Efficient lycopene synthesis strain, construction method thereof and lycopene production method

Abstract

The invention provides an efficient lycopene synthesis strain, a construction method thereof and a lycopene production method, and belongs to the technical field of metabolic engineering. According to the invention, a lycopene high-yield strain is constructed, and intracellular reducing force flow and carbon flow are used for synthesis of lycopene as much as possible by knocking out a reducing force competitive pathway, knocking out a lycopene center metabolic pathway of rhodopseudomonas palustris and strengthening a lycopene biosynthesis pathway. 119.6 mg / L of lycopene is obtained by utilizing the constructed engineering bacteria in 72-hour shake flask fermentation, and the lycopene is 4784 times higher than that of wild type TIE-1. The high-efficiency lycopene synthesis strain disclosed by the invention is suitable for producing lycopene through shake flask fermentation, and the yield of lycopene can be tested through an HPLC (High Performance Liquid Chromatography) method.

Description

Technical Field

[0001] The invention belongs to the technical field of metabolic engineering, and specifically relates to a lycopene efficient synthesis strain and a construction method thereof, and a lycopene production method. Background Art

[0002] Lycopene (C 40 H 56 ) is a tetraterpenoid carotenoid that has attracted much attention for its value in the fields of anti-oxidation, anti-aging and anti-tumor, and has been widely used in industries such as cancer prevention and treatment, cosmetics and food. The market demand for lycopene is increasing, but the chemical synthesis of lycopene has low energy efficiency, harsh reaction conditions and poor selectivity. Therefore, it is very important to build an efficient microbial cell factory to achieve efficient synthesis of lycopene.

[0003] Microbial synthesis of lycopene mainly uses glucose and glycerol as substrates, and its main synthesis pathways are the terpene biosynthesis pathway and the carotenoid biosynthesis pathway. Most non-photosynthetic eukaryotic organisms synthesize isoprene pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), the main intermediates of terpene synthesis, through the mevalonate (MVA) pathway, while plant chloroplasts and some autotrophic prokaryotic microorganisms synthesize IPP and DMAPP through the methylerythrose phosphate (MEP) pathway. IPP and DMAPP combine to produce vanillyl diphosphate (GPP), which is the main precursor for terpene synthesis. Finally, GPP is synthesized into lycopene through the carotenoid biosynthesis pathway.

[0004] Existing technologies mainly open up the lycopene biosynthesis pathway by modifying common synthetic biology chassis such as Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica. There are also studies on constructing lycopene high-yield strains based on certain bacteria that can self-synthesize carotenoids. Lycopene is an important intermediate substance in the carotenoid biosynthesis pathway and a key precursor for the biosynthesis of rhodopsin, β-carotene and other carotenoids. Therefore, blocking the downstream metabolic pathway of lycopene is crucial for the construction of lycopene high-yield strains. It is calculated that for every 1 mole of lycopene synthesized, the MEP pathway and the Carotenoid synthesis pathway consume a net of 4 moles of NADPH, so balancing the intracellular reducing power is of great significance to the biosynthesis of lycopene. The above two points, together with the accumulation of harmful intermediates in the reaction pathway, constitute the main challenges in constructing efficient lycopene synthesis strains. Summary of the invention

[0005] The purpose of the present invention is to construct a lycopene high-yield strain and apply it to shake flask fermentation to synthesize lycopene. The metabolic engineering of the strain is based on Rhodopseudomonas palustris, specifically Rhodopseudomonas palustris TIE-1 (hereinafter referred to as TIE-1). By knocking out the reducing power competition pathway, knocking out the central metabolic pathway of lycopene in Rhodopseudomonas palustris, and strengthening the lycopene biosynthetic pathway, the intracellular reducing power flow and carbon flow are used as much as possible for the synthesis of lycopene. The engineered bacteria obtained by the construction obtained 119.6 mg / L lycopene in 72h shake flask fermentation, which is 4784 times higher than the wild-type TIE-1.

[0006] The knockout of the two genes nifH (SEQ ID No. 21) and crtC (SEQ ID No. 22) described in the present invention effectively reduced the metabolic burden of TIE-1, significantly accelerated cell growth under the same conditions, and greatly improved the efficiency and yield of TIE-1 in synthesizing lycopene. Overexpression of the two endogenous genes crtE (SEQ ID No. 23) and crtB (SEQ ID No. 24) had no significant effect on the cell growth rate, and further increased the yield of lycopene.

[0007] Specifically, the construction of the lycopene efficient synthesis strain of the present invention ( Figure 4 ):

[0008] (1) By knocking out the nifH gene on the TIE-1 genome, the nitrogen fixation pathway is blocked and the competition for reducing power consumption is reduced.

[0009] (2) By knocking out the crtC gene on the TIE-1 genome, the central metabolism of lycopene was blocked and the accumulation of lycopene was increased.

