Method for increasing yield of betulinic acid synthesized by saccharomyces cerevisiae

Abstract

The invention discloses a method for increasing the yield of betulinic acid synthesized by saccharomyces cerevisiae, and belongs to the field of bioengineering and metabolic engineering. According to the invention, a synthetic route of betulinic acid is constructed in saccharomyces cerevisiae. Through overexpression of key genes such as tHMG1, IDI, ERG20, ERG9 and ERG1, construction and optimization of an IUP pathway and adoption of a multi-copy integration and promoter optimization strategy, the expression level of RoCYP01 is enhanced, the RoCYP01 gene is further subjected to rational design and directional modification, the substrate specificity and catalytic efficiency of the RoCYP01 gene are optimized, and the yield of the target product betulinic acid reaches 118.65 mg / L. In addition, by increasing supply of cofactors, conditions in the fermentation process are optimized, and the final yield of betulinic acid is successfully increased to 425.2 mg / L by adopting a continuous feeding culture strategy.

Description

Technical Field

[0001] This invention relates to a method for increasing the production of betulinic acid by brewer's yeast, belonging to the field of bioengineering and metabolic engineering. Background Technology

[0002] Betulinic acid (BA) is a natural pentacyclic triterpenoid compound with significant pharmacological activity, widely found in birch bark and some medicinal plants. It exhibits significant bioactivity in antitumor, antiviral, and anti-inflammatory activities, thus showing broad application prospects in drug development and functional foods. However, the natural source of betulinic acid mainly relies on plant extraction, which suffers from low extraction efficiency, resource constraints, long cycles, and environmental unsustainability, making industrial-scale production difficult. In recent years, heterologous synthesis using microbial cell factories has become an important alternative to plant extraction, offering advantages such as being green and environmentally friendly, highly controllable, and capable of large-scale production. However, currently reported microbial synthesis systems for betulinic acid still face bottlenecks such as insufficient precursor supply leading to limited metabolic flux, low catalytic efficiency of key P450 enzymes in the synthesis pathway, and insufficient supply of cofactors (such as NADPH and heme) affecting oxidation reaction efficiency. Therefore, a systematic metabolic engineering strategy is urgently needed to achieve efficient and stable synthesis of betulinic acid by Saccharomyces cerevisiae. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a method for increasing the production of betulinic acid by Saccharomyces cerevisiae using metabolic engineering strategies. By comprehensively optimizing the metabolism of key pathways, key enzymes, and cofactors, efficient and stable production of betulinic acid can be achieved.

[0004] This invention provides a cytochrome P450 monooxygenase mutant, which, based on the parental form shown in SEQ ID NO.1, mutates leucine at position 78 to alanine, tryptophan at position 106 to alanine, and glycine at position 420 to phenylalanine.

[0005] The present invention also provides a gene encoding the cytochrome P450 monooxygenase mutant.

[0006] The present invention also provides a recombinant Saccharomyces cerevisiae expressing the cytochrome P450 monooxygenase mutant.

[0007] In one embodiment, the brewing yeast includes, but is not limited to, brewing yeast BY4742.

[0008] The present invention also provides a recombinant brewer's yeast capable of synthesizing betulinic acid, wherein the brewer's yeast expresses lupin alcohol synthase. At LUP, cytochrome oxidase RoCYP01 and cytochrome reductase At CPR01.

[0009] In one implementation, promoter P is used. FBA1 Or promoter P TEF1 Regulation Ro Expression of CYP01; using promoter P FBA1 Or promoter P ICL1 or regulation At The expression of CPR01; using promoter P FBA1 or regulation At The expression for LUP.

[0010] In one embodiment, the recombinant brewer's yeast also overexpressed the gene. tHMG1, IDI, ERG20, ERG9, ERG1 To improve precursor supply.

