An engineered yeast strain for expressing cytochrome p450 and application thereof

Abstract

The application discloses an engineered yeast strain for expressing cytochrome P450 and application thereof, and belongs to the technical field of bioengineering. The application constructs a method for constructing a Saccharomyces cerevisiae platform strain for improving cytochrome P450 expression by using a Saccharomyces cerevisiae MAGIC whole genome screening library, uses a yeast strain containing a betalain biosensor and a Saccharomyces cerevisiae MAGIC whole genome screening gRNA library, and successfully screens a target gene capable of improving CYP76AD1 expression in Saccharomyces cerevisiae by means of high-throughput screening of whole genome perturbation and fluorescence coupling with the aid of flow cytometry. Meanwhile, the microenvironment reconstruction target genes are applied to improve the functional expression of other P450 enzymes, the universality is proved, and it is proved that the method can effectively establish a platform engineered yeast strain for high-activity expression of various P450 enzymes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering, and particularly relates to an engineered yeast strain for expressing cytochrome P450 and application thereof. BACKGROUND

[0002] Cytochrome P450 enzymes (P450) are a superfamily of heme enzymes with cysteine as the axial ligand, which widely exist in animals, plants and microorganisms. Due to the multiple biological catalytic activities such as hydroxylation, epoxidation, dealkylation and dehalogenation, P450 enzymes play an important role in the synthesis of plant natural products. And since most eukaryotic P450 enzymes are membrane proteins located in the endoplasmic reticulum, it is difficult to express them heterologously, which often becomes the rate-limiting step for the synthesis of plant natural products. Due to its unique advantages, especially the post-translational modification ability and the existence of the inner membrane system, Saccharomyces cerevisiae is one of the preferred hosts for heterologous expression of P450 enzymes and synthesis of natural products. Although a variety of P450 enzymes have been successfully expressed in Saccharomyces cerevisiae, for example, the application with the publication number CN106987533A discloses a method for constructing an engineered Saccharomyces cerevisiae strain capable of synthesizing glycyrrhetinic acid. The β-amyrin synthase gene GgbAS, cytochrome P450 oxidase genes CYP88D6 and CYP72A154 derived from Pachysandra glabra, and cytochrome P450 oxidoreductase genes CPR1 and CPR2 derived from Arabidopsis thaliana are respectively constructed to form gene expression cassettes, and then the gene expression cassettes are co-transformed into Saccharomyces cerevisiae CEN.PK2-1C. The yeast homologous recombination ability is used to assemble an engineered Saccharomyces cerevisiae strain with complete glycyrrhetinic acid biosynthetic pathway, realizing the artificial synthesis of glycyrrhetinic acid in Saccharomyces cerevisiae.

[0003] However, how to improve the expression level and activity of P450 enzymes in yeast cells still faces great challenges. In recent years, researchers have used various strategies to improve the functional expression of P450 enzymes in Saccharomyces cerevisiae, including using codon optimization or N-terminal sequence modification to improve P450 expression level, molecular modification of P450 enzymes through protein engineering, co-expression of P450 enzymes and CPR, combination optimization of P450-CPR pairs, and construction of yeast chassis cells that can improve the expression and folding microenvironment of P450 enzymes. However, these strategies are limited to specific P450 enzymes, lack of universality, and thus there is still great uncertainty in the efficient and active expression of P450 enzymes in Saccharomyces cerevisiae (Jiang L., Huang L., Cai J., et al. (2021). Functional expression of eukaryotic cytochrome P450s in yeast. Biotechnol. Bioeng. 118, 1050-1065.). Therefore, it is necessary to develop a universal method to construct an engineered yeast strain to improve the functional expression of P450 enzymes in yeast.

[0004] Saccharomyces cerevisiae has a complex inner membrane system and metabolic regulation network, and has strong plasticity. It is speculated that by multi-mode regulation (up-regulation, down-regulation and knockout) of more than 6000 genes in the whole genome of Saccharomyces cerevisiae, the intracellular microenvironment can be adjusted, which in turn helps the functional expression of P450 enzymes in Saccharomyces cerevisiae. SUMMARY

[0005] In view of the above problems, the present application provides an intracellular microenvironment modification for functional expression of cytochrome P450 and its application.

