Clostridium thermocellum engineering strain with high nitrogen utilization rate and construction and application of clostridium thermocellum engineering strain
By constructing an engineered strain of Clostridium thermocellum with high nitrogen utilization efficiency and precisely overexpressing the NitR regulatory protein, the problem of insufficient nitrogen source utilization efficiency of Clostridium thermocellum was solved, achieving efficient growth and lignocellulose saccharification under nitrogen-poor conditions and reducing the cost of culture medium.
Patent Information
- Application Number
- CN202511622819.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-06
- Publication Date
- 2026-01-23
AI Technical Summary
Clostridium thermophilum has insufficient nitrogen source utilization efficiency, resulting in high culture medium costs and limiting its industrial application.
A high nitrogen utilization rate Clostridium thermocellum strain was constructed. The regulatory protein NitR was precisely overexpressed, with its expression level being 1.2-2.0 times that of the wild type. This protein was then introduced into Clostridium thermocellum GB2 using genetic engineering techniques and expressed using the appropriate promoter p2033.
Under nitrogen-poor conditions, the growth capacity and biomass accumulation of the engineered strains were significantly enhanced, nitrogen source utilization efficiency was increased, nitrogen source addition costs were significantly reduced, and lignocellulose saccharification efficiency was improved.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of microbial technology, and particularly relates to a Clostridium thermocellum engineering strain with high nitrogen source utilization rate, construction of the Clostridium thermocellum engineering strain, and application of the Clostridium thermocellum engineering strain in whole-cell saccharification of lignocellulose. BACKGROUND
[0002] Lignocellulose is the most abundant renewable biomass resource in nature, mainly composed of cellulose, hemicellulose and lignin, and can be used as raw material for producing biofuels (such as ethanol, butanol) and high-value chemicals. Lignocellulose saccharification is a key step for converting lignocellulose biomass (such as straw, wood, bagasse, etc.) into fermentable sugars, and is an important part of biofuel and bio-based chemical production. Clostridium thermocellum, as an important cellulosome-producing bacterium, exhibits excellent cellulase and hemicellulase production capacity in lignocellulose degradation, and is a potential industrial strain in the field of biorefining. At present, Clostridium thermocellum has been widely used in lignocellulose bioconversion, and as a high-temperature chassis in synthetic biology research. Lee Rybeck Lynd of Dartmouth College in the United States proposed a consolidated bioprocessing (CBP) method to produce cellulose ethanol using Clostridium thermocellum; the inventor's team proposed a consolidated bio-saccharification (CBS) method to use a strain producing an extracellular enzyme complex, cellulosome (such as Clostridium thermocellum), as a whole-cell catalyst, which can saccharify lignocellulose and interface with various downstream fermentation to produce various bio-based products.
[0003] The natural advantage of Clostridium thermocellum lies in its ability to synthesize a large amount of extracellular cellulosome protein complex, thereby achieving efficient degradation of lignocellulose. It can be seen that the biomass of the strain and the synthesis level of its cellulosome are the premise for ensuring its efficient degradation of lignocellulose. In order to achieve this purpose, it is usually necessary to add nitrogen sources and other nutrients in the culture medium. This is because Clostridium thermocellum cannot fix nitrogen and lacks a complete nitrate assimilation pathway; it mainly relies on organic nitrogen sources (such as proteins, peptides, amino acids) and inorganic nitrogen sources (ammonium / ammonia salts, urea) as nitrogen sources. As is known, in industrial fermentation, the cost of the culture medium accounts for a large part of the total cost, and nitrogen source is one of the core components of the culture medium. Therefore, although increasing the amount of nitrogen source added to a certain extent improves the biomass and the synthesis level of the cellulosome, it increases the cost of cultivation, thereby limiting its application in industry. The inventor's previous research work has optimized the culture medium formula, but there is still a need to add more nitrogen sources than needed for growth. This indicates that the nitrogen source utilization efficiency of Clostridium thermocellum is insufficient. It is very important to increase nitrogen utilization capacity and optimize nitrogen assimilation efficiency, but there are few reports on related research.
