Nucleic acid molecules, enzyme mutants encoded thereby, recombinant vectors containing the same, engineered microorganisms and methods of construction, methods of betulinic acid production

Abstract

The application belongs to the technical field of synthetic biology, metabolic engineering and industrial microorganism, and particularly relates to a nucleic acid molecule, an enzyme mutant coded by the nucleic acid molecule, a recombinant vector containing the nucleic acid molecule, an engineered microorganism and a construction method, and a production method of betulinic acid. The nucleic acid molecule codes an enzyme mutant, and the enzyme mutant has one or more of the following mutations: T293A, L357A, F117A and P110A, relative to an enzyme with an amino acid sequence shown in SEQ ID NO. 1. The nucleic acid molecule is expressed in an engineered microorganism, and particularly further combined with a lupinol synthase and a P450, so that the yield of betulinic acid can be obviously improved. The technical route is clear, the effect is stable, and the technical route is suitable for the efficient synthesis and industrial development of betulinic acid.

Description

Technical Field

[0001] This application belongs to the fields of synthetic biology, metabolic engineering and industrial microbiology, specifically relating to nucleic acid molecules, enzyme mutants encoded by them, recombinant vectors containing them, engineered microorganisms and their construction methods, and methods for producing betulinic acid. Background Technology

[0002] Betulinic acid (BA) is a natural lupinane-type pentacyclic triterpenoid compound, also known as betulinic acid, with the chemical formula C0. 30 H 48 O3. Betulinic acid is widely distributed in natural plants such as Syzygium aromaticum leaves, birch bark, and jujube seeds. It possesses various biological and pharmacological activities, including anti-inflammatory, antibacterial, antitoxic, antidiabetic, antimalarial, anti-HIV, and antitumor effects, making it highly valuable for clinical use. Studies have shown that betulinic acid can exhibit significant toxicity to various tumor cells, including lung cancer, colon cancer, breast cancer, melanoma, and chronic myeloid leukemia, effectively inhibiting their growth with minimal side effects. Despite the enormous potential of betulinic acid in clinical applications, the insufficient supply of betulinic acid (BA) in its natural host is a major challenge to the commercialization of this compound.

[0003] Traditional plant extraction methods suffer from problems such as limited raw material sources, low extraction efficiency, high costs, and heavy environmental burden. Therefore, achieving green bio-manufacturing of betulinic acid is of great significance. Summary of the Invention

[0004] Based on this, one or more embodiments of this application provide nucleic acid molecules, enzyme mutants encoded by them, recombinant vectors containing them, engineered microorganisms and construction methods, and methods for producing betulinic acid. The technical solutions include the following:

[0005] One or more embodiments of this application provide an enzyme mutant having one or more of the following mutations relative to the enzyme with the amino acid sequence shown in SEQ ID NO.1: T293A, L357A, F117A, and P110A.

[0006] In some embodiments of this application, the enzyme mutant has at least two of the following mutations: T293A, L357A, F117A, and P110A.

[0007] In some embodiments of this application, the enzyme mutant has any of the following combinations of mutations:

[0008] Mutation combination 1 consists of T293A and L357A;

[0009] Mutation combination 2 consists of T293A, L357A, and F117A;

[0010] Mutation combination 3 consists of T293A, L357A, F117A, and P110A.

[0011] One or more embodiments of this application provide a nucleic acid molecule that encodes the enzyme mutant.

[0012] One or more embodiments of this application provide a recombinant vector, wherein the nucleotide sequence of the recombinant vector contains the nucleotide sequence of the nucleic acid molecule.

[0013] One or more embodiments of this application provide an engineered microorganism that satisfies the following conditions (A1) or (A2):

[0014] (A1) Contains the nucleic acid molecule or the recombinant vector, or the target enzyme expressed by the engineered microorganism includes the enzyme mutant;

[0015] (A2) The target enzymes expressed by the engineered microorganisms include lupin alcohol synthase, cytochrome P450 and cytochrome P450 oxidoreductase; wherein the species source of lupin alcohol synthase is olive, the species source of cytochrome P450 is periwinkle, and the species source of cytochrome P450 oxidoreductase is Arabidopsis or grape.

[0016] In some embodiments of this application, the engineered microorganisms satisfy any one of the conditions shown in (B1) to (B2) below:

[0017] (B1) The engineered microorganisms include Saccharomyces cerevisiae;

[0018] (B2) If the engineered microorganism meets the conditions shown in (A1), then the target enzyme expressed by the engineered microorganism further includes the expression of lupin alcohol synthase and / or cytochrome P450 oxidoreductase; if the engineered microorganism meets the conditions shown in (A2), then the cytochrome P450 is encoded by the nucleic acid molecule.

[0019] In some embodiments of this application, the engineered microorganisms satisfy the conditions shown in (A1), the species source of the lupin alcohol synthase is olive, and the species source of the cytochrome P450 oxidoreductase is Arabidopsis thaliana or grape.

[0020] One or more embodiments of this application provide a method for constructing the engineered microorganisms described above, the method comprising the following steps:

[0021] The recombinant vector expressing the target enzyme is introduced into the microorganism to be modified, thereby constructing the engineered microorganism.

[0022] One or more embodiments of this application provide a method for producing betulinic acid, the method comprising the following steps:

[0023] The engineered microorganisms were fermented and cultured; and betulinic acid was isolated from the resulting fermentation product.

[0024] Compared to traditional technologies, this application offers the following advantages: This application provides a nucleic acid molecule encoding a specific mutant of CrCYP716A154. Expression of this nucleic acid molecule in engineered microorganisms, particularly in combination with lupin synthase and P450, significantly increases betulinic acid yield. This technical route is well-defined and provides stable results, making it suitable for the efficient microbial synthesis and industrialization of betulinic acid. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 A schematic diagram showing the yield of lupeol produced by lupeol synthase from different plant sources.

[0027] Figure 2 A schematic diagram showing the yield of betulinic acid catalyzed by C28-position oxidases of P450 from different plant sources.

[0028] Figure 3 This is a schematic diagram showing the effect of cytochrome P450 reductase CPR from different plant sources on the synthesis of betulinic acid catalyzed by P450.

[0029] Figure 4 A schematic diagram showing the results of the synthesis of betulinic acid catalyzed by different CrCYP716A154 mutants.

[0030] Figure 5 A schematic diagram illustrating the construction process of the S4-8 high-yield betulinic acid yeast chassis.

[0031] Figure 6 Chromatograms for GC-MS detection of lupeol, betulinol and betulinic acid in S4-8 yeast fermentation broth.

[0032] Figure 7 A schematic diagram of the mass spectrum of lupinol in S4-8 yeast fermentation broth detected by GC-MS.

[0033] Figure 8A schematic diagram of the betulinol mass spectrum detected by GC-MS in the fermentation broth of S4-8 yeast.

[0034] Figure 9 A schematic diagram of the mass spectrum of betulinic acid in S4-8 yeast fermentation broth detected by GC-MS. Detailed Implementation

[0035] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0037] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0038] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0039] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0040] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0041] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0042] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0043] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0044] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0045] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0046] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0047] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0048] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0049] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0050] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0051] Saccharomyces cerevisiae, as a model eukaryotic microorganism, possesses advantages such as a clear genetic background, mature metabolic engineering tools, well-developed fermentation processes, and strong industrial adaptability, making it an important host for constructing cell factories for triterpenoid natural products. The biosynthesis of betulinic acid typically begins with 2,3-oxidized squalene as a precursor, which is catalyzed by lupin synthase to produce lupeol, followed by continuous oxidation catalyzed by cytochrome P450 to betulinic acid. However, significant differences in enzyme activity and interaction adaptation exist among lupin synthases, P450s, and CPRs from different plant sources, resulting in generally low betulinic acid synthesis efficiency in Saccharomyces cerevisiae. Therefore, screening for the optimal combination of lupin synthase, P450, and CPR suitable for Saccharomyces cerevisiae expression systems, and further enhancing the catalytic efficiency of the key P450 through protein engineering, are crucial technical issues for improving the biosynthesis level of betulinic acid.

