CN113999783A - Recombinant saccharomyces cerevisiae for expressing heterpenoid compounds and construction method and application thereof - Google Patents
Recombinant saccharomyces cerevisiae for expressing heterpenoid compounds and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant saccharomyces cerevisiae for expressing a heterpenoid compound and a construction method and application thereof, wherein exogenous cannabichromene synthetase is introduced into saccharomyces cerevisiae for expressing a cannabigerol acid, and the recombinant saccharomyces cerevisiae for expressing the heterpenoid compound is obtained by transforming into a plasmid carrying the exogenous cannabichromene synthetase gene or integrating the exogenous cannabichromene synthetase gene on a saccharomyces cerevisiae chromosome, for example, a product obtained by PCR amplification of an upstream sequence of 416d, a downstream sequence of 416d, a promoter sequence of Gal1, an ADH1 terminator sequence and a ProA-CBCAS sequence optimized by a codon and plasmid pCUT-416d are transformed into the saccharomyces cerevisiae together. The recombinant saccharomyces cerevisiae has the advantage of efficiently expressing the triterpenoids, and can be used for producing the triterpenoids through fermentation.
Description
Technical Field
The invention relates to a gene engineering modification technology in the technical field of synthetic biology, in particular to recombinant saccharomyces cerevisiae for expressing a heterpenoid compound, a construction method thereof and application thereof in industrial production of the heterpenoid compound.
Background
The heterepenoid (Merotepenoid) is a prerequisite of a plurality of natural product drugs or the drugs, and is widely applied to the fields of medicine, chemical industry and the like. Saccharomyces cerevisiae (Saccharomyces cerevisiae), an important eukaryotic model organism, is widely used as an important underpan cell for natural product biosynthesis, and is widely used for biosynthesis for expressing various natural products including heteroterpenoids and analogues thereof. Compared with the prokaryotic Escherichia coli, the complex intracellular tissue structure has a complex intracellular tissue structure similar to that of other eukaryotes, and is beneficial to physically isolating different biochemical reaction types so as to improve the orthogonality and efficiency of biochemical reaction. Compared with pichia pastoris, saccharomyces cerevisiae is an internationally recognized food-grade safe microorganism, does not produce toxins, and an expression product does not need to be subjected to a large number of safety experiments. Therefore, the construction of the saccharomyces cerevisiae strain with the high-efficiency expression of the triterpenoid has important application value and practical significance for industrial production. The expression of the triterpenoids in the saccharomyces cerevisiae is usually achieved by combining endogenous and exogenous synthetic pathways, however, the expression level of various natural products including the triterpenoids is poor due to the complex regulation mode of the endogenous pathway and the deficiency or poor efficiency of enzymes of the exogenous pathway, which becomes the bottleneck problem of the efficient expression of the compounds in the saccharomyces cerevisiae.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides the recombinant saccharomyces cerevisiae for expressing the triterpenoid and the construction method and the application thereof.
In order to solve the technical problem, one embodiment of the present invention adopts the following technical solutions:
in a first aspect, the invention provides a construction method of recombinant saccharomyces cerevisiae for expressing a triterpenoid, which introduces exogenous cannabichromene synthetase into the saccharomyces cerevisiae for expressing the cannabigerolic acid to obtain the recombinant saccharomyces cerevisiae for expressing the triterpenoid.
In the construction method of the recombinant saccharomyces cerevisiae for expressing the triterpenoid, the method for introducing the exogenous cannabis pigment synthetase comprises the following steps: by transformation into a plasmid carrying an exogenous cannabis pigment synthase gene, or by integration of an exogenous cannabis pigment synthase gene on the saccharomyces cerevisiae chromosome.
In the construction method of the recombinant saccharomyces cerevisiae for expressing the triterpenoid, the exogenous cannabis pigment synthase gene at least comprises a product obtained by PCR amplification of a ProA-CBCAS sequence optimized by a codon.
In the construction method of the recombinant saccharomyces cerevisiae for expressing the triterpenoid, a product obtained by amplifying a codon-optimized ProA-CBCAS sequence through PCR and a plasmid pCUT-416d can be jointly transformed into the saccharomyces cerevisiae.
In the construction method of the recombinant saccharomyces cerevisiae for expressing the triterpenoids, products obtained by amplifying the 416d upstream sequence, the 416d downstream sequence, the Gal1 promoter sequence and the ADH1 terminator sequence by using PCR and products obtained by amplifying the ProA-CBCAS sequence optimized by codon can be used for transformation during transformation.
In the construction method of the recombinant saccharomyces cerevisiae for expressing the triterpenoid, the transformation step comprises the following steps:
saccharomyces cerevisiae cells were activated and then cultured to OD in YEPD medium600Raising the temperature to between 0.7 and 1.0, collecting cells, mixing a transformation solution containing the product and the plasmid pCUT-416d with the cells, incubating at 30 ℃, thermally shocking at 42 ℃, and collecting the cells.
In the construction method of the recombinant saccharomyces cerevisiae for expressing the triterpenoid, one transformation amount of the transformation liquid comprises: 120uL 50% PEG3350, 18uL1mol/L LiAC, 5uL 10mg/mL single-stranded milt DNA, 35uL sterile water and 2ug of the product and plasmid pCUT-416d mixed fragment to be transformed.
