A method for total synthesis of crocin in microalgae

Abstract

This invention discloses a method for the total synthesis of crocin in microalgae, belonging to the field of crocin synthesis technology. The method includes the following steps: constructing... (The text abruptly ends here, so the translation stops as well.) GjCCD4a and GjALDH2C3 The dual-gene co-expression engineered strain ΔAB and GjCCD4a, GjALDH2C3 and GjUGT74F8 The three-gene co-expression engineered strain ΔABC was introduced into engineered strains ΔAB and ΔABC. CrtrB The engineered algal strains ΔEAB and ΔEABC were obtained through gene synthesis and cultured under a dual stress condition of high initial cell concentration coupled with nitrogen deficiency and high light intensity. The advantages of this invention are: for the first time, a complete direct synthesis system of crocin was constructed in *Syntrophus cytogenes* PCC 6803, achieving heterologous synthesis of crocin and crocin in cyanobacteria.

Description

Technical Field

[0001] This invention relates to a method for synthesizing crocin, specifically a method for the total synthesis of crocin in microalgae, belonging to the field of biosynthesis technology. Background Technology

[0002] Crocin is a natural, water-soluble carotenoid derivative with a unique structure and various biological benefits. Studies have shown that crocin exhibits significant biological activity in areas such as antioxidation, anti-inflammation, neuroprotection, antitumor activity, and metabolic regulation, and it is widely used in medicine, functional foods, health products, and cosmetics.

[0003] Currently, the main source of crocin still relies on extraction from higher plants such as saffron stigmas and gardenia fruits. These traditional methods suffer from significant drawbacks, including low content of active ingredients, complex extraction processes, and high costs, making it difficult to meet the demand for large-scale, sustainable raw material supply. Therefore, developing efficient, stable, and green new production routes for crocin is of great value and significance.

[0004] In recent years, the biosynthetic pathway of crocin has been clearly elucidated in higher plants. Crocin's precursor is zeaxanthin, a component of the carotenoid synthesis pathway, and the synthesis is completed through a three-step enzymatic reaction involving carotenoid cleavage dioxygenase (CCD), aldehyde dehydrogenase (ALDH), and UDP-glycosyltransferase (UGT). Several key enzyme genes involved in crocin synthesis have been identified in plants such as saffron, gardenia, and Buddleja officinalis, providing abundant genetic resources for heterologous crocin synthesis.

[0005] The development of synthetic biology and metabolic engineering has provided new ideas and tools for the heterologous synthesis of natural products. In plant cell factories, heterologous accumulation of crocin has been achieved in tobacco, tomato, and other plants, but problems remain, such as large land area requirements for cultivation and complex extraction and purification processes. Significant progress has also been made in microbial cell factories using *E. coli* and *Saccharomyces cerevisiae*. However, these heterologous systems still face challenges such as the need for artificially constructed synthetic pathways for the substrate (zeaxanthin), high carbon consumption during fermentation, and high carbon emissions, which restrict their large-scale application.

[0006] Microalgal chassis cells, with their advantages of photosynthetic autotrophy, rapid growth, and mature genetic manipulation systems, have become an important platform for green biomanufacturing. Synechocystis PCC 6803, a classic chassis strain in photosynthetic biology, efficiently drives carotenoid synthesis via its endogenous methyl erythritol phosphate (MEP) pathway and stably accumulates zeaxanthin, a precursor to crocin, laying a natural metabolic foundation for the heterologous synthesis of crocin. However, there are currently no research reports on the total synthesis of crocin using microalgae as chassis cells, both domestically and internationally. The feasibility and efficiency of heterologous crocin synthesis in cyanobacteria still require systematic exploration. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method for the total synthesis of crocin in microalgae using Synechocystis PCC 6803 as the chassis cell, thereby achieving the light-driven carbon fixation green bio-manufacturing of crocin.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A method for the total synthesis of crocin in microalgae includes the following steps: (1) Based on the carotenoid cleavage dioxygenase gene from gardenia GjCCD4a Crocin dialdehyde dehydrogenase gene GjALDH2C3 and UDP-glucuronyltransferase gene GjUGT74F8 The sequence is directly synthesized. GjCCD4a , GjALDH2C3 as well as GjALDH2C3-GjUGT74F8 Fragment; (2) Using Synechocystis PCC 6803 as the starting strain, construct GjCCD4a and GjALDH2C3 The dual-gene co-expression engineered strain ΔAB; (3) Using Synechocystis PCC 6803 as the starting strain, construct GjCCD4a、GjALDH2C3 and GjUGT74F8 The three-gene co-expression engineered strain ΔABC; (4) Introduce the β-carotene hydroxylase gene into engineered plants ΔAB and ΔABC. CrtrB The engineered algal strains ΔEAB and ΔEABC were obtained. (5) The engineered algal strains ΔEAB and ΔEABC were cultured under a high initial cell concentration coupled with nitrogen deficiency and high light dual stress conditions.

[0009] Preferably, in step (2), construct GjCCD4a and GjALDH2C3 The specific method for the dual-gene co-expression engineered strain ΔAB is as follows: (i) Using a homologous recombination kit, linearized vector A was combined with the target fragment containing homologous arms. GjCCD4a and GjALDH2C3 Ligation was performed to construct the pΔAB recombinant plasmid; (ii) pΔAB recombinant plasmid was transformed into Synechocystis PCC 6803 via natural transformation to obtain GjCCD4a and GjALDH2C3 The dual-gene co-expression engineered strain ΔAB.

[0010] Preferably, in step (3), construct GjCCD4a、GjALDH2C3 and GjUGT74F8 The specific method for co-expressing the three genes in the engineered strain ΔABC is as follows: (i) Using a homologous recombination kit, linearized vector A was combined with the target fragment containing homologous arms. GjCCD4a and GjALDH2C3-GjUGT74F8 Ligation was performed to construct the pΔABC recombinant plasmid; (ii) pΔABC recombinant plasmid was transformed into Synechocystis PCC 6803 via natural transformation to obtain GjCCD4a、GjALDH2C3 and GjUGT74F8 The three-gene co-expression engineered strain ΔABC.

[0011] Preferably, the linearized vector A is pCrtO-Kan, and the specific construction method is as follows: (a) Cloning of the β-carotene oxygenase gene from the genome of Synechocystis PCC 6803 by PCR crtO Upstream and downstream homologous flanking sequences crtO-U and crtO-D ; (b) From those carrying the kanamycin resistance gene Can Cloning the kanamycin resistance gene in a plasmid Can ; (c) By fusion PCR crtO-U , crtO-D and Can Fusion, Acquisition crtO-U / Can / crtO-D Fragment; (d) Cloning via TA crtO-U / Can / crtO-D The fragment was assembled into the linearized pMD18T vector to obtain the linearized vector pCrtO-Kan.

[0012] Preferably, in step (4), the following steps are performed: ΔAB and ΔABC are introduced into the engineered plants. CrtrB The specific gene-based approach is as follows: (i) Based on the β-carotene hydroxylase gene CrtR-B and streptomycin sulfate resistance gene SmR direct synthesis of sequences CrtrB-SmR Fragment fusion; (ii) Using a homologous recombination kit, linearized vector B was combined with... CrtrB-SmR Fragment fusion was performed to construct the pΔE recombinant plasmid; (iii) The engineered algal strains ΔEAB and ΔEABC were obtained by transforming the pΔE recombinant plasmid in engineered strains ΔAB and ΔABC through natural transformation.