[0052] In a first aspect, this application provides an enzyme mutant, which, relative to an enzyme with an amino acid sequence as shown in SEQ ID NO.1 or an enzyme with an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with SEQ ID NO.1, has one or more of the following mutations: T293A, L357A, F117A, and P110A.

[0053] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, in optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made when aligning two sequences to give the maximum percentage of identity. For example, comparisons can be performed using the BLAST algorithm, where the algorithm's parameters are chosen to give the maximum match between the sequences over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used in sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al., (1990) J.Mol.Biol.215:403-410; Gish, W. et al., (1993) NatureGenet.3:266-272; Madden, T.L. et al., (1996) Meth.Enzymol.266:131-141; Altschul, S.F. et al., (1997) Nucleic Acids Res.25:3389-3402; Zhang, J. et al., (1997) Genome Res.7:649-656. Other common BLAST algorithms, such as those provided by NCBI BLAST, are also well-known to those skilled in the art.

[0054] In some examples of this application, the enzyme mutant has at least two (e.g., 2, 3, or 4) of the following mutations: T293A, L357A, F117A, and P110A.

[0055] In some examples of this application, the enzyme mutant has any of the following combinations of mutations:

[0056] Mutation combination 1 consists of T293A and L357A;

[0057] Mutation combination 2 consists of T293A, L357A, and F117A;

[0058] Mutation combination 3 consists of T293A, L357A, F117A, and P110A.

[0059] In some examples of this application, the amino acid sequence of the enzyme mutant is as shown in any one of SEQ ID NO. 2 to 8, or has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with any of these sequences.

[0060] A second aspect of this application provides a nucleic acid molecule that encodes the enzyme mutant.

[0061] As used herein, the term "nucleic acid molecule" refers to DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0062] The nucleic acid molecules in this application primarily refer to isolated nucleic acid molecules. "Isolated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds described herein.

[0063] A third aspect of this application provides a recombinant vector, wherein the nucleotide sequence of the recombinant vector contains the nucleotide sequence of the nucleic acid molecule.

[0064] The term "vector," also known as "expression vector," refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and augmented mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-augmented mammalian vectors). In general, vectors may be selected from, but are not limited to, mammalian cell viruses, bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, or combinations thereof.

[0065] A fourth aspect of this application provides an engineered microorganism that satisfies the following conditions (A1) or (A2):

[0066] (A1) Contains the nucleic acid molecule or the recombinant vector, or the target enzyme expressed by the engineered microorganism includes the enzyme mutant;

[0067] (A2) The target enzymes expressed by the engineered microorganisms include lupin alcohol synthase, cytochrome P450, and cytochrome P450 oxidoreductase; wherein the species source of the lupin alcohol synthase is olive, the species source of the cytochrome P450 is periwinkle, and the species source of the cytochrome P450 oxidoreductase is Arabidopsis thaliana or grape. In some examples of this application, the engineered microorganisms satisfy any one of the conditions shown in (B1) to (B2) below:

[0068] (B1) The engineered microorganisms include Saccharomyces cerevisiae;

[0069] (B2) If the engineered microorganism meets the conditions shown in (A1), then the target enzyme expressed by the engineered microorganism further includes the expression of lupin alcohol synthase and / or cytochrome P450 oxidoreductase; if the engineered microorganism meets the conditions shown in (A2), then the cytochrome P450 is encoded by the nucleic acid molecule.

[0070] In some examples of this application, the engineered microorganisms meet the conditions shown in (A1), the species source of the lupin alcohol synthase is olive, and the species source of the cytochrome P450 oxidoreductase is Arabidopsis thaliana or grape.

[0071] In some examples of this application, the engineered microorganisms satisfy the following conditions:

[0072] rDNA sites integrate AiSQE, SptHMGR, OeOEW, VvCPR, and the aforementioned nucleic acid molecules;

[0073] AiSQE, VvCPR, and the nucleic acid molecule are integrated at the TY3 site;

[0074] SptHMGR1, TAL1, GND1, ZWF1, POS5 and TKL1 are integrated at the Deleta site;

[0075] The DOS2 site integrates Cas9, VvCPR, AiSQE, OeOEW, and the aforementioned nucleic acid molecule;

[0076] The X2 site integrates and fuses VvCPR, ERG10, ERG9, ERG13, OeOEW of the peroxisome localization signal peptide (sequence including but not limited to SEQ ID NO.61) and the nucleic acid molecule thereon;

[0077] The N-terminus of ERG7 is inserted with a ubiquitination tag Ubi4 and the protein degrader shown in SEQ ID NO. 62;

[0078] Overexpression of EfMvaE, EfMvaS, Erg12, Erg8, IDI1, SptHMGR, Erg19, and Erg20; knockout of SSM4, ROX1, LPP1, and DPP1.

[0079] A fifth aspect of this application provides a method for constructing the engineered microorganisms described above, the method comprising the following steps:

[0080] The recombinant vector expressing the target enzyme is introduced into the microorganism to be modified, thereby constructing the engineered microorganism.

[0081] A sixth aspect of this application provides a method for producing betulinic acid, the method comprising the following steps:

[0082] The engineered microorganisms are fermented and cultured; and,

[0083] Betulinic acid was isolated from the obtained fermentation products.

[0084] In some examples of this application, the fermentation culture includes steps (C1) and (C2):

[0085] (C1) The engineered microorganisms are inoculated into the amplification medium and amplified to OD. 600 Once the preset values ​​are reached, the seed solution is prepared.

[0086] (C2) The seed culture is inoculated into a fermentation medium and fermented.

[0087] Optionally, in step (C1), the amplification medium is suitable for the proliferation of Saccharomyces cerevisiae, including but not limited to uracil-deficient yeast culture media.

[0088] Optionally, in step (C1), the preset value refers to OD. 600 It should reach between 0.8 and 1.2, for example, 0.8, 0.9, 1, 1.1, 1.2.

[0089] Optionally, in step (C2), the fermentation medium is suitable for the metabolism of *Saccharomyces cerevisiae*, including but not limited to fermentation media containing glucose, galactose, and uracil-deficient yeast media. Optionally, the concentration of glucose in the fermentation medium can be, but is not limited to, 1.0 g / L to 2.0 g / L, for example, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 g / L. Optionally, the concentration of galactose in the fermentation medium can be, but is not limited to, 15 g / L to 18 g / L, for example, 15, 16, 17, or 18 g / L.

[0090] Optionally, in step (C2), the fermentation culture can adopt conditions suitable for the metabolism of Saccharomyces cerevisiae, including but not limited to: a temperature of 28℃~32℃, for example 28, 29, 30, 31, 32℃; a rotation speed of 180 rpm~220 rpm, for example 180, 185, 190, 195, 200, 205, 210, 215, 220 rpm; and a time of 48 h~96 h, for example 48, 60, 72, 84, 96 h.

[0091] In some examples of this application, the fermentation culture includes the following steps (D1) and (D2):

[0092] (D1) Provide the seed culture of the engineered microorganism;

[0093] (D2) The seed liquid is inoculated into the fermentation medium and the first stage fermentation and the second stage fermentation are carried out in sequence; the temperature of the first stage fermentation is 29.5℃~30.5℃, and the temperature of the second stage fermentation is 27.5℃~28.5℃; wherein, the time of inoculation is recorded as 0 h, the first stage fermentation is from 0 h to 24 h, and the second stage fermentation is after 24 h. The temperatures for the first stage of fermentation are, for example, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30, 30.1, 30.2, 30.3, 30.4, and 30.5℃, and the temperatures for the second stage of fermentation are 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8, 27.9, 28, 28.1, 28.2, 28.3, 28.4, and 28.5℃.

