CN116891808A

CN116891808A - Construction method and application of saccharomyces cerevisiae strain of cannabidiol synthase with subcellular structure positioning

Info

Publication number: CN116891808A
Application number: CN202310852672.4A
Authority: CN
Inventors: 陈雨彤; 谢玉萍; 李林; 刘成英; 胡瑾瑜; 罗小舟
Original assignee: Senris Biotechnology Shenzhen Co ltd
Current assignee: Senris Biotechnology Shenzhen Co ltd
Priority date: 2023-07-12
Filing date: 2023-07-12
Publication date: 2023-10-17

Abstract

The invention discloses a construction method and application of a saccharomyces cerevisiae strain of cannabidiol synthase with subcellular structure positioning, belonging to the technical field of synthetic biology and the field of genetic engineering. According to the invention, saccharomyces cerevisiae capable of synthesizing CBDA is taken as an initial strain, specific subcellular structure locating proteins and CBDAS are fused and expressed through a linker, subcellular structure locating proteins which effectively improve the yield of CBDA are obtained by screening and integrated in the same saccharomyces cerevisiae strain, the expression level of CBDAS coding genes and the enzyme activity level of CBDAS are optimized, and the yield of CBDA is greatly improved. Furthermore, the multi-copy integrated expression CBDAS is used for exploring the influence of multi-copy expression of the CBDAS with different positioning and single positioning on the yield of the CBDA, so that the recombinant saccharomyces cerevisiae with the yield improved compared with that of the single-copy expression CBDA is obtained, and the yield reaches more than 400 mu M after shaking culture for a certain time.

Description

Construction method and application of saccharomyces cerevisiae strain of cannabidiol synthase with subcellular structure positioning

Technical Field

The invention relates to a construction method and application of a saccharomyces cerevisiae strain of cannabidiol synthase with subcellular structure positioning, belonging to the technical field of synthetic biology and the field of genetic engineering.

Background

Hemp has been grown worldwide for thousands of years due to its medicinal properties, and more than 100 phytocannabinoids have been isolated from hemp to date. Cannabinoids have potential medical uses (antibacterial, anti-inflammatory, anti-tumor, anxiolytic, antidepressant etc.), and can be used for the treatment of various human diseases (epilepsy, diabetes, parkinsonism etc.). Wherein, the cannabigerolic acid (CBGA) is a basic compound generated by cannabis plants, has a protective effect on the growth of cannabis, and the CBGA exists in the hairy body of cannabis flowers and triggers the necrosis of targeted plant cells, so that the cannabis leaves are naturally 'trimmed', and more growth energy is provided for the flowers. CBGA can be converted to other various cannabinoids, for example, by enzymes of cannabis itself to three other cannabinoids: and after decarboxylation of THCA, cannabidiol (CBDA) and CBCA, CBGA, THCA and CBDA, respectively obtaining CBG, tetrahydrocannabinol (THC) and Cannabidiol (CBD). CBDA is converted from CBGA. The de-novo biosynthetic pathway of CBDA is shown in figure 1, where Cannabidiol Synthase (CBDAs) converts CBGA to CBDA and decarboxylation of CBDA gives CBD. CBDA is an important class of cannabinoids, like CBD, which activate the 5-HT1AA serotonin receptor, and is involved in modulating mood, anxiety, insomnia and nausea, and can be used in pharmaceuticals, health products and cosmetics.

Many cannabinoids are present in plants at low levels and coexist with other relatively more abundant cannabinoids, making it difficult to obtain a clean sample from the plant. Similarly, the process of chemically synthesizing cannabinoids and their derivatives is cumbersome, expensive, and low in yield. Thus, there is a need for further methods of preparing pure cannabinoids, precursors of cannabinoids, derivatives of cannabinoids or derivatives of precursors of cannabinoids, such as biosynthesis. The synthetic route to CBDA has been previously patented to be introduced into s.cerevisiae, where CBDA is produced but in lower yields.

Disclosure of Invention

[ technical problem ]

The invention aims to solve the technical problems of optimizing the expression level of cannabidiol synthase and improving the yield of cannabidiol.

Technical scheme

The invention provides a recombinant saccharomyces cerevisiae, which takes yeast which expresses enzymes of a synthetic CBDA pathway and can synthesize CBDA as an initial strain to express CBDAS, wherein the CBDAS is connected with subcellular structure positioning genes through a linker to form fusion proteins, the subcellular structure positioning genes comprise partial endogenous subcellular structure positioning genes and heterogenous self-assembled into subcellular structure genes, the partial endogenous genes comprise Aga2, PLN1, SED1, SAG1 or Atg13, and the partial heterogenous self-assembled into subcellular structure genes comprise Zera or DeltaVP 1.

In one embodiment, the cannabidiol synthase is N-terminally linked to a signal peptide.

