The invention discloses 3apos for enhancing upstream gene expression of zymomonas mobilis. UTR element and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a 3 'UTR element for enhancing upstream gene expression of zymomonas mobilis and application of the 3' UTR element. The 3 'UTR element provided by the invention can stably improve the expression level of upstream genes. When the 3 'UTR element is applied to a target gene for coding beta-glucosidase, the beta-glucosidase activity of zymomonas mobilis recombinant engineering bacteria can be remarkably improved, so that the requirement of the zymomonas mobilis recombinant engineering bacteria on efficient utilization of cellobiose substrates is met, and the yield of ethanol is finally improved. The invention provides a new key tool for realizing fine regulation and control and metabolic flux optimization of multiple genes in a metabolic pathway, and further effectively improves the application potential of the Zymomonas mobilis as a microbial cell factory.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a 3'UTR element that enhances the expression of upstream genes in motile fermentation mononucleosis and its applications. Background Technology

[0002] In the fields of synthetic biology and metabolic engineering, *Fermentomonas motilityis* has become an ideal microbial chassis cell due to its efficient ethanol production capacity, high ethanol tolerance, and wide environmental adaptability. It anaerobicly ferments sugars via the ED (Entner-Doudoroff) pathway, exhibiting significantly higher sugar utilization than strains using the EMP pathway. However, current metabolic engineering faces significant technical bottlenecks: gene manipulation systems suffer from low efficiency, high false-positive rates, difficulties in manipulating large plasmids, and limited cloning sites, hindering the reconstruction of complex metabolic pathways; metabolic regulation relies excessively on a few strong promoters, and sustained high expression can easily lead to metabolic imbalances, while existing inducible promoters cannot meet the demands for element diversity and dynamic regulatory precision; research on non-coding regulatory elements lags behind, especially the insufficient elucidation of the functional mechanisms of the 3'UTR (3' Untranslated Region), and the lack of standardized characterization platforms and compatible regulatory toolkits, resulting in insufficient precision in gene expression regulation and difficulty in achieving fine redirection of metabolic flux.

[0003] As a core mediator of posttranscriptional regulation, the 3'UTR holds significant potential for precise control of gene expression by regulating mRNA stability, translation efficiency, and subcellular localization. However, systematic screening, functional validation, and application strategies for 3'UTR elements in *Fermentomonas motilityis* remain lacking. Therefore, there is an urgent need to develop *Fermentomonas motilityis* 3'UTR elements to enrich the genetic manipulation toolkit, achieve precise regulation of gene expression, and further enhance its application potential as a highly efficient biomanufacturing chassis cell. Summary of the Invention

[0004] In view of this, the present invention proposes a 3'UTR element for enhancing the expression of upstream genes in motile fermentation monoclonal bacteria and its application.

[0005] The technical solution of this invention is implemented as follows:

[0006] In a first aspect, the present invention provides a 3'UTR element for enhancing the expression of upstream genes in *Mammotrophic motility-enhancing* bacteria, the nucleotide sequence of which is shown in SEQ ID NO:4.

[0007] Secondly, the present invention provides an application of the 3'UTR element, wherein the 3'UTR element is inserted between the coding sequence of the target gene and the transcription terminator to enhance the expression level of the target gene.

[0008] Furthermore, the 3'UTR element is used to construct a polycistronic nucleic acid molecule, which comprises, from the 5' end to the 3' end, the coding sequence of the target gene, the 3'UTR element, and the coding sequence of at least one non-target gene; wherein the 3'UTR element specifically enhances the expression level of the target gene.

[0009] Thirdly, the present invention provides an expression carrier comprising the aforementioned 3'UTR element.

[0010] Fourthly, the present invention provides a recombinant nucleic acid construct comprising the aforementioned 3'UTR element, wherein the 3'UTR element is operatively linked downstream of the coding sequence of a target gene. The operative linking is performed using enzyme digestion-ligation or seamless cloning techniques.