[0006] The present application provides an engineered yeast strain for expressing cytochrome P450, wherein the engineered yeast strain is a yeast strain in which at least one of the following target genes is inhibited: QDR3 gene, CUE1 gene, DEF1 gene, GET3 gene, BUG1 gene, SNF2 gene, MAC1 gene, QDR3 gene, CYC8 gene, DOA1 gene, SIT4 gene, GET2 gene, TCO89 gene, SWI1 gene, CAC2 gene, BRO1 gene, AMN1 gene, PHA2 gene, SPF1 gene, UBC7 gene, VPS41 gene, AOS1 gene, MNP1 gene, SSM4 gene, NAP1 gene, HTA1 gene, GTA1 gene, LAM6 gene, INO2 gene, CDC48 gene; and / or, at least one of the following genes is knocked out: EOS1 gene; and / or, at least one of the following genes is activated: TOA2 gene, YEL076C gene.

[0007] Preferably, the engineered yeast strain is one in which at least one of the following target genes is suppressed: DEF1 gene, BUG1 gene, SNF2 gene, MAC1 gene, CYC8 gene, DOA1 gene, BRO1 gene, QDR3 gene, CUE1 gene, GET2 gene, TCO89 gene, UBC7 gene, VPS41 gene, MNP1 gene, LAM6 gene, SSM4 gene, INO2 gene.

[0008] Preferably, the yeast strain is a Saccharomyces cerevisiae strain. The cytochrome P450 is betaxanthin, Z-a-santalol, vindoline, fumariline or stipulatine. Preferably, when the target gene CYC8 gene is suppressed, the yield is increased most effectively, and the yield of Z-a-santalol and vindoline is increased by 2.05 times and 1.16 times, respectively; when the target gene combination CYC8 gene and GET2 gene is suppressed, the yield of Z-a-santalol, vindoline, fumariline and stipulatine is increased by 1.42, 1.64, 1.83 and 2.11 times, respectively.

[0009] Preferably, the yeast strain further has introduced therein a gene in the synthetic pathway for expressing cytochrome P450.

[0010] The GenBank number of the DEF1 gene is NM_001179620.1, the GenBank number of the GET3 gene is NM_001180159.1, the GenBank number of the BUG1 gene is NM_001180158.1, the GenBank number of the SNF2 gene is NM_001183709.3, the GenBank number of the MACI gene is NM_001182517.1, the GenBank number of the CYC8 gene is NM_001178460.3, the GenBank number of the DOA1 gene is NM_001179778.1, the GenBank number of the SIT4 gene is NM_001180106.1, the GenBank number of the CAC2 gene is NM_001182464.1, the GenBank number of the BRO1 gene is NM_001183898.1, the GenBank number of the AMN1 gene is NM_001178506.1, the GenBank number of the PHA2 gene is NM_001183154.1, the GenBank number of the SPF1 gene is NM_001178846.3, the GenBank number of the QDR3 gene is NM_001178391.2, the GenBank number of the CUE1 gene is NM_001182771.1, the GenBank number of the GET2 gene is NM_001178974.1, the GenBank number of the TCO89 gene is NM_001183994.1, the GenBank number of the SWI1 gene is NM_001183830.2, the GenBank number of the UBC7 gene is NM_001182518.1, the GenBank number of the VPS41 gene is NM_001180388.3, the GenBank number of the AOS1 gene is NM_001184277.1, the GenBank number of the MNP1 gene is NM_001180933.1, the GenBank number of the NAP1 gene is NM_001179838.1, the GenBank number of the YEL076C gene is NM_001178891.1, the GenBank number of the HTA1 gene is NM_001180533.3, the GenBank number of the GTA1 gene is NM_001178858.3, the GenBank number of the LAM6 gene is NM_001181959.1, the GenBank number of the SSM4 gene is NM_001179380.3, the GenBank number of the CDC48 gene is NM_001180185.1, the GenBank number of the INO2 gene is NM_001180431.1, the GenBank number of the EOS1 gene is NM_001182918.1, and the GenBank number of the TOA2 gene is NM_001179624.1。.

[0011] Preferably, the gRNA expression sequence targeting said target gene is integrated into the genome of the yeast strain.