[0013] Preferably, the linearized carrier B is pCrtO, and the specific construction method is as follows: (a) Cloning of the β-carotene oxygenase gene from the genome of Synechocystis PCC 6803 by PCR crtO Upstream and downstream homologous flanking sequences crtO-U and crtO-D ; (b) From those carrying the kanamycin resistance gene Can Cloning the kanamycin resistance gene in a plasmid Can ; (c) By fusion PCR crtO-U , crtO-D and Can Fusion, Acquisition crtO-U / Can / crtO-D Fragment; (d) Cloning via TA crtO-U / Can / crtO-D The fragment was assembled into the linearized pMD18T vector to obtain the linearized vector pCrtO-Kan; (e) Using the linearized vector pCrtO-Kan as a template and the nucleotide sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15 as primers, the linearized vector pCrtO was obtained by PCR amplification.

[0014] Preferably, in step (5), the high initial cell concentration refers to OD 730 ≥0.35, where nitrogen deficiency refers to cultivation in BG11 medium with NaNO3 removed, and high light refers to a photon flux density ≥300 μmol / m 2 / s.

[0015] The advantages of this invention are as follows: For the first time, a complete direct synthesis system for crocin was constructed in *Synostemma pentaphyllum* PCC 6803, achieving heterologous synthesis of crocin and crocin in cyanobacteria. This demonstrates the feasibility and potential of *Synostemma pentaphyllum* PCC6803 as a microbial chassis cell factory for crocin production. The research results provide important theoretical basis and experimental support for the metabolic engineering modification and culture process optimization of crocin synthesis in microalgal cell factories, laying a solid foundation for promoting the green biomanufacturing and industrial application of crocin. Attached Figure Description

[0016] Figure 1 This is a diagram of the crocin synthesis pathway of Synechocystis PCC 6803 designed in this invention. In the diagram, the enzymes shown in green are from the internal source of Synechocystis PCC 6803, the enzymes shown in yellow are introduced from the outside, the substances shown in black are endogenous products, and the substances shown in blue are the expected products of introduced exogenous enzymes. Figure 2 This is a schematic diagram of the construction of various homologous recombinant plasmids of Synechocystis PCC 6803; Figure 3 This is an image showing the electrophoresis verification results of each target gene and each linearized vector. In this image, A represents pCrtO-Kan and... CsUGT709G1 The electrophoresis verification results are shown in Figures 1 and 2, respectively, for pCrtO-Kan. CsUGT709G1 B is pCrtO-Kan and GjUGT94E13 The electrophoresis verification results are shown in Figures 1 and 2, respectively, for pCrtO-Kan. GjUGT94E13 C is pCrtO-Kan and CsUGT74AD1 The electrophoresis verification results are shown in Figures 1 and 2, respectively, for pCrtO-Kan. CsUGT74AD1 D is pCrtO-Kan and GjUGT74F8 The electrophoresis verification results are shown in Figures 1 and 2, respectively, for pCrtO-Kan. GjUGT74F8 E is pCrtO-Kan and GjCCD4a and GjALDH2C3 The electrophoresis verification results are shown in Figures 1, 2, and 3, respectively, for pCrtO-Kan, GjCCD4a , GjALDH2C3 F is pCrtO-Kan and GjCCD4a and GjALDH2C3-GjUGT74F8 The electrophoresis verification results are shown in Figures 1, 2, and 3, respectively, for pCrtO-Kan, GjCCD4a , GjALDH2C3-GjUGT74F8 G is pCrtO and CrtrB-SmR The electrophoresis verification results are shown in Figures 1 and 2 respectively. CrtrB-SmR pCrtO; M is an 8000bp DNA Marker; Figure 4 These are the electrophoresis verification results of each colony. Among them, A, B, C, D, E, and F are the electrophoresis results of colonies pΔC1, pΔC2, pΔC3, pΔC4, pΔAB, and pΔABC, respectively. G and H are the electrophoresis results of colony pΔE. 1-12 are different transformants. M is an 8000bp DNA marker. Figure 5 These are the electrophoresis verification results of each recombinant plasmid. Among them, A is the electrophoresis result of recombinant plasmids pΔC1 (1-4), pΔC2 (5-8), pΔC3 (9-12), and pΔC4 (13-17). B, C, and D are the electrophoresis result of recombinant plasmids pΔAB, pΔABC, and pΔE, respectively. 1-17 are different transformants. M is an 8000bp DNA marker. Figure 6 This is a schematic diagram of the genotypes of the homologous recombination engineered algal strains of Synechocystis PCC 6803, which are the series ΔC1, ΔC2, ΔC3, ΔC4, ΔAB, ΔABC, ΔEAB, and ΔEABC. Figure 7 This is a plate culture phenotypic diagram of the homologous recombination engineered algal strains of Synechocystis PCC 6803, ΔC1, ΔC2, ΔC3, and ΔC4 series, after 28 days of culture; Figure 8This is a diagram showing the liquid culture verification results of positive transformants from the ΔC1, ΔC2, ΔC3, and ΔC4 series Synechocystis PCC 6803 homologous recombination engineered algal strains after 28 days of culture. Figure 9 Phenotypic images of wild-type Synechocystis PCC 6803 (WT) and the ΔAB, ΔABC, ΔEAB, and ΔEABC series of Synechocystis PCC6803 homologous recombination engineered algal strains after 28 days of culture on plate. Figure 10 This is a liquid culture verification result of positive transformants from wild-type (WT) Synechocystis PCC 6803 and the ΔAB, ΔABC, ΔEAB, and ΔEABC series of Synechocystis PCC 6803 homologous recombination engineered algal strains after 28 days of culture. Figure 11 This is a graph showing the PCR identification results of wild-type (WT) Synechocystis PCC 6803 strain and mutant strains ΔC1, ΔC2, ΔC3, ΔC4, ΔAB, ΔABC, ΔEAB and ΔEABC after one month of resistance screening. Figure 12 This is a graph showing the growth curves of Synechocystis PCC 6803 wild-type strain (WT) and various engineered strains ΔC1, ΔC2, ΔC3, ΔC4, ΔAB, ΔABC, ΔEAB, and ΔEABC. Figure 13 The dry weight and OD of wild-type Synechocystis PCC 6803 strain 730 Graph showing the results of plotting the standard curve of the value relationship; Figure 14 This is a graph showing the results of standard curve plotting for crocinic acid, crocin dialdehyde, crocin I, crocin II, crocin I II, crocin IV, and crocin V standards. Figure 15 This is a dynamic diagram showing the changes in algal solution color and growth status of wild-type strain WT and engineered strains ΔEAB and ΔEABC of Synechocystis PCC 6803 during the light quality and intensity induced culture process. Figure 16 This is a graph showing the qualitative and quantitative detection results of the products of Synechocystis PCC 6803 wild-type strain WT and engineered strains ΔEAB and ΔEABC. In the graph, A is the HPLC chromatogram of the standard, 1 is crocin I, 2 is crocin II, 3 is crocin III, 4 is crocin IV, 5 is crocin V, 6 is crocin acid, and 7 is crocin dialdehyde. B, C, and D are the HPLC chromatograms of wild-type strain WT, engineered strain ΔEAB, and engineered strain ΔEABC, respectively. E and F are the mass spectra of crocin acid standard and crocin V standard, respectively. G and H are the mass spectra of the products of engineered strains ΔEAB and ΔEABC, respectively. Figure 17This figure shows the HPLC qualitative and quantitative detection results of the products of wild-type WT and engineered algal strains ΔEAB and ΔEABC of Synechocystis PCC 6803 cultured under nitrogen-deficient and high-light dual stress conditions with high initial cell concentration. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0018] I. Design of the crocin synthesis pathway in Synthocodile PCC 6803 Synechocystis PCC 6803 possesses a complete upstream precursor synthesis system for the MEP pathway—the endogenous carotenoid synthesis pathway. However, it suffers from multiple defects at the key enzyme level. First, the endogenous carotenoid cleavage dioxygenase (CCD) lacks specific cleavage function and cannot generate crocin dialdehyde. Second, the endogenous crocin dialdehyde dehydrogenase (ALDH) has poor substrate adaptability and lacks a suitable substrate to exert its catalytic activity. Third, it lacks a functional UDP-glucuronyl transferase (UGT), thus failing to complete the terminal glycosylation modification of crocin. Furthermore, compared with crocin synthases derived from saffron, gardenia, and Buddleja officinalis, the endogenous key enzymes exhibit mismatched catalytic characteristics and substrate selectivity, and its own metabolic network lacks a natural crocin synthesis branch. Ultimately, Synechocystis PCC 6803 is completely unable to synthesize crocin dialdehyde, crocin acid, and crocin in its natural state. Therefore, heterologous synthesis of crocin using Synthoctomyces PCC 6803 as a photosynthetic chassis requires breaking down endogenous metabolic barriers, introducing specific enzymes exogenously through molecular engineering, and supplementing with functional UGT to open up a complete heterologous crocin synthesis pathway and achieve efficient accumulation of the target product.

