Novel nuclear localization sequence mutant and method for improving biosynthesis efficiency thereof

Abstract

The invention discloses a novel nuclear localization sequence mutant and a method for improving biosynthesis efficiency thereof, and belongs to the field of bioengineering. According to the invention, a new nuclear localization sequence is screened, and the recombinant yeast with improved metabolite synthesis efficiency is constructed by using the nuclear localization sequence obtained by screening, so that a key technical support is provided for developing a high-yield, stable and economic yeast biosynthesis platform; and large-scale industrial biological manufacturing by using the recombinant yeast as a cell factory is facilitated.

Description

Technical Field

[0001] The invention relates to a novel nuclear localization sequence mutant and a method for improving biosynthesis efficiency thereof, and belongs to the field of bioengineering. Background Art

[0002] With the rapid development of synthetic biology, metabolic engineering to transform microbial cell factories to produce valuable compounds instead of traditional production methods is a more environmentally sustainable solution. However, designing functional metabolic pathways faces multiple challenges, including controlling enzyme expression intensity or changing the product preference of key enzymes, suboptimal physicochemical reaction environments, intermediate loss caused by endogenous competing pathways, and metabolite toxicity. Among them, Saccharomyces cerevisiae is a eukaryotic microorganism that grows vigorously at low pH, has simple nutritional requirements, is not susceptible to viral contamination, and has high tolerance to substrate and product toxicity. In addition, Saccharomyces cerevisiae has a complete whole genome sequence, detailed gene annotation resources, simple gene manipulation methods, easy post-translational modification, and the ability to express complex heterologous enzymes, making it a popular industrial microorganism.

[0003] Although most current strategies for optimizing metabolic pathways, including protein fusion and synthetic protein scaffolds, can improve the productivity of certain metabolic pathways, as metabolic pathways become increasingly complex, the number of enzymes that can be built into scaffolds without competition is limited by the available binding domains, and both protein fusion and scaffolds can negatively impact enzyme activity. Leveraging the functional characteristics of various organelles to address these metabolic flux-hindering issues is a new hotspot in metabolic engineering of Saccharomyces cerevisiae, compartmentalizing metabolic pathways within subcellular organelles. The unique physicochemical environment (e.g., pH and redox potential), enzymes, metabolites, and cofactors within each organelle provide favorable conditions for different metabolic pathways. By confining pathways to smaller subcellular compartments, the local concentrations of substrates and enzymes can be increased, thereby accelerating reaction rates and improving productivity. Confining intermediates within organelles can also inhibit their conversion to byproducts and reduce their toxic effects on cellular processes.

[0004] Current organelle engineering mainly focuses on organelles such as peroxisomes, mitochondria, endoplasmic reticulum and lipid droplets. Many studies have shown that pathway compartmentalization in yeast organelles can produce significant improvements compared to cytoplasmic pathways, but this improvement can only be targeted at certain specific pathways and is not very universal. In addition, the nuclear field has not yet been developed. Due to unfavorable physiological environments and insufficient supply of essential cofactors or precursors, enzyme activity may be reduced. Therefore, the selection of target organelles for metabolic pathways is very critical. The development of new organelles can provide new opportunities to increase the output of metabolic pathways. Summary of the Invention

[0005] The present invention provides a novel yeast nuclear localization sequence mutant and a method for improving biosynthesis efficiency by using the yeast nuclear localization sequence. The method first constructs a metabolic pathway for a target product, then adds a nuclear localization sequence to proteins related to the metabolic pathway, and finally produces the target product by fermentation.

[0006] The present invention provides the following technical solutions to achieve the above objectives:

[0007] The present invention provides a novel yeast nuclear localization sequence, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides a nucleic acid construct containing the yeast nuclear localization sequence shown in SEQ ID NO.1.

[0009] In one embodiment, the nucleic acid construct contains at least one nuclear localization sequence and at least one gene related to a biological metabolic pathway; the nuclear localization sequence is connected to the upstream or downstream of the gene related to the biological metabolic pathway to guide the gene expression product to be directional and localized to the host cell nucleus.

[0010] In one embodiment, the protein encoded by the biological metabolic pathway-related gene is involved in the synthesis pathway of the target metabolite in the microorganism.