In the construction method of the recombinant saccharomyces cerevisiae for expressing the triterpenoid, the transformed saccharomyces cerevisiae is further co-cultured by a YEPD solid culture medium and an auxotrophic solid culture medium SC-URA3, and the saccharomyces cerevisiae which is introduced with the cannabichromene synthetase and is removed with the pCUT-416d plasmid is screened.
In a second aspect, the invention provides the recombinant saccharomyces cerevisiae for expressing the triterpenoid obtained by the construction method.
In a third aspect, the invention provides an application of the recombinant saccharomyces cerevisiae in fermentation production of the triterpenoid.
The technical solution of the present invention is described in more detail below.
The invention uses the Saccharomyces Cerevisiae expressing the cannabigerolic acid to construct the recombinant Saccharomyces Cerevisiae expressing the triterpenoid compounds, the Saccharomyces Cerevisiae expressing the cannabigerolic acid can select a strain which is preserved by Euroscarf center and has the strain number of CEN.PK2-1C, the strain name is Saccharomyces Cerevisiae, the latin name is Saccharomyces Cerevisiae, and the preservation center has the registration number: 30000A, the central address of Euroscarf is SRD-Scientific Research and Development GmbH
20,61440 obeursel, Germany. The European Scarf center is generally referred to as European Saccharomyces cerevisiae Archive for functional analysis.
First, in the case of cannabis pigment synthase (CBCAS) derived from cannabis sativa (c.sativa), the function of CBCAS is to cyclize cannabigerolic acid (CBGA) to CBCA.
We first prepared the relevant knock-in fragment. One optional scheme is that five products obtained by PCR amplification of the 416d upstream sequence, the 416d downstream sequence, the Gal1 promoter sequence, the ADH1 terminator sequence and the codon-optimized ProA-CBCAS sequence are five knock-in fragments to be recombined into saccharomyces cerevisiae, and one method for obtaining the five knock-in fragments is to perform PCR amplification through primers 1 to 10 and amplification sequences of corresponding PCR templates, wherein the specific PCR amplification contents are shown in table 1, the primers are shown in table 2, and the sequences of the amplified products are shown in a sequence table.
TABLE 1 PCR amplification information
TABLE 2 primer sequences
| Primer 1 | TATCGTCCAACTGCATGGAGATGA |
| Primer 2 | GATATGTATATGGTGGTAATGCCATGTGGGTCCGGTTAAACGGATCTC |
| Primer 3 | CTCATCAATGCGAGATCCGTTTAACCGGACCCACATGGCATTACCACCATATACATATCC |
| Primer 4 | TCGTGGTCCCATCGAAAATCATTATAGTTTTTTCTCCTTGACGTTAAAGTATAGAGGTA |
| Primer 5 | CCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGATTTTCGATGGGACCACGATG |
| Primer 6 | TTAATAATAAAAATCATAAATCATAAGAAATTCGCCTAATGATGGTGGGGGGGTAGTGG |
| Primer 7 | ACCCCCCCACCATCATTAGGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAG |
| Primer 8 | TGTTAAAATAGTGAAGGAGCATGTTCGGGGAGTTAGCATATCTACAATTGGGTGAA |
| Primer 9 | TTCACCCAATTGTAGATATGCTAACTCCCCGAACATGCTCCTTCACTATTTTAAC |
| Primer 10 | ATTTTTCAATTGAGGAAACTTGAAAGGTG |
After obtaining the sequences 1 to 5, the five fragments were transformed together into a strain of saccharomyces cerevisiae with high yield of cannabigerolic acid by the steps of:
activating Saccharomyces cerevisiae strain producing cannabigerolic acid in YEPD culture medium, and culturing in YEPD culture medium to obtain OD600From 0.2 up to 0.7-1.0, the cells were centrifuged, and the transformation fluid containing the five fragments and plasmid pCUT-416d was added to the cell suspension, incubating at 30 deg.C and heat-shocking at 42 deg.C, centrifuging to obtain cells, spreading on auxotrophic solid culture medium SC-URA3, culturing, selecting 1 clone, activating in YEPD medium, diluting, inoculating to YEPD solid medium, picking several clones, each of which is spread on YEPD solid medium and auxotrophic solid medium SC-URA3, selecting the clone which grows on YEPD solid medium but not on SC-URA, the strain without pCUT-416d plasmid is verified by PCR, and the saccharomyces cerevisiae with the CBCAS knocked in and without pCUT-416d plasmid, namely the recombinant saccharomyces cerevisiae for expressing the triterpenoids is obtained.
Compared with the prior art, the invention has at least the following beneficial effects: a recombinant saccharomyces cerevisiae strain capable of efficiently secreting and expressing a triterpenoid compound, namely, cannabichrome acid, is obtained by modifying a saccharomyces cerevisiae synthesis way and introducing a novel exogenous gene. The invention discovers for the first time that a recombinant strain obtained by introducing a heterologous gene (cannabichromene synthase, CBCAS) into the modified saccharomyces cerevisiae can efficiently express CBCA and CBC, and the recombinant strain has very important value in the industrial production of the heteroterpenoid.
