A furfural-tolerant recombinant expression transformant and its application

Abstract

The present invention provides a recombinant expression transformant tolerant to furfural and its application. These furfural-tolerant recombinant expression transformants respectively contain genes carrying mutation sites, and these genes include PP_RS18130<supgt;T65A / A67G< / supgt>, PP_RS02880<supgt;T517A< / supgt>, PP_RS19785<supgt;C20A< / supgt>, PP_RS20740<supgt;G1012T< / supgt>, PP_RS02385<supgt;A1046G< / supgt>. The recombinant expression transformants containing these genes are applied to the detoxification of pretreated lignocellulosic hydrolysate, which can effectively reduce the inhibitory effect of toxic by-products in the hydrolysate on fermentation microorganisms, thereby improving the efficient production of lignocellulosic raw materials into bio-based products. The present invention also provides a preparation method for these recombinant expression transformants, including the construction of a recombinant expression vector, the transformation of the expression vector, and the toxicity tolerance and transformation ability of the recombinant expression transformant to furfural.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to a furfural-tolerant recombinant expression transformant and its application. Background Art

[0002] With the rapid growth of the world's population and the shortage of fossil fuels, there is an urgent need to produce fuels and chemicals using renewable energy. As a renewable resource, lignocellulosic biomass has an annual output of approximately 10 12 tons worldwide. It is widely sourced, abundantly supplied, and inexpensive, but only a small amount of fiber raw materials are effectively utilized. Lignocellulosic raw materials are composed of cellulose, hemicellulose, and lignin, and their structure is stable and firm. Microorganisms need to be pretreated to obtain fermentable sugars before using them for fermentation. However, the pretreatment of lignocellulosic raw materials will inevitably produce a toxic complex mixture that severely inhibits microbial metabolism, including furfural, 5-hydroxymethylfurfural, formic acid, acetic acid, etc.

[0003] These inhibitors pose obvious obstacles to the production of lignocellulosic raw materials for fuels and bulk chemicals. Furfural and 5-hydroxymethylfurfural are two typical inhibitors in the pretreatment solution with relatively high contents and strong toxicity. Reducing or eliminating the inhibition of these two inhibitors on fermentation strains is the key to the efficient biorefinery of lignocellulosic raw materials.

[0004] Existing studies have shown that furfural is a more toxic inhibitor than 5-hydroxymethylfurfural. To overcome the inhibition of furfural on microbial fermentation, it is usually necessary to detoxify the pretreated hydrolysate to remove the inhibitors. However, this process is not only time-consuming and complex, increasing the operating cost, but also causes the loss of a part of fermentable sugars. The ideal method is to develop strains that can tolerate furfural to improve their tolerance to inhibitors in the pretreatment solution.

[0005] Pseudomonas putida KT2440 is a Gram-negative, non-pathogenic, environmentally safe strain. Due to its characteristics such as metabolic diversity, genetic tractability, and strong tolerance to chemical and oxidative stress, it has a wide range of applications in the fields of biotechnology and synthetic biology, including the production of a series of chemicals. Although the strain has inherent robustness, further improving the strain's toxicity tolerance to inhibitors and exploring the stress-resistant elements therein to construct a detoxified strain with high furan aldehyde inhibitor tolerance is of great significance for the high-value utilization of lignocellulose. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a furfural-tolerant recombinant expression transformant in view of the deficiencies of the prior art, so as to improve the transformant's own tolerance to inhibitors in the pretreatment solution.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned furfural-tolerant recombinant expression transformant.

[0008] To achieve the above object, the present invention provides a furfural-tolerant recombinant expression vector, and the furfural-tolerant recombinant expression vector is a vector inserted with one of the following genes carrying mutation sites:

[0009] PP_RS18130 T65A / A67G ,

[0010] PP_RS02880 T517A ,

[0011] PP_RS19785 C20A ,

[0012] PP_RS20740 G1012T ,

[0013] PP_RS02385 A1046G gene.

[0014] Among them, the amino acid sequence of the protein encoded by the nucleotide sequence of the PP_RS18130 T65A / A67G gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the nucleotide sequence of the PP_RS02880 T517A gene is shown in SEQ ID NO.2; the amino acid sequence of the protein encoded by the nucleotide sequence of the PP_RS19785 C20A gene is shown in SEQ ID NO.3; the amino acid sequence of the protein encoded by the nucleotide sequence of the PP_RS20740 G1012T gene is shown in SEQ ID NO.4; the amino acid sequence of the protein encoded by the nucleotide sequence of the PP_RS02385 A1046G gene is shown in SEQ ID NO.5.

