Lycopene synthesis gene co-expression strain, construction method and application thereof

Abstract

The embodiment of the present application discloses a lycopene synthetic gene co-expression strain and a construction method and application thereof, and relates to the technical field of biology. pnCrtE , pnCrtB , pnCrtP and pnCrtZ Four genes from Antarctic moss are connected by vectors petDuet-1 and pACYC-Duet1 to construct double plasmids, and finally the double plasmids are simultaneously transformed into an engineering bacterium to obtain co-expression. The product lycopene expressed by the co-expression strain has strong absorption at ultraviolet wavelengths of 440nm-550nm, and has characteristic three-finger peaks (442nm, 467nm, 499nm) of carotenoids compared with the empty plasmid expression strain without the connected target genes. The present application firstly carries out heterologous expression of lycopene genes from moss, and provides theoretical research and reference for development of lycopene from moss.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a lycopene synthesis gene co-expression strain, its construction method, and its application. Background Technology

[0002] Lycopene is a fat-soluble vitamin C. 40 Carotenoids, with the molecular formula C 40 H 56 It possesses a straight-chain structure with 11 conjugated double bonds and 2 non-conjugated double bonds. This unique structure endows it with strong antioxidant capabilities, effectively scavenging singlet oxygen and free radicals. Lycopene is abundant in fruits such as tomatoes, watermelons, and grapefruits. However, due to the presence of multiple unsaturated double bonds in its molecular structure, it is highly sensitive to light, heat, and oxygen, and is easily degraded. Its main applications are in the food industry as a natural pigment and functional food additive; in medicine and health: studies have shown that it possesses various physiological functions, including anti-cancer (e.g., inhibiting the PSA pathway in prostate cancer), anti-inflammatory properties, cardiovascular protection, and neurotoxicity mitigation (e.g., counteracting DEHP-induced neurotoxicity through the gut-brain axis mechanism); in the cosmetics industry: utilizing its antioxidant properties in anti-aging products; and in novel materials: developing active packaging materials with antioxidant and UV-blocking functions, such as composite films for preserving blueberries.

[0003] The biosynthesis of lycopene mainly occurs through the methyl erythritol phosphate (MEP) pathway and the methanogenic acid (MVA) pathway, producing the precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). These five-carbon precursors undergo a series of enzymatic reactions to synthesize lycopene. IPP and DMAPP condense under the catalysis of geraniol geraniol pyrophosphate synthase (GGPPS, encoded by genes such as crtE or gps) to form geraniol geraniol pyrophosphate (GGPP). Two molecules of GGPP condense under the catalysis of phytoene synthase (PSY, encoded by the crtB gene) to form colorless phytoene. Phytoene then undergoes multiple dehydrogenation reactions under the catalysis of phytoene dehydrogenase (PDS, encoded by the crtI gene), introducing multiple double bonds, ultimately producing the red lycopene.

[0004] Currently, most methods for obtaining lycopene involve extraction from natural plants, but the chemical reagents used pose certain pollution and hazards. Biotechnology methods offer advantages such as low cost, high yield, and environmental friendliness. (E. coli) Escherichia coliDue to its clear genetic background, ease of operation, and rapid growth, it has become a classic chassis microorganism for heterologous lycopene synthesis. Currently, research on obtaining lycopene expression gene clusters from prokaryotes and tandemly introducing lycopene genes from different organisms into expression strains to obtain lycopene is relatively extensive. However, expression systems for de novo synthesis of lycopene expression genes from mosses are still in their infancy. Therefore, this study is of great significance for developing the diversity of lycopene sources and biosynthetic pathways. Summary of the Invention

