Method for synthesizing lycopene based on multi-gene tandem isopentenol utilization way

Abstract

The invention discloses a multi-gene tandem recombinant strain as well as a construction method and application thereof. The invention also discloses a method for synthesizing lycopene based on an isopentenol utilization approach. The strain comprises a recombinant strain of seven key genes, namely mtipk, thim, ispA, idi, crtE, crttheB and crtI, for synthesizing lycopene in an IUP (Inhibitory Urinary Protein) way. According to the invention, the isopentenol is introduced into the escherichia coli in a way to synthesize the lycopene; according to the approach, only two-step phosphorylation reaction is used for replacing the original 2-C-methyl-D-erythritol-4-phosphoric acid approach and mevalonic acid approach which need more than seven steps to synthesize precusor substances pentadienyl pyrophosphoric acid and dimethyl allyl pyrophosphate ester of lycopene, so that the metabolic pathway is greatly shortened, and competition with central carbon metabolism in cells is avoided; the toxicity caused by accumulation of intermediate products in the metabolic process is reduced, and lycopene can be efficiently synthesized by using a cheap substrate.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for synthesizing lycopene based on a multi-gene tandem isopentenol utilization pathway. Background Art

[0002] Terpenoids, also known as isoprenoids, are a class of natural compounds with complex structures and numerous functions. All terpenoids are derived from two C5 compounds - isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Lycopene belongs to tetraterpenoids (C40) and is a natural carotenoid that is widely found in many vegetables and fruits. It has extremely strong antioxidant activity and reactive oxygen scavenging ability, which can prevent the oxidation of proteins, lipids and DNA, and can also protect the cardiovascular system by regulating human cholesterol metabolism.

[0003] The biosynthetic pathway for lycopene primarily synthesizes the precursors IPP and DMAPP via the 2-methyl-D-erythritol-4-phosphate (MEP) pathway or the mevalonate (MVA) pathway, which are then catalyzed by a series of enzymes. Because the synthesis of natural isoprenoids begins with metabolites from central carbon metabolism, it competes with essential metabolic pathways for substrates, energy, and reducing equivalents, and these pathways are also limited by other intrinsic metabolic regulations. Currently, among the various challenges in isoprenoid biosynthesis, producing sufficient amounts of the substrates IPP and DMAPP is a major bottleneck. Both the MEP and MVA pathways are complex cascade reactions involving multiple enzymes. Starting from the central carbon metabolism pathway of glucose, the synthesis of the substrates IPP and DMAPP requires more than 17 enzymatic steps and various cofactors. Furthermore, the coenzymes inhibit each other, and these cofactors and some intermediate metabolites inhibit the enzymes involved in the pathway catalysis and can also be toxic to cells. Due to the accumulation of toxic intermediates and feedback inhibition, these complex enzyme regulations and competition with central carbon metabolism pathways lead to the complexity of the MEP or MVA pathway, resulting in low yields of IPP and DMAPP, which directly affects the yield and efficiency of the synthesis of various downstream isoprenoid compounds. Therefore, to overcome this difficulty, researchers have attempted to introduce a two-step alternative method to enhance the production of IPP and DMAPP. This pathway is the isopentenol utilization pathway (IUP). Through a two-step phosphorylation process involving hydroxyethylthiazolidine kinase (THIM) and isopentenyl phosphate kinase (IPK), the isopentenol isomers isopentenol or isopentenediol are phosphorylated to form isopentenyl monophosphate (IP) or dimethylallyl monophosphate (DMAP), respectively. IP and DMAP are further phosphorylated to generate the isoprenoid precursors IPP and DMAPP, providing sufficient precursors for lycopene synthesis. Fermentation experiments using Escherichia coli have shown that the IUP pathway can significantly increase lycopene production.

[0004] When introducing these enzyme genes and modifying multiple metabolic pathways, the efficiency of lycopene synthesis is highly dependent on the coordinated expression of multiple genes—the order of gene expression, spatial location, and translation efficiency directly influence the distribution and balance of metabolic flux. Imbalances in metabolic flux across pathway steps can lead to the accumulation of toxic intermediate metabolites and the production of byproducts, thereby impacting host cell growth and product yield. Precisely controlling the sequence of multiple genes in vectors or genomes is a critical foundational requirement in fields such as synthetic biology, gene therapy, and metabolic engineering. Genetic sequence regulation is often applied to the synthesis of multigene expression products, profoundly impacting multiple aspects of gene expression regulation. First, the combination of different regulatory elements and multiple genes can affect the transcription and translation of different downstream proteins, leading to differential enzyme concentrations and, consequently, product synthesis. Sequence can also influence promoter strength, ribosome binding site (RBS) strength, intergenic spacer sequences, and the stability of mRNA secondary structures. Second, when constructing multi-gene pathways (such as metabolic pathways), the order of gene arrangement in the operon will directly affect the transcription and translation efficiency of each gene, thereby affecting the stoichiometric ratio of downstream proteins and the efficiency of the entire pathway. Third, insufficient termination signals or promoter leakage of a gene may affect the expression of adjacent genes, resulting in unexpected background expression or interference with fine regulatory circuits. Finally, for proteins that need to form complexes, the physical proximity of their encoding genes may affect the correct assembly and function of the complex through mechanisms such as co-transcription and co-translation. These effects are difficult to predict for the expression of multiple genes and the synthesis efficiency of the final product. Complex combination construction and multi-step verification are required to understand the mechanism of multi-gene synergy in different synthetic products. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a recombinant strain with multiple genes in series and its application.

