Thermophilic L-arabinose inducible gene regulation system and application thereof

Abstract

The invention provides a thermophilic L-arabinose inducible gene regulation and control system suitable for clostridium thermocellum as well as construction and application of the thermophilic L-arabinose inducible gene regulation and control system. The inducible gene regulation system comprises (a) an AraR inhibitory protein and a strong promoter for controlling the expression of the protein, (b) an inducible promoter regulated by the AraR inhibitory protein, and (c) a reporter gene operably connected to the inducible promoter. As the transformation of an expression promoter in the AraR inhibitory protein significantly improves the induction multiple and reduces leakage expression, the thermophilic inducible gene expression regulation system disclosed by the invention has excellent leakproofness, inductivity and dynamic range; the technical blank that no high-performance induction system suitable for high-temperature strains exists in the prior art is filled. In addition, an inducer adopted by the induction system is L-arabinose; the inducer simultaneously meets two conditions of low cost and stable inducible expression, and has important significance for practical application. The invention further provides application of the inducible gene expression regulation system in other strains, the strains are high-temperature strains or medium-temperature strains, and the application range of the inducible gene expression regulation system is further expanded.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and relates to an inducible gene regulation system, in particular to a Clostridium thermocellum l-arabinose-inducible gene regulation system and construction and application thereof. BACKGROUND

[0002] Precise regulation of gene expression is one of the core problems in life science research, and researchers often need to specifically control the expression of target genes. Although traditional constitutive expression systems can drive high-level expression of genes, they lack controllability in time and dosage, and are difficult to meet the needs of fine research. Therefore, inducible gene regulation systems have emerged as the times require, and have become an important tool in modern molecular biology and bioengineering. At present, although inducible systems have been widely used in model organisms, there is still a lack of inducible systems suitable for non-model microorganisms, especially thermophilic bacteria.

[0003] Clostridium thermocellum (also known as Ruminiclostridium thermocellum, Hungateiclostridium thermocellum, and Acetivibrio thermocellus in the literature) is a thermophilic, anaerobic, lignocellulose-degrading bacterium that grows optimally at 60 °C; it can utilize a unique cellulase complex and metabolic pathway to convert cellulose into energy compounds, including ethanol, hydrogen, and various organic acids. Therefore, Clostridium thermocellum is one of the most promising host organisms for consolidated bioprocessing (CBP) and consolidated bio-enzymatic saccharification (CBS). The inventors' team has previously conducted several studies on Clostridium thermocellum, developing Clostridium thermocellum engineering strains for lignocellulose saccharification (Bioresour. Technol. 2021, 337: 125441; Biotechnol. Biofuels 2019, 12:35), and developing whole-cell catalysts for PET plastic degradation based on Clostridium thermocellum (J. Hazard. Mater. 2025, 488:137441). However, to develop high-performance Clostridium thermocellum strains that can degrade plant biomass and produce industrial-related chemicals, it is inevitable to advance the genetic and metabolic engineering of Clostridium thermocellum.

[0004] In recent years, researchers have developed some genetic modification tools suitable for Clostridium thermocellum, and the study of induction systems has also begun to start. However, among the different Clostridium induction systems reported, most are strain-specific and mesophilic, which limits their application in Clostridium thermocellum. Currently, only two induction systems suitable for Clostridium thermocellum have been reported: (1) One is based on its natural celC operon, using laminaribiose as an inducer (2015 Appl. Microbiol. Biotechnol. 99:7589−7599); however, since the inducer laminaribiose can be well metabolized by Clostridium thermocellum (2022 mBio 13(5):e01476-22), its dynamic range is narrow, its induction stability is poor, and the inducer is expensive, with high use cost. (2) The other is a 2-amino purine induction system based on a thermophilic purine riboswitch (ACSSynth. Biol. 2019, 8, 633−640), which only records that it induces sfGFP production in Geobacillus thermoglucosidasius to change by 10-30 times, and the dynamic range is also narrow. In addition, the literature also records its use in Clostridium thermocellum to produce ethanol, but does not record the induction performance, and the person skilled in the art reasonably speculates that its performance parameters in Clostridium thermocellum are not better than the former. In summary, in the prior art, the induction systems suitable for high-temperature strains such as Clostridium thermocellum all have the problems of low dynamic range and poor induction stability.

