Gene combination, recombinant plasmid combination and strain for synthesizing delphinidin and method for heterologous biosynthesis of delphinidin

Abstract

The invention belongs to the field of gene engineering, and relates to a gene combination for synthesizing delphinidin, a recombinant plasmid combination, a strain and a method for heterologous biosynthesis of delphinidin. The gene combination comprises the following genes: a flavanone 3-hydroxylase gene TbF3H, a P450 cytochrome oxidase gene TbCYP, a flavanonol 4-reductase TbDFR, an anthocyanin synthetase TbANS and a P450 cytochrome reductase gene LjCPR, and the flavanone 3-hydroxylase gene TbF3H, the P450 cytochrome oxidase gene TbCYP, the flavanonol 4-reductase TbDFR, the anthocyanin synthetase TbANS and the P450 cytochrome reductase gene The invention provides a potential method for replacing traditional artificial cultivation or submerged fermentation acquisition, and lays a foundation for further transforming efficient biosynthesis through metabolic engineering.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and specifically relates to a gene combination, a recombinant plasmid combination, a strain, and a method for heterologous biosynthesis of delphinidin. Specifically, the invention relates to the flavanone-3-hydroxylase gene (TbF3H), the P450 cytochrome oxidase gene (TbCYP), the dihydroflavonol 4-reductase gene (TbDFR), the anthocyanidin synthase gene (TbANS), and the P450 cytochrome reductase gene (LjCPR) involved in the synthesis of delphinidin in water chestnuts, a recombinant expression vector containing the gene fragments, a genetically engineered Saccharomyces cerevisiae, and a method for heterologous biosynthesis of delphinidin. Background Art

[0002] Delphinidin (C15H11O7, Dp), with its R1=R2=OH substitution group within the nucleus, is the primary active monomeric component of anthocyanidins. Its three precursors, dihydroflavonols, are primarily extracted from plants, as are most flavonoids. However, this method suffers from the drawbacks of requiring large amounts of biomass, a cumbersome extraction process, and low yields. Furthermore, adverse reactions during anthocyanidin extraction, such as browning and decomposition under high temperatures and light, can lead to anthocyanidin losses and reduce production efficiency. Furthermore, due to the complex structure of anthocyanidins and the involvement of various toxic reagents and extreme reaction conditions, the synthesis of anthocyanidins using simple compounds through multi-step chemical reactions has been unsatisfactory. With the advancement of synthetic biology, the production of natural products using microbial cell factories has become a research hotspot both domestically and internationally, and numerous studies have demonstrated the great potential of microbial heterologous synthesis of plant secondary metabolites.

[0003] Water chestnut (Trapabispinosa Roxb.) is an annual aquatic floating herb in the family Trapaeidae, Myrtales. Its husks and leaves are used in traditional Chinese medicine to treat astringent, purgative, antipyretic, esophageal cancer, dysentery, and other ailments. The husks and leaves contain a variety of bioactive compounds, including phenolic acids, flavonoids, terpenes, steroids, and polysaccharides. Water chestnut shell extracts have demonstrated promising blood sugar-lowering, liver cell-protecting, antibacterial, and anti-tumor properties.

[0004] Key enzyme genes for delphinidin biosynthesis include flavanone-3-hydroxylase (F3H), P450 cytochrome oxidase (CYP), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and P450 cytochrome reductase (CPR). While the number and variety of related functional enzyme genes have been continuously updated based on the research foundation of microbial metabolic engineering synthesis of dihydroflavonols and delphinidin, few reports exist on enzyme genes involved in the biosynthesis of dihydroflavonols and delphinidin in water chestnuts. The presence of multiple key enzyme genes for delphinidin biosynthesis in water chestnuts could expand the number and variety of these genes, laying the foundation for large-scale production of delphinidin.

[0005] In summary, in order to enrich the flavonoid enzyme gene library, pathway genes were obtained by analyzing the transcriptome data of different water chestnut tissues, and the key enzyme genes for delphinidin synthesis were cloned, screened and identified, laying the foundation for the subsequent laboratory production of dihydroflavonols and delphinidin through microbial fermentation engineering. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a gene combination, a recombinant plasmid combination, a strain and a method for heterologous biosynthesis of delphinidin.

[0007] The first aspect of the present invention provides a gene combination for synthesizing delphinidin, comprising the following gene fragments:

[0008] Flavanone 3-hydroxylase gene TbF3H, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the amino acid sequence of which is shown in SEQ ID NO: 2;

[0009]

[0010] MAPVSTLTALAEEKTLQARFIRDEDERPKVAYNNFSGDIPVISLHGIDDESGAGRAEICRKIVEACEDWGVFQVVDHGVDTKLVEDMTRHARDFFALPPEEKLRFDMSGGKKGGFIVSSHLQGEAVQDWREIVTYFSYPIRARDYSRWPDKPEGWKSVTEEYSERLMDLACKLLGVLSEAMG LEKEALTKACVDMDQKVVVNYYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSNCSRLSIATFQNPAPEATVYPLQIREGEKPILEEPITFAEMYRRKMSKDLELAKLKKQQKIEEVEKAKLEAKPLEQILA(SEQ IDNO: 2).