Further, the fusion protein is selected from any one of (a) to (k):

(a) Aga2 and CBDAS are expressed through linker fusion,

(b) PLN1 and CBDAS are expressed by linker fusion,

(c) CBDAS of SED1 and the connecting signal peptide SED1sp is expressed through linker fusion,

(d) SAG1 and CBDAS connected with signal peptide SAG1sp are expressed through linker fusion,

(e) Zera and CBDAS are expressed through linker fusion,

(f) Zera and the linking signal peptide Zera based on (e) _SP The CBDAS of (c) is expressed by linker fusion,

(g) DeltaVP 1 is expressed in fusion with CBDAS or CBDAS with tag protein,

(h) Based on (g), VP2C and CBDAS or CBDAS connected with tag protein are expressed through linker fusion,

(i) Over-expressing Aga2, PLN1 or SAG1 fusion protein with CBDAS based on (a), (b) or (d).

In one embodiment, (i) when the subcellular structure localization gene is SAG1, the cannabidiol synthase is N-terminally linked to the signal peptide SAG1sp.

In one embodiment, the SED1 comprises a truncated SED1 (C226).

In one embodiment, the Atg13 includes truncations Atg13 (571-700) and Atg13 (640-686) and truncate-based mutants Atg13 (571-700) ^D651R 、Atg13(571-700) ^F641G/I645G 、Atg13(571-700) ^{D651R/Q666R/P667R} 、Atg13(640-686) ^D651R 、Atg13(640-686) ^F641G/I645G Or Atg13 (640-686) ^{D651R/Q666R/P667R} 。

In one embodiment, the truncated SED1 (C226) is truncated to amino acid positions 111 to 339 of the SED1 amino acid sequence.

In one embodiment, the truncated Atg13 (571-700) is truncated to position 571 to position 700 of the Atg13 amino acid sequence, and the truncated Atg13 (640-686) is truncated to position 640 to position 686 of the Atg13 amino acid sequence.

In one embodiment, the CBDAS gene encodes cannabidiol synthase with an amino acid sequence as shown in SEQ ID NO. 1.

In one embodiment, expressing cannabidiol synthase CBDAS refers to inserting a heterologous CBDAS gene into the saccharomyces cerevisiae genome, and either promoting expression by an endogenous pGal1 promoter or by a pTDH3 promoter.

In one embodiment, the insertion site on the Saccharomyces cerevisiae genome is selected from 1021b, XI_2, i32, and/or 106a.

In one embodiment, the sequence of the linker is shown in any one of SEQ ID NO 12-SEQ ID NO 14.

In one embodiment, the amino acid sequence of Aga2 encoded protein, PLN1 encoded protein, SAG1 encoded protein, and VP2C encoded protein are shown in SEQ ID NO. 3, 4, 6, 8, 9, 10, 11, respectively.

In one embodiment, the amino acid sequences of the subcellular structure localization proteins encoded by Atg13 (571-700) and Atg13 (640-686) are shown in SEQ ID NO. 15 and SEQ ID NO. 16, respectively.

In one embodiment, the amino acid sequence of SED1 (C226) is shown in SEQ ID NO. 17, respectively.

In one embodiment, the nucleotide sequence of the signal peptide SED1sp is shown as SEQ ID NO. 5, the nucleotide sequence of SAG1sp is shown as SEQ ID NO. 7, and Zera _SP The nucleotide sequence of (2) is shown as SEQ ID NO. 18.

In one embodiment, the cannabidiol synthase CBDAS may also be expressed by fusion of a linker with a tag protein.

In one embodiment, the tag protein is a tnsp having the amino acid sequence shown in SEQ ID NO. 19.

In one embodiment, the subcellular structure-localization gene is located upstream of cannabidiol synthase or the subcellular structure-localization gene is located downstream of cannabidiol synthase.

In one embodiment, the starting strain comprises saccharomyces cerevisiae Saccharomyces cerevisiae ySC366 or saccharomyces cerevisiae Saccharomyces cerevisiae ySC594a.

In one embodiment, the Saccharomyces cerevisiae Saccharomyces cerevisiae ySC594a was deposited with the China center for type culture Collection, having a accession number CCTCC M20231031, at month 15 of 2023.

The invention also provides a method for constructing the recombinant saccharomyces cerevisiae, which comprises the following steps:

(1) PCR amplification to obtain an expression cassette of a gene to be over-expressed, and integrating the expression cassette onto a saccharomyces cerevisiae genome; or, carrying out PCR amplification to obtain a homologous fragment for knocking out the gene, and replacing the gene to be knocked out on the saccharomyces cerevisiae genome by using the homologous fragment;

gene knockout and insertion on the saccharomyces cerevisiae genome is achieved using CRISPR-Cas9 technology;

(2) Positive clones were obtained by screening.

The invention also provides an application of the recombinant saccharomyces cerevisiae in producing cannabidiol, which comprises the following steps:

(1) Activating and culturing recombinant Saccharomyces cerevisiae to obtain recombinant Saccharomyces cerevisiae seed solution,

(2) Transferring the recombinant saccharomyces cerevisiae seed liquid into a culture medium for fermentation culture to prepare cannabidiol.

The recombinant saccharomyces cerevisiae can also be used for producing cannabidiol, in particular, the recombinant saccharomyces cerevisiae expresses decarboxylase, and cannabidiol is obtained by decarboxylation of cannabidiol, or the cannabidiol obtained by the recombinant saccharomyces cerevisiae is separated and purified, and then is subjected to decarboxylation by using an enzyme catalyst or a chemical catalyst in vitro to obtain cannabidiol.