[0094] Optionally, in step (D2), the fermentation medium comprises: 20 g / L to 30 g / L yeast extract, 10 g / L to 20 g / L peptone, 20 g / L to 30 g / L glucose, 0.05 wt% to 0.15 wt% K2HPO4, 0.8 wt% to 1.2 wt% KH2PO4, 0.05 wt% to 0.15 wt% MgSO4·7H2O, 0.08 wt% to 0.12 wt% NaCl, and water. The concentrations of yeast extract in the fermentation medium are, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 g / L; the concentrations of peptone are, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 g / L; the concentrations of glucose are, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 g / L; the concentrations of K₂HPO₄ are, for example, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%, 0.13 wt%, 0.14 wt%, and 0.15 wt%; and the concentrations of KH₂PO₄ are, for example, 0.8 wt%, 0.9 wt%, 1 wt%, and 1.1 wt%. The concentrations of MgSO4·7H2O are, for example, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%, 0.13 wt%, 0.14 wt%, and 0.15 wt%, and the concentrations of NaCl are, for example, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, and 0.12 wt%.

[0095] Optionally, in step (D2), the fermentation process maintains a pH of 5.3 to 5.7 (e.g., 5.3, 5.4, 5.5, 5.6, 5.7) and dissolved oxygen at an air saturation of 25% to 35% (e.g., 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%).

[0096] Optionally, in step (D2), during fermentation, under the condition that the glucose concentration in the obtained fermentation broth drops to below 2 g / L (e.g., 0, 0.2, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 g / L), an inducer is added to induce and ethanol is added to produce betulinic acid.

[0097] Further optionally, the inducer includes D-(+)-galactose; optionally, the final concentration of the inducer in the fermentation broth is 15 g / L to 25 g / L, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 g / L;

[0098] Further optionally, ethanol is added in the form of anhydrous ethanol; optionally, ethanol is added to maintain the final concentration of ethanol in the resulting fermentation system below 10 g / L (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g / L).

[0099] Optionally, in step (D2), during the fermentation process, yeast extract is added to the fermentation system at 11 h~13 h, 23 h~25 h or / and 47 h~49 h; further optionally, the final concentration of the yeast extract in the obtained fermentation system is 18~23 g / L, for example 18, 19, 20, 21, 22, 23 g / L.

[0100] Optionally, in step (D2), the aeration rate of the first stage fermentation is set to 1.4 vvm to 1.6 vvm (e.g., 1.4, 1.5, 1.6 vvm), and the stirring speed is set to 350 rpm to 450 rpm (e.g., 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450 rpm).

[0101] Optionally, in step (D2), the aeration rate of the first stage fermentation is set to 1.9 vvm to 2.1 vvm, and the stirring speed is set to 750 rpm to 850 rpm (e.g., 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850 rpm).

[0102] Optionally, in step (D2), the total fermentation time is 160 h to 178 h, for example, 160, 162, 168, 170, 172, 174, 176, or 178 h.

[0103] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0104] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0105] This application belongs to the field of synthetic biology and microbial metabolic engineering technology, specifically relating to an engineered Saccharomyces cerevisiae and its method and application for producing betulinic acid. This application first introduces lupin alcohol synthases from olive (Olea europaea), dandelion (Taraxacum mongolicum), bruguiera gymnorhiza, lettuce (Lactuca sativa), Celastrus orbiculatus, Rhizophora stylosa, Arabidopsis thaliana, and Artemisia annua into Saccharomyces cerevisiae in plasmid form for screening, determining that the lupin alcohol synthase from olive has the optimal ability to produce lupinol. Subsequently, using Arabidopsis-derived cytochrome P450 oxidoreductase AtCPR as a partner, cytochrome P450 from Glycyrrhizauralensis, Medicago truncatula, Catharanthus roseus, and Malus domestica were co-expressed, and the Catharanthus roseus-derived P450 was screened to find that the Catharanthus roseus-derived P450 had the best ability to produce betulinic acid. Furthermore, among lupeol-producing chassis strains, based on the aforementioned periwinkle-derived P450, co-expression of cytochrome P450 oxidoreductases with AaCPR from Artemisia annua, AtCPR1 and AtCPR2 from Arabidopsis thaliana, AiCPR from Azadirachta indica, CrCPR from periwinkle, GuCPR from Glycyrrhiza uralensis, GmCPR from soybean (Glycine max), TwCPR1 and TwCPR2 from Tripterygium wilfordii, VvCPR from Vitis vinifera, and PgCPR from Panax ginseng was conducted. Screening revealed that the Vitis vinifera-derived CPR showed the best effect in promoting betulinic acid synthesis. Finally, the periwinkle-derived P450 was mutated to further increase betulinic acid production. The engineered brewer's yeast constructed in this application has the advantages of high betulinic acid synthesis efficiency, clear combination, and easy industrial scale-up, and can be used for the biomanufacturing of betulinic acid.

[0106] Example 1: Screening of lupin alcohol synthases from different plant sources

[0107] I. Sources of Candidate Genes

[0108] Lupin alcohol synthase genes were selected from the following plants:

[0109] Using fresh olives (Olea europaea), dandelion (Taraxacum mongolicum), tree olive (Bruguiera gymnorhiza), lettuce (Lactuca sativa), Celastrus orbiculatus, Rhizophora stylosa, Arabidopsis thaliana, and Artemisia annua as materials, RNA was extracted and cDNA was obtained by reverse transcription. Design primers for the following genes: OeOEW (AB025343.1), ToTRW (AB025345.1), BgLUS (AB289586.1), CoOSC7 (MT832730.1), RsM1 (AB263203.1), AaLUS (KM670094.1), AtLUP1 (NM_001334865), and LsOSC5 (MN107546.1). Amplify the above fragments.

[0110] II. Construction of Expression Carrier

[0111] The aforementioned lupin alcohol synthase gene was ligated into the Saccharomyces cerevisiae expression vector pESC-ura using seamless ligation technology. This vector carries the promoter Gal1 and the terminator Cyc1. Taking OeOEW ligation as an example, OeOEW amplification primers F1 and R1 were designed, and the OeOEW fragment was amplified using Novizuma Taq Plus Master Mix. Reverse amplification primers F2 (SEQ ID NO.25): ATCCGCTCTAACCGAAAAGGAAG, and R2 (SEQ ID NO.26): GTTTTTCTCTCTTGACGTTAAAGTATAGAGG were designed to amplify the pESC-ura vector template. The two fragments were ligated using the Novizuma ClonExpressUltraOneStepCloning Kit and transformed into competent DH5α cells in the coli, yielding positive clones containing pESC-ura-Pgal1-OeOEW-Tcyc1.

[0112] Similarly, using the above method, a total of 7 recombinant expression plasmids were obtained: pESC-ura-Pgal1-ToTRW-Tcyc1, pESC-uraPgal1-BgLUS-Tcyc1, pESC-ura-Pgal1-CoOSC7-Tcyc1, pESC-ura-Pgal1-RsM1-Tcyc1, pESC-ura-Pgal1-AaLUS-Tcyc1, pESC-ura-Pgal1-AtLUP1-Tcyc1, and pESC-ura-Pgal1-LsOSC5-Tcyc1.

[0113] The amplification primers are as follows:

[0114] Table 1 Primers for amplifying lupin alcohol cyclase from different plant sources

[0115]

[0116] The PCR amplification system is as follows:

[0117] Table 2 PCR amplification system

[0118]

[0119] The PCR amplification procedure is as follows:

[0120] Table 3 PCR amplification program

[0121]

[0122] III. Host Strains and Transformation

[0123] 1. Preparation of competent cells (to prepare sufficient competent cells for 8 bacterial strains simultaneously)

[0124] A single colony of CEN.PK2-1D was picked from a fresh YPD plate and inoculated into 3 mL of YPD liquid. The colony was incubated overnight at 30°C and 200 rpm. The next day, it was transferred to 50 mL of YPD (initial OD). 600 (≈ 0.1~0.2), incubate at 30℃ and 200 rpm until OD 600=0.5~0.8 (logarithmic growth phase, approximately 4~6 h). Collect the bacterial culture, centrifuge at 4000 rpm for 5 min at 4℃, and discard the supernatant. Resuspend and wash with 30 mL of sterile deionized water, centrifuge (4000 rpm, 5 min), and discard the supernatant. Resuspend with 1.5 mL of 100 mM LiAc, transfer to a 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant. Add 1.0 mL of 100 mM LiAc to resuspend, and aliquot 100 μL / tube into sterile 1.5 mL centrifuge tubes for later use.