[0006] The technical problem that the present invention also aims to solve is to provide a method for constructing a recombinant strain with multiple genes in series.

[0007] The technical problem that the present invention also aims to solve is to provide the use of the multi-gene tandem recombinant strain in the synthesis of lycopene in vivo.

[0008] The technical problem that the present invention also aims to solve is to provide a method for synthesizing lycopene based on the isopentenol utilization pathway of multiple genes in series.

[0009] Technical solution: In order to solve the above technical problems, the present invention provides a recombinant strain with multiple genes in series, wherein the recombinant strain with multiple genes in series is obtained by introducing a recombinant plasmid containing the gene fragments mtipk, ispA, thim and idi into a recombinant strain containing the gene fragments crtE, crtB and crtI.

[0010] The recombinant plasmid is obtained by sequentially connecting the gene fragments mtipk, idi, thim and ispA and introducing them into a vector. Preferably, the vector is a pRSFDuet-1 vector.

[0011] The recombinant strain is obtained by sequentially connecting the gene fragments crtI, crtE and crtB, introducing the vector and then introducing the vector into a host bacterium. Preferably, the vector is a pET-22b plasmid, and the host bacterium is Escherichia coli.

[0012] The present invention also includes a method for constructing the multi-gene tandem recombinant strain, comprising the following steps:

[0013] 1) Construction of a recombinant plasmid containing the gene fragments mtipk, ispA, thim, and idi: The gene fragments mtipk, idi, thim, and ispA were sequentially ligated and introduced into a vector;

[0014] 2) Construction of a recombinant strain containing gene fragments crtE, crtB and crtI: The gene fragments crtI, crtE and crtB are sequentially connected and introduced into a vector and then introduced into the host bacteria.

[0015] Preferably, the ipk gene of isopentenyl phosphate kinase from the Methanolobus tindarius strain and the crtB gene of phytoene synthase from the Thermus thermophilus strain were codon-optimized and synthesized by a genetics company. The thim gene of hydroxyethylthiazole kinase was amplified using the Escherichia coli BL21 (DE3) genome as a template, the ispA gene of farnesyl pyrophosphate synthase and the idi gene of isopentenyl pyrophosphate isomerase were amplified using the Escherichia coli MG1655 genome as a template, and the crtE and crtI genes were amplified using the Deinococcus wulumuqiensis R12 genome as a template; the gene fragments mtipk, thim, ispA, idi, crtE, crttheB, and crtI were obtained, respectively.

[0016] After gel recovery of the mtipk (A), ispA (B), thim (C), and idi (D) fragments, overlap PCR was used to ligate the two gene fragments following each promoter in a specific sequence (e.g., AB, BA, AC, etc.). After gel recovery and purification of the PCR products, double digestion with restriction endonucleases was performed. The pRSFDuet-1 vector was double digested to create restriction sites matching the gene fragments in each of the two MCSs, which were then ligated using T4 DNA ligase. An SD1 sequence (5'-TAAGGAGGATATACA-3') was introduced between the two gene fragments following the same promoter to ensure efficient translation of downstream genes. Twelve multigene co-expression plasmids were constructed.

[0017] Among them, after the crtE, crtB and crtI gene fragments were recovered from the gel, the three gene fragments were connected together in a specific order (crtE-crtB-crtI, crtE--crtI-crtB, crtB-crtE-crtI, crtB-crtI-crtE, crtI-crtE-crtB, crtI-crtB-crtE) by overlap PCR. After gel recovery and purification of the PCR products, they were double-digested with restriction endonucleases. The pET-22b expression vector plasmid was double-digested and connected using T4 DNA ligase. An SD+AS sequence (5'-TAAGGAGGATATACA-3') was added between each gene to construct six multi-gene co-expression plasmids. After the six multi-gene co-expression plasmids were transformed into E. coli BL21 (DE3), recombinant strains S1-S6 were obtained.

[0018] Furthermore, the recombinant strains S1-S6 were cultured in LB medium at 37°C and 200 rpm for 16 h, and 1 mL of seed solution was inoculated into fresh 2YT medium (containing ampicillin) and cultured at 37°C and 200 rpm until the absorbance value OD 600 When the lycopene production rate reached 0.6–0.9, the inducer IPTG was added to a final concentration of 1 mM and induced at 30°C for 48 hours. The lycopene production capacity of strains S1–S6 was then determined. After obtaining the optimal strain S5, the 12 recombinant plasmids constructed using pRSFDuet-1 were transformed into the optimal competent cell S5 to construct fermentation strains W1–W12.

[0019] Furthermore, W1-W12 was fermented in 2YT medium with a 2% inoculation rate at 37°C and 200 rpm for 2 h. 25 mM substrate (isoprene diol:isoprene alcohol = 1:1), 0.1 mM IPTG, and 10% n-dodecane were then added, and the mixture was transferred to 30°C in the dark for preliminary fermentation experiments. Under the same conditions, strain S1 was used as a control to ferment and produce lycopene.