[0011] P450 cytochrome oxidase gene TbCYP, the nucleotide sequence of which is shown in SEQ ID NO: 3, and the amino acid sequence of which is shown in SEQ ID NO: 4;

[0012]

[0013] MGRFLIVEVAVAALLFFVTRLVVRSLLLRLQNRKLPPGPRGWPIIGALPLLGTMPHVALAKLAKKYGSVMYLKMGTCDMVVASTPDAARAFLKTLDINFSNRPPNAGATHLAYNAQDMVFAHYGPRWKLLRKLSNLHMLGGKALEDWAGVREDEVGHMLRAMCETSDRGEDVVVPEMLTYAMANMIGQVILSRRVFATKGSESNEFKDMVVELMTSSGYFNIGDFIPSIAWMDLQGIEKGMKRLHVKFDKLITEMIEQHSATAYRRKGKPDFLDQVMANSEMSDGERLNMTNVKALLLNLFTAGTDTSSSIIEWALAEMMKNPKIFRRAHEEMDRVIGRGRRLKESDIPNLPYLQAISKETMRKHPSTPLNLPRVSSEPCEVNGYYIPRGTRLSVNIWAIGRDPDVWESPLEFKPERFLEDERLSKIDPRGNSFELIPFGAGRRICAGTRMGIVLVEYVLGSLIHSFEWGLPPGTDDLDMNESFGLALQKKVPLTAVLTRRLSPTAYSS(SEQ ID NO:4)。

[0014] 二氢黄酮醇4-还原酶TbDFR,其核苷酸序列如SEQ ID NO:5所示,氨基酸序列如SEQID NO:6所示;

[0015]

[0016] MGQGGEIVCVTGASGFIGSWLVMRLLEHGYTVRATVRDPNNTKKVKHLLDLPNAKTHLSLWKADLNEEGSFDEPIQGCNGVFHVATPMDFESKDPENEVIKPTIEGMLSIMESCVKAKVRRVVFTSSAGTVNGQPVQKPVYDETSWSDLEFIRSTRMTGWMYFVSKTLAEQAAWKFAEENNLDFISI IPTLVVGPFLMPSFPPSLITALSPITGNKAHYSIIRQGQFVHLDDLVMSHLFLYQNPEAKGRYICSSHAENITHISKLLKEKYPEYDIPTEFEGVDEKLEKVSFSSEKLTELGFVFKYSLEDMFVEAVETCREKGLLPLSHEKKQQKQKDVINGKSVSLCHEKGSAEAVGKSSMAKNDHRLN(SEQID NO: 6).

[0017] Anthocyanidin synthase TbANS, the nucleotide sequence of which is shown in SEQ ID NO: 7, and the amino acid sequence of which is shown in SEQ ID NO: 8;

[0018]

[0019] MAEVAAAAGPTTGSAVRVQSISLSGQSSVPPQYIQPLQIRPGNHADSFDNIPSVDLSDFDPAHRDRIREEIGRACRDWGAFHVTGHCVPSGLLERVKAIGRAFFEDFSMEEKLKYACDASSSATEGYGSRMLENDDVVLDWRDYFDHHTLPLSRRNPSRWPHHPPDYRQTMAEYSDQMS LLAQKLLGLISELGLPTSCIEDAVGEFYQNITISFYPPCPQPDLTLGLQTHSDMGAVTLLIQDQVGGLQVLKDGTWVAVKPVPDAVIVMLADQTEIITNGAYRSAEHRAITNSSRARLSLATFHDPAKSKKIFPAPEIVSSSSPPRYREVVYGDYVSSWYTKGPEGKRNLDMLLI(SEQ ID NO: 8).

[0020] The nucleotide sequence of the P450 cytochrome reductase gene LjCPR is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10.

[0021]

[0022] (SEQ ID NO: 10).

[0023] In the present invention, the flavanone 3-hydroxylase gene TbF3H, the P450 cytochrome oxidase gene TbCYP, the dihydroflavonol 4-reductase TbDFR, and the anthocyanin synthase TbANS are derived from water chestnut-related genes and are obtained by performing gene base optimization according to the codon preference of Saccharomyces cerevisiae; the P450 cytochrome reductase gene LjCPR is derived from lotus-related genes and is obtained by performing gene base optimization according to the codon preference of Saccharomyces cerevisiae.

[0024] The second aspect of the present invention provides a recombinant plasmid for synthesizing delphinidin, containing gene fragments in the gene combination; the recombinant plasmid is an Escherichia coli expression vector and / or a Saccharomyces cerevisiae expression vector; preferably, the Escherichia coli expression vector uses pET-28a as the original vector, and the Saccharomyces cerevisiae expression vector uses pESC-URA and pESC-LEU as the original vector.

[0025] According to the recombinant plasmid of the present invention, the recombinant plasmids pESC-URA-TbF3H-F-TbCYP-C-LjCPR and pESC-LEU-TbDFR-D-TbANS-A-LjCPR were constructed.

[0026] The third aspect of the present invention provides a strain for synthesizing delphinidin, which contains the above-mentioned recombinant plasmid combination. The strain can be Escherichia coli or Saccharomyces cerevisiae, the Escherichia coli is preferably BL21 (DE3), and the Saccharomyces cerevisiae is preferably BY4741.

[0027] A fourth aspect of the present invention provides a method for heterologous biosynthesis of delphinidin, comprising: fermenting and culturing the above-mentioned strain to synthesize delphinidin. The fermentation conditions include: a culture medium containing 2% yeast, 2% tryptone, 8% glucose, 0.01 mM ferrous sulfate, 0.01 mM ascorbic acid, 0.01 mM α-ketoglutaric acid, and 0.01 mM CaCl2; the fermentation medium volume is expanded to 2 L, the pH is 5.5 ± 0.1, and the fermentation time is 96 hours.