[0010] (3) The efficiency of the carotenoid biosynthesis pathway was improved by overexpressing the endogenous crtE and crtB genes of TIE-1.

[0011] The lycopene efficient synthesis strain of the present invention is suitable for producing lycopene by shake flask fermentation, and the lycopene yield can be tested by HPLC. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the plasmid map of pK18-ΔnifH;

[0013] Figure 2 is the plasmid map of pK18-ΔcrtC;

[0014] Figure 3 is the plasmid map of pYYDT-Ptac-crtEB;

[0015] Figure 4 The present invention provides a construction process for the efficient lycopene synthesis strain;

[0016] Figure 5 is the standard curve of lycopene concentration;

[0017] Figure 6 Lycopene production of different strains. DETAILED DESCRIPTION

[0018] To further illustrate the present invention, the lycopene efficient synthesis strain and its construction method and the lycopene production method provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0019] Example 1 Construction of a highly efficient lycopene synthesis strain:

[0020] The knockout of the related genes of the present invention adopts homologous recombination method, and the vector used is suicide plasmid pK18mobsacB; the overexpression of the related genes adopts pYYDT plasmid.

[0021] 1. Gene Knockout

[0022] 1.1 DNA fragment amplification: Using TIE-1 genome as template, pK18-nifH-up was amplified with primers pK18-nifH-up-F and pK18-nifH-up-R; pK18-nifH-down was amplified with primers pK18-nifH-down-F and pK18-nifH-down-R; pK18-crtC-up was amplified with primers pK18-crtC-up-F and pK18-crtC-up-R; pK18-crtC-down was amplified with primers pK18-crtC-down-F and pK18-cr pK18-crtC-down was amplified using primer tC-down-R; pK18-nifH-up and pK18-nifH-down were used as templates, and pK18-nifH-up-F and pK18-nifH-down-R were used as primers to perform overlapping PCR to obtain pK18-nifH; pK18-crtC-up and pK18-crtC-down were used as templates, and pK18-crtC-up-F and pK18-crtC-down-R were used as primers to perform overlapping PCR to obtain pK18-crtC.

[0023] Phanta enzyme PCR amplification system (50 μL): DNA template 2 μL, upstream primer (10 μmol / L) 2 μL, downstream primer (10 μmol / L) 2 μL, 2×PhantaMix 25 μL and ddH 2 O 19 μL.

[0024] Table 1 Primer sequences used in the present invention

[0025]

[0026] 1.2 Plasmid linearization: EcoRI and BamHI restriction endonucleases were used to digest the vector plasmid pK18mobsacB stored in the laboratory of Tianjin University. The reaction conditions were 37°C, 30 min;

[0027] Enzyme digestion system (50 μL): purified plasmid (5000 ng), endonuclease 1 2.5 μL, endonuclease 2 2.5 μL, 10× Buffer 5 μL, ddH 2 O up to 50μL.

[0028] 1.3 Plasmid recombination: Use the amplified fragments obtained in steps 1.1 and 1.2 and the linearized vector as templates for seamless cloning and recombination reaction to construct pK18-ΔnifH ( Figure 1 ), pK18-ΔcrtC plasmid ( Figure 2 ). Recombination reaction conditions: 50°C, 10 min;

[0029] Recombination system (10 μL): 2×CE II Buffer 5 μL, linearized cloning vector 1 μL, insert fragment cloning vector 2 μL and ddH 2 2 μL of HO.

[0030] 1.4 Plasmid verification

[0031] After the reaction in 1.3, transfer the recombinant system into 100 μL E. coli DH5α competent cells, pipette and mix, place in ice bath for 20 minutes, heat shock at 42℃ for 90 seconds, place in ice bath for 2-3 minutes, add 900 μL SOC, and recover at 34℃ for 1 hour. Centrifuge at 6000×g for 2 minutes, discard most of the supernatant, keep about 200 μL for resuspending the bacteria and spread on LB agar medium containing 100 mg / L kanamycin, and culture at 34℃ overnight. Select positive recombinant single colonies through colony PCR identification. Inoculate PCR-positive colonies into LB medium containing 100 mg / L kanamycin, culture overnight, and preserve the bacteria to extract plasmids.

[0032] Colony PCR system (15 μL): upstream primer (10 μmol / L) 0.5 μL, downstream primer (10 μmol / L)

[0033] 0.5μL, 2x Rapid Taq MasterMix 7.5μL and ddH 2 O 6.5 μL.

