Bacteria-material heterozygote, construction method thereof and method for efficient carbon sequestration synthesis of lycopene

Abstract

The invention discloses a bacterium-material heterozygote, a construction method thereof and a method for efficient carbon sequestration synthesis of lycopene, and belongs to the technical field of microbial electrosynthesis. According to the invention, the surface of engineering strain photosynthetic bacteria is modified with tannic acid modified ZIF-8 (TA ZIF-8) by using an in-situ growth method, so that the photosynthetic bacteria are promoted to capture CO2 in MES reaction and synthesize lycopene. According to the invention, the cell heterozygote which takes CO2 as a substrate and is used for synthesizing lycopene by providing reducing power through a biological cathode is constructed, so that the efficiency and yield of producing lycopene by MES carbon sequestration are effectively improved; mES reaction and yield test results show that under the optimal performance, 72h MES reaction can generate 2.062 mg / L lycopene, and the yield is increased by 29.2% compared with that of an unmodified engineering strain.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial electrosynthesis, and specifically relates to a bacteria-material hybrid and a construction method thereof and a method for efficiently fixing carbon and synthesizing lycopene, and specifically relates to a method for constructing a bacteria-material hybrid for efficiently fixing carbon and synthesizing lycopene in microbial electrosynthesis. Background Art

[0002] Microbial electrosynthesis (MES) is a green synthesis technology that uses electrodes to provide reducing equivalents and converts simple compounds into high-value-added chemicals through microbial catalysis. 2 Compared with traditional electrocatalytic carbon fixation, capture, storage and conversion have the advantages of mild conditions, high product selectivity, and the ability to synthesize high-carbon compounds, and have good application prospects. 2 Low solubility in the liquid phase results in cellular uptake of CO 2 The low rate is one of the main factors limiting the carbon fixation efficiency of MES.

[0003] Metal organic frameworks (MOFs) have good carbon dioxide adsorption properties and are widely used to improve the CO2 absorption capacity of the microenvironment around cells. 2 Some studies have been conducted to improve the carbon fixation efficiency of MES by using MOFs nanoparticles for the functionalization of bioelectrodes or mixing them into the reactor to modify the electrodes and bacterial surfaces of the MES reactor. However, in these methods, there is less effective contact between MOFs and bacteria, and the adsorption between cells and nanoparticles is weak, which limits the improvement of the carbon fixation efficiency of MES. Summary of the invention

[0004] The bacteria involved in the present invention are a metabolic engineering strain (RP), and the metabolic engineering chassis bacteria is Rhodopseudomonas palustris, specifically Rhodopseudomonas palustris TIE-1. TA@ZIF-8 is synthesized on the surface of RP using an in-situ growth method to achieve cell surface modification and construct a cell-material hybrid; a three-electrode monopolar chamber reactor is used to carry out the MES reaction; the lycopene yield is determined using an ELISA instrument and a standard curve method, and the optimal precursor concentration for constructing a cell-material hybrid for MES carbon fixation and lycopene synthesis is determined. The present invention uses an in-situ growth method to modify tannic acid-modified ZIF-8 (TA@ZIF-8) on the surface of engineered photosynthetic bacteria to promote photosynthetic bacteria to capture CO in the MES reaction. 2 And synthesize lycopene.

[0005] Specifically, the present invention provides the following contents:

[0006] A cell-material hybrid for efficiently fixing carbon and synthesizing lycopene is provided, which uses Rhodopseudomonas palustris as a basic bacterium, and the surface of the basic bacterium is modified with tannic acid-modified ZIF-8 (TA@ZIF-8).

[0007] Preferably, the Rhodopseudomonas palustris includes Rhodopseudomonas palustris TIE-1 strain.

[0008] The present invention also provides a method for preparing the above-mentioned cell-material hybrid, comprising the following steps: (1) culturing Rhodopseudomonas palustris to an OD of 660 When the concentration is between 0.8 and 1.2, collect the bacteria by centrifugation;

[0009] (2) Using (CH 3 COO 2 The bacteria were resuspended in Zn solution, and 2-MIM solution and HTA solution were added. After culturing, the cells were allowed to stand at room temperature for a period of time, and the cell-material hybrids were collected by centrifugation.

[0010] Preferably, the culture medium used in step (1) comprises MVNG medium, and the MVNG medium comprises the following components in the following concentrations: 10 g / L yeast extract, 3 g / L peptone, 2.09 g / L MOPS, 1 g / L KH 2 PO4, 0.5g / LMgSO 4 , 4.6mL / L glycerol.