[0015] Among them, the nucleotide sequence of the PP_RS18130 T65A / A67G gene is shown in SEQ ID NO.6; the nucleotide sequence of the PP_RS02880 T517A gene is shown in SEQ ID NO.7; the nucleotide sequence of the PP_RS19785 C20A gene is shown in SEQ ID NO.8; the nucleotide sequence of the PP_RS20740 G1012T gene is shown in SEQ ID NO.9; the nucleotide sequence of the PP_RS02385 A1046G gene is shown in SEQ ID NO.10.

[0016] Among them, the furfural-tolerant recombinant expression vector has the genes at the mutated sites obtained through adaptive domestication in a medium containing furfural and combined with high-throughput sequencing analysis. The specific experimental steps are as follows:

[0017] The parent strain was activated in a mineral salt medium containing 5 g / L sodium acetate (30 °C, 200 rpm), and then the activated cells were transferred to a 5 g / L sodium acetate medium containing a low concentration of furfural (1 mM) for cultivation. When the cells reached the logarithmic growth phase, they were transferred to a fresh medium with the same furfural concentration at an OD 600 of 0.1, and this was repeated multiple times in the same manner until the cells were stable in tolerating the same furfural concentration. Then, the cells were transferred to a medium containing a higher concentration of furfural in the same way, and the operation was repeated in the medium with continuously increasing furfural concentration. Finally, the domesticated strain P. putida Z with high-concentration furfural tolerance ability was obtained. Among them, the domesticated furfural concentrations were: 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM. Then, whole-genome resequencing was performed on this strain, and through comparative analysis of the obtained data, the five genes described in the present invention were screened out for verification.

[0018] Specifically, the parent strain is an excellent detoxifying strain, Pseudomonas putida, which has knocked out the sugar metabolism pathway, does not utilize fermentable sugars but can use inhibitory organic acids and phenolic inhibitors derived from lignocellulosic biomass as carbon sources (the detailed construction process of this strain has been publicly disclosed in Patent CN202110660995.4).

[0019] The furfural-tolerant recombinant expression transformant containing the above nucleotide sequence can be prepared by the following method in the present invention. The specific steps are as follows:

[0020] (1) Amplification of the target gene: Using Max DNA Polymerase to perform PCR amplification on the gene described in the present invention to obtain a nucleic acid product;

[0021] (2) Construction of the recombinant expression vector: After purifying the product obtained in step (1), it was cloned into an expression vector using the pEASY-Basic Seamless Cloning and Assembly Kit (TransGen Biotech) to obtain a recombinant expression plasmid containing the gene described in the present invention;

[0022] (3) The recombinant expression plasmid obtained in step (2) was transformed into a host cell to obtain a recombinant expression transformant.

[0023] Among them, the expression vector described in step (2) is various conventional expression vectors in the art, such as any one of commercially available plasmids, cosmids, phage or viral vectors.

[0024] Among them, the host cell described in step (3) is various conventional host cells in the art, as long as it can satisfy that the recombinant expression plasmid can stably replicate itself, and the mutant gene described in the present invention carried by it can be effectively expressed. The preferred host cell is Pseudomonas putida, and the more preferred host cell is Pseudomonas putida KT2440 (strain number ATCC47054).

[0025] Among them, the transformation in step (3) is electrotransformation, and the electrotransformation conditions are: 2 mm electroporation cuvette, 2400 V, 25 μF, 200 Ω.

[0026] The advantage of the strain of the present invention in terms of tolerance to furfural is shown in a preferred embodiment. When the concentration of furfural in the culture medium is 20 mM, compared with the control strain containing the empty plasmid pBBR1MCS2, the strain of the present invention is significantly faster than the control strain in terms of the growth of the cells and the conversion rate of furfural. Preferably, it is KTPP_RS19785 C20A and KTPP_RS20740 G1012T strains, and the most preferred is KTPP_RS19785 C20A strains, which can achieve 100% conversion of furfural within 12 h.

