Construction and application of shewanella hydrogengens-CdS nanoparticle hybrid system based on hydAson gene

Abstract

The invention discloses construction of a shewanella hydrogengens-CdS nano particle hybrid system based on a hydAson gene and an application of the shewanella hydrogengens-CdS nano particle hybrid system. The preparation method comprises the following steps: connecting a hydAson gene from Shewanella oneidensis coded hydrogenase to a vector pYYDT, carrying out ribosome optimization transformation on the vector pYYDT to obtain a plasmid pYYDT-hydAson, introducing the recombinant plasmid pYYDT-hydAson into wild shewanella oneidensis MR-1 to obtain a recombinant shewanella engineering bacterium SES with high hydrogen production efficiency, and carrying out self-assembly to obtain a CdS / SES high hydrogen production activity hybrid system. The CdS / SES hybrid system constructed by the invention endows shewanella with light capturing ability, CdS nanoparticles provide photoelectrons for engineering bacteria to promote intracellular reducing power and ATP regeneration, and the hybrid system constructed by the invention can enhance the proton reduction rate of the engineering bacteria and improve the hydrogen production efficiency.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological energy, and particularly relates to construction and application of a hydrogen-producing Shewanella-CdS nanoparticle hybrid system based on a hydAson gene. BACKGROUND

[0002] Resources and environment are important foundations for the continuous development of human society. Since the industrial revolution, the rapid growth of social productivity has led to a sharp rise in global energy demand. However, the current global energy supply system is still dominated by non-renewable energy such as coal, oil and natural gas. This unsustainable energy consumption pattern not only leads to an energy crisis, but also causes the continuous deterioration of the ecological environment. This structural contradiction not only threatens the energy security of contemporary society, but also poses a serious challenge to the carrying capacity of the Earth's ecological system.

[0003] In the face of energy and environmental crises that have become global problems, the development and utilization of clean and renewable energy have become the focus of global attention. Solar energy is a clean and inexhaustible energy source. The energy generated by one hour of solar radiation is equivalent to the energy consumed by the global economy in one year. As an ideal renewable energy source, how to achieve efficient conversion of solar energy to chemical energy is a current research hotspot.

[0004] The photocatalytic material-microorganism hybrid system is an advanced technology that combines the functions of photocatalytic materials and microorganisms. Through the synergistic effect of light-driven and biological metabolism, it realizes efficient energy conversion, pollutant degradation or high-value product synthesis.

[0005] Shewanella oneidensis MR-1 strain has become a model microorganism for microbial electrochemistry research due to its stable extracellular electron transfer characteristics and has been widely used in the development and mechanism analysis of bioelectrochemical systems. S. oneidensis MR-1 was first isolated from the anaerobic sludge at the bottom of Lake Oneida in New York, USA. Thanks to its unique bidirectional electron transfer characteristics and the first breakthrough in genome sequencing, the research on bioelectrochemical systems based on this electrochemical model microorganism has shown a significant growth trend in recent years.

[0006] The photocatalytic material-microorganism hybrid system generally has the bottleneck problem of low transmembrane photoelectron utilization efficiency, which seriously restricts the further improvement of hydrogen production performance. This problem has not been systematically solved in previous studies. To solve the problem of low electron utilization efficiency in the hydrogen production process of the photocatalytic material-microorganism hybrid system, the application enhances the expression of [Fe-Fe] hydrogenase of Shewanella oneidensis MR-1 to enhance its ability to produce hydrogen from photoelectrons and improve the hydrogen production efficiency of the hybrid system. SUMMARY

[0007] The present application aims to overcome the deficiencies of the prior art, and provides a hydrogen-producing Shewanella engineering strain and a high-hydrogen-production-efficiency cadmium sulfide-Shewanella engineering strain hybrid system.

[0008] The second object of the present application is to provide a recombinant plasmid capable of improving the hydrogenase activity of the engineering strain to improve the hydrogen production efficiency.

[0009] The third object of the present application is to provide a semiconductor-microorganism hybrid system preparation method by means of the recombinant Shewanella, which has higher hydrogen production performance.