[0011] In one implementation, promoter P is used. TDH3 Regulating ERG20 expression using the promoter P FBA1 Regulating ERG9 expression using the promoter P ECM13 Regulating ERG1 expression using the promoter P ECM13 Regulating tHMGR expression using the promoter P TEF1 Regulate IDI expression.

[0012] In one embodiment, the recombinant brewer's yeast further has at least one of the following improvements, which further enhances the biosynthesis capacity of betulinic acid: (1) rDNA integration into genes at multiple copy sites Ro CYP01, At CPR01 At LUP; (2) Using promoter P FBA1 Or promoter P TEF1 Regulation Ro Expression of CYP01; using promoter P FBA1 Or promoter P ICL1 or regulation At The expression of CPR01; using promoter P FBA1 or regulation At The expression of LUP; (3) Expressing the cytochrome P450 monooxygenase mutant L78A / W106A / G420F; (4) Overexpressed genes ZWF1 , HEM1 The gene enhances the supply of cofactor heme and NADPH, thereby increasing the production of betulinic acid.

[0013] In one implementation, promoter P is used.FBA1 Regulate the expression of gene HEM1; using promoter P PCK1 It regulates the expression of the gene ZWF1.

[0014] The present invention also provides a method for preparing betulinic acid by fermentation, wherein the Saccharomyces cerevisiae strain is activated and cultured in a seed culture medium to obtain a seed liquid, and then the seed liquid is inoculated into a fermentation culture medium for fermentation culture.

[0015] In one embodiment, the fermentation medium uses glucose as a carbon source and peptone as a nitrogen source.

[0016] In one embodiment, metal ions Ca are also added to the fermentation medium. 2+ Fe 2+ .

[0017] In one embodiment, the brewing yeast is activated at 28-30 °C and 200-220 rpm to prepare a seed culture, which is then transferred to a fermentation medium and fermented at 28-30 °C and 200-220 rpm.

[0018] In one embodiment, the seed culture medium contains peptone, yeast extract, and 20 g / L glucose.

[0019] In one embodiment, the fermentation medium contains peptone, yeast extract, glucose, calcium chloride, ferrous sulfate, and oleic acid.

[0020] The present invention also provides the application of the cytochrome P450 monooxygenase mutant, the recombinant Saccharomyces cerevisiae, or the method in the preparation of betulinic acid or products containing betulinic acid.

[0021] Beneficial effects: (1) The present invention relates to cytochrome P450 monooxygenase Ro CYP01 was used for enzyme molecular design, optimizing its substrate specificity and catalytic efficiency, thereby reducing the accumulation of intermediate products in Saccharomyces cerevisiae and obtaining the mutant L78A / W106A / G420F. By in situ integrating the encoding gene of the mutant L78A / W106A / G420F, the recombinant strain produced a higher yield of betulinic acid than the wild type. Ro CYP01 increased by 2.3 times.

[0022] (2) This invention constructs a brewing yeast with increased betulinic acid production through multiple engineering strategies, integrating exogenous genes into the genome. Ro CYP01, At CPR01 At LUP successfully constructed a synthetic pathway for betulinic acid. This was achieved through overexpression... tHMG1, IDI, ERG20, ERG9 and ERG1By identifying key genes and improving the synthesis of related precursors, the yield of betulinic acid was successfully increased to 25.5 mg / L.

[0023] (3) This invention also enhances performance through multi-copy integration and promoter optimization strategies. Ro The expression level of CYP01 increased the production of betulinic acid to 90.39 mg / L.

[0024] (4) This invention further enhances the metabolic capacity of yeast cells by optimizing the supply of cofactors such as NADPH and heme and adjusting the composition of the fermentation medium. It also further improves the synthesis efficiency of betulinic acid by optimizing fermentation conditions (such as carbon source, nitrogen source, metal ions, fatty acids, etc.), achieving a yield of 215.8 mg / L at the shake flask level. Furthermore, by adopting a continuous fed culture strategy, the final yield of betulinic acid was successfully increased to 425.2 mg / L. Attached Figure Description

[0025] Figure 1 To construct a chassis strain for betulinic acid synthesis in Saccharomyces cerevisiae and to optimize the betulinic acid fermentation effect of recombinant strains with endogenous pathways.