[0027] The strain obtained by the present invention contains the gene with a mutation site. Any strain obtained by further modifying other irrelevant positions of these genes on the basis of the present invention has the same essence as the present invention and still belongs to the protection scope of the present invention.

[0028] The application of the above-mentioned recombinant expression transformant in detoxifying the hydrolysate obtained from biomass pretreatment is also within the protection scope of the present invention. The biomass is preferably corn stover. The present invention discloses the application of a furfural-tolerant Pseudomonas putida in detoxifying lignocellulosic raw materials. In one embodiment, by culturing with the strain of the present invention in a real hydrolysate containing inhibitors, the toxic inhibitors furfural and 5-hydroxymethylfurfural in the hydrolysate can be effectively eliminated, thereby reducing the overall toxicity of the hydrolysate. Preferably, it is KTPP_RS19785 C20A and KTPP_RS20740 G1012T strains, and the most preferred is KTPP_RS19785 C20A strains, which can convert 68% of furfural within 12 h and achieve 96% conversion of furfural within 24 h.

[0029] The application of the above-mentioned recombinant expression transformant in the biorefinery of lignocellulosic raw materials is also within the protection scope of the present invention.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0031] (1) The present invention adaptively domesticates the strain using a culture medium containing furfural and combines high-throughput sequencing analysis to discover 5 genes carrying mutation sites, and uses them to construct a furfural-tolerant recombinant expression vector, and further constructs furfural-tolerant recombinant expression transformants.

[0032] (2) The strain obtained by the present invention has good tolerance and conversion ability to furfural. Under the stress of the same concentration of furfural, the growth and conversion ability of the strain obtained by the present invention to furfural are significantly better than those of the control strain containing the empty plasmid pBBR1MCS2.

[0033] (3) The strain of the present invention has the advantages of low nutritional requirements, rapid growth, strong stress resistance, mature genetic operation system, simple preparation method, and easy operation.

[0034] (4) The present invention improves the toxic tolerance of Pseudomonas putida KT2440 to inhibitors and explores the stress resistance elements therein to construct a detoxified strain with high tolerance to furanaldehyde inhibitors, which is of great significance for achieving high-value utilization of lignocellulose.

[0035] (5) The strain of the present invention has great application prospects in the biorefining of wood fiber raw materials. It can alleviate or eliminate the inhibitory effect of toxic side reaction products of the pretreatment liquid on fermentation microorganisms, thereby improving the efficiency of biorefining. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0037] Figure 1 For PP_RS18130 T65A / A67G Diagram of the construction of the recombinant expression vector plasmid.

[0038] Figure 2 For PP_RS02880 T517A Diagram of the construction of the recombinant expression vector plasmid.

[0039] Figure 3 For PP_RS19785 C20A Diagram of the construction of the recombinant expression vector plasmid.

[0040] Figure 4 For PP_RS20740 G1012T Diagram of the construction of the recombinant expression vector plasmid.

[0041] Figure 5 For PP_RS02385 A1046G Diagram of the construction of the recombinant expression vector plasmid.

[0042] Figure 6 For PP_RS02880 T517A and PP_RS18130 T65A / A67G Electrophoretogram of recombinant expression vector. Among them, lanes 1 - 5: PP_RS02880T 517A Electrophoretic bands of recombinant expression vector; lanes 6 - 10: PP_RS18130 T65A / A67G Electrophoretic bands of recombinant expression vector.

[0043] Figure 7 For PP_RS19785 C20A Electrophoretogram of recombinant expression vector.

[0044] Figure 8 For PP_RS20740 G1012T and PP_RS02385 A1046G Electrophoretogram of recombinant expression vector. Among them, lanes 1 - 3: PP_RS20740 G1012T Electrophoretic bands of recombinant expression vector; lanes 4 - 5: PP_RS02385 A1046G Electrophoretic bands of recombinant expression vector.

[0045] Figure 9 Growth history diagram of recombinant expression transformant tolerant to furfural in inorganic salt medium containing furfural.

[0046] Figure 10 Conversion distribution diagram of furfural by recombinant expression transformant tolerant to furfural in inorganic salt medium containing furfural.

[0047] Figure 11 Conversion history diagram of furan aldehyde by recombinant expression transformant tolerant to furfural in real hydrolysate. Detailed implementation mode

[0048] The present invention will be further described in detail below with reference to specific specification drawings and examples. Detailed implementation modes and specific operation procedures are given. The examples will help to understand the present invention, and the described content is only used to illustrate the present invention. However, the protection scope of the present invention is not limited to the following examples. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed.

