US-20260125689-A1 - Novel Nuclear Localization Sequence Mutant and Method for Improving Biosynthetic Efficiency Using Same

Abstract

Disclosed are a novel nuclear localization sequence (NLS) mutant and a method for improving biosynthetic efficiency using same, belonging to the field of bioengineering. According to the present disclosure, a novel NLS is screened out, and recombinant yeast with improved metabolite synthesis efficiency is constructed by applying the NLS obtained through screening, which provides key technical support for developing a high-yield, stable and cost-effective yeast biosynthesis platform, and facilitates the use of recombinant yeast as a cell factory for large-scale industrial biomanufacturing.

Inventors

• Ye Li
• Zhonghu BAI
• Zhenhao Fu
• Pingxin LIN

Assignees

• JIANGNAN UNIVERSITY

Dates

Publication Date

20260507

Application Date

20251219

Priority Date

20250624

Claims (10)

1. 1 . A nuclear localization sequence (NLS), wherein the nucleotide sequence of the NLS is set forth in SEQ ID NO: 1.
2. 2 . A biological material comprising the NLS according to claim 1 .
3. 3 . The biological material according to claim 2 , wherein the biological material is a nucleic acid construct.
4. 4 . The biological material according to claim 3 , wherein the nucleic acid construct comprises at least one NLS and at least one gene related to a biological metabolic pathway; and the NLS is linked upstream or downstream of the gene related to the biological metabolic pathway for guiding a directional localization of a gene expression product to a host cell nucleus.
5. 5 . The biological material according to claim 4 , wherein a protein encoded by the gene related to the biological metabolic pathway is involved in a synthetic pathway of a target metabolite in a microorganism.
6. 6 . The biological material according to claim 4 , wherein the host is a eukaryotic microorganism; and the eukaryotic microorganism comprises Saccharomyces cerevisiae.
7. 7 . The biological material according to claim 2 , wherein the biological material is recombinant S. cerevisiae , comprising a gene localized under the guidance of the NLS.
8. 8 . The biological material according to claim 7 , comprising any one of (a) to (c): (a) the recombinant S. cerevisiae comprises an Acc1 gene with a nucleotide sequence as set forth in SEQ ID NO: 3 and a 2-Pyrone synthase (Gh2-PS) gene with a nucleotide sequence set forth in SEQ ID NO: 4, and 3′ ends of the Acc1 gene and the Gh2-PS gene are separately linked to the NLS; (b) the recombinant S. cerevisiae comprises the Acc1 gene with the nucleotide sequence set forth in SEQ ID NO: 3 and an RppA gene with the nucleotide sequence as set forth in SEQ ID NO: 10, and 3′ ends of the Acc1 gene and the RppA gene are separately linked to the NLS; and (c) the recombinant S. cerevisiae comprises an ARO4 gene with the nucleotide sequence set forth in SEQ ID NO: 5, an ARO7 gene with the nucleotide sequence set forth in SEQ ID NO: 6, a TyrA gene with the nucleotide sequence as set forth in SEQ ID NO: 7, a PaHpaB gene with the nucleotide sequence as set forth in SEQ ID NO: 8 and an EcHpaC gene with the nucleotide sequence as set forth in SEQ ID NO: 9, and 3′ ends of the ARO4 gene, the ARO7 gene, the TyrA gene, the PaHpaB gene and the EcHpaC gene are separately linked to the NLS.
9. 9 . A method for improving biosynthetic efficiency, comprising following steps: (a) constructing a recombinant nucleic acid construct, wherein the construct comprises: at least one nuclear localization sequence (NLS), and at least one gene encoding a key enzyme in a synthetic pathway of a target metabolite; the NLS is operably linked upstream or downstream of the gene; and the nucleotide sequence of the NLS is set forth in SEQ ID NO: 1; (b) introducing the recombinant nucleic acid construct in (a) into a eukaryotic microbial host cell; (c) culturing the host cell in (b) under suitable conditions to enable the expression and localization of the key enzyme within a cell nucleus; and (d) collecting the target metabolite.
10. 10 . The method according to claim 9 , wherein the target metabolite comprises products of the metabolic pathways of triacetic acid lactone (TAL), mevalonic acid (MVA), tryptophol, methyl anthranilate (Me-AA), 2-phenylethanol (2-PE), tyrosol, or hydroxytyrosol (HT).