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FIG. 1 is a comparison of CBCA standard sample and biosynthesis sample, wherein the first row shows the HPLC-MS peak of CBCA standard sample, the second row shows the HPLC-MS peak of recombinant Saccharomyces cerevisiae expression sample, and the third row shows negative control, and it can be seen that the recombinant Saccharomyces cerevisiae expression sample contains CBCA.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The raw material sources are as follows:
YEPD medium: the culture medium is used for enrichment culture of yeast, and comprises the following components: 10.0g/L of yeast extract, 20.0g/L of peptone, 20.0g/L of glucose and 0.03g/L of adenine sulfate; weighing 50g of the product, heating and dissolving in 1000ml of distilled water, subpackaging, and autoclaving at 116 ℃ for 30min for later use. In a case where no specific explanation is given, the YEPD medium is a liquid medium, and if a solid medium is to be prepared, 20.0g/L agar powder is added to the medium.
100mmol/L LiAC: 0.1mol/L LiAc, adjusted pH to 7.5 with acetic acid, sterilized at 121 ℃ for 20 min.
Single-stranded milt DNA: salmon sperm DNA (Solebao, H1060) was denatured in a metal bath at 95 ℃ for 5min and stored at-20 ℃.
Preparation method of auxotrophic solid culture medium SC-URA3 (the following reagents are purchased from sigma except special instructions):
(1) amino acid mother liquor (abbreviation: 5 AA):
TABLE 3 amino acid mother liquor starting material
(2)10 g/L of yeast nitrogen source mother liquor (10 YNB) (from Solebao Y8040).
(3) 50g/L of 10 ammonium sulfate mother liquor (10 AS).
(4) The 10 leucine mother liquor (10 Leu for short) is 3.6 g/L.
(5) 7.6g/L of 100 histidine mother liquor (100 His for short).
(6)20 uracil mother liquor (abbreviated as 20Ura) is 1.52 g/L.
(7) 200g/L of 10 glucose mother liquor.
(8) 1L of 5AA, 500mL of 10YNB, 500mL of 10AS, 1000mL of 10Leu, 100mL of 100His, 500mL of 10 glucose, and autoclaved at 121 ℃ for 20min were prepared, respectively.
(9) Preparation of SC-URA3(500 mL): weighing 10g of agar, adding pure water to 175mL, and autoclaving at 121 ℃ for 20 min; and (3) adding the following components into the sterilized agar solution while the agar solution is hot: 5AA 100mL, 10YNB 50mL, 10AS 50mL, 10Leu 100mL, 100His 5mL, 10 glucose 50 mL. Mixing, pouring the mixture into a flat plate while the mixture is hot, and storing the flat plate in a refrigerator at 4 ℃ for later use after the flat plate is solidified.
1mmol/L OA: OA is synthesized by hangzhou kengying chemical company limited.
CBCA standards: cerilliant company (C-150-1 ML).
And (3) amplifying the ProA-CBCAS sequence optimized by the codon by using the sequence 6 in the sequence table as a PCR template and using the primer 5 and the primer 6 to obtain a sequence 3 in the sequence table as a fragment to be knocked in. Sequence 6 is a sequence synthesized by IDT corporation.
A saccharomyces cerevisiae CEN.PK2-1C genome is used as a PCR template, a sequence 1 is obtained by amplifying a 416d upstream sequence by using a primer 1 and a primer 2, a sequence 2 is obtained by amplifying a Gal1 promoter sequence by using a primer 3 and a primer 4, a sequence 4 is obtained by amplifying an ADH1 terminator sequence by using a primer 7 and a primer 8, and a sequence 5 is obtained by amplifying a 416d downstream sequence by using a primer 9 and a primer 10. The sequence 1, the sequence 2, the sequence 4 and the sequence 5 are fragments to be knocked in, and are shown in a sequence table.
Next, the five knock-in fragments obtained above were transformed together into the strain of Saccharomyces cerevisiae CEN. PK2-1C, which produces cannabigerolic acid in high yield. The method comprises the following specific steps:
preparing a transformation solution, taking the amount of one transformation as an example: 120uL of 50% PEG3350 was mixed well, then 18uL of 1mol/L LiAC, 5uL of 10mg/mL single-stranded milt DNA (boiling at 100 ℃ for 5min and then rapidly placing on ice), 35uL of sterile water and the fragment to be transformed (fragment 1, fragment 2, fragment 3, fragment 4, fragment 5 and plasmid pCUT-416d total 2ug) were added and mixed well. Amount of one conversion: 100ul of yeast cells were used to transform one plasmid, i.e., one reaction.
Selecting a Saccharomyces cerevisiae strain CEN. PK2-1C capable of highly producing cannabigerolic acid into a 96-hole deep-hole plate containing 300uL YEPD culture medium, activating at 30 ℃ for 36h, transferring the activated Saccharomyces cerevisiae strain CEN. PK2-1C into a medium containing 500uL YEPD, and diluting by 20 times to enable initial OD600About 0.2, cultured at 30 ℃ to OD600Centrifuging at 5000rpm for 5min to remove YEPD culture medium between 0.7-1.0, resuspending the cell precipitate with 500uL sterile water, centrifuging at 5000rpm for 5min to remove supernatant, then resuspending the cell precipitate with 500uL 100mmol/L LiAC, and centrifuging at 5000rpm for 5min to remove supernatant to obtain yeast cells.