[0011] In one embodiment, the biological metabolic pathway related genes include but are not limited to: 2-pyrone synthase gene Gh2-PS, acetyl-CoA carboxylase gene Acc1 mut , 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene ARO4, chorismate mutase gene ARO7, prephenate dehydrogenase gene TyrA, 4-hydroxyphenylacetate-3-monooxygenase gene PaHpaB, flavin reductase gene EcHpaC, 1,3,6,8-tetrahydroxynaphthalene synthase gene RppA.

[0012] In one embodiment, the host is a eukaryotic microorganism, including but not limited to Saccharomyces cerevisiae.

[0013] The present invention also provides recombinant Saccharomyces cerevisiae, which contains a gene whose localization is guided by the yeast nuclear localization sequence.

[0014] In one embodiment, the recombinant Saccharomyces cerevisiae contains the Acc1 gene shown in nucleotide sequence SEQ ID NO.3 and the Gh2-PS gene shown in nucleotide sequence SEQ ID NO.4, and the 3' ends of the Acc1 gene and Gh2-PS gene are respectively connected to the nuclear localization sequence SV4040 shown in SEQ ID NO.1.

[0015] In one embodiment, the recombinant Saccharomyces cerevisiae contains the Acc1 gene shown in nucleotide sequence SEQ ID NO.3 and the RppA gene shown in nucleotide sequence SEQ ID NO.10; the 3' ends of the Acc1 gene and RppA gene are respectively connected to the nuclear localization sequence SV4040 shown in SEQ ID NO.1.

[0016] In one embodiment, the recombinant Saccharomyces cerevisiae contains the ARO4 gene shown in the nucleotide sequence of SEQ ID NO.5, the ARO7 gene shown in the nucleotide sequence of SEQ ID NO.6, the TyrA gene shown in the nucleotide sequence of SEQ ID NO.7, the PaHpaB gene shown in the nucleotide sequence of SEQ ID NO.8, and the EcHpaC gene shown in the nucleotide sequence of SEQ ID NO.9; the 3' ends of the ARO4 gene, ARO7 gene, TyrA gene, PaHpaB gene, and EcHpaC gene are respectively connected to the nuclear localization sequence SV4040 shown in SEQ ID NO.1.

[0017] The present invention also provides a method for improving biosynthesis efficiency, comprising the following steps:

[0018] (a) constructing a recombinant nucleic acid construct, the construct comprising: at least one nuclear localization sequence, and at least one gene encoding a key enzyme in a target metabolite synthesis pathway; the NLS is operably linked to the upstream or downstream of the gene;

[0019] (b) introducing the recombinant nucleic acid construct into a eukaryotic microbial host cell;

[0020] (c) culturing the host cell under suitable conditions so that the key enzyme is expressed and localized in the cell nucleus;

[0021] (d) collecting the target metabolites.

[0022] In one embodiment, the target metabolites include but are not limited to products of metabolic pathways such as triacetic acid lactone, mevalonic acid, tryptophan, methyl anthranilate, phenylethyl alcohol, tyrosol, and hydroxytyrosol.

[0023] In one embodiment, the expression in step (3) includes integrated expression and free expression.

[0024] In one embodiment, the nuclear localization sequence comprises SV4040 with a nucleotide sequence as shown in SEQ ID NO.1, SV40 with a nucleotide sequence as shown in SEQ ID NO.2, or cMyc with a nucleotide sequence as shown in SEQ ID NO.11.

[0025] The present invention also provides the application of the yeast nuclear localization sequence, the recombinant microbial cell, or the method in the fermentation field.

[0026] In one embodiment, the application includes preparing food, medicine or health care products containing one or more substances selected from hydroxytyrosol, triacetic acid lactone, mevalonic acid and tryptophan.

[0027] Beneficial effects:

[0028] (1) The present invention screened a new nuclear localization sequence, which significantly improved the biosynthesis efficiency compared with the existing nuclear localization sequence. It can increase the production of triacetic acid lactone-producing Saccharomyces cerevisiae engineered bacteria TAL (SV4040) by 26.7% and the production of flaviolin (SV4040) by 39% and the production of hydroxytyrosol-producing Saccharomyces cerevisiae engineered bacteria HT (SV4040) by 103%.

[0029] (2) The present invention uses the nuclear localization sequence obtained by screening to construct a recombinant yeast with improved metabolite synthesis efficiency, which provides key technical support for the development of a high-yield, stable and economical yeast biosynthesis platform, and helps to use recombinant yeast as a cell factory for large-scale industrial biomanufacturing. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the triacetic acid lactone cytoplasmic pathway expression cassette.

