Application of recombinant plasmid, recombinant strain and preparation in improvement of methyltransferase activity and production of neohesperidin

Abstract

The invention discloses application of a recombinant plasmid, a recombinant strain and a preparation in improvement of methyltransferase activity and production of neohesperidin, and belongs to the technical field of bioengineering. The invention aims to solve the technical problems of insufficient YLOMT activity, high eriodictyol residual quantity, low synthesis efficiency, low yield and the like when the yarrowia lipolytica is used for synthesizing neohesperidin in the prior art. The key point of the technical scheme is that a recombinant plasmid is provided, the recombinant plasmid comprises a fusion fragment, and the fusion fragment comprises a coding sequence of a mitochondrial localization signal peptide, a Kozak sequence and a YLOMT coding gene; the amino acid sequence of the mitochondrial positioning signal peptide is shown as SEQ ID NO. 36; the nucleotide sequence of the Kozak sequence is as shown in SEQ ID NO. 37; and the sequence of the YLOMT coding gene is as shown in SEQ ID NO. 38.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and specifically relates to the application of a recombinant plasmid, a recombinant strain and a preparation in improving the activity of methyltransferase and producing neohesperidin. BACKGROUND

[0002] Technical content for understanding the present application: Neohesperidin is a natural and novel nutritional sweetener. The dihydrochalcone sweetener obtained by hydrogenation of neohesperidin is 1500-2000 times sweeter than sucrose. It also has the characteristics of low heat, long-acting sweet taste and good stability, and has a wide application prospect in food science and medicine. At present, neohesperidin is mainly obtained by purification and extraction of natural plants. However, its content in natural plants is generally low, and the production process is complex and easily affected by climate. Although chemical synthesis method can produce neohesperidin, it often needs to use expensive starting materials and complicated synthesis procedures, and there are safety hazards.

[0003] In recent years, microbial production of natural products has attracted widespread attention due to its short process cycle, high efficiency and environmental friendliness. Yarrowia lipolytica, as a typical unconventional yeast, has become one of the non-model microbial chassis cells in metabolic engineering and synthetic biology research due to its characteristics of broad substrate spectrum, high-efficiency protein secretion and low growth condition requirement. However, there are still problems such as insufficient methyltransferase activity and residual eriodictyol in Yarrowia lipolytica in synthesizing neohesperidin, which limits the efficient synthesis of neohesperidin.

[0004] In order to solve the above problems, researchers have been exploring strategies to improve the activity of methyltransferase. Among them, the methods of locating the enzyme to mitochondria, introducing Kozak sequence in front of the enzyme gene sequence and optimizing yeast metabolic engineering have shown good application prospects. However, how to comprehensively utilize these strategies to significantly improve the yield of neohesperidin is still the focus and difficulty of current research.

[0005] Retrieved relevant patent documents: A method for synthesizing neohesperidin from glycerol in Yarrowia lipolytica is disclosed in the document CN117987286A published on May 7, 2024. The method introduces Yarrowia lipolytica-derived flavone O-methyltransferase (YLOMT) and other plant-derived flavone synthase into Yarrowia lipolytica to construct a chassis cell that can de novo synthesize neohesperidin and hydrogenate neohesperidin to obtain dihydrochalcone. However, the ability of the chassis cell to synthesize neohesperidin and dihydrochalcone still needs to be improved.

[0006] The prior art represented by the foregoing documents has at least the following unsolved technical problems or defects: (1) The activity of the methyltransferase in Yarrowia lipolytica is insufficient during the synthesis of neohesperidin, resulting in a high residual amount of eriodictyol, which affects the synthesis efficiency and product quality of neohesperidin; (2) In existing methods for synthesizing neohesperidin, the synthesis capacity of the chassis cell is limited, which is difficult to meet the needs of industrial production and is easily affected by environmental factors; (3) Current improvement strategies, such as single enzyme localization to mitochondria or introduction of a Kozak sequence in front of the enzyme gene sequence, have improved the situation to some extent, but the problem of insufficient methyltransferase activity has not been fully solved; (4) The catalytic efficiency and expression level of the methyltransferase in the prior art need to be further improved, which limits the synthesis yield and process efficiency of neohesperidin; (5) Due to the lack of effective synergistic strategies, it is difficult to fully utilize the advantages of each improvement measure, resulting in the synthesis efficiency and yield of neohesperidin not reaching the optimal state.

[0007] In solving the above problems or overcoming the above defects, the present application has encountered the following difficulties and obstacles: (1) The balance problem between the targeting efficiency of the signal peptide and the enzyme activity: different sources of signal peptides (such as mitochondrial targeting sequence MTS) have significant differences in the subcellular localization efficiency of methyltransferase (YLOMT), and sequences that can efficiently guide YLOMT into mitochondria need to be screened. However, the fusion of signal peptide and YLOMT may change the spatial structure of the enzyme, leading to a decrease in its catalytic activity. Although some signal peptides can improve the targeting efficiency, they may reduce the conversion efficiency of eriodictyol due to interference with the enzyme structure, and a large number of mutations and screening are needed to balance the targeting efficiency and enzyme activity.

[0008] (2) The compatibility problem of Kozak sequence and host translation system: Kozak sequence can improve the expression level of YLOMT by enhancing the translation initiation efficiency, but the translation mechanism of Yarrowia lipolytica differs from that of model organisms, and the compatibility of different Kozak sequences with ribosome binding efficiency is difficult to predict. Although some Kozak sequences theoretically meet the conservative characteristics, they may conflict with the secondary structure of the 5' end of Yarrowia lipolytica mRNA, resulting in a decrease in translation initiation efficiency, and need to be customized and verified according to the characteristics of the host.

[0009] (3) The synergistic effect of signal peptide and Kozak sequence is difficult to regulate: Optimizing signal peptide or Kozak sequence alone can improve yield to a certain extent, but the synergistic effect when the two are combined is uncertain. The combination of some signal peptide and Kozak sequence may lead to decreased mRNA stability or abnormal post-translational processing (such as incomplete cleavage of signal peptide) due to base complementarity or steric hindrance between the sequences, which may reduce the effective expression level of YLOMT. Summary of the Invention

[0010] The purpose of this invention is to provide: The application of a recombinant plasmid, recombinant strain, and preparation in improving methyltransferase activity and producing neohesperidin, and related technologies, to solve the technical problems existing in the synthesis of neohesperidin using Yersinia lipolytica, such as insufficient YLOMT activity, high senna residue, low synthesis efficiency and yield, or combinations thereof.