[0005] Therefore, it is of important practical application value to develop a high-performance (wide dynamic range, stable induction expression, low-cost inducer, low leakage, etc.) induction system suitable for high-temperature strains such as Clostridium thermocellum. SUMMARY

[0006] In view of the problems of the existing induction-type gene regulation system suitable for high-temperature strains such as Clostridium thermocellum, the present application provides a thermophilic l-arabinose induction-type gene regulation system suitable for Clostridium thermocellum and its construction and application. The promoter regulating the expression of AraR inhibitor protein in the thermophilic gene regulation system is the promoter P CbpB - araRT1 or P CbpB-araRT2 Through the foregoing modification, not only the induction fold is significantly improved, but also the leakage expression is reduced, so that the system has a wide dynamic range; at the same time, the regulation system also has the advantages of high host orthogonality, stable induction expression, low-cost inducer, etc., and has important practical application value.

[0007] Technical scheme of the present application:

[0008] The present application first provides an isolated promoter regulating expression of AraR repressor protein, the promoter being P CbpB - araRT1 or P CbpB-araRT2 ; the nucleotide sequence of the promoter P CbpB - araRT1 is shown as SEQ ID NO: 1, and the nucleotide sequence of the promoter P CbpB - araRT2 is shown as SEQ ID NO: 2. By modifying the promoter regulating expression of AraR repressor protein, the induction fold is significantly improved and the leaky expression is reduced, thereby having a wide dynamic range.

[0009] According to the literature, the repressor protein AraR in Geobacillus stearothermophilus T-6 can specifically bind to promoters (P araD and P abnE ), and the addition of l-arabinose can effectively prevent such binding. Therefore, l-arabinose can be used as a molecular inducer to regulate gene expression. However, the inventors tried to use the expression cassette of the promoter P araD or P abnE and the repressor protein AraR, and constructed an arabinose induction system in Clostridium thermocellum DSM1313. Although the strain showed obvious induced expression, the leaky activity was too high, the dynamic range was too narrow (only 5.4-fold), and the practical application value was not high. The inventors first tried to modify the inducible promoters P araD or P abnE , but neither of them could solve the above technical problems. After modifying the promoter P araR controlling the expression of AraR, it was found that the modified promoter P CbpB - araRT2 with the nucleotide sequence shown as SEQ ID NO: 2 achieved a significant improvement in induction fold and a significant reduction in leaky expression, thereby having a wide dynamic range, and the technical effect was outstanding. The modified promoter P CbpB - araRT1 with the nucleotide sequence shown as SEQ ID NO: 1, although the effect was not as significant as P CbpB - araRT2 , also achieved a significant improvement compared with the prior art.

[0010] The present application also provides an AraR repressor protein, the expression of which is controlled by the expression promoter P CbpB - araRT1 or P CbpB-araRT2; the amino acid sequence of the AraR inhibitor protein is shown as SEQ ID NO: 3; the AraR inhibitor protein can specifically bind to the promoter P araD or P abnE ; wherein, the nucleotide sequence of the promoter P araD is shown as SEQ ID NO: 4, and the nucleotide sequence of the promoter P abnE is shown as SEQ ID NO: 5. The promoters P araD and P abnE are both induced to express in Clostridium thermocellum; wherein, P abnE has a relatively higher dynamic range.

[0011] Preferably, the codon-optimized nucleotide sequence of the AraR inhibitor protein is shown as SEQ ID NO: 23.

[0012] The application also provides a thermophilic inducible gene expression regulation system, comprising: (a) an AraR expression cassette (comprising an expression promoter P CbpB - araRT1 or P CbpB-araRT2 and a gene encoding an AraR inhibitor protein) as described above, (b) an inducible promoter regulated by the AraR inhibitor protein, and (c) a reporter gene operably linked to the inducible promoter or a target gene regulated by the inducible promoter. Wherein, the inducible promoter is P araD or P abnE ; the reporter gene is taken as a heat-resistant beta-glucuronidase GusB, and the nucleotide sequence thereof is shown as SEQ ID NO: 6. Since the modification of the expression promoter in the AraR inhibitor protein significantly improves the induction fold and reduces the leaky expression, the thermophilic inducible gene expression regulation system described in the application performs well in terms of leakage, inducibility and dynamic range, filling the technical gap in the prior art that there is no high-performance inducible system suitable for high-temperature strains. In addition, the promoters and AraR proteins used in the application are mainly from heterologous bacteria different from Clostridium thermocellum (except that P CbpB - araRT1 and P CbpB-araRT2 each contain 69 nucleotides from Clostridium thermocellum), which have higher host orthogonality.