[0019] The optimal functional elements for constructing the heterogeneous synthesis pathway determined by this invention are as follows: (1) In the cleavage reaction, GjCCD4a from gardenia was selected as the key enzyme. This enzyme can specifically recognize the endogenously stable accumulation of zeaxanthin in Synechocystis PCC 6803 and precisely cleave it at the C7-C8 site to generate crocin dialdehyde. (2) In the oxidation reaction, given the poor substrate specificity and low catalytic efficiency of endogenous SynALDH, GjALDH2C3 derived from gardenia is preferentially introduced. This enzyme has high catalytic activity for crocin dialdehyde and can be directionally converted into crocin. (3) In the glycosylation reaction, GjUGT74F8 or GjUGT94E13 from gardenia is selected as the core element, or CsUGT709G1 and CsUGT74AD1 from saffron is selected as the core element. These enzymes have multi-step glycosylation ability and can directly complete the saffron series glycosylation modification with crocin acid as substrate, which significantly simplifies the genetic operation.

[0020] This invention proposes a three-level progressive rational design strategy to build a complete artificial synthesis system for crocin in the PCC 6803 chassis of Synthoctomyces cerevisiae, referring to... Figure 1 Specifically: First-level design: Using wild-type Synechocystis PCC 6803 as the chassis, only exogenous substances are introduced. UGT Gene( CsUGT709G1 (GenBank: KX385186) GjUGT94E13 (GenBank: MN944055) CsUGT74AD1 (GenBank: MF596166) or GjUGT74F8 (GenBank: MN944054), respectively denoted as C1 , C2 , C3 , C4 (Genes) to verify whether the endogenous CCD enzyme system and ALDH enzyme system have compensatory cleavage and oxidation functions and can autonomously complete the upstream conversion of zeaxanthin to crocin.

[0021] Second-level design: Based on the inability of Synechocystis PCC 6803 endogenous enzyme system to compensate. CCD , ALDH Preliminary determination of the carotenoid cleavage function of the gene was conducted, and two differentially expressed gene combination systems were constructed. The first group was... GjCCD4a (GenBank: KY631925, denoted as) A (genes) and GjALDH2C3 (GenBank: KY631926, denoted as) B (Gene) Dual-gene co-expression engineered strains were used to verify heterologous genes. CCD and ALDH The ability of crocin to synthesize during gene co-expression; the second group is... GjCCD4a、 GjALDH2C3 and GjUGT74F8 The three-gene co-expression engineered strain was used to verify the feasibility and effectiveness of the complete crocin synthesis pathway after heterologous core gene combination.

[0022] Level 3 design: Based on the clear limitations of the endogenous enzyme system's inability to compensate for cleavage and oxidation functions and the insufficient flux of endogenous zeaxanthin, the previously constructed co-expression recombinant algal strain was used as the starting strain to further introduce... CrtrB Gene (GenBank: AF162276.1, denoted as) E Genes that specifically enhance the anabolistic flux of zeaxanthin in the MEP pathway.

[0023] II. Construction of a cell factory from Synechocystis suis producing safflower glycosides PCC 6803 The original source of *Synostemma pentaphyllum* PCC 6803 used in this invention is the Pasteurella Culture Collection (PCC), numbered PCC6803. The direct source is a wild-type algal strain preserved in our laboratory, currently held in the Key Laboratory of Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences. *Synostemma pentaphyllum* PCC 6803 was cultured on BG11 medium at a temperature of 30±2℃, with a photon flux density of 40 µmol / m³. 2 / s.

[0024] 1. Design of a safflowerin expression system To construct a heterologous expression system suitable for Synesthesia cystis PCC 6803, based on the codon bias of Synesthesia cystis PCC 6803, the key enzyme gene for crocin synthesis was targeted. CsUGT709G1 , GjUGT94E13 , CsUGT74AD1 , GjUGT74F8 Codon optimization for Synechocystis was carried out.

[0025] To construct a heterologous expression system suitable for Escherichia coli, based on the codon bias of E. coli, the key enzyme gene for crocin synthesis was targeted. GjUGT74F8 , GjCCD4a , GjALDH2C3 Codon optimization for E. coli was carried out.

[0026] To evaluate the expression potential of endogenous homologous genes from *Synostemma pentaphyllum* PCC 6803, the linearized vector pCrtO-Kan was obtained by PCR amplification using the endogenous strong promoter cpc560 as the driving element. The linearized vector pCrtO-Kan was then combined with four gene sequences containing homologous arms using a homologous recombination kit. UGT Gene( CsUGT709G1 , GjUGT94E13 , CsUGT74AD1 , GjUGT74F8 The plasmids were connected separately to construct a series of recombinant plasmids, namely pΔC1, pΔC2, pΔC3, and pΔC4.

[0027] To assess the source of gardenia CCD , ALDH and UGT The expression efficiency of the gene in Synechocystis PCC 6803 was assessed by using a homologous recombination kit to combine the linearized vector pCrtO-Kan with the target fragment containing homologous arms. GjCCD4a、GjALDH2C3 or GjALDH2C3-GjUGT74F8 The recombinant plasmids pΔAB and pΔABC were constructed by linking them together. Furthermore, the β-carotene hydroxylase gene (… CrtrB ) and streptomycin resistance gene ( SmR ) constituted fusion fragments CrtrB-SmR The pΔE recombinant plasmid was constructed by ligating it with the linearized vector pCrtO. Details on the construction of various homologous recombinant plasmids from Synechocystis PCC 6803 can be found in [link to documentation]. Figure 2 .

[0028] 2. Codon optimization CsUGT709G1 , GjUGT94E13 , CsUGT74AD1 , GjUGT74F8 Four types UGT The optimized codon sequences of the gene from Synechocystis are shown in SEQ ID NO: 1 to SEQ ID NO: 4. GjUGT74F8 , GjCCD4a , GjALDH2C3 The optimized codon sequences of the three genes in E. coli are shown in SEQ ID NO: 5 to SEQ ID NO: 7.

[0029] 3. Primer design Based on the genome sequence and target gene of Synechocystis PCC 6803 published in the NCBI database. CsUGT709G1 , GjUGT94E13 , CsUGT74AD1 , GjUGT74F8 The optimized codon sequence of *Synostemma pentaphyllum* was used to design specific amplification primers using Primer 5.0 software. The nucleotide sequences of the designed primers are shown in SEQ ID NO: 8 and SEQ ID NO: 9.

[0030] 4. Obtaining the target gene and the linearized vector Using strains containing the target gene as templates and the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9 as specific amplification primers, four glycosyltransferase genes were obtained by PCR amplification: CsUGT709G1 , CsUGT74AD1 , GjUGT94E13 and GjUGT74F8 .

[0031] The PCR amplification reaction system is as follows: 2×Phanta® Max Master Mix 25μL, primer-F 2μL, primer-R 2μL, template 1μL, ddH2O 20μL.