[0026] Figure 2 To improve the supply of betulinic acid for fermentation by constructing an IUP pathway.

[0027] Figure 3 To optimize the synthesis of betulinic acid by recombinant bacteria using substrate concentrations in the IUP pathway.

[0028] Figure 4 Optimizing key genes for multiple strategies Ro The expression level of CYP01 balances the effect of the second-step continuous oxidation reaction from lupeol to betulinic acid.

[0029] Figure 5 For rational design Ro CYP01 achieves the equilibrium effect of the second-step continuous oxidation reaction.

[0030] Figure 6 The effects of different highly active mutants on betulinic acid synthesis.

[0031] Figure 7 The effect of betulinic acid biosynthesis after optimization of the fermentation process.

[0032] Figure 8 The fermentation process curve for preparing betulinic acid via fed-batch fermentation. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0034] (a) Culture medium: Seed culture medium: liquid YPD medium, consisting of 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose.

[0035] Fermentation medium: 20 g / L peptone, 10 g / L yeast extract, 30 g / L glucose, 0.2 g / L calcium chloride, 2 mM ferrous sulfate, 10 mM oleic acid.

[0036] SD medium: 20 g / L glucose, 6.7 g / L YNB, 40 mg / L lysine, 20 mg / L histidine, 60 mg / L leucine.

[0037] YPGE medium: 10 g / L yeast extract, 10 g / L peptone, 5 g / L glucose and 3% (v / v) anhydrous ethanol.

[0038] (II) Product extraction and detection methods: Betulinic acid extraction: Take 1 ml of fermentation broth and add an equal volume of ethyl acetate for extraction. Take the upper organic phase, dry it with a nitrogen blower, add 200 μL of methanol to dissolve it, and then perform HPLC detection.

[0039] Betulinic acid determination: The betulinic acid yield of the recombinant bacteria was determined using high-performance liquid chromatography (HPLC). An XDB-C18 analytical column was used. Mobile phase A was acetonitrile (0.1% formic acid), and mobile phase B was water (0.1% formic acid). Isocratic elution was performed with 70% A to 30% B for 35 min at a flow rate of 1 mL / min. -1 The detection wavelength is 203 nm.

[0040] Table 1. Strains involved in this invention

[0041] Example 1: Preparation of competent Escherichia coli cells Escherichia coli was streaked onto antibiotic-free LB agar and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB liquid medium, and cultured at 37°C with shaking at 220 rpm until the logarithmic growth phase (OD50) was reached. 600 =0.4 0.6). Transfer the bacterial culture to a pre-chilled 50 mL centrifuge tube and incubate on ice for 10 min, then centrifuge at 2000 rpm for 10 min and discard the supernatant. Next, add 1 / 10 of the bacterial culture volume of pre-chilled 0.1 M CaCl2 to resuspend, incubate on ice for 20 min, and centrifuge at 4000 rpm to discard the supernatant. Add 500 μL of pre-chilled 0.1 M CaCl2 containing 15% glycerol to fully resuspend, place on ice, aliquot 100 μL into 1.5 mL centrifuge tubes, and store at -80°C. E. coli transformation: Take... Competent cells stored at 80℃ were incubated on ice for 5 min, then 1 μL of plasmid or 10 μL of recombinant product was added, and the cells were incubated on ice for 30 min. After heat shock at 42℃ for 90 s, the cells were incubated on ice for 3 min, then 900 μL of LB medium was added, and the cells were incubated at 37℃ and 180 rpm for 60 min. The bacterial culture was then spread on LB solid medium containing antibiotics and cultured at 37℃ for 12–16 h.

