CN116814517A - Escherichia coli capable of reducing carbon dioxide in light driving manner and application of escherichia coli in organic acid production - Google Patents
Escherichia coli capable of reducing carbon dioxide in light driving manner and application of escherichia coli in organic acid production Download PDFInfo
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- CN116814517A CN116814517A CN202310782457.1A CN202310782457A CN116814517A CN 116814517 A CN116814517 A CN 116814517A CN 202310782457 A CN202310782457 A CN 202310782457A CN 116814517 A CN116814517 A CN 116814517A
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- escherichia coli
- carbon dioxide
- coli
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- organic acid
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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Abstract
The invention discloses escherichia coli capable of reducing carbon dioxide by light driving and application thereof in organic acid production, and belongs to the technical field of biology. According to the invention, through screening metal ion transport proteins, quantum dot types and formate dehydrogenase, a transport protein EcMntH coding gene with the number of UniProt: P0A769 is overexpressed in a host, quantum dot PbS is synthesized by culturing in a culture medium containing a lead source, and the formate dehydrogenase FDH coding gene with the nucleotide sequence shown as SEQ ID NO.1 is heterologously expressed, a recombinant strain capable of efficiently driving carbon dioxide reduction by utilizing light is obtained, and the strain is used as a chassis strain for genetic engineering transformation, so that the recombinant strain can be used for preparing various organic acids, and lays a foundation for green production of the organic acids.
Description
Technical Field
The invention relates to escherichia coli capable of reducing carbon dioxide by light driving and application thereof in organic acid production, and belongs to the technical field of biology.
Background
Carbon dioxide (CO) 2 ) Is one of the main greenhouse gases for global warming, and is based on CO of biological carbon fixation technology 2 Resource utilization technology is also receiving more and more attention, and CO is utilized 2 CO as a raw material 2 The resource utilization is used for producing biological fuel, chemicals, food and other substances, and is used for constructing a new energy system with low carbon and green resource utilization.
At present, energy crisis and environmental crisis become global problems, and development and utilization of renewable energy are important schemes for alleviating energy and environmental problems. The use of wind energy, solar energy, water energy, biomass energy, geothermal energy, etc. as renewable energy sources helps to alleviate the global crisis described above. The solar energy is taken as an effective renewable clean energy source, and the realization of efficient conversion from solar energy to chemical energy is always a hot spot for research in recent years. In natural photosynthesis, after light energy is captured by a pigment molecule and electrons are transferred to PSI by photosystem I (PSI) and photosystem II (PSII), a dark reaction is performed in chloroplast stroma. However, the conversion efficiency of natural photosynthesis is very low and generally does not exceed 6% (most plants are at 0.1% and crops are generally at 1% -2%). Recently, an artificial photosynthetic system is established by combining synthetic biology with nano materials, and a semiconductor material captures light energy to realize electron-hole separation. The organisms bind to the semiconductor material and receive photo-generated electrons of the semiconductor, thereby generating reducing power to supply the cell growth metabolism.
Formic acid is an important chemical raw material, widely exists in nature, is the simplest carboxylic acid, and has a special structure. Because the aldehyde group contained in the molecule has reducibility, the aldehyde group is used as an acidic reducer for bleaching straw hat, leather and the like in the textile and printing industry, and can remove ink spots and rust on clothes; the coagulant used as lactic acid in the leather industry is a rubber coagulant; formic acid is also a raw material for preparing oxalic acid; formic acid is also a printing and dyeing mordant, a metal surface treating agent, a disinfection preservative and the like. In conclusion, formic acid is one of basic organic chemical raw materials, and is widely used in pesticide, leather, textile, printing and dyeing, medicine, rubber industry and the like, and various solvents, plasticizers, rubber coagulants, animal feed additives, insulin synthesis by a new process and the like can be prepared. In the consumption of formic acid in China, the medical industry accounts for about 45%, the chemical industry accounts for about 30%, and other departments such as light industry, spinning and the like account for about 25%. Formic acid is one of important export chemical products in China, compared with the traditional fermentation production of formic acid, the optical drive engineering strain is utilized to metabolize and produce formic acid, so that the optical energy can be efficiently utilized, a new idea is developed for relieving energy crisis, and CO can be promoted 2 Becomes a win-win strategy. The present invention has been made accordingly.
