Kluyveromyces marxianus promoter-terminator combinations and uses thereof

Abstract

This invention discloses a promoter-terminator combination for *Kluyveromyces martensii*, wherein the promoter... P STF2 The nucleotides are as shown in SEQ ID NO.1, and the terminator is... T STF2 The nucleotide sequence is shown in SEQ ID NO.2. The application of the *Kluyveromyces martensii* promoter-terminator combination in driving exogenous protein expression. This invention uses transcriptome sequencing technology to screen highly expressed genes from *Kluyveromyces martensii* transcriptome sequencing data using FPKM value as an indicator, obtaining an endogenous, highly efficient promoter-terminator combination. P STF2 ‑T STF2 .through egfp The reporter gene was validated and found to have stronger transcriptional activity, making it an ideal combination of regulatory elements for the efficient expression of exogenous proteins in *Kluyveromyces martensii*. Its application to the exogenous expression of myrosinase and xylanase resulted in efficient expression of both enzymes, demonstrating the good versatility and application value of this regulatory element combination.

Description

Technical Field

[0001] This invention relates to a promoter-terminator combination of *Kluyveromyces martensii* and its applications, belonging to the fields of genetic engineering and synthetic biology. Background Technology

[0002] Max Kluyveromycin ( Kluyveromyces marxianus, K.marxianus Kluyveromyces martensii, belonging to the genus Kluyveromyces in the family Yeastae of the order Yeastales in the class Yeastae of the subphylum Ascomycota in fungal taxonomy, is a food-grade, non-traditional yeast widely found in dairy products (such as cheese and kefir), fruits, plant secretions, and certain soil environments. Its superior fermentation performance gives it great potential in the production of edible and medicinal proteins. First, in terms of metabolic characteristics, Kluyveromyces martensii is a Crabtree-negative yeast, meaning aerobic conditions are more conducive to respiration than alcoholic fermentation. Second, it exhibits excellent heat resistance; some strains have an optimal growth temperature of 40–45°C and can even survive for short periods at 50°C. At 40°C, its growth rate reaches 0.86–0.99 g / h, making it considered one of the fastest-growing eukaryotes. Furthermore, Kluyveromyces martensii possesses a broad spectrum of carbon source utilization capabilities, not only utilizing glucose and galactose but also efficiently hydrolyzing and utilizing renewable or waste carbon sources such as lactose, cellobiose, xylose, and inulin (e.g., whey and lignocellulose hydrolysates), demonstrating a significant advantage in reducing raw material costs.

[0003] As the core regulatory sequence for gene transcription, the promoter's strength directly determines the transcriptional level of the target gene, thus profoundly affecting protein expression levels. Therefore, discovering promoter-terminator combinations that are more conducive to achieving efficient expression of exogenous genes is of great significance for constructing efficient protein expression systems. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a Max Kluyveromyces promoter-terminator combination and its application in driving the efficient expression of exogenous proteins.

[0005] This invention is achieved through the following technical solution: A Kluyveromyces martensii promoter-terminator combination, promoter P STF2 The nucleotides are as shown in SEQ ID NO.1, and the terminator is... T STF2 The nucleotide sequence is shown in SEQ ID NO.2. It is derived from *Kluyveromyces martensii*. STF2 Upstream and downstream sequences of gene (KLMA_40045).

[0006] The application of the Max Kluyveromyces promoter-terminator combination in driving the expression of exogenous proteins, in constructing exogenous protein expression vectors, and in constructing recombinant engineered bacteria that express exogenous proteins.

[0007] Furthermore, the exogenous protein is selected from myrosinase or xylanase.

[0008] An expression vector comprising the above-described Max Kluyveromyces promoter-terminator combination.

[0009] Furthermore, the backbone of the expression vector is the pUC19 plasmid.

[0010] The expression vector is used in the construction of recombinant engineered bacteria that express exogenous proteins.

[0011] Furthermore, the exogenous protein is selected from myrosinase or xylanase.

[0012] A recombinant engineered bacterium whose genome contains the aforementioned Max Kluyveromyces promoter-terminator combination, as well as the gene for the foreign protein.

[0013] Furthermore, the exogenous protein is selected from myrosinase or xylanase.

[0014] Application of the recombinant engineered bacteria in the preparation of exogenous proteins.