[ advantageous effects ]

According to the invention, saccharomyces cerevisiae capable of synthesizing CBDA is taken as an initial strain, specific subcellular structure locating proteins and CBDAS are fused and expressed through a linker, subcellular structure locating proteins which effectively improve the yield of CBDA are obtained by screening and integrated in the same saccharomyces cerevisiae strain, the expression level of CBDAS coding genes and the enzyme activity level of CBDAS are optimized, and the yield of CBDA is greatly improved. Furthermore, the multi-copy integrated expression CBDAS is used for exploring the influence of multi-copy expression of the CBDAS with different positioning and single positioning on the yield of the CBDA, so that the recombinant saccharomyces cerevisiae with the yield improved compared with that of the single-copy expression CBDA is obtained, and the yield reaches more than 400 mu M after shaking culture for a certain time. Further optimization, the concentration and conversion rate of CBDA final products are 7mM and 78% respectively, and the accumulation of CBGA is about 1mM by taking 10mM Olive Acid (OA) as a substrate.

Drawings

FIG. 1 is a synthetic pathway of cannabidiol in Saccharomyces cerevisiae;

FIG. 2 is the effect of linker on CBDA yield;

FIG. 3 is the effect of subcellular structure-localization genes on CBDA production; .

FIG. 4 is a ySC644B 10L fermenter fill test.

Biological material

Saccharomyces cerevisiae ySC594a, taxonomic designation Saccharomyces cerevisiae, was deposited with the China center for type culture Collection, accession number CCTCC M20231031, at 2023, month 06 and 15.

Detailed Description

In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the invention, whereby the invention is not limited to the specific embodiments disclosed below.

Terminology:

CBDAS refers to a heterologous cannabidiol synthase.

CBDA refers to heterologous cannabidiol.

SED1 encodes a differentially expressed cell wall protein which is thought to be transiently secreted first as GPI (glycosylated derivative of phosphoinositide) anchored form onto the plasma membrane and then transferred to the glucan layer of the cell wall.

PLN1 is one of the core members of the lipid-associated protein family, is a highly phosphorylated protein localized on the surface of lipid droplets, has a dual regulation effect on the metabolism of triglycerides in adipose tissue, and can promote lipolysis in the basal state by preventing lipase from approaching lipid droplets, and promote hormone-stimulated lipolysis.

Zera is an n-terminal self-assembled domain from Zein, a maize storage protein, capable of inducing proteome formation in different plant tissues and heterologous systems.

Atg13 is the regulatory subunit of the Atg1p signal complex; stimulating Atg1p kinase activity; vesicle formation and cytoplasmic to vacuole targeting pathways are necessary during autophagy; comprising autophagy and the HORMA domain required for recruiting the phosphatidylinositol 3-kinase complex subunit Atg14p to the autophagy pre-structure.

SAG1 is an α -lectin of α cells; binding to Aga1p during agglutination, the N-terminal half is homologous to the immunoglobulin superfamily, contains a-lectin binding sites, and the C-terminal half is highly glycosylated, containing GPI anchors.

Δvp1 refers to the absence of a putative nuclear localization signal on VP1, which is part of the murine polyomavirus virus like particle MPyV system, which can self-assemble into a chamber shell.

VP2C is a component of the MPyV system of the mouse polyomavirus virus-like particle, mediating the encapsulation of cargo proteins by binding to the inner surface of the VP1 shell.

The experimental method comprises the following steps:

overexpression refers to up-regulating the expression of a gene, i.e., the gene is transcribed and translated excessively, and the final gene expression product exceeds normal levels.

The knockout is to make the DNA fragment with a certain known sequence undergo homologous recombination with the gene whose sequence is identical or similar to that in the genome of receptor cell so as to make the specific gene function in the genome of receptor cell lose action.

The PCR amplification method, fusion method of different fragments, gene knockout and over-expression method used in the following examples can employ common technical means in the art, such as fusion PCR, homologous recombination and CRISPR-Cas9 technology. The enzymes and the kits are commercially available products.

The conversion was performed using lithium acetate/PEG 3350. The transformation methods used in the following examples were: the host strain was first activated in 1 XYPD medium and incubated overnight at 30℃and 200 rpm. Then inoculating to a new 2 XYPD culture medium to make the initial OD value be 0.2, continuously culturing at 30 ℃ for 4-4.5h, taking 5OD bacterial liquid, centrifuging at normal temperature of 3000rcf for 5min, discarding the supernatant, and washing twice with sterilized ultrapure water to obtain yeast cells; preparing DNA mixtures, obtaining cells from 5OD of each construct, and mixing with 50. Mu.L DNA mixture from 2. Mu.g of the insert, 250ng of tool plasmid and a sufficient amount of ddH to resuspend the cells ₂ And mixing O. Adding lithium acetate conversion mixture into suspended cells, culturing to obtain cells, coating the cells on a screening plate, obtaining single colony, namely recombinant saccharomyces cerevisiae, and preserving the recombinant saccharomyces cerevisiae after sequencing and verifying that the conversion is successful.