[0028] The technical route of the present invention is as follows Figure 1 As shown in the following figure: In this study, using the yeast Saccharomyces cerevisiae BY4741 strain as a base cell, key enzyme genes for delphinidin synthesis were screened from water chestnuts. Flavanone-3-hydroxylase (F3H), P450 cytochrome oxidase (CYP), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) genes related to delphinidin synthesis were selected for prokaryotic and eukaryotic heterologous cloning and expression, and bioinformatics analysis of the candidate enzyme genes was performed. An engineered Saccharomyces cerevisiae strain was constructed to produce dihydroflavonols and delphinidin using naringenin as a substrate. Enzyme genes with corresponding activities were screened by adding substrate and detecting fermentation products. Dihydroflavonols and delphinidin were then fermented in Saccharomyces cerevisiae. Metabolic strategies such as specific gene optimization, promoter adjustment, fermentation condition optimization, and transcriptional activation were used to increase dihydroflavonol and delphinidin production.

[0029] The heterologous expression described in the present invention specifically refers to: using the cDNA obtained by reverse transcription of water chestnut total RNA as a template, amplifying the candidate TbF3H, TbCYP, TbDFR and TbANS expression sequence fragments by PCR, connecting the various TbF3H, TbCYP, TbDFR and TbANS expression fragments, the Escherichia coli expression vector pET-28a and the Saccharomyces cerevisiae expression vectors pESC-URA and pESC-LEU by enzyme cutting to obtain a series of recombinant expression vectors, which are then transferred into the Escherichia coli BL21 or Saccharomyces cerevisiae BY4741, BY4742 expression system to heterologously express the candidate enzyme genes.

[0030] The gene optimization described in the present invention specifically refers to: gene optimization is to optimize the base adaptability of genes with enzyme activity based on the codon preference of Saccharomyces cerevisiae.

[0031] The promoter adjustment described in the present invention refers to: screening 6 constitutive promoters from a Saccharomyces cerevisiae promoter library, constructing expression vectors with the eGFP reporter gene respectively, and then transforming them into Saccharomyces cerevisiae, and comparing the strengths of the 6 constitutive promoters by fluorescence microscopy and flow cytometry; then, connecting the 6 constitutive promoters with different strengths with the TbF3H gene respectively to construct constitutive yeast expression vectors, and transforming them into Saccharomyces cerevisiae for fermentation to compare DHK production.

[0032] The fermentation condition optimization described in the present invention refers to optimizing the amount of carbon and nitrogen sources added to the yeast YPD fermentation medium. In addition, the enzymes related to the synthesis of flavonoids require the action of multiple cofactors to exert their catalytic activity. Therefore, it is necessary to conduct experiments on the exogenous addition of cofactors to find the optimal amount of cofactors and explore the optimal fermentation conditions suitable for the target strain.

[0033] The metabolic strategy of transcriptional activation described in the present invention refers to: constructing a CRISPR-dCas9 transcriptional activation system to regulate the transcriptional activation of key enzyme genes in the Dp synthesis pathway, thereby improving the activation level of endogenous targets.

[0034] Compared with the existing technology, the present invention screened the key enzyme genes for delphinidin synthesis from water chestnuts, and screened out the flavanone 3-hydroxylase (F3H), P450 cytochrome oxidase gene (CYP), dihydroflavonol-4-reductase (DFR) and anthocyanidin synthase (ANS) related to delphinidin synthesis. In addition, a Saccharomyces cerevisiae strain that synthesizes delphinidin using naringenin as a substrate was constructed. The fermentation results without promoter showed that P SCS2 -P PTC3 The strain had the highest product production, with a Dp yield of 21.4 mg / L; S-PThe -FCDA strain was scaled up in a 5-L fermentor, achieving a Dp yield of 36.2 mg / L, 1.69 times that of shake flask production. This provides a potential alternative to traditional artificial cultivation or submerged fermentation, and lays the foundation for further metabolic engineering to achieve efficient biosynthesis.

[0035] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0037] Figure 1 This is the technical roadmap of the present invention.

[0038] Figure 2 This is a diagram of the biosynthetic pathway of delphinidin according to the present invention.

[0039] Figure 3 The electrophoresis results of the PCR amplification products of the genes described in the present invention, wherein A: RNA nucleic acid quality detection diagram; B: F3H gene PCR amplification diagram; C: CYP gene PCR amplification diagram; D: DFR gene PCR amplification diagram; E: ANS gene PCR amplification diagram, M: DNA marker.

[0040] Figure 4 This is the E. coli expression plasmid map of the gene described in the present invention (taking TbF3H as an example).

[0041] Figure 5 This is the expression plasmid map of the Saccharomyces cerevisiae gene of the present invention (taking TbF3H as an example).

[0042] Figure 6 This is the SDS-PAGE result diagram of the prokaryotic expression of the gene recombinant plasmid vector of the present invention (M: Marker; 1 / 2: pET-28a empty vector expression diagram; 4 / 6: F3H protein expression diagram; 8 / 10: DFR protein expression diagram; 12 / 14: ANS protein expression diagram; 16 / 18: CYP protein expression diagram).

[0043] Figure 7 This is an HPLC detection chart of the fermentation product of the whole gene vector of the Saccharomyces cerevisiae strain that synthesizes delphinidin in the present invention.

[0044] Figure 8These are the PCR electrophoresis diagrams of the water chestnut optimized gene positive clones of the present invention, wherein A: pESC-TbF3H-F colony PCR electrophoresis diagram; B: B1 / B2: pESC-URA-TbF3H-F-TbCYP-C-LjCPR and pESC-URA-TbDFR-D-TbANS-A-LjCPR colony PCR electrophoresis diagram; C: P PTC3 -LjCPR-T TDH1 Electropherogram of expression cassette.

[0045] Figure 9 These are the electrophoresis diagrams of the recombinant plasmids digested with double enzymes by the optimized water chestnut gene of the present invention, wherein A: double enzyme digestion diagram of the recombinant plasmid pESC-URA-TbF3H-F; B: double enzyme digestion diagram of the recombinant plasmid pESC-LEU-TbDFR-D-TbANS-A-LjCPR; C: 1 / 2: double enzyme digestion diagram of the recombinant plasmid pESC-URA-TbF3H-F-TbCYP-C-LjCPR.