Disclosure of Invention
In order to solve the problems, the invention provides a construction method of an optical drive carbon dioxide reduction system, and a chassis strain which can be used for producing organic acid by optical drive is successfully obtained.
The first object of the invention is to provide an escherichia coli capable of reducing carbon dioxide in a light-driven way, wherein the escherichia coli is obtained by over-expressing a transporter EcMntH coding gene with the number of UniProt: P0A769 in a host, culturing and synthesizing quantum dots PbS in a lead-source-containing culture medium, and heterologously expressing a formate dehydrogenase FDH coding gene with a nucleotide sequence shown as SEQ ID NO. 1.
Furthermore, the escherichia coli also overexpresses ACS coding genes of acetyl-CoA synthetase, ACDH coding genes of acyl acetaldehyde dehydrogenase, FLS coding genes of formaldehyde lyase and DHAK coding genes of dihydroxyacetone kinase.
Further, the E.coli also overexpresses the lactate dehydrogenase LDH-encoding gene.
Further, the lead source is a soluble lead salt.
Further, the lead source concentration in the culture system is not more than 3mM.
Further, the amount of cells in the system reaches OD 600 At=18-22, the lead source was added.
Further, the culture medium is LB culture medium.
Further, the nucleotide sequence of the ACS encoding gene of the acetyl-CoA synthetase is shown as SEQ ID NO.2, the nucleotide sequence of the ACDH encoding gene of the acylated acetaldehyde dehydrogenase is shown as SEQ ID NO.3, the nucleotide sequence of the FLS encoding gene of the formaldehyde lyase is shown as SEQ ID NO.4, the nucleotide sequence of the DHAK encoding gene of the dihydroxyacetone kinase is shown as SEQ ID NO.5, and the nucleotide sequence of the LDH encoding gene of the lactic acid dehydrogenase is shown as SEQ ID NO. 6.
Further, E.coli BL21 is used as a host.
The second object of the present invention is to provide a method for producing organic acid by driving light, wherein the method adopts the escherichia coli to produce under illumination, or extracts enzyme and PbS in the escherichia coli as catalysts to produce.
Further, the organic acids include, but are not limited to, formic acid, lactic acid, pyruvic acid, and the like.
Further, the production system contains substances which provide a source of carbon dioxide, such as sodium carbonate and the like.
Further, illumination with a light source of 400-430nm is preferred.
The invention has the beneficial effects that:
the invention provides a recombinant escherichia coli which utilizes light energy to efficiently drive organic acid production, wherein three stages are adopted to synthesize a photocatalyst in vivo, namely, nanometer quantum dots are synthesized in cells after thalli reach a stationary phase, the photoelectric performance of an inorganic-biological hybrid is verified, and finally, formic acid, pyruvic acid and lactic acid are produced by combining with metabolic pathways in engineering strains. The strain obtained by the invention has huge subsequent transformation potential, and the method for producing the organic acid by fermentation has low cost, and is suitable for industrial production.
Drawings
FIG. 1 shows the results of confocal imaging of recombinant plasmid pEt-28a-EcMntH membrane protein.
FIG. 2 is an ion tolerance test chart of E.coli.
Fig. 3 is a scanning electron microscope image of quantum dot synthesis.
FIG. 4 shows E.coli and Pb (NO) 3 ) 2 EDS Mapping graph after incubation.
FIG. 5 shows E.coli and Pb (NO) 3 ) 2 XRD pattern after incubation.
Fig. 6 is an HFLS path diagram.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The materials and methods involved in the following examples are as follows:
LB medium: 10g/L of beef extract, 10g/L of peptone, 5g/L of yeast extract, 3g/L of NaCl, 30mg/L of nystatin (nystatin), 30mg/L of cycloheximide, 4mL/L of 0.2% bromothymol blue, 15g/L of agar powder and p H7.0.0-7.2.