Description

REFERENCE TO SEQUENCE LISTING The instant application contains a Sequence Listing in XML format as a file named “YGHY-2025-22-SEQ.xml”, created on Dec. 11, 2025, of 50576 Bytes in size, and which is hereby incorporated by reference in its entirety. TECHNICAL FIELD The present disclosure relates to a novel nuclear localization sequence (NLS) mutant and a method for improving biosynthetic efficiency using same, belonging to the field of bioengineering. BACKGROUND With the rapid development of synthetic biology, producing valuable compounds by engineering microbial cell factories through metabolic engineering as an alternative to conventional production methods has emerged as a more environmentally friendly and sustainable solution. However, designing functional metabolic pathways faces multiple challenges, including controlling the expression intensity of enzymes, or altering the product tendency of key enzymes, suboptimal physicochemical reaction environments, intermediate loss caused by endogenous competing pathways, and metabolite toxicity. Among eukaryotic microorganisms, Saccharomyces cerevisiae grows vigorously at low pH, is simple in nutritional requirements, is not susceptible to viral contamination, and has high tolerance to substrate and product toxicity. Additionally, S. cerevisiae possesses a complete whole genome sequence, is detailed in gene annotation resources and simple in gene manipulation methods, facilitates post-translational modification, and can express complex heterologous enzymes, making it favored for industrial applications. Although most current strategies for optimizing metabolic pathways, including protein fusion and synthesis of protein scaffolds, can enhance 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 available binding domains, and both protein fusion and scaffolds will exert adverse effects on enzyme activity. Leveraging the functional characteristics of various organelles to address these obstacles to metabolic flux has emerged as a new focus in metabolic engineering modification of S. cerevisiae, which involves compartmentalizing metabolic pathways within subcellular organelles. The unique physicochemical environment (e.g., pH and redox potential), enzymes, metabolites, and cofactors in each organelle provide favorable conditions for different metabolic pathways. By confining pathways within smaller subcellular compartments, the local concentrations of substrates and enzymes can be increased, thereby accelerating reaction rates and improving productivity. Restricting intermediates within organelles may also inhibit their conversion to byproducts and reduce their toxic effects on cellular processes. Current organelle engineering mainly focuses on organelles such as peroxisomes, mitochondria, endoplasmic reticulum, and lipid droplets. Numerous studies have demonstrated that pathway compartmentalization in yeast organelles can yield significant improvement compared to cytoplasmic pathways, but this improvement is only applicable to certain specific pathways and lacks strong universality, with the nuclear compartment remains underexplored to date. 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 crucial, and the development of novel organelles offers new opportunities to increase the yield of metabolic pathways. SUMMARY The present disclosure provides a novel yeast NLS mutant and a method for improving biosynthetic efficiency using a yeast NLS. According to the method, metabolic pathways for target products are first constructed, an NLS is then added to proteins related to the metabolic pathways, and finally, the target products are produced by fermentation. The present disclosure provides the following technical solutions to achieve the above objective: The present disclosure provides a novel yeast NLS, a nucleotide sequence of the novel yeast NLS is as set forth in SEQ ID NO. 1. The present disclosure further provides a nucleic acid construct including the yeast NLS as set forth in SEQ ID NO. 1. In one embodiment, the nucleic acid construct includes at least one NLS and at least one gene related to a biological metabolic pathway; and the NLS is linked upstream or downstream of the gene related to the biological metabolic pathway for guiding a directional localization of a gene expression product to a host cell nucleus. In one embodiment, a protein encoded by the gene related to the biological metabolic pathway is involved in a synthetic pathway of a target metabolite in a microorganism. In one embodiment, the genes related to the biological metabolic pathways include but are not limited to: 2-pyrone synthase gene (Gh2-PS), acetyl-CoA carboxylase gene (Acc1mut), 3-deoxy-D-arab