[0015] The *Kluyveromyces martensii* promoter-terminator combination of this invention is obtained based on transcriptome sequencing screening. The screening process is as follows: Transcriptome sequencing of *Kluyveromyces martensii* was performed to analyze gene expression levels at different culture times. The FPKM value was used as the basis for assessing transcription levels to screen for highly expressed genes. The transcription start sites were predicted using bioinformatics methods, and the upstream promoter and downstream terminator sequences of the highly expressed genes were cloned from the *Kluyveromyces martensii* genome. Subsequently, using enhanced green fluorescent protein as a reporter gene, a promoter-terminator combination was constructed containing the promoter and terminator sequences. egfp The gene and terminator expression cassette were integrated into a multicopy site in the *Kluyveromyces martensii* genome (an 18S rDNA sequence, specifically integrated via homologous recombination, using a genotypic resistance gene as a selection marker) to obtain recombinant strains. The transcriptional activity of the promoter was verified by measuring the relative fluorescence intensity of the recombinant strains at different culture times, and compared with known strong promoters. The results show that the promoter-terminator combination of this invention— P STF2 -T STF2 It is a powerful combination of expression elements. Finally, the promoters obtained from this screening are used to drive the expression of exogenous target genes (black myrosinase gene and xylanase gene), using endogenous... INU1ssSignal peptides were used to construct highly efficient expression strains, and the driving effect of the promoter was evaluated by measuring the enzyme activity in the fermentation broth.

[0016] This invention utilizes transcriptome sequencing technology to screen highly expressed genes from *Kluyveromyces martensii* transcriptome sequencing data using FPKM values ​​as an indicator, resulting in a novel, highly efficient endogenous promoter-terminator combination. P STF2 -T STF2 .through egfp Reporter gene validation, the gene screened in this invention P STF2 -T STF2 The relative fluorescence intensity of the combination at 12 h, 24 h, and 36 h was significantly higher than that of commonly reported strong promoter-terminator combinations. P ADH1 -T ADH1 (P < 0.05), demonstrating its stronger transcriptional activity, making it an ideal combination of regulatory elements for efficient expression of exogenous proteins in *Kluyveromyces martensii*. This invention utilizes this... P STF2 - T STF2 The combination was successfully applied to the exogenous expression of myrosinase and xylanase, with both enzymes achieving high-efficiency expression, demonstrating the good versatility and application value of this regulatory element combination. The promoter-terminator combination of this invention, derived from the same highly expressed gene, exhibits good compatibility and adaptability, strong transcriptional activity, and can significantly improve the expression level of exogenous proteins in *Kluyveromyces martensii*, providing an efficient expression tool for the application of this host in industrial enzyme preparations, pharmaceutical proteins, and other fields.

[0017] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0018] Figure 1 Comparison of FPKM values ​​of genes corresponding to different promoters at 12 h, 24 h and 36 h.

[0019] Figure 2 Map of Plasmid 1 recombinant plasmid.

[0020] Figure 3 Map of Plasmid 2 recombinant plasmid.

[0021] Figure 4 Comparison of relative fluorescence intensities of KM5 and KMA at different incubation times.

[0022] Figure 5 : Map of Plasmid 3 recombinant plasmid.

[0023] Figure 6 : P STF2 -T STF2 Results of transformant enzyme activity assay for driving black mustard enzyme expression.

[0024] Figure 7 : Map of Plasmid 4 recombinant plasmid.

[0025] Figure 8 : P STF2 -T STF2 Results of enzyme activity assay for transformants driving xylanase expression. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0027] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0028] Traditional promoter screening methods typically rely on the inference or cloning of known genes. However, with the rapid development of high-throughput sequencing technology, transcriptome sequencing has become a powerful tool for analyzing the global gene expression profile of organisms. By analyzing transcriptome data from strains under specific conditions, endogenous genes with the highest transcription levels can be directly identified, allowing the acquisition of their upstream promoter and downstream terminator sequences as candidate combinations of highly efficient regulatory elements. This omics-based screening strategy offers advantages such as high efficiency, accuracy, and specificity. Furthermore, since the promoter and terminator originate from the same highly expressed gene, they exhibit good compatibility and adaptability, which is more conducive to achieving efficient expression of exogenous genes. Therefore, this invention attempts to mine endogenous promoter-terminator combinations in *Kluyveromyces martensii* using transcriptome sequencing technology, aiming to obtain promoter-terminator combinations for highly efficient protein expression.