[0049] In the following examples, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0050] Example 1: Mining of mutant genes

[0051] 1. Adaptive domestication of the parental strain: The parental strain is an excellent detoxifying strain, Pseudomonas putida, which has knocked out the sugar metabolism pathway, does not utilize fermentable sugars but can use inhibitory organic acids and phenolic inhibitors derived from lignocellulosic biomass as carbon sources (the detailed construction process of this strain has been published in Patent CN202110660995.4). To further improve the stress resistance of this strain to furfural, the parental strain was used for adaptive domestication, and a strain with high furfural tolerance, named P. putida Z, was obtained. The tolerance and conversion ability of this strain to furfural are significantly higher than those of the parental strain.

[0052] Steps for the adaptive domestication of the parental strain:

[0053] The parental strain was activated in an inorganic salt medium containing 5 g / L sodium acetate (30 °C, 200 rpm), and then the activated cells were transferred to a 5 g / L sodium acetate medium containing a low concentration of furfural (1 mM) for cultivation. When the cells reached the logarithmic growth phase, they were transferred at an OD 600 of 0.1 to a fresh medium with the same furfural concentration, and this was repeated multiple times in the same way until the cells were stable in their tolerance to the same furfural concentration. Then, the cells were transferred to a medium with a higher furfural concentration in the same way, and the operation was repeated in the medium with continuously increasing furfural concentrations until a domesticated strain with the ability to tolerate high concentrations of furfural was finally obtained. Among them, the domesticated furfural concentrations were: 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM.

[0054] The above medium formulation: 1 - 25 mM furfural, 5 g / L sodium acetate, 4.25 g / L Na2HPO4·2H2O, 1.5 g / L KH2PO4, 0.25 g / L NaCl, 0.5 g / L NH4Cl, and 2 mM MgSO4; trace element solution 2.5 mL / L.

[0055] Trace element solution: 300 mg / L H3BO3, 50 mg / L ZnCl2, 30 mg / L MnCl2·4H2O, 200 mg / L CoCl2, 10 mg / L CuCl2·2H2O, 20 mg / L NiCl2·6H2O, 30 mg / L Na2MoO4·2H2O.

[0056] 2. Screening of mutant genes

[0057] Analyze the response of P. putida Z to furfural inhibitors by genome re-sequencing. The genomic DNA of P. putida Z was extracted using the MiniBEST Bacteria Genomic DNA Extraction Kit from Takara. Whole-genome re-sequencing was completed on the Illunima Hiseq X10 PE150 sequencing platform. The specific method is as follows: After the genomic DNA of P. putida Z was randomly fragmented into short DNA fragments by enzymes, blunt-end repair was performed. Then, dA tails were ligated to both ends of the DNA fragments, and sequencing adapters were ligated. The DNA fragments with adapters were purified using AMPure XP magnetic beads, and fragments in the range of 300-400 bp were selected for PCR amplification. The constructed library was purified and library-tested, and then sequenced on the Illunima Hiseq X10 PE150.

[0058] The original image data obtained by sequencing was converted into sequence data, i.e., raw reads, through base calling. A total of 1139720400 bp of Clean date, 7574382 Total Reads, and 7324681 Clean Reads were obtained. This data volume meets the requirements and has good quality. The data was aligned to the reference genome (NC_002947.4) using the alignment software bwa, and the total alignment rate was over 99%. The UnifiedGenotyper module of the software GATK (3.4-46) was used to perform variant detection on multiple samples of the processed alignment file. ANNOVAR was used to perform functional annotation on the detected variants. Functional analysis was performed on the non-synonymous mutant structural genes of the obtained SNPs (Single nucleotide polymorphisms) occurring in exons, and 5 genes carrying mutation sites were screened out, including: PP_RS18130 T65A / A67G , PP_RS02880 T517A , PP_RS19785 C20A , PP_RS20740 G1012T , PP_RS02385 A1046G , which were used to investigate the tolerance and transformation of 5 mutant genes to furfural in the follow-up.

[0059] Example 2: Construction of recombinant expression vector

[0060] 1. Amplification of target genes: For the 5 mutant genes obtained above, corresponding primers were designed and synthesized according to their nucleotide sequences (SEQ ID NO.6-SEQ ID NO.10). Using the genome of the domesticated strain as a template, Max DNA Polymerase was used to amplify 5 mutant genes respectively.

