-DG.

FIG. 3 growth of Y Δ SNF3 in the presence of glucose or xylose. A, growing by using glucose; and B, utilizing xylose to grow.

Figure 4. change in xylose concentration during growth of Y Δ SNF3 in the presence of xylose.

Detailed Description

The invention is further described below with reference to specific examples, but it will be understood by those skilled in the art that the invention is not limited to these specific examples.

Reagents and strains: all of the reagents of the present invention are practically commercially available reagents of a grade higher than that of the reagent. Wherein, glucose, xylose, sucrose, raffinose, glycerol, galactose, yeast basic nitrogen source, uracil, tryptophan, leucine, a gel recovery kit and all restriction endonucleases are all sourced from Shanghai Biotech engineering company. PrimeSTAR HS DNA polymerase, T4DNA ligase was purchased from Dalibao Bio, pGEM-T-Easy vector from Promega. Escherichia coli XL10-gold strain (Stratagene, Calif.) and DH 5. alpha. strain were used as host bacteria for DNA manipulation, and Luria-Bertani (LB) medium containing 100. mu.g/ml ampicillin was used for E.coli culture. Glucose synthesis medium (glucose 20g/l, yeast basic nitrogen source 6.7g/l, tryptophan 20mg/l, leucine 30mg/l) was used mainly for transformation. YPD medium (10g/l yeast extract, 20g/l Bacto peptone, 20g/l glucose) was used for yeast pre-culture.

RNA seq is completed by Shanghai Meiji biological medicine science and technology, general gene cloning and sequencing in plasmid construction process are completed by Shanghai biological engineering, Inc., and primer synthesis company, Shanghai biological engineering, Inc.

Primer information relating to the present invention

Example 1 construction of a Kluyveromyces marxianus SNF3 knock-out mutant

Construction of SNF3 Gene knockout cassette:

1) construction of plasmid pKMSNF 3.

The genomic DNA of Kluyveromyces marxianus was extracted, KmSNNF 3(GenBank accession No.: BAP73238.1) was amplified from the Kluyveromyces marxianus genome and inserted into pGEM-T easy vector, and the obtained plasmid was named pKMSNNF 3.

The specific operation is as follows:

(1) extraction of genomic DNA

a) The frozen and deposited bacteria are streaked and cultured in an incubator at 37 ℃ for 24 hours.

b) Single clones were picked, inoculated into 5ml of liquid medium, shaken at 37 ℃ and incubated overnight at 250 rpm.

c) The cells were centrifuged at 12000rpm for 1 minute, and the supernatant was discarded to collect the cells.

d) The cells were resuspended in 500. mu.l of sterile water, transferred to a 1.5ml centrifuge tube, centrifuged at 12000rpm for 1 minute, the supernatant was discarded, and the cells were collected.

e) The cells were resuspended by adding 200. mu.l of lysis buffer (EDTA (1mM), TritonX-100 (2% (w/v)), NaCl (100mM), SDS (1% (w/v)), Tris-Cl (10mM, pH 8.0)).

f) To this were added 0.3g of glass beads (425 nm, sigma, USA) and 200. mu.l of phenol chloroform solution (25: 24: 1, pH 8.0), vortexed at high speed for 3 minutes.

g) An additional 200. mu.l of 1 XTE buffer (1mM EDTA,10mM Tris-Cl, pH 8.0) was added and gently shaken rapidly for 30 seconds.

h) After centrifugation at 12000rpm for 5 minutes, the upper aqueous phase was transferred to a new 1.5ml centrifuge tube, and 1ml of ice-cold absolute ethanol was added thereto, and mixed by inversion.

i) Centrifuge at 12000rpm for 5 min at 4 ℃, discard the supernatant and resuspend the pellet in 400. mu.l of 1 × TE buffer.

j) Mu.l of 2ng/ml RNase A (Shanghai, China, Biopsis) was added thereto and incubated at 37 ℃ for 5 minutes.

k) Add 40. mu.l of 3M sodium acetate (pH 5.2) and 1ml of ice-cold absolute ethanol and mix by inversion.

l) centrifugation was carried out at 12000rpm for 10 minutes at 4 ℃ and the supernatant was discarded, and the precipitate was washed with 1ml of ice-cold 75% ethanol and centrifuged again to remove the supernatant.

m) drying the precipitate at room temperature for 10 minutes, and resuspending the precipitate with 40-100. mu.l of 1 × TE buffer to obtain the extracted yeast genomic DNA.