[0125] 2. Preparation of the conversion mixture (single reaction)

[0126] In a centrifuge tube containing 100 μL of competent cells, add the following in sequence:

[0127] Table 4 Yeast Conversion Solution System

[0128]

[0129] Gently whisk to mix; do not shake violently.

[0130] 3. Conversion Procedure

[0131] Incubate in a 30°C water bath for 30 min (tap gently to mix every 10 min). Heat shock in a 42°C water bath for 30 min (tap gently to mix every 10 min).

[0132] 4. Recovery and Coating

[0133] Centrifuge (12000 rpm, 15 s), discard the supernatant, and resuspend in 1 mL of YPD Plus. Recover at 30℃, 200 rpm for 60 min (significantly improves transformation efficiency). Centrifuge (12000 rpm, 15 s), discard the supernatant, and resuspend the precipitate in 100 μL of sterile water. Spread the entire sample onto selective YPD plates containing the appropriate antibiotic (e.g., G418 final concentration 200–400 μg / mL). Invert the plates in a 30℃ incubator and incubate for 2–5 days until single colonies emerge.

[0134] 5. Validation of positive clones

[0135] Single clones were picked and cultured in selective liquid medium. Genomic DNA was extracted, and the plasmid integration / presence status was verified by PCR (using plasmid-specific primers) or restriction enzyme digestion. The verified clones were named S1-1 (Oe), S1-2 (To), S1-3 (Bg), S1-4 (Co), S1-5 (Rs), S1-6 (Aa), S1-7 (At), and S1-8 (Ls), respectively.

[0136] IV. Fermentation Culture

[0137] The fermentation method in this application is as follows: Multiple single colonies are randomly picked from a yeast auxotrophic agar plate and inoculated into 400 μL of uracil-deficient yeast medium (Pangeno, catalog number YGM003A-3) for activation culture until the strains reach OD500. 600 Once the concentration reaches between 0.8 and 1.2, 200 μL of the culture medium is transferred to a liquid culture medium containing 20 mL of uracil-deficient yeast, and 100 μL of glucose and 900 μL of galactose are added to make the glucose and galactose contents in the culture medium 2 g / L and 18 g / L, respectively. Each strain is subjected to three parallel fermentations. Finally, after fermentation at 200 rpm in a shaker at 30℃ for 3 days, the bacterial solution is broken up and extracted.

[0138] V. Treatment of bacterial solution

[0139] The sample and detection method for betulinic acid and branched metabolites produced by yeast cells in this application are as follows:

[0140] After the fermentation of the recombinant Saccharomyces cerevisiae strain, terpenoid products were extracted from the fermentation broth. First, 1 mL of each fermentation broth was centrifuged at 12000 rpm for 1 min to obtain cell precipitate. 300 µL of ethyl acetate and 0.5 g of glass beads were added to the precipitate, and the mixture was homogenized at 65 Hz in a shaking grinder for 15 min to disrupt the cells. Then, 700 µL of ethyl acetate was added, the mixture was vortexed, and sonicated for 30 min. The mixture was then centrifuged at 12000 rpm for 8 min. The supernatant was filtered through a 0.22 µm organic filter membrane, and 50 µL of the filtrate was concentrated by evaporation at 60 °C in a rotary evaporator. Then, 50 µL of N,O-bis(trimethylsilyl)acetamide was added, and the mixture was reacted in a metal bath at 80 °C for 30 min to prepare the sample for GC-MS analysis.

[0141] VI. Product Testing

[0142] GC-MS chromatographic separation conditions: An HP-5 ms capillary column was used for component separation. High-purity helium was used as the carrier gas, and a constant flow rate of 1.5 mL / min was employed. The injection port temperature was set to 300℃, and the injection mode was splitless injection with a single injection volume of 1 μL. The column oven temperature program was as follows: initial column temperature 80℃, held for 1 min; linearly increased to 280℃ at a rate of 20℃ / min, held at this temperature for 15 min; finally, the temperature was increased to 300℃ and held for 5 min to complete the entire chromatographic separation process.

[0143] Mass spectrometry detection parameters: Sample ionization was performed using an electron impact ion source (EI), the mass-to-charge ratio (m / z) range for ion scanning was set to 40~600, and data acquisition was performed in multiple reaction monitoring (MRM) mode to improve the sensitivity and specificity of target component detection.

[0144] VII. Results

[0145] The results showed that, under the same fermentation conditions, lupin alcohol synthase derived from olive oil produced the highest lupin alcohol yield, at 7.42 mg / L, significantly higher than other candidate lupin alcohol synthases, such as... Figure 1 As shown. Therefore, subsequent experiments selected lupin synthase from olive as the key upstream enzyme in the betulinic acid synthesis module.

[0146] Example 2: Screening of P450 from different plant sources

[0147] I. Sources of Candidate P450

[0148] Using fresh Ural licorice (Glycyrrhiza uralensis), alfalfa (Medicago truncatula), periwinkle (Catharanthus roseus), and apple (Malus domestica) as materials, RNA was extracted and cDNA was obtained by reverse transcription, following the method in Example 1. Primers were designed to amplify the GuCYP716A179 (LC157867.1), MtCYP716A12 (DQ335781.1), CrCYP716A154 (JN565975.1), and MdCYP716A175 genes (XM_008392874) to amplify the above fragments. The amplification primers are as follows:

[0149] Table 5 Primers for amplifying P450 from different plant sources

[0150]

[0151] II. Construction of Expression Carrier

[0152] Following the procedure in Example 1, the cytochrome P450 gene was ligated into the Saccharomyces cerevisiae expression vector pESC-his, which carries the promoter Gal1 and the terminator Cyc1, resulting in four recombinant expression plasmids: pESC-his-Pgal1-GuCYP716A179-Tcyc1, pESC-his-Pgal1-MtCYP716A12-Tcyc1, pESC-his-Pgal1-CrCYP716A154-Tcyc1, and pESC-his-Pgal1-MdCYP716A175-Tcyc1.

[0153] III. Cytochrome P450 reductase CPR settings

[0154] The cytochrome P450 oxidoreductase AtCPR1 (NM001203894.1) derived from Arabidopsis thaliana was used as an electron transport chaperone. Primers F3 (SEQ ID NO.35): TTTCAAAAATTCTTACTTTTTTTTTGGATG and R3 (SEQ ID NO.36): GCGAATTTCTTATGATTTATGAT were used to back amplify pESC-ura-Gal1-P450-cyct1 of different P450 groups via primers F4 (SEQ ID NO.37): ATCATAAATCATAAGAAATTCGCTCACCAGACATCTCTGAGGTATC and R4 (SEQ ID NO.36): ATCATAAATCATAAGAAATTCGCTCACCAGACATCTCTGAGGTATC and R .... NO.38): CATCCAAAAAAAAAGTAAGAATTTTTGAAAATGACTTCTGCTTTGTATGCTTC amplifies AtCPR1. Following the method in Example 1, AtCPR1 is used to construct the pESC-ura-Gal1-P450-cyct1 plasmid. Between the Gal10 promoter and Adh1 terminator, plasmids pESC-his-Pgal1-GuCYP716A179-Tcyc1-PGal10-AtCPR1-Tadh1, pESC-his-Pgal1-MtCYP716A12-Tcyc1-PGal10-AtCPR1-Tadh1, pESC-his-Pgal1-CrCYP716A154-Tcyc1-PGal10-AtCPR1-Tadh1, and pESC-his-Pgal1- MdCYP716A175-Tcyc1-PGal10-AtCPR1-Tadh1.