[0032] The PCR amplification reaction cycle program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 1 min 30 s, 35 cycles; 72℃ final extension for 3 min; store at 16℃.

[0033] CsUGT709G1 , GjUGT94E13 , CsUGT74AD1 and GjUGT74F8 Four types UGTThe expected sizes of the PCR amplification products of the gene were 2291 bp, 2084 bp, 2123 bp, and 2117 bp, respectively. The amplification products were verified by 1% agarose gel electrophoresis. After confirming that the fragment size was consistent with the expectation, the fragments were excised, recovered, and purified.

[0034] according to GjCCD4a , GjALDH2C3 and GjUGT74F8 Optimized E. coli codon sequences were directly synthesized. GjCCD4a , GjALDH2C3 as well as GjALDH2C3-GjUGT74F8 Fragment. Among them, GjCCD4a and GjALDH2C3 The fragment was used to construct a dual-gene expression system (denoted as AB, 3507bp). GjCCD4a and GjALDH2C3-GjUGT74F8 The fragment was used to construct a three-gene expression system (denoted as ABC, 4881bp).

[0035] Based on the β-carotene hydroxylase gene CrtR-B (SEQ ID NO: 12) and streptomycin sulfate resistance gene SmR The sequence of (SEQ ID NO: 13) was directly synthesized. CrtrB-SmR Fusion fragment (denoted as E, 2639bp).

[0036] Using the β-carotene oxygenase gene from Synechocystis crtO To identify the homologous recombination site of exogenous DNA, a gene containing a 1500bp homologous flanking sequence and a kanamycin resistance gene was constructed. Kan The starting plasmid pCrtO-Kan was specifically cloned from the genome of Synechocystis by PCR. crtO Upstream and downstream homologous flanking sequences crtO-U and crtO-D (SEQ ID NO: 10 and SEQ ID NO: 11), from those with Kan Cloning in plasmids Kan Subsequently, fusion PCR was used to... crtO-U , crtO-D and Kan Fusion, Acquisition crtO-U / Kan / crtO-D The fragment, ultimately cloned via TA, will crtO-U / Kan / crtO-D The fragment was assembled into the linearized pMD18T vector to obtain the linearized vector pCrtO-Kan, with an expected size of approximately 7000 bp. The amplified product was verified by 1% agarose gel electrophoresis. After confirming that the fragment size was consistent with the expectation, the fragment was excised, recovered, and purified.

[0037] Using the linearized vector pCrtO-Kan as a template and pCrtO-F / pCrtO-R (SEQ ID NO: 14, SEQ ID NO: 15) as primers, the linearized vector pCrtO was obtained by PCR amplification.

[0038] The PCR amplification reaction system is as follows: 2×Phanta® Max Master Mix 25μL, primer-F 2μL, primer-R 2μL, template 1μL, ddH2O 20μL.

[0039] The PCR amplification reaction cycle program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 4 min 30 s, 35 cycles; 72℃ final extension for 10 min; store at 16℃.

[0040] The linearized vector pCrtO was expected to be 4200 bp in size. The amplified product was verified by 1% agarose gel electrophoresis, and after confirming that the fragment size was consistent with the expectation, it was excised, recovered, and purified.

[0041] Electrophoresis verification results of each target gene and each linearized vector are shown below. Figure 3 From A to D, it can be seen that the linearized carrier pCrtO-Kan is compatible with four types of... UGT The gene bands were single and clear, matching the expected size, indicating that the product quality was satisfactory. E and F show that the linearized vector pCrtO-Kan and the target fragment... GjCCD4a , GjALDH2C3 and GjALDH2C3-GjUGT74F8 The bands are single and clear, meeting the expected size, and the product quality is qualified. From G, we can see that the linearized vector pCrtO and the target fragment... CrtrB-SmR The amplified bands were clear and met the expected size, indicating that the product quality was up to standard.

[0042] 5. Restructuring System Construction Homologous recombination technology was used to combine the linearized vector pCrtO-Kan with the target fragment. CsUGT709G1 , GjUGT94E13 , CsUGT74AD1 , GjUGT74F8 , GjCCD4a , GjALDH2C3 , GjALDH2C3-GjUGT74F8 The linearized vector pCrtO is connected to the target fragment. CrtrB-SmR connect.

[0043] The recombination reaction system is as follows: linearized carrier pCrtO-Kan or pCrtO 1 μL, target fragment 1 μL, 2×ClonExpress Mix 5 μL, ddH2O 3 μL.

[0044] Single segment ( CsUGT709G1 , GjUGT94E13 , CsUGT74AD1 , GjUGT74F8 , CrtrB-SmR Recombination reaction: Incubated at 50℃ for 10 min, then immediately cooled on ice. A series of recombinant vectors, pΔC1, pΔC2, pΔC3, pΔC4, and pΔE, were constructed.

[0045] Multiple segments ( GjCCD4a and GjALDH2C3 , GjCCD4a and GjALDH2C3-GjUGT74F8 Recombination reaction: Incubate at 50℃ for 15 min, then immediately cool on ice. pΔAB and pΔABC recombinant vectors were constructed respectively.

[0046] 6. Transformation of competent Escherichia coli cells The recombinant vectors (pΔC1, pΔC2, pΔC3, pΔC4, pΔE, pΔAB, pΔABC) were transformed into Escherichia coli DH5α competent cells for amplification and identification. Specifically: 100 μL of DH5α competent cells were thawed in an ice bath, 10 μL of recombinant reaction solution was added, and the mixture was gently mixed and incubated in an ice bath for 30 min; the cells were then heat-shocked in a water bath at 42℃ for 90 s, and then quickly transferred to 4℃ for 2 min of cooling; 900 μL of antibiotic-free LB liquid medium was added, and the cells were cultured at 37℃ with shaking at 200 rpm for 1 h to allow the bacteria to recover and express the resistance gene; 100 μL of the bacterial culture was spread on BG11 solid medium containing the corresponding antibiotic (100 μg / mL kanamycin or 50 μg / mL streptomycin sulfate), and incubated overnight at 37℃ with the cells inverted (12-16 h) until single colonies were formed, resulting in pΔC1, pΔC2, pΔC3, pΔC4, pΔE, pΔAB, and pΔABC colonies, respectively.

[0047] 7. Colony PCR identification and sequencing verification Positive single colonies were picked and identified by PCR using primers shown in SEQ ID NO: 16 and SEQ ID NO: 17 for pΔAB and pΔABC, primers shown in SEQ ID NO: 18 and SEQ ID NO: 19 for pΔE, and primers shown in SEQ ID NO: 20 and SEQ ID NO: 21 for pΔC1, pΔC2, pΔC3, and pΔC4.

[0048] The PCR amplification reaction system is as follows: 10 μL of 2×Rapid Taq Master Mix, 1 μL of primer-F, 1 μL of primer-R, 2 μL of template, and 6 μL of ddH2O.

[0049] The PCR amplification reaction cycle program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 2 min, 35 cycles; 72℃ final extension for 5 min; store at 16℃.

[0050] The expected size of the PCR product from pΔE was 3000 bp, and the expected size of the PCR products from the other colonies was 4000 bp. The products were detected by 1% agarose gel electrophoresis, and clones showing fragments of the expected size were preliminarily identified as positive recombinants.

[0051] Positive clones were further sequenced, and the sequencing results were compared with the optimized gene sequence. If the sequence homology was ≥99%, it was considered a correct recombinant.

[0052] The electrophoresis verification results for each colony are shown in the figure. Figure 4 .Depend on Figure 4 It can be seen that the fragments of colonies pΔC1 and pΔC3 are approximately 3500 bp, and the fragments of colonies pΔC2 and pΔC4 are approximately 3000 bp. The band sizes are as expected, and the target gene has been successfully inserted. The fragments of colonies pΔAB and pΔABC are approximately 3000 bp, and the fragment of colony pΔE is approximately 2700 bp. The sequencing alignment of all three is consistent with the known sequences.

