Clostridium thermocellum engineering strain for highly expressing cellulosome as well as construction and application of clostridium thermocellum engineering strain

Abstract

The invention creatively provides the application of the gene of the inactivated coded protein HtrA1Ct in improving the expression quantity of the cellulosome of the clostridium thermocellum, and the sequence of the protein coded by the gene is as shown in SEQ ID NO: 1. The method not only has an unexpected technical effect, but also has an important leading value for the subsequent research on the HtrA family protein and the clostridium thermocellum. On the basis, the invention also provides a clostridium thermocellum engineering strain delta htrA1 for highly expressing the cellulosome, the engineering strain delta htrA1 is constructed by taking clostridium thermocellum DSM1313 as a chassis strain, and a gene clo13130972 for coding HtrA1Ct protein in the engineering strain delta htrA1 is inactivated. Compared with a control strain, the engineering strain delta htrA1 has a higher cellulosome expression level and higher cellulase activity, and the technical effect is remarkably improved and unpredictable. Moreover, the yield of the engineering strain delta htrA1 is remarkably increased under the condition that the specific enzyme activity of the cellulosome is not changed, so that the efficiency of the engineering strain in degrading cellulose substrates is greatly improved, and the engineering strain delta htrA1 has important industrial application value.

Description

Technical Field

[0001] The invention belongs to the field of microbial technology and relates to the field of genetic engineering, and specifically relates to an engineered strain of Clostridium thermocellum capable of highly expressing cellulosomes, and a construction method and application thereof. Background Art

[0002] Lignocellulosic biomass is the most abundant renewable biomass energy on earth. Through biological transformation pathways, lignocellulose can be converted into bioenergy such as bioethanol and biobutanol. Clostridium thermocellum has the ability to degrade cellulose efficiently. It achieves efficient degradation of cellulose by secreting a multi-enzyme complex called cellulosome. Cellulosomes are composed of non-catalytic units (non-catalytic subunits) such as scaffold proteins and enzyme units (catalytic subunits) with different catalytic activities. Through non-covalent interactions between multi-level scaffold proteins and enzyme assembly modules, these subunits assemble into supramolecular multi-enzyme complexes with a molecular weight exceeding megadaltons. Among them, the scaffold proteins of cellulosomes integrate multiple enzymes to form a spatial proximity effect between enzymes; cellulosomes have cell wall binding modules on some scaffold proteins or enzymes, which anchor the cellulosomes to the cell wall so that the enzymatic hydrolysis products are quickly absorbed by the cell; cellulosomes have carbohydrate binding modules on some scaffold proteins or enzymes, which enable the cellulosomes to quickly recognize substrates; the assembly and interaction of these functional modules realize multi-level synergy between enzymes, enzymes and cells, and enzymes and substrates.

[0003] It is well known to those skilled in the art that the expression level of cellulosomes determines the ability of Clostridium thermocellum to degrade substrates. As a thermophilic anaerobic bacterium, Clostridium thermocellum has a significant deficiency in its ability to produce exocrine proteins compared to aerobic microorganisms, and the amount of cellulosomes produced is still difficult to meet the needs of industrial applications. Increasing the expression level of cellulosomes can increase the degradation rate of substrates, thereby reducing the cost requirements during practical applications. Therefore, increasing the expression level of cellulosomes, thereby effectively improving the cellulose degradation ability of Clostridium thermocellum, is of great significance for practical applications.

[0004] As mentioned above, the structure and components of the cellulosome are extremely complex, containing dozens of enzymes and multiple scaffold proteins, and the expression levels of each subunit are not exactly the same and are subject to complex regulation. At present, people's understanding of the expression and regulation mechanism of cellulosomes is mainly concentrated at the transcriptional level; for example, studies on growth rate coupling have shown that the expression level of cellulosomes is negatively correlated with the rate of cell growth, and cellulosomes are also subject to multiple substrate coupling regulation and negative feedback inhibition. According to prior art records, in other species, increasing the expression level of a single protein can usually be achieved by enhancing the promoter of the corresponding gene, optimizing the codons of the protein-coding gene, optimizing the signal peptide of the exogenous protein, and fusing it with a highly expressed gene. However, cellulosomes involve dozens of genes, and the components and regulatory mechanisms are extremely complex, and these solutions are difficult to implement. Therefore, improving the expression level of cellulosomes by modifying Clostridium thermocellum is still a technical problem that needs to be solved urgently.

[0005] HtrA (high-temperature requirement A) family proteins are a class of highly conserved heat shock serine proteases that are widely present in prokaryotes, plants and mammals. HtrA proteins contain signal peptides or transmembrane proteins, and the protease domain is located outside the membrane, usually involved in the post-translational regulation of extracellular proteins. According to literature reports, the functions of HtrA family proteins vary to a certain extent in different strains, but there are no reports that HtrA proteins have adverse effects on bacterial protein expression or excretion. Summary of the invention

[0006] In view of the problems existing in the prior art in the expression of cellulosomes of Clostridium thermocellum, the present invention first provides the application of inactivating the gene encoding the protein HtrA1_Ct in increasing the expression of cellulosomes of Clostridium thermocellum. On this basis, the present application also provides an engineered strain of Clostridium thermocellum ΔhtrA1 with high expression of cellulosomes and its construction method and application. The present application constructs an engineered strain ΔhtrA1 by inactivating the gene (clo1313_0972) of the HtrA family protein HtrA1_Ct in Clostridium thermocellum, significantly increasing the expression of cellulosomes and achieving unexpected technical effects.