[0020] Furthermore, the strains for synthesizing lycopene based on Escherichia coli are S1, S2, S3, S4, S5, S6; W1, W2, W3, W4, W5, W6, W7, W8, W9, W10, W11, and W12; the substrates are isopentanediol and isopentenol at a concentration of 0-50 mM; the substrate ratio is 0:1, 1:1, 1:2, 1:3, 1:4, 4:1, 3:1, 2:1, and 1:0.

[0021] The present invention also includes the use of the multi-gene tandem recombinant strain in synthesizing lycopene.

[0022] The present invention also includes a method for synthesizing lycopene based on the isopentenol utilization pathway of the recombinant bacteria, comprising the following steps: adding the multi-gene tandem recombinant strain to a substrate to induce fermentation and produce lycopene.

[0023] The substrate comprises isopentanediol and isopentenol, wherein the substrate concentration is 2.5-50 mM, and wherein the molar concentration ratio of isopentanediol to isopentenol is 1:1 to 4:1.

[0024] Wherein, the concentration of the inducer IPTG is 0-2mM; preferably, the fermentation step also includes adding a carbon source or a nitrogen source, preferably, the carbon source includes glucose, sucrose, lactose, galactose, glycerol, sodium pyruvate or maltose; preferably, the carbon source concentration is 0-30g / L; preferably, the nitrogen source includes corn steep liquor, beef extract, urea, ammonium oxalate or ammonium sulfate; preferably, the nitrogen source concentration is 0-2g / L; wherein, the fermentation is a two-stage fermentation method, the time of the first stage is 0-16h; the fermentation temperature is 15-37°C; and the fermentation time is 6-72h.

[0025] Preferably, the fermentation strain is W10; the substrate concentration is 7.5 mM; the ratio of isopentanediol to isopentenol is 1:4; the IPTG concentration is 0.01 mM; the carbon source is sodium pyruvate at a concentration of 20 g / L; the nitrogen source is not added additionally; the cell growth time is 4 h; the fermentation temperature is 25° C.; and the fermentation time is 36 h.

[0026] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: The present invention constructs a recombinant strain containing the seven key genes for lycopene synthesis in the IUP pathway: mtipk, thim, ispA, idi, crtE, crttheB, and crtI. Fermentation optimization is carried out using Escherichia coli as the base strain, and the isopentenol utilization pathway is introduced into E. coli for lycopene synthesis. This pathway replaces the original 2-C-methyl-D-erythritol-4-phosphate pathway and mevalonate pathway, which require more than seven steps to synthesize the lycopene precursors pentadienyl pyrophosphate and dimethylallyl pyrophosphate, with only two phosphorylation reactions. This significantly shortens the metabolic pathway, avoids competition with intracellular central carbon metabolism, reduces the toxicity caused by the accumulation of intermediates during the metabolic process, and enables efficient, safe, and high-throughput synthesis of lycopene using inexpensive substrates. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a diagram of the construction and fermentation of the recombinant plasmids of the key lycopene genes in Examples 1 and 2;

[0028] Figure 2 The diagram of the construction and fermentation of the lycopene fermentation strain in Example 3;

[0029] Figure 3 Graph showing the effect of substrate concentration on lycopene synthesis in Example 4;

[0030] Figure 4 This is a graph showing the effect of substrate ratio on lycopene synthesis in Example 4;

[0031] Figure 5 This is a graph showing the effect of IPTG concentration on lycopene synthesis in Example 4;

[0032] Figure 6 This is a graph showing the effect of carbon source type on lycopene synthesis in Example 4;

[0033] Figure 7 This is a graph showing the effect of carbon source concentration on lycopene synthesis in Example 4;

[0034] Figure 8 This is a graph showing the effect of nitrogen source type on lycopene synthesis in Example 4;

[0035] Figure 9 This is a graph showing the effect of cell growth time on lycopene synthesis in Example 4;

[0036] Figure 10 This is a graph showing the effect of fermentation temperature on lycopene synthesis in Example 4. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0038] Example 1 Construction of recombinant plasmids for key enzymes in lycopene synthesis via the IUP pathway

[0039] 1. Synthesis and cloning of seven key enzyme genes in the IUP pathway:

[0040] Isopentenyl phosphate kinase (IPK): The ipk gene (mtipk, GenBank accession number WP_023846034.1) from the Methanolobus tindarius strain was codon-optimized according to Escherichia coli codon preference (the optimized nucleotide sequence is shown in SEQ ID NO. 41). The gene fragment mtipk was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The synthesized mtipk DNA fragment was used as a template for PCR amplification using the following primers.

[0041] The GenBank accession number of the Thim gene of hydroxyethylthiazole kinase (THIM) is HEI1802564.1. The gene fragment thim was amplified by PCR using Escherichia coli BL21 (DE3) as a template and the following primers.

[0042] The GenBank accession number of the ispA gene of farnesyl pyrophosphate synthase (ISPA) is ELM7833996.1. The ispA gene fragment was amplified by PCR using Escherichia coli MG1655 as a template and the following primers.