[0046] Figure 10 The CRISPR-dCas9-VPR vector map and colony PCR map. A: dCas9-VPR and dCas9-VPR-eGFP vector map; B: Cas9 domain D10A and H840A site-directed mutagenesis electrophoresis; C: VPR vector electrophoresis; D: P2A-VPR-T2A-eGFP vector electrophoresis; E: P PGK1 -gRNAscaffold-P TDH3 Fragment electrophoresis; F: Reporter gene fluorescence microscopy detection results.

[0047] Figure 11 This is a comparison chart of the production of DHK, a fermentation product produced by a constitutive promoter.

[0048] Figure 12 The changes in DHK production at different carbon and nitrogen source concentrations.

[0049] Figure 13 The following is a graph showing the effects of cofactors on DHK production. A: Effect of ascorbic acid addition on DHK; B: Effect of FeSO addition on DHK; C: Effect of α-ketoglutaric acid addition on DHK; D: Effect of CaCl addition on DHK.

[0050] Figure 14 A comparison chart of fermentation products using constitutive promoters.

[0051] Figure 15 P S-P -Image of optimized fermentation culture of FCDABY4741 strain in a 5-L tank. DETAILED DESCRIPTION

[0052] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0053] In the examples, if the specific conditions are not specified, all experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0054] Example 1

[0055] By screening the candidate enzyme genes of water chestnut, including flavanone 3-hydroxylase gene TbF3H, P450 cytochrome oxidase gene TbCYP, dihydroflavonol 4-reductase TbDFR and anthocyanin synthase TbANS, each gene was cloned, expressed and enzyme activity identified, laying the foundation for the subsequent biosynthesis of flavonoids and delphinidin.

[0056] 1.1 Sequencing of various parts of water chestnut

[0057] Water chestnut tissues were obtained from freshly harvested, unfrozen samples. The research team initially sequenced RNA from various parts of water chestnut using the DNBSEQ sequencing platform (BGI, Shenzhen, China), constructing 12 cDNA libraries (FR_1, FR_2, FR_3, LF_1, LF_2, LF_3, ST_1, ST_2, ST_3, RT_1, RT_2, and RT_3). From these libraries, they screened for key genes involved in the biosynthesis of delphinidin, a key metabolite in water chestnut. Based on transcriptome analysis, they explored the biosynthetic pathway for delphinidin, a key secondary metabolite in water chestnut, and identified candidate enzyme genes involved in this pathway through data screening, laying the foundation for the subsequent synthesis of delphinidin using water chestnut enzyme genes.

[0058] 1.2 Cloning of candidate genes for delphinidin synthesis

[0059] Based on transcriptome sequencing data previously obtained by our laboratory, this study screened candidate enzyme genes involved in delphinidin biosynthesis, including: F3H candidate genes TbF3H-CL3333 and TbF3H-CL744; CYP candidate genes TbCYP75A-CL8351 and TbCYP75A-1434; DFR candidate genes TbDFR-CL7215 and TbDFR-16509; and ANS candidate genes TbANS-CL9282 and TbANS-CL5063. Primers were designed based on the gene sequences, and the sequences of each gene were obtained by polymerase chain reaction (PCR).

[0060] Using PCR technology, the cDNA obtained by reverse transcription of water chestnut total RNA and stored at -20°C was used as a template. According to the sequence information of the candidate enzyme gene obtained by water chestnut transcriptome sequencing, primers were designed and restriction sites were added to the 5' end of the primers. The specific primer sequences are shown in Table 1:

[0061] Table 1: Specific primer sequences used to amplify the F3Hs, CYPs, DFRs, and ANSs genes in water chestnuts

[0062] Primer name Sequence (5'-3') TbF3H-CL3333_F CGCGGATCCATGGCTCCCACGGTCTCGAC, SEQ ID NO: 11 TbF3H-CL3333_R GCAAGCTTGTCGACAGCTAAGATCTGTTCC, SEQ ID NO: 12 TbF3H-CL744_F GATCCGAATTCATGCAAGAGGTAGCGAGGG, SEQ ID NO: 13 TbF3H-CL744_R ATAAGAAGCGGCCGCCTGCGGCGATCTTCG, SEQ ID NO: 14 TbCYP75A-CL8351_F CGCGGATCCATGGGAAGGTTCCTCATCGT, SEQ ID NO: 15 TbCYP75A-CL8351_R CAAGCTTGTCGACAGATGAGTAAGCGGTG, SEQ ID NO: 16 TbCYP75A-1434_F CGGATCCGAATTCATGAAGAGGCTGAGCAGTCA, SEQ ID NO: 17 TbCYP75A-1434_R ATAAGAAGCGGCCGCTGATTTCTCATATGCAG, SEQ ID NO: 18 TbDFR-CL7215_F CGCGGATCCATGGGGCAGGGGGGTGAGAT, SEQ ID NO: 19 TbDFR-CL7215_R GCTTGTCGACATTCAACCGATGGTCATTTTTC, SEQ ID NO: 20 TbDFR-16509_F CGCGGATCCCGTTTGAACATTCTTCACCT, SEQ ID NO: 21 TbDFR-16509_R GCTTGTCGACTGAGACGGGGAAATGCTTTTC, SEQ ID NO: 22 TbANS-CL9282_F CGCGGATCCATGGCCGAAGTAGCTGCAG, SEQ ID NO: 23 TbANS-CL9282_R GCTTGTCGACAATGAGAAGCATATCTAAATTTC, SEQ ID NO: 24 TbANS-CL5063_F GATCCGAATTCCAGCCTCACACTTCAGCAAT, SEQ ID NO: 25 TbANS-CL5063_R ATAAGAATGCGGCCGCTTCGGATTTCTTGGCCTC, SEQ ID NO: 26

[0063] The PCR reaction system is shown in Table 2. The total PCR reaction system is 50 μL. The PCR reaction program is as follows: 95°C, 10 s; 98°C, 10 s; Tm, 5 s; 72°C extension, 5 s / kb; cycle amplification 30 times, and then 72°C, 5 min to end.