[0011] Fifthly, the present invention provides a gene expression unit comprising, from the 5' end to the 3' end, a promoter, a coding sequence of a target gene, the 3'UTR element, and a terminator.

[0012] In a sixth aspect, the present invention provides a recombinant cell comprising the aforementioned 3'UTR element.

[0013] In a seventh aspect, the present invention provides a recombinant bacterium comprising the aforementioned 3'UTR element.

[0014] Eighthly, the present invention provides a method for improving the β-glucosidase activity and / or ethanol yield of recombinant engineered *Saccharomyces cerevisiae*, comprising operably linking the 3' end of the coding sequence of the β-glucosidase gene to the 3'UTR element and introducing it into the engineered bacteria. Further, the operably linked linking is performed using enzyme digestion-ligation or seamless cloning techniques.

[0015] The beneficial effects of the present invention include at least the following:

[0016] The 3'UTR element provided by this invention can stably enhance the expression levels of its upstream genes. Its application is independent of specific promoters, providing a new key tool for fine-tuning the regulation of multiple genes in metabolic pathways and optimizing metabolic flux. Specifically, this 3'UTR element significantly enhances the β-glucosidase activity of recombinant engineered *Saccharomyces cerevisiae*, thereby meeting the demand for efficient utilization of cellobiose substrates. Simultaneously, the increased synthesis of β-glucosidase leads to its extracellular secretion via signal peptide mediation, effectively increasing the enzyme concentration in the fermentation broth. This enables the engineered bacteria to rapidly degrade cellobiose in the system, avoiding metabolic inhibition caused by substrate accumulation, ultimately increasing ethanol production.

[0017] Furthermore, this invention establishes an efficient screening strategy by integrating functional genomics, transcriptomics, and proteomics data. This strategy can rapidly and accurately identify UTR sequences with expression-enhancing functions across the entire genome of *Fermentomonas motilityis*. Simultaneously, the dual-luciferase reporter gene system is used to precisely quantify the functions of the screened elements. This method effectively eliminates interference from complex factors such as metabolic pathways, directly and objectively assessing the regulatory capacity of UTR sequences. This ensures that the functions of the elements possess good predictability and reproducibility, providing a reliable data foundation for rational design in synthetic biology. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The diagram shows the structure of a monocistronic vector and the fluorescence intensity of the reporter gene GFP in the recombinant strain. A represents pEZ-GFP; B represents pEZ-GFP-UTR, where EGFP indicates the GFP reporter gene; C represents the fluorescence intensity of the reporter gene GFP in the recombinant strain, with the x-axis representing strains Z. mobilis-GFP-1 to Z. mobilis-GFP-8, and C representing the control strain Z. mobilis-GFP.

[0020] Figure 2 The diagram shows the structure of a polycistronic vector and the fluorescence intensity of the reporter genes GFP and mCherry in the recombinant strain. A represents pEZ-GFP-mCherry; B represents pEZ-GFP-UTR-mCherry; C represents the fluorescence intensity of the reporter genes GFP and mCherry in the recombinant strain. The horizontal axis 1-8 represent strains Z. mobilis-GFP-mCherry-1 to Z. mobilis-GFP-mCherry-8, respectively, and C represents the control strain Z. mobilis-GFP-mCherry.

[0021] Figure 3The construction and performance characterization of engineered cellobiose metabolism bacteria are shown in Figures A, B, and C. Figure A is a schematic diagram of the recombinant vector pEZ-Tfu0937; Figure B is a schematic diagram of the recombinant vector pEZ-Tfu0937-UTR with the introduction of the 3'UTR element; Figure C shows the results of ethanol production and extracellular β-glucosidase activity of the recombinant strain. Asterisks in the figure indicate statistically significant differences between groups (*p<0.05, **p<0.01, ***p<0.001, ****p<0.001). Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0023] Example 1: UTR Sequence Screening