[0053] 8. Extraction and quality testing of recombinant plasmids using alkaline lysis method The recombinant plasmid was extracted using a conventional alkaline lysis method, and the plasmid concentration was determined. The plasmid concentration was found to be above 200 ng / μL.

[0054] The electrophoresis verification results of each recombinant plasmid are shown in the figure. Figure 5 .Depend on Figure 5 It can be seen that among the pΔC series recombinant plasmids, pΔC1 and pΔC2 bands are clear and have high concentrations, consistent with expected sizes; pΔC3 and pΔC4 bands are darker and partially diffuse, indicating lower concentrations or slight degradation. The dual-gene recombinant plasmid pΔAB shows clear and uniform bands with no obvious extraneous bands or degradation, indicating product stability. The tri-gene recombinant plasmid pΔABC shows good band consistency, demonstrating the reliability of the large plasmid extraction process. The first eight samples of recombinant plasmid pΔE show blurred bands, while the last eight samples show band sizes consistent with expectations, indicating batch-to-batch variation.

[0055] 9. Obtaining engineered algal strains producing saffron through natural transformation. Recombinant plasmids were transformed using the natural transformation method to obtain homologous recombinant engineered algal strains of Synechocystis PCC 6803, namely ΔC1, ΔC2, ΔC3, ΔC4, ΔAB, ΔABC, ΔEAB, and ΔEABC. Genotypic diagrams are shown below. Figure 6 .

[0056] The specific methods for recombinant plasmid transformation are as follows: (1) Select wild-type strain of Synechocystis PCC 6803 and streak it on BG11 plate. Incubate at 30±2℃ and a photon flux density of 40µmol / m 2 Cultivate under / s conditions. Select single algal colonies and expand the culture into 100mL of BG11 liquid medium, then incubate statically in a light incubator until OD reaches [value missing]. 730 =0.6-0.8. Collect algal cells by centrifugation at 5000 rpm at room temperature, wash twice with fresh BG11 liquid medium, and then dilute the algal precipitate to OD0.6. 730 =2.5, as a transformation receptor.

[0057] (2) Take 500 μL of the above algal solution and add recombinant plasmids (pΔC1, pΔC2, pΔC3, pΔC4, pΔAB or pΔABC) at a ratio of 1 μg of recombinant plasmid per 100 μL of algal solution. After gently mixing, place the solution in a light incubator and incubate under low light for 6-8 h. Spread the incubated algal solution evenly on a BG11 plate with a nitrocellulose membrane. After incubating in a light incubator for 18 h, transfer the nitrocellulose membrane to a BG11 plate containing antibiotics (50 μg / mL kanamycin). After two weeks of continuous culture, green engineered algal strains will appear. Use an inoculation loop to pick up the engineered algal strains grown on the BG11 plate and streak them on a BG11 plate containing antibiotics (50 μg / mL kanamycin) for secondary screening. The isolated engineered algal strains were then streaked and passaged again every 7 days, with the antibiotic concentration gradually increased (50 μg / mL kanamycin on day 7, 60 μg / mL on day 14, and 70 μg / mL on day 21). After multiple subcultures, stable engineered algal strains were selected and transferred to BG11 liquid medium for large-scale culture. The engineered algal strains obtained from the last isolation were inoculated into BG11 liquid medium (48-well plate) containing antibiotic (50 μg / mL kanamycin) and cultured under light. The degree of homogeneity of the engineered algal strains (i.e., homologous recombination of the genome in all cells) was confirmed by PCR. Engineered algal strains ΔC1, ΔC2, ΔC3, ΔC4, ΔAB, and ΔABC were obtained.

[0058] (3) Using some engineered algal strains ΔAB and ΔABC as transformation recipients, the recombinant plasmid pΔE was transformed into the recipients using the same method as in step (2). In addition to kanamycin, streptomycin sulfate (25 μg / mL) was added to the antibiotics. During the streak passage operation of the isolated engineered algal strains every 7 days, the streptomycin sulfate concentration was 25 μg / mL on day 7, 30 μg / mL on day 14, and 35 μg / mL on day 21. Engineered algal strains ΔEAB and ΔEABC were obtained respectively.

[0059] After the recombinant plasmids pΔC1, pΔC2, pΔC3, and pΔC4 were transformed into the wild-type *Syneocystis* PCC 6803, homologous recombinant engineered algal strains of *Syneocystis* PCC 6803 were obtained, namely the ΔC1, ΔC2, ΔC3, and ΔC4 series. The phenotypic characteristics of these homologous recombinant engineered algal strains after 28 days of plate culture are shown in [the table below]. Figure 7 The results of liquid culture verification of positive transformants are shown in [link to relevant documentation]. Figure 8 .Depend on Figure 7 and Figure 8 It can be seen that 14 days after transformation, most of the algae on the plate gradually turned yellow and died under antibiotic pressure; at 28 days of culture, smooth and uniform green single clones began to appear on the plate, indicating that the recombinant vector carrying the kanamycin resistance gene had been successfully integrated into the genome of Synechocystis, and the positive transformants acquired resistance and resumed growth. Figure 7 The monoclonal algal colonies obtained from the initial screening were subjected to continuous resistance streaking culture for at least one month to obtain mutant strains that could stably grow in kanamycin-resistant medium. Figure 8 , ΔC1, ΔC2, ΔC3, ΔC4).

[0060] After the recombinant plasmids pΔAB and pΔABC were transformed into the wild-type strain of *Syneocystis* PCC 6803, homologous recombinant engineered algal strains of *Syneocystis* PCC 6803 were obtained, namely the ΔAB and ΔABC series. The phenotypic characteristics of these homologous recombinant engineered algal strains after 28 days of plate culture are shown in the figure. Figure 9 The results of liquid culture verification of positive transformants are shown in [link to relevant documentation]. Figure 10 .Depend on Figure 9 and Figure 10 It can be seen that: 14 days after transformation, the algae gradually turned from green to yellow; 28 days later, smooth, uniform green single clones began to appear on the plate, indicating that the recombinant vector carrying the kanamycin resistance gene had been successfully integrated into the genome of Synechocystis, and the positive transformants acquired resistance and resumed growth. Figure 9 (Left side). The monoclonal algal colonies obtained from the initial screening were subjected to continuous resistance streaking culture for at least one month to obtain mutant strains that could be stably inherited in kanamycin-resistant medium ( Figure 10 (Left side).

[0061] After the recombinant vector pΔE was transformed into the engineered algal strains ΔAB and ΔABC, homologous recombinant engineered algal strains of Synechocystis PCC 6803 were obtained, namely ΔEAB and ΔEABC series. The phenotypic characteristics of these homologous recombinant engineered algal strains after 28 days of plate culture are shown in the figure. Figure 9 The results of liquid culture verification of positive transformants are shown in [link to relevant documentation]. Figure 10 .Depend on Figure 9 and Figure 10It can be seen that: 14 days after transformation, the algae gradually turned from green to yellow; 28 days later, smooth, uniform green monoclonal colonies began to appear on the plate, indicating that the recombinant vector carrying the kanamycin and streptomycin resistance genes had been successfully integrated into the genome of Synechocystis, and the positive transformants acquired resistance and resumed growth. Figure 9 (Right side). The monoclonal algal colonies obtained from the initial screening were subjected to continuous resistance streaking culture for at least one month to obtain mutant strains that could be stably inherited in a medium containing both kanamycin and streptomycin sulfate resistance. Figure 10 (Right side).