[0064] The reaction solution was used for electrophoresis under the following conditions: 120 V, 35 min.

[0065] Table 2: PCR reaction system

[0066] Reagent name Dosage Forward Primer (10 μM) 2μL Revers Primer (10 μM) 2μL 2×KeyPoMasterMix(DyePlus) 25 μL Template 100ng Nuclease-freeddH2O up to 50 μL

[0067] Using the cDNA obtained by reverse transcription as a template, bands of about 1000 bp and 800 bp were amplified respectively ( Figure 3 -B), bands around 1500 bp and 1000 bp ( Figure 3 -C), a band of about 1000 bp ( Figure 3 -D), a band of about 1000 bp ( Figure 3 -E). Transcriptome results for the candidate genes showed that the F3Hs enzyme candidate gene was approximately 1000 bp long, the CYPs enzyme candidate gene was approximately 1500 bp long, the DFRs enzyme candidate gene was approximately 1100 bp long, and the ANSs enzyme candidate gene was approximately 1000 bp long. These banding results were generally consistent with the theoretical results. Transcriptome sequencing results for the candidate genes showed that the banding results for each gene were roughly similar to the theoretical results. Samples were sent to Sangon for sequencing, which confirmed the correct results and successful amplification of the candidate genes.

[0068] 1.3 Construction of prokaryotic and eukaryotic expression vectors for delphinidin synthesis candidate genes

[0069] [1] Use restriction endonucleases (selected according to the restriction endonuclease sites of the candidate gene) to digest the target gene fragment of the empty plasmid. The enzyme digestion reaction system is shown in Table 3. Add each component to a 1.5 mL centrifuge tube, vortex to mix, and react in a 37°C water bath for 2-3 hours.

[0070] Table 3 Enzyme digestion reaction system

[0071] Components Dosage Restriction endonucleases (2 types) 2μL DNA 2 μg 0.1% BSA 4μL 10×HBuffer 4μL RNasedH2O up to 40 μL

[0072] [2] Purification of enzyme digestion products: Use PCR purification kit (OMEGA) to purify enzyme digestion products and remove other interfering reagents in the enzyme digestion system. Add 4 times the sample volume of CPBuffer to the above system, vortex mix for 1 minute, install the purification column and collection tube, transfer the mixed solution to the purification column, 12000rpm, 1min, discard the collection tube filtrate; add 700μL DNA Wash Buffer, 12000rpm, 1min, discard the collection tube filtrate, repeat this step twice; 12000rpm, 3min idling to remove the residual cleaning solution in the purification column matrix; transfer the pure column to a new 1.5mL centrifuge tube, place it in a 37℃ oven to dry for 10min to remove the residual anhydrous ethanol in the purification column, then add 30μL Elution Buffer preheated in a 60℃ water bath, let it stand at room temperature for 2min, and centrifuge it at 12000rpm for 2min; use an ultra-micro UV spectrophotometer to detect the concentration and purity of the eluate, and store the sample at -20℃.

[0073] [3] The enzyme-linked plasmid and target gene fragments were successfully digested, and the reaction system is shown in Table 4. The enzyme-linked reaction system was briefly centrifuged to mix, and then connected to a 16-degree centrifuge for 30 minutes.

[0074] Table 4: Enzyme-linked reaction system

[0075] Components Dosage DNA Ligation Kit Add equal volume Target gene fragment Target gene: vector fragment = 6:1 Vector fragment 1μL (molar ratio)

[0076] [4] The ligation mixture and the empty plasmid vector were transformed into Escherichia coli and Saccharomyces cerevisiae, respectively. E. coli was transformed by heat shock, and S. cerevisiae was transformed by electroporation. Single colonies were picked for colony PCR reaction, and the reaction solution was subjected to agarose gel electrophoresis and imaging. Positive colonies were cultured and the plasmids were extracted and sent to a sequencing company for sequencing and verification.

[0077] Figure 4 This is the E. coli expression plasmid map of the gene described in the present invention (taking TbF3H as an example). Figure 5 This is the expression plasmid map of the Saccharomyces cerevisiae gene of the present invention (taking TbF3H as an example).

[0078] 1.4 Construction of Escherichia coli and Saccharomyces cerevisiae expression strains for candidate genes related to delphinidin synthesis

[0079] The recombinant plasmids pET-28a-TbF3H-3333F, pET-28a-TbF3H-744H, pET-28a-TbCYP-8351C, pET-28a-TbCYP-1434Y, pET-28a-TbDFR-7215D, pET-28a-TbDFR-16509R, pET-28a-TbANS-9282A and pET-28a-TbANS-5063S verified correctly in 1.3 were transformed into BL21 (DE3) Escherichia coli expression strains. The recombinant expression strains were screened by colony PCR verification and 0.5 mM IPTG was added to induce the expression of the target protein.

[0080] The yeast expression vectors pESC-RUA-TbF3H-3333F, pESC-RUA-TbF3H-744H, pESC-RUA-TbCYP-8351C-LjCPR, and pESC-RUA-TbCYP-1434Y-LjCPR successfully constructed in 1.3 were transformed into the BY4741 Saccharomyces cerevisiae strain, and positive strains were screened by colony PCR for fermentation culture.