[0013] An engineered strain carrying an inducible gene expression regulation system, wherein the strain is integrated or transformed with the thermophilic inducible gene expression regulation system as described above; the strain is a high-temperature strain or a mesophilic strain. As described above, the addition of l-arabinose can release the inducible promoter P araD or P abnE;Therefore, the inducing agent adopted by the engineering strain is l-arabinose. Moreover, l-arabinose is abundant in hemicellulose of plants, and a cheap food-grade product is easy to purchase on the market, and cannot be utilized by Clostridium thermocellum; therefore, the inducing agent adopted by the present application also meets the two conditions of low cost and stable induction expression, which is of great significance for practical application. The concentration of the inducing agent is 2-4 g / L, and the induction time is 5.5-6.5 h. Under the foregoing induction conditions, the reporter gene GusB achieves the highest induced activity.

[0014] The high-temperature strain is Clostridium thermocellum or a bacterium with similar growth temperature (45-65°C); and the mesophilic strain is Escherichia coli, Clostridium cellulolyticum or a bacterium with similar growth temperature (25-45°C). Although the original intention of constructing the induction and regulation system in the present application is to solve the technical problem that there is no high-performance induction system in high-temperature strains such as Clostridium thermocellum, the inventors have found that the induction and regulation system constructed in the present application is not only suitable for the foregoing high-temperature strains, but also suitable for the foregoing mesophilic strains. This further expands the application range of the induction and regulation system.

[0015] A biosynthesis or biodegradation system based on the engineering strain as described above is used for inducible production of target proteins, which can be recombinant proteins as target products, enzymes for synthesizing target compounds in a biosynthesis system, or enzymes for degrading specific compounds in a biodegradation system. The present application specifically verifies that the thermophilic inducible gene expression regulation system realizes the induced expression of BGL in the process of lignocellulose saccharification and the induced expression of leafcutter compost cutinase (LCC) in PET degradation. This fully demonstrates that the biosynthesis system using the engineering strain as described above has good application in whole-cell saccharification of cellulose substrate and degradation of amorphous PET film.

[0016] The present application has the following beneficial effects:

[0017] (1) The present application provides a thermophilic gene inducible expression regulation system composed of an AraR inhibitory protein specifically combined with l-arabinose, an isolated promoter regulating expression of the AraR inhibitory protein, and an inducible promoter P araD or P abnE , which realizes significant increase in induction fold and significant reduction in leaky expression, thereby having a wide dynamic range.

[0018] (2) The regulation system described in the present application not only has low leakage and high dynamic range, but also can be applied to high-temperature strains or mesophilic strains, thereby expanding the application range of the induction and regulation system and solving the problem of insufficient performance of the induction and expression system suitable for high-temperature strains in the prior art.

[0019] (3) The inducer used in the regulatory system described in the application is L-arabinose, which meets the two conditions of low cost and stable induction expression, and has important significance for practical application. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 Figure 1 (a) Schematic diagram of the inducible expression plasmid, (b) LacZ activity of wild-type (DSM1313) and two engineered strains of G. stearothermophilus induced by 4 g / L L-arabinose and uninduced, (c) GusB activity of wild-type (DSM1313) and engineered strains AraR-P e GusB activity of GusB.

[0021] Figure 2 Figure 2 Structure of the L-arabinose utilization gene cluster of G. stearothermophilus T-6 used in plasmid construction.

[0022] Figure 3 Figure 3 Optimization of the L-arabinose inducible promoter of G. stearothermophilus in Example 2.

[0023] Figure 4 Figure 4 Effect of different concentrations of L-arabinose (0-10 g / L) on GusB activity of the engineered strain AraR T1 P C P e GusB activity. The induction time was 6 h.

[0024] Figure 5 Figure 5 Optimization of the L-arabinose induction concentration (a) and induction time (b) of ThermoARAi in Example 3.

[0025] Figure 6 Figure 6 GusB activity of G. stearothermophilus strains uninduced in Example 2.

[0026] Figure 7 Figure 7 Specificity of the inducer and potential inhibition of other sugars of G. stearothermophilus ThermoARAi in Example 4.