[0042] Example 2: Construction of the gene expression cassette of Saccharomyces cerevisiae Integrating heterologous genes using Saccharomyces cerevisiae BY4742 as the starting strain Ro CYP01, At LUP, At CPR01. The amino acid sequences of enzymes involved in the betulinic acid synthesis pathway were optimized according to the codon preference of Saccharomyces cerevisiae, and the entire genome was synthesized, yielding the sequences shown in SEQ ID NO.1 to SEQ ID NO.3. Ro CYP01, At LUP, At The CPR01 gene. Using a synthetically produced gene and the Saccharomyces cerevisiae genome as templates, vectors, promoters, terminators, homologous arms, and fragments of the target gene were obtained via PCR. These fragments were then used to construct gene expression cassettes using plasmid construction methods, for example, P... TEF1 - Ro CYP01-T ADH2 The promoter, gene, and terminator fragments were amplified by PCR, with each fragment carrying an 18 bp homologous arm. The resulting fragments were then assembled using a seamless cloning enzyme. The ligation products were subsequently transformed and identified by colony PCR. Verified single colonies were recultured, and plasmids were extracted. Using this plasmid as a template, upstream primers were designed for the upstream homologous arm, and downstream primers for the downstream homologous arm. The fragments used for yeast transformation were amplified by PCR.

[0043] Example 3: Conversion of brewing yeast Integrative expression of the *Saccharomyces cerevisiae* genome was performed using homologous recombination editing technology. The genome amplified in Example 2 containing... Ro CYP01, At LUP, At The expression cassette of the CPR01 gene was transformed into *Saccharomyces cerevisiae* BY4742. The main transformation procedure was as follows: 240 μL of 50% PEG3350, 36 μL of 1M lithium acetate, 10 μL of salmon sperm DNA, and 1-3 μg of plasmid or fragment were added sequentially. After mixing with a single yeast colony by shaking, the mixture was heat-shocked at 30°C for 30 min in a metal bath, followed by a 42°C for 15 min water bath. After heat shock, the supernatant was discarded by centrifugation, and the culture was resuspended in 1 ml of YPD medium and incubated at 30°C for 2 h on a shaker. The culture was then plated on plates corresponding to the auxotype and grown for 3-5 days. Single colonies were picked for colony PCR verification. The correctly verified strain was named SCBA1 and preserved in glycerol tubes. The transformation of other strains was performed using the same procedure.

[0044] Example 4: Production of betulinic acid by shake-flask fermentation of recombinant brewing yeast Pick a portion of strain SCBA1 constructed in Example 3 from a glycerol tube and streak it onto a YPD solid plate. Incubate at 30°C. o Incubate in a C4 incubator for 3-5 days. After clear single colonies have grown on the solid agar plate, pick a single colony from the plate and inoculate it into a glass test tube containing 5 mL of YPD liquid medium. Incubate at 30°C. o C. Cultured at 220 rpm for 24 h to obtain the seed culture. Then, inoculated at a rate of 5% (v / v) into a 250 mL shake flask containing 100 mL of fermentation medium, and incubated at 30°C. o C. After fermenting at 220 rpm for 72 h, add isoprene alcohol substrate to a final concentration of 20 mM and continue fermentation for 7 days. Then, determine the betulinic acid content in the fermentation system.

[0045] Example 5: Construction of a chassis strain for betulinic acid synthesis in Saccharomyces cerevisiae and optimization of endogenous pathways to enhance betulinic acid biosynthesis. Chassis strain SC1 was constructed according to the methods described in Examples 1-3. 、 SC2 、 SC3 、 SCBA1.

[0046] Based on strain BY4742, strain SC1 was constructed by integrating and expressing the ERG20 gene (as shown in SEQ ID NO.8), ERG9 (as shown in SEQ ID NO.10), and ERG1 (as shown in SEQ ID NO.9) at the YML059C site (the upstream and downstream homologous arm sequences of the recognition site are shown in SEQ ID NO.13 and SEQ ID NO.14).