[0024] The NCBI database contains complete genomic information for various *M. motile fermentomonas* strains, including ZM4 (ATCC 31821), ZM401 (ATCC 31822), ATCC 31823, NCIMB 11163, ATCC 10988, ATCC 29191, ATCC 29192, CP4 (NRRL B-14023), ATCC 29192, NRRL B-12526, and NRRL B-1960 (see https: / / www.ncbi.nlm.nih.gov / datasets / genome / ?taxon=542,120045). Based on public database resources and a self-built dataset, multidimensional omics information from 22 microarrays, 32 RNA-seq datasets, and 4 proteomics experiments was integrated to construct a UTR sequence screening workflow. The method is as follows:

[0025] 1. Multi-omics data preprocessing and integration

[0026] To address the differences in data types across various omics datasets, a specific standardization strategy was adopted. For RNA-seq data, the median ratio method was used to eliminate technical bias between samples. Background correction and probe summarization were performed on microarray data. For proteomics data, quantile normalization was used to unify expression level distribution, ensuring data comparability. Using gene IDs as the link, UTR sequences (extracted from genomic data, with a length focused at 100-300 bp, matching mRNA transcript structure) were correlated with the transcriptome expression levels (microarray / RNA-seq quantification values) and proteome expression levels of corresponding genes, constructing a three-dimensional dataset of DNA sequences, mRNA, and proteins.

[0027] 2. Feature Extraction and Fusion

[0028] Nucleotide composition (GC content, pyrimidine / purine ratio), conserved motifs (e.g., Kozak sequence CCACC), secondary structure (predicted via RNAfold), and minimum free energy (MFE, reflecting structural stability) of UTRs were extracted. Using gene transcription abundance / protein expression level under normal culture conditions as a benchmark, the regulatory efficacy of UTRs on downstream genes was quantified, and the gene expression deviation corresponding to candidate UTRs was calculated as an initial characterization of regulatory activity. Correlation analysis was used to screen UTRs corresponding to genes with a significant positive correlation between transcription abundance and protein expression level (r>0.5, p<0.01), excluding post-transcriptional regulatory interference and focusing on UTR sequences that directly affect translation.

[0029] 3. Multi-level sequence filtering

[0030] UTRs corresponding to the top 20% of genes in mRNA transcription abundance (RNA-seq, MicroArray) and the top 20% of genes in protein expression at the translational level were screened. Further screening was conducted by obtaining the intersection of these UTRs to ensure that the genes associated with the UTRs possessed high transcriptional-translational characteristics. Conserved sequences that consistently appeared under different metabolic states were identified, ultimately determining eight candidate 3' UTR sequences. 100-300 bp UTR sequences were extracted using bioinformatics analysis to establish a UTR sequence database for future research.

[0031] 4. Screening Results

[0032] The eight 3'UTR sequences selected in this application are as follows:

[0033] Example 2

[0034] In this embodiment, *Escherichia coli* DH5α was used as the cloning strain. ZM4-Δ0028 (see patent CN118652307A), which has higher transformation efficiency due to the knockout of the ZMO0028 gene expressing the type IV restriction modification system, was used as the host strain. The shuttle plasmid vector pEZ15Asp (hereinafter also abbreviated as pEZ15A, which contains the spectinomycin resistance gene and the origin of replication of *Zymomonas mobilis*, constructed according to the literature "Yang S, Mohagheghi A, Franden MA, et al. Metabolic engineering of *Zymomonas mobilis* for 2,3-butanediol production from lignocellulosic biomass sugars[J]. Biotechnol Biofuels, 2016, 9(1):189.") was used as the cloning and host expression vector. Green fluorescent protein (GFP) and red fluorescent protein (mCherry) were used as reporter genes. After codon optimization, the gene sequences were synthesized and integrated into the pEZ15A vector, then introduced into host cells via electroporation. The specific method is as follows:

[0035] 1. Constructing the vector pEZ-GFP

[0036] Using the synthesized GFP gene as a template, PCR amplification was performed to obtain the GFP gene fragment. The reaction system and procedure are shown in Tables 2 and 3.