[0004] The regulatory protein NitR is a typical transcriptional regulator, mainly existing in some bacteria, for responding to nitrogen-containing compounds (especially nitrite) in the environment and regulating the expression of related genes. However, there is no nitrite in the culture system of Clostridium thermocellum, so it is unpredictable whether the regulatory protein NitR can play a role and under what conditions it can play a positive role in solving the problem of insufficient nitrogen source utilization efficiency of Clostridium thermocellum. There is no related report in the prior art. SUMMARY
[0005] To solve the problem of insufficient nitrogen source utilization efficiency of Clostridium thermocellum in the prior art, the present application provides a Clostridium thermocellum engineering strain with high nitrogen source utilization efficiency and a construction method thereof. The present application constructs a Clostridium thermocellum engineering strain by precisely expressing a specific regulatory protein NitR. Compared with the wild strain, the growth ability and biomass accumulation of the engineering strain under nitrogen-poor conditions are significantly improved, thereby realizing the increase of nitrogen source utilization efficiency. Based on this, the present application also provides the application of the engineering strain in lignocellulose saccharification, which provides an effective solution to the nitrogen limitation bottleneck in the process of lignocellulose saccharification.
[0006] Technical scheme of the present application:
[0007] The present application provides a Clostridium thermocellum engineering strain with high nitrogen utilization efficiency, which precisely overexpresses the NitR regulatory protein. The precise overexpression is that the expression level of the regulatory protein NitR is 1.2-2.0 times that of the wild type. The amino acid sequence of the NitR regulatory protein is shown in SEQ ID NO: 1. The chassis strain of the strain is Clostridium thermocellum GB2 with a glucosidase expression frame on the genome, and the coding gene of the glucosidase is shown in SEQ ID NO: 2. The Clostridium thermocellum GB2 is an engineering strain expressing BGL in Clostridium thermocellum DSM1313. The inventors unexpectedly found that when the expression level of the regulatory protein NitR is controlled to be 1.2-2.0 times that of the wild type, the growth ability and biomass accumulation of the engineering strain under nitrogen-poor conditions are significantly improved, thereby realizing the increase of nitrogen source utilization efficiency and achieving unexpected technical effects.
[0008] Preferably, the promoter regulating the expression of the NitR protein is a p2033 promoter with a nucleotide sequence as shown in SEQ ID NO: 4. The p2033 promoter used in the present application has a suitable expression strength, meets the requirement of precisely overexpressing the regulatory protein NitR, and does not affect the growth of the strain. The maximum biomass of the strain described in the present application is increased by more than 30% compared with the wild strain, whether in a nitrogen-poor medium (the nitrogen addition amount in the medium is less than 0.1 g / L) or in a nitrogen-rich medium (the nitrogen addition amount in the medium is more than 0.1 g / L). Moreover, the biomass of the engineered strain under the nitrogen-poor condition is comparable to the biomass level of the wild strain in the nitrogen-rich medium, and the technical effect is remarkable.
[0009] The present application also provides a method for constructing the Clostridium thermocellum engineered strain as described above, which comprises introducing a coding gene of a NitR regulatory protein into Clostridium thermocellum GB2 by genetic engineering means, and selecting a suitable promoter to control the expression level at 1.2-2 times of the wild type, so as to obtain a Clostridium thermocellum engineered strain with high nitrogen utilization rate. The nucleotide sequence of the NitR regulatory protein is as shown in SEQ ID NO: 3. The genetic engineering means specifically comprises: using an expression based on plasmid pHK as a skeleton or using a method of homologous recombination to realize expression based on genomic integration.
[0010] Preferably, the suitable promoter is a p2033 promoter with a nucleotide sequence as shown in SEQ ID NO: 4.