[0012] The gRNA expression sequences targeting the QDR3 gene are as shown in SEQ ID No. 2, SEQ ID No. 9, SEQ ID No. 21 and SEQ ID No. 22, the gRNA expression sequences targeting the CUE1 gene are as shown in SEQ ID No. 3, SEQ ID No. 19, SEQ ID No. 25, SEQ ID No. 29 and SEQ ID No. 32, the gRNA expression sequences targeting the DEF1 gene are as shown in SEQ ID No. 4, the gRNA expression sequences targeting the GET3 gene are as shown in SEQ ID No. 5 and SEQ ID No. 27, the gRNA expression sequences targeting the BUG1 gene are as shown in SEQ ID No. 6, SEQ ID No. 28 and SEQ ID No. 37, the gRNA expression sequences targeting the SNF2 gene are as shown in SEQ ID No. 7, the gRNA expression sequences targeting the MAC1 gene are as shown in SEQ ID No. 8, the gRNA expression sequences targeting the CYC8 gene are as shown in SEQ ID No. 10, the gRNA expression sequences targeting the DOA1 gene are as shown in SEQ ID No. 12, the gRNA expression sequences targeting the SIT4 gene are as shown in SEQ ID No. 13, the gRNA expression sequences targeting the GET2 gene are as shown in SEQ ID No. 14, SEQ ID No. 33 and SEQ ID No. 35, the gRNA expression sequences targeting the TCO89 gene are as shown in SEQ ID No. 15 and SEQ ID No. 16, the gRNA expression sequences targeting the SWI1 gene are as shown in SEQ ID No. 17, the gRNA expression sequences targeting the CAC2 gene are as shown in SEQ ID No. 18, the gRNA expression sequences targeting the BRO1 gene are as shown in SEQ ID No. 20, the gRNA expression sequences targeting the AMN1 gene are as shown in SEQ ID No. 23, the gRNA expression sequences targeting the PHA2 gene are as shown in SEQ ID No. 24, SEQ ID No. 38 and SEQ ID No. 48, the gRNA expression sequences targeting the SPF1 gene are as shown in SEQ ID No. 26, the gRNA expression sequences targeting the UBC7 gene are as shown in SEQ ID No. 30, the gRNA expression sequences targeting the VPS41 gene are as shown in SEQ ID No. 31, the gRNA expression sequences targeting the AOS1 gene are as shown in SEQ ID No. 34, the gRNA expression sequences targeting the MNP1 gene are as shown in SEQ ID No. 36, the gRNA expression sequences targeting the SSM4 gene are as shown in SEQ ID No. 40, the gRNA expression sequences targeting the NAP1 gene are as shown in SEQ ID No.42, the gRNA expression sequence targeting the HTA1 gene is shown as SEQ ID No. 43, the gRNA expression sequence targeting the GTA1 gene is shown as SEQ ID No. 44, the gRNA expression sequence targeting the LAM6 gene is shown as SEQ ID No. 45, the gRNA expression sequence targeting the INO2 gene is shown as SEQ ID No. 46, the gRNA expression sequence targeting the CDC48 gene is shown as SEQ ID No. 47, the gRNA expression sequence targeting the EOS1 gene is shown as SEQ ID No. 39, the gRNA expression sequence targeting the TOA2 gene is shown as SEQ ID No. 11, and the gRNA expression sequence targeting the YEL076C gene is shown as SEQ ID No. 41.

[0013] The application also provides the use of the engineered yeast strain in expressing cytochrome P450.

[0014] The application has the following beneficial effects: the method for constructing the Saccharomyces cerevisiae platform strain for improving the expression of cytochrome P450 by using the Saccharomyces cerevisiae MAGIC whole genome screening library, which uses the betaxanthin biosensor yeast strain and the Saccharomyces cerevisiae MAGIC whole genome screening gRNA library, and successfully screens the target genes capable of improving the expression of CYP76AD1 in Saccharomyces cerevisiae by whole genome perturbation and high-throughput screening coupled with fluorescence, with the aid of flow cytometry. The application of these microenvironment reconstruction target genes to improve the functional expression of other P450 enzymes proves their universality, and it is proved that the method can effectively establish a platform engineering yeast strain for the high-activity expression of various P450 enzymes. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A flow chart for screening target points for improving the expression of cytochrome P450 enzymes by using the MAGIC whole genome gRNA library.

[0016] Figure 2 A process for collecting high-fluorescence-intensity cells by using flow cytometry fluorescence sorting technology.