[0011] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] The definition of the standard chemical term can be found in the reference "Genetic Engineering; Higher Education Press; August 1, 2013; 2nd Edition".

[0014] Unless otherwise specified, conventional methods within the scope of the art, such as lithium acetate conversion, PCR amplification, recombinant strain construction, strain culture, vector linearization, plasmid linearization, and high-performance liquid chromatography, shall be used.

[0015] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0016] The terms "optional / arbitrary" or "optionally / arbitrarily" mean that the event or situation subsequently described may or may not occur, including both the occurrence and non-occurrence of the event or situation. For example, according to the definition below: competent cells are selected from any one or more of TG1 competent cells, DH-5α competent cells, and JM109 competent cells, which means that competent cells are selected from TG1 competent cells, or DH-5α competent cells, or JM109 competent cells, or TG1 competent cells and DH-5α competent cells, or DH-5α competent cells and JM109 competent cells.

[0017] As used in this article, the terms “engineered,” “recombinant,” “mutant,” “modified,” or “non-natural” refer to organisms, microorganisms, cells, nucleic acid molecules, or vectors that have been modified by introducing heterologous nucleic acid molecules, or cells or microorganisms that have been genetically engineered through human intervention, i.e., by introducing heterologous nucleic acid molecules, or cells or microorganisms whose expression of endogenous nucleic acid molecules or genes has been altered to the point that it is controlled, dysregulated, or constitutive, where such alterations or modifications can be introduced through genetic engineering.

[0018] The term "genetic engineering" used in this article, also known as gene splicing technology and DNA recombination technology, refers to a genetic technology that uses molecular genetics as its theoretical basis and modern methods of molecular biology and microbiology as its means to construct hybrid DNA molecules in vitro according to a pre-designed blueprint using genes from different sources, and then introduce them into living cells to alter the original genetic characteristics of organisms, obtain new varieties, and produce new products. The term "nucleotide" as used in this article refers to a small molecule composed of a pentose sugar, a phosphate group, and a nitrogenous base. It is the basic unit of nucleic acids (DNA and RNA) and plays a crucial role in genetic information storage, energy metabolism, and cell signal transduction. In a nucleotide: the pentose sugar provides the backbone structure for RNA or DNA; DNA contains deoxyribose, and RNA contains ribose. The phosphate group is used to link sugar molecules (pentose sugars) to form phosphodiester bonds in the nucleic acid chain. The nitrogenous bases are purines (adenine, guanine) and pyrimidines (cytosine, thymine, uracil), which pair through hydrogen bonds (such as AT, CG). Different nucleotides are sometimes represented by the nitrogenous bases on them.

[0019] As used in this article, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid. Vectors can be, for example, plasmids, granules, viruses, or bacteriophages. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells.

[0020] As used in this article, the term "expression vector" refers to a vector that, when present in a suitable environment, can direct the expression of one or more transgene-encoded proteins carried by the vector.

[0021] The term “expression cassette” as used in this article refers to a nucleotide sequence that enables the expression of structural genes (i.e., protein-coding sequences) in a host compatible with such sequences.

[0022] The term "plasmid vector" as used in this article refers to a circular DNA molecule with a relatively small molecular weight that exists independently of chromosomal DNA. Plasmids can be transferred, replicate independently, or integrate into chromosomal DNA and replicate along with it.

[0023] The term "host cell" as used in this article refers to a cell that can receive foreign genes (target genes) and express them within itself to produce the required proteins.

[0024] The term "stable expression" used in this article refers to the phenomenon where a foreign gene is integrated into the host cell genome through transfection technology, forming a stable and heritable transformed line. Its core characteristics are long-lasting and heritable expression of the foreign gene. This process often faces obstacles such as gene silencing, involving molecular mechanisms such as repetitive sequences, high copy numbers, and methylation levels. Currently, stable expression efficiency is mainly improved through techniques such as genome editing, gene structure modification, and enhancer addition, which have significant application value in gene library construction and functional screening.

[0025] The term "methyltransferases" as used in this article refers to an important class of enzymes that are widely found in plants, animals, and microorganisms. They are mainly responsible for catalyzing methylation reactions in organisms to form methylated products of nucleic acids, proteins, polysaccharides, and lipids.

[0026] The term "YLOMT" as used in this article refers to yeast derived from Yeast lipolytica (YLOMT). Yarrowia lipolytica The flavonoid O-methyltransferase is a key enzyme in the synthesis of neohesperidin, which catalyzes the conversion of the precursor sennaol into the product neohesperidin through a methyl transfer reaction.

[0027] As used herein, the term "homologous arm" refers to a DNA fragment that shares a homologous sequence with a specific region of the host genome. In genetic engineering, homologous arms mediate the integration of exogenous genes into target locations in the host genome via homologous recombination, ensuring stable integration of exogenous genes into specific sites. In this invention, a "homologous arm-signal peptide-YLOMT fusion fragment" is constructed to precisely integrate the signal peptide and YLOMT gene into the genome of *Yarrowia lipolytica* via homologous recombination.

[0028] The term "signal peptide" as used herein refers to a short peptide sequence at the N-terminus of a protein that functions to guide the directional transport of nascent proteins to specific organelles (such as mitochondria). In this invention, MTS1, MTS2, and MTS3 are all signal peptides, belonging to mitochondrial targeting sequences (MTS), used to guide YLOMT into the mitochondria, improving the enzyme's localization efficiency in specific organelles and enhancing catalytic reactions.

[0029] The term "MTS" used in this article refers to a class of signal peptides responsible for guiding proteins to mitochondria. In this invention, MTS1 (COX4-SC source), MTS2 (COX4-YL source), and MTS3 (MTS-OMT source) are all MTSs that guide YLOMT into mitochondria, optimizing enzyme spatial localization and improving catalytic efficiency.

[0030] As used herein, the term "OMT" refers to O-methyltransferase, which catalyzes the transfer of a methyl group from a donor molecule to an oxygen atom in an acceptor molecule. In this invention, "MTS-OMT" refers to a mitochondrial targeting sequence (MTS3) derived from a certain O-methyltransferase, having the amino acid sequence shown in SEQ ID NO. 36, which guides YLOMT into the mitochondria.

[0031] As used herein, the term "COX4" refers to cytochrome c oxidase subunit 4, the core subunit of mitochondrial respiratory chain complex IV, whose N-terminus typically contains a mitochondrial targeting sequence (MTS). In this invention, "COX4-SC" and "COX4-YL" refer to COX4 derived from different species.