[0047] Based on strain BY4742, strain SC2 was constructed by integrating and expressing the tHMGR gene (as shown in SEQ ID NO. 6), IDI gene (as shown in SEQ ID NO. 7), ScCK gene (as shown in SEQ ID NO. 5), and AtIPK gene (as shown in SEQ ID NO. 4) at the YDR44W site (the upstream and downstream homologous arm sequences of the recognition site are shown in SEQ ID NO. 15 and SEQ ID NO. 16).

[0048] Based on strain SC2, strain SC3 was constructed by integrating and expressing the ERG20 gene (SEQ ID NO. 8), ERG9 gene (SEQ ID NO. 10), and ERG1 gene (SEQ ID NO. 9) at the YML059C site (the upstream and downstream homologous arm sequences of the recognition site are shown in SEQ ID NO. 13 and SEQ ID NO. 14).

[0049] Based on the strain SCBA1 constructed in Example 3, the gene was integrated and expressed at the YDR44W site (the upstream and downstream homologous arm sequences of the recognition site are shown in SEQ ID NO.15 and SEQ ID NO.16). tHMG1 (SEQ ID NO.6) IDI1 The strain SCBA2 (SEQ ID NO.7)

[0050] Furthermore, an expression gene was constructed based on strain SCBA1. ERG20 (SEQ ID NO.8) ERG1 (SEQ ID NO.9) and ERG9 The strain SCBA3 (SEQ ID NO.10)

[0051] Furthermore, based on strain SCBA3, an overexpression gene t was constructed. HMG1 (SEQ ID NO.6) IDI1 (SEQ ID NO.7) ERG20 (SEQ ID NO.8) ERG1 (SEQ ID NO.9) and ERG9 The strain SCBA4 (SEQ ID NO.10)

[0052] The strain was cultured in shake flasks using the method described in Example 4, and the highest yield of strain SCBA4 reached 12.43 mg / L. Figure 1 ).

[0053] Example 6: Improving the supply of IPP / DMAPP through the construction of an IUP pathway for efficient production of betulinic acid. To further improve precursor supply and optimize betulinic acid synthesis, an IUP pathway was constructed. Genes were introduced based on the strain SCBA4 constructed in Example 5. At IPK (SEQ ID NO.4) and Sc CK (SEQ ID NO.5), the constructed strain SCBA5. The strain was cultured in shake flasks using the culture method described in Example 4, with the substrate isoprene alcohol added 72 h after inoculation.

[0054] The results showed that the yield of betulinic acid increased to 20.6 mg / L. Figure 2 This indicates that the IUP pathway plays a positive role in improving the synthesis of target products.

[0055] The strain SCBA5 was cultured in shake flasks using the method described in Example 4. After 72 h of inoculation, isoprene alcohol was added at concentrations of 10 mM, 15 mM, 20 mM, 25 mM, and 30 mM. The results showed that the highest betulinic acid yield (25.5 mg / L) was achieved when 20 mM isoprene alcohol was added, representing a 1.8-fold increase compared to the control group without the added exogenous precursor. Figure 3 ).

[0056] Example 7: Multi-strategy optimization Ro The expression level of CYP01 balances the second-step continuous oxidation reaction from lupeol to betulinic acid. Using the gene expression cassette construction method described in Example 2, rDNA sites in the *Saccharomyces cerevisiae* genome were designated as homologous arms. The expression cassette carrying these homologous arms was transformed into the strain SCBA5 constructed in Example 6, resulting in multi-copy integrated strains SCBA6-SCBA15. Then, P was selected. ADH3 (nucleotide sequence as shown in SEQ ID NO.17), P TEF1 (nucleotide sequence as shown in SEQ ID NO.18), P PET9 (nucleotide sequence as shown in SEQ ID NO.19), P CCW14 (nucleotide sequence as shown in SEQ ID NO. 20), P ECM13 (nucleotide sequence as shown in SEQ ID NO.21), P CYC1 (nucleotide sequence as shown in SEQ ID NO.22), P PGI1 (nucleotide sequence as shown in SEQ ID NO.23) or P GAL1(The nucleotide sequence is shown in SEQ ID NO. 24) Promoters of different strengths were screened. The main process was to construct expression cassettes containing different promoters according to the method in Example 2, and to construct strains SCBA16-SCBA23 by homologous recombination at the YPRC15 site. Then, a second round of multi-copy integration optimization was performed to construct strains SCBA24-SCBA29. The strains were cultured in shake flasks using the culture method in Example 4. The results showed that after optimizing the expression levels of key enzymes, strain SCBA28 produced the highest betulinic acid yield of 90.39 mg / L after fermentation. Figure 4 ).