[0017] Figure 3 A result graph of the first round of MAGIC whole genome evolution target point screening.

[0018] Figure 4 A result graph of the second round of MAGIC whole genome evolution target point screening.

[0019] Figure 5 A result graph of the influence of the target points screened by the first round of MAGIC whole genome evolution on the synthesis of Z-alpha-amyrin.

[0020] Figure 6Figure for the effect of target points screened by the second round of MAGIC whole genome evolution on the synthesis of Z-alpha-amyrin.

[0021] Figure 7 Figure for the effect of target points screened by the MAGIC whole genome evolution on the synthesis of vindoline.

[0022] Figure 8 Figure for the effect of target points screened by the MAGIC whole genome evolution on the synthesis of fargesin.

[0023] Figure 9 Figure for the effect of target points screened by the MAGIC whole genome evolution on the synthesis of strictosidine. DETAILED DESCRIPTION

[0024] Escherichia coli DH5a was purchased from Beijing Genki Biological Technology Co., Ltd. Saccharomyces cerevisiae BY4741 was purchased from China Industrial Microbial Culture Collection Center (CICC). DNA polymerase, restriction endonuclease and T4 ligase were purchased from NEB Company. Escherichia coli plasmid extraction kit was purchased from AXYGEN Company, yeast plasmid extraction kit was purchased from Zymo Research Company, and PCR product nucleic acid purification kit was purchased from Thermo Scientific Company.

[0025] The culture medium of Escherichia coli used LB medium, the formula was: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L. The culture of Saccharomyces cerevisiae used YPD medium or SCD-URA screening medium. The formula of YPD medium was yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L. The formula of SCD-URA medium was: amino acid-free yeast nitrogen source (Difco, Boom, The Netherlands) 1.7 g / L, ammonium sulfate 5 g / L, CSM-URA (MP Biomedicals, Solon, Ohio) 0.77 g / L, glucose 20 g / L.

[0026] Figure 1 Figure for the process of creating Saccharomyces cerevisiae platform strain capable of improving the expression of cytochrome P450 enzyme activity by the MAGIC whole genome library screening of the present application. Figure 1 The gene sequences used in the present application are shown in Table 1.

[0027] Table 1 Gene sequences used in the present application

[0028] Gene name Gene number / GenBank number Gene name Gene number / GenBank number SAS (from Santalum album L.) SEQ ID No. 1 AtCPR2 KC842188.1 CYP76AD1 KU644144.1 AtCPR1 NM 001203894.1 DOD KM502867.1 T16H2 JF742645.1 CYP736A167 KU169302.1 16OMT EF444544.1 tHMG1 NM_001182434.1 T3O KP122967.1 ERG8 NM_001182727.1 T3R KP122966.1 ERG13 NM_001182489.1 NMT HM584929.1 ERG20 NM_001181600.1 D4H U71605.1 ERG12 NM_001182715.1 DAT AF053307.1 ERG10 NM_001183842.1 CFS AB434654 MVD1 NM_001183220.1 SPS EU882969 IDI1 NM_001183931.1

[0029] Embodiment 1: A method for constructing a Saccharomyces cerevisiae platform strain for high-throughput screening of target points for improving the functional expression of P450 enzymes using a MAGIC multifunctional library

[0030] 1. A betalain biosensor yeast strain comprising the following steps:

[0031] Starting from Saccharomyces cerevisiae BY4741, a MAGIC tri-functional Cas protein was integrated for subsequent gene regulation (Lian J, et al. (2017) Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system. Nat Commun., 8(1): 1-9.), and then the betalain synthesis pathway genes CYP76AD1 and DOD were integrated into the genome to construct a betalain-producing yeast strain. The GenBank numbers of the CYP76AD1 gene sequence and the DOD gene sequence are KU644144.1 and KM502867.1, respectively. Betalains are yellow and fluorescent (excitation wavelength of 498 nm and emission wavelength of 533 nm), and high-throughput screening can be performed by flow cytometry through the enhancement of color and fluorescence.