178uL of the transformation medium was added to the yeast cells and the cells were resuspended. The cell suspension was incubated at 30 ℃ for 30min and heat-shocked in a 42 ℃ water bath for 25 min. Then, the mixture was centrifuged at 5000rpm for 1min to remove the transformation solution, 500. mu.L of sterile water was added for resuspension, and centrifuged at 5000rpm for 1min to remove the supernatant, and 200. mu.L of sterile water was added for resuspension and applied to an auxotrophic solid medium SC-URA3, followed by culturing at 30 ℃ for three days.
1 clone was picked from the auxotrophic solid medium SC-URA3 and transferred to a test tube containing 5mL of YEPD medium after activation at 30 ℃ for 36 hours, diluted 20-fold, plated on YEPD solid medium after 36 hours, and cultured for two days. Selecting 4 clones from the culture medium, distributing and coating the clones on a YEPD solid culture medium and an auxotrophic solid culture medium SC-URA3, culturing for three days, selecting the clones which grow on the YEPD solid culture medium but do not grow on SC-URA3, namely the strains without pCUT-416d plasmids, verifying the strains by utilizing PCR, amplifying genomes by using a sequence 3 and a sequence 2, and obtaining the saccharomyces cerevisiae knocked in CBCAS and without the pCUT-416d plasmids after sequencing is verified to be correct.
In order to realize high CBCA yield, three Saccharomyces cerevisiae strains knocked in CBCAS are selected and repeatedly activated in a 24-hole deep-hole plate containing 3mLYEPD liquid culture medium for 36h, diluted in the 24-hole deep-hole plate containing 3mL YEPG liquid culture medium according to a ratio of 20, added with 1mmol/L OA, cultured at 30 ℃ for 96h to obtain a bacterial liquid containing CBCA, and then the next CBCA content determination is carried out.
Firstly, determining an LC-MS spectrum of a standard sample, and specifically comprising the following steps: yeast lyase was added to the culture broth containing the CBCA standards at a concentration of 2U/OD, followed by addition of ethyl acetate/formic acid (0.05% (v/v)) at a volume ratio of 2:1 and lysis by bead beating for 3 minutes (30 s-1). The organic and aqueous phases were then separated by centrifugation. The organic phase was evaporated in a vacuum oven at 50 ℃ and the remaining solid was dissolved in acetonitrile/water/formic acid (80%/20%/0.05% (v/v/v)) and filtered through a PVDF membrane to give the final solution to be assayed. The solution to be detected was analyzed using a hplc-ms equipped with a reversed C18 separation column, using a 0.05% formic acid in water as mobile phase a and a 0.05% formic acid in acetonitrile as mobile phase B, eluting with the following gradient:
0-3.5min,45％B–62.5％B；
3.5-8.0min,62.5％B,0.2mL/min；
8.0-8.5min,62.5％B–97％B,0.2mL/min；
8.5-12.5min,97％B,0.2mL/min；
12.5-12.7min,97％B-45％B,0.2-0.4mL/min；
12.7-15.5min,45％B,0.4mL/min；
the LC-MS curve of the finally obtained CBCA standard sample is shown in figure 1. By using the same method, an LC-MS curve of a sample to be detected containing the CBCA recombinant saccharomyces cerevisiae liquid can be obtained, as can be seen from figure 1, the recombinant saccharomyces cerevisiae can efficiently express CBCA, and the peak-appearing time of the CBCA is consistent with that of a standard sample, which indicates that the saccharomyces cerevisiae strain inserted with CBCAS can efficiently produce CBCA.
Although the invention has been described herein with reference to illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More specifically, various variations and modifications may be made to the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure herein. In addition to variations and modifications in the component parts and/or arrangements, other uses will also be apparent to those skilled in the art.