[0031] Figure 2 Qualitative analysis of triacetic acid lactone (TAL); A: detection result of triacetic acid lactone (TAL) fermentation broth; B: detection result of triacetic acid lactone (TAL) fermentation broth by mass spectrometry.

[0032] Figure 3 Schematic diagram of the triacetic acid lactone nuclear pathway expression cassette.

[0033] Figure 4 This is the fermentation result of triacetic acid lactone (TAL) engineered bacteria.

[0034] Figure 5 Schematic diagram of the flavolin cytoplasmic pathway expression cassette.

[0035] Figure 6 Schematic diagram of the flavolin nuclear pathway expression cassette.

[0036] Figure 7 This is the result of fermentation of flaviolin by engineered bacteria.

[0037] Figure 8 These are the fermentation results of the cytoplasmic pathway engineered bacteria Flaviolin (Cytosol) and the nuclear pathway engineered bacteria Flaviolin (BPSV40) and Flaviolin (HEH2).

[0038] Figure 9 Schematic diagram of the cytoplasmic pathway expression cassette for hydroxytyrosol.

[0039] Figure 10 Qualitative analysis of hydroxytyrosol (HT); wherein, A: detection result of hydroxytyrosol (HT) fermentation broth; B: detection result of hydroxytyrosol (HT) fermentation broth by mass spectrometry.

[0040] Figure 11 Schematic diagram of the hydroxytyrosol nuclear pathway expression cassette.

[0041] Figure 12 This is the fermentation result of hydroxytyrosol (HT) engineered bacteria. DETAILED DESCRIPTION

[0042] (1) Culture medium:

[0043] Selective SC medium: 6.7 g / L yeast nitrogen base, 1.27 g / L amino acid dropout mix, 20 g / L glucose.

[0044] YTD medium: 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose.

[0045] (2) Detection method:

[0046] TAL detection method: At the end of fermentation, the culture was centrifuged at 12,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter, and a 10 μL sample was injected into the HPLC system. Metabolites were separated on a Shimadzu Shim-pack GIST C18 column (5 μm, 150 mm × 4.6 mm). The column oven was maintained at 30°C. The sample was separated using a gradient of two solvents: 1% acetic acid in H₂O (A) and 1% acetic acid in CH₃CN (B). The procedure began with a linear change of solvent B from 2% to 7% (0–5.0 min), 7% to 95% (5.0–7.0 min), 95% (7.0–9.0 min), 95% to 2% (9.0–11.0 min), and 2% (11.0–20.0 min). The flow rate was 1 mL min⁻¹, and the detection wavelength was 280 nm. TAL concentrations were quantified using a standard curve of TAL standards.

[0047] Hydroxytyrosol detection method: Detection was performed using high-performance liquid chromatography (HPLC). The fermentation broth was centrifuged, the supernatant collected, and filtered through a microporous filter. Analytical conditions were as follows: the mobile phase consisted of 80% (v / v) formic acid (0.1% (w / v)) and 20% (v / v) pure methanol at a flow rate of 1 mL / min; separation was performed on a Shimadzu Shim-pack GIST C18 column (5 μm, 150 mm × 4.6 mm), at a column temperature of 30°C; the detection wavelength was 280 nm; and the injection volume was 10 μL.

[0048] Flaviolin detection method: The fermentation broth was centrifuged and the supernatant was collected. The absorbance value at OD380 was measured using a microplate reader as the flavilin yield.

[0049] (3) Expression elements and sequences.

[0050] The sequences involved in the construction of the expression cassette in the specific embodiment are shown in Table 1.

[0051] Table 1 Sequences of gene regulatory expression elements

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] Example 1 Improving the yield of triacetic acid lactone by nuclear localization sequences SV40 and SV4040

[0058] (1) Construction and verification of triacetic acid lactone biological metabolic pathway: The key enzymes required for the production of triacetic acid lactone include Acc1 mut (nucleotide sequence shown in SEQ ID NO.3) and Gh2-PS (nucleotide sequence shown in SEQ ID NO.4). First, homologous recombination was performed in E. coli JM109 using ABclonal 2X MultiF Seamless Assembly Mix to construct a yeast expression cassette, as shown in FIG. Figure 1 As shown. The plasmid was linearized using restriction enzyme Not1 and then transformed into yeast CENPK2-1D. The constructed recombinant Saccharomyces cerevisiae was cultured on a selective SC plate at 30°C for 48 hours to screen for the correct engineered bacteria. The results were verified by liquid chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Figure 2As shown, the engineered strain was able to heterologously produce triacetic acid lactone, and the cytoplasmic metabolic pathway was successfully constructed. This engineered strain was named TAL (Cytosol) as a cytoplasmic pathway engineered strain.