[0032] The term "SC" used in this article refers to: Saccharomyces cerevisiae (SC). Saccharomyces cerevisiae COX4 is a commonly used model yeast. In this invention, "COX4-SC" indicates that the COX4 protein and its corresponding MTS (MTS1) are derived from Saccharomyces cerevisiae and have the amino acid sequence shown in SEQ ID NO.34. This signal peptide guides YLOMT into the mitochondria.

[0033] The term "YL" used in this article refers to: Yersinia lipophila (YL). Yarrowia lipolyticaCOX4 is an industrial yeast commonly used in metabolic engineering and synthetic biology, possessing strong exogenous gene expression capabilities and metabolic regulation potential. In this invention, "COX4-YL" indicates that the COX4 protein and its corresponding MTS (MTS2) originate from *Yarrowia lipolytica*, have the amino acid sequence shown in SEQ ID NO. 35, and that this signal peptide guides YLOMT into the mitochondria.

[0034] The term "fusion gene" as used in this article refers to a recombinant gene formed by linking DNA fragments of two or more different genes through molecular cloning technology.

[0035] The term "chassis cell" as used herein refers to a genetically modified host cell with a stable genetic background and suitable for the expression of exogenous genes, serving as the basis for constructing functional strains in synthetic biology. In this invention, *Yarrowia lipolytica* NHP-6 is used as the chassis cell to integrate the YLOMT fusion gene containing a signal peptide and a kozak sequence, thereby constructing a recombinant strain that efficiently produces novel hesperidin.

[0036] As used herein, the term "promoter" refers to a DNA sequence in which RNA polymerase binds and initiates gene transcription, determining gene expression initiation and transcription efficiency. The TEFIN used in this invention is a strong endogenous promoter from *Yarrowia lipophila*, used to drive efficient transcription of the YLOMT gene and increase gene expression levels.

[0037] The term "Kozak" as used in this article refers to a conserved nucleotide sequence on eukaryotic mRNA surrounding the start codon ATG. This sequence, by binding to ribosomes, influences translation initiation efficiency and is a crucial element in regulating protein synthesis levels. In this invention, Kozak3, Kozak7, and Kozak9 are different Kozak sequences used to optimize the translation initiation of YLOMT, increase its protein expression level, and thereby enhance catalytic efficiency.

[0038] In a first aspect, the present invention provides: a recombinant plasmid comprising a fusion fragment, the fusion fragment containing a coding sequence for a mitochondrial localization signal peptide, a Kozak sequence, and a YLOMT coding gene.

[0039] These include technical features such as recombinant plasmids, fusion fragments, mitochondrial localization signal peptides, Kozak sequences, and YLOMT-encoding genes.

[0040] The amino acid sequence of the mitochondrial localization signal peptide, a technical feature, is shown in SEQ ID NO.36.

[0041] The nucleotide sequence of the technical feature Kozak sequence is shown in SEQ ID NO.37.

[0042] The sequence of the YLOMT encoding gene, a technical feature, is shown in SEQ ID NO.38.

[0043] The technical feature fusion fragment also includes homologous arm sequences and terminators.

[0044] Among them, the homologous arm will pair and recombine with the homologous sequence of the preset integration site in the host genome, and insert the exogenous expression cassette into a specific location in the host genome.

[0045] The terminator is selected from any one or more of the following: tLIP2 terminator, tXPR2 terminator, tLEU2 terminator, tURA3 terminator, and tICL1 terminator.

[0046] The preferred terminator is tLIP2 terminator.

[0047] Among them, the technical feature recombinant plasmid also includes promoter fragments.

[0048] The promoter is selected from any one or more of the following: TEFIN promoter, TEF1 promoter, LIP2 promoter, and XPR2 promoter.

[0049] The preferred promoter is the TEFIN promoter.

[0050] The promoter is further preferably the endogenous strong promoter TEFIN of Yersinia lipophila.

[0051] The preparation method of the recombinant plasmid with technical characteristics includes: cloning and constructing the promoter fragment, fusion fragment and linearized vector backbone fragment, transforming them into competent cells, screening for positive integration plasmids, and obtaining the recombinant plasmid.

[0052] The linearized vector backbone fragments are derived from plasmids p20-AXP, p21-XPR, pUC19, pBluescript IIKS (+), pET-28a (+), or pGEM-T Easy.

[0053] The linearized vector backbone fragment is preferably derived from plasmid p20-AXP or p21-XPR.

[0054] Among them, the linearized vector backbone fragment is further preferably derived from plasmid p20-AXP.

[0055] In some embodiments, the linearized vector backbone fragment is a linearized p20-AXP backbone fragment, and the preparation method includes: linearizing p20-AXP using primers.

[0056] In some embodiments, the linearized vector backbone fragment is a linearized p21-XPR backbone fragment, and the preparation method includes: linearizing p21-XPR using primers.

[0057] The competent cells are selected from any one or more of TG1 competent cells, DH-5α competent cells, and JM109 competent cells.

[0058] Among them, the competent cells are preferably DH-5α competent cells.

[0059] The method for preparing the promoter fragment includes: using the genome of Yersinia lipolyticis strain W29Δku70 as a template, amplifying the endogenous strong promoter TEFIN of Yersinia lipolyticis to obtain the promoter fragment.

[0060] The preparation method of the fusion fragment includes: using the YLOMT gene as a template, performing a first round of amplification to obtain the kozak9-YLOMT fusion fragment; performing a second round of amplification to obtain the MTS-OMT-kozak9-YLOMT fragment; and performing a third round of amplification to obtain the homologous arm-MTS-OMT-kozak9-YLOMT fusion fragment.

[0061] The primers used in the first round of amplification were koazk9-II-F and YlOMT-tLIP2-R.

[0062] The primers used for the second round of amplification were MTS-OMT-II-F and YlOMT-tLIP2-R.

[0063] The primers used in the three rounds of amplification were MTS-pTEFIN-F and YlOMT-tLIP2-R.

[0064] The molar ratio of the promoter fragment, fusion fragment, and linearized vector backbone fragment is 1:1:2.

[0065] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred embodiment: the amino acid sequence of the mitochondrial localization signal peptide is shown in SEQ ID NO.36; the nucleotide sequence of the Kozak sequence is shown in SEQ ID NO.37; and the sequence of the YLOMT encoding gene is shown in SEQ ID NO.38. This technical solution, based on solving the technical problems of "insufficient YLOMT activity, high succinylcholine residue, and low synthesis efficiency and yield in the prior art when using *Yersinia lipolytica* to synthesize new hesperidin," further solves the technical problems of "insufficient YLOMT activity, high succinylcholine residue, and low synthesis efficiency and yield in the prior art when using *Yersinia lipolytica* to synthesize new hesperidin."