[0011] Preferably, the culturing in step (1) is accompanied by shaking, the shaking frequency is 220 rpm, and the temperature is 34°C.

[0012] Preferably, in step (2) (CH 3 COO 2 The volume ratio of Zn solution, 2-MIM solution, and HTA solution was 100:100:1;

[0013] And the (CH 3 COO 2 The concentration of the Zn solution was 0.3 mM, the concentration of the 2-MIM solution was 24 mM, and the concentration of the HTA solution was 6 mM.

[0014] Preferably, the culture temperature in step (2) is 34° C. and the culture time is 10 min.

[0015] The present invention also provides a method for synthesizing lycopene by the cell-material hybrid, comprising the following steps: in a MES reactor, using CO 2The carbon source was used, the reaction conditions were 30°C, the constant voltage mode was adopted, the applied voltage was -0.5V, and the reaction time was 72h.

[0016] Preferably, the MES reactor comprises an Ag / AgCl reference electrode, a platinum electrode and an acidified carbon felt electrode, with the platinum electrode serving as the anode and the carbon felt electrode serving as the cathode.

[0017] Preferably, the assembly method of the MES reactor comprises: adding NFM culture medium and trace element mother solution into a sterilized screw bottle, inoculating the cell-material hybrid suspension into the culture medium, and the concentration after inoculation is OD 660 ~1;

[0018] Insert the electrodes, making sure that the platinum mesh and carbon felt are completely submerged in the culture medium; insert the sterilized extended needle and 5mL syringe needle respectively, making sure that the extended needle is below the liquid surface and the 5mL syringe needle does not touch the liquid surface at all, and the ends of both needles are connected to a 0.22μm water-based polyethersulfone sterile filter membrane;

[0019] CO was introduced into the reactor through a sterile extension needle. 2 / N 2 Mixed gas, component is CO 2 : 20%, N 2 : 80%.

[0020] The present invention constructs a CO 2 The cell-material hybrid with TA@ZIF-8 as substrate and reducing power provided by the biocathode for the synthesis of lycopene effectively improved the efficiency and yield of lycopene produced by MES carbon fixation. The in situ growth of TA@ZIF-8 on the RP surface produced a cell-material hybrid with TA@ZIF-8 uniformly distributed, which significantly increased the CO2 around the bacteria. 2 concentration, which increased the lycopene yield of the MES reaction; the results of the MES reaction and its yield test showed that the final concentration of the suspension was 0.15mM Zn 2+ The cell-material hybrid produced by the in situ growth method of modifying RP with 12mM 2-MIM and 0.03mM HTA had the best performance of MES carbon fixation and lycopene synthesis, producing 2.062mg / L lycopene in a 72h MES reaction, an increase of 29.2% compared with the unmodified engineered strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Synthesis of TA@ZIF-8 on RP surface by in-situ growth method Schematic diagram of the principle;

[0022] Figure 2 is a scanning electron microscopy image of unmodified RP;

[0023] Figure 3This is the distribution image of Zn element in unmodified RP measured by energy spectrometer;

[0024] Figure 4 Scanning electron microscopy image of TA@ZIF-8 modified RP;

[0025] Figure 5 The distribution image of Zn element in RP modified with TA@ZIF-8 measured by energy spectrometer;

[0026] Figure 6 X-ray diffraction spectra of TA@ZIF-8, RP, and TA@ZIF-8 modified RP;

[0027] Figure 7 Fourier transform infrared absorption spectra of TA@ZIF-8, RP, and TA@ZIF-8 modified RP;

[0028] Figure 8 BET for RP and TA@ZIF-8 modified RP Adsorption curve, adsorbed gas is CO 2 ;

[0029] Fig. 9 Zn 2+ Concentrator, the effect of modifying RP using different precursor concentrations on yield. DETAILED DESCRIPTION

[0030] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] The culture medium and composition used in the present invention are as follows:

[0032] (1) MVNG medium: 10 g / L yeast extract, 3 g / L Peptone, 2.09 g / L MOPS, 1 g / L KH 2 PO 4 , 0.5 g / L MgSO 4 , 4.6mL / L glycerol, add 50mg / L kanamycin sulfate after sterilization;

[0033] (2)NFM medium: 18 g / L Na 2 HPO4·12H 2 O, 6g / LKH 2 PO 4 ,1g / L(NH 4 ) 2 SO 4 ,0.58g / LMgSO 4 ,0.067g / L CaCl 2, after sterilization, add 50 mg / L kanamycin sulfate;

[0034] (3) Trace element mother solution: 2.5g / L EDTA, 10.95g / L ZnSO 4 7H 2 O,5g / LFeSO 4 7H 2 O,1.54g / LMnSO 4 ·H 2 O,0.392g / L CuSO 4 ·5H 2 O,0.248g / L Co(NO 3 ) 2 6H 2 O,0.177g / LNa 2 B 4 O 7 10H 2 O.

