CN114517203A

CN114517203A - Macleaya cordata berberine bridge enzyme gene optimization sequence and application thereof

Info

Publication number: CN114517203A
Application number: CN202210268913.6A
Authority: CN
Inventors: 曾建国; 黄鹏; 徐子璇
Original assignee: Micolta Bioresource Inc ltd
Current assignee: Micolta Bioresource Inc ltd
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-05-20

Abstract

The invention discloses a macleaya cordata berberine bridge enzyme gene optimization sequence and application thereof, wherein macleaya cordata berberine bridge enzyme genes (McBBE) are optimized according to codon preference of saccharomyces cerevisiae to obtain McBBE1, McBBE2, McBBE3, McBBE4 and McBBE5, then five optimized macleaya cordata berberine bridge enzyme gene sequences are respectively transformed into saccharomyces cerevisiae to be expressed to obtain strains which are YH01, YH02, YH03, YH04 and YH05 respectively, then after shake flask fermentation and precursor feeding, the yield of the golden violaxanthin is determined, the strain with the highest yield is YH03, and finally the McBBE3 sequence corresponding to the strain YH03 is integrated into a saccharomyces cerevisiae genome to obtain the saccharomyces cerevisiae engineering bacteria with high yield of the golden violaxanthin, catalytic efficiency of the bbe in the saccharomyces cerevisiae is improved, and the yield of the golden violaxanthin is improved.

Description

Macleaya cordata berberine bridge enzyme gene optimization sequence and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and biological engineering, and particularly relates to a macleaya cordata berberine bridge enzyme gene with optimized codons and application thereof.

Background

Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a unicellular eukaryote, has a protein modification processing function and a homologous recombination mechanism, has a clearer and simpler genetic background compared with other eukaryotes, and is a model organism in basic research due to easy operability and safety. In synthetic biology, yeast is often used as a microbial system for expressing natural products.

tRNA is an important molecule in the translation of proteins, which specifically and directly links the triplet of codons to the corresponding amino acids. Therefore, the interaction between tRNA and codon in ribosome has important influence on the speed and accuracy of protein translation. Of the 64 codons, the 20 standard amino acids of the synthetic protein were translated by the 61 codon code, and all other amino acids except methionine (Met) and tryptophan (Trp) had 2 to 6 synonymous codons, i.e., different codons encoding the same amino acid. Codon-biased presence is found in almost all species: the choice of codons by the Coding sequence (CDS) is non-random, the frequency of usage of synonymous codons varies, and the propensity to choose varies in different genomes.

Codon optimization is an important factor affecting protein translation and function. The target gene is optimized according to the codon preference of an expression host, and the expression quantity can be obviously improved. In 1982, Bennettzem et al analyzed codon usage preference of highly expressed genes in Saccharomyces cerevisiae, and compared with other genes of Saccharomyces cerevisiae, found that these genes have certain similarity to codon usage, and found out 22 codons preferred by Saccharomyces cerevisiae. Provides a theoretical basis for the codon optimization of heterologous genes in the saccharomyces cerevisiae. The whole sequence sequencing work of the saccharomyces cerevisiae genome was completed in 1996, and genome information provides more basis for the research of yeast codon bias.

The CRISPR/Cas system is firstly found in escherichia coli, is an acquired immune system of bacteria for exogenous DNA such as bacteriophage, and mainly relies on a ribonucleoprotein complex formed by crRNA and Cas protein to recognize a PAM structure on a target sequence, so that the specificity cutting is carried out on the invasive bacteriophage or plasmid. There are three main types of CRISPR systems, of which the type II system requires only one Cas9 protein, crRNA and tracrRNA to function. There are studies that show that integration of crRNA and tracrRNA into sgRNA does not affect the effect of CRISPR/Cas9 system. Dicarlo et al used the CRISPR-Cas9 system in Saccharomyces cerevisiae for the first time in 2013, and they transferred artificially designed sgRNA plasmid and donor DNA into Saccharomyces cerevisiae to realize the editing of arginine permease gene, and at the same time, showed that the CRISPR-Cas9 system has no obvious toxicity to Saccharomyces cerevisiae. The CRISPR-Cas9 system is gradually widely applied to saccharomyces cerevisiae.

The McBBE gene is short for macleaya cordata berberine bridge enzyme gene, and the berberine bridge enzyme participates in catalyzing the conversion of Reticuline ((S) -Reticuline) into corydaline ((S) -Scoulerine), and is a key enzyme in the sanguinarine synthesis pathway. At present, the functional verification of McBBE is carried out by using a Saccharomyces cerevisiae system, but the obtained McBBE has extremely low catalytic efficiency in Saccharomyces cerevisiae. Therefore, the improvement of the catalytic efficiency of McBBE in Saccharomyces cerevisiae and the improvement of the yield of the aureovioline are urgently needed.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention provides a macleaya cordata berberine bridge enzyme gene with optimized codons and application thereof.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the first aspect of the invention provides a macleaya cordata berberine bridge enzyme gene optimization sequence, which comprises: McBBE1, McBBE2, McBBE3, McBBE4, McBBE5, wherein: the nucleotide sequence of McBBE1 is shown as SEQ ID NO.1, the nucleotide sequence of McBBE2 is shown as SEQ ID NO.2, the nucleotide sequence of McBBE3 is shown as SEQ ID NO.3, the nucleotide sequence of McBBE4 is shown as SEQ ID NO.4, and the nucleotide sequence of McBBE5 is shown as SEQ ID NO. 5.