[0011] The application of the Clostridium thermocellum engineered strain as described above in saccharification of lignocellulosic raw materials. The application specifically comprises: using the Clostridium thermocellum engineered strain as a whole-cell catalyst to perform saccharification treatment of lignocellulosic under nitrogen-poor conditions. The nitrogen-poor conditions specifically comprise: the nitrogen content is <0.08 g / L according to the calculation of nitrogen element equivalent, and the nitrogen source is ammonium bicarbonate. Based on this, the lignocellulosic used in the application can preferentially select lignocellulosic raw materials (such as corn stalks, xylose residues) with low nitrogen content, which not only improves the saccharification efficiency but also significantly reduces the nitrogen source addition cost, and has important significance for industrial application.
[0012] The present application has the following beneficial effects:
[0013] (1) The present application provides a Clostridium thermocellum engineered strain with high nitrogen source utilization rate; compared with the wild strain, the growth ability and biomass accumulation of the engineered strain under nitrogen-poor conditions are significantly improved, thereby realizing the increase of nitrogen source utilization efficiency.
[0014] (2) The application provides a construction method of the Clostridium thermocellum engineering strain, and the construction method is used for constructing the Clostridium thermocellum engineering strain by precisely expressing a specific regulatory protein NitR, and is simple and easy to operate.
[0015] (3) The application also provides application of the engineering strain in lignocellulose saccharification; the engineering strain is particularly suitable for saccharification treatment of lignocellulose raw materials (such as corn straw and wood sugar residue) with low nitrogen content, significantly reduces the cost of nitrogen source addition, improves the saccharification efficiency, and has important significance for industrial application.
[0016] It should be noted that the increase of nitrogen source utilization efficiency realized by the engineering strain and the construction method of the application is obvious to those skilled in the art, and the beneficial effect is not limited to the application in the examples in the application, and other applications based on the beneficial effect of the engineering strain and the construction method are also included in the scope of the patent. BRIEF DESCRIPTION OF DRAWINGS
[0017] Appendix Figure 1 The relative expression amount of the NitR gene of the GB2 and PNitR engineering strains in Example 2;
[0018] Appendix Figure 2 The relative expression amount of the NitR gene of the GB2 and GNitR engineering strains in Example 3. DETAILED DESCRIPTION
[0019] The application will be further described below in combination with examples.
[0020] Example 1: Construction of NitR expression vector
[0021] First, the NitR gene (SEQ ID NO: 3) and the p2033 promoter (SEQ ID NO: 4) are amplified by PCR using the C. thermocellum DSM1313 genomic DNA as a template, then the two fragments are connected into a fusion fragment p2033-NitR. Finally, the p2033-NitR is connected to the plasmid backbone pHK by using a seamless cloning kit to obtain the pHK-p2033-NitR expression vector.
[0022] Example 2: Construction of engineering strain by plasmid overexpression of NitR
[0023] The pHK-p2033-NitR expression vector constructed in Example 1 was transformed into Clostridium thermocellum GB2 by electroporation and cultured on a plate containing methionine sulfoximine. The obtained transformants were subjected to colony PCR and sequencing verification to obtain the plasmid overexpression NitR engineering strain PNitR. The GB2 strain was used as a control, and the expression level of the NitR gene in PNitR was analyzed by fluorescence quantitative PCR (qRT-PCR). The results are shown in Figure 1 .
[0024] As can be seen from Figure 1 , the expression level of the NitR gene in the engineering strain PNitR is about 2 times higher than that of the wild type strain, indicating that the PNitR strain is successfully constructed and the overexpression of the NitR gene is achieved.
[0025] Example 3: Construction of engineering strain overexpressing NitR by genomic integration
[0026] First, the p2033-NitR expression cassette was amplified by PCR using the pHK-p2033-NitR constructed in Example 1 as a template, and the p2033-NitR fusion fragment was ligated into the middle of the two homologous arm sequences of the pHK homologous recombination plasmid using a seamless cloning kit. The two homologous arm sequences are the sequences of 1000 bases upstream and downstream of the insertion site. The pHK homologous recombination plasmid also has an expression frame of tdk for homologous recombination reverse screening. Subsequently, the pHK homologous recombination plasmid carrying the p2033-NitR expression frame was transformed into Clostridium thermocellum GB2 by electroporation and cultured on a plate containing methionine sulfoximine. The obtained transformants were cultured in a medium containing FUDR and no uracil and solid plate screening, and the genomic integration overexpression NitR engineering strain GNitR was obtained by methionine sulfoximine resistance loss and genomic PCR verification.