[0005] Therefore, embodiments of the present invention provide a lycopene synthesis gene co-expression strain, its construction method, and its application. The present invention utilizes Antarctic moss... Pohlia nutans M211 transcriptome acquisition pnCrtE , pnCrtB , pnCrtP and pnCrtZ After gene optimization, a dual-plasmid petDuet-1- was constructed. pnCrtE+B+P+Z -pACYC-Duet1 was simultaneously transferred into engineered bacteria at a certain volume ratio for co-expression, which enabled the production of lycopene from mosses.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions: According to a first aspect of the present invention, the present invention provides a strain co-expressing a lycopene synthesis gene, wherein the lycopene synthesis gene... pnCrtE, pnCrtB, pnCrtP and pnCrtZ After codon optimization, the cells were transformed into competent cells; among them, the optimized cells... pnCrtE The nucleotide sequence of the gene is shown in SEQ ID No. 2, and the optimized sequence is as follows. pnCrtB The nucleotide sequence of the gene is shown in SEQ ID No. 4, and the optimized sequence is as follows. pnCrtP The nucleotide sequence of the gene is shown in SEQ ID No. 6, and the optimized sequence is as follows. pnCrtZ The nucleotide sequence of the gene is shown in SEQ ID No. 8.

[0007] Furthermore, the lycopene synthesis gene pnCrtE, pnCrtB, pnCrtP and pnCrtZ It originates from Antarctic moss.

[0008] According to a second aspect of the present invention, the present invention provides a method for constructing a lycopene synthesis gene co-expression strain as described above, the method comprising: (1) The optimized pnCrtE The gene was ligated with the petDuet-1 plasmid by enzyme digestion to obtain petDuet-1- pnCrtE plasmids; (2) The optimized pnCrtBGenes and the petDuet-1- pnCrtE The plasmid was ligated after enzyme digestion to obtain petDuet-1- pnCrtE+B plasmids; (3) pnCrtP The gene and pACYC-Duet1 plasmid were ligated by enzyme digestion to obtain pACYC-Duet1- pnCrtP plasmids; (4) pnCrtZ Genes and pACYC-Duet1- pnCrtP The plasmid was ligated after enzyme digestion to obtain pACYC-Duet1- pnCrtP+Z plasmids; (5) Put petDuet-1- pnCrtE+B plasmids and pACYC-Duet1- pnCrtP+Z Plasmids were co-transformed into competent cells, and positive strains were screened to obtain strain petDuet-1- pnCrtE+B+P+Z -pACYC-Duet1 is a strain that co-expresses the lycopene synthesis gene.

[0009] Furthermore, in step (1), the restriction enzyme site is BamHI / SalI; In step (2), the restriction site is Kpn I / Avr II; In step (3), the restriction site is EcoRI / SalI; In step (4), the restriction site is Kpn I / Avr II.

[0010] Furthermore, in step (4), petDuet-1- pnCrtE+B plasmid and pACYC-Duet1- pnCrtP+Z The volume ratio is 1:1.2.

[0011] According to a third aspect of the present invention, the present invention provides the application of the lycopene synthesis gene co-expression strain described above in lycopene production.

[0012] According to a fourth aspect of the present invention, the present invention provides a method for producing lycopene, the method comprising: Under light-protected conditions, the lycopene synthesis gene co-expression strain described above was inoculated at 1.5% into a solution containing 50 μg / ml Amp. + and 30 μg / ml Cmr +In LB medium, the culture was expanded to the logarithmic growth phase at 37°C and 200 rpm. The resulting bacterial culture was inoculated into the above LB medium at 1-1.5% and cultured for another 6-8 hours at 37°C and 200 rpm. 0.5 mM IPTG was added and the culture was induced overnight at 4°C and 150 rpm. The supernatant was removed by centrifugation, the bacterial cells were freeze-dried, ethyl acetate was added and mixed by pipetting, grinding beads were added and homogenized at low temperature, and the supernatant was collected by low-temperature centrifugation.

[0013] Further, the method includes: during the induction process, adding 100 mg / L of amino acids, wherein the amino acids are glutamic acid and / or proline, to the LB medium in batches, and adding them 4 times.