[0059] (2) In the key enzyme Acc1 mut The C-terminus of Gh2-PS was fused with the nuclear localization sequence SV4040 (nucleotide sequence shown in SEQ ID NO.1) and SV40 (nucleotide sequence shown in SEQ ID NO.2), respectively: Based on the expression cassette constructed in step (1), the nuclear localization sequence was added to Acc1 by overlap extension PCR. mut and the C-terminus of Gh2-PS were transformed into E. coli JM109 to obtain a new yeast expression cassette (e.g. Figure 3 The involved sequences are shown in Table 1). The plasmid was linearized with the restriction endonuclease Not1 and then transformed into yeast CENPK2-1D, so that the two key enzymes in yeast CENPK2-1D were localized to the nucleus as nuclear pathway engineering bacteria, named TAL(SV40) and TAL(SV4040), respectively.

[0060] (3) Fermentation performance verification: The three genetically engineered yeast strains obtained in steps (1) and (2) were inoculated into 2 mL of selective SC medium in a 24-well deep-well plate and cultured at 30°C and 220 rpm for 24 h. Then, 100 μL of seed culture was inoculated into a new 24-well deep-well plate, each well containing 2 mL of YTD medium, and grown at 30°C and 220 rpm for 48 h. First, the cell density (OD) was measured by UV-visible spectrophotometer 759S. 600 The culture was then centrifuged at 12000 rpm for 5 min. Finally, the supernatant was filtered using a 0.22 μm filter membrane and transferred to a liquid phase vial for liquid phase analysis. Figure 4 As shown, the triacetic acid lactone production of the nuclear pathway engineered bacteria TAL (SV40) was 41.2% higher than that of the cytoplasmic pathway engineered bacteria TAL (Cytosol), indicating that the triacetic acid lactone synthesis efficiency of yeast was successfully improved by the nuclear localization sequence. In addition, the triacetic acid lactone production of the nuclear pathway engineered bacteria TAL (SV4040) was 26.7% higher than that of the nuclear pathway engineered bacteria TAL (SV40), indicating that the new nuclear localization sequence mutant SV4040 has more advantages in improving synthesis efficiency.

[0061] Example 2: Improving the production of flavolin by nuclear localization sequences SV40, SV4040, and cMyc

[0062] (1) Construction and verification of the bio-metabolism pathway of flavolin: The key enzymes required for the production of triacetic acid lactone include Acc1mut (nucleotide sequence shown in SEQ ID NO.3) and RppA (nucleotide sequence shown in SEQ ID NO.10). First, homologous recombination was performed in E. coli JM109 using ABclonal 2X MultiF Seamless Assembly Mix to construct a yeast expression cassette (such as Figure 5 The plasmid was linearized using the restriction endonuclease Not1 and then transformed into the yeast CENPK2-1D. The recombinant Saccharomyces cerevisiae was cultured on selective SC plates at 30°C for 48 hours to screen for the correct engineered strain. This engineered strain, designated as a cytoplasmic pathway engineered strain, was named Flaviolin (Cytosol).

[0063] (2) In the key enzyme Acc1 mut The nuclear localization sequences SV4040 (nucleotide sequence shown in SEQ ID NO.1), SV40 (nucleotide sequence shown in SEQ ID NO.2) and cMyc (nucleotide sequence shown in SEQ ID NO.11) were fused to the C-terminus of RppA respectively: Based on the expression cassette constructed in step (1), the nuclear localization sequences were added to Acc1 by overlap extension PCR. mut and the C-terminus of RppA, and transformed into E. coli JM109 to obtain a new yeast expression cassette (e.g. Figure 6 The plasmid was linearized with the restriction endonuclease Not1 and then transformed into yeast CENPK2-1D, so that two key enzymes in yeast CENPK2-1D were localized to the nucleus as nuclear pathway engineering bacteria, and were named Flaviolin (SV40), Flaviolin (SV4040), and Flaviolin (cMyc), respectively.