[0032] 2. Screening of MAGIC whole genome targets for improving the expression of P450 (CYP76AD1), which comprises the following steps:

[0033] When using the MAGIC library (Lian J et al. (2019) Multi-functional genome-wide CRISPR system for high throughput genotype-phenotype mapping. Nat Commun., 10(1): 5794.) for whole genome evolution, first, the three plasmid libraries p416-gsLibA, p416-gsLibI and p416-gsLibD of the MAGIC were respectively transformed into the above-constructed betalain biosensor strain, and after the completion of yeast transformation, 0.5% of the cells were spread on SED-URA / G418 plates to calculate the transformation efficiency (the total number of each transformation should reach about 10 6 After overnight culture, the remaining cells were transferred to a new 50 mL SED-URA / G418 shake flask for culture. When the strain grew to the logarithmic growth phase, 1 ml of bacterial cells was collected, washed twice with 1M pH 7.5 PBS buffer, and an appropriate amount of PBS buffer was taken to dilute the cell liquid to 1×10 5~1 x 10 6 cells / ml, and then loaded into a flow cytometry sorter (BD, FACS Melody), in which the 488 nm laser tube channel was selected according to the fluorescence properties of betalain. The fluorescence intensity of the top 2% of cells was collected in SED-URA / G418 test tubes and recovered overnight on a shaker (30°C, 250 rpm). After recovery, an appropriate amount of cell liquid was spread on SED-URA / G418 plates, and high-throughput screening was performed using the color change of the colonies in the plates. The colonies with a clear color change were selected and inoculated in 96-well plates containing SED-URA / G418 medium for 24 h. Then, the seed liquid in the 96-well plates was transferred to new 96-well plates containing SED-URA / G418 medium at an initial OD600 of 0.1, and the cells in the logarithmic phase were collected for fluorescence intensity detection. Subsequently, the plasmids of the colonies with significantly increased fluorescence intensity were sequenced and extracted, and the extracted plasmids were transferred into DH5a for amplification. Finally, the amplified plasmids were extracted and transferred back into the sensor strain for verification, in order to screen for target points that can improve the expression of CYP76AD1 in yeast and increase the production of betalain Figure 1

[0034] Using a betalain biosensor yeast strain and a MAGIC whole genome library, and using flow sorting Figure 2 and double screening of colony color, a total of 25 target points that can improve the accumulation of betalain in S. cerevisiae to different extents were screened in the first round of MAGIC whole genome evolution, as shown in Figure 3 Table 2 and Table 3. Among them, the target points with the highest betalain yield improvement multiples were 10-32, 10-62, 12-75 and 10-44, which were inhibitory gRNAs targeting genes MAC1, DOA1, BRO1 and CYC8, respectively. Compared with the starting betalain biosensor strain, these target points increased the yield of betalain by 3.02, 2.61, 2.59 and 2.39 times, respectively. In addition, target points 5-38, 8-58, 10-9, 10-10, 10-77, 12-19, 12-69, 17-58 and 25-15 all increased the accumulation of betalain by more than 1.5 times.

[0035] Table 2 First round of MAGIC whole genome evolution screening target point information summary

[0036] Target Type Gene Fold increase Target Type Gene Fold increase 5-2 Inhibition QDR3 1.39 11-70 Inhibition TCO89 1.32 5-38 Inhibition CUE1 2 12-19 Inhibition TCO89 1.54 8-58 Inhibition DEF1 1.55 12-39 Inhibition SWI1 1.22 10-5 Inhibition GET3 1.15 12-57 Inhibition CAC2 1.29 10-9 Inhibition BUG1 1.83 12-69 Inhibition CUE1 2.21 10-10 Inhibition SNF2 1.57 12-75 Inhibition BRO1 2.59 10-32 Inhibition MAC1 3.02 17-52 Inhibition QDR3 1.37 10-39 Inhibition QDR3 1.32 17-58 Inhibition QDR3 1.68 10-44 Inhibition CYC8 2.39 18-10 Inhibition AMN1 1.31 10-58 Activation TOA2 1.27 23-28 Inhibition PHA2 1.48 10-62 Inhibition DOA1 2.61 25-15 Inhibition CUE1 1.67 10-65 Inhibition SIT4 1.47 25-47 Inhibition SPF1 1.39 10-77 Inhibition GET2 2.28

[0037] Table 3 First round of MAGIC whole genome evolution screening target point gRNA sequence