[0027] The GNitR was subjected to fluorescence quantitative PCR (qRT-PCR) analysis, and the wild type strain was used as a control to analyze the expression level of the NitR gene. The results are shown in Figure 2 . As shown in Figure 2 , the expression level of the NitR gene in the engineering strain GNitR is about 1.2 times higher than that of the wild type strain, indicating that the GNitR strain is successfully constructed. Since the NitR gene carried by it is in a single copy form, the expression level is relatively small compared with the PNitR strain.
[0028] Example 4: Growth phenotype analysis of engineering strain in nitrogen-rich / nitrogen-poor medium
[0029] To verify the growth performance of the engineered strains constructed in Example 2 and Example 3 under different nitrogen supply levels, the PNitR strain and the GNitR strain were inoculated into a nitrogen-poor medium (0.1 g / L of ammonium bicarbonate was added to the medium) and a nitrogen-rich medium (1 g / L of ammonium bicarbonate was added to the medium) for culture. The optical density (OD 600 ) in the culture process was determined to evaluate the growth of the bacterial cells, and the maximum OD 600 value was recorded as an index of the maximum biomass of the bacterial cells.
[0030] Table 1. Maximum biomass of the engineered strains PNitR and GNitR under different nitrogen addition conditions
[0031]
[0032] As shown in Table 1, under different nitrogen addition conditions, the maximum biomass (OD 600 ) of the engineered strains PNitR and GNitR was higher than that of the control strain GB2, indicating that the growth performance of the engineered strains was significantly improved. Specifically, the following was observed.
[0033] (1) In the nitrogen-poor medium (0.1 g / L of ammonium bicarbonate was added), the maximum OD 600 of the PNitR strain was 1.30, which was about 1.7 times that of the GB2 strain (OD 600= 0.75), and the OD 600 of the GNitR strain was 0.95, which was about 27% higher than that of the GB2 strain. This indicates that both of the engineered strains maintain a high growth level under low nitrogen source conditions, and the PNitR strain has a more significant improvement effect.
[0034] (2) In the nitrogen-rich medium (1 g / L of ammonium bicarbonate was added), the maximum biomass of the PNitR and GNitR strains reached 2.47 and 2.28, respectively, which was about 30% and 20% higher than that of the GB2 strain (OD 600= 1.90). This result indicates that the PNitR and GNitR engineered strains constructed in the application have a high biomass accumulation capacity under nitrogen-rich conditions and have good nitrogen source utilization performance.
[0035] In summary, the growth performance of the PNitR and GNitR strains constructed in the application is better than that of the control strain GB2 under different ammonium bicarbonate addition conditions. Among them, the biomass level of the PNitR strain under nitrogen-poor conditions is close to that of the GB2 strain under nitrogen-rich conditions, indicating that the strain has a higher nitrogen utilization efficiency and a significant technical effect.
[0036] Example 5: Application of the engineered strain in lignocellulose saccharification (low nitrogen condition)
[0037] To verify the saccharification performance of the Clostridium thermocellum engineering strains constructed in Example 2 and Example 3 under low nitrogen conditions, corn stalks and xylose residues were used as substrates for saccharification experiments. The PNitR strain and GNitR strain described in Example 4 were used as whole-cell catalysts, and the unmodified Clostridium thermocellum chassis strain GB2 was used as a control strain. The saccharification reaction was carried out under low nitrogen conditions (0.08 g / L of nitrogen content calculated by nitrogen element equivalent, and ammonium bicarbonate as the nitrogen source).
[0038] The pretreated corn stalks or xylose residues were added to the saccharification system as substrates, and the PNitR, GNitR, or GB2 strain was used as the whole-cell catalyst, and the reaction was carried out at 60°C and 200 rpm for a period of time. After the reaction was completed, the reducing sugar content in the saccharification liquid was determined by the DNS method to evaluate the saccharification efficiency.