[0155] IV. Construction of Recombinant Strains

[0156] The plasmid pESC-ura-Pgal1-OeOEW-Tcyc1, which produced the highest amount of lupeol, was then coupled with pESC-his-Pgal1-GuCYP716A179-Tcyc1-PGal10-AtCPR1-Tadh1, pESC-his-Pgal1-MtCYP716A12-Tcyc1-PGal10-AtCPR1-Tadh1, pESC-his-Pgal1-CrCYP716A154-Tcyc1-PGal10-AtCPR1-Tadh1, and pESC-his-Pgal1- The plasmid MdCYP716A175-Tcyc1-PGal10-AtCPR1-Tadh1 was simultaneously introduced into the laboratory-preserved Saccharomyces cerevisiae chassis strain CEN.PK2-1D using the lithium acetate conversion method, resulting in four corresponding Saccharomyces cerevisiae strains producing betulinic acid: GuC, MtC, CrC, and MdC, which are designated as S2-1 to S2-4, respectively.

[0157] V. Fermentation Culture and Bacterial Liquid Treatment

[0158] Each strain was fermented and cultured according to the fermentation method in Example 1, and the resulting bacterial broth was also treated according to the method in Example 1.

[0159] VI. Product Testing

[0160] The yield of betulinic acid in the fermentation broth of each strain was tested according to the detection method in Example 1.

[0161] VII. Results

[0162] The results showed that the periwinkle-derived P450 CrCYP716A154 had the highest betulinic acid production capacity, with a betulinic acid yield of 3.52 mg / L, which was superior to the P450 derived from Ural licorice, alfalfa, and apple. Figure 2 As shown. Therefore, CrCYP716A154 was selected as the target oxidase in subsequent experiments.

[0163] Example 3: Screening of CPR from different plant sources

[0164] I. Sources of Candidate CPRs

[0165] Following the procedure in Example 2, the recombinant expression plasmid pESC-his-Pgal1-CrCYP716A154-Tcyc1 was constructed. This recombinant expression plasmid was introduced into a lupeol-producing chassis strain, namely recombinant Saccharomyces cerevisiae Oe. In the resulting lupeol-producing chassis strain (denoted as S3-0), using CrCYP716A154 as the fixed P450 gene, the following 11 different plant-derived CPRs were co-expressed:

[0166] AaCPR from Artemisia annua, AtCPR1 and AtCPR2 from Arabidopsis thaliana, AiCPR from Azadirachta indica, CrCPR from Vinca periwinkle, GuCPR from Glycyrrhiza uralensis, GmCPR from Glycine max, TwCPR1 and TwCPR2 from Tripterygium wilfordii, VvCPR from Vitis vinifera, and PgCPR from Panax ginseng.

[0167] RNA was extracted from fresh Artemisia annua, Arabidopsis thaliana, Azadirachta indica, Catharanthus roseus, Glycyrrhizauralensis, Glycine max, Tripterygium wilfordii, Vitis vinifera, and Panax ginseng, following the method described in Example 1, and cDNA was obtained by reverse transcription. Design amplification primers for AaCPR (EF197890.1), AtCPR1 (NM001203894.1), AtCPR2 (NM119167.4), AiCPR (KM108319.1), CrCPR (X69791.1), GuCPR (KY798117.1), GmCPR (AY170374.1), TwCPR1 (MF135233.1), TwCPR2 (MF135235.1), VvCPR (XM002270696.4), and PgCPR (MT1601791.1) to amplify the above fragments.

[0168] The amplification primers are as follows:

[0169] Table 6 Primers for amplifying CPR from different plant sources

[0170]

[0171] II. Construction of expression vectors and construction of recombinant strains

[0172] Following the procedure in Example 1, the cytochrome P450 reductase CPR gene was ligated into the Saccharomyces cerevisiae expression vector pESC-trp, which carries the promoter Gal1 and the terminator Cyc1, resulting in pESC-trp-Pgal1-AaCPR-Tcyc1, pESC-trp-Pgal1-AiCPR-Tcyc1, pESC-trp-Pgal1-AtCPR1-Tcyc1, pESC-trp-Pgal1-AtCPR2-Tcyc1, and pESC-trp-Pgal1-AtCPR2-Tcyc1. Eleven recombinant expression plasmids, including p-Pgal1-CrCPR-Tcyc1, pESCtrp-Pgal1-GmCPR-Tcyc1, pESC-trp-Pgal1-GuCPR-Tcyc1, pESC-trp-Pgal1-PgCPR-Tcyc1, pESC-trp-Pgal1-TwCPR1-Tcyc1, pESC-trp-Pgal1-TwCPR2-Tcyc1, and pESC-trp-Pgal1-VvCPR-Tcyc1, were introduced into lupeol-producing chassis strains expressing CrCYP716A154, resulting in Saccharomyces cerevisiae strains AaR, AiR, At1R, At2R, CrR, GmR, GuR, PgR, Tw1R, Tw2R, and VvR, designated S3-1 to S3-11, respectively.

[0173] III. Fermentation culture and bacterial culture treatment, product detection

[0174] Each strain was fermented and cultured according to the fermentation method of Example 1, and the resulting bacterial broth was also treated according to the method of Example 1.

[0175] IV. Product Testing

[0176] The yield of betulinic acid was detected and analyzed according to the method in Example 1.

[0177] V. Results

[0178] The results showed that the combination of grape-derived VvCPR and CrCYP716A154 yielded the highest betulinic acid production, reaching 7.21 mg / L, which was significantly better than the other 10 CPRs. Figure 3 As shown, this indicates that VvCPR and CrCYP716A154 have a better electron transfer matching relationship, which can effectively improve the synthesis efficiency of betulinic acid.

[0179] Example 4: Mutation optimization of CrCYP716A154

[0180] I. Mutation Methods

[0181] 1. CrCYP716A154 was modified by site-directed mutagenesis using a full-size gold spot mutagenesis kit (catalog number: FM111-01) to construct a mutation library. The preferred mutation sites were located in the substrate recognition region.

[0182] SEQ ID NO.1 (480aa):

[0183] MEIFYVTTLLSLFVLLVSLSFHFLFYKNKSTLPGPLPPGRTGWPMVGESLQFLSAGWKGHPEKFIFDRMAKYSSNVFRSHLLGEPAAVFCGAIGNKFLFSNENKLVQAWWPDSVNKVFPSS NQTSSKEEAIKMRKMLPNFLKPEALQRYIGLMDQIAQKHFSSGWENREQVEVFPLAKNYTFWLASRLFVSVEDPIEVAKLLEPFNVLASGLISVPIDLPGTPFNRAIKASNQVRKMLISI IKQRKIDLAEGKASPTQDILSHMLLTSDENGKFMHELDIADKILGLLIGGHDTASSACTFIVKFLGELPEIYEGVYKEQMEIANSKAPGEFLNWEDIQKMKYSWNVACEVLRLAPPLQGA FREALNDFMFHGFSIPKGWKIYWSVNSTHRNPECFPDPLKFDPSRFDGSGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEKIIPNEKIVVDPMPIPEKGLPVRLYPHINA.

[0184] SEQ ID NO.2 (480aa):

[0185] MEIFYVTLLSLFVLLVSLSFHFLFYKNKSTLPGPLPPGRTGWPMVGESLQFLSAGWKGHPEKFIFDRMAKYSSNVFRSHLLGEPAAVFCGAIGNKFLFSNENKLVQAWWPDSVNKVFPSSNQTSSKEEAIKMRKMLPNFLKPEALQRYIGLMDQIAQKHFSSGWENREQVEVFPLAKNYTFWLASRLFVSVEDPIEVAKLLEPFNVLASGLISVPIDLPGTPFNRAIKASNQVRKMLISIIKQRKIDLAEGKASPTQDILSHMLLTSDENGKFMHELDIADKILGLLIGGHDAASSACTFIVKFLGELPEIYEGVYKEQMEIANSKAPGEFLNWEDIQKMKYSWNVACEVLRLAPPLQGAFREALNDFMFHGFSIPKGWKIYWSVNSTHRNPECFPDPLKFDPSRFDGSGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEKIIPNEKIVVDPMPIPEKGLPVRLYPHINA。

[0186] SEQ ID NO.3(480aa):