[0038] ​

[0039] Based on the results of the first round of MAGIC whole genome evolution screening, a second round of iterative screening was carried out to further improve the functional expression of P450 enzymes in S. cerevisiae and obtain more general target points that can improve the cell microenvironment for the expression and folding of P450 enzymes. The gRNA (SpSg-CYC8) expression frame that inhibits the expression of CYC8 gene in target points 10-44 was selected and integrated into the sensor strain to construct the starting strain for the second round of MAGIC whole genome evolution. After the MAGIC plasmid library was again introduced into the starting strain, iterative whole genome evolution and target screening were again carried out according to the process of the first round of whole genome evolution screening, and the results are shown in Figure 4 As shown, compared with the control strain, 22 target points were further screened to improve the production of betalain in different degrees, and the target point information is shown in Table 4, and the target gRNA sequence is shown in Table 5. Among them, the highest yield of several target points is 25-15, 30-7, 31-7 and 33-72, which further improves the accumulation of betalain in S. cerevisiae by 2.62, 2.7, 2.23 and 2.53 times, respectively. Among them, target points 25-15 and 31-7 are transcriptional inhibition gRNAs targeting CUE1 gene, 25-15 has been identified as a target point in the first round of screening, and 31-7 is an inhibition gRNA targeting CUE1 gene identified in the second round. In addition, target points 10-5, 10-9, 30-61, 31-47, 31-65, 32-20, 33-8 and 34-40 further improve the yield of betalain by more than 1.5 times, which are inhibition gRNAs targeting genes GET3, BUG1, VPS41, GET2, MNP1, BUG1, SSM4 and INO2, respectively.

[0040] Table 4 Summary of target points in the second round of MAGIC whole genome evolution screening

[0041]

[0042]

[0043] Table 5 gRNA sequence of target points in the second round of MAGIC whole genome evolution screening

[0044]

[0045] Example 1: Effect of MAGIC whole genome evolution screening target points on santalol synthesis

[0046] The Z-α-santalol synthesis strain catalyzed by P450 (CYP736A167 from Santalum album) was selected to verify the universality of the screened microenvironment reconstruction target points.

[0047] (a.) Select the laboratory constructed with Saccharomyces cerevisiae BY4741 as the starting strain, integrated with the MAGIC three-function Cas protein for subsequent gene regulation (Lian J, et al. (2017). Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system. Nat Commun., 8. (1): 1-9.), namely BY4741-AID6 strain. Integrate four copies of santalene synthase (SAS) in the BY4741-AID6 strain genome X1 VI, IX1, XIII1 and X1 sites to produce santalene yeast strain as the starting strain (Dong C, et. al. (2020). A single Cas9-VPR nuclease for simultaneous gene activation, repression, and editing in Saccharomyces cerevisiae. ACS Synth. Biol., 9: 2252-2257), and overexpress the MVA pathway related gene expression frame, tHMG1-ERG8-ERG13-ERG20-ERG12 and EGR10-MVD1-IDI1-tHMG1 to improve the accumulation of Z-α-santalol precursor santalene, the GenBank numbers of the expression frame genes of tHMG1, ERG8, ERG13, ERG20, ERG12, EGRI0, MVD1 and IDI1 are NM_001182434.1, NM_001182727.1, NM_001182489.1, NM_001181600.1, NM_001182715.1, NM_001183842.1, NM_001183220.1 and NM_001183931.1, respectively.

[0048] (b.) Integrate CYP736A167 and AtCPR2 genes into the yeast genome to construct Z-α-santalol synthesis strain, the GenBank numbers of CYP736A167 and AtCPR2 genes are as shown in KU169302.1 and KC842188.1, respectively.

[0049] (c.) Integrate gRNA expression sequences targeting each target gene into the Z-α-santalol producing yeast genome, either alone or in combination, and use GCMS to determine the yield of santalene and Z-α-santalol.

[0050] As Figure 5 and Figure 6As shown, compared with the control strain, the transcriptional inhibition gRNAs targeting CYC8, SIT4, GET2, TCO89, AMN1 and SPF1, i.e. target points 10-44, 10-65, 10-77, 12-19, 18-10 and 25-47, increased the production of Z-a-santalol by 2.05, 1.16, 1.26, 1.32, 1.17 and 1.52 times, respectively. Among the target points screened in the second round of the MAGIC whole genome evolution screening, 12 target points could further increase the production of Z-a-santalol based on the target point 10-44 (SpSg-CYC8), among which the target points 31-24 (targeting GET2), 31-47 (targeting GET2) and 33-24 (targeting NAP1) further increased the accumulation of Z-a-santalol by 1.37, 1.42 and 1.37 times, respectively.