[0057] Example 8: Rational Design Ro CYP01 achieves equilibrium for the second-step continuous oxidation reaction. The nucleotide sequence is as shown in SEQ ID NO.1 Ro The CYP01 gene was constructed on plasmid pESC-URA according to the method in Example 2 and transformed into Saccharomyces cerevisiae WAT11. Seed culture was performed in SD medium using the method in Example 4, followed by transfer to 500 ml YPGE medium and cultured at 30°C for 12 h. Then, 20 g / L galactose was added and the culture was induced at 30°C. After 12 h of induction, cells were collected. Cells were resuspended in 0.5 g / ml TEK buffer (50 mM Tris HCl, 1 mM EDTA, 100 mM KCl, pH 7.4) and allowed to stand for 5 min. After centrifugation, cells were resuspended in 50 ml of ice-cold TES B buffer (50 mM Tris HCl, 1 mM EDTA, 600 mM sorbitol, pH 7.4). The cells were homogenized using a high-pressure homogenizer, centrifuged, and the supernatant was collected. 150 mM NaCl and 0.1 g / ml PEG-4000 were added to precipitate microsomes. After centrifugation, the microparticles were resuspended in 1 mL of TEG buffer (50 mM Tris HCl, 1 mM EDTA, 20% (v / v) glycerol, pH 7.4). In vitro reaction conditions for microsomes: anhydrous ethanol: Tween 80: betulinol solution (2.5 mM) = 8:1:2.25 (v / v), (DMSO not exceeding 5%), NADPH 2 mM. After the reaction, the reaction solution was dissolved in methanol by HPLC using an equal volume of ethyl acetate and then analyzed.

[0058] By designing different strategies to construct mutants, and further combining highly active mutants for mutation, the correspondence between the constructed recombinant bacteria and the mutants is as follows: CM1: L78A / W106A; CM2: W106A / F359A; CM3: L78A / F359A; CM4: I210Y / I286F; CM5: I210R / I286F; CM6: F414A / V415; CM7: F414A / G420F; CM8: V415A / G420F; CM9: F414A / V415A / G420F; CM10: L78A / F359A / W106A; CM11: L78A / W106A / I210R; CM12: L78A / W106A / I210Y; CM13: L78A / W106A / I286F; CM14: L78A / W106A / F414A; CM15: L78A / W106A / V415A; CM16: L78A / W106A / G420F; CM17: L78A / W106A / F359A / I210R; CM18: L78A / W106A / F359A / 210Y.

[0059] Recombinant bacteria expressing each mutant were cultured according to the method described in this embodiment. The results showed that the mutants... Ro Under the same conditions, CYP01-L78A / W106A / G420F catalyzed the production of betulinic acid from betulin alcohol to the wild type, achieving 2.3 times the yield. Figure 5 ).