[0066] The second preferred option uses the tLIP2 terminator. This option addresses the existing technical problems of insufficient YLOMT activity, high senna residue, and low synthesis efficiency and yield when synthesizing new hesperidin using *Yersinia lipolytica*. Furthermore, it solves the problem of low expression efficiency of the exogenous gene (YLOMT) in *Yersinia lipolytica* and uncoordinated transcription / translation processes, leading to insufficient synthesis of the target protein.

[0067] The third preferred option is to use the endogenous strong promoter TEFIN from *Yersinia lipolytica* as the promoter. This approach addresses the existing technical problems of insufficient YLOMT activity, high senna residue, and low synthesis efficiency and yield when synthesizing new hesperidin using *Yersinia lipolytica*. Furthermore, it solves the problem of low expression efficiency of the exogenous gene (YLOMT) in *Yersinia lipolytica* and uncoordinated transcription / translation processes, leading to insufficient synthesis of the target protein.

[0068] In a second aspect, the present invention provides: a recombinant strain, wherein the genome of the recombinant strain integrates an exogenous expression cassette, the exogenous expression cassette being prepared by linearizing any of the recombinant plasmids described above.

[0069] These include technical features such as recombinant strains and exogenous expression cassettes.

[0070] Among them, the technical feature is that the exogenous expression cassette linearizes the recombinant plasmid described in any of the above items through primers.

[0071] The technical features of the recombinant strain preparation method include the following steps: co-transforming exogenous expression cassettes and gRNA plasmids into Yersinia lipolyticis chassis cells, achieving genome integration through homologous recombination, screening for positive transformants, and obtaining recombinant strains.

[0072] Among the technical features, the recombinant strain is preferably a lipophilic yeast strain ( Yarrowia lipolyticaYO1, with accession number CGMCC NO.34491.

[0073] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the second aspect of the present invention includes: First preferred option: The recombinant strain is preferably a lipophilic yeast strain ( Yarrowia lipolytica YO1, with accession number CGMCC NO.34491. This technical solution, based on solving the technical problems of "insufficient YLOMT activity, high succinylcholine residue, and low synthesis efficiency and yield in the synthesis of new hesperidin using Yersinia lipolytica in the prior art", further solves the technical problems of "insufficient YLOMT activity, high succinylcholine residue, and low synthesis efficiency and yield in the synthesis of new hesperidin using Yersinia lipolytica in the prior art".

[0074] Thirdly, the present invention provides: a genetically engineered preparation.

[0075] Among its technical features is the use of genetically engineered agents.

[0076] Among them, the technical features are: genetically engineered preparations, including one or more of the following: cultures of recombinant strains, culture extracts, cell fragments, bacterial cells, fermentation broth, fermentation broth precipitates, and lyophilized powders.

[0077] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the third aspect of the present invention includes: The first preferred option: the genetically engineered preparation includes one or more of the following: culture of the recombinant strain described above, culture extract, cell fragments, bacterial cells, fermentation broth, fermentation broth precipitate, and lyophilized powder. This technical solution, while addressing the existing problems of insufficient YLOMT activity, high senna residue, and low synthesis efficiency and yield in the synthesis of neohesperidin using *Yarrowia lipolytica*, further solves the problems of difficulty in storing, transporting, and scaling up the recombinant strains and their metabolites, as well as the poor stability and low ease of use of active ingredients in actual production.

[0078] Fourthly, the present invention provides the application of any of the recombinant plasmids, recombinant strains, or genetically engineered agents described above.

[0079] This includes technical features: applications.

[0080] Among them, the technical features include any one or more of the following: (1) Application in enhancing methyltransferase activity; (2) Application in the production of new hesperidin.

[0081] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the fourth aspect of the present invention includes: The first preferred option: The application includes any one or more of the following: (1) application in improving methyltransferase activity; (2) application in the production of neohesperidin. This technical solution solves the technical problem of "insufficient YLOMT activity, high senna residue, low synthesis efficiency and yield in the synthesis of neohesperidin using Yersinia lipolytica in the prior art" and further solves the technical problem of "the lack of specific tools in the prior art that can efficiently improve the activity of methyltransferase (YLOMT) and be used for the production of neohesperidin, which makes it difficult to accurately control the related biosynthetic process and limit its industrial application".

[0082] Fifthly, the present invention provides a method for improving methyltransferase activity.

[0083] Among them is a technical feature: a method for improving methyltransferase activity.

[0084] Among them, the technical feature is that the method for improving methyltransferase activity includes using any of the recombinant plasmids, recombinant strains or genetically engineered agents described above.

[0085] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the fifth aspect of the present invention includes: The first preferred approach: Methods for improving methyltransferase activity include using any of the recombinant plasmids, recombinant strains, or genetically engineered agents described above. This approach addresses the existing technical problems of insufficient YLOMT activity, high senna residue, and low synthesis efficiency and yield when synthesizing neohesperidin using *Yarrowia lipolytica*. Furthermore, it solves the problem that existing methods for achieving high-efficiency methyltransferase (YLOMT) activity are complex to operate, have unstable efficiency, and lack specific enhancement methods suitable for the *Yarrowia lipolytica* system, making it difficult to achieve targeted optimization of YLOMT activity in a simple and feasible manner.

[0086] In a sixth aspect, the present invention provides: a method for producing novel hesperidin.

[0087] Among them is a technical feature: a method for producing novel hesperidin.

[0088] Among them, the technical feature is that the method for producing new hesperidin includes using any of the recombinant plasmids, recombinant strains or genetically engineered agents described above.

[0089] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the sixth aspect of the present invention includes: The first preferred option: A method for producing novel hesperidin includes using any of the recombinant plasmids, recombinant strains, or genetically engineered agents described above. This technical solution addresses the existing problems of insufficient YLOMT activity, high senna residue, and low synthesis efficiency and yield in the synthesis of novel hesperidin using *Yarrowia lipolytica*. Furthermore, it solves the problems of cumbersome procedures, low product purity, reliance on chemical synthesis or inefficient biotransformation systems, leading to high production costs and difficulties in large-scale production.

[0090] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: Compared with existing technologies, this invention has better technical effects in improving YLOMT activity, reducing senna residue, and increasing neohesperidin yield. According to experimental tests, this invention reduces senna residue to 107.2 mg / mL. According to experimental tests, this invention increases neohesperidin yield to 412.1 mg / mL.