[0010] The technical solutions of the present application are summarized as follows:

[0011] A recombinant plasmid, in which a promoter and a hydA gene encoding hydrogenase derived from Shewanella oneidensis are connected to a basic vector plasmid. son The target gene is connected to the vector pYYDT to obtain a recombinant plasmid pYYDT-hydA son .

[0012] First, the recombinant plasmid is introduced into the Escherichia coli WM3064 strain by conventional chemical transformation method, and then the Shewanella oneidensis MR-1 is used as the final host strain, and the conjugation transfer technology is used to successfully introduce the recombinant plasmid into the host strain, and finally the hydrogen-producing Shewanella engineering strain SES is constructed. Through colony PCR verification and agarose gel electrophoresis analysis, the target band is clearly visible, which confirms that the target gene has been effectively integrated into the host strain genome and realizes stable amplification.

[0013] A semiconductor-microorganism hybrid system preparation method, in which CdS nanoparticles are introduced into the intracellular and extracellular membrane of the hydrogen-producing Shewanella engineering strain.

[0014] After the engineering strain SES is activated and cultured to the stable phase, the bacterial cells are collected by centrifugation at 4℃ and 5000rpm for 5 minutes, and washed with 20mM HEPES solution for three times. The bacterial cells are resuspended in 100mL anaerobic mineral medium containing 1mM Na2S2O3 and 20mM sodium lactate, so that the initial OD 600 is 0.1, and then 1mM CdCl2 is added after the cells are incubated at 30℃ and 200rpm for 24 hours to form CdS nanoparticles. The whole incubation process needs to be carried out under anaerobic conditions, and high-purity nitrogen gas is used to purge for 30min to completely remove oxygen.

[0015] Use of the above hydrogen-producing semiconductor-microorganism hybrid system CdS / SES for photocatalytic hydrogen production.

[0016] The washed CdS / SES hybrid system was transferred to a hydrogen production medium and subjected to photocatalytic hydrogen production under anaerobic conditions. The volume of the hydrogen production medium was set to 30 mL, and the headspace volume of the photocatalytic reactor was 55 mL. Visible light was provided by a 300 W xenon lamp equipped with a 420 nm filter to filter ultraviolet light, and the light intensity was maintained at 100 mW / cm 2 , and kept at a constant temperature and anaerobic conditions. Samples were taken every 12 hours, and the hydrogen production was monitored using a gas chromatograph.

[0017] The advantages of the present application are as follows:

[0018] A recombinant plasmid constructed in the present application is pYYDT-hydA son , which can enable the engineered Shewanella oneidensis SES to express [Fe-Fe] hydrogenase HydA son strongly, enhance the proton reduction capacity of the engineered bacteria, and further enhance the utilization efficiency of intracellular reducing power, thereby improving the hydrogen production efficiency of the semiconductor-microorganism hybrid system.

[0019] The semiconductor-microorganism hybrid system constructed in the present application is CdS / SES. CdS nanoparticles are excited by light to generate photoelectrons, which are taken up by the hydrogenase HydA son located in the periplasm through a direct electron transfer pathway, thereby enhancing the hydrogen production efficiency of the hybrid system.

[0020] The semiconductor / engineered bacteria hybrid system constructed in the present application has the advantages of simple construction method, low cost of raw materials, independent regulation of target genes, and high hydrogen production efficiency. BRIEF DESCRIPTION OF DRAWINGS Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are included to provide a description of the preferred embodiments, and are not intended to limit the scope of the application. Moreover, the same reference numerals are used throughout the same figures. In the drawings:

[0021] Figure 1 SEM image of the CdS / SERP hybrid system

[0022] Figure 2 TEM image of the CdS / SERP hybrid system

[0023] Figure 3 Structure of the recombinant plasmid

[0024] Figure 4The hydrogen production amount of the hybrid system CdS / SERP constructed by the hydrogen-producing Shewanella recombinant strain is compared with that of the hybrid system CdS / MR-1 constructed by Shewanella.