[0060] Furthermore, the catalytically enhanced agents were respectively... Ro The CYP01 mutant genes I210R (SEQ ID NO.25), W106A (SEQ ID NO.26), F414A (SEQ ID NO.27), L78A / W106A (SEQ ID NO.28), and L78A / W106A / G420F (SEQ ID NO.29) were integrated in situ into the genome. Homologous recombination fragments were designed based on the homologous arms at both ends of the wild-type gene to construct... Ro The integration frame of the CYP01 mutant was transformed according to the method in Example 3, replacing the wild-type genome of the *Saccharomyces cerevisiae* strain SCBA28 constructed in Example 7, to construct strains SCBA30 to SCBA34. The strains were cultured in shake flasks according to the method in Example 4. The results showed that strain SCBA28 had the highest betulinic acid yield after fermentation, at 118.65 mg / L, which was 31.2% higher than the rate-limiting enzyme expression optimization strategy and 4.65 times higher than strain SCBA5. Figure 6 ).

[0061] Example 9 improves efficiency by regulating the supply of cofactors. Ro The catalytic efficiency of CYP01 promotes the efficient biosynthesis of betulinic acid.

[0062] The gene expression cassette was constructed according to Example 2, and the gene was overexpressed in Saccharomyces cerevisiae SCBA35. ZWF1 (SEQ ID NO.11) HEM1 (SEQ ID NO.12). First, through overexpression HEM1 (SEQ ID NO.12) The heme biosynthesis pathway was introduced into strain SCBA34 constructed in Example 8 to construct SCBA35. Then, by overexpressing heme in SCBA35... ZWF1 The NADPH regeneration pathway was reconstructed to obtain strain SCBA36.

[0063] Example 10 Optimization of betulinic acid fermentation conditions Based on YPD medium, the carbon source, nitrogen source, metal ions, and fatty acids were optimized respectively.

[0064] (1) Carbon source optimization Based on YPD fermentation medium, the carbon sources were replaced with glucose, sucrose, maltose, mannitol, and glycerol at concentrations of 20 g / L, respectively. The SCBA36 strain was fermented at 30℃ and 220 rpm, and the results showed that glucose as the carbon source yielded 164.1 mg / L.

[0065] (2) Nitrogen source optimization Based on YPD fermentation medium, the nitrogen source was replaced with peptone, soybean peptone, corn paste, ammonium sulfate, and fish peptone at a concentration of 20 g / L, respectively. The SCBA36 strain was fermented at 30℃ and 220 rpm. The results showed that the highest fermentation yield (164.1 mg / L) was achieved when peptone was still used as the nitrogen source.

[0066] (3) Optimization of metal ions Based on YPD fermentation medium, ZnSO4, CaCl2, MgSO4K2HPO4, or 1 mM FeSO4 were added at a concentration of 0.2 g / L. The SCBA36 strain was fermented at 30℃ and 220 rpm. The results showed that adding 0.2 g / L CaCl2 or 1 mM FeSO4 increased the yield, with the highest betulinic acid concentration (171.2 mg / L) achieved when 0.2 g / L CaCl2 was added.

[0067] (4) Fatty acid optimization Based on a fermentation medium containing 20 g / L peptone, 10 g / L yeast extract, 30 g / L glucose, 0.2 g / L calcium chloride, and 2 mM ferrous sulfate, oleic acid at concentrations of 5, 10, 15, 20, and 25 mM was added. The SCBA36 strain was fermented at 30°C and 220 rpm. The results showed that adding 10 mM oleic acid could increase the yield.

[0068] Fermentation results showed that increasing heme production increased betulinic acid (BA) yield by 24%. Compared to SCBA35, using the Shanghai Sangon Biotech NADPH assay kit, an increase of 10% in intracellular NADPH and an 11% increase in BA yield were observed. Finally, after optimizing fermentation conditions in shake flasks, the highest betulinic acid yield was achieved at 215.8 mg / L. Figure 7 ).