[0064] (3) Fermentation verification performance: The four genetically engineered yeast strains obtained in steps (1) and (2) were inoculated into 2 mL of selective SC medium in a 24-well deep-well plate and cultured at 30°C and 220 rpm for 24 h. Then 100 μL of seed culture was inoculated into a new 24-well deep-well plate, each well containing 2 mL of YTD medium, and grown at 30°C and 220 rpm for 48 h. First, the cell density (OD600) was measured using a UV-visible spectrophotometer 759S. The culture was then centrifuged at 12,000 rpm for 5 min. The supernatant was taken and the absorbance value at OD380 was detected by a microplate reader as the yield of flavonoids, as shown in FIG. Figure 7As shown, the flaviolin production of nuclear pathway engineered bacteria Flaviolin (SV40) and Flaviolin (cMyc) was 36.8% and 47.4% higher than that of cytoplasmic pathway engineered bacteria Flaviolin (Cytosol), respectively, indicating that the nuclear localization sequence was successfully used to improve the flaviolin synthesis efficiency of yeast. In addition, the flaviolin production of nuclear pathway engineered bacteria Flaviolin (SV4040) was 39% and 29% higher than that of nuclear pathway engineered bacteria Flaviolin (SV40) and Flaviolin (cMyc), respectively, indicating that the new nuclear localization sequence mutant SV4040 has more advantages in improving synthesis efficiency.

[0065] Example 3: Increasing the production of hydroxytyrosol by nuclear localization sequence SV4040

[0066] (1) Construction and verification of hydroxytyrosol metabolic pathway: The key enzymes required for the production of triacetic acid lactone include ARO4 (nucleotide sequence shown in SEQ ID NO.5), ARO7 (nucleotide sequence shown in SEQ ID NO.6), TyrA (nucleotide sequence shown in SEQ ID NO.7), PaHpaB (nucleotide sequence shown in SEQ ID NO.8), and EcHpaC (nucleotide sequence shown in SEQ ID NO.9). First, homologous recombination was performed in Escherichia coli JM109 using ABclonal 2X MultiF Seamless Assembly Mix to construct a yeast expression cassette (such as Figure 9 The plasmid was linearized with Not1 and then transformed into yeast CENPK2-1D. The recombinant Saccharomyces cerevisiae was cultured on YTD plates containing 200 μg / ml hygromycin B at 30°C for 48 hours to screen for the correct engineered bacteria. The results were verified by liquid chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Figure 10 As shown, the engineered strain can heterologously produce hydroxytyrosol, and the cytoplasmic metabolic pathway is successfully constructed. This engineered strain is named HT (Cytosol) as a cytoplasmic pathway engineered strain.

[0067] (2) Fusion of SV4040 (nucleotide sequence shown in SEQ ID NO. 1) to the C-termini of key enzymes ARO4, ARO7, TyrA, PaHpaB, and EcHpaC: Based on the expression cassette constructed in step (1), the nuclear localization sequence was added to the C-termini of ARO4, ARO7, TyrA, PaHpaB, and EcHpaC by overlap extension PCR and transformed into Escherichia coli JM109 to obtain new yeast expression cassettes. The results are shown in FIG. Figure 11As shown, the plasmid was linearized with Not1 and then transformed into yeast CENPK2-1D, so that the five key enzymes in yeast CENPK2-1D were localized to the nucleus as a nuclear pathway engineering strain, named HT (SV4040).

[0068] (3) Fermentation performance verification: The two genetically engineered yeast strains obtained in step (1) and step (2) were inoculated into 2 mL of YTD medium containing 200 μg / ml hygromycin B in a 24-well deep-well plate and cultured at 30°C and 220 rpm for 24 h. Then 100 μL of seed culture was inoculated into a new 24-well deep-well plate, each well containing 2 mL of YTD medium, and grown at 30°C and 220 rpm for 48 h. First, the cell density (OD) was measured by UV-visible spectrophotometer 759S. 600 The culture was then centrifuged at 12000 rpm for 5 min. Finally, the supernatant was filtered using a 0.22 μm filter membrane and transferred to a liquid phase vial for liquid phase analysis. Figure 12 As shown, the hydroxytyrosol production of the nuclear pathway engineered bacteria HT (SV4040) was 103% higher than that of the cytoplasmic pathway engineered bacteria TAL (Cytosol), indicating that the nuclear localization sequence was successfully used to improve the hydroxytyrosol synthesis efficiency of yeast.