[0187] MEIFYVTLLSLFVLLVSLSFHFLFYKNKSTLPGPLPPGRTGWPMVGESLQFLSAGWKGHPEKFIFDRMAKYSSNVFRSHLLGEPAAVFCGAIGNKFLFSNENKLVQAWWPDSVNKVFPSSNQTSSKEEAIKMRKMLPNFLKPEALQRYIGLMDQIAQKHFSSGWENREQVEVFPLAKNYTFWLASRLFVSVEDPIEVAKLLEPFNVLASGLISVPIDLPGTPFNRAIKASNQVRKMLISIIKQRKIDLAEGKASPTQDILSHMLLTSDENGKFMHELDIADKILGLLIGGHDTASSACTFIVKFLGELPEIYEGVYKEQMEIANSKAPGEFLNWEDIQKMKYSWNVACEVLRLAPPAQGAFREALNDFMFHGFSIPKGWKIYWSVNSTHRNPECFPDPLKFDPSRFDGSGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEKIIPNEKIVVDPMPIPEKGLPVRLYPHINA。

[0188] SEQ ID NO.4(480aa):

[0189] MEIFYVTLLSLFVLLVSLSFHFLFYKNKSTLPGPLPPGRTGWPMVGESLQFLSAGWKGHPEKFIFDRMAKYSSNVFRSHLLGEPAAVFCGAIGNKFLFSNENKLVQAWWPDSVNKVAPSSNQTSSKEEAIKMRKMLPNFLKPEALQRYIGLMDQIAQKHFSSGWENREQVEVFPLAKNYTFWLASRLFVSVEDPIEVAKLLEPFNVLASGLISVPIDLPGTPFNRAIKASNQVRKMLISIIKQRKIDLAEGKASPTQDILSHMLLTSDENGKFMHELDIADKILGLLIGGHDTASSACTFIVKFLGELPEIYEGVYKEQMEIANSKAPGEFLNWEDIQKMKYSWNVACEVLRLAPPLQGAFREALNDFMFHGFSIPKGWKIYWSVNSTHRNPECFPDPLKFDPSRFDGSGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEKIIPNEKIVVDPMPIPEKGLPVRLYPHINA。

[0190] SEQ ID NO.5(480aa):

[0191] MEIFYVTLLSLFVLLVSLSFHFLFYKNKSTLPGPLPPGRTGWPMVGESLQFLSAGWKGHPEKFIFDRMAKYSSNVFRSHLLGEPAAVFCGAIGNKFLFSNENKLVQAWWADSVNKVFPSSNQTSSKEEAIKMRKMLPNFLKPEALQRYIGLMDQIAQKHFSSGWENREQVEVFPLAKNYTFWLASRLFVSVEDPIEVAKLLEPFNVLASGLISVPIDLPGTPFNRAIKASNQVRKMLISIIKQRKIDLAEGKASPTQDILSHMLLTSDENGKFMHELDIADKILGLLIGGHDTASSACTFIVKFLGELPEIYEGVYKEQMEIANSKAPGEFLNWEDIQKMKYSWNVACEVLRLAPPLQGAFREALNDFMFHGFSIPKGWKIYWSVNSTHRNPECFPDPLKFDPSRFDGSGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEKIIPNEKIVVDPMPIPEKGLPVRLYPHINA。

[0192] SEQ ID NO.6(480aa):

[0193] MEIFYVTLLSLFVLLVSLSFHFLFYKNKSTLPGPLPPGRTGWPMVGESLQFLSAGWKGHPEKFIFDRMAKYSSNVFRSHLLGEPAAVFCGAIGNKFLFSNENKLVQAWWPDSVNKVFPSSNQTSSKEEAIKMRKMLPNFLKPEALQRYIGLMDQIAQKHFSSGWENREQVEVFPLAKNYTFWLASRLFVSVEDPIEVAKLLEPFNVLASGLISVPIDLPGTPFNRAIKASNQVRKMLISIIKQRKIDLAEGKASPTQDILSHMLLTSDENGKFMHELDIADKILGLLIGGHDAASSACTFIVKFLGELPEIYEGVYKEQMEIANSKAPGEFLNWEDIQKMKYSWNVACEVLRLAPPAQGAFREALNDFMFHGFSIPKGWKIYWSVNSTHRNPECFPDPLKFDPSRFDGSGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEKIIPNEKIVVDPMPIPEKGLPVRLYPHINA。

[0194] SEQ ID NO.7(480aa):

[0195] MEIFYVTLLSLFVLLVSLSFHFLFYKNKSTLPGPLPPGRTGWPMVGESLQFLSAGWKGHPEKFIFDRMAKYSSNVFRSHLLGEPAAVFCGAIGNKFLFSNENKLVQAWWPDSVNKVAPSSNQTSSKEEAIKMRKMLPNFLKPEALQRYIGLMDQIAQKHFSSGWENREQVEVFPLAKNYTFWLASRLFVSVEDPIEVAKLLEPFNVLASGLISVPIDLPGTPFNRAIKASNQVRKMLISIIKQRKIDLAEGKASPTQDILSHMLLTSDENGKFMHELDIADKILGLLIGGHDAASSACTFIVKFLGELPEIYEGVYKEQMEIANSKAPGEFLNWEDIQKMKYSWNVACEVLRLAPPAQGAFREALNDFMFHGFSIPKGWKIYWSVNSTHRNPECFPDPLKFDPSRFDGSGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEKIIPNEKIVVDPMPIPEKGLPVRLYPHINA。

[0196] SEQ ID NO.8(480aa):

[0197] MEIFYVTTLLSLFVLLVSLSFHFLFYKNKSTLPGPLPPGRTGWPMVGESLQFLSAGWKGHPEKFIFDRMAKYSSNVFRSHLLGEPAAVFCGAIGNKFLFSNENKLVQAWWADSVNKVAPSS NQTSSKEEAIKMRKMLPNFLKPEALQRYIGLMDQIAQKHFSSGWENREQVEVFPLAKNYTFWLASRLFVSVEDPIEVAKLLEPFNVLASGLISVPIDLPGTPFNRAIKASNQVRKMLISI IKQRKIDLAEGKASPTQDILSHMLLTSDENGKFMHELDIADKILGLLIGGHDAASSACTFIVKFLGELPEIYEGVYKEQMEIANSKAPGEFLNWEDIQKMKYSWNVACEVLRLAPPAQGA FREALNDFMFHGFSIPKGWKIYWSVNSTHRNPECFPDPLKFDPSRFDGSGPAPYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEKIIPNEKIVVDPMPIPEKGLPVRLYPHINA.

[0198] 2. Screening Strategy

[0199] The mutant was co-expressed with lupin synthase from olive and VvCPR from grape in Saccharomyces cerevisiae CEN.PK2-1D. Mutants with increased betulinic acid production were screened using primary and secondary screening methods.

[0200] 3. Selecting the best mutants

[0201] The mutants CYPM-1, CYPM-2, CYPM-3, CYPM-4, CYPM-5, CYPM-6, and CYPM-7 were obtained. Recombinant strains S4-1 to S4-7 were constructed accordingly. Among them, the recombinant strain constructed by co-expressing wild-type CrCYP716A154 with lupin alcohol synthase from olive and VvCPR from grape in Saccharomyces cerevisiae CEN.PK2-1D was designated S4-0.

[0202] Table 7 CrCYP716A154 mutants and their corresponding mutations

[0203]

[0204] Among them, the superposition of the preferred mutants yielded CYPM-7, which increased betulinic acid production by more than three times compared to the wild-type CrCYP716A154, reaching 25.7 mg / L. This mutant efficiently catalyzes the conversion of betulinol to betulinic acid.

[0205] In this embodiment, each strain was fermented and cultured according to the fermentation method of Example 1, and the resulting bacterial solution was also processed according to the method of Example 1. The yield of betulinic acid was also detected and analyzed according to the method of Example 1.

[0206] II. Results

[0207] The results showed that mutating the periwinkle-derived strain P450 CrCYP716A154 effectively enhanced its ability to catalyze the oxidation of lupeol to betulinic acid, thereby further increasing the betulinic acid yield of the engineered strain. Figure 4 As shown.

[0208] Example 5

[0209] The obtained CYPM-7 mutant was used to construct a high-yield betulinic acid yeast chassis. The following genome integration strategy was employed to gradually increase the yield of the target product, betulinic acid. Figure 5 .