(2) Amplification of a fragment comprising the gene KmSNF3 and upstream and downstream thereof: using KmSNF3F (Seq ID No.1) and KmSNF3R (Seq ID No.2) as primers and YHJ010 genome DNA as a template, amplifying a target gene fragment by using high fidelity PrimeSTAR HS DNA polymerase, wherein PCR reaction is as follows:

reaction conditions are as follows:

thus, the PCR product obtained by PCR amplification contains the KmSNF3 gene and fragments upstream and downstream thereof.

(3) After a reaction system is prepared according to the following steps by Taq DNA polymerase, the reaction is carried out for 30 minutes at 72 ℃, and the A treatment is carried out on the tail end of the PCR product fragment. The reaction system for adding A is as follows:

(4) and (4) connecting carriers. After A was added to the end of the PCR product, the PCR product was directly ligated to pGEM-T easy vector, and after preparing a ligation reaction system as follows, the ligation reaction was carried out overnight at 16 ℃. The ligation reaction system is as follows:

(5) and (4) transforming the escherichia coli. The next day, the ligation product is transformed into escherichia coli, and after the clone grows out, the monoclonal antibody is selected and inoculated into a test tube containing a fresh LB liquid culture medium for overnight culture; after overnight culture, plasmid was extracted from the culture broth, and sequencing was performed to confirm sequence information, thereby obtaining plasmid pKmSNNF 3 containing KmSNNF 3 gene.

2) Construction of a plasmid containing a SNF3 Gene knockout cassette

A partial fragment (middle 991bp) of the ORF of the gene in the plasmid pKMSNF3 obtained by the above procedure was deleted by PCR to obtain the complete expression cassette of ScURA3 (GenBank: AM697670.1, base 2061 to base 3158). And carrying out enzyme digestion on the ScURA3 complete gene expression frame DNA by utilizing Sma I, and connecting the obtained product with the obtained linear pKMSNF3 vector to obtain a plasmid containing a gene knockout frame, wherein the plasmid is named as plasmid pKMSNF 3-U.

The method comprises the following specific steps:

(1) amplification of the ScURA3 expression cassette fragment: plasmid YEUGAP (the gift from professor Shangdu university of Jingdu, Jade. Shangdu.) (Zhang et al, 2014) is used as a template for amplification reaction, and a fragment containing the expression frame of SCURA3 is amplified by using high fidelity enzyme PrimeSTAR HS DNA polymerase and primers SCURA3-SMAI-F (SEQ ID No.3) and SCURA3-SMAI-R (SEQ ID No. 4).

(2) Reverse amplification reaction is carried out by using primers dKmSNNF 3F (SEQ ID No.5) and dKmSNNF 3R (SEQ ID No.6) and using a plasmid pKmSNNF 3 as a template, an amplification product comprises a whole T vector framework and a partial sequence of a corresponding gene, a PCR amplification system is as follows, and the reaction program is set to be the same as that of KmSNNF 3 gene amplification.