In a second aspect, the invention provides a recombinant expression vector comprising the optimized sequence of the gene designated McBBE1, McBBE2, McBBE3, McBBE4 or McBBE5 according to claim 1.

Further, the air conditioner is provided with a fan,

the expression vector is pYES 2-Ura.

Further, the air conditioner is provided with a fan,

recombinant expression vectors comprising optimized gene sequences for McBBE1, McBBE2, McBBE3, McBBE4, and McBBE5 are pYES2-N1, pYES2-N2, pYES2-N3, pYES2-N4, and pYES2-N5, respectively.

In a third aspect, the present invention provides a genetically engineered host bacterium comprising the recombinant expression vector described above.

Further, the air conditioner is characterized in that,

the host bacteria is saccharomyces cerevisiae.

The fourth aspect of the invention provides the use of the macleaya cordata berberine bridge enzyme gene optimized sequence, the recombinant expression vector or the genetic engineering host bacterium in preparation of the corydaline aurantium.

The fifth aspect of the invention provides a method for preparing saccharomyces cerevisiae engineering bacteria for high-yield production of golden yellow corydaline by using macleaya cordata berberine bridge enzyme gene optimized sequences, which comprises the following steps:

(1) the optimized gene sequences McBBE1, McBBE2, McBBE3, McBBE4 and McBBE5 are respectively connected to expression vectors, and the recombinant vectors which are obtained after transformation and are identified by PCR are respectively named as: pYES2-N1, pYES2-N2, pYES2-N3, pYES2-N4, pYES 2-N5;

(2) respectively transforming the recombinant vectors obtained in the step (1) into saccharomyces cerevisiae for expression, screening positive clones, and identifying correct strains which are respectively named as: YH01, YH02, YH03, YH04, YH 05;

(3) respectively carrying out shake flask fermentation and precursor feeding on the strains obtained in the step (2), collecting bacterial liquid, and then carrying out golden viologen extraction and yield determination to obtain a saccharomyces cerevisiae strain capable of generating the golden viologen;

(4) and (4) finding out the optimized gene corresponding to the strain obtained in the step (3), integrating the optimized gene into a saccharomyces cerevisiae genome by using a CRISPR-Cas9 technology, and identifying correctly to obtain the saccharomyces cerevisiae engineering bacteria with high yield of the corydaline.

The sixth aspect of the invention provides a saccharomyces cerevisiae engineering bacterium prepared by the method for preparing the saccharomyces cerevisiae engineering bacterium for high-yield production of the golden yellow corydaline by adopting the macleaya cordata berberine bridge enzyme gene optimized sequence, wherein the saccharomyces cerevisiae engineering bacterium is YPR01 containing an McBBE3 gene sequence.

CRISPR/Cas9 is an abbreviation for Clustered regulated Short Palindromic Repeats (Regularly Clustered Short Palindromic Repeats)/CRISPR-associated protein 9 (CRISPR-associated protein 9), and is the third generation "genome-directed editing technology".

PAM: (Protosporacer adjacenttont motif) prepro-spacer sequence, a short nucleotide motif found in crRNA molecules, can be specifically recognized and cleaved by Cas9 protein.

The invention has the following beneficial effects:

the method comprises the steps of optimizing macleaya cordata berberine bridge enzyme genes (McBBE) according to codon preference of saccharomyces cerevisiae to obtain McBBE1, McBBE2, McBBE3, McBBE4 and McBBE5, then respectively converting five optimized macleaya cordata berberine bridge enzyme gene sequences into saccharomyces cerevisiae to express to obtain strains of YH01, YH02, YH03, YH04 and YH05, measuring the yield of chrysogenin after shake flask fermentation and precursor cordierite feeding, obtaining a strain YH03 with the highest yield, and finally integrating McBBE3 sequences corresponding to the strain 03 into a saccharomyces cerevisiae genome to obtain the saccharomyces cerevisiae engineering bacteria of high-yield chrysogenin, improving catalytic efficiency of McBBE in the saccharomyces cerevisiae, and realizing improvement of the yield of the chrysogenin.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a gene amplification electrophoretogram of McBBE1, McBBE2, McBBE3, McBBE4 and McBBE 5;

FIG. 2 is a schematic diagram of the construction of a macleaya cordata berberine bridge enzyme gene (McBBE) expression vector;

FIG. 3 is a graph of the standard curve for reticuline;

FIG. 4 is a standard graph of the preparation of corydaline;

FIG. 5 is a schematic diagram of a gRNA plasmid;

FIG. 6 shows the sequencing of gRNA at YPR1 site;

FIG. 7 is a gene amplification electrophoretogram of YPR1up, YPR1down, GAL1p, CYC1t and McBBE 3;

FIG. 8 is a plasmid map of pUC19-YPR 1-N3.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or to implicitly indicate the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

In the description herein, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

1. Design of codon optimization sequence of macleaya cordata berberine bridge enzyme gene (McBBE) and primer synthesis

According to the codon preference of saccharomyces cerevisiae, the macleaya cordata berberine bridge enzyme gene (McBBE) is optimized, 5 codon optimization schemes are designed, the genes are named as McBBE1, McBBE2, McBBE3, McBBE4 and McBBE5 respectively, and the synthesis of the genes is completed by Nanjing Kinssry Biotech company.