[0027] Figure 8 Figure 8 BGL activity (a) and whole-cell saccharification of pretreated xylose residue substrate (b) of the ThermoARAi-BGL strain in Example 5.

[0028] Figure 9 Figure 9 Concentration change of L-arabinose during the 10-day whole-cell saccharification process using pretreated xylose residue as the substrate in Example 5.

[0029] Figure 10Figure 10 To perform whole-cell degradation of amorphous PET flake by ThermoARAi-LCC system in Example 6.

[0030] Attached Figure 11 To perform induced expression of ThermoARAi system in E. coli in Example 7.

[0031] Attached Figure 12 To perform induced expression of ThermoARAi system in C. cellulolyticum in Example 8. DETAILED DESCRIPTION

[0032] The application is further described below in conjunction with examples.

[0033] 1. Strains and culture methods

[0034] The strains used in this study are listed in Table S1. E. coli was cultured aerobically in Luria-Bertani (LB) medium with ampicillin (100 μg / mL) or chloramphenicol (17 μg / mL) as needed. Since removal of plasmid Dcm methylation facilitates transformation in T. thermosaccharolyticum, we selected E. coli BL21 (DE3) as the cloning host for all plasmid constructions. T. thermosaccharolyticum strains were routinely anaerobically cultured at 55 ℃ in GS-2 medium with 5 g / L cellobiose as the carbon source, unless otherwise stated. Thiamphenicol (Tm) and l-arabinose were added at concentrations of 3 μg / mL and 0 ~ 10 g / L, respectively, as needed for plasmid maintenance and induction.

[0035] 2. Plasmid constructions

[0036] All plasmids constructed in this study are listed in Table 1, and the primer sequences are listed in Table 2. The schematic diagram of the induced expression plasmid is shown in Figure 1 a. Genetic elements derived from a 38 kb gene cluster (GenBank No. DQ868502.2) encoding l-arabinose and arabinose degradation metabolic enzymes in G. stearothermophilus T-6 were used to construct the inducible system, and the positions of the genetic elements are shown in Figure 2 . Among them, the letter P represents the promoter: Pr represents the promoter of the l-arabinose repressor gene araR, Pd and Pe represent the inducible promoters P araD and P abnE , respectively, of the araD gene encoding l-ribulose epimerase and the abnE gene encoding arabinose oligosaccharide binding protein. The letter Ω represents the transcription terminator, T araT represents the 45 bp terminator used in this study.

[0037] The construction of plasmid is divided into three parts: preparation of plasmid backbone, assembly of genetic elements and ligation. The endogenous promoter P groEL was replaced by the exogenous P Teth514_1306 and Amp R expression cassette was added to get the pHKm vector. The pHKm vector was linearized by PCR to get the plasmid backbone. The genetic elements of the inducible expression system mainly include the inducible promoter (P araD or P abnE ), the repressor protein AraR expression cassette (AraR coding sequence was optimized according to the codon bias of Clostridium stercorarium) and the 45 bp terminator (the terminator region of AraT, T AraT ). The above genetic elements were synthesized by GenScript. The reporter genes lacZ (Thebr_0629) encoding thermophilic β-galactosidase (LacZ) or gusB (SSO3036) encoding hyperthermophilic β-glucuronidase (GusB) were amplified by PCR using the genomic DNA of Thermoanaerobacter brockii subsp. finnii Ako-1 or Sulfolobus solfataricus P2 as template, respectively. The genetic elements of the inducible plasmid and the reporter genes were assembled by fusion PCR, and the assembled fragments were ligated with the linearized plasmid backbone using the Novagen Seamless Cloning Kit. The ligation mixture was transformed into E. coli BL21 (DE3) according to the chemical transformation protocol, and the positive clones were verified by colony PCR and sequencing to obtain the inducible plasmids pHKm-AraR-P d LacZ, pHKm-AraR-P e LacZ and pHKm-AraR-P e GusB, respectively.