[0062] 10. PCR identification of engineered algal strains Genomic DNA from engineered algal strains ΔC1, ΔC2, ΔC3, ΔC4, ΔAB, ΔABC, ΔEAB, and ΔEABC in the logarithmic growth phase was used as templates. PCR amplification was performed using target gene-specific primers and 2×Rapid Taq Master Mix. Wild-type strain (WT) of Synechocystis PCC 6803 was used as a negative control.

[0063] After one month of resistance screening, several mutant strains of each of the following types were randomly selected for PCR identification: ΔC1, ΔC2, ΔC3, ΔC4, ΔAB, ΔABC, ΔEAB, and ΔEABC. The identification results are shown below. Figure 11 .Depend on Figure 11 It can be known that: (1) Engineered algal strains (ΔC1, ΔC2, ΔC3, ΔC4) that successfully integrated recombinant plasmids pΔC1, pΔC2, pΔC3, and pΔC4 could amplify a characteristic band of about 5000 bp. Wild-type strain PCC 6803 of Synechocystis showed no amplification products, indicating that the foreign gene integration was successful. The positive rates of mutant strains of ΔC1, ΔC2, ΔC3, and ΔC4 were 73.3%, 87.5%, 73.3%, and 50%, respectively. Some surviving algal strains without the target band were false positives, which were presumably due to spontaneous mutations that acquired kanamycin resistance. (2) The engineered algal strains (ΔAB and ΔABC) that successfully integrated the recombinant plasmids pΔAB and pΔABC could amplify a characteristic band of about 4000 bp, while the wild-type strain (WT) of Synechocystis PCC 6803 did not have any amplification products, indicating that the foreign gene integration was successful; the positive rates of the ΔAB and ΔABC mutant strains were 90.9% and 91.7%, respectively. (3) The engineered algal strains (ΔEAB and ΔEABC) that successfully integrated the recombinant plasmid pΔE all amplified a characteristic band of about 4000bp. The wild-type strain PCC 6803 of Synechocystis showed no amplification product, indicating that the foreign gene was successfully integrated. The positive rates of the ΔEAB and ΔEABC mutant strains were 81.8% and 72.7%, respectively.

[0064] (4) Compared with the single-gene transformation system, the false positive rate of the multi-gene transformation system is significantly reduced, suggesting that the increased length of the homologous flanking sequence effectively improves the efficiency of homologous recombination integration.

[0065] 11. Growth curve determination To evaluate the growth characteristics of engineered algal strains, engineered algal strains (ΔC1, ΔC2, ΔC3, ΔC4, ΔAB, ΔABC, ΔEAB, ΔEABC) grown to the logarithmic growth phase in BG11 liquid medium, as well as wild-type Synechocystis PCC 6803, were used. Initial OD values ​​were adjusted using fresh BG11 liquid medium. 730 =0.1, inoculated into BG11 liquid medium, and placed in a light incubator for static culture. The culture conditions were: temperature 30℃, photon flux density 40 μmol / m 2 The light-dark cycle was 12h:12h. The absorbance of the culture at 730nm was measured every 2 days using a UV-Vis spectrophotometer for 15 consecutive days. The culture time was plotted as the x-axis, and the OD value was... 730 The value is used as the ordinate to plot the growth curve.

[0066] The growth curves of wild-type *Synostemma pentaphyllum* PCC 6803 and various engineered strains (ΔC1, ΔC2, ΔC3, ΔC4, ΔAB, ΔABC, ΔEAB, ΔEABC) are shown in the figure. Figure 12 .Depend on Figure 12 It can be seen that the wild-type strain of Synechocystis PCC 6803 and the engineered strains (ΔC1, ΔC2, ΔC3, ΔC4, ΔAB, ΔABC, ΔEAB, ΔEABC) all exhibit a typical delayed-logarithmic-steady-state growth pattern. Compared with the wild-type strain of Synechocystis PCC 6803, the growth rates of the engineered strains are basically consistent, and the growth performance of some engineered strains is even significantly improved.

[0067] 12. Determination of algal biomass standard curve Wild-type *Synostemma pentaphyllum* PCC 6803 was selected as the research object. Five concentration gradients of algal solutions (15 mL each) were prepared, and the absorbance of each gradient at 730 nm was measured using a UV-Vis spectrophotometer. A 0.45 μm aqueous filter membrane was dried to constant weight, and its initial dry weight was recorded. The algal solutions of each gradient were vacuum filtered, and the filter membranes containing the retained algae were dried again to constant weight. The dry weight of the algae was calculated (algae dry weight = total weight of dried filter membrane - initial dry weight of filter membrane). OD values ​​were used as the basis for the analysis. 730 Plot the dry weight and OD of wild-type Synechocystis PCC 6803 strain on the x-axis and the dry weight of the algae on the y-axis. 730 Standard curve of value relationship.

[0068] Dry weight and OD of wild-type Synthoctopus PCC 6803 730The results of plotting the standard curve of the value relationship are shown in the figure. Figure 13 .Depend on Figure 13 It can be seen that the OD of wild-type strain PCC 6803 of Synthocodile is... 730 The value shows a very strong positive linear correlation with the dry weight concentration, and the fitted equation is y = 1.02989x - 0.06293, with a coefficient of determination R0. 2 =0.9943, indicating OD 730 The values ​​can effectively explain the differences in dry weight concentration. This standard curve can be used in subsequent experiments to analyze OD values. 730 This method allows for rapid estimation of algal biomass, providing a basis for normalized calculations of product synthesis efficiency.

[0069] 13. Determination of crocin content in Synechocystis PCC 6803 (1) Prepare standard working solutions of crocin, crocin dialdehyde, and crocin I / II / III / IV / V. Crocin acid standard working solution: A series of standard working solutions with concentrations of 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, 1.0 μg / mL, 2.0 μg / mL and 10.0 μg / mL were prepared using methanol as solvent.

[0070] Crocin dialdehyde standard working solution: A series of standard working solutions with concentrations of 0.4 μg / mL, 0.8 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL and 10.0 μg / mL were prepared using methanol as the solvent.

[0071] Crocin I standard working solution: A series of standard working solutions with concentrations of 0.4 μg / mL, 4.0 μg / mL, 8.0 μg / mL, 40 μg / mL and 80 μg / mL were prepared using methanol as solvent.

[0072] Crocin II, III, IV, and V standard working solutions: A series of standard working solutions with concentrations of 0.4 μg / mL, 0.8 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, and 20 μg / mL were prepared using methanol as the solvent.

[0073] (2) Plot the HPLC standard curve Take the prepared standard working solutions of each concentration (three replicates per group) and perform HPLC analysis. Chromatographic conditions: A Thermo reversed-phase C18 column (4.6 mm × 250 mm, 5 μm) was used. The column temperature was set at room temperature, the detection wavelength was 440 nm, mobile phase A was 0.1% acetate-ammonium acetate buffer (pH 4.0), mobile phase B was 100% acetonitrile, the flow rate was 1.5 mL / min, and the gradient elution program was as follows: 0-1 min, maintain phase B at 20%; 1-8 min, phase B linearly increases from 20% to 60%; 8-8.1 min, phase B linearly increases from 60% to 70%; 8.1-13 min, phase B linearly increases from 70% to 98%; 13-15 min, phase B linearly decreases from 98% to 20%; 15-20 min, maintain phase B at 20% for column equilibration.

[0074] The absorption peak area at 440 nm was recorded using a UV detector. Standard curves were plotted with standard concentration (μg / mL) on the x-axis and peak area on the y-axis, and linear regression equations were fitted to obtain standard curves for crocinic acid, crocin dialdehyde, crocin I, crocin II, crocin III, crocin IV, and crocin V, respectively. (See attached diagram.) Figure 14 .