[0014] The embodiments of the present invention have the following advantages: 1. Gene sequences used in this invention pnCrtE , pnCrtB , pnCrtP and pnCrtZ Derived from Antarctic moss Pohlia nutans M211 The transcriptome was analyzed, and then, based on host preference, artificial codons were used for optimization. pnCrtE and pnCrtB The gene was constructed by homologous recombination with MCS1 and MCS2 of the vector petDuet-1 through BamHI / SalI and KpnI / AvrII restriction sites to form petDuet-1- pnCrtE+B plasmid, optimized pnCrtP and pnCrtZ The gene was constructed by homologous recombination with the MCS1 and MCS2 of pACYC-Duet1 through EcoRI / Sal I and Kpn I / Avr II restriction sites to form pACYC-Duet1- pnCrtP+Z Plasmids, and then both plasmids are co-transformed into E. coli. E. coli BL21 (DE3) in (petDuet-1- pnCrtE+B+P+Z The lycopene synthesis gene from Antarctic moss was co-expressed with pACYC-Duet1, and the resulting dual plasmids could stably coexist within a single expression strain. This strain could simultaneously express the artificially codon-optimized version. pnCrtE , pnCrtB , pnCrtP and pnCrtZ Four types of genes.

[0015] 2. The co-expression strain constructed in this invention not only contains two plasmids, but also verifies for the first time the synthetic pathway of moss carotenoids, which can produce red lycopene with three finger peaks of absorption.

[0016] 3. This invention uses an improved fermentation medium and adds extra amino acids in batches at regular intervals to ensure the concentration of the substrate for product synthesis. Compared with the medium without added amino acids, the expression level of lycopene is increased by about 40%.

[0017] 4. This invention is the first to demonstrate heterologous expression of the lycopene gene from moss, providing theoretical research and reference for the development of lycopene from moss. Attached Figure Description

[0018] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0019] Figure 1 for pnCrtE , pnCrtB , pnCrtP and pnCrtZ Predicted sequence domains of four genes; where A is... pnCrtE B is pnCrtB C is pnCrtP D is pnCrtZ;

[0020] Figure 2 For PetDuet-1- pnCrtE + B A schematic diagram of the gene map constructed from plasmids.

[0021] Figure 3 pACYC-Duet1- pnCrtP + Z A schematic diagram of the gene map constructed from plasmids.

[0022] Figure 4 For PetDuet-1- pnCrtE (A), PetDuet-1- pnCrtB (B), pACYC-Duet1- pnCrtP (C) and pACYC-Duet1- pnCrtZ (D) Results of double enzyme digestion of plasmid, where: the left side is the marker and the right side is the target gene.

[0023] Figure 5 SDS-PAGE analysis of pnCrtE, pnCrtB, pnCrtP, and pnCrtZ proteins, where: 1: Protein Marker; 2: petDuet-1- pnCrtE + B +P + Z -pACYC-Duet1 co-expressed protein; 3:petDuet-1-pACYC-Duet1 empty plasmid protein.

[0024] Figure 6 For petDuet-1- pnCrtE + B + P + Z -pACYC-Duet1 dual-plasmid E. coli fermentation broth color.

[0025] Figure 7 For petDuet-1- pnCrtE + B + P + Z The cell color after introducing the -pACYC-Duet1 double plasmid (A) and the petDuet-1-pACYC-Duet1 empty plasmid (B) into E. coli for expression.

[0026] Figure 8 The UV full-wavelength spectrum of the expression product of dual plasmid E. coli (with the extract of empty plasmid E. coli as the baseline). Detailed Implementation

[0027] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Antarctic moss pnCrtE , pnCrtB , pnCrtP and pnCrtZ Construction of expression plasmids In this embodiment pnCrtE , pnCrtB , pnCrtP and pnCrtZ All genes were derived from the Antarctic moss transcriptome published on NCBI (Pohlia nutans M211, Submission ID: SUB12092531; BioProject ID: PRJNA889080); the gene expression vectors were petDuet-1 (Amp+) and pACYC-Duet1 (Cmr+), respectively. Figure 1 As shown, the amino acid functional domain prediction results indicate pnCrtE , pnCrtB ,pnCrtP and pnCrtZ They respectively possess the functions of geraniol-geraniol pyrophosphate synthase, phytoene synthase, phytoene dehydrogenase, and ζ-carotene desaturase.