[0210] (1) The gene fragments VvCPR (XM002270696.4), AiSQE (OP896241.1), OeOEW (AB025343.1), two copies of SptHMGR (WLJ58900.1) and CrCYP716A154 (CYPM-7, the nucleic acid encoding enzyme mutant shown in SEQ ID NO.8) were integrated into the rDNA site of the chromosome of the starting yeast strain (CEN.PK2-1D), and the screening tag was tryptophan Trp, to obtain yeast strain BA-1;

[0211] (2) Two copies of AiSQE, two copies of CrCYP716A154 (CYPM-7, the nucleic acid encoding the enzyme mutant shown in SEQ ID NO.8) and VvCPR (XM002270696.4) gene fragments were integrated into the TY3 site of the chromosome of yeast strain BA-1, and the selection tag was leucine (Leu) to obtain yeast strain BA-2;

[0212] (3) The gene fragments of GND1 (NC_001140.6), ZWF1 (NC_001146.8), TKL1 (NC_001148.4), POS5 (NC_001148.4), TAL1 (NC_001144.5) and SptHMGR1 from Saccharomyces cerevisiae were integrated into the Delta site of chromosome BA-2, and the screening tag was histidine His, to obtain yeast strain BA-3;

[0213] (4) The gene fragments of Cas9, VvCPR (XM002270696.4), two copies of OeOEW (AB025343.1) and AiSQE (OP896241.1), and CrCYP716A154 (CYPM-7, the nucleic acid encoding enzyme mutant shown in SEQ ID NO.8) were integrated into the DOS2 site of the chromosome of Saccharomyces cerevisiae BA-3. The selection tag was genimycin G418, and yeast strain BA-4 was obtained.

[0214] (5) In the yeast strain BA-4, the gene fragments VvCPR (XM002270696.4), OeOEW (AB025343.1), ERG9 (NC_001140.6), CrCYP716A154 (CYPM-7, the nucleic acid encoded by the enzyme mutant shown in SEQ ID NO.8), ERG10 (NC_001148.4) and ERG13 (NC_001145.3) fused with the peroxisome localization signal peptide ePTS1 (SEQ ID NO.61:TTGGGAAGAGGTAGAAGATCCAAATTGTAG), ERG10 (NC_001148.4) and ERG13 (NC_001145.3) were integrated into the X2 site of the yeast chromosome BA-5, and the screening tag was hygromycin Hyg, to obtain yeast strain BA-5;

[0215] (6) In the yeast BA-5, using CRISPR / Cas technology, the ubiquitination tag Ubi4 (NC_001144.5) and the protein degrader (SEQ ID NO.62:FHKSGAWLLPVSLV) were inserted into the N-terminus of the gene encoding lanosterol synthase ERG7 to obtain yeast strain BA-6.

[0216] (7) Overexpress the mevalonate pathway-related genes EfMvaE and EfMvaS (AF290092.1), Erg8 (NC_001145.3), Erg12 (NC_001145.3), IDI1 (NC_001148.4), SptHMGR (WLJ58900.1), Erg19 (NC_001146.8) and downstream Erg20 (NC_001142.9) in the yeast BA-6 to increase squalene production; at the same time, knock out genes SSM4, ROX1, LPP1 and DPP1 to obtain yeast strain BA-7, i.e. S4-8.

[0217] In this embodiment, strain S4-8 was fermented and cultured according to the fermentation method of Example 1. The resulting bacterial broth was also treated according to the method of Example 1. The yield of betulinic acid and other substances was also detected and analyzed according to the method of Example 1. The final betulinic acid yield of strain S4-8 was 637 mg / L. Figures 6 to 9 As shown.

[0218] Table 8 Primer sequences used for constructing strain S4-8

[0219]

[0220] Table 8 (continued)

[0221]

[0222] Table 8 (continued)

[0223]

[0224] Example 6: Production of betulinic acid by fermentation

[0225] The high-yield engineered Saccharomyces cerevisiae S4-8 strain obtained in Example 5 was used for the production of betulinum esters, as follows:

[0226] I. Seed Liquid Preparation

[0227] Preparation of primary seed culture: Select engineered *Saccharomyces cerevisiae* strains preserved on YPD slant agar. Using an inoculation loop, take a small amount of cells in a sterile environment and inoculate into a 250 mL Erlenmeyer flask containing 50 mL of optimized YPD liquid medium (containing: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, and water). Incubate at 30℃ and 200 rpm for 16–20 h until the OD of the culture reaches the target concentration. 600 Achieving a pH of 4.0 to 6.0 indicates it is considered a first-grade seed solution.

[0228] Preparation of secondary seed culture: Transfer the above primary seed culture at an inoculation rate of 10% (v / v) to a 500 mL Erlenmeyer flask containing 200 mL of optimized YPD liquid medium, and continue to incubate at 30℃ and 200 rpm for 12-16 h with shaking, until the bacterial OD of the culture is reached. 600 The pH value reaches 8.0~12.0. This secondary seed culture can then be used as the inoculum for the next step in a 1.5 L fermenter.

[0229] II. Fermentation Production

[0230] 1. A 1.5 L mechanically stirred fermenter was used for fed-batch fermentation with a loading volume of 1 L. The fermentation medium was optimized YPD medium. The components and contents of the optimized YPD medium were as follows: 25 g / L yeast extract, 15 g / L peptone, 25 g / L glucose, with added K2HPO4 0.12 wt%, KH2PO4 0.9 wt%, MgSO4·7H2O 0.1 wt%, and NaCl 0.1 wt%.

[0231] 2. The secondary seed culture was inoculated at a rate of 10% (v / v). The fermentation process employed a two-stage temperature control: maintaining 30℃ for 0–24 h (stage 1), and then lowering the temperature to 28℃ after 24 h (stage 2). Throughout the process, the pH was maintained at a constant 5.5 ± 0.2 online using 25% ammonia and 2 M phosphoric acid. Dissolved oxygen (DO) was set to 30% air saturation, controlled by a cascade of aeration rate and stirring speed. During the strain cultivation period (0–24 h), the aeration rate was set to 1.5 vvm and the stirring speed to 400 rpm; during the product synthesis stage (after 24 h), the aeration rate was set to 2.0 vvm and the stirring speed to 800 rpm. In the second stage, with the glucose concentration in the fermentation broth reduced to below 2 g / L, D-(+)-galactose at a final concentration of 20 g / L was added as an inducer, and anhydrous ethanol was used as the carbon source. Anhydrous ethanol was added at a rate of approximately 4 mL / h to maintain its final concentration below 10.0 g / L. Additionally, yeast extract solution at a final concentration of 20 g / L was added to the bioreactor at 12 h, 24 h, and 48 h. The total fermentation period was 168 h, and samples were taken periodically to detect cell density and betulinic acid production.

[0232] III. Production Detection

[0233] After fermentation, the product was extracted and the betulinic acid yield was determined according to the method in Example 1.

[0234] IV. Results

[0235] The results showed that at the end of the entire fermentation cycle, the OD of the engineered strain... 600The concentration was 120, and the betulinic acid yield reached 3.23 g / L, indicating good potential for biomanufacturing.

[0236] Example 7: Fermentation production of betulinic acid based on the CrCYP716A154 mutant

[0237] The S4-0, S4-1, S4-2, S4-3, S4-4, S4-5, S4-6 and S4-7 in Example 4 were fermented to produce betulinic acid according to the operation method of Example 6, and the product was extracted and the betulinic acid yield was determined according to the method of Example 1.

[0238] The results showed that at the end of the fermentation cycle, the yields of betulinic acid from fermentation of S4-0, S4-1, S4-2, S4-3, S4-4, S4-5, S4-6 and S4-7 were 52.2 mg / L, 67.2 mg / L, 72.6 mg / L, 55.4 mg / L, 85.7 mg / L, 189.5 mg / L, 230.6 mg / L and 289.4 mg / L, respectively.