[0029] Example 1: Kluyveromyces martensii transcriptome sequencing and screening of highly expressed genes Transcriptome sequencing was performed on *Kluyveromyces martensii* KM0 (a publicly known strain available from the China General Microbiological Culture Collection Center, accession number CGMCC NO.21978). Samples were taken at 12 h, 24 h, and 36 h to extract total RNA, construct cDNA libraries, and sequenced using the Illumina platform. The FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) value, calculated using featureCounts, was used to assess gene transcription levels. Simultaneously, comparisons were made with previously reported strong promoters. ADH1 , FBA1 , GAP3 , PGK and ADH2 Compare them.

[0030] Sequencing results showed that the KLMA_40045 gene had the highest FPKM value at 12 h, reaching 20191.29. The FPKM values ​​of genes corresponding to commonly used strong promoters reported so far are as follows: ADH1 (2483.48) FBA1 (968.91) GAP3 (1209.92) PGK (1340.26) ADH2 (939.82). A comparison of FPKM values ​​of genes corresponding to different promoters at 12 h, 24 h, and 36 h is shown in the figure below. Figure 1 As shown, the FPKM values ​​of KLMA_40045 at 12 h, 24 h, and 36 h are significantly higher than those of previously reported strong promoters. ADH1 , FBA1 , GAP3 , PGK and ADH2 This indicates that the regulatory elements of this gene may be a combination of potent expression elements, and have value for further development.

[0031] Example 2 Cloning and Sequence Analysis of the STF2 Gene Promoter and Terminator Through whole genome sequence analysis of *Kluyveromyces martensii*, and by using the NCBI website... K. marxianusUsing DMKU3-1042 as a template, the KLMA_40045 gene sequence and its upstream and downstream sequences were searched. The BDGP website (http: / / www.fruitfly.org / ) was used to predict the promoter transcription start site of this gene. The results showed a possible transcription start site 458 bp upstream of the gene, with a confidence level of 0.99. Therefore, a 500 bp sequence upstream of the gene was selected as a candidate promoter, and a 500 bp sequence downstream was selected as a candidate terminator.

[0032] BLAST multiple sequence alignment of the gene sequence on the NCBI website revealed that it is an ATPase stabilizing factor gene. STF2 . STF2 The nucleotide sequence of the promoter is shown in SEQ ID NO.1. STF2 The nucleotide sequence of the terminator is shown in SEQ ID NO.2.

[0033] Example 3 egfp Construction of reporter gene validation expression vector For comparison STF2 Gene promoter-terminator combination ( P STF2 -T STF2 ) and reported strong promoter-terminator combinations P ADH1 -T ADH1 The transcriptional activity of the protein was verified by constructing green fluorescent protein expression vectors Plasmid 1 and Plasmid 2. egfp The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the plasmid information is shown in Table 1.

[0034] Table 1 Plasmid information and genotypes

[0035] The specific steps are as follows: Using Kluyveromyces martensii KM0 genomic DNA as a template, PCR amplification was performed separately. STF2 promoter ( P STF2 ), STF2 terminator ( T STF2 ), ADH1 promoter ( P ADH1 ) and ADH1 terminator ( T ADH1 Using pPIC9K plasmid as a template, the egfp gene was amplified by PCR. Using pUC19 plasmid as a backbone, the gene was amplified according to… rDNA1 -Promoter- egfp-Terminator- G418T - rDNA2 The recombinant plasmid was constructed in the order of "". rDNA1 and rDNA2 The ribosomal DNA sequence of *Kluyveromyces martensii* serves as a flanking arm for homologous recombination integration. G418T The gene for mycotoxin resistance was used as a selection marker. Two recombinant plasmids were successfully constructed: PUC19- P STF2 - egf p- T STF2 and PUC19- P ADH1 - egfp - T ADH1 The plasmid 1 recombinant plasmid map is shown below. Figure 2 As shown, the Plasmid 2 recombinant plasmid map is as follows: Figure 3 As shown.

[0036] The specific primers used for amplification are as follows: rDNA1 The nucleotide sequence of the upstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6.

[0037] P STF2 The nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8.

[0038] egfp The nucleotide sequence of the upstream primer is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.10.

[0039] T STF2 The nucleotide sequence of the upstream primer is shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.12.

[0040] G418T: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.14.

[0041] P ADH1 The nucleotide sequence of the upstream primer is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.16.

[0042] TADH1 The nucleotide sequence of the upstream primer is shown in SEQ ID NO.17, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.18.