[0081] 1.5SDS-PAGE assay

[0082] Use Biosharp's SDS-PAGE kit to prepare protein gel. The specific method is as follows:

[0083] [1] Pick a single colony of E. coli expression strain that has been successfully identified from the LB resistance plate, inoculate it into 5 mL of LB corresponding resistance medium, and culture it at 37°C overnight;

[0084] [2] Inoculate the seed solution into LB liquid medium containing 1% glucose at a 2% inoculum volume, culture for about 4 hours, and then draw an appropriate amount of bacterial solution and store it in a 1.5 mL centrifuge tube;

[0085] [3] IPTG was added to the remaining bacterial solution to induce the bacterial culture at a final concentration of 0.5 mM. After culturing for 4 h, 1 mL of bacterial culture was aspirated and the precipitate was collected by centrifugation.

[0086] [4] Resuspend and wash the cells with 400 μL PBS, centrifuge at 12000 rpm for 1 min, repeat the wash twice, add 200 μL PBS to resuspend the cells, and vortex to mix;

[0087] [5] Pipette 20 μL of bacterial mixture into a 1.5 mL centrifuge tube, add 4 μL of protein loading buffer (5×), mix well and boil for 10 minutes. After cooling, detect the expression of the target protein by SDS-PAGE electrophoresis. The electrophoresis conditions are set to 90 V for 30 minutes, then 120 V for 60-70 minutes. The protein gel configuration is shown in Table 5.

[0088] Protein expression results Figure 6 As shown in the figure, M: Marker; 1 / 2: pET-28a empty vector expression; 4 / 6: F3H protein expression; 8 / 10: DFR protein expression; 12 / 14: ANS protein expression; 16 / 18: CYP protein expression. Compared with the blank control group, specific protein bands appeared in the experimental group (in the black box in the figure). The actual value of the target enzyme gene recombinant protein was close to the predicted value, indicating that each recombinant protein was successfully expressed in E. coli.

[0089] Table 5: SDS-PAGE protein gel recipe

[0090] Element 10% separation gel (mL) 5% stacking gel (mL) H2O 4.9 2.6 4×Tris / SDS separation gel buffer pH 8.8 2.5 - 40% Acr-Bis (29:1) 2.5 0.4 4×Tris / SDS stacking gel buffer pH 6.8 - 1 TEMED 0.01 0.004 10% SDS 0.1 0.04

[0091] 1.6 Identification of candidate genes for delphinidin synthesis and functional characterization using yeast expression strains

[0092] [1] Pick a single colony of Saccharomyces cerevisiae BY4741 carrying the target gene vector and inoculate it into 5 mL of liquid YPD medium. Incubate overnight at 28°C in a shaking incubator.

[0093] [2] Inoculate 10% of the inoculum into a 250 mL shake flask containing 25 mL of sugar-free YPD, add 4% galactose and the substrate of the target gene, and ferment at 28°C in a shaker for 3-4 days.

[0094] [3] Take 800 μL of bacterial solution in a 1.5 mL centrifuge tube, add an equal volume of 1% hydrochloric acid methanol, vortex mix for 2 minutes, and then place it in an ultrasonic cleaner for 30 minutes;

[0095] [4] The mixture was centrifuged at 12000 rpm for 3 min, the supernatant was filtered through a 0.22 μm filter membrane, and the sample was sent to Science Compass for LC-MS analysis.

[0096] [5] HPLC liquid phase detection method: separation was performed using a solvent system of 0.5% (v / v) formic acid / water (A) and 0.5% (v / v) formic acid / methanol (B), with a linear gradient from 15% to 60% B for 20 minutes, followed by a 5-minute hold at 60% B and a 10-minute equilibrium at 15% B. The injection volume was 100 μL, the detection wavelength was 290 nm, the column temperature was 30°C, the flow rate was 0.8 mL / min, and the DAD detector was used. The chromatographic column was a ZORBAX Eclipse Plus C18 (4.6×250 mm, 5 μm).

[0097] The candidate genes were expressed using the engineered Saccharomyces cerevisiae strain constructed in Example 1. The candidate genes TbF3H-3333F and TbCYP-8351C-LjCPR, which had corresponding enzyme activities according to the LC-MS detection results, were ligated to pESC-URA to construct a three-gene co-expression vector. This vector was then co-transformed with the pESC-LEU-TbDFR-7215D-TbANS-9282A recombinant vector into a BY4741 strain to form the pESC-URA-TbF3H-3333F-TbCYP-8351C-LjCPR and pESC-LEU-TbDFR-7215D-TbANS-9282A yeast strains, achieving de novo synthesis of delphinidin in the same Saccharomyces cerevisiae strain.

[0098] HPLC experimental results are as follows Figure 7 As shown. The test showed that the PGFCLDA-BY4741 yeast strain can synthesize delphinidin using naringenin as a substrate, among which the intermediate product eriodictyol accumulated in large quantities, with a yield of 927.5 mg / L, DHK yield of 29.3 mg / L, DHQ yield of 36.6 mg / L, and DYM yield of only 2.0 mg / L. Since the yield of the delphinidin precursor dihydromyricetin was not high, the yield of the final product delphinidin was only 7.1 mg / L. The reason may be that the CYP enzyme activity consumed a lot of enzyme activity in the early stage of catalyzing NAR to produce ERI, and the catalytic activity of F3H on the substrate eriodictyol was not strong, resulting in a low yield of dihydroquercetin, and the galactose-inducible promoter did not drive gene expression with high intensity, which affected the protease activity. In addition, the expression and fermentation conditions of Saccharomyces cerevisiae in the current experiment were not optimal for the production of delphinidin.