[0029] Since the gene originates from bryophytes, this study aims to achieve better expression in Escherichia coli. pnCrtE (GC content: 56.49%; CAL: 0.93%) pnCrtB (GC content: 58.60%; CAL: 0.92%) pnCrtP (GC content: 55.20%; CAL: 0.93) and pnCrtZ (GC content: 48.86%; CAL: 0.52) The genes were codon optimized according to the expression preferences of E. coli. The optimized gene sequences are shown in Table 1. The optimized pnCrtE (GC content: 58.64%; CAL: 0.93), pnCrtB (GC content: 58.60%; CAL: 0.92), pnCrtP (GC content: 55.20%; CAL: 0.93), and pnCrtZ (GC content: 56.79%; CAL: 0.94) have GC contents close to 60% and higher GAL content, which is more suitable for expression. Therefore, the optimized genes are more suitable for expression in E. coli.

[0030] Table 1. Original and optimized sequences of pnCrtE, pnCrtB, pnCrtP, and pnCrtZ SEQ ID No.1 SEQ ID No.2 SEQ ID No.3 SEQ ID No.4 SEQ ID No.5 SEQ ID No.6 SEQ ID No.7 SEQ ID No.8

[0031] Optimized genes pnCrtE The petDuet-1- was constructed by linking the BamHI / SalI restriction site to the MCS1 site of petDuet-1. pnCrtE Plasmid (forward amplification primers: see SEQ ID No. 9, reverse amplification primers: see SEQ ID No. 10); Optimized gene pnCrtB Linked to petDuet-1 via the Kpn I / Avr II restriction site. pnCrtE petDuet-1 was constructed at the MCS2 site in the plasmid. pnCrtE+B Plasmid (forward amplification primers: see SEQ ID No. 11, reverse amplification primers: see SEQ ID No. 12) (constructed plasmid map as shown) Figure 2 As shown); the optimized gene pnCrtP pACYC-Duet1 was constructed by linking the EcoRI / SalI restriction site to the MCS1 site of pACYC-Duet1. pnCrtPPlasmid (forward amplification primers: see SEQ ID No. 13, reverse amplification primers: see SEQ ID No. 14); Optimized gene pnCrtZ Linked to pACYC-Duet1- via the Kpn I / Avr II restriction site. pnCrtP pACYC-Duet1- was constructed at the MCS2 site. pnCrtP+Z Plasmid (forward amplification primers: see SEQ ID No. 15, reverse amplification primers: see SEQ ID No. 16) (constructed plasmid map as shown) Figure 3 (As shown).

[0032] To further confirm the optimized target sequence pnCrtE , pnCrtB , pnCrtP and pnCrtZ To confirm successful ligation into the petDuet-1 and pACYC-Duet1 vectors, the ligation products were double-digested with restriction endonucleases. Specifically, BamH1, Sal1, Kpn1, Avr II, and EcoRI were used, and the reaction was carried out in a 37°C water bath for 2 hours to ensure specific cleavage at the restriction sites. The digested products were then purified and enriched using a QIAquick gel extraction kit. Undigested plasmids, enzymes, and impurities in the reaction buffer were removed by column centrifugation to obtain pure digested fragments. Finally, the results were detected by dextran gel electrophoresis using a 1% agarose gel and TAE buffer at 120V for 30 minutes. Figure 4 As shown, the presence of both the target band and the vector band on gel electrophoresis indicates that the target sequence was successfully ligated.

[0033] Example 2: Construction and expression of a dual-plasmid expression strain Will carry the target gene pnCrtE , pnCrtB , pnCrtP and pnCrtZ The two plasmids (petDuet-1- pnCrtE+B and pACYC-Duet1- pnCrtP+Z Simultaneously transferred to E. coli at a volume ratio of 1:1.2 via heat transfer. E. coli In BL21(DE3) competent cells, the cells were cultured overnight on plates containing both Amp+ and Cmr+ antibiotics to screen for positive strains that had successfully undergone double plasmid transformation (petDuet-1-). pnCrtE+B+P+Z (pACYC-Duet1), and finally, single colonies were selected and cultured at 37℃ and 200 rpm for 4-6 hours. 150 mL of the bacterial culture was then sent to Sangon Biotech (Shanghai) Co., Ltd. for gene sequencing. Based on the sequencing results, the successfully constructed dual-plasmid expression strain was preserved.