[0037] The GFP gene and its amplification primers are as follows:

[0038]

[0039] pEZ15A vector backbone amplification, the reaction system is shown in Table 2, the reaction program is as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 2 min (30 cycles), 72℃ final extension for 5 min, to obtain the vector backbone.

[0040] Using the ClonExpress II One Step Cloning Kit (Vazyme), the GFP gene fragment and the vector backbone were mixed at a molar ratio of 3:1 and reacted at 50°C for 30 min. The mixture was then transformed into E. coli DH5α, plated on LB medium containing 100 μg / mL spectinomycin, and cultured overnight at 37°C. Single clones were picked, plasmids were extracted, and after the sequence was verified by Sanger sequencing, it was named pEZ-GFP.

[0041] 2. Construction of the monocistronic recombination vector pEZ-GFP-UTR

[0042] 3' UTR sequences (3' UTR sequences 1-8 in Table 1) were amplified by PCR, with the reaction system and procedure the same as in Tables 2 and 3. Using pEZ-GFP as a template, the pEZ-GFP vector was amplified in reverse using reverse amplification primer pairs. After the PCR products were recovered by agarose gel electrophoresis, they were seamlessly cloned and ligated (method as above). The UTR fragments were mixed with the vector backbone at a molar ratio of 3:1, reacted at 50℃ for 30 min, transformed into E. coli DH5α, plated on LB medium containing 100 μg / mL spectinomycin, and cultured overnight at 37℃. Single clones were picked, plasmids were extracted, and after Sanger sequencing verification, they were named pEZ-GFP-1, pEZ-GFP-2, pEZ-GFP-3, pEZ-GFP-4, pEZ-GFP-5, pEZ-GFP-6, pEZ-GFP-7, and pEZ-GFP-8. The primers are as follows:

[0043]

[0044] 3. Construction of the polycistronic recombination vector pEZ-GFP-mCherry

[0045] Using the synthesized mCherry gene as a template, PCR amplification was performed. Using pEZ-GFP as a template, the pEZ-GFP vector was amplified in reverse (reaction conditions and system conditions were the same as above). After the PCR product was recovered by agarose gel electrophoresis, it was seamlessly cloned and ligated (method as above). The mCherry fragment was mixed with the pEZ-GFP vector backbone at a molar ratio of 3:1, reacted at 50℃ for 30 min, transformed into E. coli DH5α, plated on LB medium containing 100 μg / mL spectinomycin, and cultured overnight at 37℃. Single clones were picked, plasmids were extracted, and after Sanger sequencing verification, the plasmid was named pEZ-GFP-mCherry. The primers for the pEZ-GFP reverse amplification are as follows;

[0046]

[0047] 4. Constructing the polycistronic recombination vector pEZ-GFP-UTR-mCherry

[0048] The corresponding 3' UTR sequences were amplified by PCR using the primers in Table 4. Using pEZ-GFP-mCherry as a template, pEZ-GFP-mCherry was amplified in reverse. After the PCR products were recovered by agarose gel electrophoresis, they were seamlessly cloned and ligated. The UTR fragments were mixed with the vector backbone at a molar ratio of 3:1 and incubated at 50℃ for 30 min. The mixture was then transformed into E. coli DH5α, plated on LB medium containing 100 μg / mL spectinomycin, and cultured overnight at 37℃. Single clones were picked, plasmids were extracted, and after Sanger sequencing verification, they were named pEZ-GFP-mCherry-1, pEZ-GFP-mCherry-2, pEZ-GFP-mCherry-3, pEZ-GFP-mCherry-4, pEZ-GFP-mCherry-5, pEZ-GFP-mCherry-6, pEZ-GFP-mCherry-7, and pEZ-GFP-mCherry-8. The reverse amplification primers for pEZ-GFP-mCherry are as follows;

[0049]

[0050] 5. Transformation and positive clone screening

[0051] (1) Preparation of Z. mobilis ZM4-Δ0028 electrotransformation competent cells: Centrifuge the logarithmic phase bacterial culture at 4℃ and 5000×g for 5 min, wash 3 times with pre-cooled 10% glycerol, and resuspend in 1 / 50 of the original volume of 10% glycerol.