[0043] The PCR reaction system consisted of 2 μL of each primer, 1 μL of template, 20 μL of sterile water, and 25 μL of 2×PhantaMax MasterMix (Dye Plus) enzyme, for a total volume of 50 μL.

[0044] The PCR reaction system was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, Tm-5℃ annealing for 15 s, 72℃ extension for 60 s, 30 cycles, followed by a 10 min extension at 72℃.

[0045] The amplified fragments were subjected to agarose gel electrophoresis for purification and recovery.

[0046] The target gene fragment and the PUC19 cloning vector were ligated using seamless cloning technology. The ligation product was transformed into E. coli DH5α competent cells, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated at 37°C for 15 hours. Single clones were picked and transferred to LB liquid medium containing 100 μg / mL ampicillin, and incubated at 37°C and 220 rpm for 12 hours. Sequencing was performed after positive verification.

[0047] Example 4 Construction and Validation of Recombinant Strains The recombinant plasmid successfully constructed in Example 3 was linearized by PCR, and the linearized fragment was recovered. Competent cells of *Kluyveromyces martensii* KM0 were prepared, and the linearized fragment was transformed into the competent cells by electroporation. The transformation product was plated on YPD plates containing 0.2 mg / mL genimycin resistance and incubated at 30°C for 2–3 days. Single colonies grown on the plates were picked, genomic DNA was extracted, and PCR verification of positive clones was performed. P STF2 -T STF2 Driven converter ( P STF2 - egfp-T STF2 It was named KM5. P ADH1 -T ADH1 Driven converter ( P ADH1 - egfp-T ADH1 It was named KMA.

[0048] Example 5: Determination of relative fluorescence intensity Kluyveromyces martensii strains KM0, KM5, and KMA were inoculated into 10 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for 18–24 h to serve as seed culture. Then, 1% of the inoculum was transferred to 50 mL of fresh YPD fermentation medium and cultured at 30℃ and 220 rpm. Samples were taken at 12 h, 24 h, and 36 h to determine the OD (octane rating) of the bacterial culture. 600nm The fluorescence intensity was measured using a fluorescence spectrophotometer (excitation wavelength 488 nm, emission wavelength 510 nm) on a suitable amount of bacterial culture. The copy number of RNA extracted from the bacterial culture was also determined. The relative fluorescence intensity was calculated using the following formula: Relative fluorescence intensity = .

[0049] The comparison of the relative fluorescence intensities of KM5 and KMA at different incubation times is shown in the figure below. Figure 4 As shown, the results indicated that the relative fluorescence intensity of strain KM5 was significantly higher than that of strain KMA at 12 h, 24 h, and 36 h (P < 0.05), indicating that... P STF2 -T STF2 The combined transcriptional activity was significantly stronger than P ADH1 -T ADH1 The combination is a high-performance, efficient combination of endogenous expression regulatory elements.

[0050] Example 6 Based on P STF2 -T STF2 Construction of engineered strains for expressing black mycosesase To further verify P STF2 -T STF2 The ability of combined-drive exogenous protein expression was investigated by constructing an engineered strain using the black myrosinase gene (Rmy) (the amino acid sequence of black myrosinase is shown in SEQ ID NO.4) as the target gene. The specific steps are as follows: With artificially synthesized black mustard enzyme ( Rmy Using the *Kluyveromyces martensii* KM0 genome as a template, the target gene fragment was amplified by PCR (the nucleotide sequence of the upstream primer is shown in SEQ ID NO.19, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.20). PCR amplification was performed using the *Kluyveromyces martensii* KM0 genome as a template. P STF2 -T STF2 and for integration rDNA1 and rDNA2Fragment. Using pUC19 plasmid as a backbone, according to " rDNA1-P STF2 -Rmy-T STF2 -bleoR-rDNA2 The recombinant plasmid PUC19-Rmy was constructed using the sequence of "". Here, bleoR is the bleomycin resistance gene, used as a selection marker. The Plasmid 3 recombinant plasmid map is shown below. Figure 5 As shown.

[0051] The constructed PUC19-Rmy recombinant plasmid was linearized by PCR and then electroporated into KM0Δ. ku70 (Knockout based on strain KM0) ku70 Genes (preserved in the laboratory) were collected in competent cells. The transformed bacterial culture was plated on YPD plates containing 0.2 mg / mL bleomycin resistance and incubated at 30°C for 2–3 days. Single colonies were picked for PCR verification to obtain positive transformants.