[0007] The technical solution of the present invention:

[0008] The application of inactivating the gene encoding protein HtrA1_Ct in increasing the expression of cellulosomes in Clostridium thermocellum, the protein sequence encoded by the gene is shown in SEQ ID NO: 1. It is well known to those skilled in the art that the 8 strains of Clostridium thermocellum that have completed genome sequencing in the NCBI database all have genes encoding HtrA1_Ct proteins, and the amino acid sequences of HtrA1_Ct are 100% identical. It can be seen that HtrA1_Ct protein is ubiquitous in Clostridium thermocellum and its function is conservative; therefore, the technical effects produced by inactivating the gene encoding protein HtrA1_Ct in Clostridium thermocellum are consistent, and both can significantly increase the expression of cellulosomes, which not only fills the gap in the prior art on the influence of HtrA family proteins on bacterial protein expression, but also produces unexpected technical effects.

[0009] The engineered strain ΔhtrA1 of Clostridium thermocellum highly expressing cellulosomes is constructed with Clostridium thermocellum DSM1313 as the chassis strain, and the gene clo1313_0972 encoding the HtrA1_Ct protein in the engineered strain ΔhtrA1 is inactivated. The cellulosome expression and cellulase activity of the engineered strain ΔhtrA1 of Clostridium thermocellum are significantly increased, thereby significantly improving the saccharification efficiency of the whole bacteria, producing unexpected technical effects. This is because the inventors accidentally discovered in the process of studying the cellulosome regulation mechanism that the HtrA family protein HtrA1_Ct of Clostridium thermocellum may be detrimental to the exocytic expression of cellulosomes, and its inactivation can significantly increase the amount of cellulosomes secreted by Clostridium thermocellum, thereby having the effect of enhancing the ability of Clostridium thermocellum to degrade cellulose substrates, and has application prospects in lignocellulose saccharification and bioconversion.

[0010] As described above, the method for constructing the engineered strain ΔhtrA1 of Clostridium thermocellum achieves the inactivation of the gene clo1313_0972 encoding the HtrA1_Ct protein by including but not limited to gene knockout, gene targeting insertion, site-directed mutagenesis or small RNA-mediated gene silencing. The aforementioned method for inactivating a gene is well known to those skilled in the art. The present invention uses a gene knockout method to inactivate the gene encoding the protein HtrA1_Ct, including step (1) constructing a knockout plasmid and step (2) transforming Clostridium thermocellum and knocking out the gene clo1313_0972. Specifically:

[0011] (1) The homology arm sequence of the coding gene clo1313_0972 of Clostridium thermocellum HtrA1_Ct was designed, and the homology arm sequence was connected to the homologous recombination plasmid by gene cloning to obtain a knockout plasmid.

[0012] (2) The aforementioned knockout plasmid was transformed into Clostridium thermocellum, and the knockout of the gene encoding HtrA1_Ct was achieved and verified by homologous recombination screening.

[0013] A bacterial agent of the engineered strain ΔhtrA1 of Clostridium thermocellum as described above.

[0014] The application of the aforementioned engineered strain of Clostridium thermocellum ΔhtrA1 in degrading lignocellulose is specifically as follows:

[0015] (1) Preparing seed solution: inoculating the engineered strain ΔhtrA1 into a fermentation medium under anaerobic conditions, and culturing under certain conditions until the mid-logarithmic growth period to obtain a seed solution. The initial pH of the fermentation medium is 5.6-7.5, and the culture conditions are: a temperature of 55-65° C. and a rotation speed of 150-250 rpm.

[0016] (2) Saccharification: The seed liquid is then inoculated into a saccharification medium containing a lignocellulosic substrate, and a hydrolysis reaction is carried out at a certain temperature and rotation speed to obtain a sugar solution containing glucose. The solid-liquid weight volume ratio of the lignocellulosic substrate to the saccharification medium is 2-10%, and the volume ratio of the seed liquid to the saccharification medium is 10%. The conditions for the hydrolysis reaction are: a temperature of 55-65°C and a rotation speed of 150-250rpm.

[0017] Preferably, the saccharification medium also includes β-glucosidase; the dosage of the β-glucosidase is 40-200U β-glucosidase per 100mL saccharification system. By adding additional β-glucosidase, the feedback inhibition of cello-oligosaccharides on cellulosomes is relieved, the reducing sugar yield and the maximum sugar production rate are significantly increased, thereby further improving the saccharification effect of the system.

[0018] Beneficial effects of the present invention:

[0019] (1) The present invention first and foremost provides the use of inactivating the gene encoding the protein HtrA1_Ct in increasing the expression of cellulosomes in Clostridium thermocellum, which not only produces unexpected technical effects, but also has important guiding value for subsequent research on HtrA family proteins and Clostridium thermocellum.

[0020] (2) The present invention also provides an engineered strain of Clostridium thermocellum ΔhtrA1 that highly expresses cellulosomes. The engineered strain ΔhtrA1 is constructed by inactivating the gene encoding the protein HtrA1_Ct. Compared with the control strain, the engineered strain ΔhtrA1 has a higher cellulosome expression level and a higher cellulase activity, and the technical effect is significantly improved and unexpected.

[0021] (3) Compared with the prior art, the engineered strain ΔhtrA1 of Clostridium thermocellum described in the present application achieves a significant increase in yield while maintaining the specific enzyme activity of cellulosomes, thereby greatly improving the efficiency of the engineered strain in degrading cellulose substrates, thereby improving production efficiency and reducing production costs, and has important industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Attached Figure 1 Schematic diagram of the plasmid pHK-homo used to construct the highly expressed cellulosome engineering strain ΔhtrA1 in Example 1 of the present invention.