[0043] The GenBank accession number of the isopentenyl pyrophosphate isomerase (IDI) gene is ELT9670440.1. The gene fragment idi was amplified by PCR using Escherichia coli MG1655 as a template and the following primers.

[0044] The seven key enzyme genes in the IUP pathway of the present invention, the names of the plasmids in which they are located, and the names of the corresponding primers are shown in Table 1.

[0045] Table 1 Primer comparison table of seven key enzyme genes in the IUP pathway

[0046]

[0047]

[0048] The forward primer W1AF has the nucleotide sequence shown in SEQ ID NO. 1, and a SacI restriction site, specifically: 5'-GAGCTCGATGGATAATAATAATATTACCA-3';

[0049] The reverse primer W1A R has the nucleotide sequence shown in SEQ ID NO. 2, specifically: 5′-ATTATTCCTCCTATATGTTTAAATGGTGCCTG-3′;

[0050] The forward primer W1B F has the nucleotide sequence shown in SEQ ID NO. 3, specifically: 5′-GCAGGCACCATTTAATAAGGAGGATATACAATGGACTTTCCGCAGCAA-3′;

[0051] The reverse primer W1B R has the nucleotide sequence shown in SEQ ID NO. 4 and a NotI restriction enzyme cleavage site, specifically: 5′-CGACTTAAGCATTATGCGGCCGCTTATTT-3′;

[0052] The forward primer W1C F has the nucleotide sequence shown in SEQ ID NO. 5, and the NdeI restriction site is specifically: 5'-CGCCATATGATGCAAGTCGACCT-3';

[0053] The reverse primer W1C R has the nucleotide sequence shown in SEQ ID NO. 6, specifically: 5′-ATTCCTCCTATATGTTCATGCCTGCACCT-3′;

[0054] The forward primer W1D F has the nucleotide sequence shown in SEQ ID NO. 7, specifically: 5′-GAGGTGCAGGCATGATAAGGAGGATATACACAAACGGAACACGT-3′;

[0055] The reverse primer W1D R has the nucleotide sequence shown in SEQ ID NO. 8 and an XhoI restriction enzyme cleavage site, specifically 5'-GGTTTCTTTACCAGACTCGAGTTATTTAAGCTGGGTAAA-3';

[0056] The forward primer W2D F has the nucleotide sequence shown in SEQ ID NO. 9, and the NdeI restriction site is specifically: 5'-CATATGCAAACGGAACACGT-3';

[0057] The reverse primer W2D R has the nucleotide sequence shown in SEQ ID NO. 10, specifically: 5′-ATTCCTCCTATATGTTTATTTAAGCTGGGTAAA-3′;

[0058] The forward primer W2C F has the nucleotide sequence shown in SEQ ID NO. 11, specifically: 5′-TAAGGAGGATATACAATGCAAGTCGACCT-3′;

[0059] The reverse primer W2C R has the nucleotide sequence shown in SEQ ID NO. 12 and an XhoI restriction enzyme cleavage site, specifically: 5'-CTCGAGTCATGCCTGCACCT-3';

[0060] The forward primer W3B F has the nucleotide sequence shown in SEQ ID NO. 13, and the SacI restriction site is specifically: 5'-GAGCTCATGGACTTTCCGCAGCAA-3';

[0061] The reverse primer W3B R has the nucleotide sequence shown in SEQ ID NO. 14, specifically: 5′-ATTCCTCCTATATGTTTATTTATTACGCTGGAT-3′;

[0062] The forward primer W3A F has the nucleotide sequence shown in SEQ ID NO. 15, specifically: 5′-TAAGGAGGATATACAGATGGATAATAATAATATTACCA-3′;

[0063] The reverse primer W3AR has the nucleotide sequence shown in SEQ ID NO. 16 and a NotI restriction enzyme cleavage site, specifically: 5′-GCGGCCGCTTAAATGGTGCCTG-3′;

[0064] The reverse primer W5C R has the nucleotide sequence shown in SEQ ID NO. 17 and a NotI restriction enzyme cleavage site, specifically: 5'-CCGCTCGAGTCATGCCTGCACCT-3';

[0065] The forward primer W5B F has the nucleotide sequence shown in SEQ ID NO. 18, and the NdeI restriction site is specifically: 5'-CATATGATGGACTTTCCGCAGCAA-3';

[0066] The reverse primer W6B R has the nucleotide sequence shown in SEQ ID NO. 19 and an XhoI restriction enzyme cleavage site, specifically: 5′-CTCGAGTTATTTATTACGCTGGAT-3′;

[0067] The forward primer W7C F has the nucleotide sequence shown in SEQ ID NO. 20, and the SacI restriction site is specifically: 5'-CATATGATGGACTTTCCGCAGCAA-3';

[0068] The forward primer W9D F has the nucleotide sequence shown in SEQ ID NO. 21, and a NotI restriction enzyme cleavage site, specifically: 5'-GCGGCCGCTTATTTAAGCTGGGTAAA-3';

[0069] The forward primer W11D F has the nucleotide sequence shown in SEQ ID NO. 22, and a SacI restriction enzyme cleavage site, specifically: 5'-GAGCTCGCAAACGGAACACGT-3'.