[0091] Furthermore, based on the present invention: 1. Based on the comparison of three different signal peptides MTS1, MTS2, and MTS3 in Example 1, the recombinant strain NHP-7-3 containing the recombinant plasmid of this invention, constructed from MTS3, showed a 13.59% increase in neohesperidin production and a 25.30% reduction in sennae residue compared to the control Yeast Rice NHP-6. The effects of increasing neohesperidin production and reducing sennae residue were the most significant.

[0092] 2. Based on the comparison of three different kozak sequences in Example 2, the recombinant strain NHP-8-3 containing the recombinant plasmid of this invention, constructed from kozak9, showed a 16.6% increase in neohesperidin production and a 22.0% decrease in sennae residue compared to the control Yeast Extract NHP-6. The effects of increasing neohesperidin production and reducing sennae residue were the most significant.

[0093] 3. Based on Examples 1-3, the comparison of different recombinant strains. The recombinant strain containing the recombinant plasmid of the present invention had a hesperidin residue of 107.2 mg / mL and a neohesperidin yield of 412.1 mg / mL. Compared with the control Yersinia lipolytica NHP-6, the recombinant strain NHP9 showed a 33.36% increase in neohesperidin yield and a 35.42% decrease in hesperidin residue.

[0094] Preservation Instructions Preserved strain: Lipolysaccharidase strain YO1; Classification and nomenclature: Yersinia lipophila ( Yarrowia lipolytica ; Accession number: CGMCC No. 34491; Preservation date: May 9, 2025; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0095] Figure 1 The image shows the p20-(AXP)-MTS-OMT-YLOMT plasmid.

[0096] Figure 2 This is the map of plasmid p21-kozak-9-YLOMT.

[0097] Figure 3 The image shows the p20-MTS-OMT-kozak9-YLOMT plasmid. Detailed Implementation

[0098] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0099] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0100] The *E. coli* DH5α competent cells of this invention were purchased from Sangon Biotech (Shanghai) Co., Ltd.; all gene, primer synthesis and sequencing services were provided by Suzhou Genewiz Biotechnology Co., Ltd.; Phanta® Max Super-Fidelity DNA Polymerase was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0101] Basic Example 1 The PCR amplification procedure used in this invention is as follows: the target fragment is amplified using Phanta® Max Super-Fidelity DNA Polymerase. The specific reaction system and amplification procedure are shown in Table 1.

[0102] Table 1

[0103] Basic Implementation Example 2 This invention uses a lithium acetate conversion method for conversion. The specific operation steps are as follows: 1. Pick a single colony and incubate it in the corresponding culture medium overnight at 30°C and 200 rpm for 12 hours. 2. Transfer the yeast culture to 50 mL of fresh YPDA medium with an initial OD600 value of 0.2 and activate it for 4-5 hours to allow the yeast to multiply for two generations, so that the OD600 value of the culture is between 0.8 and 0.9.

[0104] 3. Transfer the bacterial culture into a sterile 50 mL centrifuge tube, centrifuge at 3600 rpm for 10 min to collect the bacterial cells, and wash twice with 35 mL of sterile ddH2O.

[0105] 4. Resuspend the bacterial cells in 900µL of sterile ddH2O, then aliquot 100µL into each 1.5mL centrifuge tube for transformation. 5. Place the dispensed centrifuge tubes into a handheld centrifuge, centrifuge for 20 seconds, discard the supernatant, and add the conversion system (all the above operations must be completed in a laminar flow hood to ensure a sterile environment).

[0106] 6. Screening and identification of positive transformants. Spread the transformed yeast cells on selection medium containing YPD and incubate at 30°C for 2-3 days to screen for resistant clones.

[0107] Basic Example 3 The primer information used in this invention is shown in Table 2: Table 2

[0108] The alkaline hydrolysis sequence of the signal peptide of this invention is shown in Table 3: Table 3

[0109] The kozak sequence base sequence of this invention is shown in Table 4: Table 4

[0110] Example 1 The following plasmids containing methyltransferase expression peptides were constructed: p20-(AXP)-COX4-SC-YLOMT, p20-(AXP)-COX4-YL-YLOMT, and p20-(AXP)-MTS-OMT-YLOMT. Genome integration: The fusion gene containing the homologous arm-signal peptide-methyltransferase was transformed into the chassis of *Yarrowia lipolytica*.

[0111] 1. Amplify the fusion unidirectional promoter fragment Using the genome of Yersinia lipolyticis strain W29Δku70 (RefSeq: GCF_001761485.1 (ASM176148v1)) as a template, the endogenous strong promoter TEFIN of Yersinia lipolyticis was amplified using primers P-T1-pTEFIN-F and P-T1-pTEFIN-R to obtain the TEFIN fragment.

[0112] 2. Assembly of fusion fragments containing homologous arms-signal peptide-YLOMT (1) Using the methyltransferase (YLOMT) gene as a template, the YLOMT fragment was obtained by one round of amplification using primers YLOMT-F and YLOMT-R; The methyltransferase (YLOMT) gene sequence is shown in SEQ ID NO.38: .

[0113] (2) Using the YLOMT fragment as a template, a second round of amplification was performed with upstream primer COX4-SC-F and downstream primer YlOMT-tLIP2-R to obtain the COX4-SC-YLOMT fragment; a third round of amplification was performed with upstream primer COX4-SC-pTEFIN-F and downstream primer YlOMT-tLIP2-R to obtain the fusion fragment containing homologous arm-signal peptide-YLOMT: COX4-SC-YLOMT fusion fragment.

[0114] (3) Using the YLOMT fragment as a template, a second round of amplification was performed with upstream primer COX4-YL-F and downstream primer YlOMT-tLIP2-R to obtain the COX4-YL-YLOMT fragment; a third round of amplification was performed with upstream primer COX4-YL-pTEFIN-F and downstream primer YlOMT-tLIP2-R to obtain the fusion fragment containing homologous arm-signal peptide-YLOMT: COX4-YL-YLOMT fusion fragment.

[0115] (4) Using the YLOMT fragment as a template, a second round of amplification was performed with upstream primer MTS-OMT-F and downstream primer YlOMT-tLIP2-R to obtain the MTS-OMT-YLOMT fragment; a third round of amplification was performed with upstream primer MTS-pTEFIN-F and downstream primer YlOMT-tLIP2-R to obtain the fusion fragment containing homologous arm-signal peptide-YLOMT: MTS-OMT-YLOMT fusion fragment.

[0116] 3. Construction of positive integration plasmid (1) The plasmid p20-AXP was linearized using primers Gibson-F and Gibson-R to obtain the linearized p20-AXP backbone fragment.