[0023] Attached Figure 2 The growth condition of the highly expressed cellulosome engineered strain ΔhtrA1 in Example 2 of the present invention under the conditions of cellobiose (A) and microcrystalline cellulose (B) as the sole carbon sources.

[0024] Attached Figure 3 The expression of cellulosomes in the highly expressed cellulosome engineering strain ΔhtrA1 in Example 3 of the present invention when cultured with microcrystalline cellulose as the carbon source.

[0025] Attached Figure 4 This is the test result of the cellulase activity in the supernatant of the highly expressed cellulosome engineered strain ΔhtrA1 in Example 4 of the present invention when cultured with microcrystalline cellulose as the sole carbon source.

[0026] Attached Figure 5 This is a verification of the transcription of cellulosome-related genes in the cellulosome-highly expressed engineered strain ΔhtrA1 in Example 5 of the present invention. The relative transcription level in the figure is the ratio of the transcription level of the engineered strain ΔhtrA1 to the transcription level in the control strain.

[0027] Attached Figure 6 The saccharification results of the highly expressed cellulosome engineered strain ΔhtrA1 in Example 6 of the present invention at a substrate concentration of 4% (w%v) microcrystalline cellulose. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the embodiments.

[0029] Example 1: Construction of engineered strain ΔhtrA1

[0030] It is well known to those skilled in the art that the eight strains of Clostridium thermocellum that have completed genome sequencing in the NCBI database all have genes encoding HtrA1_Ct proteins, and the strain numbers and corresponding HtrA1_Ct protein numbers are: DSM 1313 (ADU74040.1), ATCC 27405 (ABN52518.1), AD2 (ALX07978.1), DSM 26360 (ANV75724.1), BC1 (CDG35956.1), YS (EIC06176.1), M3 (UWV47875.1), PAL5 (XDO42405.1). The amino acid sequences of HtrA1_Ct in all the above strains are 100% identical, which indicates that HtrA1_Ct protein is ubiquitous in Clostridium thermocellum and its function is conserved. Therefore, the technical effect of inactivating the gene encoding protein HtrA1_Ct in Clostridium thermocellum is consistent, and both can significantly increase the expression of cellulosomes. In this example, Clostridium thermocellum DSM 1313 strain is used as an example to construct an engineered strain.

[0031] In addition, it is well known to those skilled in the art that gene inactivation can be achieved by gene knockout, gene targeting insertion, site-directed mutagenesis or small RNA-mediated gene silencing. In view of this, the present invention uses gene knockout as an example to construct an engineered strain ΔhtrA1. The open reading frame of HtrA1_Ct of Clostridium thermocellum (encoded by the nucleic acid sequence 1131697 to 1133226 in the genome CP002416.1) is used as the gene knockout site. Firstly, the upstream and downstream homologous arms of the target gene were amplified from the genomic DNA of Clostridium thermocellum. The homologous arm sequences were ligated to the pHK-homo plasmid vector according to the seamless cloning protocol of Novagen (Nanjing). The constructed plasmid was modified to remove the Dcm methylation status and then electroporated into the C. thermocellum strain ΔpyrF. Positive transformants were identified by PCR and continuously screened on MJ solid medium containing FUDR (5-fluoro-2-deoxyuracil, 10 μg / mL) and GS-2 solid medium containing 5-FOA (5-fluoroacetic acid, 500 μg / mL) to obtain the engineered strain ΔhtrA1.

[0032] The specific steps are:

[0033] (1) The homologous recombination plasmid pHK-homo( Figure 1) was transformed into the control strain ΔpyrF (pyrF deleted strain), and screened on GS-2 semisolid medium (KH2PO4 1.5 g / L, K2HPO4·3H2O 3.8 g / L, urea 2.1 g / L, MgCl2·6H2O 1.0 g / L, CaCl2·2H2O 150 mg / L, FeSO4·6H2O 1.25 mg / L, cysteine ​​hydrochloride 1.0 g / L, MOPS sodium salt 10 g / L, yeast extract 6.0 g / L, cellobiose 5.0 g / L, trisodium citrate dihydrate 3.0 g / L, resazurin 0.1 mg / L, high temperature agar 0.03 g / L, pH 7.4) plates containing chloramphenicol methyl sulfoxide to obtain plasmid transformants.

[0034] (2) The obtained transformants were transferred for three generations in MJ liquid medium (KH2PO4 1.5 g / L, K2HPO4·3H2O 3.8 g / L, urea 2.1 g / L, MgCl2·6H2O 1.0 g / L, CaCl2·2H2O 150 mg / L, FeSO4·6H2O 1.25 mg / L, cysteine ​​hydrochloride 1.0 g / L, MOPS sodium salt 10 g / L, cellobiose 5.0 g / L, trisodium citrate dihydrate 3.0 g / L, resazurin 0.1 mg / L, pyridoxamine hydrochloride 2 mg / L, biotin 0.2 mg / L, p-aminobenzoic acid 0.4 mg / L, vitamin B12 0.2 mg / L, pH 7.4) and then coated with MJ semisolid medium containing 10 μg / mL 5-fluorodeoxyuridine (FUDR) for the first homologous recombination screening. In this step, Tdk encoded on the plasmid can convert FUDR into F-dUMP, which is toxic to cells. At the same time, the chassis cells must rely on the PyrF protein encoded by the sequence between the two homologous arms on the plasmid to synthesize uracil nucleotides in order to survive in MJ medium. Therefore, this screening strategy ensures that the strain can survive only when the homologous recombination module on the plasmid undergoes homologous recombination with the genome (thus integrating the PyrF encoding gene into the genome) and the recombined plasmid with the Tdk encoding gene is lost from the strain. According to the principle that long homologous arms preferentially undergo homologous recombination, the two long homologous arms before and after first undergo homologous recombination with the genome, and the recombinants obtained at this time are restored to the uracil auxotrophy type of the starting strain.