[0070] Geranylgeranyl pyrophosphate synthase (GGPPS) and phytoene dehydrogenase (PDS): PCR amplification of the crtE and crtI gene fragments was performed using the following primers, using Deinococcus wulumuqiensis R12 strain (derived from Chinese patent ZL201410346854.5, deposit number CGMCC 1.8884), respectively. The GenBank accession numbers for the crtE and crtI genes are KP319019 and KP319021, respectively.

[0071] Phytoene synthase (PSY): The crtB gene (GenBank accession number BAD71897.1) of the phytoene synthase from Thermus thermophilus strain was codon-optimized according to Escherichia coli codon preference (the optimized sequence is shown in SEQ ID NO. 42) and then synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. PCR amplification was performed using the following primers using the synthesized DNA as a template.

[0072] S1E F has the nucleotide sequence shown in SEQ ID NO. 23, and the NdeI restriction site is specifically: 5'-CGCCATATGCGTCCCG-3';

[0073] S1E R has the nucleotide sequence shown in SEQ ID NO. 24, and the EcoRI restriction site is specifically: 5'-CCGATTCCTCCTATATGTGAATTCTCACTTCTCCCG-3';

[0074] S1B F has the nucleotide sequence shown in SEQ ID NO. 25, and the EcoRI restriction site is specifically: 5'-CCGGAATTCTAAGGAGGATATACATCGCATGCCGGCG-3';

[0075] S1B R has the nucleotide sequence shown in SEQ ID NO. 26, and a HindIII restriction enzyme cleavage site, specifically: 5'-CCCATTCCTCCTATATGTAAGCTTCGGGCTGCCTTCCG-3';

[0076] S1I F has the nucleotide sequence shown in SEQ ID NO. 27, and a HindIII restriction enzyme cleavage site, specifically: 5'-CCCAAGCTTTAAGGAGGATATACATATGACATCCCCTCT-3';

[0077] S1I R has the nucleotide sequence shown in SEQ ID NO. 28, and an XhoI restriction enzyme cleavage site, specifically: 5'-CCG ATTCCTCCTATATGTCTCGAGTCAGCGCCGGATGT-3';

[0078] S2I F has the nucleotide sequence shown in SEQ ID NO. 29, and the EcoRI restriction site is specifically: 5'-GAATTCTAAGGAGGATATACATATGACATCCCCTCT-3';

[0079] S2I R has a nucleotide sequence as shown in SEQ ID NO. 30, and a HindIII restriction enzyme cleavage site, specifically: 5'-ATTCCTCCTATATGTAAGCTTTCAGCGCCGGATGT-3';

[0080] S2B F has the nucleotide sequence shown in SEQ ID NO. 31, and a HindIII restriction enzyme cleavage site, specifically: 5'-TAAGGAGGATATACAAAGCTTTCGCATGCCGGCG-3';

[0081] S2B R has the nucleotide sequence shown in SEQ ID NO. 32, and an XhoI restriction enzyme cleavage site, specifically: 5'-CTCGAGTCAGCGCCGGATGT-3';

[0082] S3B F has the nucleotide sequence shown in SEQ ID NO. 33, and the NdeI restriction site is specifically: 5'-CATATGTCGCATGCCGGCG-3';

[0083] S3B R has the nucleotide sequence shown in SEQ ID NO. 34, and the EcoRI restriction site is specifically: 5'-ATTCCTCCTATATGTGAATTCCGGGCTGCCTTCCG-3';

[0084] S3E F has the nucleotide sequence shown in SEQ ID NO. 35, and the EcoRI restriction site is specifically: 5'-GAATTCTAAGGAGGATATACACGTCCCG-3';

[0085] S3E R has the nucleotide sequence shown in SEQ ID NO. 36, and a HindIII restriction enzyme cleavage site, specifically: 5'-ATTCCTCCTATATGTAAGCTTTCACTTCTCCCG-3';

[0086] S4E F has the nucleotide sequence shown in SEQ ID NO. 37, and a HindIII restriction enzyme cleavage site, specifically: 5'-AAGCTTTAAGGAGGATATACACGTCCCG-3';

[0087] S4ER has a nucleotide sequence as shown in SEQ ID NO.38, and an XhoI restriction enzyme cleavage site, specifically: 5'-CTCGAGTCACTTCTCCCG-3'.

[0088] S5I F has the nucleotide sequence shown in SEQ ID NO. 39, and the NdeI restriction site is specifically: 5'-CATATGTATGACATCCCCTCT-3';

[0089] S5I R has a nucleotide sequence as shown in SEQ ID NO. 40, and an EcoRI restriction enzyme cleavage site, specifically: 5'-ATTCCTCCTATATGTGAATTCTCAGCGCCGGATGT-3'.