Colony PCR and sequencing verification: after monoclonal colonies grow on the screening plate, colony PCR and sequencing verification are carried out, and the specific steps are as follows: a small amount of cells are picked by a gun head and respectively placed in 20 mu L of 20mmol/LNaOH solution, vortex mixing is carried out, incubation is carried out for 20min at the temperature of 95 ℃ of a metal bath, vortex mixing is carried out, 1 mu L of bacterial liquid is taken as a template to carry out colony PCR reaction, the reaction primers are primer 7 and primer 8, the sizes of cloning bands and negative cloning bands are compared, and bacterial liquid of colony PCR positive clones is selected to be sent to Jin Weizhi company for sequencing verification. The strain with correct sequence is subjected to streak preservation and glycerol cryopreservation.

Culturing recombinant saccharomyces cerevisiae strains: after the single colony was cultured overnight in 3ml of a1 XYPD 24-well plate at 30℃in a shaker at 200rpm for 16 hours, the bacterial liquid was diluted 10 times with 1 XYPD and the bacterial liquid OD was measured by an ultraviolet spectrophotometer and the wavelength was set at 600nm. Then, the initial OD was 0.2 and transferred to 3mL of 1 XYPG medium for cultivation. The culture mode is as follows: after transfer, 10. Mu.L of 0.1M OA, 300. Mu.L of 20% galactose was added every 24 h. After 72 hours of cultivation, 200. Mu.L of bacterial liquid was collected as a sample.

The method for measuring the enzymatic activity of the cannabidiol synthase comprises the following steps:

under YPG medium conditions, the activity of cannabidiol synthase was judged from CBDA production by addition of substrate caproic acid or OA fermentation.

The method for detecting the CBDA yield of the recombinant saccharomyces cerevisiae comprises the following steps: after sample collection, according to the sample OD ₆₀₀ Incubation with wall breaking enzyme 2U/OD at 30℃and 200rpm shaker for 60min followed by 0.2mL of 0.5mm glass beads and 0.4mL of ethyl acetate: formic acid (0.05%) was treated in a high speed tissue mill at 65Hz for 180s at 30s intervals, repeated three times, each treatment followed by cooling the mill tray on ice for 1min, shaking for 15-30s, instantaneous centrifugation followed by taking the upper organic layer into a 0.28mL to 1.5mL centrifuge tube, repeating twice, and combining the collected upper organic layers. The organic layer of the three extractions was evaporated at 45℃for 1H to free of solvent residues, and resuspended in AHF (acetonitrile: H) ₂ O: formic acid=80:20:0.05%, 140 μl of the internal standard PHB (propyl p-hydroxybenzoate solution standard, 15 μΜ) was resuspended, and 0.22 μΜ pvdf filter membrane was filtered into the cannula in the liquid phase detection vial as the detection sample. Three of each sample were parallel. After sample preparation, the assay was performed by HPLC and the assay conditions are shown in table 1.

Table 1: HPLC detection conditions

2 XYPD medium: yeast extract 20.0g/L, peptone 40.0g/L, and glucose 40.0g/L.

Lithium acetate conversion mixture: 50% W/V PEG 3350260. Mu.L, 1mol/L LiOAc 36. Mu.L, denatured salmon sperm DNA 10. Mu.L (denatured salmon sperm DNA was denatured in a metal bath at 95℃for 5min before use), ddH ₂ O 4μL。

Screening plates lacking uracil: 1.7g/L of YNB yeast nitrogen source mother solution (without amino acid), 5g/L of ammonium sulfate, 20g/L of agar, 20g/L of glucose and the like, wherein the various amino acids are shown in Table 1, and the following steps are added: glucose is sterilized separately.

YPG medium: 10g/L yeast extract, 20g/L peptone, 20g/L galactose.

TABLE 2 screening for the content of various amino acids in plates

Amino acids	(mg/L)	Amino acids	(mg/L)
				Adenine hemisulfate	18	L-phenylalanine	76
L-alanine	76	L-proline	76
				L-arginine	76	L-threonine	76
L-aspartic acid	76	L-serine	76
				L-asparagine	76	L-tryptophan	76
L-cysteine	76	L-tyrosine	76
				L-glutamic acid	76	L-valine	76
L-Glycine	76	L-methionine	76
				L-isoleucine	76	L-lysine	76
L-glutamine	76	L-leucine	360
				L-histidine	76

TABLE 3 primer sequences

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TABLE 4 genotype of strains

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Example 1 preparation of Saccharomyces cerevisiae ySC594a

Based on the strain Saccharomyces cerevisiae ySC366 contained in the patent CN114657078A, after continuous passage on YPD culture media with different concentrations of glucose, a growth dominant strain is obtained, the metabolic rate of glucose is measured, and a strain Saccharomyces cerevisiae ySC594a with a fast glucose metabolic rate is selected and is preserved in China center for type culture collection (CCTCC M20231031) at the year of 2023 and 15.