[0035] (3) The recombinants obtained after the first homologous recombination were first passaged three times in GS-2 liquid medium, and the bacterial solution was gradiently diluted with the same medium and then coated with GS-2 semi-solid medium containing 500 μg / mL 5-fluoroorotic acid (FOA) for screening to obtain the target gene knockout strain, namely the engineered strain ΔhtrA1. In this step, the reverse screening effect of PyrF will promote the second homologous recombination of the upstream long homologous arm and the short homologous arm, removing the pyrF expression frame from the genome. After the second homologous recombination, the mutant strain mutated from the prototrophic type to the uracil auxotrophic type, thereby achieving the knockout of the gene at the target site on the genome. The obtained engineered strain ΔhtrA1 was verified by PCR and DNA sequencing to confirm that the htrA1 gene had been knocked out.

[0036] Example 2: Determination of growth phenotype of engineered strain ΔhtrA1

[0037] The growth phenotype of the engineered strain ΔhtrA1 obtained in Example 1 was determined, mainly for the growth when cellobiose and microcrystalline cellulose were the sole carbon sources ( Figure 2 ). The specific steps are as follows:

[0038] 1) Seed liquid activation: The engineered strain ΔhtrA1 and the control strain ΔpyrF were cultured in GS-2 medium containing 5 g / L cellobiose as carbon source at 55°C and 170 rpm horizontal shaking for three generations until the activity was the best and the OD 600 Transfer after about 1.0.

[0039] 2) Growth curve determination: 1% of the activated seed solution was inoculated into anaerobic bottles containing 5 g / L cellobiose or microcrystalline cellulose as carbon source GS-2 liquid culture medium, and cultured at 55°C with horizontal shaking at 170 rpm. Three independent fermentations were performed for each strain. 1.5 mL samples were collected from these cultures every 4-6 hours.

[0040] 3) Determination of cellobiose carbon source growth curve: Monitoring the optical density at 600 nm (OD 600 ) was used to determine the growth of the strain in cellulose sugar medium.

[0041] 4) Determination of the growth curve of microcrystalline cellulose carbon source: Since cells will bind to the solid surface of microcrystalline cellulose through the surface fibrils, the cell growth is monitored by the amount of precipitated protein. The Bradford method is used to quantitatively determine the protein in the cell particles. The bacterial solution is centrifuged at 12000g for 2 minutes, and the culture medium supernatant is discarded. The obtained cell pellet is rinsed with sterile distilled water, then resuspended in 0.2M NaOH and concentrated 5 times. Boil for 10 minutes, centrifuge twice, and take the supernatant for protein quantification using Bradford protein Assay Kit (Beyotime Biotech, Shanghai).

[0042] Depend on Figure 2 It can be seen that in the presence of cellobiose ( Figure 2 A) or microcrystalline cellulose ( Figure 2 B) During the carbon source culture process, there was no obvious growth difference between the ΔhtrA1 mutant and the control ΔpyrF. This shows that the HtrA1_Ct protein is not essential for the growth of Clostridium thermocellum, and the inactivation of htrA1 does not affect the normal life activities of Clostridium thermocellum.

[0043] Example 3: Analysis of the expression of cellulosomes in the engineered strain ΔhtrA1

[0044] (1) Quantitative analysis of supernatant protein

[0045] In order to analyze the effect of HtrA1_Ct on cellulosome expression, this example first quantitatively analyzed the proteins secreted into the culture supernatant of the engineered strain ΔhtrA1 during the growth of Avicel culture. The specific steps are as follows:

[0046] According to the established growth curve, the culture fluid of the engineered strain ΔhtrA1 at different growth stages in the medium with microcrystalline cellulose as the carbon source was collected. The supernatant was collected by centrifugation at 12000g, and the supernatant protein was filtered with a 0.22μm syringe filter and then concentrated 20 times with an ultrafiltration concentrator with a molecular weight cutoff of 10.0kDa. The protein concentration was determined using the Bradford method, and the results are shown in Figure 3 As shown in A.

[0047] Depend on Figure 3A shows that secreted proteins can be detected in the culture supernatant from the beginning of the logarithmic growth phase of the strain (after 10h), but compared with the growth curve, after the strain growth enters the plateau phase (after 20h), the exocytotic protein concentration is still increasing, and it does not level off until 30h later. It can be seen that the supernatant protein concentration of the engineered strain ΔhtrA1 has increased significantly compared with the control strain ΔpyrF. Among them, the supernatant protein of the engineered strain ΔhtrA1 in the late logarithmic phase increased by about 50% compared with the control strain ΔpyrF. This shows that the amount of exocytotic protein in the engineered strain ΔhtrA1 has been significantly increased.

[0048] (2) SDS-PAGE analysis of supernatant proteins

[0049] Since the exoproteins of Clostridium thermocellum can be divided into cellulosome components and non-cellulosome components, this example uses SDS-PAGE to detect the supernatant proteins in the early, logarithmic and plateau phases of growth with Avicel as the carbon source to analyze the expression changes of cellulosomes. SDS-PAGE was performed using a 4-20% gradient precast gel (Yeasen, Shanghai) to ensure that there was an equal amount of culture supernatant in each well. ImageJ was used to determine the intensity of the relevant bands in the extracellular protein, using the band intensity of CipA as a reference and marking the main protein band of the cellulosome with a red arrow. The results are shown in Figure 2. Figure 3 As shown in B.