[0052] (2) Mix 100 μL of competent cells with 1 μg of recombinant plasmid, add to a 0.2 cm electroporation cuvette, set the parameters to 2.5 kV, 25 μF, 200 Ω for electroporation, immediately add 1 mL of RM medium, incubate at 28℃ for 1 h, spread on RM medium (containing spectinomycin (100 μg / mL) and glucose (50 g / L), incubate upside down at 28℃ for 48 h, pick single clones for colony PCR verification, the verification primers are as follows:

[0053]

[0054] (3) The validated recombinant strains and plasmids are as follows:

[0055]

[0056] 6. Characterization of recombinant strains

[0057] (1) Culture of strains

[0058] The strains were cultured in rich medium (RMG5, 10 g / L yeast extract, 50 g / L glucose, 2 g / L KH2PO4) at 28 °C. All strains were supplemented with spectinomycin 100 μg / mL as needed.

[0059] (2) Flow cytometry analysis of fluorescence intensity

[0060] First, wash the cells twice with phosphate-buffered saline (PBS), then resuspend them in PBS to a concentration of OD. 600 Approximately 0.1 OD 600 (approximately 10 per milliliter) 7 (10,000 cells) were analyzed, and then the constructed recombinant strain was subjected to fluorescence detection and analysis using a CytoFLEX FCM flow cytometer (Beckman Coulter, Inc., USA) with PBS as the sheath solution. EGFP fluorescence was excited at 488 nm and detected using the FITC channel, while mCherry fluorescence was excited at 488 nm and detected using the PC5.5 channel. To avoid the possibility of random events affecting the cell population distribution results, at least 20,000 events were analyzed for each sample, and three biological replicates were set for each sample. The measured data were processed using FlowJo software (FlowJo, LLC, USA).

[0061] (3) Results

[0062] In the monocistronic system, the fluorescence intensity of the recombinant strains is as follows:

[0063]

[0064] The results show that ( Figure 1 (See Tables C and 5). Statistical analysis showed that the sequences containing P1360 (sequence 2), P1779 (sequence 4), and P1351 (sequence 6) differed significantly from the control group. In the monocistronic system, the fluorescence intensity of the reporter genes upstream of the 3'UTR sequences P1360 (sequence 2) and P1779 (sequence 4) was more than 1.5 times higher than that of the single promoter, with the fluorescence intensity of P1779 reaching more than 2.3 times that of the control group.

[0065] In the polycistronic system, the fluorescence intensity of the recombinant strains is as follows:

[0066]

[0067] The results show that ( Figure 2(See Tables C and 6). In the polycistronic system, the fluorescence intensity of the upstream reporter gene (GFP) of P0177 (sequence 1), P1779 (sequence 4), P1351 (sequence 6), P1194 (sequence 7) and P0300 (sequence 8) was more than 1.5 times higher than that of the single promoter, and the fluorescence intensity of P1779 (sequence 4) was more than 2.2 times that of the control group.

[0068] Experimental results show that P1779 (sequence 4) can stably increase the expression of the upstream gene by more than 2-fold in both monocistronic (pEZ-GFP-UTR) and polycistronic (pEZ-GFP-UTR-mCherry) vectors. This indicates that its enhancement effect is independent of specific vector configurations and has strong versatility and robustness.