[0099] Example 2 Water chestnut optimized gene cloning and vector construction

[0100] 2.1 Optimized gene cloning of water chestnut

[0101] The candidate genes identified as having the corresponding enzyme activity in Example 1: TbF3H-3333F, TbCYP-8351C, TbDFR-7215D, and TbANS-9282A were selected and sent to Nanjing GenScript Biotechnology Co., Ltd. for codon optimization. Primers were designed using SnapGene software, and the optimized genes were cloned. The galactose promoter P was expressed in the cloned genes. GAL1 / P GAL10 The primers were designed as shown in Table 6.

[0102] Table 6: Codon-optimized gene primer sequence list

[0103]

[0104] 2.2 Construction of Saccharomyces cerevisiae expression vector and recombinant plasmid for optimized gene

[0105] TbF3H-3333F was constructed into the pESC-URA vector to obtain a single gene expression vector; and the LjCPR reductase expression cassette gene was connected to TbF3H-3333F-TbCYP-8351C and TbDFR-7215D-TbANS-9282A respectively, and cloned by enzyme digestion and homologous recombination method, and transformed into the competent cells of large intestine. The positive clones were screened by colony PCR. Figure 8 shown.

[0106] The plasmid was extracted, double enzyme digested and sent for sequencing verification. The enzyme digestion results were as follows Figure 9 As shown, the recombinant plasmid pESC-URA-TbF3H-F ( Figure 9 -A), pESC-URA-TbF3H-F-TbCYP-C-LjCPR( Figure 9 -C) and pESC-LEU-TbDFR-D-TbANS-A-LjCPR( Figure 9 -B).

[0107] Example 3 CRISPR-dCas9-VPR vector construction and verification

[0108] CRISPR-dCas9-VPR activation vector was constructed as follows Figure 10 As shown in the figure, site-directed mutagenesis primers (D10A and H840A) are designed to make Cas9 lose its nuclease cleavage activity (dCas9), so that it cannot cut the DNA double strand and can only bind to DNA. By carrying the activation factor and designing the corresponding promoter gRNA, the target gene is activated ( Figure 10 -B / C).

[0109] Design specific primers and connect the tracRNA expression cassette to the VPR activator and eGFP reporter gene with the dCas9 linearized vector through homologous recombination to construct an activation expression vector. Colony PCR verification was performed as follows: Figure 10 -D / E, the plasmid was extracted and sent for sequencing verification to obtain pESC-TRP-P PGK1 -gRNAscaffold-P TDH3 -dCas9-P2A-VPR and pESC-TRP-P PGK1 -gRNAscaffold-P TDH3 -dCas9-P2A-VPR-T2A-eGFP vector.

[0110] Then the two vectors were transformed into Saccharomyces cerevisiae, positive strains were screened, and the expression of reporter genes was observed under a fluorescence microscope. Figure 10-F, the eGFP reporter gene was successfully expressed, indicating that the dCas9-VPR protein was successfully expressed.

[0111] Example 4 Promoter Adjustment to Increase DHK Yield

[0112] Six constitutive promoters were screened from the Saccharomyces cerevisiae promoter library and expression vectors were constructed with the eGFP reporter gene. The promoters were then transformed into Saccharomyces cerevisiae. The strengths of the six constitutive promoters were compared by fluorescence microscopy and flow cytometry. Six constitutive promoters with different strengths were then connected to the TbF3H gene to construct constitutive yeast expression vectors. The vectors were then transformed into Saccharomyces cerevisiae for fermentation and the DHK production was compared. The results are shown in Figure 2. Figure 11 As shown, P PTC3 The DHK production of the promoter reached 66 mg / L, which was 2.4 times that of the galactose promoter.

[0113] Example 5 Optimization of Saccharomyces cerevisiae fermentation conditions

[0114] The specific steps for optimizing fermentation conditions for DHK production by Saccharomyces cerevisiae strains are as follows:

[0115] First, the concentration of each component of the fermentation medium YPD was optimized, the inoculation amount was kept consistent, the concentration of each component of the medium was controlled for fermentation, and the fermentation production of the product dihydrokaempferol was detected. Figure 12 As shown in the figure, when the yeast concentration is 2%, the DHK production is the highest, and as the yeast concentration increases, the DHK production decreases slightly; when the tryptone concentration is 2%, the DHK production is the highest, and as the tryptone concentration increases, the DHK production gradually decreases; when the glucose concentration is 8%, the DHK production is the highest, and when the sugar concentration exceeds 8%, the DHK production begins to decrease, which may be due to the high sugar concentration in the culture medium, and the high osmotic pressure of sugar will cause the growth of brewer's yeast to stagnate.

[0116] Then, by adjusting the amount of cofactors: α-ketoglutarate, ascorbic acid, FeSO4 and CaCl2, 8 concentration gradients were set up, and fermentation experiments were carried out under the above-determined medium component concentrations. Figure 13 shown.

[0117] 1. The best effect was achieved when the ascorbic acid addition was 0.01 mM, with a DHK yield of 87.6 mg / L. Further increase in the ascorbic acid concentration had no significant effect on the DHK yield. Figure 13 -A);

[0118] 2. The FeSO4 addition amount has a good effect in the range of 0.005mM to 0.01mM ( Figure 13 -B);

[0119] 3. The best effect was achieved when the addition amount of α-ketoglutarate was 0.01mM, and the DHK production reached 188.04mg / L. When the addition amount was greater than 0.5mM, the bacterial growth was severely inhibited, resulting in a serious decrease in DHK production ( Figure 13 -C);

[0120] 4. The addition of CaCl2 has a good effect in the range of 0.01mM to 0.05mM. It is the cofactor that has the greatest effect on DHK production, with the highest DHK production reaching 216.7mg / L ( Figure 13 -D).