[0038] The different truncations of P abnE and P araR in the promoter-optimized plasmids were achieved by PCR amplification using the pHKm-AraR-P e GusB plasmid as template. The endogenous promoters P 815 (the promoter of clo1313_0815) and P CbpB (the promoter of clo1313_1194) were amplified from the Clostridium stercorarium DSM1313 genome. The assembly, ligation and transformation steps were the same as above, and the pHKm-AraR T2 P C -P eGusB as the final ThermoARAi plasmid. Subsequently, gusB was replaced by synthetic bglA (GenBank No. KY418041) and lcc (GenBank No. HQ704839.1, the coding sequence was optimized according to the codon bias of Clostridium thermocellum) respectively to obtain pHKm-AraR T2 P C -P e BGL and pHKm-AraR T2 P C -P e LCC plasmid. In order to achieve exogenous expression, a signal peptide (from the gene clo1313_2747) was fused to the N-terminus of bglA and lcc.

[0039] 3. Electroporation and screening of Clostridium thermocellum

[0040] The plasmid constructed in E. coli BL21 (DE3) was extracted by Omega plasmid DNA mini kit, and then electroporated into Clostridium thermocellum DSM1313. After electroporation, the cells were recovered in 4 mL GS-2 medium at 51 °C for 8-10 h. About 1 mL of the recovered culture was pipetted and spotted on an empty plate, mixed with GS-2 solid medium containing Tm. The plate was placed in an anaerobic jar and cultured at 51 °C for 5-7 days, and then the positive clones were verified by colony PCR and sequencing. The colonies with complete expression cassette were picked and inoculated in fresh GS-2 medium, and the bacterial solution grown to late logarithmic phase was re-verified for the integrity of the expression cassette by PCR and sequencing.

[0041] Example 1: Initial construction of a thermophilic l-arabinose inducible system

[0042] In view of the fact that the repressor protein AraR in Geobacillus stearothermophilus T-6 can specifically bind to the promoters (P araD and P abnE ), the addition of l-arabinose can effectively prevent this binding. In this embodiment, the inventors used the promoter P araD (nucleotide sequence as shown in SEQ ID NO: 4) or P abnE (nucleotide sequence as shown in SEQ ID NO: 5) to pair with the expression cassette of the repressor protein AraR, and initially constructed a plasmid-based arabinose inducible system in Clostridium thermocellum DSM1313 Figure 1 a). Figure 1 In a, Pi is an inducible promoter that regulates the expression of the reporter gene; Pr is a promoter that controls the expression of the AraR repressor protein; T araT is the terminator of araT; Amp RSelective markers for constructing Escherichia coli plasmids; CAT is a selective marker for maintaining Clostridium thermophilum plasmids.

[0043] β-galactosidase (LacZ) has been developed as a common reporter gene in thermophilic anaerobes. Therefore, in order to verify P araD or P abnE To determine whether it could serve as an inducible promoter, the inventors first selected LacZ from *T. brockii* subsp. *finnii* Ako-1 as the reporter gene. P was then used... araD or P abnE As an inducible promoter containing AraR-P d LacZ or AraR-P e The plasmid of the LacZ expression cassette was transformed into Clostridium thermocellum DSM1313. The β-galactosidase activity of LacZ was then determined by adding or not adding 4 g / L of l-arabinose to the culture medium.

[0044] The specific method is as follows: 2 mL of cell culture was centrifuged at 12000 rpm for 3 min. The collected cells were resuspended twice in 500 μL Z-buffer (100 mM Na2HPO4, 1 mM MgCl2·6H2O, pH 7.5). Cells were disrupted by sonication, and the disruption solution was centrifuged at 14000 rpm for 10 min at 4 ℃ to remove cell debris. The resulting supernatant was used for protein concentration and LacZ enzyme activity determination. Bovine serum albumin was used as a standard, and the protein concentration of the supernatant was determined using the Bradford method. The enzymatic reaction system consisted of different volumes of supernatant and 8 μL of o-nitrobenzene-β-D-galactopyranoside (ONPG) stock solution (13.1 mg / mL aqueous solution), and Z-buffer was added to bring the total volume to 100 μL. After incubating the activity assay system at 55 ℃ for 10 min, 40 μL of the reaction solution was added to 200 μL of 1 M Na2CO3 to terminate the reaction. The absorbance at 420 nm increased with the release of the yellow cleavage product 2-nitrophenol (ONP). A LacZ enzyme activity unit (U) is defined as the amount of β-galactosidase required to release 1 μmol of ONP per minute. Enzyme activity was normalized to protein concentration. The induction effect was observed based on LacZ activity; results are detailed below. Figure 1 b. For example Figure 1 As shown in b, AraR-P d LacZ and AraR-P e The LacZ strain exhibited induced expression levels of 1.2 U / mg and 2.5 U / mg, respectively, with leakage expression levels of 0.36 U / mg and 0.31 U / mg, approximately three times the background activity level of DSM1313. This indicates that: (1) P araD and PabnE Promoters in Clostridium thermocellum all have induced expression, and can be used as inducible promoters; (2) P araD In comparison, P abnE has a higher dynamic range of 8.2-fold (dynamic range = induced expression / leaky expression).