[0090] 2. Construction of a dual-plasmid system for lycopene synthesis based on the IUP pathway

[0091] like Figure 1As shown, PCR amplification was performed according to the above primer pairs to obtain gene fragments crtE (999 bp), crtB (870 bp) and crtI (1647 bp), respectively. The PCR products were separated by 1% agarose gel electrophoresis and recovered. By overlap PCR, the three gene fragments were respectively divided into crtE-SD+AS-crtB-SD+AS-crtI (S1), crtE-SD+AS-crtI-SD+AS-crtB (S2), crtB--SD+AS-crtE--SD+AS-crtI (S3), and crtB--SD+AS-crtE--SD+AS-crtB (S4). Six gene fragments were ligated in specific sequences: S-crtI (S3), crtB-SD+AS-crtI-SD+AS-crtE (S4), crtI-SD+AS-crtE-SD+AS-crtB (S5), and crtI-SD+AS-crtB-SD+AS-crtE (S6). PCR products were separated and recovered by 1% agarose gel electrophoresis. After size confirmation by spotting, they were double-digested with the pET-22b vector using the corresponding restriction endonucleases. After gel recovery, the vector and the target fragments were ligated using T4 DNA ligase. The ligation solution was transformed into Escherichia coli DH5α competent cells using the heat shock method. Recombinant transformants were screened on LB plates containing ampicillin and selected for colony PCR. Recombinant plasmids were extracted from transformant colonies that showed the target bands and double-digested with restriction endonucleases for verification. The recombinant plasmids were then sent to Jinweizhi Biotechnology Co., Ltd. for sequencing, resulting in six recombinant plasmids.

[0092] like Figure 2As shown, PCR amplification using the above primer pairs yielded bands of gene fragments with different restriction sites: mtipk (777 bp), thim (789 bp), ispA (930 bp), and idi (576 bp). After gel recovery, the two gene fragments following each promoter, to be cloned into the pRSFDuet-1 vector (Novagen 71341), were ligated together in a specific sequence by overlap PCR. The primers between the two gene fragments carried the SD1 sequence, as shown in Table 2. After gel recovery and purification, the PCR products were double-digested with the corresponding restriction endonucleases, SacI-NotI or NdeI-XhoI, and ligated with the pRSFDuet-1 vector digested with the restriction endonucleases SacI-NotI and NdeI-XhoI using T4 DNA ligase. The ligation solution was transformed into competent E. coli DH5α cells using the heat shock method. Recombinant transformants were screened on LB plates containing kanamycin and selected for colony PCR. Recombinant plasmids were extracted from transformant colonies showing the target band and verified by double digestion with restriction endonucleases. The cells were sent to Genewise Biotechnology Co., Ltd. for sequencing, and 12 recombinant plasmids constructed on the pRSFDuet-1 vector were obtained.

[0093] Table 2 Combinations of 12 recombinant plasmids constructed on the pRSFDuet-1 vector

[0094]

[0095] Example 2 Construction of Lycopene Synthetic Chassis Strain

[0096] 1. Construction of S1-S6 chassis strains

[0097] The six recombinant plasmids constructed in Example 1 on the pET22b vector were transformed into E. coli BL21 (DE3) to obtain recombinant strains S1-S6. After culturing in LB medium at 37°C and 200 rpm for 16 h, 1 mL of seed solution was inoculated into fresh 2YT medium (10 g / L yeast powder, 16 g / L tryptone, 5 g / L sodium chloride, autoclaved at 121°C for 20 min) (containing ampicillin) and cultured at 37°C and 200 rpm until the absorbance value OD 600When the pH value reached 0.6–0.9, the inducer IPTG was added to a final concentration of 1 mM, and the mixture was induced at 30°C for 48 hours to determine the ability of strains S1-S6 to produce lycopene. To detect the content of lycopene in the sample, acetone was used to extract the lycopene in the reaction system, and the mixture was centrifuged at 12,000 rpm and 4°C for 20 minutes. The supernatant was taken as the sample. A C18 column (4.6×150 mm; 5 μm; Agilent Technologies) was used, the column temperature was 30°C, and the signal at 474 nm was detected. The sample was eluted with solvent A (90% aqueous acetonitrile) and solution B (methanol-isopropanol (3:2, v / v) at a flow rate of 1.0 mL / min: 0-15 min, 100%-10% solvent A and 0-90% solvent B; 15-30 min, 10% solvent A and 90% solvent B; 30-35 min, 10%-100% solvent A and 90%-0 solvent B. Figure 1 As shown, the S5 strain has the ability to produce lycopene and is far superior to other strains. The lycopene production reaches the maximum value of 20.07 mg / L at 48 h.

[0098] 2. Preparation of S1-S6 competent cells

[0099] LB+A (ampicillin) liquid medium: tryptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, ampicillin 50 μg / mL.