[0069] Example 3

[0070] 1. Construction of control strain ZM-Tfu0937

[0071] Based on the β-glucosidase protein sequence of *Thermobifida fusca* YX (GenBank: AAZ54975.1), the gene Bgl-Tfu0937 was artificially synthesized after codon optimization and integrated into the pEZ15A vector. The recombinant plasmid was introduced into the host bacterium *Z. mobilis* ZM4-Δ0028 via electroporation. The control strain ZM-Tfu0937 was obtained after spectinomycin resistance screening. The specific methods are as follows:

[0072] (1) The Bgl-Tfu0937 gene was amplified by PCR using primers Bgl-Tfu0937-F and Bgl-Tfu0937-R. The reaction system and procedure are shown in Table 2 and Table 3.

[0073] (2) The linearized vector pEZ15A was amplified by reverse PCR using primers pEZ15A-Rev-F and pEZ15A-Rev-R. The reaction system is shown in Table 2; the reaction program was: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 40 s (30 cycles), and 72℃ final extension for 5 min. The pEZ15A fragment was obtained.

[0074] (3) Using seamless cloning technology, the Bgl-Tfu0937 gene fragment with homologous arms of pEZ15A at both ends was inserted into pEZ15A: The gene fragment and the vector backbone were mixed at a molar ratio of 3:1 using the ClonExpress II One Step Cloning Kit (Vazyme) and reacted at 50℃ for 30 min; transformed into E. coli DH5α competent cells and plated on LB medium containing 100 μg / mL spectinomycin, cultured overnight at 37℃, and obtained the recombinant plasmid pEZ-Tfu0937 by colony PCR and sequencing verification.

[0075] (4) Plasmid pEZ-Tfu0937 was introduced into competent strain Z. mobilis ZM4-Δ0028 using electroporation; it was plated on RM medium containing spectinomycin (100 μg / mL) (containing 50 g / L glucose) and incubated upside down at 28°C for 48 h. Positive clones were screened. The presence of the Bgl-Tfu0937 gene in the recombinant strain was verified by colony PCR, and the recombinant strain was named ZM-Tfu0937. The relevant elements and primers are as follows:

[0076]

[0077] 2. Construction of the recombinant strain ZM-Tfu0937-UTR containing the 3'UTR sequence

[0078] Using plasmid pEZ-Tfu0937 as a template, P1779 (sequence 4) was fused to the 3' end of the Bgl-Tfu0937 gene. Reverse overlap PCR was performed using primers Tfu0937-F and Tfu0937-R to obtain the linearized product pEZ-Tfu0937. Homologous sequences to pEZ-Tfu0937 were introduced upstream and downstream of the UTR sequence, respectively, and PCR amplification was performed using primers UTR-F and UTR-R to obtain the linearized DNA product. The linearized product was then ligated and circularized using seamless cloning technology. The product was transformed into E. coli DH5α competent cells, and positive clones were screened on LB agar plates containing 100 μg / mL spectinomycin. Colony PCR and sequencing confirmed the successful construction of the recombinant plasmid pEZ-Tfu0937-UTR (structural diagram shown in Figure 1). Figure 3(B) The recombinant plasmid was introduced into competent Z. mobilis ZM4-Δ0028 using electroporation. The plasmid was plated on RM medium (containing spectinomycin (100 μg / mL) and incubated upside down at 28°C for 48 h. Positive clones were screened. Colony PCR confirmed the presence of the Bgl-Tfu0937 gene in the recombinant strain, and the resulting recombinant strain was named ZM-Tfu0937-UTR. The relevant primers are as follows:

[0079]

[0080] 3. Cellobiose fermentation test

[0081] (1) Determination of ethanol content in fermentation broth supernatant: The seed culture of the test strain (OD) was prepared by adding the supernatant to the fermentation broth. 600nm Approximately 0.8 g / L) of the culture medium was inoculated at a volume ratio of 10% into RM medium (20 g / L cellobiose, 10 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, final concentration 100 μg / mL spectinomycin, and water) using 20 g / L cellobiose as the sole carbon source. After static fermentation at 28°C for 48 hours, samples were collected. The samples were centrifuged at 12000 rpm for 1 min, and the supernatant was aspirated using a 1 mL syringe, filtered through a 0.22 μm aqueous filter, and added to a sample vial. High-performance liquid chromatography (HPLC) was used to analyze the ethanol yield in the fermentation broth. The column temperature was set at 60°C, the mobile phase was 5 mM H₂SO₄, and the flow rate was 0.5 mL / min. The ethanol content was quantified using a standard curve (n=3).