The invention uses In-Fusion cloning technology to construct a vector, and according to the technical principle, primers of McBBE1, McBBE2, McBBE3, McBBE4 and McBBE5 genes are designed and synthesized by Beijing Optimala biology Limited company, and specific primer sequences are shown In Table 1.

TABLE 1

2. Design and Synthesis of identifying primers

The primers shown in Table 1 are used for amplifying McBBE1, McBBE2, McBBE3, McBBE4 and McBBE5, and the amplified genes are connected to pYES2-Ura vector plasmid, so that the primers are designed about 300-400bp respectively at the upstream and downstream of the pYES2-Ura vector connection site and are mainly used for identifying the vector construction result, and the specific primer sequences are shown in Table 2.

TABLE 2

The preparation method comprises the following specific steps:

(1) the pYES2-Ura plasmid is cut into a linearized plasmid by KpnI-HF and XBAI double enzyme under the reaction condition of 37 ℃ for 2 hours, 1 percent agarose gel electrophoresis is carried out under the electrophoresis condition of 120V for 20min, a target fragment is recovered by cutting gel, a target DNA is purified by a gel recovery kit, and the size of the fragment is about 5768 bp.

(2) The target gene was amplified using synthetic McBBE1, McBBE2, McBBE3, McBBE4, McBBE5 gene sequences as templates, PrimerSTAR Max DNA Polymerase and primers in Table 1. PCR reaction procedure: pre-denaturation at 98 ℃ for 30s, annealing at 55 ℃ for 10s, extension at 72 ℃ for 30s/kb (25 cycles), final extension at 72 ℃ for 7min, and holding at 4 ℃. After completion, the detection was carried out electrophoretically on agarose gels, the results are shown in FIG. 1, where M represents DL 2000DNA Marker, 1 represents McBBE1, 2 represents McBBE2, 3 represents McBBE3, 4 represents McBBE4, and 5 represents McBBE 5. The correct target band was recovered using the Zymocleian Gel DNA Recovery Kit.

(3) Connecting the 5 amplified target genes in the step (2) with pYES2-Ura linearized vector through ligase respectively, wherein the construction schematic diagram of the expression vector is shown in figure 2, calculating the concentration required by connection according to the lengths of target genes and linear vector fragments according to the instruction of the Infusion homologous recombinase, adding the fragments and the Infusion enzyme into a reaction system, carrying out vortex mixing, and placing at 50 ℃ for reaction for 20 min.

3. Transformation of E.coli

After the construction of the expression vector is completed, the expression vector is transferred into escherichia coli, and the specific method comprises the following steps:

(1) the ligation product was pipetted at 1. mu.L and transferred to pre-cooled 50. mu.L E.coli competent DH 5. alpha. and after mixing, the mixture was allowed to stand on ice for 5 min.

(2) The heat shock was applied to the water bath at 42 ℃ for 45s, and the mixture was rapidly transferred to ice and allowed to stand for 2 min.

(3) 200. mu.L of non-resistant LB medium was added. Shaking culture at 37 ℃ for 1 hour.

(4) Spread onto LB plates with Amp resistance. Incubation was carried out overnight at 37 ℃. After the colonies grow on the LB plate, selecting a single colony to carry out colony PCR identification and screening on positive clones, and respectively naming the correctly identified recombinant vectors as: pYES2-N1, pYES2-N2, pYES2-N3, pYES2-N4, and pYES 2-N5.

4. Saccharomyces cerevisiae transformation

(1) Firstly, taking out a CEN.PK2-1C saccharomyces cerevisiae strain preserved in a laboratory at the temperature of-80 ℃, carrying out streak culture on a YPD culture medium, and activating the saccharomyces cerevisiae. And (3) selecting a single colony of the CEN, PK2-1C strain to be cultured in 3mL YPD medium at 30 ℃ overnight, sucking 200 mu L of bacterial liquid the next day, transferring the bacterial liquid to 5mL YPD medium, putting the culture in a shaking table at 30 ℃ for 4-5 hours, and preparing competent cells when the OD value of the bacterial liquid reaches about 0.8.

(2) Sucking 1mL of bacterial liquid into a 1.5mL sterile EP tube, centrifuging for 1min at 4000rpm of a 4 ℃ centrifuge, and discarding the supernatant; washing with 1mL of sterile water once, centrifuging again and discarding the supernatant; resuspending with 0.1M LiAc, standing on ice for 10min, centrifuging to remove supernatant, and retaining the thallus for transformation.