<110> Sendzein Biotech (Shenzhen) Limited
<120> recombinant saccharomyces cerevisiae for expressing triterpenoids and construction method and application thereof
<160>16
<210>1
<211>1027
<212>DNA
<213> Saccharomyces cerevisiae
<400>1
TATCGTCCAA CTGCATGGAG ATGAGTCGTG GCAAGAATAC CAAGAGTTCC TCGGTTTGCC
AGTTATTAAA AGACTCGTAT TTCCAAAAGA CTGCAACATA CTACTCAGTG CAGCTTCACA
GAAACCTCAT TCGTTTATTC CCTTGTTTGA TTCAGAAGCA GGTGGGACAG GTGAACTTTT
GGATTGGAAC TCGATTTCTG ACTGGGTTGG AAGGCAAGAG AGCCCCGAAA GCTTACATTT
TATGTTAGCT GGTGGACTGA CGCCAGAAAA TGTTGGTGAT GCGCTTAGAT TAAATGGCGT
TATTGGTGTT GATGTAAGCG GAGGTGTGGA GACAAATGGT GTAAAAGACT CTAACAAAAT
AGCAAATTTC GTCAAAAATG CTAAGAAATA GGTTATTACT GAGTAGTATT TATTTAAGTA
TTGTTTGTGC ACTTGCCTGC AGGCCTTTTG AAAAGCAAGC ATAAAAGATC TAAACATAAA
ATCTGTAAAA TAACAAGATG TAAAGATAAT GCTAAATCAT TTGGCTTTTT GATTGATTGT
ACAGGAAAAT ATACATCGCA GGGGGTTGAC TTTTACCATT TCACCGCAAT GGAATCAAAC
TTGTTGAAGA GAATGTTCAC AGGCGCATAC GCTACAATGA CCCGATTCTT GCTAGCCTTT
TCTCGGTCTT GCAAACAACC GCCGGCAGCT TAGTATATAA ATACACATGT ACATACCTCT
CTCCGTATCC TCGTAATCAT TTTCTTGTAT TTATCGTCTT TTCGCTGTAA AAACTTTATC
ACACTTATCT CAAATACACT TATTAACCGC TTTTACTATT ATCTTCTACG CTGACAGTAA
TATCAAACAG TGACACATAT TAAACACAGT GGTTTCTTTG CATAAACACC ATCAGCCTCA
AGTCGTCAAG TAAAGATTTC GTGTTCATGC AGATAGATAA CAATCTATAT GTTGATAATT
AGCGTTGCCT CATCAATGCG AGATCCGTTT AACCGGACCC ACATGGCATT ACCACCATAT
ACATATC
<210>2
<211>654
<212> DNA
<213> Saccharomyces cerevisiae
<400>2
CTCATCAATG CGAGATCCGT TTAACCGGAC CCACATGGCA TTACCACCAT ATACATATCC
ATATCTAATC TTACTTATAT GTTGTGGAAA TGTAAAGAGC CCCATTATCT TAGCCTAAAA
AAACCTTCTC TTTGGAACTT TCAGTAATAC GCTTAACTGC TCATTGCTAT ATTGAAGTAC
GGATTAGAAG CCGCCGAGCG GGCGACAGCC CTCCGACGGA AGACTCTCCT CCGTGCGTCC
TCGTCTTCAC CGGTCGCGTT CCTGAAACGC AGATGTGCCT CGCGCCGCAC TGCTCCGAAC
AATAAAGATT CTACAATACT AGCTTTTATG GTTATGAAGA GGAAAAATTG GCAGTAACCT
GGCCCCACAA ACCTTCAAAT TAACGAATCA AATTAACAAC CATAGGATGA TAATGCGATT
AGTTTTTTAG CCTTATTTCT GGGGTAATTA ATCAGCGAAG CGATGATTTT TGATCTATTA
ACAGATATAT AAATGGAAAA GCTGCATAAC CACTTTAACT AATACTTTCA ACATTTTCAG
TTTGTATTAC TTCTTATTCA AATGTCATAA AAGTATCAAC AAAAAATTGT TAATATACCT
CTATACTTTA ACGTCAAGGA GAAAAAACTA TAATGATTTT CGATGGGACC ACGA
<210>3
<211>1456
<212> DNA
<213> Artificial sequence
<400>3
CCTCTATACT TTAACGTCAA GGAGAAAAAA CTATAATGAT TTTCGATGGG ACCACGATGT CCATTGCGAT AGGGCTACTT TCAACGCTGG GCATAGGCGC AGAAGCGAAT CCGCAGGAAA ATTTTTTGAA GTGCTTTAGC GAATATATTC CAAACAACCC CGCAAATCCG AAGTTTATCT ATACTCAGCA TGATCAGCTG TACATGAGTG TGCTTAACAG CACGATACAG AATCTGAGAT TTACCTCCGA TACGACGCCT AAACCGCTGG TGATAGTAAC GCCGTCTAAT GTTTCACACA TACAAGCTAG TATTCTATGC AGCAAGAAGG TCGGCCTGCA GATTCGTACC CGTTCTGGAG GACACGACGC AGAGGGTCTG AGCTACATCT CACAGGTGCC GTTCGCAATA GTGGACTTAC GTAATATGCA CACCGTCAAG GTGGACATTC ATTCCCAGAC GGCCTGGGTC GAGGCTGGTG CCACGTTGGG GGAGGTATAC TACTGGATTA ATGAGATGAA CGAAAACTTT TCTTTCCCAG GAGGTTATTG CCCCACGGTG GGTGTCGGTG GGCACTTCAG CGGCGGTGGA TACGGTGCTC TGATGCGTAA CTACGGACTT GCTGCAGACA ACATTATAGA CGCCCACCTT GTCAATGTAG ACGGAAAAGT GTTGGACAGA AAGTCTATGG GCGAGGACCT GTTTTGGGCT ATCAGAGGAG