TB medium: 10g/L tryptone, 5g/L yeast extract powder and 0.5g/L NaCl.
Organic acid production conditions: after growth to log phase at 37℃culture, protein expression was induced by switching to 25 ℃. And (3) biosynthesis of PbS in the protein induction expression process, and finally, illumination is carried out by a light source of 410-420nm to provide NADH for producing organic acid.
Formic acid and lactic acid detection method: the chromatographic column is HPX-87H (Bio-Rad Hercules), and the column temperature is 52 ℃; the detector is a differential refraction detector; mobile phase 5mM H 2 SO 4 The flow rate was 0.6mL/min. The fermentation supernatant was directly used for formic acid and lactic acid after removing impurities by 0.22 μm microfiltration. During intracellular formic acid and lactic acid measurement, ultrasonic crushing is first adopted, and then the supernatant is centrifugally taken for detection。
Cell concentration determination: after properly diluting the fermentation broth, the OD was measured by an ultraviolet spectrophotometer at a wavelength of 600nm 600 。
Example 1: escherichia coli biosynthesis nanometer quantum dot
(1) Screening of semiconductor nanomaterials
In order to realize NADH regeneration in the escherichia coli, an NADH regeneration system for synthesizing the photocatalytic semiconductor nanomaterial in vivo is designed. And finally, the lead sulfide (PbS) quantum dots are biosynthesized by using escherichia coli through a pre-experiment test. During synthesis, the addition of raw materials triggers intracellular metabolic pathways to lead the sulfhydryl group of cysteine and Pb 2+ The coupling forms PbS, and the escherichia coli becomes an excellent choice of biological carriers due to the fast passage, short growth period and clear metabolic network, which is beneficial to metabolic engineering transformation.
(2) Metal ion transporter expression
The lead ion transport was performed using DMT1 (SLC 11A2: uniProt: P49281, homo sapiens (Human)) protein, and since the transport proteins have been reported to be Human, we selected homologous proteins SaCnt (uniProt: Q2FVE 7), ecMntH (uniProt: P0A 769), ppmntH (uniProt: A0A2C5WDR 2) and BsMntH (uniProt: P96593) derived from microorganisms by homology alignment, respectively, to amplify the gene fragments of interest from the genomes of E.coli, B.subtilis, S.aureus and P.putida.
The PCR amplification parameters were: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 53℃for 30s, extension at 72℃for 30s,30 cycles, and extension at 72℃for 10min. PCR products were sequenced by Souzhou Jin Weizhi Biotechnology Co.
PET-28a is taken as a plasmid vector to construct bacteria, and then the bacteria are connected to plasmids in a way of enzyme digestion (BglII and XhoI) connection to obtain recombinant plasmids pEt-28a-DMT1, pEt-28a-SaCnt, pEt-28a-EcMntH, pEt-28a-PpMntH and pEt-28a-BsMntH, and the recombinant plasmids are preserved for subsequent experiments.
Analysis is carried out by evaluating the filming condition of the transport protein based on the laser confocal imaging result, and the filming condition of the recombinant plasmid pEt-28a-EcMntH protein is found to be better, so that the recombinant plasmid pEt-28a-EcMntH is selected for subsequent experiments (figure 1).
(3) Escherichia coli Strain vs Pb 2+ Tolerance identification of (2)
Gradient spot plate identification: inoculating E.coli BL21 into LB culture medium to obtain seed solution, inoculating onto culture medium plate, and setting Pb in 50mL system of test group 2+ The OD was measured after 12 hours of incubation at 37℃with concentrations of 1mM, 2mM, 3mM, 4mM and 5mM, respectively 600 And the colony growth state was observed. Meanwhile, the growth state of the strain was observed under an optical microscope. Finally, E.coli BL21 was found to be tolerant to 3mM Pb (NO 3 ) 2 (FIG. 2).