[0034] The positive strain petDuet-1- pnCrtE+B+P+Z -pACYC-Duet1 was cultured at a 1.5% inoculation ratio into 600 ml LB medium, with the addition of antibiotics Amp+ (working concentration: 50 μg / ml) and Cmr+ (working concentration: 30 μg / ml). The culture was incubated at 37°C and 200 rpm in a shaker until the logarithmic growth phase, i.e., the OD of the bacterial culture was determined. 600 The value was 0.8. IPTG inducer (working concentration: 0.5 mM) was then added. Fermentation induction continued for 10-16 hours at 16°C and 150 rpm in a shaker, with all operations conducted in the dark. The induced fermentation broth was centrifuged at 4°C to remove the supernatant. The remaining cells were resuspended in 20 mL of sterile PBS and sonicated for 30 minutes (50 Hz) until the suspension was nearly transparent. The precipitate was removed by centrifugation, and the supernatant was retained. The protein concentration of the supernatant was determined using a BSA protein concentration assay kit. The blank plasmid (petDuet-1-pACYC-Duet1) and the plasmid carrying the target gene (petDuet-1-) were then compared. pnCrtE +B+P+Z The protein concentration of pACYC-Duet1 was adjusted to a uniform level (50 mg / mL). Then, 4X loading buffer was added, and the mixture was boiled in water for 10 minutes and cooled before SDS-PAGE electrophoresis. The loading volume was 10 μL. After electrophoresis, Coomassie Brilliant Blue was used for staining for 40 minutes, followed by destaining with destaining solution for 1-2 hours.

[0035] The results are as follows Figure 5 As shown, the molecular weights of pnCrtE, pnCrtB, pnCrtP, and pnCrtZ proteins are 40 kDa, 39.9 kDa, 61.6 kDa, and 40 kDa, respectively. The molecular weights of many proteins show some deviation on SDS-PAGE, especially the peptide chains modified by post-transcriptional translation. In addition, due to the addition of protein tags, promoters, and enzyme sites on the plasmid, the relative molecular weights of the proteins expressed by the above four genes are relatively high. The results indicate that all four proteins can be expressed in a soluble manner.

[0036] Example 3: Fermentation of the strain and extraction of the product The expression strain petDuet-1- pnCrtE+B+P+Z pACYC-Duet1 and the blank control strain (petDuet-1-pACYC-Duet1) were cultured to the logarithmic growth phase. The logarithmic growth phase bacterial culture was then inoculated at a ratio of 1.5% into 1000 ml of a solution containing double antibiotics (Amp). + (50 μg / ml) and Cmr +In LB medium (30 μg / ml), the culture conditions were: 37°C at 200 rpm for 6-8 h; induction conditions: Isopropylβ-D-1-thiogalactopyranoside (IPTG) (freshly prepared, working concentration 0.5 mM) was added for low-temperature induction, followed by overnight induction at 150 rpm at 4°C. During induction, sterile, freshly prepared 100 mg / L glutamate and proline (equal amounts of both amino acids) were added every 4 hours, and the entire process was carried out in the dark. Figure 6 As shown, the final fermentation broth turned red, indicating that the two-plasmid strain (petDuet-1-) was present. pnCrtE+B+P+Z After induced fermentation, the strain -pACYC-Duet1 turned red ( Figure 7 ).

[0037] The supernatant of the fermentation broth was removed by high-speed centrifugation at 4°C. The remaining precipitated bacterial cells were washed with PBS 3-5 times and then centrifuged again at low temperature. The clean bacterial cells were enriched and freeze-dried. About 0.2g of the dried bacterial strain with all moisture removed was weighed, 5ml of ethyl acetate was added and mixed by pipetting. The grinding beads were added and the mixture was homogenized at low temperature for 5-10 minutes. After grinding, the supernatant was collected by low-temperature centrifugation.