[0061] Table 1-1 Primers for Amplifying 5 Mutant Genes

[0062]

[0063]

[0064] PCR reaction system:

[0065]

[0066] PCR amplification conditions: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s; annealing at 69.6°C for 15 s; extension at 72°C for 30 s, for 30 cycles; final extension at 72°C for 5 min.

[0067] 2. Construction of Recombinant Expression Vectors

[0068] After purifying the amplification products of the above 5 mutant genes, the 5 nucleic acid purification products were respectively reverse amplified with the expression vector pBBR1MCS2 (Addgene) using the pEASY-Basic Seamless Cloning and Assembly Kit (TransGen Biotech), and the purified products were ligated by gel recovery to obtain the recombinant expression vectors. The plasmid construction diagram of the recombinant expression vectors is as shown in Figures 1 to 5 shown, and the electrophoresis diagram of the recombinant expression vectors is as shown in Figures 6 to 8 shown.

[0069] Table 1-2 Reverse Amplification Primers

[0070]

[0071] Example 3: Preparation of Recombinant Expression Transformants Tolerant to Furfural (the Strains of the Present Invention)

[0072] The 5 recombinant expression vectors obtained in Example 2 were transformed into the competent cells of the host strain Pseudomonas putida KT2440 (strain number ATCC47054) by electrotransformation (electroporation conditions: 2 mm electroporation cuvette, 2400 V, 25 μF, 200 Ω). Method for preparing competent cells: (1) Inoculate the bacterial liquid in the glycerol tube into a shake tube containing 5 mL of LB liquid medium according to an inoculation amount of 1%, and culture at 30°C and 200 rpm for 10 - 12 h until the mid-logarithmic phase; (2) Transfer the activated bacteria to two 250 mL conical flasks containing 50 mL of LB liquid medium according to an inoculation amount of 1%, and culture on a shaker at 30°C and 200 rpm until the cell OD 600When it reaches 0.6 - 0.8 (0.75 is optimal), immediately perform an ice bath for 10 min; (3) Transfer the cells after the ice bath to two pre-cooled 50 mL centrifuge tubes, balance them, and centrifuge at 4°C, 6000 rpm for 10 min. Discard the supernatant and collect the cells; (4) Wash the cells twice with 5 mL of pre-cooled electroporation buffer; (5) Resuspend every 50 mL of cells with 300 μL of electroporation buffer, and aliquot the resuspended competent cells into 1.5 mL centrifuge tubes (100 μL - 150 μL per tube). Add 10 μL of the recombinant expression transformant to every 100 μL of competent cells. Immediately add 1 mL of culture medium after electroporation and resuscitate at 30°C, 200 rpm for 1 h; Take 100 μL of the resuscitated cell suspension and spread it on an LB agar solid plate medium containing 50 μg / mL kanamycin for overnight culture. Pick a single colony on the plate and transfer it to an LB liquid medium containing 50 μg / mL kanamycin, and culture at 30°C, 200 rpm. Then extract the plasmid, perform PCR verification on the recombinant plasmid, and perform DNA sequencing on the verified correct recombinant plasmid. Preserve the correctly sequenced recombinant expression transformant with glycerol for subsequent experiments. The control plasmid pBBR1MCS2 was electrotransformed into the strain Pseudomonas putida KT2440 as the control strain.

[0073] Example 4: Examine the conversion of furfural by the recombinant expression transformant tolerant to furfural in an inorganic salt medium containing furfural

[0074] The Pseudomonas putida tolerant to furfural prepared in Example 3 was activated overnight in LB medium and then inoculated at an initial inoculum OD 600 0.2 into an M9 inorganic salt medium (pH 7.0) containing 10 g / L glucose and 20 mM furfural, and cultured at 30°C, 200 rpm for 48 h. The experimental results are as shown in the appendix Figure 9 and 10 It can be seen that the 5 strains containing the mutant genes described in the present invention are superior to the control strain in terms of growth and furfural conversion performance. Among them, the overexpressed mutant gene PP_RS19785 C20A of the KTPP_RS19785 C20A strain exhibits the best growth and furfural conversion ability, and the strain can convert 100% of furfural within 12 h. The strain KTPP_RS20740 G1012T can also achieve 100% furfural conversion within 24 h, while the control strain only achieved 49% furfural conversion within 24 h. Compared with the control strain, the strains KTPP_RS18130 T65A / A67G , KTPP_RS02880 T517A , and KTPP_RS02385 A1046G overexpressing the other three mutant genesThe conversion rates of furfural were increased by 28%, 29% and 36% respectively. The experimental results show that the recombinant expression transformant described in the present invention can significantly improve the stress resistance of the strain to furfural.