[0082] (2) β-glucosidase activity assay: 100 μL of fermentation broth supernatant was added to 0.4 mL of acetate buffer (pH=6.3) containing 10 mM CaCl2, followed by 100 μL of 5 mM p-nitrophenyl-β-D-glucopyranoside (PNPG). After incubation at 37°C for 10 min, 0.2 mL of 2 M Na2CO3 solution was immediately added, and the reaction was terminated by thorough mixing. 200 μL of supernatant was used to measure the absorbance at 405 nm. Enzyme activity was calculated using a standard curve (n=3). The amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol per minute was defined as 1 U.

[0083] HPLC quantitative results showed that ( Figure 3The ethanol yield of the ZM-Tfu0937 fermentation system was 0.76 g / L, while the recombinant strain ZM-Tfu0937-UTR produced 1.49 g / L under the same conditions, representing a 96.05% increase compared to the control. Statistical analysis showed a significant increase in ethanol yield (p<0.05). Enzyme activity assays showed that the β-glucosidase activity in the fermentation broth of ZM-Tfu0937 was 54.13 U / mL, while the activity of ZM-Tfu0937-UTR was 85.66 U / mL, representing a 58.25% increase compared to the control. Statistical analysis showed a significant increase in enzyme activity (p<0.001).

[0084] In summary, the efficient cellobiose conversion system constructed based on the 3'UTR sequence (P1779) in this application significantly enhances extracellular β-glucosidase activity, accelerates the hydrolysis of cellobiose into glucose, and promotes a significant increase in ethanol production.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A 3'UTR element for enhancing the expression of upstream genes in Zymomonas mobilis, characterized in that, The nucleotide sequence of the 3'UTR element is shown in SEQ ID NO:

4.

2. The application of the 3'UTR element according to claim 1, characterized in that, The application is as follows: the 3'UTR element is inserted between the coding sequence of the target gene and the transcription terminator to enhance the expression level of the target gene.

3. The application according to claim 2, characterized in that, This is used to construct a polycistronic nucleic acid molecule, which comprises, from the 5' end to the 3' end, the coding sequence of a target gene, the 3'UTR element, and the coding sequence of at least one non-target gene; wherein the 3'UTR element specifically enhances the expression level of the target gene.

4. An expression carrier, characterized in that, It includes the 3'UTR element as described in claim 1.

5. A recombinant nucleic acid construct, characterized in that, It includes the 3'UTR element as described in claim 1, wherein the 3'UTR element is operatively linked downstream of the coding sequence of the target gene.

6. A gene expression unit, characterized in that, From the 5' end to the 3' end, it sequentially includes: a promoter, the coding sequence of the target gene, the 3'UTR element as described in claim 1, and a terminator.

7. A recombinant cell, characterized in that, It includes the 3'UTR element as described in claim 1.

8. A recombinant bacterium, characterized in that, It includes the 3'UTR element as described in claim 1.

9. A method for improving the β-glucosidase activity and / or ethanol yield of recombinant engineered *Saccharomyces cerevisiae*, characterized in that, The 3' end of the coding sequence of the β-glucosidase gene is operatively linked to the 3'UTR element of claim 1 and introduced into the engineered bacteria.

10. The method of claim 9, characterized in that, The operative ligation is performed using enzyme digestion-ligation or seamless cloning techniques.

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