[0038] The UV spectrophotometer was adjusted to baseline using the extract of the blank control strain, with the UVB wavelength range set to 200-700 nm. The dual-plasmid expression strain (petDuet-1-) was then measured. pnCrtE+B+P+Z Spectrum of the extract (-pACYC-Duet1). Results are as follows. Figure 8 As shown, the extract of the expression strain carrying the target gene exhibits a three-finger peak characteristic of carotenoids, with absorbances of 442 nm, 467 nm, and 499 nm, indicating that the expression product is lycopene. The expression level of lycopene was measured using a 1 cm optical diameter cuvette, with the absorbance at 467 nm as a reference. The absorbance of the product obtained during induction fermentation using modified LB medium (with added glutamic acid and proline) was 0.4823, compared to 0.2893 for the product obtained from fermentation in ordinary LB medium (without added glutamic acid and proline). The modified LB medium can increase lycopene yield by approximately 40%.

[0039] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for producing lycopene, characterized in that, The method includes: Under light-protected conditions, the lycopene synthesis gene co-expressing strain was inoculated at 1.5% into a solution containing 50 μg / ml Amp. + and 30 μg / ml Cmr + In LB medium, the culture was expanded to the logarithmic growth phase at 37°C and 200 rpm; the obtained bacterial culture was inoculated into the above LB medium at 1-1.5% and cultured for another 6-8 hours at 37°C and 200 rpm; 0.5 mM IPTG was added and induced overnight at 4°C and 150 rpm; the supernatant was removed by centrifugation; the bacterial cells were freeze-dried; ethyl acetate was added and mixed by pipetting; grinding beads were added and homogenized at low temperature; the supernatant was collected by low-temperature centrifugation. The method further includes: during the induction process, adding 50 mg / L glutamic acid and 50 mg / L proline to the LB medium in batches, with the addition occurring 4 times; The method for constructing the lycopene synthesis gene co-expression strain includes the following steps: (1) The optimized pnCrtE The gene was ligated with the petDuet-1 plasmid by enzyme digestion to obtain petDuet-1- pnCrtE plasmids; (2) The optimized pnCrtB Genes and the petDuet-1- pnCrtE The plasmid was ligated after enzyme digestion to obtain petDuet-1- pnCrtE+B plasmids; (3) The optimized pnCrtP The gene and pACYC-Duet1 plasmid were ligated by enzyme digestion to obtain pACYC-Duet1- pnCrtP plasmids; (4) Optimize pnCrtZ Genes and pACYC-Duet1- pnCrtP The plasmid was ligated after enzyme digestion to obtain pACYC-Duet1- pnCrtP+Z plasmids; (5) Put petDuet-1- pnCrtE+B plasmids and pACYC-Duet1- pnCrtP+Z The plasmids were co-transformed into E. coli at a volume ratio of 1:1.

2. E. coli Positive strains were screened from BL21(DE3) competent cells to obtain strain petDuet-1- pnCrtE+B+P+ Z -pACYC-Duet1 is a strain that co-expresses the lycopene synthesis gene; Among them, the optimized pnCrtE The nucleotide sequence of the gene is shown in SEQ ID No. 2, and the optimized sequence is as follows. pnCrtB The nucleotide sequence of the gene is shown in SEQ ID No. 4, and the optimized sequence is as follows. pnCrtP The nucleotide sequence of the gene is shown in SEQ ID No. 6, and the optimized sequence is as follows. pnCrtZ The nucleotide sequence of the gene is shown in SEQ ID No. 8; The lycopene synthesis gene pnCrtE, pnCrtB, pnCrtP and pnCrtZ It originates from Antarctic moss.

2. The method for producing lycopene according to claim 1, characterized in that, In step (1), the restriction enzyme site is BamHI / SalI; In step (2), the restriction site is Kpn I / Avr II; In step (3), the restriction site is EcoRI / SalI; In step (4), the restriction site is Kpn I / Avr II.

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