EXAMPLE 2 construction of recombinant Yeast Strain expressing cell surface display localized cannabidiol synthase

(1) Construction of Strain ySC629

The strain ySC594a was used as a host, the genome of Saccharomyces cerevisiae CEN.PK2-1C was used as a template, the fragment 1021b-up was amplified by using the primer 1021b-up-F/1021b-up-R, the fragment LEU2 was amplified by using the primer LEU2-F/LEU2-R, the fragment 1021b gRNA-1021b Down was amplified by using the primers 1021b gRNA-1021b Down-F and 1021b Down-R, and all the fragments were transformed into the host Saccharomyces cerevisiae ySC594a prepared in example 1, and gRNA at 1021b site was complemented back by the expression cassette 1021b up-pLEU2-LEU2-tLEU2-1021b gRNA-1021b Down to obtain the strain ySC629.

(2) Construction of cell surface display positioned recombinant Yeast Strain of cannabidiol synthase

The expression cassette is amplified by PCR using primers to create an integrated expression cassette (typically comprising two flanking homology regions, a promoter, a synthase gene sequence, a subcellular structure localization gene, a terminator and a linker, with or without signal peptide sequences attached to the synthase gene) comprising flanking homology regions targeting the selected genomic locus and then co-transformed into yeast cells with Cas9-gRNA plasmid pCUT 1021b ura targeting the gene. In addition, the primers provide homology arms between fragments, so that homologous recombination self-assembly can be performed on 1-5 individual fragments of yeast. The pCUT 1021b ura plasmid targeting the genomic locus 1021b site is assembled from a linear backbone pCUT and a linear fragment comprising the gRNA sequence. sgrnas were generated by an online sgRNA design tool.

(a) Recombinant Saccharomyces cerevisiae carrying a synthase gene to which a signal peptide is not linked

The integrated fragment was amplified by PCR with 2X Phanta Max Master Mix (Phanta DNA polymerase). The upstream homology arm 1021b-Up fragment of integration site 1021b was obtained by amplification with 1021b-UP-F and 1021b-UP-R in Table 3, the downstream homology arm 1021b-Down fragment of integration site 1021b was obtained by amplification with 1021b-Down-F and 1021b-Down-R, and the subcellular structure localized gene fragment Aga2-Gly6 was obtained by amplification with pGAL1-Aga2-F and Aga2-Gly6-R using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template; the synthase fragment CBDAS-tADH1 was obtained by PCR amplification using the existing strain ySC594a as template and the primers Gly6-CBDAS-F and tADH 1-R.

The amplified upstream homology arm 1021b-Up fragment, downstream homology arm 1021b-Down fragment, fragment Aga2-Gly6, promoter pGAL1, fragment CBDAS-tADH1 (heterologous cannabidiol synthase and yeast terminator) was constructed to obtain 1021b-Up

-pGal1-Aga2-Gly6-CBDAS-tADH1-1021b-Down expression cassette fragment; the 1021b-Up obtained

Transforming the pGal1-Aga2-Gly6-CBDAS-tADH1-1021b-Down expression cassette fragment and the knockout plasmid pCUT 1021b ura into the host Saccharomyces cerevisiae ySC629 constructed in the step (1), so as to obtain a strain ySC630; the primers 1021b seq-F and 1021b seq-R in Table 3 were used to perform PCR on strain ySC630 to obtain a bacterial colony of PCR positive clones, which were used for gene sequencing, and the results showed successful integration of the above expression cassette into 1021b site of Saccharomyces cerevisiae ySC629.

By the same method, recombinant Saccharomyces cerevisiae (strain genotypes are shown in Table 4) carrying different Saccharomyces cerevisiae endogenous subcellular structure localization genes (Aga 2, PLN1, atg13 (571-700), atg13 (640-686) and different linker (Gly 6, GSA or EAAAK) are respectively constructed according to the primer sequences in Table 3 by taking Saccharomyces cerevisiae ySC629 as a host.

(b) Recombinant Saccharomyces cerevisiae carrying a signal peptide-linked synthetase gene

The integrated fragment was amplified by PCR with 2X Phanta Max Master Mix (Phanta DNA polymerase). The upstream homology arm 1021b-Up fragment of integration site 1021b was obtained by amplification using 1021b-UP-F and 1021b-UP-R in Table 3, the downstream homology arm 1021b-Down fragment of integration site 1021b was obtained by amplification using 1021b-Down-F and 1021b-Down-R, and the signal peptide fragment pGAL1-SEDsp was obtained by amplification using 1021b-pGAL1-F and pGAL1-SEDsp-R, using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template; the existing strain ySC594a is used as a template, a synthetase fragment SED1sp-CBDAS-Gly6 is obtained through PCR amplification by using primers SED1sp-CBDAS-F and CBDAS-Gly6-R, an SED1 fragment is obtained through PCR amplification by using primers Gly6-SED1-F and SED1-tADH1-R, and a terminator fragment tADH1 is obtained through PCR amplification by using tADH1-F and tADH 1-R.