[0050] Depend on Figure 3 B shows that during the logarithmic growth phase of the bacteria, protein excretion can be detected in the culture supernatant. Thereafter (logarithmic phase and plateau phase), the supernatant protein concentration of ΔhtrA1 increased significantly compared with the control strain ΔpyrF. Among them, in the late logarithmic phase, the supernatant protein of ΔhtrA1 increased by about 50% compared with the control strain ΔpyrF. Under the condition that each well contains the same equivalent volume of culture supernatant, the protein bands of the main components of the cellulose body of the ΔhtrA1 mutant are significantly thickened, including the four highest content components of the cellulose body: the only primary scaffolding protein CipA, the main secondary scaffolding protein OlpB, and the main exonucleases Cel48S and Cel9K. This shows that the protein concentration of the above cellulose body components in the culture supernatant has increased significantly.

[0051] Furthermore, the four main fibrillar components in the supernatant were quantitatively analyzed using the gel grayscale analysis software ImageJ. The results are as follows: Figure 3 C. Figure 3C shows that: (a) in the middle or early logarithmic phase (sampling at 15h of culture), the differences between the bands in the supernatant protein are not large; (b) in the supernatant protein cultured to the late logarithmic phase (20h), the target protein bands of the engineered strain ΔhtrA1 all showed an increase of more than 2 times, especially the fibrosome scaffolding proteins OlpB and CipA, which increased by 4.96 times and 2.63 times that of the wild type, respectively; (c) in the supernatant protein cultured to the stationary phase (30h), the engineered strain ΔhtrA1 also showed an increase of 0.5-0.7 times. It can be seen that the results of ImageJ grayscale analysis are basically consistent with the results of supernatant protein concentration determination, both indicating that the expression of fibrosome components in the engineered strain ΔhtrA1 has increased significantly.

[0052] In summary, it can be preliminarily speculated that HtrA1_Ct anchored in the periplasmic space plays the role of a protease, regulating the protein level of the cellulosome components secreted into the extracellular space, thereby limiting the expression of the cellulosome. Therefore, its inactivation significantly increases the expression of the cellulosome.

[0053] Example 4: Analysis of enzyme activity of cellulose in the supernatant of engineered strain ΔhtrA1

[0054] In order to analyze whether the inactivation of htrA1 affects the activity of cellulosomes, or whether the cellulosomes increased in the supernatant of the engineered strain ΔhtrA1 have normal cellulosome activity, the cellulose degradation ability and specific enzyme activity of the culture supernatant of the engineered strain ΔhtrA1 were measured in this example ( Figure 4 ). The specific steps are as follows:

[0055] 1) According to the established growth curve, the culture fluid of the engineered strain ΔhtrA1 at different growth stages cultured in a medium with microcrystalline cellulose as a carbon source was collected, and the obtained supernatant was mainly composed of cellulose and was directly used to determine the cellulase activity.

[0056] 2) For the saccharification reaction of the supernatant, 1 mL of the supernatant was used as the crude enzyme solution, 40 mg of microcrystalline cellulose was used as the substrate, and thiamphenicol (34 μg / ml) was added to inhibit cell growth in a 1.0 ml reaction system. The hydrolysis experiment was carried out on a horizontal shaker at 170 rpm and 55 °C for 17 h. A substrate-free reaction system was prepared as a negative control. The 3,5-dinitrosalicylic acid (DNS) method was used to determine the reducing sugar to quantify the hydrolysis product. One unit of enzyme activity is defined as the volume (mL) or amount (μg) of the supernatant that releases 1 nmol of reducing sugar (glucose equivalent) per minute.

[0057] Depend on Figure 4 A shows that, with microcrystalline cellulose as substrate, the yield of reducing sugar produced by degrading microcrystalline cellulose per unit volume of culture supernatant of the engineered strain ΔhtrA1 is significantly increased compared with that of the control strain ΔpyrF; Figure 4 B shows that the specific enzyme activity per unit mass of the culture supernatant of the engineered strain ΔhtrA1 is basically the same as that of ΔpyrF. This indicates that: (1) the inactivation of htrA1 increases the expression of cellulosomes; (2) the cellulosomes have the same specific enzyme activity as the control strain ΔpyrF, and the inactivation of htrA1 does not affect the quality of cellulosomes.

[0058] In summary, HtrA1_Ct inactivation can not only increase the expression of effective cellulosomes, but also does not affect the activity of cellulosomes, which is of great significance for the saccharification of lignocellulose using Clostridium thermocellum.

[0059] Example 5: Analysis of transcriptional levels of engineered strain ΔhtrA1

[0060] In order to further analyze the relationship between HtrA1_Ct and cellulosome regulation, this example used qRT-PCR to analyze the transcription of some cellulosome-related genes in ΔhtrA1 using microcrystalline cellulose as the carbon source ( Figure 5 ). The specific steps are as follows:

[0061] (1) According to the growth curve when microcrystalline cellulose was the sole carbon source, the cells cultured to the early logarithmic phase (15 hours) and the late logarithmic phase (20 hours) were collected and centrifuged at 10,000 g at 4°C. The supernatant was discarded and the cells were frozen at -80°C for RNA extraction.