[0025] Figure 5 The intracellular reducing power equivalent NADH / NAD of the hybrid system CdS / SERP constructed by the hydrogen-producing Shewanella recombinant strain is compared with that of the hybrid system CdS / MR-1 constructed by Shewanella. + Comparison chart

[0026] Figure 6 The intracellular ATP content of the hybrid system CdS / SERP constructed by the hydrogen-producing Shewanella recombinant strain is compared with that of the hybrid system CdS / MR-1 constructed by Shewanella. DETAILED DESCRIPTION

[0027] The original strain is wild-type Shewanella oneidensis MR-1, which is referred to as wild-type Shewanella MR-1, and is purchased from the ATCC (USA, https: / / www.atcc.org / ) company in September 2010 as ATCC 700550 strain.

[0028] The application will be further described below in combination with specific examples.

[0029] Example 1: Strain construction of hydrogen-producing Shewanella engineering strain SES

[0030] A method for constructing a hydrogen-producing Shewanella recombinant strain, comprising the following steps: constructing a recombinant plasmid pYYDT-hydA son , and transferring the above plasmid into wild-type Shewanella MR-1 to obtain a hydrogen-producing Shewanella engineering strain SES.

[0031] The specific construction method comprises the following steps:

[0032] Recombinant plasmid pYYDT-hydA son Construction:

[0033] The gene hydA son encoding HydA son hydrogenase derived from Shewanella oneidensis is connected to the vector pYYDT to obtain a recombinant plasmid pYYDT-hydA son .

[0034] The nucleotide sequence of the gene hydA son is shown in SEQ ID NO. 2;

[0035] The nucleotide sequence of the recombinant plasmid pYYDT-hydA son is shown in SEQ ID NO. 1.

[0036] The obtained recombinant plasmid pYYDT-hydA son The plasmid was transformed into E. coli WM3064 competent cells, and the WM3064 competent cells were prepared as follows:

[0037] 1. Take the WM3064 glycerol bacteria, and streak inoculate on the LD (LB medium added with 0.3M / mL 2,6-diaminoheptanedioic acid) solid medium;

[0038] 2. Place the solid medium in a 37°C incubator and culture for 24h;

[0039] 3. Use a loop to pick single colonies on the solid medium, and transfer to a test tube containing LD medium without antibiotics for primary inoculation and culture, and place the test tube in a 37°C, 220rpm shaker for 12h,

[0040] 4. Take 1mL of the bacterial solution from the test tube, and transfer to a flask containing LD medium without antibiotics for secondary culture, and place the flask in a 37°C, 220rpm shaker until the OD 600 value is 0.6

[0041] 5. Ice-bath the culture medium for 20min, centrifuge the bacterial solution at 5000rpm, 4°C for 10min,

[0042] 6. Pour off the supernatant, resuspend the precipitate using the competent preparation reagent, aliquot the resuspended solution, and store in a -80°C refrigerator.

[0043] The specific steps of the WM3064 chemical transformation are as follows:

[0044] 1. Take 50μL of WM3064 competent cells from a -80°C refrigerator and thaw on ice;

[0045] 2. Mix the pYYDT-hydA son plasmid with the WM3064 competent cells in a 1.5mL centrifuge tube, and ice-bath for 30min;

[0046] 3. Place the ice-bathed centrifuge tube in a 42°C water bath for 90s, and ice-bath again for 2min;

[0047] 4. Add 1mL of LD medium to the above system, and culture at 37°C, 180rpm for 45-60min;

[0048] 5. Place the centrifuge tube after shaker culture in a centrifuge, centrifuge at 5000rpm for 5min, pour off the supernatant, resuspend the bacterial precipitate with about 100μL of LB culture solution, spread the resuspended solution on an LB plate (containing 1mM / mL kanamycin), and culture at 30°C for 24h;

[0049] 6. Use colony PCR to verify the transformants, and then extract plasmids from the strains with correct bands and perform sequencing verification. Save the recombinant WM3064 strain with correct sequencing verification result for subsequent conjugation experiments.