The amplified upstream homology arm 1021b-Up fragment, downstream homology arm 1021b-Down fragment, signal peptide fragment pGAL1-SEDsp, fragment SED1sp-CBDAS-Gly6, SED1 fragment and terminator fragment tADH1 are constructed to obtain

1021b-Up-pGal1-SED1sp-CBDAS-Gly6-SED1-tADH1-1021b-Down expression cassette fragment; transforming the 1021b-Up-pGal1-SED1sp-CBDAS-Gly6-SED1-tADH1-1021b-Down expression cassette fragment and the knockout plasmid pCUT 1021b ura into the host Saccharomyces cerevisiae ySC629 constructed in the step (1), and obtaining a strain ySC636; the primers 1021b seq-F and 1021b seq-R in Table 3 were used to perform PCR on strain ySC636 to obtain a bacterial solution of colony PCR positive clones for gene sequencing, which showed successful integration of the above expression cassette into 1021b site of Saccharomyces cerevisiae ySC629.

By the same method, recombinant Saccharomyces cerevisiae (strain genotypes are shown in Table 4) carrying different Saccharomyces cerevisiae endogenous subcellular structure localization genes (SED 1, SED1 (C226), SAG1 and different linker (Gly 6, GSA or EAAAK) was constructed according to the primer sequences in Table 3 by using Saccharomyces cerevisiae ySC629 as a host.

(c) Recombinant saccharomyces cerevisiae carrying exogenous subcellular structure localization gene

Construction of recombinant Saccharomyces cerevisiae carrying exogenous subcellular structure localized gene DeltaVP 1, amplification of the synthesized gene fragment DeltaVP 1 using primers pTDH 3-DeltaVP 1-F and DeltaVP 1-R to obtain subcellular structure localized gene fragment DeltaVP 1, amplification of the genome of Saccharomyces cerevisiae CEN.PK2-1C using primers tTDH1-F and tTDH1-R in Table 3 to obtain the upstream homology arm XI_2-Up fragment of integration site XI_2, amplification of the downstream homology arm XI_2-Down fragment of XI_2 using XI_2-Down-F and XI_2-Down-R to obtain promoter fragment pTDH3 using primers pTDH3-F, pTDH-R, amplification of the existing strain ySC594a using primers tTDH1-F and tTDH1-R to obtain the terminator fragment tTDH1, and subsequent transformation of the expression cassette into Saccharomyces cerevisiae 6283 using the same method to obtain the recombinant Saccharomyces cerevisiae 6283.

Similarly, recombinant Saccharomyces cerevisiae ySC824 and ySC825 (strain genotypes are shown in Table 4) were constructed by the same method using the synthesized gene fragments DeltaVP 1-Tnsp-CBDAS and DeltaVP 1-CBDAS as templates.

Likewise, the synthetic gene fragments Zera, zera _SP The recombinant Saccharomyces cerevisiae (strain genotypes are shown in Table 4) is constructed by using the same method with CBDAS-GSA-Zera as a template.

By the same method, in Saccharomyces cerevisiae ySC823 as a host, recombinant Saccharomyces cerevisiae ySC826 and ySC827 with or without tag proteins integrated at the i32 site of the Saccharomyces cerevisiae genome were constructed according to the primer sequences in Table 3, respectively.

By the same method, in Saccharomyces cerevisiae ySC824 as a host, recombinant Saccharomyces cerevisiae ySC828 and ySC829 were constructed according to the primer sequences in Table 3, respectively, with or without tag proteins integrated at the i32 site of the Saccharomyces cerevisiae genome.

By the same method, in Saccharomyces cerevisiae ySC825 as a host, recombinant Saccharomyces cerevisiae ySC and ySC831 were constructed according to the primer sequences in Table 3, respectively, with or without tag proteins integrated at the i32 site of the Saccharomyces cerevisiae genome.

(d) Recombinant saccharomyces cerevisiae carrying subcellular structure positioning mutant genes

Carrying subcellular structure localization mutant gene Atg13 (571-700) ^D651R 、Atg13(571-700) ^F641G/I645G 、Atg13(571-700) ^{D651R/Q666R/P667R} 、Atg13(640-686) ^D651R 、Atg13(640-686) ^F641G/I645G Or Atg13 (640-686) ^{D651R/Q666R/P667R} ) Is used, respectively, with a mutation primer (e.g., atg 13571-700) F641G/I645G-R/Atg13 (571-700) F641G/I645G-F, atg (571-700) D651R-R/Atg13 (571-700) D651R-F, atg (571-700)) D651R/Q666R/P667R/Atg 13 (571-700)) D651R/Q666R/P667R-F, atg (640-686) F641G/I645G-R/Atg13 (640-686) F641G-F, atg (640-686) D651R-R/Atg13 (640-686) D651R-F,

(640-686) D651R/Q666R/P667R-R/(640-686) D651R/Q666R/P667R-F) taking the subcellular structure locating gene fragment of the starting gene as a template, carrying out PCR amplification to obtain the subcellular structure locating gene fragment of the mutant gene, further constructing to obtain an expression cassette fragment, and adopting the same method in the step (a) to obtain the recombinant saccharomyces cerevisiae carrying the subcellular structure locating mutant gene and different linker.

Shake flask cultivation ySC, 630, and detection of CBDA content as shown in fig. 2, the CBDA content of the starting strain ySC594a (CBDA yield 105 μm) was significantly lower than ySC630 (CBDA yield 302 μm).