[0239] Example 8

[0240] This embodiment is a variation of Embodiment 6, and the only difference from Embodiment 6 is that:

[0241] In step 1, the fermentation medium used contains: 20 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, with the addition of 0.15 wt% K2HPO4, 0.8 wt% KH2PO4, 0.12 wt% MgSO4·7H2O, 0.08 wt% NaCl, and water.

[0242] 2. The secondary seed culture was inoculated at a rate of 10% (v / v). The fermentation process employed a two-stage temperature control: maintaining 29.5℃ for 0–24 h (stage 1), and then lowering the temperature to 27.5℃ after 24 h (stage 2). Throughout the process, the pH was maintained at a constant 5.5 ± 0.2 online using 25% ammonia and 2 M phosphoric acid. Dissolved oxygen (DO) was set to 25% air saturation, controlled by a cascade of aeration rate and stirring speed. During the strain cultivation period (0–24 h), the aeration rate was set to 1.5 vvm and the stirring speed to 400 rpm; during the product synthesis stage (after 24 h), the aeration rate was set to 2.0 vvm and the stirring speed to 800 rpm. Under the condition that the glucose concentration of the obtained fermentation broth drops below 2 g / L, in the second stage, D-(+)-galactose with a final concentration of 15 g / L was added as an inducer, and anhydrous ethanol was used as the carbon source. Ethanol was added at a rate of approximately 4 mL / h and its concentration was maintained below 10.0 g / L. In addition, yeast extract solution with a final concentration of 20 g / L was added to the bioreactor at 12 h, 24 h, and 48 h. The total fermentation period was 168 h, and samples were taken at regular intervals to detect cell density and betulinic acid production.

[0243] The rest is the same as in Example 6.

[0244] Using the method of this embodiment, the fermentation cycles of S4-0, S4-1, S4-2, S4-3, S4-4, S-5, S4-6, S4-7, and S4-8 were completed, and the betulinic acid yields were 41.3 mg / L, 56.5 mg / L, 69.1 mg / L, 60.4 mg / L, 78.1 mg / L, 178.9 mg / L, 220.8 mg / L, 256.9 mg / L, and 2.15 g / L, respectively.

[0245] Example 9

[0246] This embodiment is a variation of Embodiment 6, and the only difference from Embodiment 6 is that:

[0247] In step 1, the fermentation medium used contains: 20 g / L yeast extract, 10 g / L peptone, 30 g / L glucose, 0.05 wt% K2HPO4, 1.2 wt% KH2PO4, 0.08 wt% MgSO4·7H2O, 0.12 wt% NaCl, and water.

[0248] 2. The secondary seed culture was inoculated at a rate of 10% (v / v). The fermentation process employed a two-stage temperature control: maintaining 30.5℃ for 0–24 h (stage 1), and then lowering the temperature to 28.5℃ after 24 h (stage 2). Throughout the process, the pH was maintained at a constant 5.5 ± 0.2 online using 25% ammonia and 2 M phosphoric acid. Dissolved oxygen (DO) was set to 35% air saturation, controlled by a cascade of aeration rate and stirring speed. During the strain cultivation period (0–24 h), the aeration rate was set to 1.5 vvm and the stirring speed to 400 rpm; during the product synthesis stage (after 24 h), the aeration rate was set to 2.0 vvm and the stirring speed to 800 rpm. Under the condition that the glucose concentration of the obtained fermentation broth drops below 2 g / L, in the second stage, D-(+)-galactose with a final concentration of 25 g / L was added as an inducer, and anhydrous ethanol was used as the carbon source. Ethanol was added at a rate of approximately 4 mL / h and its concentration was maintained below 10.0 g / L. In addition, yeast extract solution with a final concentration of 20 g / L was added to the bioreactor at 12 h, 24 h, and 48 h. The total fermentation period was 168 h, and samples were taken at regular intervals to detect cell density and betulinic acid production.

[0249] The rest is the same as in Example 6.

[0250] Using the method of this embodiment, the fermentation cycles of S4-0, S4-1, S4-2, S4-3, S4-4, S4-5, S4-6, S4-7, and S4-8 were completed, and the yields of betulinic acid obtained were 57.2 mg / L, 74.8 mg / L, 80.2 mg / L, 63.5 mg / L, 95.1 mg / L, 227.9 mg / L, 263.0 mg / L, 297.1 mg / L, and 3.52 g / L, respectively.

[0251] This application, through a stepwise screening of lupin synthase, P450, and CPR, identified an optimal combination for betulinic acid synthesis in *Saccharomyces cerevisiae*: lupin synthase from olive oil + CrCYP716A154 from periwinkle + VvCPR from grape. Based on this, CrCYP716A154 was further optimized through mutation, resulting in increased betulinic acid yield. This technical route is well-defined, exhibits stable results, and is suitable for the efficient microbial synthesis and industrialization of betulinic acid.

[0252] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0253] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. An enzyme mutant, characterized in that, The enzyme mutant has any of the following combinations of mutations relative to the enzyme with the amino acid sequence shown in SEQ ID NO.1: Mutation combination 1 consists of T293A and L357A; Mutation combination 2 consists of T293A, L357A, and F117A; Mutation combination 3 consists of T293A, L357A, F117A, and P110A.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the enzyme mutant of claim 1.

3. A recombinant vector, characterized in that, The nucleotide sequence of the recombinant vector contains the nucleotide sequence of the nucleic acid molecule as described in claim 2.

4. Engineered microorganisms, characterized in that, The engineered microorganisms meet the following conditions (A1) or (A2): (A1) Contains the nucleic acid molecule of claim 2 or the recombinant vector of claim 3, or the target enzyme expressed by the engineered microorganism includes the enzyme mutant of claim 1; (A2) The target enzymes expressed by the engineered microorganisms include lupin alcohol synthase, cytochrome P450 and cytochrome P450 oxidoreductase; wherein the species source of lupin alcohol synthase is olive, the species source of cytochrome P450 is periwinkle, and the species source of cytochrome P450 oxidoreductase is Arabidopsis or grape.

5. The engineered microorganism according to claim 4, characterized in that, The engineered microorganisms satisfy any one of the following conditions (B1) to (B2): (B1) The engineered microorganisms include Saccharomyces cerevisiae; (B2) If the engineered microorganism satisfies the conditions shown in (A1), then the target enzyme expressed by the engineered microorganism further includes the expression of lupin alcohol synthase and / or cytochrome P450 oxidoreductase; if the engineered microorganism satisfies the conditions shown in (A2), then the cytochrome P450 is encoded by the nucleic acid molecule described in claim 2.

6. The engineered microorganism according to claim 5, characterized in that, The engineered microorganisms meet the conditions shown in (A1), the species source of the lupin alcohol synthase is olive, and the species source of the cytochrome P450 oxidoreductase is Arabidopsis thaliana or grape.

7. The method for constructing engineered microorganisms according to any one of claims 4 to 6, characterized in that, The construction method includes the following steps: The recombinant vector expressing the target enzyme is introduced into the microorganism to be modified, thereby constructing the engineered microorganism.

8. A method for producing betulinic acid, characterized in that, The production method includes the following steps: The engineered microorganisms of any one of claims 4 to 6 are fermented and cultured; and betulinic acid is isolated from the resulting fermentation product.

9. The method for producing betulinic acid according to claim 8, characterized in that, Fermentation culture includes steps (C1) and (C2): (C1) The engineered microorganisms are inoculated into the amplification medium and amplified to OD. 600 Once the preset values ​​are reached, the seed solution is prepared. (C2) The seed culture is inoculated into a fermentation medium and fermented.

10. The method for producing betulinic acid according to claim 8, characterized in that, The fermentation culture includes the following steps (D1) and (D2): (D1) Provide the seed culture of the engineered microorganism; (D2) The seed culture is inoculated into the fermentation medium, and the first stage fermentation and the second stage fermentation are carried out in sequence; The temperature for the first stage of fermentation is 29.5℃~30.5℃, and the temperature for the second stage of fermentation is 27.5℃~28.5℃. The inoculation time is recorded as 0 h, the first stage of fermentation is from 0 h to 24 h, and the second stage of fermentation is after 24 h.

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