[0039] Table 2. Saccharification effect of engineering strains PNitR and GNitR under low nitrogen culture conditions
[0040]
[0041] As shown in Table 2, under low nitrogen culture conditions, the reducing sugar yields of the engineering strains PNitR and GNitR in the saccharification reaction of lignocellulosic substrates (corn stalks and xylose residues) were significantly higher than that of the control strain GB2, indicating that the engineering strains still maintained strong saccharification catalytic ability under low nitrogen conditions. Specifically:
[0042] (1) When corn stalks were used as the substrate, the reducing sugar yield of the PNitR strain was 33.0 g / L, which was about 32% higher than that of the GB2 strain (25.0 g / L); the reducing sugar yield of the GNitR strain was 30.0 g / L, which was about 20% higher than that of the GB2 strain. (2) When xylose residues were used as the substrate, the reducing sugar yield of the PNitR strain reached 37.8 g / L, which was about 35% higher than that of the GB2 strain (28.0 g / L), and the GNitR strain was 35.0 g / L, which was about 25% higher.
[0043] In summary, the two engineering strains constructed in the present application showed high saccharification efficiency under different lignocellulosic substrates, and the PNitR strain showed more significant improvement. This indicates that the engineering strains not only can effectively promote the decomposition of lignocellulose and the accumulation of saccharification products under low nitrogen conditions, but also can significantly reduce the amount of nitrogen source added, and have good industrial application potential.
Claims
1. A high-nitrogen utilization Clostridium thermocellum engineered strain, characterized in that: The NitR regulatory protein is precisely overexpressed in the engineering strain; the precise overexpression is that the expression level of the regulatory protein NitR is 1.2-2.0 times of the wild type; the amino acid sequence of the NitR regulatory protein is shown as SEQ ID NO:
1.
2. The engineered strain of Clostridium thermocellum of claim 1, characterized in that: The promoter for regulating the expression of the NitR protein is a p2033 promoter with a nucleotide sequence shown as SEQ ID NO:
4.
3. The engineered strain of Clostridium thermocellum of claim 1 or 2, wherein: The chassis strain of the strain is Clostridium thermocellum GB2 with a glucosidase expression frame on the genome, the coding gene of the glucosidase is shown as SEQ ID NO: 2, and the Clostridium thermocellum GB2 is an engineering strain of Clostridium thermocellum DSM1313 expressing BGL.
4. The method for constructing engineered strain of Clostridium thermocellum according to any one of claims 1-3, wherein: By means of genetic engineering, the coding gene of the NitR regulatory protein is introduced into Clostridium thermocellum GB2, and a suitable promoter is selected to control the expression level at 1.2-2 times of the wild type, thereby obtaining the Clostridium thermocellum engineering strain with high nitrogen utilization rate.
5. The method for constructing engineered strain of Clostridium thermocellum according to claim 4, characterized in that: The nucleotide sequence of the NitR regulatory protein is shown as SEQ ID NO:
3.
6. The method for constructing engineered strain of Clostridium thermocellum according to claim 4 or 5, characterized in that: The genetic engineering means is specifically: expression based on plasmid pHK as a backbone or expression based on genomic integration by homologous recombination.
7. The method for constructing engineered strain of Clostridium thermocellum according to claim 6, characterized in that: The suitable promoter is a p2033 promoter with a nucleotide sequence shown as SEQ ID NO:
4.
8. The Clostridium thermocellum engineering strain according to any one of claims 1-3 is applied in saccharification of lignocellulosic raw materials.
9. Use according to claim 8, characterized in that: The Clostridium thermocellum engineering strain is used as a whole cell catalyst to perform saccharification treatment of lignocellulosic under nitrogen-poor conditions.
10. Use according to claim 9, characterized in that: The nitrogen-poor conditions are specifically: according to the calculation of nitrogen element equivalent, the nitrogen content is <0.08 g / L, and the nitrogen source is ammonium bicarbonate.