[0035] The solutions involved in the present invention are all sterilized by membrane, including:

[0036] (1) 0.3 mM (CH 3 COO 2 Zn solution;

[0037] (2) 24 mM 2-MIM solution;

[0038] (3) 6 mM HTA solution.

[0039] Example 1

[0040] according to Figure 1 The process shown in the figure shows that the operation of synthesizing TA@ZIF-8 on the cell surface by in situ growth method is as follows:

[0041] (1) Add 5 mL of MVNG medium to the shaking tube, pick a single colony from the agar medium and inoculate it, and culture it at 34°C and 220 rpm for 24 h to obtain the primary bacterial solution;

[0042] (2) Inoculate 2 mL of primary bacterial solution into 200 mL of MVNG medium and culture at 34°C and 220 rpm for 24 h until the OD660 is approximately 1.0;

[0043] (3) Centrifuge the bacterial solution at 6000 × g for 5 min to collect the cells, resuspend the cells in sterile 1× PBS and centrifuge again, repeating once;

[0044] (4) Use 100 mL of 0.3 mM (CH 3 COO 2Resuspend the cells in Zn solution, immediately add 100 mL of 24 mM 2-MIM solution and 1 mL of 6 mM HTA solution to the resuspended suspension, incubate at 34 °C, 220 rpm for 10 min, remove and let stand at room temperature for 30 min;

[0045] (5) Centrifuge the suspension at 6000 × g for 5 min to collect the cell-material hybrids, resuspend the cells in sterile 1× PBS and centrifuge, and repeat once.

[0046] The MES settings and operations used in this embodiment are:

[0047] (1) The MES reactor mentioned in this method is a monopolar chamber three-electrode reactor, which consists of a 100 mL screw-cap bottle with a rubber stopper, an Ag / AgCl reference electrode, a 1 cm×1 cm platinum electrode, and a 2.5 cm×3 cm acidified carbon felt electrode. The platinum electrode is used as the anode and the carbon felt electrode is used as the cathode.

[0048] (2) The carbon source of MES is CO 2 The gas supply method is continuous gas flow. The gas enters the reactor through a 100mm extended needle and flows out of the reactor through a 5mL syringe needle. The gas used is 20% CO 2 +80%N 2 Mixed gas, gas flow rate is 4cm 3 s -1 .

[0049] (3) The MES reactor assembly method is as follows: add 100 mL of NFM culture medium and 100 μL of trace element stock solution to a sterilized screw-cap bottle. Inoculate the cell-material hybrid suspension into the culture medium and make its OD 660 The pH value is controlled at about 1.0. Insert the electrodes and ensure that the platinum mesh and carbon felt are completely submerged in the culture medium. Insert the sterilized extended needle and 5mL syringe needle respectively, ensuring that the extended needle extends below the liquid surface and the 5mL syringe needle does not touch the liquid surface at all. The ends of both needles are connected to a 0.22μm water-based polyethersulfone sterile filter membrane. Strictly seal the reactor.

[0050] (4) The MES reaction conditions mentioned in this method are 30°C, constant voltage mode, an applied voltage of -0.5 V, and a reaction time of 72 h.

[0051] Lycopene yield determination method in this embodiment:

[0052] (1) Take 2 mL of bacterial solution from the shaken MES reactor and divide it into two 1.5 mL centrifuge tubes, 1 mL in each tube. Centrifuge at 6000 × g for 5 min, discard the supernatant and retain the bacterial cells.

[0053] (2) Resuspend the bacteria in a centrifuge tube with 1 mL of 1× PBS and measure the absorbance of the bacterial solution in the reactor.

[0054] (3) 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 minutes, and then extract lycopene in a metal bath at 56°C for 15 minutes. During this period, take out the suspension every 5 minutes and vortex for 1 minute.

[0055] (4) The suspension after the extraction product was centrifuged at 10,000 × g for 5 min, 200 μL of the supernatant was transferred to a quartz ELISA plate, and the absorbance at a wavelength of 472 nm was measured in an ELISA instrument. The lycopene content in the extract was calculated using the standard curve method. The concentrations of the lycopene standard solutions used to draw the standard curve were 2.5, 5, 10, 20, 40, and 80 mg / L.