GTGGCGGCGA GAATTTCGGT ATCATTGCTG CATGGAAGAT AAAATTAGTC GTTGTACCAT CTAAGGCGAC GATCTTTTCA GTAAAGAAAA ACATGGAAAT CCACGGTCTG GTAAAATTAT TTAATAAGTG GCAAAACATC GCATACAAGT ATGATAAAGA TTTAATGCTT ACGACGCATT TTAGGACAAG AAATATTACT GACAATCATG GCAAGAACAA AACCACCGTT CACGGGTATT TTTCTAGCAT CTTTCTAGGG GGGGTCGATT CCTTGGTAGA CCTGATGAAT AAGAGTTTCC CCGAATTAGG CATTAAAAAA ACGGACTGCA AAGAGTTGTC CTGGATAGAC ACCACGATCT TTTATTCCGG GGTAGTAAAT TACAACACGG CCAATTTTAA GAAGGAGATC TTATTAGATA GATCTGCAGG CAAGAAGACG GCTTTCTCCA TAAAACTTGA CTATGTCAAG AAATTGATCC CGGAGACTGC CATGGTGAAG ATTCTTGAAA AGTTGTATGA GGAGGAAGTA GGGGTCGGGA TGTATGTTTT GTATCCATAC GGAGGCATAA TGGATGAAAT CTCTGAAAGC ACCATACCGT TTCCGCATAG AGCCGGTATT ATGTACGAAC TTTGGTATAC TGCTACCTGG GAAAAACAGG AAGATAACGA AAAGCATATC AACTGGGTAA GATCTGTCTA TAACTTCACG ACTCCTTATG TGAGCCAAAA TCCGCGTTTA GCTTACCTAA ACTACAGGGA CCTTGACTTA GGGAAAACAA ATCCCGAATC ACCGAACAAT TACACACAAG CCAGGATCTG GGGGGAAAAA TATTTCGGTA AGAACTTCAA TCGTCTAGTA AAGGTAAAAA CAAAGGCGGA CCCAAATAAC TTCTTCAGAA ACGAGCAGTC AATCCCACCA CTACCCCCCC ACCATCATTA GGCGAATTTC TTATGATTTA
TGATTTTTAT TATTAA
<210>4
<211>297
<212> DNA
<213> Saccharomyces cerevisiae
<400>4
ACCCCCCCAC CATCATTAGG CGAATTTCTT ATGATTTATG ATTTTTATTA TTAAATAAGT
TATAAAAAAA ATAAGTGTAT ACAAATTTTA AAGTGACTCT TAGGTTTTAA AACGAAAATT
CTTATTCTTG AGTAACTCTT TCCTGTAGGT CAGGTTGCTT TCTCAGGTAT AGCATGAGGT
CGCTCTTATT GACCACACCT CTACCGGCAT GCCGAGCAAA TGCCTGCAAA TCGCTCCCCA
TTTCACCCAA TTGTAGATAT GCTAACTCCC CGAACATGCT CCTTCACTAT TTTAACA
<210>5
<211>968
<212> DNA
<213> Saccharomyces cerevisiae
<400>5
TTCACCCAAT TGTAGATATG CTAACTCCCC GAACATGCTC CTTCACTATT TTAACATGTG
GAATTCTTGA AAGAATGAAA TCGCCATGCC AAGCCATCAC ACGGTCTTTT ATGCAATTGA
TTGACCGCCT GCAACACATA GGCAGTAAAA TTTTTACTGA AACGTATATA ATCATCATAA
GCGACAAGTG AGGCAACACC TTTGTTACCA CATTGACAAC CCCAGGTATT CATACTTCCT
ATTAGCGGAA TCAGGAGTGC AAAAAGAGAA AATAAAAGTA AAAAGGTAGG GCAACACATA
GTATGAATAC AAACGTTCCA ATATTCAGTT CTCCGGTCAG AGATTTACCA AGGTCTTTCG
AACAAAAACA TTTAGCGGTT GTAGATGCAT TTTTCCAAAC ATACCATGTC AAACCTGATT
TTATCGCTAG GTCTCCTGGC AGAGTAAATC TGATTGGTGA GCATATAGAT TATTGCGATT
TTTCAGTTTT GCCATTAGCC ATTGATGTGG ATATGCTTTG CGCAGTTAAA ATTTTAGACG
AAAAAAATCC ATCCATTACC TTAACAAATG CGGACCCTAA ATTTGCTCAG CGAAAGTTTG
ATCTGCCTTT AGATGGTTCC TACATGGCCA TAGATCCGTC TGTGTCGGAA TGGTCGAATT
ACTTTAAATG CGGACTACAT GTGGCACATT CATACTTGAA AAAAATTGCT CCGGAAAGAT
TTAATAATAC ACCCTTAGTA GGTGCGCAGA TCTTTTGCCA GAGCGATATT CCTACTGGTG
GTGGACTCTC ATCTGCATTT ACTTGCGCGG CAGCACTAGC CACAATTAGA GCCAATATGG
GAAAAAATTT TGATATTTCC AAAAAAGACT TGACCCGCAT CACAGCGGTT GCTGAGCACT
ATGTTGGAGT CAATAATGGT GGTATGGATC AAGCAACGTC TGTTTATGGG GAAGAAGATC
ATGCTCTATA CGTAGAGTTT AGGCCAAAAC TAAAGGCCAC ACCTTTCAAG TTTCCTCAAT
TGAAAAAT
<210>6
<211>1626
<212> DNA
<213> Artificial sequence
<400>6
ATGATTTTCG ATGGGACCAC GATGTCCATT GCGATAGGGC TACTTTCAAC GCTGGGCATA
GGCGCAGAAG CGAATCCGCA GGAAAATTTT TTGAAGTGCT TTAGCGAATA TATTCCAAAC
AACCCCGCAA ATCCGAAGTT TATCTATACT CAGCATGATC AGCTGTACAT GAGTGTGCTT
AACAGCACGA TACAGAATCT GAGATTTACC TCCGATACGA CGCCTAAACC GCTGGTGATA