[0100] The correct recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells using the heat shock method; a single colony was picked and cultured in LB+A liquid medium at 37°C, 200 rpm, overnight (12-14 h); 1 mL of the overnight culture was transferred to 100 mL of LB+A liquid medium and cultured at 37°C, 250 rpm, with vigorous shaking, and samples were taken every 0.5 h to measure the OD value. 600 , until OD 600 When the value reaches 0.35-0.4, stop culturing and set aside; pre-cool 0.1M CaCl2 solution on ice; (all the following steps are performed on a clean bench and on ice) draw 1.5mL of cultured bacterial liquid into a 1.5mL centrifuge tube and cool on ice for 10min; centrifuge at 3000g at 4℃ for 5min; discard the supernatant, add 100μL of pre-cooled 0.1M CaCl2 solution, gently blow evenly with a pipette to resuspend the cells, and place on ice for 20min; centrifuge at 3000g at 4℃ for 5min; discard the supernatant, add 100μL of pre-cooled 0.1M CaCl2 solution, gently blow evenly with a pipette to resuspend the cells, and place on ice for 20min; the cell suspension can be used immediately for transformation experiments or added with a cryoprotectant (15%-20% glycerol) and placed in an ultra-low temperature refrigerator for storage (-80℃).

[0101] Example 3 Synthesis of lycopene based on the IUP pathway

[0102] The 12 recombinant plasmids successfully sequenced and constructed on the pRSFDuet-1 vector were transformed into competent cells of the S5 strain using the heat shock method. The cells were evenly plated on LB+A (amphenicol)+K (kanamycin) plates and cultured overnight at 37°C. Colony PCR and sequencing confirmed the availability of recombinant strains W1, W2, W3, W4, W5, W6, W7, W8, W9, W10, W11, and W12 capable of producing lycopene. W1-W12 were fermented in 2YT medium with a 2% inoculum at 37°C and 200 rpm for 2 hours. After addition of 25 mM substrate (1:1 isopentenyldiol:isopentenol), 0.1 mM IPTG, and 10% n-dodecane, the culture was heated to 30°C and fermented in the dark. The products were then detected using the S5 strain as a control under the same conditions. Lycopene content in samples was determined by extracting lycopene with acetone. The reaction mixture was centrifuged at 12,000 rpm and 4°C for 20 min, and the supernatant was used as the sample. A C18 column (4.6×150 mm, 5 μm; Agilent Technologies) was used at 30°C, and the signal was detected at 474 nm. The sample was eluted with solvent A (90% aqueous acetonitrile) and solution B (methanol-isopropanol (3:2, v / v)) at a flow rate of 1.0 mL / min: 0-15 min, 100% to 10% solvent A and 0-90% solvent B; 15-30 min, 10% solvent A and 90% solvent B; 30-35 min, 10% to 100% solvent A and 90% to 0% solvent B.

[0103] like Figure 2 As shown, strain W10 has the ability to produce lycopene and is far superior to strain S5. The lycopene production reaches the maximum value of 50.44 mg / L at 48 h.

[0104] Example 4 Optimization of the Lycopene Synthesis Culture Conditions in Example 3

[0105] Based on the research of Example 3, this example optimizes the substrate concentration, substrate ratio, IPTG concentration, carbon source, nitrogen source, addition time of substrate and inducer, and fermentation temperature and time.

[0106] 1. Effects of substrate concentration and substrate ratio on lycopene production

[0107] Based on the determination of the fermentation base strain W10, we explored the effects of substrate concentration and ratio. Figure 3The results showed that the maximum yield of lycopene reached 72.35 mg / L when the substrate concentration was 7.5 mM (the ratio of isopentanediol to isopentenol was 3:1). As the substrate concentration increased, the yield of lycopene gradually decreased. It is speculated that the reason may be that excessive isopentanol / isopentanediol is toxic to Escherichia coli, thus affecting the biomass. Then, considering the influence between the two substrates, we took the total substrate concentration of 7.5 mM as the basis and explored the molar concentration ratio of isopentanediol and isopentenol as 0:1, 1:1, 2:1, 3:1, 4:1, 1:0, 1:2, 1:3, 1:4, Figure 4 The results showed that when the ratio of isopentanediol to isopentenol was 1:3, the maximum lycopene production was 76.9 mg / L.

[0108] 2. Effect of IPTG concentration on lycopene production

[0109] In order to study whether the addition of inducers would affect the synthesis of lycopene, we designed to add 0mM, 0.005mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM, and 2mM IPTG. The results are as follows Figure 5 It can be seen that when 0.01 mM IPTG was added, the yield was much higher than other concentrations, at 108.4 mg / L.

[0110] 3. Effects of exogenous carbon source addition and carbon source concentration on lycopene production

[0111] We tried to increase the production of lycopene by adding other carbon sources to 2YT medium. Figure 6 As shown in the figure, compared with the control group (2YT medium only, no other carbon sources added), the addition of sodium pyruvate as a carbon source can significantly increase the production of lycopene. Then, in order to determine the optimal concentration of sodium pyruvate, 0g / L, 5g / L, 10g / L, 15g / L, 20g / L, 25g / L, and 30g / L of sodium pyruvate were selected and added to the basal medium 2YT. The results are shown in the figure. Figure 7 When sodium pyruvate concentration was less than 10 g / L, lycopene production gradually increased with increasing concentration. However, as the concentration continued to increase, production gradually decreased. Therefore, 10 g / L was the optimal sodium pyruvate concentration, with lycopene production reaching a maximum of 262.41 g / L after 36 hours.