[0117] (2) The linearized p20-AXP backbone fragment, the fusion unidirectional promoter fragment, and the COX4-SC-YLOMT fusion fragment were assembled using the Gibson assembly method at a ratio of 2:1:1. The assembly was transformed into Escherichia coli DH5α competent cells and cultured overnight. Single colonies that grew in the selection plate (50µg / mL ampicillin) were sent for sequencing (sequencing primers pTEFIN-cexu and tTLI2-cexu). After the sequencing was successful, the positive integration plasmid p20-(AXP)-COX4-SC-YLOMT was returned.

[0118] (3) The linearized p20-AXP backbone fragment, the fusion unidirectional promoter fragment, and the COX4-YL-YLOMT fusion fragment were assembled using the Gibson assembly method at a ratio of 2:1:1. The assembly was transformed into Escherichia coli DH5α competent cells and cultured overnight. Single colonies that grew in the selection plate (50µg / mL ampicillin) were sent for sequencing (sequencing primers pTEFIN-cexu and tTLI2-cexu). After the sequencing was successful, the positive integration plasmid p20-(AXP)-COX4-YL-YLOMT was returned.

[0119] (4) The linearized p20-AXP backbone fragment, the fusion unidirectional promoter fragment, and the MTS-OMT-YLOMT fusion fragment were assembled using the Gibson assembly method at a ratio of 2:1:1, transformed into E. coli DH5α competent cells, and cultured overnight. Single colonies growing in the selection plate (50 µg / mL ampicillin antibiotic) were sent for sequencing (sequencing primers pTEFIN-cexu and tTLI2-cexu). After successful sequencing, the positive integration plasmid p20-(AXP)-MTS-OMT-YLOMT was returned. Figure 1 ).

[0120] 4. Construction of recombinant strains (1) Construction of gRNA20 plasmid (abbreviated as plasmid G20): The existing gRNA plasmid (purchased from Addgene) in the laboratory was linearized using primers gRNA20-F and gRNA20-R to obtain the linearized gRNA20 fragment. The above linearized gRNA20 fragment was transformed into E. coli DH5α competent cells and cultured overnight. Single colonies that grew in the selection plate (50µg / mL ampicillin antibiotic) were sent for sequencing (sequencing primer gRNA-cexu-F). After the sequencing was successful, the positive integration plasmid gRNA20 was returned.

[0121] (2) The preparation method of Yersinia lipophila NHP-6 is the same as that of the recombinant strain ZYL-4 in CN117987286A.

[0122] (3) Using primers YL-p20-F and YL-p20-R, linearize p20-(AXP)-COX4-SC-YLOMT, p20-(AXP)-COX4-YL-YLOMT or p20-(AXP)-MTS-OMT-YLOMT respectively to obtain p20-(AXP)-COX4-SC-YLOMT expression cassette, p20-(AXP)-COX4-YL-YLOMT expression cassette or p20-(AXP)-MTS-OMT-YLOMT expression cassette.

[0123] (4) The p20-(AXP)-COX4-SC-YLOMT expression cassette and its corresponding knock-in plasmid G20 were transformed into Yersinia lipophila NHP-6 by lithium acetate method to obtain recombinant strain NHP-7-1; (5) The p20-(AXP)-COX4-YL-YLOMT expression cassette and its corresponding knock-in plasmid G20 were transformed into Yersinia lipophila NHP-6 by lithium acetate method to obtain recombinant strain NHP-7-2. (6) The p20-(AXP)-MTS-OMT-YLOMT expression cassette and its corresponding knock-in plasmid G20 were transformed into Yersinia lipophila NHP-6 by lithium acetate method to obtain recombinant strain NHP-7-3; (7) 30 resistant clones of the above recombinant strains were randomly selected, genomic DNA was extracted, and PCR verification was performed (verification primers YZ-p20-F and YZ-p20-R) to screen out positive transformants.

[0124] 5. Fermentation verification (1) Streak the recombinant strains NHP-7-1, NHP-7-2 and NHP-7-3 on YPD plates to activate them. Pick single clones into test tubes containing 4 mL of YPD medium and culture them at 30℃ and 220 rpm for 48 h to obtain seed culture of recombinant strains NHP-7-1, NHP-7-2 and NHP-7-3 respectively.

[0125] (2) Seed cultures of recombinant strains NHP-7-1, NHP-7-2, and NHP-7-3 were inoculated into 30 mL of fermentation medium (YNB medium with 2% glucose added as a carbon source) at an inoculation ratio of 1% v / v. Three parallel tubes were set up, with Yersinia lipolytica NHP-6 as the control group. Fermentation was carried out at 30℃ and 220 rpm for 4 days.

[0126] (3) After the fermentation culture is completed, take 500 μL of fermentation broth, add an equal volume of anhydrous methanol and mix thoroughly. Sonicate for 30 min, centrifuge at 12000 rpm for 15 min, take the supernatant for high performance liquid chromatography analysis, and detect the residual amount of sennaol and the yield of neohesperidin.

[0127] The measurement results are shown in Table 3: Table 3

[0128] The results showed that the recombinant strain NHP-7-3 produced 351 mg / mL of neohesperidin and had a low residue of 124 mg / mL of senna. Compared with the control Yersinia lipolytica NHP-6, the recombinant strain NHP-7-3 showed a 13.59% increase in neohesperidin production and a 25.30% decrease in senna residue.

[0129] Example 2 Expression vectors p21-kozak-3-YLOMT, p21-kozak-7-YLOMT, and p21-kozak-9-YLOMT containing three different kozak sequences were constructed. Genome integration: The fusion gene containing the homologous arm-kozak-methyltransferase was transformed into the chassis of Yersinia lipolytica.

[0130] 1. Construction of fused bidirectional promoter segments The fused bidirectional promoter fragment was obtained by referring to the method in Example 1.

[0131] 2. Construction of a methyltransferase (YLOMT) fusion fragment containing three different Kozak sequences (1) Using the methyltransferase (YLOMT) gene as a template, the YLOMT fragment was obtained by one round of amplification using primers YLOMT-F and YLOMT-R; (2) Using the YLOMT fragment as a template, a second round of amplification was performed with upstream primer kozak3-F and downstream primer YlOMT-tLIP2-R to obtain a fusion fragment containing the homologous arm -kozak-YLOMT: kozak-3-YLOMT fusion fragment.

[0132] (3) Using the YLOMT fragment as a template, a second round of amplification was performed with upstream primer kozak7-F and downstream primer YlOMT-tLIP2-R to obtain a fusion fragment containing the homologous arm -kozak-YLOMT: kozak-7-YLOMT fusion fragment.