[0062] (2) RNA extraction process: Use lysozyme and β-mercaptoethanol to resuspend the bacteria and let it stand at 37°C for 10 minutes. Then add 1mL Trizol to the bacterial lysate, mix it thoroughly with a pipette tip, and then use a vortex machine to shake for 1 minute, and let it stand at room temperature for 5-10 minutes. Continue to add 200μL of chloroform, mix it upside down, and shake it vigorously on a vortex machine for 1 minute to emulsify the mixture into milky white as much as possible. After standing at room temperature for 5 minutes, centrifuge at low temperature and centrifuge at 12000g for 15 minutes to obtain the RNA, protein, and organic phase layer liquid. Pipette 800μL of RNA layer liquid and place it on ice, and add 431μL of pre-cooled anhydrous ethanol. The mixed solution was transferred into a centrifugal column (Qiagen), centrifuged at 12000g for 15s at room temperature, and the waste liquid was discarded; 700μL Buffer RW1 (Qiagen) was added, and the solution was centrifuged at 10000g for 15s at room temperature, and the waste liquid was discarded; 500μL Buffer RPE was added, and the solution was centrifuged at 12000g for 15s at room temperature, and the waste liquid was discarded. After repeating the step of adding Buffer RPE, the centrifugal column was emptied and centrifuged at 12000g for 3min. 30μL RNase free ddH2O was used to elute the centrifugal column. The final eluate was the extracted total RNA.

[0063] (3) Reverse transcription of cDNA: RevertAid First Strand cDNA Synthesis Kit (ThermoScientific) was used to reverse transcribe cDNA from the RNA extracted in 2). The reaction system was as follows: 1-5 μg RNA (DNA removed), 1 μL Random Hexamer primer, 2 μL 10 mM dNTP Mix, 4 μL 5× Reaction Buffer, 1 μL RiboLock RNase Inhibitor (20 U / μL), 1 μL RevertAid M-MuLV RT (200 U / μL), and RNase free ddH2O was added to make up the reaction volume to 20 μL. The reaction system was prepared on ice. The reaction procedure was as follows: 25°C for 5 min, 42°C for 60 min, 70°C for 5 min, and 4°C for insulation. After reverse transcription, qPCR analysis can be performed immediately, or the synthesized cDNA can be frozen at -80°C for later use.

[0064] (4) Real-time fluorescence quantitative PCR reaction: The cDNA obtained in (3) was subjected to real-time fluorescence quantitative PCR reaction. The reaction system used was QuantiNova SYBR Green PCR Kit (Qiagen). The reaction system was: 1 μg cDNA, 10 μM Primer A, 10 μM Primer B, 10 μL 2×SYBR Green PCR Master Mix, and RNase free ddH2O was added to make up the reaction volume to 20 μL. Specific detection primers were designed according to the base sequence of the gene to be tested. The real-time fluorescence quantitative PCR reaction was measured in a fully automatic fluorescence quantitative PCR system to obtain C T The housekeeping gene gapDH (glyceraldehyde-3-phosphate dehydrogenase, Clo1313_2095) was used as the reference gene and the Livak method (2 -ΔΔCT Method) to calculate the transcription level of the gene.

[0065] (5) Calculation of relative transcription levels: The gene transcription levels of the engineered strain ΔhtrA1 and the control strain ΔpyrF were first measured as described above, and then the transcription level of each gene in the engineered strain ΔhtrA1 was divided by its transcription level in the control strain ΔpyrF to obtain the relative transcription level of the gene in the engineered strain ΔhtrA1 and the control strain ΔpyrF.

[0066] Depend on Figure 5A shows that in the early logarithmic phase (15h), except for the OlpB gene, the relative transcription levels of the main components of the cellulosome, such as CipA, Cel48S, Cel9K, and three SigI factors (known regulatory proteins that regulate cellulosome expression), were not much different between the engineered strain ΔhtrA1 and the control strain ΔpyrF (all p values ​​were greater than 0.5). Figure 5 B shows that in the late logarithmic phase (20h), the relative transcription levels of the main components of the cellulosomes of the engineered strain ΔhtrA1 increased by 2-3 times compared with the early logarithmic phase; at the same time, the transcription levels of the three SigI factors did not change significantly. This shows that the inactivation of htrA1 has a certain transcriptional regulation on the cellulosome components, but it has little to do with the known SigI / RsgI regulatory system, and there may be unknown regulatory mechanisms.

[0067] Example 6: Whole-cell saccharification based on engineered strain ΔhtrA1

[0068] In view of the fact that there have been detailed reports on the use of Clostridium thermocellum for whole-cell saccharification, the present application uses the engineered strain ΔhtrA1 constructed in Example 1 to perform a saccharification experiment using 4% (w / v) microcrystalline cellulose as a substrate and cultured at 55°C as an example. At the same time, a group of experimental groups (Examples 7 and 9) in which β-glucosidase was added to the saccharification system was set up to further relieve the feedback inhibition of cellobiose during the saccharification process and improve the saccharification ability of Clostridium thermocellum ( Figure 6 ). This example uses β-glucosidase CaBgl (GenBank database number JX030398.1) from Caldicellulosiruptor sp. F32; the addition of BGL can relieve the feedback inhibition of cellobiose on cellulosomes (Biotechnol Biofuels (2017) 10: 124; Patent 201710324806.X) has been disclosed. The specific steps are as follows:

[0069] (1) Seed liquid activation: Under anaerobic conditions, the engineered strain ΔhtrA1 was cultured three times in a medium containing 5 g / L cellobiose as the sole carbon source at a 1% volume fraction, at 55°C and 170 rpm horizontal shaking. Determination of the OOD of the third generation bacterial liquid 600 , in OD 600 The bacterial liquid with a concentration of 1.0 was transferred to a culture medium containing 5 g / L microcrystalline cellulose carbon source at a 1% volume fraction inoculation ratio and cultured at 55°C and 170 rpm for 30 h. At this time, the expression of cellulosomes in the culture medium was the highest and the cellulosome activity was the best, and this was used as the seed liquid for inoculation of the saccharification medium.