[0069] Example 11: Production of betulinic acid by continuous fed-batch fermentation of strain SCBA36 in a 7 L fermenter A single colony of *Saccharomyces cerevisiae* SCBA36 constructed in Example 9 was picked and cultured in 5 mL of YPD medium at 30°C and 220 rpm for 24 h to obtain a primary seed culture. This primary seed culture was then transferred at an inoculum rate of 10% (v / v) to a 2 L flat-bottomed flask containing 400 mL of seed medium and cultured at 30°C and 220 rpm for 18 h. 24 h. Subsequently, it was inoculated into a 7 L fermenter containing 4 L of fermentation medium (20 g / L peptone, 10 g / L yeast extract, 30 g / L glucose, 0.2 g / L calcium chloride, 2 mM ferrous sulfate, 10 mM oleic acid) and fermented under controlled conditions of 30℃, pH 5.5, and dissolved oxygen greater than 30%. The initial fermentation period (72 h) was the cell growth stage. Fed medium (500 g / L glucose, 160 g / L peptone, and 80 g / L yeast extract) was used to maintain a glucose concentration of 0.8-1.2 g / L to promote efficient cell growth. After 72 h of fermentation, the product production stage began. At this point, 20 mM isoprenol was added as a substrate, and ethanol was fed in as a carbon source to induce the synthesis of the target product, betulinic acid, while maintaining an ethanol concentration of 1-5 g / L in the fermentation system. The entire fermentation process lasted 7 days, with regular sampling to monitor cell density and product content. The fermentation process is shown in Figure 6. Biomass increased rapidly in the first 48 hours, then increased slowly thereafter. Betulinic acid production accumulated over time, reaching a peak of 425.2 mg / L at 132 hours. Figure 8 ).

[0070] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A cytochrome P450 monooxygenase mutant, characterized in that, Based on the parental line shown in SEQ ID NO.1, leucine at position 78 was mutated to alanine, tryptophan at position 106 was mutated to alanine, and glycine at position 420 was mutated to phenylalanine.

2. The gene encoding the cytochrome P450 monooxygenase mutant of claim 1.

3. A recombinant Saccharomyces cerevisiae expressing the cytochrome P450 monooxygenase mutant of claim 1 or containing the gene of claim 2.

4. The recombinant brewing yeast according to claim 3, characterized in that, The brewing yeast includes, but is not limited to, brewing yeast BY4742.

5. A recombinant brewing yeast, characterized in that, Lupin alcohol synthase was expressed. At LUP, cytochrome oxidase Ro CYP01 and cytochrome reductase At CPR01; and overexpressed the gene. tHMG1, IDI, ERG20, ERG9, ERG1 .

6. The recombinant brewing yeast according to claim 5, characterized in that, The cytochrome oxidase Ro CYP01 includes, but is not limited to, the enzyme shown in SEQ ID NO.1 or the mutant described in claim 1.

7. A method for increasing the yield of betulinic acid in brewing yeast, characterized in that, At least one of the following improvements is made to the brewing yeast: (1) rDNA integration into genes at multiple copy sites Ro CYP01, At CPR01 At LUP; (2) Using promoter P FBA1 Or promoter P TEF1 Regulation Ro Expression of CYP01; using promoter P FBA1 Or promoter P ICL1 or regulation At The expression of CPR01; using promoter P FBA1 or regulation At The expression of LUP; (3) Expressing the cytochrome P450 monooxygenase mutant L78A / W106A / G420F as described in claim 1; (4) Overexpressed genes ZWF1 , HEM1 Gene.

8. A method for preparing betulinic acid by fermentation, characterized in that, The *Saccharomyces cerevisiae* strain described in claim 5 or 6 is activated and cultured in a seed culture medium to obtain a seed liquid, which is then inoculated into a fermentation culture medium for fermentation culture.

9. The method according to claim 8, characterized in that, The fermentation medium uses glucose as the carbon source and peptone as the nitrogen source; optionally, metal ions Ca2+ are also added to the fermentation medium. 2+ Fe 2+ .

10. The cytochrome P450 monooxygenase mutant of claim 1, the recombinant Saccharomyces cerevisiae of claim 5 or 6, or the method of any one of claims 7 to 9 in the preparation of betulinic acid or products containing betulinic acid.

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