[0075] The above-mentioned inorganic salt medium formula containing furfural is as follows: 10 g / L glucose, 20 mM furfural, 4.25 g / L Na2HPO4·2H2O, 1.5 g / L KH2PO4, 0.25 g / L NaCl, 0.5 g / L NH4Cl and 2 mM MgSO4; trace element solution 2.5 mL / L.

[0076] Trace element solution: 300 mg / L H3BO3, 50 mg / L ZnCl2, 30 mg / L MnCl2·4H2O, 200 mg / L CoCl2, 10 mg / L CuCl2·2H2O, 20 mg / L NiCl2·6H2O, 30 mg / L Na2MoO4·2H2O.

[0077] Example 5: Conversion of furfural by the recombinant expression transformant tolerant to furfural in real hydrolysate

[0078] The Pseudomonas putida tolerant to furfural prepared in Example 3 was activated overnight in LB medium and then inoculated into the corn stover pretreatment liquor at an initial inoculum OD 600 0.2, and cultured at 30 °C and 200 rpm for 48 h. Samples were taken every 12 h in the middle. The samples were centrifuged at 10000 rpm for 5 min to obtain the supernatant. The supernatant was diluted by an appropriate multiple, filtered through a 0.22 μm filter membrane, and the content of furfural in the sample was analyzed by liquid chromatography to investigate the furfural conversion ability of the strain in the complex real hydrolysate. As shown in the appendix Figure 11 As shown, compared with the control strain containing the empty plasmid pBBR1MCS2, the strains carrying the mutant site genes described in the present invention all showed better furfural conversion ability in the complex hydrolysate. Among them, the overexpressed mutant gene PP_RS19785 C20A of the KTPP_RS19785 C20A strain showed the best furfural conversion ability, and could convert 68% of furfural in 12 h and complete 96% of furfural conversion in 24 h. Followed by the KTPP_RS20740 G1012T strain, which could convert 42.91% of furfural in 12 h and complete 96.4% of furfural conversion in 24 h. The strains KTPP_RS20740 G1012T , KTPP_RS18130 T65A / A67G , KTPP_RS02385 A1046G and KTPP_RS02880 T517A, they can also achieve 92.4%, 50.3% and 54% furfural conversion respectively within 24 h, while the control strain only converted 49% of furfural after 48 h of cultivation.

[0079] The above-mentioned medium formula for corn stover pretreatment liquid is: 10 g / L glucose, 16.94 g / L xylose, 2.29 g / L arabinose, 0.99 g / L formic acid, 3.04 g / L acetic acid, 0.68 g / L levulinic acid, 1.22 g / L furfural, 0.26 g / L HMF and other inhibitors, 17.16 g / L Na2HPO4·12H2O, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2.

Claims

1. A furfural-tolerant recombinant expression vector, characterized in that, The furfural-tolerant recombinant expression vector is a vector into which the following gene carrying a mutation site is inserted: PP_RS19785 C20A gene; Among them, the nucleotide sequence of the PP_RS19785 C20A gene is shown in SEQ ID NO.

8.

2. The furfural-tolerant recombinant expression vector according to claim 1, wherein The gene carrying the mutation site is obtained by adaptive domestication in a medium containing furfural and combined with high-throughput sequencing analysis.

3. The furfural-tolerant recombinant expression vector according to claim 1, characterized in that, The vector is any one of plasmid, cosmid, phage or viral vector.

4. A recombinant expression transformant, characterized in that, It is constructed by transforming the recombinant expression vector according to any one of claims 1 to 3 into a host cell; Among them, the host cell is Pseudomonas putida Pseudomonas putida .

5. The recombinant expression transformant according to claim 4, characterized in that, The transformation is electrotransformation.

6. Use of the recombinant expression transformant according to claim 4 in tolerating furfural.

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