EXAMPLE 3 construction of recombinant Saccharomyces cerevisiae expressing multiple copies of Single subunit cell-localized genes

On the basis of example 2, the integrated fragment was amplified by PCR with 2X PhantaMax Master Mix (PhantaDNA polymerase). The upstream homology arm 106a-Up fragment of integration site 106a was amplified using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and 106a-UP-F and 106a-UP-R in Table 3, and the downstream homology arm 106a-Down fragment of integration site 106a was amplified using 106a-Down-F and 106 a-Down-R; the pGal1-Aga2-Gly6-CBDAS-tADH1 fragment was obtained by amplification using the genome of strain ySC630 as a template and 106a-UP-pGAL1-F and tADH1-106a-Down-R in Table 3, and then all fragments were transformed into Saccharomyces cerevisiae strain ySC630 together with plasmid pCUT 106aura to obtain strain ySC630A. The primers 106a seq-F and 106a seq-F in the table were used for PCR reaction of strain ySC630A to obtain bacterial liquid of colony PCR positive clone for gene sequencing.

The pCUT 106aura plasmid targeting the genomic locus 106a site was assembled from a linear backbone pCUT and a linear fragment comprising the gRNA sequence. sgrnas were generated by an online sgRNA design tool.

By the same method, recombinant Saccharomyces cerevisiae carrying multiple copies of the expressed single subunit cell-localized genes was constructed separately from the primer sequences in Table 3.

Shake flask culture ySC, 630A, ySC, 635, A, ySC, 644A and examined for CBDA content as shown in fig. 3, multiple copies of recombinant saccharomyces cerevisiae ySC a (CBDA yield 257 μm), ySC, 635A (CBDA yield 117 μm), ySC, 644A (CBDA yield 250 μm) were inferior to single copies of recombinant saccharomyces cerevisiae ySC (CBDA yield 302 μm), ySC, 635 (CBDA yield 200 μm), ySC, 644 (CBDA yield 259 μm) expressing single subunit cell-localized genes.

EXAMPLE 4 construction of recombinant Saccharomyces cerevisiae carrying multiple subunit cell-localized genes

On the basis of example 2, the integrated fragment was amplified by PCR with 2X PhantaMax Master Mix (PhantaDNA polymerase). The upstream homology arm 106a-Up fragment of integration site 106a was amplified using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and 106a-UP-F and 106a-UP-R in Table 3, and the downstream homology arm 106a-Down fragment of integration site 106a was amplified using 106a-Down-F and 106 a-Down-R; the pGal1-Aga2-Gly6-CBDAS-tADH1 fragment was obtained by amplification using the genome of strain ySC630 as a template and 106a-UP-pGAL1-F and tADH1-106a-Down-R in Table 3, and then all the fragments were transformed into Saccharomyces cerevisiae strain ySC644 to obtain strain ySC630C. Primers 106a seq-F and 106a seq-R in the table were used to perform PCR reaction on strain ySC630C to obtain bacterial liquid of colony PCR positive clone for gene sequencing.

By the same method, recombinant Saccharomyces cerevisiae carrying a plurality of subunit cell-localized genes was constructed separately from the primer sequences in Table 3.

Shake flask culture ySC, 630B, ySC, 630C, ySC, 644B and examined for CBDA content as shown in fig. 3, recombinant saccharomyces cerevisiae ySC B (CBDA yield 389 μm), ySC630C (CBDA yield 577 μm), ySC644B (CBDA yield 483 μm) were higher in CBDA yield than recombinant saccharomyces cerevisiae ySC (CBDA yield 302 μm), ySC635 (CBDA yield 200 μm), ySC644 (CBDA yield 259 μm) expressing a single subunit cell-localized gene in single copy.

Shake flask fermentation was performed using the recombinant Saccharomyces cerevisiae of examples 2-4 and the starting strain ySC594a, using 250ml shake flasks, three strains each in parallel, 1mM OA, 20ml YPG medium for 96h, 100. Mu.l 0.1M OA was added at 24h, 48h, 200. Mu.l bacterial liquid was extracted at 72h and 96h, and CBDA production was measured, 25. Mu.l bacterial liquid was measured for OD ₆₀₀ Values.

In summary, as can be seen from fig. 2-3, the present invention uses different linkers to fusion express specific subcellular structure localization proteins and cannabidiol synthase CBDAS, and the CBDA yield of partial localization is significantly improved compared with that of the original strain ySC594a, and the CBDA yield is not significantly different from that of different linkers. The integration of the CBDAS with different positioning is better than the multi-copy expression of the CBDAS with single positioning. According to the invention, the CBDAS of the cannabidiol synthase is positioned on a specific subcellular structure through a linker, so that the expression level of the CBDAS of the heterologous cannabidiol synthase is optimized, and the yield of the cannabidiol is improved.