(3) And (3) connecting the amplification product part obtained in the step (2) with a ScURA3 expression frame by using blunt ends to obtain a pKMSNF3-U plasmid. In the plasmid pKMSNF3-U, the two ends of the expression frame of ScURA3 are the residual segments of the corresponding genes, and can be used as homologous arms to carry out homologous recombination with genes in yeast, so that the target genes are knocked out, the connection reaction system is as follows, and the reaction is carried out overnight at 16 ℃;

(4) the ligation product is transformed into Escherichia coli, and sequence information is confirmed through colony PCR (cells of a small number of colonies are selected as a template, other reaction conditions are the same as the diffusion of SNF3) and sequencing to obtain a plasmid pKMSNF3-U containing a KmSNF3 gene knockout frame.

Knock-out of KmSNF3 Gene

Knock out KmSNNF 3 gene, and the starting strain is Kluyveromyces marxianus YHJ 010.

(1) A plasmid pKMSNF3-U containing a gene knockout frame is taken as a template, and KmSNF3F (SEQ ID No.1) and KmSNF3R (SEQ ID No.2) are taken as primers to amplify a knockout frame fragment of the gene; then concentrating and purifying the amplified knockout frame segment of the gene by an ethanol precipitation method;ethanol precipitation method

1) The obtained DNA solution (e.g., PCR reaction solution) was pooled into a clean 1.5ml centrifuge tube, and one-tenth volume of 3M sodium acetate solution (pH 5.2) was added and mixed well;

2) adding 2-3 times volume of the precooled absolute ethyl alcohol into the centrifuge tube by using a pipettor, reversing the mixture, fully mixing the mixture, and then placing the mixture in a refrigerator at the temperature of-20 ℃ for standing for about 20 minutes; at the moment, opening the low-temperature refrigerated centrifuge for precooling to 4 ℃;

3) after the mixed solution is completely stood, taking out the mixed solution, and centrifuging the mixed solution by a centrifuge at the temperature of 4 ℃ for 10 minutes under the centrifugation condition of 16000 Xg;

4) after the centrifugation is finished, taking out the centrifugal tube, carefully removing the supernatant, adding 1ml of precooled 75% ethanol into the centrifugal tube by using a liquid transfer device, and centrifuging for 5 minutes at the rotating speed of 16000 Xg;

5) after the centrifugation is finished, removing the supernatant, washing the supernatant once by using 1ml of precooled 75% ethanol, wherein the centrifugation condition is still 16000 Xg for 5 minutes;

6) carefully discarding the supernatant after centrifugation, carefully sucking residual liquid by using a pipettor, opening the cover, placing at room temperature, and volatilizing residual ethanol;

7) after leaving for about 10 minutes, the DNA precipitate obtained was dissolved by adding an appropriate volume of double distilled water, and then stored in a refrigerator at-20 ℃.

(2) The knock-out fragment of each gene was transformed into Kluyveromyces marxianus YHJ010, and screened by a synthetic medium plate supplemented with leucine and tryptophan, and only a strain whose fragment successfully transferred restored uracil-synthesizing ability to the strain YHJ010 survived on the plate.

Lithium acetate conversion of yeast

i) Kluyveromyces marxianus strain YHJ010 was streaked on YPD agar medium plate, and cultured in an incubator at 37 ℃ for 24 hours.

ii) Single colonies were picked from the streaking plates and inoculated into 5ml of liquid YPD medium, shaking at 37 ℃ and incubated overnight at 250 rpm.

iii) 500. mu.l of overnight-cultured broth was inoculated into 5ml of fresh YPD medium and cultured at 37 ℃ for 4 hours at 250rpm on a shaker.

iv) centrifuging the bacterial solution at 5000rpm for 3 minutes, discarding the supernatant, and collecting the bacterial cells.

v) preparation of 1ml of transformation buffer:

50％PEG4000 800μl

2M lithium acetate 100. mu.l

1M DTT 100μl

vi) aspirate 150 μ l of transformation buffer to resuspend the cells and transfer to a 1.5ml centrifuge tube, centrifuge rapidly at 5000rpm for 5 seconds, and discard the supernatant.

vii) aspirate 100. mu.l of transformation buffer and resuspend the cells again, add 5. mu.l of linearized plasmid to it, and mix well.

viii) placing the mixed solution in a water bath kettle at 47 ℃ and carrying out heat shock in the water bath for 15 minutes.

ix) the mixture was removed, 100. mu.l of SD broth was added thereto, and vortexed for 30 seconds.

x) uniformly coating the mixed solution on a screened synthetic culture medium, and performing inverted culture in an incubator at 37 ℃ for 3-4 days.

xi) streaking single clones on the plate to a new identical plate, and secondary screening.

xii) streaked monoclonal antibodies were picked and cultured in liquid YPD medium as selected strain Y Δ SNF 3.