[0051] Application Example 2: Influence of the target points screened by the MAGIC whole genome evolution screening on the synthesis of ventilidine

[0052] Strains for the synthesis of ventilidine catalyzed by multiple P450s (T16H2, T3O and D4H) were selected to verify the universality of the microenvironment modification target points screened.

[0053] Starting from the BY4741-AID6 constructed in Application Example 1, AtCPR1, T16H2 (tabersonine-16-hydroxylase 2), 16OMT (16-hydroxytabersonine-O-methyltransferase), T3O (tabersonine-3-oxidase), T3R (tabersonine-3-reductase), NMT (3-hydroxy-16-methyl-2,3-dihydrotabersonine-N-methyltransferase), D4H (deacetoxyventilidine-4-hydroxylase) and DAT (deacetoxyventilidine-4-O-acetyltransferase) genes were integrated into the genome, and the GenBank numbers of the AtCPR1, T16H2, 16OMT, T3O, T3R, NMT, D4H and DAT genes were NM_001203894.1, JF742645.1, EF444544.1, KP122967.1, KP122966.1, HM584929.1, U71605.1 and AF053307.1, respectively, to construct a ventilidine-producing strain; then several target points 10-44 (targeting CYC8), 25-47 (targeting SPF1), 31-24 (targeting GET2), 31-47 (targeting GET2) and 33-24 (targeting NAP1) with better effects were introduced into the strain to further verify the influence of these target points on the expression of P450 enzymes in the ventilidine synthesis pathway.

[0054] As shown in Table 6, compared with the control strain, the transcriptional inhibition gRNAs targeting CYC8, SIT4, GET2, TCO89, AMN1 and SPF1, i.e. target points 10-44, 10-65, 10-77, 12-19, 18-10 and 25-47, increased the production of Z-a-santalol by 2.05, 1.16, 1.26, 1.32, 1.17 and 1.52 times, respectively. Among the target points screened in the second round of the MAGIC whole genome evolution screening, 12 target points could further increase the production of Z-a-santalol based on the target point 10-44 (SpSg-CYC8), among which the target points 31-24 (targeting GET2), 31-47 (targeting GET2) and 33-24 (targeting NAP1) further increased the accumulation of Z-a-santalol by 1.37, 1.42 and 1.37 times, respectively. Figure 7As shown, multiple targets can improve the yield of vindoline. Among them, when the target 10-44 (targeting CYC8) is expressed alone, the yield of vindoline can be increased by 1.16 times, which is slightly lower than 1.2 times of the OPI1 knockout strain (OPI1 knockout strain is widely used to improve the activity expression of P450 enzyme in yeast intracellular). But on the basis of targeting CYC8, the expression of target points 25-47 (targeting SPF1), 31-24 (targeting GET2), 31-47 (targeting GET2) and 33-24 (targeting NAP1) is further increased, and the yield of vindoline is increased by 1.36, 1.38, 1.64 and 1.22 times respectively than the starting strain, which is higher than the OPI1 knockout strain. It is proved that the functional expression of P450 in vindoline synthesis pathway is further enhanced.

[0055] Application Example 4: Screening of target points by MAGIC whole genome evolution to affect the functional expression of P450 cobave alkaloid synthase (CFS)

[0056] The cobave alkaloid synthesis strain catalyzed by P450 cobave alkaloid synthase (CFS) is selected to verify the universality of the microenvironment reconstruction target points screened. In this application example, the substrate flavosvine by P450 enzyme CFS is catalyzed to synthesize cobave alkaloid in a single step. Based on the BY4741-AID6 constructed in application example 1, the cobave alkaloid synthase gene (CFS) is integrated into the genome to construct a cobave alkaloid producing strain, and the GenBank number of CFS gene is AB434654; then the best co-acting target points 10-44 (targeting CYC8) and 31-47 (targeting GET2) are introduced into the strain to further verify the effect of intracellular microenvironment reconstruction target points on the expression of P450 enzyme in cobave alkaloid synthesis pathway. The substrate flavosvine of cobave alkaloid synthase is added in the fermentation medium, and the synthesis level of product cobave alkaloid is detected by LC-MS after fermentation. As shown in Figure 8 As shown, the test target points can improve the yield of cobave alkaloid. Compared with the control strain, the yield of cobave alkaloid is increased by 1.83 times when the target points 10-44 (targeting CYC8) and 31-47 (targeting GET2) are expressed at the same time. It is proved that the microenvironment reconstruction target points improve the functional expression of P450 cobave alkaloid synthase.