Example 2: coli engineering strain for constructing inorganic-biological coupling system
(1) Construction of recombinant bacterium for synthesizing PbS
According to the screening result, the bacterial body quantity in the system reaches OD 600 When=20, the lead source (here Pb (NO 3 ) 2 ) The maximum tolerance of 3mM is taken as the feeding amount to be added into the system, so as to promote the E.coli BL21 (recombinant bacterium EM-1) of the E.coli cell into which the recombinant plasmid pEt-28a-EcMntH is introduced to adsorb Pb 2+ And synthesizing PbS semiconductor quantum dots on the cell surface or in cells to obtain recombinant strain EM-2.
(2) Verification of inorganic-biological hybrid systems
The method comprises the steps of firstly determining that the biosynthesis site of the quantum dot is on a cell membrane by utilizing a scanning electron microscope (see fig. 3, wherein A has no PbS, B, C, D has PbS binding), and then confirming that the quantum dot which is biosynthesized on the cell membrane is indeed PbS by EDS (see fig. 4) and X-ray diffraction (see fig. 5, wherein A, B is purified PbS material).
(3) Optical verification of inorganic-biological hybrid systems
The photocurrent, resistance and absorption spectrum of the inorganic-biological hybrid system obtained above were examined. A three-electrode system (counter electrode: platinum electrode; reference electrode: ag/AgCl; working electrode: ITO) was used in a 0.5M Na 2 SO 4 In the solution, a xenon lamp with the wavelength of more than 400nm is used, and a detection system is circularly illuminated for 160s (the lamp 20s is turned on and the lamp 20s is turned off). Results tableIt is clear that under the illumination of a xenon lamp, the hybrid system is capable of generating photocurrent.
(4) Regeneration NADH verification of inorganic-biological hybrid system
NAD + +2H + +2e - NADH
Two electrons are consumed for each NADH generation, and after the electrons are generated by illumination, the electrons and the NAD are generated + The combined generation of NADH provides the reducing power required for the reaction.
Example 3: CO using inorganic-biological hybrid systems 2 Reduction production of organic acids
(1) Inorganic-biological hybrid optical drive CO 2 Reduction to formic acid
Formic acid has higher volume hydrogen content (53 g/L), is convenient to store and transport, is not easy to burn and explode, and is a chemical raw material which is easier to store than hydrogen. The formate dehydrogenase clFDH (UniProt: D8GNS 3) from Clostridium immortalized (Clostridium ljungdahlii) was selected for plasmid construction to give recombinant plasmid pEt28a-clFDH. The recombinant plasmid was introduced into recombinant strain EM-2 constructed in example 2 to obtain recombinant strain EM-3, and formic acid production was performed. Formic acid produced under light conditions was observed to be higher than 80% under dark conditions, reaching 0.45mg/L.
During in-vitro production, recombinant bacterium EM-3 is induced after enrichment culture in a TB culture medium, bacterial cells are collected and crushed, in-vitro purification is carried out to obtain clFDH pure enzyme, and simultaneously PbS nano particles are purified to carry out in-vitro reaction, so that formic acid can be synthesized under the illumination condition.
(2) Introduction of CO 2 Construction of genetically engineered strains of fixed pathways
By constructing CO 2 Fixing module for CO 2 Is reduced to produce lactic acid. According to the database mining, designing a piece of CO 2 The fixed pathways, over-expressed FDH, ACS, ACDH, FLS and DHAK proteins, are shown in figure 6.
Based on database screening, the formate dehydrogenase gene sequence of Clostridium ljungdahlii is selected and subjected to PCR amplification, and after gel recovery, the recombinant plasmid pER-FDH is obtained by adopting an enzyme digestion connection and one-step homologous recombination method, and the amplification primer template is shown in Table 1.
The acetyl CoA synthetase uses the genome of the escherichia coli MG1655 as a template to carry out PCR amplification, enzyme digestion connection and one-step homologous recombination method are adopted after glue recovery to obtain recombinant plasmid pER-ACS, and the primer template for ACS amplification is shown in table 1.