(3) Because the probability of non-homologous recombination in the kluyveromyces marxianus is higher, the strain Y delta SNF3 which passes the screening further determines that the transferred segment and the target gene have the homologous recombination in a colony PCR mode, thereby confirming that the strain is the strain with the corresponding gene knocked out.

Specifically, PCR amplification is carried out on the screened strain Y delta SNF3 by using primers KmSNNF 3F (SEQ ID No.1) and KmSNNF 3R (SEQ ID No.2), and the colony PCR system for confirming the knockout result of the strain Y delta SNF3 is the same as the amplification condition of the SNF3 gene. The results are shown in FIG. 1.

The results show that the KmSNNF 3 gene in FIG. 1 has a significantly enlarged amplification band in the knockout strain Y.DELTA.SNF 3 compared with the gene-knockout starting strain (YHJ 010). Since the ORF of KmNSF3 was partially deleted and the expression cassette of ScURA3 was inserted during the construction of the knock-out cassette, the length of the target gene amplified from the knock-out genome (4.0Kb) was larger than that of the target gene amplified from the wild type (2.8Kb) when the gene knock-out was successful. The successful knock-out of the corresponding gene in strain Y delta SNF3 was suggested.

Example 2 Release of glucose inhibitory Effect in Strain Y.DELTA.SNF 3

The glucose inhibitory effect of the KmNSF3 knock-out strain Y Δ SNF3 was identified.

The growth of strain Y.DELTA.SNF 3 was observed on yeast extract peptone (YP) plates supplemented with glucose analog 2-Deoxyglucose (2-DG) in xylose, galactose, sucrose and raffinose, respectively. Since KmSNNF 3 in strain Y.DELTA.SNF 3 was knocked out, in order to maintain the consistency of genetic background, strain YWD005 (inventor's construction, construction method see (Wu et al, 2020) in which the URA3 tag (i.e., the key gene for restoring the ability of the strain to synthesize uracil) was complemented back to Kluyveromyces marxianus strain YHJ010 (inventor's Kluyveromyces marxianus strain NBRC1777 as the starting strain; construction method see Hong et al, 2007) was used as the control strain.

The specific operation is as follows:

(1) streaking a to-be-detected strain Y delta SNF3 and a control strain YWD005 to a YPD solid culture medium, and culturing overnight in a biochemical incubator at 37 ℃;

(2) the following day, monoclonals were picked from the plates and cultured in tubes containing 5ml of YPG (10g/l yeast extract, 20g/l bactopeptone, 20g/l glycerol) liquid medium under conditions consistent with 37 ℃ overnight at 250 rpm.

(3) The bacterial liquid cultured overnight is measured by ultraviolet spectrophotometer to obtain bacterial density OD_600nmControl point plate sample concentration gradient by calculation and dilution with sterile water: the first concentration gradient is OD _600nm1, followed by 10-fold dilution in order, i.e. OD_600nm＝10^-1,10^-2,10^-3。

(4) Pipette up OD600 ═ 1, 10^-1,10^-2And 10^-3Dropping 1.5 μ l of diluted bacteria solution to be spotted onto the corresponding position on the plate, after the plate is spotted, putting the plate into an incubator to be cultured at 37 ℃ after the bacteria solution is dried.

(5) After 48 hours of incubation, each plate was photographed under a Tanon 1600 fully automated digital gel imaging analysis system.