[0133] (4) Using the YLOMT fragment as a template, a second round of amplification was performed with upstream primer kozak9-F and downstream primer YlOMT-tLIP2-R to obtain a fusion fragment containing the homologous arm -kozak-YLOMT: kozak-9-YLOMT fusion fragment.

[0134] 3. Construction of a methyltransferase (YLOMT) expression vector containing three different Kozak sequences (1) The plasmid p21-XPR2 was linearized using primers Gibson-F and Gibson-R to obtain the linearized p21-XPR2 backbone fragment.

[0135] (2) The linearized p21-XPR2 backbone fragment, the fusion unidirectional promoter fragment, and the kozak-3-YLOMT fusion fragment were assembled using the Gibson assembly method at a ratio of 2:1:1, transformed into Escherichia coli DH5α competent cells, cultured overnight, and single colonies grown in the selection plate (50µg / mL ampicillin antibiotic) were sent for sequencing (sequencing primers pTEFIN-cexu and tTLI2-cexu). After the sequencing was successful, the positive integration plasmid p21-kozak-3-YLOMT was returned.

[0136] (3) The linearized p21-XPR2 backbone fragment, the fusion unidirectional promoter fragment, and the kozak-7-YLOMT fusion fragment were assembled in a 1:1:1 ratio using the Gibson assembly method, transformed into Escherichia coli DH5α competent cells, cultured overnight, and single colonies grown in the selection plate (50µg / mL ampicillin antibiotic) were sent for sequencing (sequencing primers pTEFIN-cexu and tTLI2-cexu). After the sequencing was successful, the positive integration plasmid p21-kozak-7-YLOMT was returned.

[0137] (4) The linearized p21-XPR2 backbone fragment, the fusion unidirectional promoter fragment, and the kozak-9-YLOMT fusion fragment were assembled using the Gibson assembly method at a ratio of 2:1:1, transformed into E. coli DH5α competent cells, and cultured overnight. Single colonies growing in the selection plate (50 µg / mL ampicillin antibiotic) were sent for sequencing (sequencing primers pTEFIN-cexu and tTLI2-cexu). After successful sequencing, the positive integration plasmid p21-kozak-9-YLOMT was returned. Figure 2 ).

[0138] 4. Construction of recombinant strains (1) Construction of gRNA21 plasmid (abbreviated as plasmid G21): The existing gRNA plasmid in the laboratory was linearized using primers gRNA21-F and gRNA21-R to obtain the linearized gRNA21 fragment. The above linearized gRNA21 was transformed into E. coli DH5α competent cells and cultured overnight. Single colonies that grew in the selection plate (50µg / mL ampicillin antibiotic) were sent for sequencing (sequencing primer gRNA-cexu-F). After the sequencing was successful, the positive integration plasmid gRNA21 was returned.

[0139] (2) The preparation method of Yersinia lipophila NHP-6 is the same as that of the recombinant strain ZYL-4 in CN117987286A.

[0140] (3) Using primers YL-p20-F and YL-p20-R, linearize p21-kozak-3-YLOMT, p21-kozak-7-YLOMT or p21-kozak-9-YLOMT respectively to obtain p21-kozak-3-YLOMT expression cassette, p21-kozak-7-YLOMT expression cassette or p21-kozak-9-YLOMT expression cassette.

[0141] (4) The p21-kozak-3-YLOMT expression cassette and its corresponding knock-in plasmid G21 were transformed into Yersinia lipophila NHP-6 by lithium acetate to obtain the recombinant strain NHP-8-1.

[0142] (5) The p21-kozak-7-YLOMT expression cassette and its corresponding knock-in plasmid G21 were transformed into Yersinia lipophila NHP-6 by lithium acetate to obtain the recombinant strain NHP-8-2.

[0143] (6) The p21-kozak-9-YLOMT expression cassette and its corresponding knock-in plasmid G21 were transformed into Yersinia lipophila NHP-6 by lithium acetate to obtain the recombinant strain NHP-8-3.

[0144] (7) 30 resistant clones of the above recombinant strains were randomly selected, genomic DNA was extracted, and PCR verification was performed (verification primers YZ-p20-F and YZ-p20-R) to screen out positive transformants.

[0145] 5. Fermentation verification (1) Streak the recombinant strains NHP-8-1, NHP-8-2 and NHP-8-3 on YPD plates to activate them. Pick single clones into test tubes containing 4 mL of YPD medium and incubate at 30℃ and 220 rpm for 48 h to obtain seed culture of recombinant strains NHP-8-1, NHP-8-2 and NHP-8-3 respectively.

[0146] (2) The seed cultures of recombinant strains NHP-8-1, NHP-8-2, and NHP-8-3 were inoculated into 30 mL of fermentation medium (YNB medium with 2% glucose added as a carbon source) at an inoculation ratio of 1% v / v. Three parallel tubes were set up, with Yersinia lipolytica NHP-6 as the control group. Fermentation was carried out at 30℃ and 220 rpm for 4 days.

[0147] (3) After the fermentation culture is completed, take 500 μL of fermentation broth, add an equal volume of anhydrous methanol and mix thoroughly. Sonicate for 30 min, centrifuge at 12000 rpm for 15 min, take the supernatant for high performance liquid chromatography analysis, and detect the residual amount of sennaol and the yield of neohesperidin.

[0148] The measurement results are shown in Table 4: Table 4

[0149] The results showed that the recombinant strain NHP-8-3 produced 358 mg / mL of neohesperidin and had a low residue of 128 mg / mL of senna. Compared with the control Yersinia lipolytica NHP-6, the recombinant strain NHP-8-3 showed a 16.6% increase in neohesperidin production and a 22.0% decrease in senna residue.

[0150] Example 3 1. Amplify the fusion unidirectional promoter fragment The fused unidirectional promoter fragment was obtained by referring to the method in Example 1.

[0151] 2. Assembly of the fusion fragment of the homologous arm -MTS-OMT-kozak9-YLOMT (1) Using the methyltransferase (YLOMT) gene as a template, a first round of amplification was performed with primers koazk9-II-F and YlOMT-tLIP2-R to obtain the kozak9-YLOMT fusion fragment; a second round of amplification was performed with primers MTS-OMT-II-F and YlOMT-tLIP2-R to obtain the MTS-OMT-kozak9-YLOMT fragment; a third round of amplification was performed with primers MTS-pTEFIN-F and YlOMT-tLIP2-R to obtain the homologous arm-MTS-OMT-kozak9-YLOMT fusion fragment.