[0070] (2) Whole-bacteria saccharification: The seed liquid activated in step (1) was inoculated into saccharification GS-2 medium with 40 g / L microcrystalline cellulose as the sole carbon source at a volume fraction ratio of 5%. The culture was cultured at 55°C and 170 rpm with horizontal shaking. The saccharification lasted for 8 days. During this process, 0.5 mL of the culture was sampled every 12 hours for the first 2 days, and then every day. After centrifugation at 12000 g, the supernatant of the sample was analyzed for saccharification effect, and the amount of reducing sugar generated was determined using the DNS method.

[0071] (3) Saccharification curve fitting. The reducing sugar concentration obtained by saccharification in 2) and the corresponding saccharification time were used to fit the reducing sugar production curve of each full-bacteria saccharification system using the improved Gompertz equation. The improved Gompertz equation formula is:

[0072]

[0073] In the above formula, M(t) is the cumulative reducing sugar production (mg / mL), P is the expected maximum reducing sugar production (mg / mL), and R m is the maximum sugar production rate (mg / mL / d), λ is the lag time, and e is the exponential constant 2.718. The expected maximum reducing sugar yield M is calculated by dividing P by the initial substrate concentration. max (g glucose / g microcrystalline cellulose). The reducing sugar production curve of each experimental group was fitted according to the improved Gompertz equation. The parameter values ​​of the fitting curve, R 2 and obtain M max Shown in Table 1.

[0074] Example 7: Whole-cell saccharification of engineered strain ΔhtrA1 supplemented with CaBgl

[0075] The difference from Example 6 is that in step (2) the whole-cell saccharification step, 80 U of CaBgl protein was added to the saccharification GS-2 medium, and then the whole-cell saccharification was carried out under the same conditions.

[0076] Example 8: Whole-cell saccharification of control strain ΔpyrF

[0077] The difference from Example 6 is that in step (1), the control strain ΔpyrF with pyrF knocked out was used for seed liquid activation. Then, whole-cell saccharification was performed under the same conditions.

[0078] Example 9: Whole-cell saccharification of control strain ΔpyrF supplemented with CaBgl

[0079] The difference from Example 8 is that in step (2) the whole-cell saccharification step, 80 U of CaBgl protein was added to the saccharification GS-2 medium, and then the whole-cell saccharification was carried out under the same conditions.

[0080] Table 1. Results of whole cell saccharification in Examples 6-9

[0081]

[0082] * The wild-type strain M max Set to 1.

[0083] From Table 1, we can see that:

[0084] (1) The expected maximum reducing sugar yield P of the engineered strain ΔhtrA1 is 17.29 mg / mL, the maximum sugar production rate Rm is 8.50 mg / mL / d, the lag time λ is 0.28 d, and the expected maximum reducing sugar yield M max The expected maximum reducing sugar yield P of the control strain ΔpyrF is only 13.85 mg / d, the maximum sugar production rate Rm is 7.00 mg / mL / d, the lag time λ is 0.44 d, and the expected maximum reducing sugar yield M max It is 0.346g glucose / 1.00g microcrystalline cellulose. It can be seen that compared with the control strain ΔpyrF, the maximum reducing sugar yield P of the engineered strain ΔhtrA1 increased by 25%, the maximum sugar production rate Rm increased by 21%, the lag time decreased by 36%, and the maximum reducing sugar yield M max This indicates that the saccharification capacity of the engineered strain ΔhtrA1 is significantly higher than that of the control strain ΔpyrF, and the engineered strain ΔhtrA1 has a better saccharification effect.

[0085] (2) The expected maximum reducing sugar yield P of the engineered strain ΔhtrA1 with CaBgl added was 34.49 mg / mL, the maximum sugar production rate Rm was 18.56 mg / mL / d, the lag time λ was 0.34 d, and the expected maximum reducing sugar yield M max The expected maximum reducing sugar yield P of the control strain ΔpyrF with CaBgl added was 33.10 mg / mL, the maximum sugar production rate Rm was 15.18 mg / mL / d, the lag time λ was 0.79 d, and the expected maximum reducing sugar yield M max It is 0.808g glucose / 1.00g microcrystalline cellulose. It can be seen that the engineered strain ΔhtrA1 with CaBgl added has an expected maximum reducing sugar yield P increased by 4% compared with the control strain ΔpyrF with CaBgl added; the maximum sugar production rate Rm increased by 22%, the lag time decreased by 57%, and the expected maximum reducing sugar yield M maxThis shows that with the addition of CaBgl, the saccharification capacity of the engineered strain ΔhtrA1 is still significantly higher than that of the control strain ΔpyrF, and the engineered strain ΔhtrA1 has a better saccharification effect, especially in terms of saccharification rate.

[0086] (3) Regardless of the engineered strain ΔhtrA1 or the control strain ΔpyrF, the expected maximum reducing sugar yield P, maximum sugar production rate Rm, and expected maximum reducing sugar yield M of the saccharification process after adding CaBgl max Both are significantly higher than the saccharification process without adding CaBgl, which is completely consistent with the expected effect of adding β-glucosidase matched with cellosomes, that is, relieving the feedback inhibition of cellobiose on cellosomes. Therefore, in practical applications, the addition of β-glucosidase is very necessary.