The results are shown in FIG. 2.

The results show that the knock-out mutant Y.DELTA.SNF 3 of gene KmSNNF 3 has a very significant growth advantage on xylose, galactose, sucrose and raffinose plates containing 2-DG compared to the control YWD005, as shown in FIG. 2. Wherein: in the case of culturing using glucose as a carbon source, the seeded cell density (OD600) was 1 to 10^-1、10^-2、10^-3The growth of Y.DELTA.SNF 3 was almost the same as that of YWD005 regardless of the presence or absence of 2-DG, and when glycerol, galactose, xylose, sucrose, and raffinose were used as carbon sources and 2-DG was not present in the medium, the cell density of the inoculated cells was 1 or 10^-1、10^-2、10^-3The growth of the lower Y.DELTA.SNF 3 was almost not different from that of YWD005, but when 2-DG was present in the medium, the control strain showed significant growth only when inoculated with a cell density of 1, 10^-1There was weak growth, no growth at other inoculum concentrations, and Y.DELTA.SNF 3 at 1, 10^-1、10^-2、10^-3The growth is obvious under the condition.

2-DG can enter cells via glucose transporters and initiate a glucose inhibitory effect, which inhibits the utilization of other, non-glucose by cells, but cannot metabolically provide energy and carbon sources for cell metabolism, etc. Thus strain Y Δ SNF3 has a growth advantage over control strain YWD005 on 2-DG containing non-glucose carbon source plates, indicating that knock-out of SNF3 in kluyveromyces marxianus removes the inhibitory effect of glucose on the utilization of various non-glucose carbon sources, including xylose, to varying degrees.

Example 3 ability of Strain Y Δ SNF3 to utilize glucose and xylose

The KmSNNF 3 knock-out mutant Y delta SNF3 was tested for growth on glucose or xylose.

The strain Y delta SNF3 and the control strain YWD005 were cultured in a medium containing glucose or xylose as a carbon source, and the bacterial density was measured by OD 600. And further detecting the change of xylose concentration in the culture supernatant of the strain Y delta SNF 3.

The method comprises the following specific steps:

strain Y Δ SNF3 was cultured in 5ml YPD liquid medium at 42 ℃ overnight at 250rpm as a preculture;

the pre-cultured strain was used to initiate OD₆₀₀0.5 to 100g/L glucose (YPD) or 100g/L xylose (YPX), shaking at 42 ℃ and 250rpm, and taking the medium at different time points during the cultivation (sampling time shown in FIGS. 3A and 3B) to determine the OD₆₀₀And a growth curve was plotted (FIG. 3A, FIG. 3B), and the residual xylose concentration in the culture supernatant of the strains at different time points after 0, 2, 6, 10, 24, 28, 36, 50, 58 hours of culture was measured by high performance liquid chromatography [ Agilent 1260 chromatograph (Agilent) ROA-Organic Acid H + (8%) column, 0.0025M sulfuric Acid as mobile phase]The measurement was carried out (FIG. 4).

The results showed that, in the medium containing 100g/L glucose, the cell density of Y.DELTA.SNF 3 was lower than that of the control strain YWD005 at 2, 3 and 10 hours, but Y.DELTA.SNF 3 reached a higher bacterial density (OD. sup. D) than that of the control strain YWD005 at each time point after 23, 28, 36, 50 and 58 hours of culture₆₀₀). OD of Y.DELTA.SNF 3 after 58 hours of culture₆₀₀37, while the control strain YWD005 reached only 29 (FIG. 3A), the bacterial density (OD) of Y.DELTA.SNF 3₆₀₀) 27.6% higher than the control strain. In contrast, in the medium containing 100g/L xylose, Y.DELTA.SNF 3 was higher in both growth rate and final bacterial density than the control strain YWD005 at each time point (12, 16, 20, 23, 38, 58 hours), and OD of Y.DELTA.SNF 3 was found after 58 hours of culture₆₀₀Up to 35, and OD of YWD005₆₀₀Bacterial Density (OD) of only 24, Y.DELTA.SNF 3₆₀₀) 45.8% higher than the control strain (fig. 3B).