[0050] The specific steps of conjugation transfer between WM3064 and Shewanella MR-1 are as follows:

[0051] 1. Pick a single colony of the correct WM3064 strain on the solid culture medium, inoculate into LD medium (containing 1 mM / mL kanamycin), and perform primary culture at 37°C and 220 rpm.

[0052] 2. Take the MR-1 glycerol bacteria, spread on LB solid medium, and culture in a 30°C incubator for 24 h. Then pick a single colony on the solid medium, inoculate into LB medium, and perform primary culture at 30°C and 220 rpm.

[0053] 3. Take 500 μL of the above WM3064 and MR-1 primary bacterial solution, mix them in a 1.5 mL centrifuge tube, centrifuge at 5000 rpm for 5 min, discard the supernatant, resuspend with 1 mL of antibiotic-free LD medium, and then stand in a 30°C incubator for 2 h.

[0054] 4. After standing, centrifuge the centrifuge tube containing the bacterial solution at 5000 rpm for 5 min, discard the supernatant, resuspend with 1 mL of antibiotic-free LB medium, take 100 μL of the bacterial solution, spread on LB plates (containing 1 mM / mL kanamycin), and culture at 30°C for 24 h.

[0055] 5. Pick a single colony for PCR verification. The correct Shewanella is the target hydrogen-producing Shewanella engineering strain.

[0056] Example 2: CdS self-assembly of hydrogen-producing Shewanella engineering strain SES

[0057] 1. Activation of SES strain

[0058] Take the hydrogen-producing Shewanella engineering strain SES from the -80°C refrigerator, streak on LB solid medium (containing 1 mM / mL kanamycin), and stand in a 30°C incubator for 24 h for activation. Then pick a single colony from the solid culture medium, inoculate into LB medium (containing 1 mM / mL kanamycin), and culture at 30°C and 220 rpm overnight.

[0059] 2. Construction of CdS / SES hybrid system

[0060] Pick up a single SES colony from the solid culture medium, inoculate it into 5mL LB liquid culture medium with the corresponding resistance, shake and culture it at 220rpm in LB culture medium at 30℃ for 12h, and then inoculate the culture solution into 100mL LB liquid culture medium containing the corresponding resistance and inducer at an inoculation ratio of 1% (v / v), and continue to culture for 20 hours under the same temperature and rotation speed conditions to reach the stable period. Collect the bacteria by centrifugation at 4℃ and 5000rpm for 5 minutes, and wash them three times with 20mM HEPES solution. Resuspend the bacteria in 100mL anaerobic mineral culture medium containing 1mM Na2S2O3 and 20mM sodium lactate so that the initial OD 600 The cells were incubated at 30°C and 200 rpm for 24 hours, and then 1 mM CdCl2 was added and incubated for another 24 hours to form CdS nanoparticles. The entire incubation process was carried out under anaerobic conditions, using high-purity nitrogen purge for 30 minutes to completely remove oxygen.

[0061] Anaerobic mineral medium contained 5.85 g / L NaCl, 0.3 g / L NaOH, 1.498 g / L NH4Cl, 0.097 g / L KCl, 11.91 g / L HEPES, 0.658 g / L β-glycerophosphate disodium and 1 mL trace mineral solution.

[0062] 3. Results

[0063] The scanning electron microscopy (SEM) results of the hybrid system CdS / SES are as follows Figure 1 As shown in Figure 2, a large number of nanoparticles are distributed on its surface. Transmission electron microscopy (TEM) images are shown in Figure 2. Figure 2 As shown, the formed nanoparticles are mostly located in the extracellular space and tend to attach to the cell outer membrane. This confirms that the self-assembled CdS nanoparticles are mainly distributed in the extracellular space of S. oneidensis.