[0056] Morphological characterization of cells and cell-material hybrids showed that a uniform layer of crystalline material could be synthesized on the bacterial surface using the in situ growth method ( Figure 2-5 ).

[0057] The characterization results of its physical and chemical properties confirmed that the modified crystals were TA@ZIF-8. The above two characterizations confirmed that the in situ growth method can achieve uniform TA@ZIF-8 modification on the cell surface ( Figure 6-7 ).

[0058] Characterization of the gas adsorption properties of cells and cell-material hybrids showed that TA@ZIF-8 adsorbed on the cell surface significantly enhanced the cell's ability to adsorb CO2 ( Figure 8 ).

[0059] The results of MES product concentration tests on cells and cell-material hybrids showed that after optimizing the concentration of Zn2+, 0.15mM Zn 2+ The cell-material hybrid produced by the in situ growth method of RP modified by 12mM 2-MIM and 0.03mM HTA had the best performance of MES carbon fixation and lycopene synthesis, producing 2.062mg / L lycopene in a 72h MES reaction, which was 29.2% higher than that of the unmodified engineered strain ( Fig. 9 ).

[0060] 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 cell-material hybrid for efficient carbon fixation and lycopene synthesis, characterized in that: Rhodopseudomonas palustris is used as a basic bacterium, and the surface of the basic bacterium is modified with tannic acid-modified ZIF-8.

2. The cell-material hybrid according to claim 1, characterized in that: The Rhodopseudomonas palustris includes the Rhodopseudomonas palustris TIE-1 strain.

3. The method for preparing the cell-material hybrid according to claim 1 or 2, characterized in that: The following steps are involved: (1) Cultivate Rhodopseudomonas palustris to OD 660 When the concentration is between 0.8 and 1.2, collect the bacteria by centrifugation; (2) resuspending the bacterial cells using a (CH3COO)2Zn solution, and adding a 2-MIM solution and an HTA solution. After culturing, the cells are allowed to stand at room temperature for a period of time, and the cell-material hybrids are collected by centrifugation.

4. The preparation method according to claim 3, characterized in that: The culture medium used in the culture of step (1) includes MVNG culture medium, and the MVNG culture medium includes components in the following concentrations: 10 g / L yeast extract, 3 g / L peptone, 2.09 g / L MOPS, 1 g / L KH2PO4, 0.5 g / LMgSO4, and 4.6 mL / L glycerol.

5. The preparation method according to claim 3, characterized in that: The culture in step (1) is accompanied by shaking, the shaking frequency is 220 rpm, and the temperature is 34°C.

6. The preparation method according to claim 3, characterized in that: The volume ratio of the (CH3COO)2Zn solution, 2-MIM solution and HTA solution in step (2) is 100:100:1; The concentration of the (CH3COO)2Zn solution is 0.3 mM, the concentration of the 2-MIM solution is 24 mM, and the concentration of the HTA solution is 6 mM.

7. The preparation method according to claim 6, characterized in that: The culture temperature in step (2) is 34° C. and the culture time is 10 min.

8. The method for synthesizing lycopene by using a cell-material hybrid according to claim 1 or 2, characterized in that: The following steps are involved: In the MES reactor, CO2 was used as the carbon source, the reaction conditions were 30°C, the constant voltage mode was adopted, the applied voltage was -0.5V, and the reaction time was 72h.

9. The method according to claim 8, characterized in that: The MES reactor comprises an Ag / AgCl reference electrode, a platinum electrode and an acidified carbon felt electrode, with the platinum electrode serving as an anode and the carbon felt electrode serving as a cathode.

10. The method according to claim 9, characterized in that: The assembly method of the MES reactor comprises: adding NFM culture medium and trace element mother solution into a sterilized screw bottle, inoculating a cell-material hybrid suspension into the culture medium, and the concentration OD after inoculation is 660 0.8~1.2; Insert the electrodes, making sure that the platinum mesh and carbon felt are completely submerged in the culture medium; insert the sterilized extended needle and 5mL syringe needle respectively, making sure that the extended needle is below the liquid surface and the 5mL syringe needle does not touch the liquid surface at all, and the ends of both needles are connected to a 0.22μm water-based polyethersulfone sterile filter membrane; A CO2 / N2 mixed gas with the composition of CO2: 20%, N2: 80% was introduced into the reactor through a sterilized extended needle.

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