[0112] 4. Effect of exogenous nitrogen addition on lycopene production

[0113] On the basis of 2YT+10g / L sodium pyruvate, different nitrogen sources (beef extract, corn steep liquor, urea, ammonium oxalate, ammonium sulfate) were added at a concentration of 2g / L to optimize the nitrogen source. Figure 8As shown in the figure, compared with the control group (no other nitrogen sources added), the addition of either organic nitrogen sources or inorganic nitrogen had no effect on the improvement of lycopene production, so no additional nitrogen sources were added in subsequent experiments.

[0114] 5. Effect of cell growth time on lycopene production

[0115] In order to find the balance between cell growth and product accumulation during the fermentation process, we chose a two-stage fermentation method. In the first stage, the microorganisms were fully grown under the optimal temperature conditions, and in the second stage, low temperature was used to induce the expression of the target protein and synthesize the product. Therefore, in order to explore the effect of the first stage culture time (i.e., cell growth time) on lycopene production, we added substrates and inducers at different time points. The results are as follows: Figure 9 As shown in the figure, lycopene production increased with the extension of the first-stage culture time. When the first-stage culture time was 2 hours, the lycopene production reached a maximum of 340.3 mg / L at 36 hours after inoculation. When the first-stage cell growth time was further extended to 4 hours, the lycopene production began to decrease.

[0116] 6. Effect of induction temperature on lycopene production

[0117] Temperature is one of the important factors affecting the fermentation process, so we investigated the effect of different induction temperatures on lycopene synthesis. Figure 10 As shown in the figure, at lower induction temperatures (15°C, 20°C, 25°C, 30°C, and 37°C), lycopene production increased with increasing temperature. However, when the temperature exceeded 30°C, lycopene production decreased with increasing temperature. At an induction temperature of 30°C, the highest lycopene yield of 408.02 mg / L was achieved after 36 hours of fermentation.

[0118] In this example, the lycopene fermentation conditions in Example 4 were optimized. A single colony was selected and inoculated into 2YT liquid culture medium. The culture was carried out at 37°C, 200 rpm for 12 hours to obtain a seed solution. The seed solution was then inoculated into 50 mL of fresh liquid culture medium containing 2YT and 10 g / L sodium pyruvate at a 2% inoculum size. After incubation at 37°C, 200 rpm for 2 hours, 0.01 mM IPTG and a total concentration of 7.5 mM substrate (isopentenyldiol:isopentenol = 1:3) were added to the fermentation broth. The fermentation temperature was then lowered to 30°C and fermentation continued. A final yield of 408.02 ± 6.13 mg / L of lycopene was obtained, representing a 20-fold increase in yield compared to Example 2.

Claims

1. A recombinant strain with multiple genes in series, characterized in that: The multi-gene tandem recombinant strain is a strain containing gene fragments mtipk 、 ispA 、 thim and idi The recombinant plasmid containing the gene fragment crtE 、 crtB and crtI The recombinant strain was obtained.

2. The multi-gene tandem recombinant strain according to claim 1, characterized in that: The recombinant plasmid is a gene fragment mtipk 、 idi、thim and ispA The vector is preferably pRSFDuet-1.

3. The multi-gene tandem recombinant strain according to claim 1, characterized in that: The recombinant strain is the gene fragment crtI、crtE and crtB The vector is connected and introduced into the host bacteria in sequence. Preferably, the vector is a pET-22b plasmid and the host bacteria is Escherichia coli.

4. The method for constructing a recombinant strain of multiple genes in tandem according to any one of claims 1 to 3, characterized in that: The following steps are involved: 1) Constructing a gene fragment mtipk 、 ispA 、 thim and idi Recombinant plasmid: Gene fragment mtipk 、 idi、thim and ispA Sequentially connect and introduce into the vector to obtain; 2) Constructing a gene fragment crtE 、 crtB and crtI Recombinant strains: gene fragments crtI、crtE and crtB Sequentially connect and introduce the vector and then introduce it into the host bacteria.

5. Use of the recombinant strain containing multiple genes in tandem according to any one of claims 1 to 3 in synthesizing lycopene.

6. A method for synthesizing lycopene using the prenol pathway of the recombinant bacteria according to any one of claims 1 to 3, characterized in that: The following steps are involved: The multi-gene tandem recombinant strain is added to a substrate to induce fermentation to produce lycopene.

7. The method according to claim 6, characterized in that The substrates include prenyldiol and prenol.

8. The method according to claim 6, characterized in that The substrate concentration is 2.5-50 mM.

9. The method according to claim 7, characterized in that The molar concentration ratio of isopentanediol to isopentenol is 1:1 to 4:

1.

10. The method according to claim 5, characterized in that The concentration of the inducer IPTG is 0-2 mM; preferably, the fermentation step further includes adding a carbon source or a nitrogen source, preferably, the carbon source includes glucose, sucrose, lactose, galactose, glycerol, sodium pyruvate or maltose; preferably, the carbon source concentration is 0-30 g / L; preferably, the nitrogen source includes corn steep liquor, beef extract, urea, ammonium oxalate or ammonium sulfate; preferably, the nitrogen source concentration is 0-2 g / L, preferably, the fermentation is a two-stage fermentation method, the first stage time is 0-16 hours; the fermentation temperature is 15-37°C; and the fermentation time is 6-72 hours.

Images