(3) Meanwhile, the ssDNA is denatured by a PCR instrument, and is immediately embedded into an ice box for cooling after 5min at 99 ℃ for use when being converted; prepare 360. mu.l of transformation system: 240 μ l of PEG3350 (50% w/v), 10 μ l of ssDNA, 36 μ l of 1M LiAc, 2 μ l of plasmid, 72 μ l of sterile water; adding 360 μ l of the transformation system into the prepared competence, mixing uniformly by vortex, culturing at 30 deg.C for 30min, and thermally shocking in 42 deg.C water bath for 25 min; centrifuging at 4 deg.C centrifuge at 4000rpm for 5min, and removing supernatant; the corresponding SD/Dropout medium was added, resuspended, plated on the corresponding medium plate, and cultured in an incubator at 30 ℃. And (3) carrying out colony PCR identification and screening on positive clones, and identifying correct strains respectively and naming the strains as: YH01, YH02, YH03, YH04 and YH 05.

5. Saccharomyces cerevisiae shake flask fermentation and precursor feeding

The strain BHH001 containing the original McBBE gene and the strain YS001 containing the PsBBE gene derived from poppy were used as positive control groups, and shake flask fermentation and precursor feeding were carried out together with the strains YH01, YH02, YH03, YH04, YH 05. Selecting a yeast single colony from an ultraclean workbench, placing the yeast single colony in 2mL of glucose-deficient culture medium, placing the culture medium in a shaking table, carrying out overnight culture at 30 ℃, centrifuging the culture solution by using a centrifuge at 12000rpm for 1min, collecting thalli, re-suspending the thalli by using 1mL of galactose-deficient culture medium, transferring the thalli into a sterile shaking bottle containing 19mL of galactose-deficient culture medium, and carrying out shaking table culture at 30 ℃ for 24 h; measuring OD600 by using an ultraviolet spectrophotometer, diluting the OD value to 0.8 by using a galactose culture medium according to a proportion, keeping the total volume at 6mL, continuously culturing the culture solution with the OD600 being 0.8 for 7h, centrifuging the culture solution at 5000rpm for 5min by using a centrifuge, discarding supernatant, suspending the thalli in 2mL of Tris-EDTA buffer solution, adding 6.5mg/L of (S) -triticine for feeding, shaking to resuspend the thalli, and continuously culturing for 24 h.

6. Extraction of golden yellow viologen and determination of yield

(1) Extraction: after culturing the 7 strains for 24h, respectively centrifuging the culture solution at 5000rpm for 10min, transferring the supernatant to a 5mL centrifuge tube, adding 500. mu.L methanol and a proper amount of glass beads into the residual strains, and vortexing and shaking for 30 min. Centrifuging for 5min again, sucking supernatant, filtering with 0.22 μm filter membrane to obtain 7 samples, and waiting for detection on computer.

(2) Preparation of a standard curve: the (S) -reticuline and (S) -scoulerine standard substances are respectively and precisely weighed by an electronic balance and dissolved by DMSO to prepare standard substance stock solutions of the 2 alkaloids. Sucking a proper amount of standard substance stock solution, and sequentially diluting with methanol according to a proportion. (S) -reticuline is prepared into standard solutions with the concentration of 40000ng/mL, 20000ng/mL, 10000ng/mL, 5000ng/mL, 2500ng/mL and 1000ng/mL respectively. (S) -scoulerine is prepared into standard solutions with the concentrations of 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 5ng/mL, 2.5ng/mL and 1ng/mL respectively. HPLC-QQQ MS was used for the measurement, and the chromatographic peak area was recorded. And (5) drawing a standard curve by taking the peak area integral value as an ordinate (Y) and the mass concentration (ng/mL) as an abscissa (X), and fitting to obtain a linear regression equation Y which is aX + b. The obtained standard curves are shown in fig. 3 and 4, wherein fig. 3 is a standard curve of (S) -reticuline, and fig. 4 is a standard curve of (S) -troulerine.

(3) And (3) yield determination: the sample obtained in (1) was subjected to yield measurement using HPLC-QQQ-MS. The results are shown in Table 3. According to the data, the YH03 strain has the highest yield which reaches 1.12mg/L and the yield is improved to 17.4%. The BLH001 was increased by 58-fold compared to strain BLH 001. The McBBE3 shows the highest catalytic efficiency in Saccharomyces cerevisiae.

TABLE 3

6. Integration of McBBE3 gene into Saccharomyces cerevisiae genome by CRISPR-Cas9 technology

(1) Constructing a gRNA plasmid: gRNA of YPR1 site is designed through website http:// chopchopchop. cbu. uib. no/, and double-stranded nucleotide fragments are obtained by annealing and connection of upper and lower primers, and the sequences of the primers are shown in Table 4. Then, the pCRCT plasmid vector was digested with BsaI, and the fragment and the linear vector were ligated to transform E.coli, thereby obtaining a recombinant plasmid YPR1-gRNA, the plasmid schematic diagram is shown in FIG. 5. And carrying out colony PCR verification on the obtained transformant, and finding that the length of the target DNA band is consistent with that of the theoretical DNA band through gel electrophoresis detection. And then, selecting positive transformants to be cultured in an Amp resistant LB liquid culture medium overnight, and extracting plasmids on the next day to send to a sequencing company for sequencing. The sequencing result is shown in fig. 6, which indicates that the gRNA plasmid is successfully constructed.