[0152] (2) The plasmid p20-AXP was linearized using primers Gibson-F and Gibson-R to obtain the linearized p20-AXP backbone fragment.

[0153] (3) The linearized p20-AXP backbone fragment, the fusion unidirectional promoter fragment, and the homologous arm-MTS-OMT-kozak9-YLOMT fusion fragment were assembled using the Gibson assembly method at a ratio of 2:1:1, transformed into E. coli DH5α competent cells, and cultured overnight. Single colonies growing in the selection plate (50 µg / mL ampicillin antibiotic) were sent for sequencing (sequencing primers pTEFIN-cexu and tTLI2-cexu). After successful sequencing, the positive integration plasmid p20-MTS-OMT-kozak9-YLOMT was returned. Figure 3 ).

[0154] 3. Construction of recombinant strains (1) p20-(AXP)-MTS-OMT-kozak9-YLOMT was linearized using primers YL-p20-F and YL-p20-R to obtain the p20-(AXP)-MTS-OMT-kozak9-YLOMT expression cassette.

[0155] (2) The p20-(AXP)-MTS-OMT-kozak9-YLOMT expression cassette and its corresponding knock-in plasmid G20 were transformed into Yersinia lipophila NHP-6 by lithium acetate to obtain the recombinant strain NHP-9.

[0156] (3) 30 resistant clones of the above recombinant strain NHP-9 were randomly selected, genomic DNA was extracted, and PCR verification was performed (verification primers YZ-p20-F and YZ-p20-R) to screen out positive transformants.

[0157] The recombinant strain NHP-9 obtained by the above method was named YO1 and deposited at the China General Microbiological Culture Collection Center with accession number CGMCC NO.34491.

[0158] 4. Fermentation verification (1) Streak the recombinant strain NHP-9 on a YPD plate to activate it. Pick a single clone into a test tube containing 4 mL of YPD medium and culture it at 30℃ and 220 rpm for 48 h to obtain the seed liquid of the recombinant strain NHP-9.

[0159] (2) The seed culture of recombinant strain NHP9 was inoculated into 30 mL of fermentation medium (YNB medium with 2% glucose added as a carbon source) at an inoculation ratio of 1% v / v. Three parallel tubes were set up, with Yersinia lipolytica NHP-6 as the control group. Fermentation was carried out at 30℃ and 220 rpm for 4 days.

[0160] (3) After the fermentation culture is completed, take 500 μL of fermentation broth, add an equal volume of anhydrous methanol and mix thoroughly. Sonicate for 30 min, centrifuge at 12000 rpm for 15 min, take the supernatant for high performance liquid chromatography analysis, and detect the residual amount of sennaol and the yield of neohesperidin.

[0161] Results: The residue of sennaol was 107.2 mg / mL, and the yield of neohesperidin was 412.1 mg / mL.

[0162] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A recombinant plasmid, characterized in that, The recombinant plasmid includes a fusion fragment, which contains a coding sequence for a mitochondrial localization signal peptide, a Kozak sequence, and a YLOMT coding gene; The amino acid sequence of the mitochondrial localization signal peptide is shown in SEQ ID NO.36; The nucleotide sequence of the Kozak sequence is shown in SEQ ID NO.37; The sequence of the YLOMT encoding gene is shown in SEQ ID NO.

38.

2. The recombinant plasmid according to claim 1, characterized in that, The fusion fragment also includes a homologous arm sequence and a terminator.

3. The recombinant strain according to claim 2, characterized in that, The terminator is selected from any one or more of the following: tLIP2 terminator, tXPR2 terminator, tLEU2 terminator, tURA3 terminator, and tICL1 terminator.

4. The recombinant strain according to claim 3, characterized in that, The terminator is the tLIP2 terminator.

5. The recombinant plasmid according to claim 1, characterized in that, The recombinant plasmid also includes a promoter fragment, wherein the promoter is selected from any one or more of the TEFIN promoter, TEF1 promoter, LIP2 promoter, and XPR2 promoter.

6. The recombinant strain according to claim 5, characterized in that, The promoter is the TEFIN promoter, which is the endogenous strong promoter of Yersinia lipophila.

7. The recombinant plasmid according to claim 1, characterized in that, The method for preparing the recombinant plasmid includes: cloning and constructing the promoter fragment, fusion fragment and linearized vector backbone fragment, transforming them into competent cells, screening for positive integration plasmids, and obtaining the recombinant plasmid.

8. The recombinant plasmid according to claim 7, characterized in that, The linearized vector backbone fragments are derived from plasmids p20-AXP, p21-XPR, pUC19, pBluescript II KS (+), pET-28a (+), or pGEM-T Easy.

9. The recombinant plasmid according to claim 8, characterized in that, The linearized vector backbone fragment is derived from plasmid p20-AXP or p21-XPR.

10. The recombinant plasmid according to claim 7, characterized in that, The competent cells are selected from any one or more of TG1 competent cells, DH-5α competent cells, and JM109 competent cells.

11. A recombinant bacterial strain, characterized in that, The recombinant strain genome integrates a foreign expression cassette, which is prepared by linearizing the recombinant plasmid according to any one of claims 1-10.

12. The recombinant strain according to claim 11, characterized in that, The steps of the method for preparing the recombinant strain include: co-transforming the exogenous expression cassette and gRNA plasmid into Yersinia lipolyticis chassis cells, achieving genome integration through homologous recombination, screening for positive transformants, and obtaining the recombinant strain.

13. The recombinant strain according to claim 12, characterized in that, The recombinant strain is a lipophilic yeast strain ( Yarrowia lipolytica) YO1, with accession number CGMCC NO.34491.

14. A genetically engineered preparation, characterized in that, The genetically engineered preparation comprises one or more of the following: culture of the recombinant strain as described in any one of claims 11-13, culture extract, cell fragments, bacterial cells, fermentation broth, fermentation broth precipitate, and lyophilized powder.

15. The application of the recombinant plasmid according to any one of claims 1-10, the recombinant strain according to claims 11-13, or the gene-engineered preparation according to claim 14, characterized in that, The applications include any one or more of the following: (1) Application in enhancing methyltransferase activity; (2) Application in the production of new hesperidin.

16. A method for increasing methyltransferase activity, characterized in that, The method includes using the recombinant plasmid of any one of claims 1-10, the recombinant strain of claims 11-13, or the genetically engineered preparation of claim 14.

17. A method for producing novel hesperidin, characterized in that, The method includes using the recombinant plasmid of any one of claims 1-10, the recombinant strain of claims 11-13, or the genetically engineered preparation of claim 14.

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