[0057] Application Example 5: Screening of target points by MAGIC whole genome evolution to affect the functional expression of P450 stipulatine synthase (SPS)

[0058] A strain catalyzing the synthesis of stipulatine by P450 stipulatine synthase (SPS) is selected to verify the universality of the screened microenvironment remodeling targets. In this application example, the substrate scoulerine is catalyzed by P450 enzyme CFS to synthesize stipulatine in one step. Starting from the BY4741-AID6 constructed in application example 1, the stipulatine synthase gene (SPS) is integrated into the genome to construct a stipulatine-producing strain, and the GenBank number of the SPS gene is EU882969. Then the best co-acting targets 10-44 (targeting CYC8) and 31-47 (targeting GET2) are introduced into the strain to further verify the effect of the intracellular microenvironment remodeling targets on the expression of P450 enzyme in the stipulatine synthesis pathway. The substrate scoulerine of stipulatine synthase is added in the fermentation medium, and after fermentation, the synthesis level of the product stipulatine is detected by LCMS. As shown in Figure 9 , the test targets can improve the yield of stipulatine. Compared with the control strain, when the targets 10-44 (targeting CYC8) and 31-47 (targeting GET2) are expressed at the same time, the yield of stipulatine is increased by 2.11 times. It is shown that the microenvironment remodeling targets improve the functional expression of P450 stipulatine synthase.

Claims

1. An engineered yeast strain, characterized in that, The engineered yeast strain is a yeast strain containing... CYC8 Genes and GET2 Gene suppression is performed; the yeast strain in question is a Saccharomyces cerevisiae strain. The CYC8 GenBank No. NM_001178460.3 of the gene, GET2 GenBank No. NM_001178974.1 of the gene.

2. The engineered yeast strain of claim 1, wherein, Integrate the gRNA expression sequence targeting the target gene into the genome of the yeast strain.

3. The engineered yeast strain of claim 2, wherein, Targeting CYC8 The gRNA expression sequence targeting the gene is shown in SEQ ID No.

10. GET2 The gRNA expression sequence targeting the gene is shown in SEQ ID No. 14, SEQ ID No. 33 and SEQ ID No.

35.

4. Use of the engineered yeast strain of any one of claims 1-3 in the expression of betalaxanthin, wherein the genome of the engineered yeast strain further comprises integrated betalaxanthin synthesis pathway genes. CYP76AD1 With DOD , to construct a betalaxanthin-producing yeast strain. wherein CYP76AD1 GenBank accession numbers for the gene sequences are KU644144.1 and KM502867.1, respectively. DOD GenBank accession numbers for the gene sequences are KU644144.1 and KM502867.1, respectively.

5. The use of the engineered yeast strain of any one of claims 1-3 in the expression of ventolin, wherein the genome of the engineered yeast strain further integrates AtCPR1 、 T16H2 、 16OMT 、 T3O 、 T3R 、 NMT 、 D4H and DAT genes to construct a ventolin-producing strain. AtCPR1 , T16H2 , 16OMT , T3O, T3R, NMT, D4H and DAT GenBank numbers of the genes are NM_001203894.1, JF742645.1, EF444544.1, KP122967.1, KP122966.1, HM584929.1, U71605.1 and AF053307.1, respectively.

6. The application of the engineered yeast strain of any one of claims 1-3 in expressing cephalomannine, wherein the genome of the engineered yeast strain further integrates a cephalomannine synthase gene, and a cephalomannine-producing strain is constructed. The GenBank number of the cephalomannine synthase gene is AB434654.

7. The application of the engineered yeast strain of any one of claims 1-3 in expressing gaudincine, wherein the genome of the engineered yeast strain further integrates a gaudincine synthase gene, and a gaudincine-producing strain is constructed. The GenBank number of the gaudincine synthase gene is EU882969.

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