[0045] In addition, it can be known from Figure 1 b that Clostridium thermocellum has endogenous background β-galactosidase activity, which can interfere with the judgment of the leaky level of the induction system. Therefore, the inventors selected the heat-resistant β-glucuronidase GusB from the hyperthermophilic archaeon S. solfataricus as a reporter gene, which was controlled by the P abnE promoter to express, and obtained the strain AraR-P e GusB. Then, by adding or not adding 4 g / L of l-arabinose in the culture medium, the β-glucuronidase activity was determined.

[0046] The specific method is as follows: the above-mentioned cell disruption method is adopted, and the GusB buffer (50 mM Na2HPO4, 1 mM EDTA, pH 7.0) is used for resuspension. The GusB enzyme used in this paper is derived from the hyperthermophilic archaeon Sulfolobus solfataricus P2 with an optimal growth temperature of 80 °C. Due to the high stability of the enzyme, the disruption solution is incubated at 75 °C for 1 hour to eliminate endogenous enzyme activity before centrifugation at 4 °C and protein concentration determination. The reaction system is composed of 20 μL of lysate supernatant and 180 μL of 4-methylumbelliferyl-β-D-glucuronide (MUG, dissolved in fresh GusB buffer, concentration of 4 mM), and is preheated at 37 °C. The reaction solution is added to an opaque 96-well microplate, and the fluorescence kinetics curve is recorded by using an enzyme marker (SuPerMax 3000FA, Shanghai Shenshu Biotechnology Co., Ltd., Shanghai, China) at 37 °C in time scanning mode (Ex = 362 nm, Em = 439 nm), scanning once every 20 s, for 15 min. The slope of the fluorescence kinetics curve is the activity unit (U) of β-glucuronidase, which is normalized to the protein concentration for analysis. The induction effect is observed according to the GusB activity, and the results are shown in Figure 1 c. As shown in Figure 1 c, the strain AraR-P e GusB exhibits obvious induced expression, and the GusB activity is 41976.39 U / mg. However, at the same time, the leaky activity of the strain is as high as 7743.4 U / mg, which leads to a dynamic range of only 5.4-fold, compared with the AraR-P e LacZ strain does not increase but decreases.

[0047] This indicates that the dynamic range of the thermophilic l-arabinose induction system constructed in this embodiment is not high, and its practical application value is low, requiring further optimization of its performance.

[0048] Example 2: Promoter optimization to reduce leakage

[0049] Given the strain AraR-P constructed in Example 1 e The excessively high leakage activity of GusB limits its practical application value. This embodiment aims to reduce the leakage expression level by optimizing the promoter, thereby improving the dynamic range of the induction system.

[0050] (1) Optimization of the inducible promoter

[0051] To optimize the induced promoter, the inventors analyzed P using a BPROM server (http: / / www.softberry.com / berry.phtml?topic=bprom&group=programs&subgroup=gfindb). abnE For details on the main control elements of the promoter, please refer to the appendix. Figure 3 .like Figure 3 As shown, the top figure depicts the initial arabinose-induced system AraR-P e The basic regulatory elements of the GusB promoter include P araR and P abnE Promoter, immediately adjacent to terminator T araT Located on the left and right sides respectively. The -35 and -10 zones are highlighted with thick black and red lines respectively. The inventors also predicted P abnE Another set of possible -35 and -10 region sequences for the promoter (thick gray and orange lines). Furthermore, three identical inverted repeat sequences, AraO1, AraO2, and AraO3, similar to the AraR operating site (AraO), were observed, represented by dark red, blue, and light blue rectangles, respectively; of these, only the AraO2 site has been reported in the literature. Purple and green rectangles represent promoter P, respectively. 815 and promoter P CbpB The transcription start site (TSS) is indicated by an arrow. The rectangles marked with RBS and cre represent the ribosome binding site and the putative catabolic response element site, respectively.