TABLE 4

(2) And (3) expression module treatment construction: PK2-1C genome DNA of CEN is used as a template to amplify YPR1up and YPR1down gene segments, plasmid pYES2-Ura is used as a template to amplify GAL1p and CYC1t gene segments, plasmid pYES2-N3 is used as a template to amplify McBBE3 gene segments, and specific primer information is shown in Table 5. DNA bands with lengths of 499bp, 442bp, 1611bp, 248bp and 476bp, respectively, can be observed after gel electrophoresis, and the results are shown in FIG. 7, wherein M represents DL 2000DNA Marker, 1 represents YPR1up, 2 represents GAL1p, and 3 represents: the results of McBBE3, 4 indicating CYC1t and 5 indicating YPR1down, which were consistent with their theoretical band lengths, demonstrated successful gene amplification. YPR1up and GAL1p were ligated, CYC1t and YPR1down, and then the final three fragments were ligated to the pUC19 vector which had been digested by Infusion. After completion of Infusion ligation, E.coli transformation was carried out to obtain an expression module plasmid pUC19-YPR 1-N3. Colony PCR verification is carried out on the obtained transformant, and the length of the target DNA band is consistent with that of the theoretical DNA band through gel electrophoresis detection. Then, positive transformants are picked and cultured in an Amp resistant LB liquid medium overnight, and the plasmids are extracted the next day and sent to a sequencing company for full-length sequencing. The sequencing result shows that the gene sequence of the positive colony is consistent with the target gene sequence, the success of the construction of the recombinant plasmid is proved, and the plasmid map is shown in figure 8.

TABLE 5

(3) Integration of the McBBE3 gene into the genome: the plasmid pUC19-YPR1-N3 is cut by enzyme to obtain an expression module of YPR1 site, the expression module and the YPR1-gRNA plasmid are transferred into Saccharomyces cerevisiae CEN. PK2-1C together, positive clones are screened by Uracil (URA) single-defect SD/Dropout culture medium, and a single-copy strain YPR01 is obtained after PCR verification of yeast colony. The strain YPR01 is a Saccharomyces cerevisiae engineering strain containing McBBE3 gene and capable of producing corydaline with high yield.

The foregoing is a further detailed description of the invention in connection with specific preferred embodiments and is not intended to limit the invention to the specific embodiments described. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Description of the sequence:

SEQ ID Nos. 1 to 5 are the nucleotide sequences of McBBE1, McBBE2, McBBE3, McBBE4 and McBBE5, respectively.

SEQ ID Nos. 6 to 15 are nucleotide sequences of McBBE1-URA-F, McBBE1-URA-R, McBBE2-URA-F, McBBE2-URA-R, McBBE3-URA-F, McBBE3-URA-R, McBBE4-URA-F, McBBE4-URA-R, McBBE52-URA-F, McBBE5-URA-R, respectively.

SEQ ID Nos. 16 to 17 are the nucleotide sequences of pYES2-URA-F, pYES2-URA-R, respectively.

SEQ ID Nos. 18 to 19 are the nucleotide sequences of YPR1-GRNA-F, YPR1-GRNA-R, respectively.

SEQ ID Nos. 20-29 are nucleotide sequences of Y-UP-F, Y-UP-R, Y-GAL-F, GAL-McBBE3-R, McBBE3-F, McBBE3-R, CYC1-McBBE3-F, Y-CYC1-R, Y-DOWN-F, Y-DOWN-R, respectively.

SEQUENCE LISTING

<110> Hunan Meida biological resources Co., Ltd

<120> optimized sequence of macleaya cordata berberine bridge enzyme gene and application thereof

<130> 20220316

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gttccaaaga aggtgaccgt tttcagattg atgaagcacg ttaagattga agaagcttct 780