[0069] Comparative Example 1:

[0070] The specific implementation method is the same as that of Example 2, except that the nuclear localization sequence SV40 is replaced by BPSV40 (nucleotide sequence shown in SEQ ID NO.12) and HEH2 (nucleotide sequence shown in SEQ ID NO.13), respectively. The recombinant bacteria Flaviolin (BPSV40) and Flaviolin (HEH2) are constructed and fermented according to the method of Example 2. The results are shown in FIG. Figure 8 As shown, the yields of the two nuclear pathway engineered bacteria were much lower than those of the cytoplasmic engineered bacteria, indicating that only nuclear localization sequences with a binding strength similar to that of SV40 and the transport protein Kap60 can improve the biosynthesis efficiency, among which the new mutant SV4040 had the best effect.

[0071] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A nuclear localization sequence, characterized in that The nucleotide sequence is shown in SEQ ID NO.

1.

2. A nucleic acid construct comprising the nuclear localization sequence of claim 1.

3. The nucleic acid construct according to claim 2, characterized in that The nucleic acid construct contains at least one nuclear localization sequence and at least one gene related to a biological metabolic pathway; the nuclear localization sequence is connected to the upstream or downstream of the gene related to the biological metabolic pathway to guide the gene expression product to be directional and localized to the host cell nucleus.

4. The nucleic acid construct according to claim 2 or 3, characterized in that The proteins encoded by the biological metabolic pathway-related genes are involved in the synthesis pathway of target metabolites in microorganisms.

5. The nucleic acid construct according to any one of claims 2 to 4, characterized in that The host is a eukaryotic microorganism; the eukaryotic microorganism includes but is not limited to Saccharomyces cerevisiae.

6. Recombinant Saccharomyces cerevisiae, characterized in that A gene containing the nuclear localization sequence of claim 1.

7. The recombinant Saccharomyces cerevisiae according to claim 6, characterized in that Including any of (a) to (c): (a) The recombinant Saccharomyces cerevisiae contains the Acc1 gene shown in the nucleotide sequence SEQ ID NO. 3 and the Gh2-PS gene shown in the nucleotide sequence SEQ ID NO. 4, and the 3' ends of the Acc1 gene and the Gh2-PS gene are respectively connected to the nuclear localization sequence of claim 1; (b) The recombinant Saccharomyces cerevisiae contains the Acc1 gene shown in the nucleotide sequence SEQ ID NO.3 and the RppA gene shown in the nucleotide sequence SEQ ID NO.10; the 3' ends of the Acc1 gene and the RppA gene are respectively connected to the nuclear localization sequence of claim 1. (c) The recombinant Saccharomyces cerevisiae contains the ARO4 gene shown in the nucleotide sequence of SEQ ID NO.5, the ARO7 gene shown in the nucleotide sequence of SEQ ID NO.6, the TyrA gene shown in the nucleotide sequence of SEQ ID NO.7, the PaHpaB gene shown in the nucleotide sequence of SEQ ID NO.8, and the EcHpaC gene shown in the nucleotide sequence of SEQ ID NO.9; the 3' ends of the ARO4 gene, ARO7 gene, TyrA gene, PaHpaB gene, and EcHpaC gene are respectively connected to the nuclear localization sequence of claim 1.

8. A method for improving biosynthesis efficiency, characterized in that: The following steps are involved: (a) constructing a recombinant nucleic acid construct, the construct comprising: at least one nuclear localization sequence, and at least one gene encoding a key enzyme in a target metabolite synthesis pathway; the NLS is operably linked to the upstream or downstream of the gene; (b) introducing the recombinant nucleic acid construct described in (a) into a eukaryotic microbial host cell; (c) culturing the host cell described in (b) under suitable conditions so that the key enzyme is expressed and localized in the cell nucleus; (d) Collecting target metabolites.

9. The method according to claim 8, characterized in that The target metabolites include, but are not limited to, products of the metabolic pathways of triacetic acid lactone, mevalonic acid, tryptophan, methyl anthranilate, phenylethanol, tyrosol or hydroxytyrosol.

10. Use of the nuclear localization sequence according to claim 1, or the nucleic acid construct according to any one of claims 2 to 5, or the recombinant Saccharomyces cerevisiae according to any one of claims 6 to 7, or the method according to any one of claims 8 to 9 in the field of fermentation.

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