Respectively synthesizing ACDH, FLS and DHAK gene fragments by adopting a gene synthesis mode, and then respectively connecting the fragments to plasmid pER by adopting an enzyme digestion (BglII and XhoI) connection mode to respectively obtain recombinant plasmids pER-ACDH, pER-FLS and pER-DHAK; five plasmids (pER-FDH, pER-ACS, pER-ACDH, pER-FLS and pER-DHAK) obtained by the above construction were assembled stepwise into one plasmid pER-CF5A, respectively, by the same-tailed enzyme assembly technique (see paper A synthetic biology platform for engineering metabolic pathways in E.coll) (construction method, see patent publication No. CN 110951660B).
The lactic dehydrogenase LDH gene fragment is synthesized by adopting a gene synthesis mode, and then is connected to the plasmid pER by adopting a mode of enzyme digestion (BglII and XhoI) connection and one-step homologous recombination to obtain the recombinant plasmid pER-LDH. In the HFLS path, CO 2 After dihydroxyacetone phosphate is produced by immobilization, the dihydroxyacetone phosphate enters a glycolysis path and is then immobilized by CO 2 Pyruvic acid is produced, and lactic acid is produced by lactic dehydrogenase catalysis under anaerobic conditions.
Specifically, plasmid pER-CF5A and recombinant plasmid pER-LDH are transformed into competent cells of recombinant bacterium EM-2 to obtain recombinant bacterium EM-4, the recombinant bacterium EM-4 is coated on a resistance plate containing the kanamicin to obtain a transformant, and finally the transformant is subjected to an in vitro test path to verify heterologous CO 2 The effect of producing organic acid by the fixed way is that the yield of lactic acid under illumination reaches 1.65g/L, which is higher than 93% of darkness.
TABLE 1
| Primer name | Primer sequences |
| FDH-S | agatatacatatggcagatctGATGAAAAGTATACTAACTACTTGTCCTTATTGT |
| FDH-A | ggtttctttaccagactcgagTTAAGCGTCTTTACGCATACTCTTTT |
| ACS-S | agatatacatatggcagatctGATGAGCCAAATTCACAAACACACC |
| ACS-A | ggtttctttaccagactcgagTTACGATGGCATCGCGATAGC |
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. An escherichia coli capable of driving and reducing carbon dioxide, which is characterized in that: the escherichia coli is obtained by over-expressing a transporter EcMntH coding gene with the number of UniProt: P0A769 in a host, culturing and synthesizing quantum dot PbS in a culture medium containing a lead source, and heterologously expressing a formate dehydrogenase FDH coding gene with a nucleotide sequence shown as SEQ ID NO. 1.
2. The escherichia coli as set forth in claim 1, wherein: the escherichia coli also overexpresses ACS (acetyl-CoA synthetase) encoding genes, ACDH (acyl-aldehyde dehydrogenase) encoding genes, FLS (formaldehyde synthase) encoding genes and DHAK (dihydroxyacetone kinase) encoding genes.
3. The escherichia coli as set forth in claim 1, wherein: the escherichia coli also overexpresses a lactate dehydrogenase LDH-encoding gene.
4. The escherichia coli as set forth in claim 1, wherein: the lead source is a soluble lead salt.
5. The escherichia coli as set forth in claim 1, wherein: the lead source concentration in the culture system is not more than 3mM.
6. The escherichia coli as set forth in claim 1, wherein: the bacterial mass in the system reaches OD 600 At=18-22, the lead source was added.
7. The escherichia coli as set forth in claim 1, wherein: e.coli BL21 hosts.
8. A method for optically driving the production of an organic acid, characterized by: production using the E.coli of any one of claims 1 to 7 under light or extracting the enzyme and PbS from the E.coli as a catalyst.
9. The method according to claim 8, wherein: the organic acid comprises any one of formic acid, lactic acid and pyruvic acid.
10. The method according to claim 8, wherein: the production system contains substances for providing carbon dioxide sources.
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