At each time point (12, 16, 20, 23, 38, 58 hours), the residual xylose in the culture supernatant of Y Δ SNF3 strain was less than that of WD005 strain, indicating that Y Δ SNF3 has a faster ability to utilize xylose, and that 32g/L of xylose remained in the culture supernatant of Y Δ SNF3 strain, while 53g/L of xylose remained in the culture supernatant of YWD005 strain after 58 hours of culture (fig. 4). Namely, the Y delta SNF3 strain and the YWD005 strain respectively consume 68g/L and 47g/L of xylose, and the xylose consumption is increased by 44.7 percent within 58 hours.

Taken together, the results show that strain Y Δ SNF3 was able to utilize xylose faster and with higher xylose utilization capacity when cultured with xylose-containing medium compared to control strain YWD 005.

Example 4 transcriptome analysis in mixed sugar culture of strain Y Δ SNF3 glucose and xylose.

The mutant strain Y.DELTA.SNF 3 and the control strain YWD005 were cultured with a starting OD600 of 1 in a medium containing 8% glucose and 2% xylose (1% yeast extract, 2% peptone and glucose and xylose, also abbreviated as YPDX in the drawing) and cultured at 37 ℃ to OD ₆₀₀8, and detecting the incubation at that timeGlucose in the supernatant was confirmed to have not been consumed (test method same as xylose determination). The cells were collected by centrifugation at 12000rpm, and sent to Meiji organisms (Shanghai) for RNA-seq sequencing and analysis, and the results are shown in Table 1.

TABLE 1 comparison of expression levels of xylitol dehydrogenase, xylitol reductase and xylulokinase measured by RNA-seq

The results showed that the expression levels of xylose reductase, xylitol dehydrogenase and xylulokinase of the mutant strain Y Δ SNF3 were increased by 10.98-fold, 15.11-fold and 2.51-fold, respectively, and log2FC (Δ YSNF3/YWD005) was 3.71, 4.14 and 1.94, respectively, as compared with the expression of the corresponding gene of YWD 005.

It was confirmed that the mutant strain Y.DELTA.SNF 3 exhibited enhanced expression of xylose reductase, xylitol dehydrogenase, xylulokinase and the like in a mixed sugar medium containing glucose and xylose, suggesting a mode of action for relieving the glucose inhibitory effect thereof.

Reference documents:

1.Fatma,S.,Hameed,A.,Noman,M.,Ahmed,T.,Shahid,M.,Tariq,M.,Sohail,I.,Tabassum,R.2018.Lignocellulosic Biomass:A Sustainable Bioenergy Source for the Future.Protein and Peptide Letters,25(2),148-163.

2.Fonseca,G.G.,Heinzle,E.,Wittmann,C.,Gombert,A.K.2008.The yeast Kluyveromyces marxianus and its biotechnological potential.Appl Microbiol Biotechnol,79(3),339-354.

3.Hong,J.,Wang,Y.,Kumagai,H.,Tamaki,H.2007.Construction of thermotolerant yeast expressing thermostable cellulase genes.Journal of Biotechnology,130(2),114-123.

4.Hua,Y.,Wang,J.,Zhu,Y.,Zhang,B.,Kong,X.,Li,W.,Wang,D.,Hong,J.2019.Release of glucose repression on xylose utilization in Kluyveromyces marxianus to enhance glucose-xylose co-utilization and xylitol production from corncob hydrolysate.Microbial Cell Factories,18,24.

5.Kim,S.R.,Ha,S.J.,Wei,N.,Oh,E.J.,Jin,Y.S.2012.Simultaneous co-fermentation of mixed sugars:a promising strategy for producing cellulosic ethanol.Trends in Biotechnology,30(5),274-282.