[0075] (3) Extraction of crocin Collect algal cells by centrifuging 50 mL of algal solution at 5000 rpm for 5 min at room temperature. Add an appropriate amount of steel beads to the algal precipitate and perform freeze grinding at 4 °C (50 Hz, 60 s), repeating three times. Add 1 mL of pre-cooled methanol, vortex, and extract by shaking in the dark for 2 min, then incubate overnight at 4 °C. The next day, centrifuge at 7000 rpm for 5 min, collect the supernatant, and repeat the extraction until the algae turn completely white. Combine all supernatants, vacuum dry, and resuspend in 0.5 mL of methanol. Filter the pigment solution through a 0.22 μm organic phase filter membrane, transfer to a brown centrifuge tube, and store the pigment sample at -20 °C protected from light for later use.

[0076] (4) HPLC quantitative analysis The pigment samples were analyzed by HPLC. Quantitative analysis was performed by comparing the retention times of the sample chromatographic peaks with those of the standard. The content of the target product in the sample (μg / g dry weight) was calculated using the standard curve, as shown in the following formula: Target product content in sample (μg / g dry weight) = (sample concentration × extraction volume) / algal dry weight (5) LC-MS detection The HPLC parameters were set according to the above-mentioned detection method for crocin, with the flow rate adjusted to 0.4 mL / min. Mass spectrometry detection conditions: drying gas flow rate 6.0 L / min, gas temperature 325℃; sheath gas flow rate 12.0 L / min, gas temperature 350℃; capillary voltage set to 4000 V.

[0077] HPLC analysis of the pigment samples showed that, under the above culture conditions, no characteristic peaks corresponding to the standards of crocin and crocin were detected in any of the engineered algal strains, and the content of the relevant products was lower than the lower limit of quantitation of the standard curve, indicating that the target crocin product has not been detected in the engineered algal strains under the current experimental conditions.

[0078] III. Optimizing the culture conditions of Synechocystis PCC 6803 cell factories producing saffron Wild-type Synechocystis PCC 6803 and engineered strains ΔEAB and ΔEABC were cultured in BG11 liquid medium to the logarithmic growth phase. After centrifugation and concentration, the following treatments were applied for different experiments: 1. Response to light quality and intensity induction Adjust the algal solution concentration to OD using BG11 liquid culture medium. 730 =0.85, five light quality and intensity processing groups were set: normal white light (NL), red light (Re), blue light (BL), green light (Gr), and high light (HL). The photon flux density of NL, Re, BL, and Gr was 100 μmol / m². 2 / s, HL photon flux density is 300 μmol / m 2 / s. The induction culture period is 35 days. For the first 15 days, a photocycle of 12h light / 12h dark is used. From the 16th day onwards, the photocycle is adjusted to 14h light / 10h dark.

[0079] During the light quality and intensity induced culture process, the dynamic changes in algal solution color and growth status of wild-type Synechocystis PCC 6803 and engineered strains ΔEAB and ΔEABC are shown in [the table below]. Figure 15 .Depend on Figure 15It was observed that in the initial stage of cultivation, all algal solutions were blue-green, exhibiting a consistent phenotype. With prolonged cultivation, phenotypes gradually differentiated: under green light, all algal strains maintained a stable blue-green color; under blue light, ΔEAB and ΔEABC showed a deeper blue-green hue in the later stages; under red light, wild-type strains were yellowish-red, while engineered strains had a more intense color; under high light, ΔEAB turned yellow, while the wild-type strains and ΔEABC maintained relatively stable colors; under white light, wild-type strains were lighter in color. Wild-type strains showed minimal color change and stable growth under various light qualities, indicating a relatively stable photosynthetic pigment system. ΔEAB exhibited significant growth phenotypes under red, blue, and high light, demonstrating stronger light adaptability. The phenotype and growth advantage of ΔEABC fell between the two. In summary, the ΔEAB engineered algal strain possesses excellent light adaptability and growth potential under optimized light quality and intensity.

[0080] 2. Response to nitrogen deficiency stress The algae were washed with ultrapure water and resuspended in nitrogen-deficient BG11 medium (NaNO3 removed) to adjust the initial OD. 730 =0.2, the induction culture period was 14 days, 12h light / 12h dark, and the photon flux density was 40μmol / m 2 / s, during which the color change of the algal solution was observed and recorded every 48 hours.

[0081] During nitrogen-deficiency stress-induced culture, the color changes and pigment extraction characteristics of the algal solution of wild-type *Synostemma pentaphyllum* PCC 6803 and engineered strains ΔEAB and ΔEABC were as follows: After 10 days of culture, all strains faded from dark green to yellowish-green; after 20 days of continuous culture, they completely faded from green and turned orange-yellow, with the orange-yellow depth of the wild-type strain being significantly higher than that of the engineered strain. After centrifugation and collection of algal cells, the supernatant was clear and transparent. The algal precipitate was yellowish-green, and after methanol extraction, the residue color significantly faded, while the yellow pigment in the extract was effectively enriched. In summary, the engineered strain ΔEAB exhibited excellent pigment stability and stress tolerance under nitrogen-deficiency stress; the phenotype of the engineered strain ΔEABC was weaker than that of ΔEAB. The results indicate that the introduction of exogenous crocin synthesis genes significantly altered the carotenoid metabolic pathway of *Synostemma pentaphyllum*.

[0082] After nitrogen-deficiency induction culture, HPLC was used to qualitatively and quantitatively detect the products ΔEAB and ΔEABC of wild-type and engineered strains of Synechocystis PCC 6803. The results are shown in the figure. Figure 16 .Depend on Figure 16 It can be known that: (1) No characteristic peaks of crocin dialdehyde and crocin were detected in wild strain PCC 6803 of Synechocystis.

[0083] (2) The product of engineered algal strain ΔEAB showed a single characteristic peak at 10.41 min, consistent with the crocin standard, with a content of 13.37 ± 0.67 μg / g dry weight and a yield of 3.97 ± 0.20 μg / L. Mass spectrometry confirmed that the precise molecular weight of the peak component of engineered algal strain ΔEAB with a retention time of 4.77 min matched that of the crocin standard.

[0084] (3) A single characteristic peak was detected in the product of engineered algal strain ΔEABC at 7.9 min, which matched the crocin V standard, with a content of 7.52±0.17 μg / g dry weight and a yield of 1.85±0.05 μg / L. Mass spectrometry confirmed that the precise molecular weight of the peak component of engineered algal strain ΔEABC at a retention time of 7.18 min completely corresponded to the crocin V standard.

[0085] 3. Response to initial cell concentration The algal solution concentration was adjusted to OD using BG11 liquid culture medium. 730 =0.15, OD 730 =0.25, OD 730 =0.35, nitrogen deficiency stress induced culture, two light intensity treatment groups were set up: a single nitrogen deficiency stress group (photon flux density 40 μmol / m 2 / s); Nitrogen deficiency-high light dual stress group (photon flux density 150 μmol / m²); 2 / s).

[0086] Under single nitrogen deficiency stress, the wild-type strain of Synechocystis PCC 6803 and the engineered strains ΔEAB and ΔEABC showed differences in initial cell concentration OD. 730 The color changes and pigment extraction characteristics of the algal solution at concentrations of 0.15, 0.25, and 0.35 are as follows: Under single nitrogen deficiency stress, no orange-red pigment precipitation was observed in the wild-type strains, and almost no crocin was accumulated. The pigment precipitation phenotype of the engineered algal strains showed a significant dependence on the initial cell concentration. In the high-concentration group, both ΔEAB and ΔEABC of the engineered algal strains were bright yellow, indicating a high level of pigment accumulation; the overall color of the medium-concentration group became lighter, but the engineered algal strains still maintained a relatively deep bright yellow; in the low-concentration group, the pigment precipitation of all engineered algal strains was light yellow or light brown, and product synthesis was limited. As the culture time progressed, the color of each algal strain gradually transitioned from the initial green to yellow, and the rate and degree of color change showed a significant concentration dependence. The higher the initial cell concentration, the more prominent the yellowing trend.