[0087] In summary, during the 8-day saccharification process, the addition of CaBgl relieved the feedback inhibition of cello-oligosaccharides on cellulosomes, and significantly increased the reducing sugar yield and the maximum sugar production rate, which was consistent with expectations. At the same time, both in the experimental group without CaBgl and in the experimental group with CaBgl, the engineered strain ΔhtrA1 had a significant improvement in the expected maximum reducing sugar yield and maximum sugar production rate compared with the control strain ΔpyrF, and the lag time was shortened by nearly half.

[0088] In addition, the saccharification system of the engineered strain ΔhtrA1 is expected to have a maximum reducing sugar yield M max Compared with the control strain ΔpyrF, there is a 1.25-fold increase. In the saccharification system with the addition of CaBgl, the expected maximum reducing sugar yield of ΔhtrA1 is only slightly higher than that of WT (ΔpyrF); this is because part of the cellulose will be consumed by cell growth, and this expected maximum reducing sugar yield has basically reached the maximum value of sugar production by Clostridium thermocellum on the degradation of microcrystalline cellulose substrate. This result further shows that the inactivation of HtrA1 protein is conducive to the increase of effective cellulosome expression. Even when the maximum reducing sugar yield close to the limit can be obtained, the maximum sugar production rate can be increased and the lag time can be shortened, thereby showing a significant improvement in saccharification performance.

[0089] SEQ ID NO.1:

[0090] MDELNYNGFSNEENEIKSFGELNNTTGENVDANINGDVVENAAQYAAENIIENVDADVGENAAEGFGENVIENAAVGVADKRVENEKTYEGSFVDLKSASYYSESYTKPKKRKNSVLLQMILVAVMSSILGGSIVGGFFVFGVPALSPSVQSIFRNTNVQNGSNDATSGVDTDYYKKVVIENNADSSVVVAIAEKVGPSVVGISVKSTTSISDFWFFTPRDTESQGSGIIIRSDGYIMTNYHVIESALNGRTNTLLPNASINVILPSDPDTPPHPATVVGTDSKTDLAVLKIEATNLPVIEFGDSDKIRVGELAVAIGNPGGLEYMGSVTVGVISGLNRTIPITDGKELKLIQTDAAINPGNSGGALVNAEGKLIGVNTAKIGQGYEGLGFAIPVNKAKEITDSLIQYKYVRGRPSLGIQINSGYTKEIADRYGLPEGVLVYNVEIFSAAYKAGIQKDDIITEFNGVRVKNYDELEEQKNKYKPGDKVKLKIHRDGKDITVEVTLDEQK

Claims

1. Use of an inactivated gene encoding protein HtrA1_Ct in increasing the expression of cellulosomes of Clostridium thermocellum, characterized in that: The protein sequence encoded by the gene is shown in SEQ ID NO:

1.

2. An engineered strain of Clostridium thermocellum ΔhtrA1 that highly expresses cellulosomes, characterized in that: The engineered strain ΔhtrA1 is constructed by using Clostridium thermocellum DSM1313 as a base strain, and the gene clo1313_0972 encoding the HtrA1_Ct protein in the genome of the engineered strain ΔhtrA1 is inactivated.

3. The method for constructing the engineered strain ΔhtrA1 of Clostridium thermocellum according to claim 2, characterized in that: Inactivation of the gene clo1313_0972 encoding the HtrA1_Ct protein is achieved by methods including but not limited to gene knockout, gene targeted insertion, site-directed mutagenesis or small RNA-mediated gene silencing.

4. The method for constructing the engineered strain ΔhtrA1 of Clostridium thermocellum according to claim 3, characterized in that: The gene knockout comprises the steps of (1) constructing a knockout plasmid and (2) transforming Clostridium thermocellum and knocking out the gene clo1313_0972. 5 . A bacterial agent comprising the engineered strain ΔhtrA1 of Clostridium thermocellum according to claim 2 .

6. Use of the engineered strain ΔhtrA1 of Clostridium thermocellum as claimed in claim 2 in degrading lignocellulose.

7. The use according to claim 6, characterized in that: The application is specifically as follows: (1) preparing seed liquid: inoculating the engineered strain ΔhtrA1 into a fermentation medium under anaerobic conditions, culturing under certain conditions until the middle logarithmic growth period, and obtaining seed liquid; (2) saccharification: then inoculating the seed liquid into a saccharification medium containing a lignocellulose substrate, performing a hydrolysis reaction at a certain temperature and rotation speed, and obtaining a sugar liquid containing glucose.

8. The use according to claim 6, characterized in that: In step (2), the solid-liquid weight volume ratio of the lignocellulose substrate to the saccharification medium is 2-10%, and the volume ratio of the seed liquid to the saccharification medium is 10%.

9. The use according to claim 6, characterized in that: In step (1), the initial pH of the fermentation medium is 5.6-7.5, and the culture conditions are: a temperature of 55-65° C. and a rotation speed of 150-250 rpm; and in step (2), the conditions for the hydrolysis reaction are: a temperature of 55-65° C. and a rotation speed of 150-250 rpm.

10. The use according to any one of claims 7 to 9, characterized in that: β-glucosidase is added to the saccharification medium; the dosage of the β-glucosidase is 40-200 U β-glucosidase per 100 mL of saccharification system.

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