[0052] Positive transformants were inoculated into 10 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for 18–24 h. Then, they were transferred to 50 mL of YD fermentation medium at a 1% inoculation rate and cultured for another 120 h. The fermentation broth was collected, centrifuged at 4℃ and 6000 g for 20 min, and the supernatant was used to determine the activity of black mustard enzyme.

[0053] Take 100 μL of fermentation broth (using boil-inactivated enzyme as a control) and add it to 100 μL of glucosinolate solution (concentration 1 mg / mL). Incubate the mixture under standard conditions (40℃, pH 7.0) for 30 min, then terminate the reaction by boiling in a water bath for 10 min. Add 300 μL of DNS solution and mix well. Boil in distilled water for 5 min, cool to room temperature under running distilled water, centrifuge to collect the supernatant, and measure the absorbance at 540 nm using a microplate reader (200 μL). Simultaneously, determine the protein concentration using Coomassie Brilliant Blue G-250.

[0054] P STF2 -T STF2 The results of the transformant enzyme activity assay driving black mustard enzyme expression are as follows: Figure 6 As shown. The results show that, based on P STF2 -T STF2 The combined constructed black mustard enzyme expression strain exhibited good enzyme activity levels (up to 725.06 U / g), confirming... P STF2 -T STF2 The combination can effectively drive the expression of exogenous proteins.

[0055] Example 7 Based on P STF2 -T STF2 Construction of engineered strains for xylanase expression Following the method in Example 6, the target gene was replaced with the xylanase gene ( Xyn ), build based on P STF2 -T STF2 The combined xylanase expression engineered strains, plasmid map as follows: Figure 7 As shown in the figure. Positive transformants were fermented, and the fermentation supernatant was collected at 24 h, 48 h, 72 h and 96 h to determine the specific activity of xylanase.

[0056] Take 20 μL of fermentation broth (using boil-inactivated enzyme as a control) and add it to 180 μL of beech xylan solution (beech xylan concentration: 0.5%, w / v, g / ml; solvent: Tris-HCl, pH 7.0). Incubate the mixture at 55℃ for 10 min, then stop the reaction by boiling in a water bath for 10 min. Add 300 μL of DNS solution and mix well. Boil in distilled water for 5 min, cool to room temperature by running distilled water, centrifuge to collect the supernatant, and take 200 μL to measure the absorbance at 540 nm using a microplate reader. Simultaneously, determine the protein concentration using Coomassie Brilliant Blue G-250.

[0057] P STF2 -T STF2 The results of the transformant enzyme activity assay driving xylanase expression are as follows: Figure 8 As shown. The results show that, based on P STF2 -T STF2 The constructed xylanase expression strain exhibited good secretory expression performance during fermentation, reaching a high specific activity (132.11 U / mg) at 24 h and maintaining good activity throughout the fermentation process. This result further confirms... P STF2 -T STF2 The versatility and efficiency of the combination in driving the expression of different exogenous proteins.

[0058] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A promoter-terminator combination of *Kluyveromyces martensii*, characterized in that: promoter P STF2 The nucleotides are as shown in SEQ ID NO.1, and the terminator is... T STF2 The nucleotide sequence is shown in SEQ ID NO.

2.

2. The application of the Kluyveromyces martensii promoter-terminator combination as described in claim 1 in driving the expression of exogenous proteins, or in constructing exogenous protein expression vectors, or in constructing recombinant engineered bacteria that express exogenous proteins.

3. The application according to claim 2, characterized in that: The exogenous protein is selected from myrosinase or xylanase.

4. An expression vector, characterized in that: It includes the Max Kluyveromyces promoter-terminator combination as described in claim 1.

5. The expression vector according to claim 4, characterized in that: The backbone of the expression vector is the pUC19 plasmid.

6. The use of the expression vector according to claim 4 or 5 in constructing recombinant engineered bacteria expressing exogenous proteins.

7. The application according to claim 6, characterized in that: The exogenous protein is selected from myrosinase or xylanase.

8. A recombinant engineered bacterium, characterized in that: The genome contains the promoter-terminator combination of *Kluyveromyces martensii* as described in claim 1, as well as the gene for the foreign protein.

9. The recombinant engineered bacteria according to claim 8, characterized in that: The exogenous protein is selected from myrosinase or xylanase.

10. The use of the recombinant engineered bacteria according to claim 8 or 9 in the preparation of exogenous proteins.

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