[0052] To further evaluate P abnE The functions of each segment of the promoter sequence were investigated, and various truncation and analyses were performed on the -35 and -10 regions and the AraR operating site (nucleotide sequences are shown in SEQ ID NO: 10-19). See details for the results. Figure 3 The gray area at the top. (By...) Figure 3 It can be seen that, compared with the full-length construct AraR-P eGusB, P abnE The truncation of the promoter led to different degrees of reduction in the dynamic range of all constructs. Specifically, the constructs with the catabolite response element site (cre) or the predicted -10 region truncated, AraR-P e-cre GusB, AraR-P eT2 GusB and AraR-P eT3 GusB, the induced GusB activity was reduced, and the induction fold was reduced to about 2.8. Further truncation of the predicted -35 region and AraO3, AraR-P eT4 GusB and AraR-P eT5 GusB, the basal expression level of the GusB construct before induction was similar to that of the full-length construct AraR-P e GusB, but the expression level after induction of both was significantly reduced, so its dynamic range was not improved. Subsequently, the 5' end sequence of the promoter was truncated differently, and AraR-P abnE GusB, AraR-P T1e GusB, AraR-P T2e GusB and AraR-P T3e GusB, it was found that the predicted site AraO1 was crucial for the binding of the AraR inhibitor protein to P abnE The inducibility of the promoter is crucial.

[0053] In summary, in addition to the reported AraO2 site, the inventors found that (1) the AraO1 site is crucial for the binding of the AraR inhibitor protein. AraR-P T4e GusB and AraR-P T6e The results of the GusB construct showed that the -35 and -10 regions are key elements affecting transcriptional activity. Therefore, in the subsequent optimization of the induction system, it is necessary to retain the entire sequence. (2) The downstream predicted cre and AraO3 sites of the promoter have no effect on its inducibility in C. thermocellum, but affect the strength of the promoter. Therefore, in order to have the highest activity after induction, the full-length P abnE promoter is preferred. abnE promoter.

[0054] (2) Optimization of the AraR inhibitor protein expression promoter

[0055] The P araR promoter (nucleotide sequence as shown in SEQ ID NO: 7) was modified in various ways, and the results are detailed in Figure 3 the pink bottom part below. From Figure 3 it can be seen that the truncated P araR promoter 5' sequence (nucleotide sequence as shown in SEQ ID NO: 20, AraR T1 -PT6e GusB and AraR T1 -P e GusB construct) or fusion with endogenous promoter sequences (AraRP) 815 -P e Neither the GusB construct nor the induction fold of the induction system was enhanced. It is noteworthy that direct fusion of constitutive P... 815 AraRP promoter (nucleotide sequence as shown in SEQ ID NO:8) sequence 815 -P e The GusB construct exhibited reduced induction activity, likely due to the strong expression of the AraR repressor protein.

[0056] To solve this problem, the inventors modified P araR The promoters were truncated in different ways and then combined with short sequences (<100 bp in length) to form a constitutive promoter P. CbpB or P 815 Fusion (nucleotide sequences as shown in SEQ ID NO:1-2, 21-22). See details for results. Figure 3 ,Depend on Figure 3 It can be seen that these modifications significantly increased the fold induction and reduced leakage expression, thereby significantly expanding the dynamic range. Specifically, using promoter P... 815-araRT1 (nucleotide sequence as shown in SEQ ID NO:21), P CbpB - araRT1 (nucleotide sequence as shown in SEQ ID NO:1) and P CbpB - araRT2 (The nucleotide sequence is shown in SEQ ID NO:2) Constructed AraR T1 P 815 -P e GusB、AraR T1 P C -P e GusB and AraR T2 P C -P e GusB's dynamic range reached 10.3x, 24.3x, and 138.6x, respectively. While AraR... T1 P C -P e GusB and AraR T2 P C -P e GusB activity decreased after GusB induction to the levels of the construct AraR-P. e GusB was 35.6% and 70.5%, and adjusting the concentration of l-arabinose did not increase the maximum inducible level ( Figure 4 andFigure 5 a), but its leaky expression was comparable to the background expression level of DSM1313 Figure 6 ), the background-subtracted leak was almost 0, and the strain ARAi-P e GusB. Therefore, both of them achieved a significant improvement in dynamic range compared to AraR-P e GusB. Among them, AraR T2 P C -P e GusB construct was particularly outstanding, with a dynamic range of 138.6-fold, and AraR T1 P C -P e GusB construct, although weaker than AraR T2 P C -P e GusB, still had practical application value. Therefore, although the inventors made various modifications, only the constructs using P CbpB - araRT1 and P CbpB - araRT2 achieved a significant improvement in dynamic range. The inducible system using P CbpB - araRT2 was named ThermoARAi system. These results show that the use of P CbpB - araRT1 and P CbpB - araRT2 as a modified AraR repressor protein expression promoter has unexpected technical effects. In summary, by modifying the AraR repressor protein expression promoter, this embodiment achieved a significant increase in induced expression and a significant decrease in leaky expression, with a dynamic range of up to 138.6-fold. Therefore, AraR T2 P C -P e GusB construct performed well in terms of leakiness, inducibility, and dynamic range, and the ThermoARAi system host established based on an exogenous gene had high orthogonality and did not adversely affect the growth and metabolism of the strain.