GTAACGCCGT CTAATGTTTC ACACATACAA GCTAGTATTC TATGCAGCAA GAAGGTCGGC
CTGCAGATTC GTACCCGTTC TGGAGGACAC GACGCAGAGG GTCTGAGCTA CATCTCACAG
GTGCCGTTCG CAATAGTGGA CTTACGTAAT ATGCACACCG TCAAGGTGGA CATTCATTCC
CAGACGGCCT GGGTCGAGGC TGGTGCCACG TTGGGGGAGG TATACTACTG GATTAATGAG
ATGAACGAAA ACTTTTCTTT CCCAGGAGGT TATTGCCCCA CGGTGGGTGT CGGTGGGCAC
TTCAGCGGCG GTGGATACGG TGCTCTGATG CGTAACTACG GACTTGCTGC AGACAACATT
ATAGACGCCC ACCTTGTCAA TGTAGACGGA AAAGTGTTGG ACAGAAAGTC TATGGGCGAG
GACCTGTTTT GGGCTATCAG AGGAGGTGGC GGCGAGAATT TCGGTATCAT TGCTGCATGG
AAGATAAAAT TAGTCGTTGT ACCATCTAAG GCGACGATCT TTTCAGTAAA GAAAAACATG
GAAATCCACG GTCTGGTAAA ATTATTTAAT AAGTGGCAAA ACATCGCATA CAAGTATGAT
AAAGATTTAA TGCTTACGAC GCATTTTAGG ACAAGAAATA TTACTGACAA TCATGGCAAG
AACAAAACCA CCGTTCACGG GTATTTTTCT AGCATCTTTC TAGGGGGGGT CGATTCCTTG
GTAGACCTGA TGAATAAGAG TTTCCCCGAA TTAGGCATTA AAAAAACGGA CTGCAAAGAG
TTGTCCTGGA TAGACACCAC GATCTTTTAT TCCGGGGTAG TAAATTACAA CACGGCCAAT
TTTAAGAAGG AGATCTTATT AGATAGATCT GCAGGCAAGA AGACGGCTTT CTCCATAAAA
CTTGACTATG TCAAGAAATT GATCCCGGAG ACTGCCATGG TGAAGATTCT TGAAAAGTTG
TATGAGGAGG AAGTAGGGGT CGGGATGTAT GTTTTGTATC CATACGGAGG CATAATGGAT
GAAATCTCTG AAAGCACCAT ACCGTTTCCG CATAGAGCCG GTATTATGTA CGAACTTTGG
TATACTGCTA CCTGGGAAAA ACAGGAAGAT AACGAAAAGC ATATCAACTG GGTAAGATCT
GTCTATAACT TCACGACTCC TTATGTGAGC CAAAATCCGC GTTTAGCTTA CCTAAACTAC
AGGGACCTTG ACTTAGGGAA AACAAATCCC GAATCACCGA ACAATTACAC ACAAGCCAGG
ATCTGGGGGG AAAAATATTT CGGTAAGAAC TTCAATCGTC TAGTAAAGGT AAAAACAAAG
GCGGACCCAA ATAACTTCTT CAGAAACGAG CAGTCAATCC CACCACTACC CCCCCACCAT
CATTAG
<210>7
<211>24
<212> DNA
<213> Artificial sequence
<400>7
TATCGTCCAA CTGCATGGAG ATGA
<210>8
<211>48
<212> DNA
<213> Artificial sequence
<400>8
GATATGTATA TGGTGGTAAT GCCATGTGGG TCCGGTTAAA CGGATCTC
<210>9
<211>60
<212> DNA
<213> Artificial sequence
<400>9
CTCATCAATG CGAGATCCGT TTAACCGGAC CCACATGGCA TTACCACCAT ATACATATCC
<210>10
<211>59
<212> DNA
<213> Artificial sequence
<400>10
TCGTGGTCCC ATCGAAAATC ATTATAGTTT TTTCTCCTTG ACGTTAAAGT ATAGAGGTA
<210>11
<211>59
<212> DNA
<213> Artificial sequence
<400>11
CCTCTATACT TTAACGTCAA GGAGAAAAAA CTATAATGAT TTTCGATGGG ACCACGATG
<210>12
<211>59
<212> DNA
<213> Artificial sequence
<400>12
TTAATAATAA AAATCATAAA TCATAAGAAA TTCGCCTAAT GATGGTGGGG GGGTAGTGG
<210>13
<211>59
<212> DNA
<213> Artificial sequence
<400>13
ACCCCCCCAC CATCATTAGG CGAATTTCTT ATGATTTATG ATTTTTATTA TTAAATAAG
<210>14
<211>56
<212> DNA
<213> Artificial sequence
<400>14
TGTTAAAATA GTGAAGGAGC ATGTTCGGGG AGTTAGCATA TCTACAATTG GGTGAA
<210>15
<211>55
<212> DNA
<213> Artificial sequence
<400>15
TTCACCCAAT TGTAGATATG CTAACTCCCC GAACATGCTC CTTCACTATT TTAAC
<210>16
<211>29
<212> DNA
<213> Artificial sequence
<400>16
ATTTTTCAAT TGAGGAAACT TGAAAGGTG
Claims (10)
1. A construction method of recombinant saccharomyces cerevisiae for expressing a heterpene compound is characterized in that exogenous cannabis pigment synthetase is introduced into saccharomyces cerevisiae for expressing cannabigerolic acid to obtain the recombinant saccharomyces cerevisiae for expressing the heterpene compound.