aatttattgc acaagtggca attcgttgct gatgaattgg atgatgattt cactttgtcc 840

gttttgggtg gtgctgatga aaatgaagtt tggttaatgt tcttgggttt atatttaggt 900

ccaaaaaccg ttgctaagtc taccatcgac ccaaagttcc cagaattggg tttgattgaa 960

gaagaattct ctgaaatgtc ctggggtgaa tcgtttgctt acttggctgg tttgaagaca 1020

gttggtgaat tgaacaaccg ttttttgaaa tttgatgaca gagctttcaa gactaaggtc 1080

gatttcgcta aggaaccaat tccattgaag gttattaacg gtgctttgga aatcttatct 1140

aaagaaccta gaggtttcgt cgccttgaac ggtttgggtg gtatgatgtc aaaagtctct 1200

agcgacttca ctccttttcc acacagatct ggtaccagat tgatggttga atacatcatt 1260

gcttggaaca aggacgaaga ttccaaatct gaagaattca ctaactggtt gcaaagattc 1320

tacgactaca tggaaccttt tgtttccaag aacccaagag tcggttacgt aaaccacatt 1380

gatttggact tgggtggaat tgactggaag aacaagactt ccacctctaa cgctatcgaa 1440

atagcccgga cttggggtga aaagtacttc ttgaccaact acgaaagatt agtcaaggct 1500

aagactctta ttgatccaaa gaacgttttc aaccatccac aatccatccc accaatggaa 1560

ttcgaccttg aacaaaattg gggtgtcaac gtcgagggta ttgtccaatg a 1611

<210> 3

<211> 1611

<212> DNA

<213> McBBE3

<400> 3

atggacacca agatcagaaa cttgtcctct agtctgttta tcttcatctc cgttttgact 60

tgtgctttag gtgacgactt gctatcttgt ttgacctctc acggtgttca caactacact 120

actccatctt ctgattctaa ttctgactat ttgagatttt tacatgtctc catccaaaac 180

ccattgttcg aaaacccagc tactccaaag ccagccgcta ttgttatgcc acgtactaag 240

gaagaattgg cttccactgt tagatgttgt actagaggtt cctggactgt ccgtttgaga 300

tctggtggtc actcttacga aggtttgtct tacaccgcgg atactccttt cgtcttgatc 360

gatttgatga acatgaacag aatatcaatc gatgtcgaat ccgaaaccgc ttgggttgag 420

tctggtgcta cccttggtga attgtactac gctatcactg aaagtactga ctctttcggt 480

ttcactgccg gttggtgccc aacagttggt tctggtggtc acatctctgg tggtgggttc 540

ggtatgatgt ccagaaagta cggtttagct gctgacaacg tcgtcgacgc catcttgatt 600

gatggtaacg gtgttatttt ggaccgtaac tccatgggtg aagatgtttt ctgggctatt 660

agaggtggcg gtggtggtgt ctggggtgcc atctacgcct ggaaaattaa gttgttgcca 720

gttccaaaga aggtcactgt tttcagatta atgaagcacg tcaagattga agaagcttct 780

aacttgttgc acaagtggca attcgtcgct gatgaattgg atgatgattt cactttgtcc 840

gtcttgggtg gagctgacga aaatgaagtt tggttaatgt tcctaggttt gtacttaggt 900

ccaaagaccg tagctaagtc tactatcgac ccaaagttcc cagaattagg tttgattgaa 960

gaagaattct ctgaaatgtc ttggggtgaa tcatttgctt acttggctgg tttgaagacc 1020

gtgggtgaat tgaacaacag attcttgaaa ttcgatgaca gagctttcaa gaccaaggtc 1080

gatttcgcca aggaaccaat ccctttgaag gttatcaacg gtgctttgga aatcttgtct 1140

aaagaaccac gtggtttcgt tgccttgaat ggtttaggag gtatgatgtc taaggtttct 1200

tccgacttca ccccattccc acacagatcc ggtaccagat tgatggttga atacattatt 1260

gcttggaaca aagatgaaga ctccaagtct gaagaattta cgaactggtt gcaaagattc 1320

tacgactaca tggaaccatt tgtctccaag aacccaagag ttggttacgt taaccacatt 1380

gatttggacc taggtggtat cgactggaag aacaagactt caacctccaa cgctatcgaa 1440

attgccagaa cctggggtga aaagtacttc ttgactaact atgaaagatt ggttaaggct 1500

aaaaccctga tagaccctaa gaacgttttc aaccatccac aatccattcc accaatggaa 1560

ttcgacttgg aacaaaattg gggtgtcaac gttgaaggta ttgtccaatg a 1611

<210> 4

<211> 1611

<212> DNA

<213> McBBE4

<400> 4

atggacacca aaatcagaaa tttgtcttct tctttgttta ttttcatctc cgttttgact 60

tgtgctttgg gtgacgattt attgtcttgt ttgacctctc acggtgttca caactatacc 120

actccatctt ctgattccaa ctctgactac ttgagatttt tgcacgtttc catccaaaac 180

ccattgttcg aaaatccagc tactccaaag ccagctgcta ttgttatgcc acgtaccaag 240

gaagaattgg cctcgaccgt cagatgttgt actagaggtt cttggactgt taggttgaga 300

tctggtggtc actcttacga aggtttgagt tacactgctg acactccttt cgtcttgatt 360

gatttaatga acatgaacag aatctctatt gatgtcgaat ctgaaactgc ttgggttgaa 420

tccggtgcta ccttaggtga attatactac gctatcacag aaagcactga ctcctttggt 480

ttcactgccg gttggtgccc aactgttggt tccggtggtc atatctctgg tggtggtttc 540

ggtatgatgt ctagaaagta cggtttggca gccgacaacg ttgtcgatgc tatcttgatt 600

gacggtaacg gtgtcatttt ggacagaaac tccatgggtg aagacgtttt ctgggctatc 660

cgtggtggtg gtggaggcgt ttggggtgct atctacgcct ggaaaatcaa gttgttgcca 720