[0034] 1.5 Cell transformation

[0035] The wild-type TIE-1 strain (or TIE-1ΔnifH strain) was cultured in MVNG medium at 34°C until OD660 = 0.4-0.5 to prepare electroporation competent cells. 1000ng of the plasmid extracted in 1.4 was pipetted and added to 100μL competent cells, pipetted and mixed, and the mixture was transferred to a 1mm electroporation cup, ice-bathed for 20min, and then electroporated at 2kV. 900μL MVNG medium was added, resuscitated at 34°C for 2h, centrifuged at 6000×g for 2min, most of the supernatant was discarded, and about 200μL was retained for resuspending the bacteria and coating on MVNG agar medium containing 100mg / L kanamycin, and cultured at 34°C for 3 days. Single colonies with positive first homologous recombination of the genome with pK18-ΔnifH or pK18-ΔcrtC were selected by colony PCR identification. The PCR-positive bacteria were inoculated into MVNG medium and cultured overnight to OD660 ~ 1.0, and 200 μL of the culture solution was spread on MVNG medium containing 15% sucrose and cultured at 34 ° C for 5 days. The positive single colony whose genome completed the second homologous recombination was selected by colony PCR identification. The PCR-positive colony was inoculated into MVNG medium and cultured for 24 hours, and then the bacteria were preserved to obtain the TIE-1ΔnifH strain (or TIE-1ΔnifHΔcrtC strain).

[0036] 2. Gene overexpression

[0037] 2.1 DNA fragment amplification: Using TIE-1 genome as template, crtE-F and crtE-R as primers were used to amplify crtE, and crtB-F and crtB-R were used to amplify crtB; using crtE and crtB as templates, crtE-F and crtB-R as primers were used to amplify crtEB. The DNA polymerase used in PCR was Phanta enzyme, and the fragment amplification system was the same as in 1.1.

[0038] 2.2 Linearization of plasmid: XhoI and NdeI restriction endonucleases were used to digest the vector plasmid pYYDT preserved in the laboratory of Tianjin University. The enzyme digestion system was the same as 1.2.

[0039] 2.3 Plasmid recombination: Use the amplified fragments obtained in steps 2.1 and 2.2 and the linearized vector as templates for seamless cloning and recombination reaction to construct the pYYDT-Ptac-crtEB plasmid ( Figure 3 ). The recombination system is the same as 1.3.

[0040] 2.4 Plasmid verification: Same as 1.4.

[0041] 2.5 Cell transformation: TIE-1ΔnifHΔcrtC strain was cultured in MVNG medium at 34°C until OD660=0.4-0.5 to prepare electroporation competent cells. 1000ng of the plasmid extracted in 2.4 was added to 100μL competent cells, pipetted and mixed, and the mixture was transferred to a 1mm electroporation cup, ice bathed for 20min, and then electroporated at 2kV. 900μLMVNG medium was added, resuscitated at 34°C for 2h, centrifuged at 6000×g for 2min, most of the supernatant was discarded, and about 200μL was retained for resuspending the cells and smeared on MVNG agar medium containing 100mg / L kanamycin, and cultured at 34°C for 3 days. Single colonies positive for pYYDT-Ptac-crtEB transformants were selected by colony PCR identification. The PCR-positive colonies were inoculated into MVNG medium containing 100 mg / L kanamycin and cultured for 24 hours to obtain the TIE-1ΔnifHΔcrtC pYYDT-Ptac-crtEB strain.

[0042] Example 2 Method for producing lycopene by shake flask fermentation of engineered strains:

[0043] (1) Slant culture: Take the -80℃ stored strain and inoculate it on agar medium, culture it at 34℃ for 72h, and subculture it once;

[0044] (2) Shake flask seed culture: Use an inoculation loop to scrape a ring of slant seeds and inoculate it into a shake tube containing 5 mL of MVNG medium. Cultivate at 34°C and 220 rpm for 24 h.

[0045] (3) Shake flask fermentation: 1 mL of seed solution was inoculated into a 250 mL Erlenmeyer flask containing fermentation medium (final volume was 100 mL), sealed with sealing film, and cultured at 34°C, 220 rpm, with a fermentation period of 72 h. 12 h after inoculation, 50 μL of 1 M IPTG inducer was added to the culture medium.

[0046] Example 3 Lycopene yield testing method:

[0047] The lycopene yield test method of the present invention is based on high performance liquid chromatography (HP-LC), and the specific method is as follows:

[0048] (1) Lycopene extraction: Take 1 mL of the fermented bacterial solution, centrifuge at 6000×g for 5 min, discard the supernatant, and retain the bacteria. Wash the bacteria twice with 1×PBS. In a dark environment, resuspend the bacteria in the second centrifuge tube with 1 mL of 1% butylated hydroxytoluene (BHT) acetone solution, vortex for 5 min, and then extract lycopene in a metal bath at 56°C for 15 min. During this period, take out the suspension every 5 min and vortex for 1 min.