6.Neigeborn,L.,Schwartzberg,P.,Reid,R.,Carlson,M.1986.Null Mutations in the Snf3Gene of Saccharomyces-Cerevisiae Cause a Different Phenotype Than Do Previously Isolated Missense Mutations.Molecular and Cellular Biology,6(11),3569-3574.

7.Ozcan,S.2002.Two different signals regulate repression and induction of gene expression by glucose.Journal of Biological Chemistry,277(49),46993-46997.

8.Ozcan,S.,Dover,J.,Rosenwald,A.G.,Wolfl,S.,Johnston,M.1996.Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression.Proceedings of the National Academy of Sciences of the United States of America,93(22),12428-12432.

9.Pentjuss,A.,Stalidzans,E.,Liepins,J.,Kokina,A.,Martynova,J.,Zikmanis,P.,Mozga,I.,Scherbaka,R.,Hartman,H.,Poolman,M.G.,Fell,D.A.,Vigants,A.2017.Model-based biotechnological potential analysis of Kluyveromyces marxianus central metabolism.J Ind Microbiol Biotechnol,44(8),1177-1190.

10.Wu,D.,Wang,D.,Hong,J.2020.Effect of a Novel Alpha/Beta Hydrolase Domain Protein on Tolerance of K.marxianus to Lignocellulosic Biomass Derived Inhibitors.Frontiers in Bioengeering and Biotechnology,8,844.

11.Zhang,B.,Zhang,J.,Wang,D.,Han,R.,Ding,R.,Gao,X.,Sun,L.,Hong,J.2016.Simultaneous fermentation of glucose and xylose at elevated temperatures co-produces ethanol and xylitol through overexpression of a xylose-specific transporter in engineered Kluyveromyces marxianus.Bioresour.Technol.,216,227-237.

12.Zhang,J.,Zhang,B.,Wang,D.M.,Gao,X.L.,Hong,J.2014.Xylitol production at high temperature by engineered Kluyveromyces marxianus.Bioresour.Technol.,152,192-201.

Claims (8)

1. kluyveromyces marxianus YHJ010 mutant strain in which gene KmSNF3 was knocked out, released glucose inhibitory effect, maintained glucose-utilizing ability, and had high xylose-utilizing ability, wherein KmSNF3 GenBank access No.: BAP 73238.1.

2. The mutant strain of claim 1, which is Kluyveromyces marxianus (Kluyveromyces marxianus) CGMCC No. 20304.

3. A method for constructing a mutant strain having relieved glucose suppressive effect, which comprises making the gene KmSNNF 3 of Kluyveromyces marxianus, preferably a strain derived from Kluyveromyces marxianus NBRC1777, more preferably Kluyveromyces marxianus YHJ010, non-dominant, preferably by knock-out.

4. The method of claim 3, wherein the knockout is performed by constructing a vector with a knockout box comprising the expression box of ScURA3, preferably the complete expression box of ScURA3 GenBank: the sequence from base 2061 to base 3158 of AM697670.1,

preferably, the vector with the knockout frame is plasmid pKMSNF 3-U.

5. The vector used in the construction method according to claim 3 or 4, preferably the plasmid pKMSNF3 or pKMSNF 3-U.

6. Use of the vector of claim 5 for the construction of strains with a abolished glucose repression effect.

7. Use of a mutant strain according to claim 1 or 2 for the utilization or degradation of a non-glucose carbon source.

8. A method for utilizing a non-glucose carbon source, which comprises culturing the mutant strain of claim 1 or 2 or treating a solution containing a non-glucose carbon source with the strain constructed by the method of claim 3 or 4,

the non-glucose carbon source is preferably xylose, glycerol, galactose, sucrose, raffinose, more preferably xylose, and the solution is preferably biomass hydrolysate or a bio-based chemical product, or a waste residue treatment solution thereof.

Images