gtcccaaaga aggtcactgt tttcagattg atgaagcacg tcaagatcga agaagcttct 780

aacctgttgc acaagtggca attcgttgct gatgaattgg atgatgactt taccctttca 840

gttctcgggg gtgctgacga aaacgaagtc tggttaatgt tcttgggttt gtacctaggc 900

ccaaaaacag tcgccaagtc tactattgac ccaaagttcc cagaactggg tttgattgaa 960

gaagaattct ccgaaatgtc ctggggtgag tccttcgctt acttggctgg tttaaaaacc 1020

gtgggtgaat tgaacaacag attcttgaaa ttcgatgaca gagctttcaa gaccaaggtt 1080

gacttcgcca aggaaccaat ccctttgaag gttatcaacg gtgccttgga aatcttaagt 1140

aaggaaccaa gaggtttcgt cgccttgaac ggtttgggtg gtatgatgtc taaggtttct 1200

tccgacttca ctccattccc acacagatct ggtaccagac taatggttga atacatcatt 1260

gcttggaaca aggatgaaga ttcgaagtcc gaagaattta ccaactggtt gcaaagattc 1320

tacgactaca tggaaccatt tgtctccaag aacccacgtg tcggttacgt caaccacatt 1380

gatttggact tgggtggtat cgactggaag aacaagacca gcacttccaa cgctattgaa 1440

attgctagaa cctggggtga aaagtatttc ctaactaact acgaaagatt ggttaaggct 1500

aagaccttga tcgatccaaa gaatgtcttc aaccatccac aatccattcc tccaatggaa 1560

ttcgacttgg aacaaaactg gggtgttaac gtcgaaggta ttgttcaatg a 1611

<210> 5

<211> 1611

<212> DNA

<213> McBBE5

<400> 5

atggatacta aaattaggaa tttgtcttct tcattgttca tattcatttc tgttttaact 60

tgtgcattag gtgacgattt gttgtcttgt ttgacttctc atggtgttca taattacaca 120

actccatcat ctgactctaa ttcagattat ttgagattct tgcacgtctc aattcaaaat 180

ccattgttcg aaaacccagc tactcctaag ccagcagcta ttgtcatgcc aaggacaaag 240

gaagagttgg cttctacagt caggtgctgc acaagaggtt cttggacagt caggttgagg 300

tctggtggtc attcttacga aggtttatct tatacagctg atactccatt tgttttgata 360

gatttaatga acatgaacag aatttctatt gatgtcgaat ctgagactgc ttgggtcgaa 420

tcaggtgcta ctttaggtga attgtattac gctattacag aatctacaga ttctttcggt 480

ttcacagcag gttggtgccc aactgttggt tcaggtggtc acatttctgg tggtggattc 540

ggtatgatgt ctaggaagta tggtttagct gctgacaacg tcgtcgacgc tatattgatt 600

gatggtaatg gtgttatatt agatagaaat tctatgggtg aagatgtttt ttgggctatt 660

aggggtggtg gtggtggtgt ttggggtgct atttatgctt ggaaaattaa attattacca 720

gttccaaaaa aggttactgt tttcagattg atgaaacatg ttaaaattga ggaagcttca 780

aatttattgc ataaatggca attcgttgca gacgagttgg acgacgactt cactttgtct 840

gtcttgggtg gtgcagacga gaacgaagtc tggttgatgt ttttaggttt gtatttgggt 900

cctaagacag ttgctaaatc tactatagat cctaaatttc ctgaattagg tttgattgaa 960

gaagaatttt ctgaaatgtc atggggtgaa tcatttgcat acttggcagg tttgaagaca 1020

gtcggtgagt tgaataatag atttttgaag ttcgatgata gagcttttaa aactaaggtt 1080

gactttgcaa aagaacctat tccattaaaa gttattaatg gagcattaga gattttgtca 1140

aaagaaccaa ggggattcgt tgctttgaac ggtttgggtg gaatgatgtc taaagtctct 1200

tcagatttta ctccatttcc acatagatca ggtactagat tgatggttga gtatattatt 1260

gcatggaaca aagatgaaga ttctaaatca gaagaattta ctaattggtt gcaaagattt 1320

tacgactaca tggaaccatt tgtttctaaa aaccctagag ttggttatgt taatcatatt 1380

gatttagatt tgggtggtat tgactggaaa aacaagacat caacttctaa tgcaattgaa 1440

atagctagaa catggggtga aaaatacttt ttgactaatt atgaaagatt ggttaaagct 1500

aaaactttga ttgacccaaa aaacgttttt aatcatccac aatcaattcc accaatggag 1560

ttcgacttgg aacagaactg gggtgtcaat gtcgagggaa ttgtccaata a 1611

<210> 6

<211> 36

<212> DNA

<213> McBBE1-URA-F

<400> 6

ggaatattaa gcttgatgga caccaagatc agaaat 36

<210> 7

<211> 36

<212> DNA

<213> McBBE1-URA-R

<400> 7

gatgcggccc tctagtcatt ggacaatgcc ttcaac 36

<210> 8

<211> 36

<212> DNA

<213> McBBE2-URA-F

<400> 8

ggaatattaa gcttgatgga caccaagatc agaaac 36

<210> 9

<211> 36

<212> DNA

<213> McBBE2-URA-R

<400> 9

gatgcggccc tctagtcatt ggacaatacc ctcgac 36

<210> 10

<211> 36

<212> DNA

<213> McBBE3-URA-F

<400> 10

ggaatattaa gcttgatgga caccaagatc agaaac 36

<210> 11

<211> 36

<212> DNA

<213> McBBE3-URA-R

<400> 11

gatgcggccc tctagtcatt ggacaatacc ttcaac 36

<210> 12

<211> 36

<212> DNA

<213> McBBE4-URA-F

<400> 12

ggaatattaa gcttgatgga caccaaaatc agaaat 36

<210> 13

<211> 36

<212> DNA

<213> McBBE4-URA-R

<400> 13

gatgcggccc tctagtcatt gaacaatacc ttcgac 36

<210> 14

<211> 36

<212> DNA

<213> McBBE5-URA-F

<400> 14