[0049] (2) HP-LC settings: The lycopene yield test used a Waters C18 column with specifications of 4.6×150 mm; 5 μm. The detector used in the test was a Waters 2489 UV / Vis detector. The mobile phase used in the test was a mixed solution of 50% acetonitrile, 30% methanol, and 20% isopropanol. The injection volume was 20 μL, the elution time was 15 min, and the mobile phase flow rate was 1.0 mL / min. The detector temperature was 30°C, and the detection light wavelength was 472 nm.

[0050] (3) The lycopene yield was calculated using the standard curve method. The concentrations of the standard solutions included 3.125, 6.25, 12.5, 25, 50, and 100 mg / L. Figure 5 The standard curve is shown.

[0051] The lycopene production of the wild-type strain of TIE-1 after 3 days of shake flask fermentation was 0.025 mg / L, and the OD660 was 3.158; the lycopene production of TIE-1ΔnifH in 72h shake flask fermentation increased to 0.043 mg / L, and the OD660 increased to 5.542; the lycopene production of TIE-1ΔnifHΔcrtC in 72h shake flask fermentation increased to 57.98 mg / L, and the OD660 increased to 7.819; the lycopene production of TIE-1ΔnifHΔcrtC pYYDT-Ptac-crtEB in 72h shake flask fermentation increased to 119.6 mg / L, and the OD660 was 7.796, which remained basically unchanged ( Figure 6 ).

[0052] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a lycopene efficient synthesis strain, characterized in that: The method includes using Rhodopseudomonas palustris as a chassis, knocking out the nifH gene and the crtC gene in the genome of the basic strain, and overexpressing the endogenous crtE gene and the crtB gene to construct a strain that efficiently synthesizes lycopene.

2. The construction method according to claim 1, characterized in that: The base strain includes Rhodopseudomonas palustris TIE-1.

3. The construction method according to claim 1, characterized in that: The method comprises knocking out the gene by adopting homologous recombination method, and the vector used comprises suicide plasmid pK18mobsacB.

4. The construction method according to claim 3, characterized in that: When knocking out the gene, the steps include constructing a pK18-ΔnifH plasmid and a pK18-ΔcrtC plasmid and then transforming the basic strain; The primers used in the construction of the pK18-ΔnifH plasmid and the pK18-ΔcrtC plasmid include primers whose nucleotide sequences are shown in SEQ ID No.1 to SEQ ID No.8: pK18-nifH-up-F, pK18-nifH-up-R, pK18-nifH-down-F, pK18-nifH-down-R, pK18-crtC-up-F, pK18-crtC-up-R, pK18-crtC-down-F and pK18-crtC-down-R.

5. The construction method according to claim 4, characterized in that: The method comprises amplifying pK18-nifH-up with primers pK18-nifH-up-F and pK18-nifH-up-R; amplifying pK18-nifH-down with primers pK18-nifH-down-F and pK18-nifH-down-R; amplifying pK18-crtC-up with primers pK18-crtC-up-F and pK18-crtC-up-R; amplifying pK18-crtC-down with primers pK18-crtC-down-F and pK18-crtC-down-R. pK18-crtC-down was amplified from the product; pK18-nifH-up and pK18-nifH-down were used as templates, and pK18-nifH-up-F and pK18-nifH-down-R were used as primers to perform overlapping PCR to obtain pK18-nifH; pK18-crtC-up and pK18-crtC-down were used as templates, and pK18-crtC-up-F and pK18-crtC-down-R were used as primers to perform overlapping PCR to obtain pK18-crtC; The suicide plasmid pK18mobsacB was double-digested with restriction endonucleases EcoRI and BamHI to obtain a linearized vector, and pK18-nifH and pK18-crtC were seamlessly cloned and recombined with the linearized vector to construct pK18-ΔnifH plasmid and pK18-ΔcrtC plasmid.

6. The construction method according to claim 1, characterized in that: The primers used for overexpression include primers with nucleotide sequences as shown in SEQ ID No.9 to SEQ ID No.12: crtE-F, crtE-R, crtE and crtB.

7. The construction method according to claim 6, characterized in that: The basic plasmid used for the overexpression includes pYYDT.

8. The construction method according to claim 6 or 7, characterized in that: The method includes using the genome of the basic strain as a template, using crtE-F and crtE-R as primers to amplify crtE, and using crtB-F and crtB-R as primers to amplify crtB; using crtE and crtB as templates, and using crtE-F and crtB-R as primers to amplify crtEB; The plasmid pYYDT was double-digested to obtain a linearized vector, and crtEB and the linearized vector were used as templates for seamless cloning and recombination reaction to construct the pYYDT-Ptac-crtEB plasmid.

9. A lycopene-efficient synthetic strain constructed by the construction method according to any one of claims 1 to 8.

10. Use of the lycopene efficient synthesis strain according to claim 9 in the production of lycopene, characterized in that: The method of production includes shake flask fermentation.

Images