2. The method for constructing recombinant saccharomyces cerevisiae expressing triterpenoids according to claim 1, wherein the exogenous cannabis pigment synthase is introduced by transforming into a plasmid carrying the exogenous cannabis pigment synthase gene or integrating the exogenous cannabis pigment synthase gene on the saccharomyces cerevisiae chromosome.
3. The method for constructing recombinant saccharomyces cerevisiae expressing triterpenoids according to claim 2, wherein the exogenous cannabichromes synthase gene comprises at least a product obtained by PCR amplification of a codon-optimized ProA-CBCAS sequence.
4. The method for constructing recombinant Saccharomyces cerevisiae expressing triterpenes according to claim 3, wherein the product of PCR amplification of the codon-optimized ProA-CBCAS sequence is transformed into Saccharomyces cerevisiae together with plasmid pCUT-416 d.
5. The method for constructing recombinant Saccharomyces cerevisiae expressing triterpenoids according to claim 4, wherein PCR amplification of the 416d upstream sequence, 416d downstream sequence, Gal1 promoter sequence, ADH1 terminator sequence is used for transformation to obtain the product, and PCR amplification of the codon-optimized ProA-CBCAS sequence is used for transformation.
6. The method for constructing recombinant Saccharomyces cerevisiae expressing triterpenoids according to claim 3, wherein the step of transforming comprises: saccharomyces cerevisiae cells were activated and then cultured to OD in YEPD medium600Raising the temperature to between 0.7 and 1.0, collecting cells, mixing a transformation solution containing the product and the plasmid pCUT-416d with the cells, incubating at 30 ℃, thermally shocking at 42 ℃, and collecting the cells.
7. The method for constructing recombinant Saccharomyces cerevisiae expressing triterpenes according to claim 6, wherein one transformation amount of the transformation solution comprises: 120uL 50% PEG3350, 18uL1mol/L LiAC, 5uL 10mg/mL single-stranded milt DNA, 35uL sterile water and 2ug of the product and plasmid pCUT-416d mixed fragment to be transformed.
8. The method of claim 4, wherein the transformed Saccharomyces cerevisiae is cultured in YEPD solid medium and auxotrophic solid medium SC-URA3, and then selected from the group consisting of Saccharomyces cerevisiae introduced with cannabichromene synthase and without pCUT-416d plasmid.
9. The recombinant saccharomyces cerevisiae for expressing the triterpenoid obtained by the construction method of any one of claims 1 to 8.
10. Use of the recombinant saccharomyces cerevisiae according to claim 9 for the fermentative production of a triterpenoid.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107075523A (en) * | 2014-06-27 | 2017-08-18 | 加拿大国家研究委员会 | Cannabichromene acid synthase from hemp |
| WO2019071000A1 (en) * | 2017-10-05 | 2019-04-11 | Intrexon Corporation | Microorganisms and methods for the fermentation of cannabinoids |
| WO2019209885A2 (en) * | 2018-04-23 | 2019-10-31 | Renew Biopharma, Inc. | Enzyme engineering to alter the functional repertoire of cannabinoid synthases |
| CN112795495A (en) * | 2020-12-14 | 2021-05-14 | 大连理工大学 | Method for producing heterologous cannabichromene by using saccharomyces cerevisiae |
-
2020
- 2020-02-26 CN CN202010121334.XA patent/CN113999783A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107075523A (en) * | 2014-06-27 | 2017-08-18 | 加拿大国家研究委员会 | Cannabichromene acid synthase from hemp |
| WO2019071000A1 (en) * | 2017-10-05 | 2019-04-11 | Intrexon Corporation | Microorganisms and methods for the fermentation of cannabinoids |
| WO2019209885A2 (en) * | 2018-04-23 | 2019-10-31 | Renew Biopharma, Inc. | Enzyme engineering to alter the functional repertoire of cannabinoid synthases |
| CN112795495A (en) * | 2020-12-14 | 2021-05-14 | 大连理工大学 | Method for producing heterologous cannabichromene by using saccharomyces cerevisiae |
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| AMANDA REIDER APEL 等: ""A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae"", 《NUCLEIC ACIDS RESEARCH》 * |
| MATHEW M. JESSOP-FABRE 等: ""EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9"", 《BIOTECHNOLOGY JOURNAL》 * |
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