ggaatattaa gcttgatgga tactaaaatt aggaat 36

<210> 15

<211> 36

<212> DNA

<213> McBBE5-URA-R

<400> 15

gatgcggccc tctagttatt ggacaattcc ctcgac 36

<210> 16

<211> 20

<212> DNA

<213> pYES2-URA-F

<400> 16

gatcggacta ctagcagctg 20

<210> 17

<211> 20

<212> DNA

<213> pYES2-URA-R

<400> 17

taaataggga cctagacttc 20

<210> 18

<211> 32

<212> DNA

<213> YPR1-GRNA-F

<400> 18

ccaaaacaga gtcgttgata tgaagtggtt tt 32

<210> 19

<211> 32

<212> DNA

<213> YPR1-GRNA-R

<400> 19

ctctaaaacc acttcatatc aacgactctg tt 32

<210> 20

<211> 45

<212> DNA

<213> Y-UP-F

<400> 20

ttacgccaag cttgcatgcc tgcagctatg gtttaccacg caatg 45

<210> 21

<211> 38

<212> DNA

<213> Y-UP-R

<400> 21

gcggcttcta atccgacgta agagctactt aaatacac 38

<210> 22

<211> 31

<212> DNA

<213> Y-GAL-F

<400> 22

aagtagctct tacgtcggat tagaagccgc c 31

<210> 23

<211> 38

<212> DNA

<213> GAL-McBBE3-R

<400> 23

gatcttggtg tccatctcct tgacgttaaa gtatagag 38

<210> 24

<211> 36

<212> DNA

<213> McBBE3-F

<400> 24

tttaacgtca aggagatgga caccaagatc agaaac 36

<210> 25

<211> 37

<212> DNA

<213> McBBE3-R

<400> 25

taactaatta catgatcatt ggacaatacc ttcaacg 37

<210> 26

<211> 38

<212> DNA

<213> CYC1-McBBE3-F

<400> 26

ggtattgtcc aatgatcatg taattagtta tgtcacgc 38

<210> 27

<211> 34

<212> DNA

<213> Y-CYC1-R

<400> 27

gcgcgctcat tggaagcaaa ttaaagcctt cgag 34

<210> 28

<211> 30

<212> DNA

<213> Y-DOWN-F

<400> 28

aaggctttaa tttgcttcca atgagcgcgc 30

<210> 29

<211> 41

<212> DNA

<213> Y-DOWN-R

<400> 29

aacgacggcc agtgaattcg agctcgcagc cagcgttcaa c 41

Claims

1. A macleaya cordata berberine bridge enzyme gene optimized sequence is characterized in that the macleaya cordata berberine bridge enzyme gene optimized sequence comprises: McBBE1, McBBE2, McBBE3, McBBE4, McBBE5, wherein: the nucleotide sequence of McBBE1 is shown as SEQ ID NO.1, the nucleotide sequence of McBBE2 is shown as SEQ ID NO.2, the nucleotide sequence of McBBE3 is shown as SEQ ID NO.3, the nucleotide sequence of McBBE4 is shown as SEQ ID NO.4, and the nucleotide sequence of McBBE5 is shown as SEQ ID NO. 5.

2. A recombinant expression vector comprising the gene optimized sequence of claim 1 designated McBBE1, McBBE2, McBBE3, McBBE4, or McBBE 5.

3. The recombinant expression vector according to claim 2, wherein the expression vector is pYES 2-Ura.

4. A genetically engineered host bacterium comprising the recombinant expression vector of claim 2.

5. The genetically engineered host bacterium of claim 4, wherein the host bacterium is Saccharomyces cerevisiae.

6. The use of the macleaya cordata berberine bridge enzyme gene optimized sequence of claim 1, the recombinant expression vector of claim 2 or the genetically engineered host bacterium of claim 4 in the preparation of corydaline.

7. The method for preparing the saccharomyces cerevisiae engineering bacteria for high-yield golden yellow corydaline by using the macleaya cordata berberine bridge enzyme gene optimized sequence as claimed in claim 1, which comprises the following steps:

(1) the optimized gene sequences McBBE1, McBBE2, McBBE3, McBBE4 and McBBE5 are respectively connected to expression vectors, and recombinant vectors which are obtained through transformation and are correctly identified by PCR are respectively named as: pYES2-N1, pYES2-N2, pYES2-N3, pYES2-N4, pYES 2-N5;

(4) And (4) finding out an optimized gene corresponding to the strain obtained in the step (3), integrating the optimized gene into a saccharomyces cerevisiae genome by using a CRISPR-Cas9 technology, and identifying correctly to obtain the engineered saccharomyces cerevisiae for high-yield corydaline.

8. The saccharomyces cerevisiae engineering bacteria prepared by the method for preparing the high-yield corydaline-producing saccharomyces cerevisiae engineering bacteria of claim 7, wherein the saccharomyces cerevisiae engineering bacteria is YPR01 containing McBBE3 gene sequences.