Example 5ySC644B tank fermentation

The present example used a hundred organisms 4 in 10L stainless steel fermenter. Taking the recombinant Saccharomyces cerevisiae constructed in example 4 as a fermentation strain, preparing a first-stage seed solution by using ySC644B glycerin tube, culturing by using YPD liquid culture medium, shaking a bottle by using 250ml, filling 20ml of liquid, and transferring a second-stage seed solution after overnight culture at 30 ℃/200 rpm. The secondary seed liquid medium was YPD, a 2000ml shaking flask was used, the liquid loading amount was 500ml, and after culturing at 30 ℃/200rpm, the fermentation tank was inoculated, and the initial OD600 was controlled to about 0.2. YPD was used for fermentation medium and fermentation parameters were as follows: 1vvm/0.5MPa/DO not less than 50%/pH 5.0. Glucose (carbon source) is supplemented in a fed-batch feeding mode in the fermentation process, and the residual sugar is controlled to be less than 5g/L and the ethanol concentration is controlled to be less than 10g/L. OA feed was added at 5 intervals from 24hr at the beginning of the fermentation using a 1M mother liquor. Samples were taken every 24hr during fermentation to detect the OD and product of the fermentation. After fermentation, the fermentation liquor is treated by the fermentation waste liquor after in-situ high-temperature sterilization. As can be seen from FIG. 4, the final CBDA product concentration and conversion were 7mM and 78% respectively, and about 1mM CBGA was accumulated, using YPD medium and glucose as a carbon source for 144 hours with 10mM OA as a substrate. Fermentation experiments prove that the strain has potential for fermentation amplification and actual production.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A recombinant saccharomyces cerevisiae, characterized in that a yeast which expresses an enzyme for synthesizing cannabidiol pathway and can synthesize cannabidiol is taken as an original strain, cannabidiol synthase CBDAS is expressed, the cannabidiol synthase CBDAS is connected with subcellular structure positioning genes through a linker to form fusion proteins, the subcellular structure positioning genes comprise partial endogenous subcellular structure positioning genes or heterologous self-assembled into subcellular structure genes, the partial endogenous genes comprise Aga2, PLN1, SED1, SAG1 or Atg13, and the partial heterologous self-assembled into subcellular structure genes comprise Zera or DeltaVP 1; the amino acid sequence of the cannabidiol synthase CBDAS is shown as SEQ ID NO. 1.

2. The recombinant s.cerevisiae according to claim 1, wherein the cannabidiol synthase CBDAS is N-terminally linked to a signal peptide.

3. The recombinant saccharomyces cerevisiae according to claim 1 or 2, wherein said fusion protein is selected from any of (a) to (i):

(a) Aga2 and CBDAS are expressed through linker fusion;

(b) PLN1 and CBDAS are expressed by linker fusion;

(c) CBDAS of SED1 and the connecting signal peptide SED1sp is expressed through linker fusion;

(d) SAG1 and CBDAS connected with signal peptide SAG1sp are expressed through linker fusion;

(e) The Zera and the CBDAS are expressed through linker fusion;

(f) On the basis of (e), the CBDAS is connected with a signal peptide Zera at the upstream _SP ；

(g) DeltaVP 1 is expressed in fusion with CBDAS or CBDAS with tagged proteins;

(h) On the basis of (g), VP2C and CBDAS or CBDAS connected with tag protein are expressed through linker fusion;

(i) Over-expressing Aga2, PLN1 or SAG1 fusion protein with CBDAS based on (a), (b) or (d);

(i) In the method, when the subcellular structure localization gene is SAG1, the N end of the cannabidiol synthase is connected with a signal peptide SAG1sp.

4. The recombinant s.cerevisiae according to claim 3, wherein the SED1 comprises a truncated SED1 (C226);

the Atg13 comprises a truncated Atg13 (571-700) and an Atg13 (640-686) mutant Atg13 (571-700) based on the truncated Atg ^D651R 、Atg13(571-700) ^F641G/I645G 、Atg13(571-700) ^{D651R/Q666R/P667R} 、Atg13(640-686) ^D651R 、Atg13(640-686) ^F641G/I645G Or Atg13 (640-686) ^{D651R/Q666R/P667R} The truncated SED1 (C226) is cut to the 111 th to 339 th positions of the amino acid sequence of the SED 1; the truncated Atg13 (571-700) is truncated to 571 th to 700 th positions of an Atg13 amino acid sequence, and the truncated Atg13 (640-686) is truncated to 640 th to 686 th positions of the Atg13 amino acid sequence.

5. A recombinant saccharomyces cerevisiae according to claim 3 wherein the cannabidiol synthase CBDAS is expressed by fusion of a linker with a tag protein.

6. The recombinant s.cerevisiae according to claim 3, wherein the subcellular structure-localization gene is located upstream of cannabidiol synthase or the subcellular structure-localization gene is located downstream of cannabidiol synthase.

7. The recombinant s.cerevisiae according to claim 1, wherein the expression of cannabidiol synthase CBDAS means that the heterologous CBDAS gene is inserted into the s.cerevisiae genome and expression is promoted by endogenous pGal1 promoter or pTDH3 promoter.

8. The recombinant s.cerevisiae according to claim 7, wherein the insertion site on the s.cerevisiae genome is selected from 1021b, XI_2, i32 and/or 106a.

9. Use of the recombinant s.cerevisiae according to any one of claims 1 to 8 for the production of cannabidiol comprising the steps of:

10. Use of the recombinant saccharomyces cerevisiae according to any of claims 1-8 for the production of cannabidiol.