[0121] The constructed pESC-URA-TbF3H-F-TbCYP-C-LjCPR and pESC-LEU-TbDFR-D-TbANS-A-LjCPR constitutive promoter three-gene co-expression vectors were co-transformed into BY4741 cerevisiae to obtain a yeast strain that synthesizes Dp from scratch using naringenin as a substrate. After fermentation, the treated fermentation samples were detected by HPLC. The results are as follows Figure 14 As shown, the PPTC3 promoter strain had the highest product production, with a dihydroquercetin yield of 19.18 mg / L, a dihydromyricetin yield of 65.8 mg / L, and the final product delphinidin yield of 21.4 mg / L.

[0122] P S-P -FCDABY4741 was expanded and cultured under the above fermentation tank optimization conditions. The fermentation results were as follows Figure 15 As shown, after 96 hours of fermentation in the fermenter, Dp production reached a maximum of 36.2 mg / L, 1.69 times that achieved in shake flasks, marking the highest yield known to date. DYM production reached 18.1 mg / L, and DHQ production reached 178.4 mg / L. The intermediate product ERI accumulated significantly, reaching 918 mg / L after 96 hours of fermentation, while DHK production reached 83.7 mg / L after 30 hours of fermentation. During the fermentation and expansion of the full-gene vector strain, the OD600 growth rate was low and sugar consumption was rapid. Further optimization of fermenter conditions is needed to increase bacterial growth concentration and enhance product yield.

[0123] The water chestnut F3Hs, CYPs, DFRs, and ANSs obtained in this invention increase the diversity of delphinidin-synthesizing enzyme genes, laying the foundation for subsequent microbial metabolic engineering to produce delphinidin. Because Saccharomyces cerevisiae cells grow rapidly and are easily fermented, and there is a mature platform for genetic manipulation of Saccharomyces cerevisiae, future metabolic engineering combined with fermentation engineering techniques is expected to significantly increase delphinidin fermentation yields. Therefore, this invention represents a promising industrialized delphinidin biosynthesis and genetic engineering technology.

[0124] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A gene combination for synthesizing delphinidin, comprising the following gene fragments: Flavanone 3-hydroxylase gene TbF3H, the nucleotide sequence of which is shown in SEQ ID NO: 1; P450 cytochrome oxidase gene TbCYP, the nucleotide sequence of which is shown in SEQ ID NO: 3; Dihydroflavonol 4-reductase TbDFR, the nucleotide sequence of which is shown in SEQ ID NO ID NO: 5; Anthocyanin synthase TbANS, the nucleotide sequence of which is shown in SEQ ID NO ID NO: 7; The nucleotide sequence of the P450 cytochrome reductase gene LjCPR is shown in SEQ ID NO:

9.

2. The gene combination for synthesizing delphinidin according to claim 1, wherein: The flavanone 3-hydroxylase gene TbF3H, the P450 cytochrome oxidase gene TbCYP, the dihydroflavonol 4-reductase TbDFR, and the anthocyanin synthase TbANS are derived from water chestnut-related genes and are obtained by optimizing the gene bases according to the codon preference of Saccharomyces cerevisiae; the P450 cytochrome reductase gene LjCPR is derived from lotus-related genes and is obtained by optimizing the gene bases according to the codon preference of Saccharomyces cerevisiae.

3. A recombinant plasmid combination for synthesizing delphinidin, containing the gene fragments of the gene combination according to claim 1 or 2; the recombinant plasmid is an Escherichia coli expression vector and / or a Saccharomyces cerevisiae expression vector; preferably, the Escherichia coli expression vector uses pET-28a as the original vector, and the Saccharomyces cerevisiae expression vector uses pESC-URA and pESC-LEU as the original vector.

4. The recombinant plasmid combination for synthesizing delphinidin according to claim 3 was used to construct the recombinant plasmid combination of pESC-URA-TbF3H-F-TbCYP-C-LjCPR and pESC-LEU-TbDFR-D-TbANS-A-LjCPR.

5. A strain for synthesizing delphinidin, comprising the recombinant plasmid combination according to claim 3 or 4.

6. The strain according to claim 5, wherein The strain is Escherichia coli or Saccharomyces cerevisiae, the Escherichia coli is BL21 (DE3), and the Saccharomyces cerevisiae strain is BY4741.

7. The gene cloning and expression method for synthesizing delphinidin according to claim 1, characterized in that: The steps include: (1) Using cDNA obtained by reverse transcription of total RNA from Trapabispinosa Roxb. as a template, amplifying the gene fragment encoding the synthetic delphinidin according to claim 2 by PCR; (2) Clone the gene fragment obtained in step (1) into an expression vector to construct a recombinant expression vector; and then transfer the recombinant expression vector into an expression system for heterologous expression.

8. The cloning and expression method according to claim 7, characterized in that: The method for cloning the gene fragment obtained in step (1) into an expression vector to construct a recombinant expression vector comprises: enzymatically connecting the LjCPR reductase expression cassette gene with TbF3H-TbCYP and TbDFR-TbANS to construct a three-gene co-expression vector, extracting the plasmid after PCR verification of the transformed Escherichia coli colony, and then transforming each constitutive expression vector into the BY4741 cerevisiae yeast strain to construct a yeast strain that synthesizes Dp de novo using naringenin as a substrate.

9. A method for heterologous biosynthesis of delphinidin, comprising: The strain described in claim 8 is fermented and cultured to synthesize delphinidin.

10. The method for heterologous biosynthesis of delphinidin according to claim 9, wherein: The fermentation culture conditions include: a culture medium containing 2% Yeast, 2% Tryptone, 8% Glucose, 0.01mM ferrous sulfate, 0.01mM ascorbic acid, 0.01mM α-ketoglutaric acid and 0.01mM CaCl2; the volume of the expanded fermentation culture medium is 2L, the pH is 5.5±0.1, and the fermentation time is 96h.

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