[0087] Under nitrogen deficiency and high light dual stress, the wild-type strain of Synechocystis PCC 6803 and the engineered strains ΔEAB and ΔEABC showed differences in initial cell concentration OD. 730The color changes and pigment extraction characteristics of the algal solution at concentrations of 0.15, 0.25, and 0.35 are as follows: the color intensity and uniformity of pigment precipitation are better than under single stress, and the differences between strains are more pronounced. In the high concentration group, the engineered strain ΔEAB exhibits a bright orange-yellow color and performs best, while ΔEABC also shows an orange-yellow color. In the medium concentration group, ΔEAB and ΔEABC maintain a bright yellow color, with the yellow characteristic being stronger than under single stress. In the low concentration group, the overall color becomes lighter, but ΔEAB and ΔEABC still retain a slight orange-yellow hue.

[0088] In summary, under the dual stress conditions of nitrogen deficiency and high light intensity with high initial cell concentration, the engineered algal strains showed significantly better pigment accumulation capacity and stress adaptability than the wild-type strains. Among them, ΔEAB performed the best, followed by ΔEABC.

[0089] HPLC was used to qualitatively and quantitatively detect the products ΔEAB and ΔEABC of wild-type and engineered algal strains cultured under nitrogen-deficiency and high-light dual stress at high initial cell concentrations. The results are shown in the figure. Figure 17 .Depend on Figure 17 It was found that at a detection wavelength of 440 nm, no significant characteristic peaks were detected in the wild-type strain within the characteristic retention time range (10-14 min) corresponding to the crocin standard. Both the wild-type and engineered algal strains showed a significant increase in pigment yield, with the engineered algal strain showing better results. Slight peak fluctuations were detected in ΔEAB and ΔEABC around 10.4 min, indicating that ΔEAB and ΔEABC effectively synthesized and accumulated crocin. A characteristic peak was detected in ΔEABC around 7.9 min, but the peak area was generally low.

[0090] The above results indicate that a high initial cell concentration can rapidly increase biomass. Coupled with nitrogen deficiency and high light stress, this can shorten the culture period, reduce the damage to cells caused by long-term stress, and enhance precursor supply and pathway metabolic flux, thereby achieving a balance between growth and synthesis.

[0091] In summary, the optimal engineered algal strain for producing crocin is ΔEAB, and the optimal engineered algal strain for producing crocin V is ΔEABC. The optimal induction conditions for both are: high initial cell concentration combined with nitrogen deficiency and high light stress.

[0092] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A method for the total synthesis of crocin in microalgae, characterized in that, Includes the following steps: (1) Based on the carotenoid cleavage dioxygenase gene from gardenia GjCCD4a Crocin dialdehyde dehydrogenase gene GjALDH2C3 and UDP-glucuronyltransferase gene GjUGT74F8 The sequence is directly synthesized. GjCCD4a , GjALDH2C3 as well as GjALDH2C3-GjUGT74F8 Fragment; (2) Using Synechocystis PCC 6803 as the starting strain, construct GjCCD4a and GjALDH2C3 The dual-gene co-expression engineered strain ΔAB; (3) Using Synechocystis PCC 6803 as the starting strain, construct GjCCD4a, GjALDH2C3 and GjUGT74F8 The three-gene co-expression engineered strain ΔABC; (4) Introduce the β-carotene hydroxylase gene into engineered plants ΔAB and ΔABC. CrtrB The engineered algal strains ΔEAB and ΔEABC were obtained. (5) The engineered algal strains ΔEAB and ΔEABC were cultured under a high initial cell concentration coupled with nitrogen deficiency and high light dual stress conditions.

2. The method for total synthesis of crocin in microalgae according to claim 1, characterized in that, In step (2), construct GjCCD4a and GjALDH2C3 The specific method for the dual-gene co-expression engineered strain ΔAB is as follows: (i) Using a homologous recombination kit, linearized vector A was combined with the target fragment containing homologous arms. GjCCD4a and GjALDH2C3 Ligation was performed to construct the pΔAB recombinant plasmid; (ii) pΔAB recombinant plasmid was transformed into Synechocystis PCC 6803 via natural transformation to obtain GjCCD4a and GjALDH2C3 The dual-gene co-expression engineered strain ΔAB.

3. The method for total synthesis of crocin in microalgae according to claim 1, characterized in that, In step (3), construct GjCCD4a, GjALDH2C3 and GjUGT74F8 The specific method for co-expressing the three genes in the engineered strain ΔABC is as follows: (i) Using a homologous recombination kit, linearized vector A was combined with the target fragment containing homologous arms. GjCCD4a and GjALDH2C3- GjUGT74F8 Ligation was performed to construct the pΔABC recombinant plasmid; (ii) pΔABC recombinant plasmid was transformed into Synechocystis PCC 6803 via natural transformation to obtain GjCCD4a、 GjALDH2C3 and GjUGT74F8 The three-gene co-expression engineered strain ΔABC.

4. The method for total synthesis of crocin in microalgae according to claim 2 or 3, characterized in that, In step (i), the linearized vector A is pCrtO-Kan, and the specific construction method is as follows: (a) Cloning of the β-carotene oxygenase gene from the genome of Synechocystis PCC 6803 by PCR crtO Upstream and downstream homologous flanking sequences crtO-U and crtO-D ; (b) From those carrying the kanamycin resistance gene Kan Cloning the kanamycin resistance gene in a plasmid Kan ; (c) By fusion PCR crtO-U , crtO-D and Kan Fusion, Acquisition crtO-U / Kan / crtO-D Fragment; (d) Cloning via TA crtO-U / Kan / crtO-D The fragment was assembled into the linearized pMD18T vector to obtain the linearized vector pCrtO-Kan.

5. The method for total synthesis of crocin in microalgae according to claim 1, characterized in that, In step (4), the following steps are introduced into the engineered plants ΔAB and ΔABC: CrtrB The specific gene-based approach is as follows: (i) Based on the β-carotene hydroxylase gene CrtR-B and streptomycin sulfate resistance gene SmR direct synthesis of sequences CrtrB-SmR Fragment fusion; (ii) Using a homologous recombination kit, linearized vector B was combined with... CrtrB-SmR Fragment fusion was performed to construct the pΔE recombinant plasmid; (iii) The engineered algal strains ΔEAB and ΔEABC were obtained by transforming the pΔE recombinant plasmid in engineered strains ΔAB and ΔABC through natural transformation.

6. The method for total synthesis of crocin in microalgae according to claim 5, characterized in that, In step (ii), the linearized vector B is pCrtO, and the specific construction method is as follows: (a) Cloning of the β-carotene oxygenase gene from the genome of Synechocystis PCC 6803 by PCR crtO Upstream and downstream homologous flanking sequences crtO-U and crtO-D ; (b) From those carrying the kanamycin resistance gene Kan Cloning the kanamycin resistance gene in a plasmid Kan ; (c) By fusion PCR crtO-U , crtO-D and Kan Fusion, Acquisition crtO-U / Kan / crtO-D Fragment; (d) Cloning via TA crtO-U / Kan / crtO-D The fragment was assembled into the linearized pMD18T vector to obtain the linearized vector pCrtO-Kan; (e) Using the linearized vector pCrtO-Kan as a template and the nucleotide sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15 as primers, the linearized vector pCrtO was obtained by PCR amplification.

7. The method for total synthesis of crocin in microalgae according to claim 1, characterized in that, In step (5), the high initial cell concentration refers to OD 730 ≥0.35, where nitrogen deficiency refers to cultivation in BG11 medium with NaNO3 removed, and high light refers to a photon flux density ≥300 μmol / m 2 / s.

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