[0064] Example 3: Hydrogen production from a CdS / SES hybrid system

[0065] 1. System construction of photocatalytic hydrogen production

[0066] The present invention uses CdS nanoparticles generated by self-assembly of S. oneidensis to construct a biohybrid system. After multiple washings with 20mM HEPES buffer, the nanoparticles are resuspended in 30mL of anaerobic hydrogen production medium. The reaction system is placed in a photocatalytic reactor with a headspace volume set to 55mL and a 300W xenon lamp light source (light intensity 100mW / cm2) shielded from ultraviolet rays by a 420nm filter. 2) to provide visible light irradiation. The system was operated under constant temperature of 30°C with magnetic stirring at 250 rpm, and the temperature stability was maintained by a circulating water bath system. The samples were taken every 12 hours during the experiment, and the hydrogen production was periodically detected by gas chromatography to evaluate the catalytic performance.

[0067] 2. Determination of the intracellular reducing power level of the CdS / SES hybrid system

[0068] The determination of the reducing power level was performed using the Biyun Tian NAD + / NADH detection kit (WST-8 method). After the light reaction, the CdS / SES hybrid system was centrifuged at 6000 rpm for 5 minutes, and then the medium was aspirated, and pre-cooled NADH / NAD + extract was added to lyse the cells. Then the cells were centrifuged at 6000 rpm for 5 minutes at 4°C, and the supernatant was used as the sample to be tested. If the content of NAD + or NADH was to be determined alone, the sample needed to be heated in a 60°C water bath for 30 min. In a transparent 96-well plate, 20 μL of the standard or sample was added, 90 μL of ethanol dehydrogenase working solution was added, and it was incubated in a 37°C constant temperature incubator for 10 min in the dark, 10 μL of color developing solution was then added, and it was again incubated in a 37°C constant temperature incubator for 40 min in the dark. The absorbance at 450 nm was detected using an enzyme marker.

[0069] 2. Determination of the intracellular energy level of the CdS / SES hybrid system

[0070] The intracellular ATP was determined using the Biyun Tian enhanced ATP detection kit. After the light reaction, the CdS / SES hybrid system was centrifuged at 6000 rpm for 5 minutes, and then the medium was aspirated, 200 μL of lysis solution was added to the bacterial cells, and after centrifugation at 12000 g for 5 min at 4°C, the supernatant was taken for testing. In a white 96-well plate, 100 μL of ATP detection working solution was added, and after standing at room temperature for 3-5 min, 20 μL of the standard or sample was added, and after mixing, the chemiluminescence was detected using an enzyme marker in luminescence mode. The ATP concentration was normalized by the protein concentration (μg / mL), and was recorded as nmol / μg protein.

[0071] 3. Results

[0072] The amount of photocatalytic hydrogen production is shown in Table 1. Figure 4 After 96 hours of photocatalytic hydrogen production, the amount of hydrogen production of the hybrid system CdS / SES reached 302.4 ± 21.8 μmol / mg, which was higher than that of the CdS / MR-1 system, 221.6 ± 14.9 μmol / mg. In addition, Figure 5 shows that the intracellular reducing power equivalent (NADH / NAD +) is 2.30, higher than 2.05 of CdS / MR-1, Figure 6 The intracellular energy level of the hybrid system CdS / SES is 4.01 nmol / μg, higher than 3.46 nmol / μg of CdS / MR-1.

[0073] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A recombinant plasmid, characterized in that: The basic vector plasmid is connected to a plasmid containing at least one promoter and HydA from Shewanella oneidensis. son The gene expression element composed of the hydrogenase synthesis gene has a sequence shown in SEQ ID NO.

2.

2. The recombinant plasmid according to claim 1, characterized in that The basic plasmid vector is pYYDT.

3. The recombinant plasmid according to claim 1 is introduced into the wild-type Shewanella oneidensis MR-1 to obtain the hydrogen-producing Shewanella engineered strain SES.

4. A semiconductor nanoparticle, characterized in that Na2S2O3 and CdCl2 were added to Shewanella oneidensis successively, and CdS nanoparticles were self-synthesized through intracellular metabolism and attached to the bacterial outer membrane and periplasm.

5. Use of the hydrogen-producing Shewanella engineered strain according to claims 1-4 and CdS nanoparticles in photocatalytic hydrogen production in a semiconductor-microorganism hybrid system.

Images