[0057] Example 3: Optimization of ThermoARAi system induction conditions

[0058] To further explore the optimal induction conditions of the ThermoARAi system of C. thermocellum, this embodiment studied the effects of l-arabinose concentration and induction time on GusB activity, and the results are shown in Figure 5 . Among them, Figure 5a The effect of different concentrations of l-arabinose (0-10 g / L) on GusB activity, with 4 h induction time. Figure 5 b GusB activity (bar graph) and bacterial growth (line graph) over time (0-14 h) with and without 4 g / L l-arabinose induction.

[0059] As shown in Figure 5 a, with the inducer concentration ranging from 0 to 2 g / L, the GusB activity increased with the increase of the inducer concentration. However, the GusB activity did not further increase when the l-arabinose concentration was increased from 2 g / L to 10 g / L, indicating that the maximum induction of GusB activity could be achieved at 2 g / L. As shown in Figure 5 b, with the increase of the induction time, the change trend of the GusB activity was consistent with the growth cycle of the bacteria, and the addition of the inducer did not affect the growth of the bacteria. The GusB activity could be detected after 2 h of l-arabinose induction, indicating that the induction reaction of the system was fast. After 6 h of continuous induction, the bacterial amount reached the maximum, at which time the GusB had the strongest activated expression of 150±13 folds, and the dynamic range was wide. However, the induction activity of the GusB did not increase but decreased when the induction time was further prolonged, which was related to the cell lysis in the late growth stage. Therefore, the l-arabinose induction of 2-4 g / L for 68 h had the highest GusB induction activity, and the induction reaction was fast, the inducer was non-toxic, and had almost no interference to the growth of the strain.

[0060] Example 4: Verification of the inducer specificity of the ThermoARAi system

[0061] To verify the inducer specificity of the ThermoARAi system, the l-arabinose inducer specificity of the ThermoARAi in Clostridium thermocellum and the inhibitory effect of d-arabinose, d-glucose, d-xylose, d-fructose, d-galactose and d-mannose were determined. Specifically, (1) 4 g / L of various sugars, including l-arabinose (l-Ara), d-arabinose (d-Ara), d-glucose (d-Glc), d-xylose (d-Xyl), d-fructose (d-Fru), d-galactose (d-Gal) and d-mannose (d-Man) were used as potential inducers, and the specificity of the inducer was tested by adding or not adding these sugars in the culture medium and testing the β-glucuronidase activity according to the method in Example 1. (2) The inhibitory effect was determined by adding a mixture of 4 g / L l-arabinose and 2 g / L or 10 g / L of other sugars in the culture medium, and testing the β-glucuronidase activity according to the method in Example 1. The induction time was 6 h. The results are shown in Figure 7 .

[0062] As shown in Figure 7 Figure 6, l-arabinose induced 175 ± 13-fold activation of GusB expression, while other sugars did not show any induction, indicating that l-arabinose is a specific inducer of ThermoARAl. After mixing 4 g / L of l-arabinose with 2 g / L or 10 g / L of other sugars, most of the sugars showed little change in GusB activity after induction, indicating that they have little inhibitory effect; while 10 g / L of d-mannose greatly reduced the induced GusB activity, indicating that 10 g / L of d-mannose significantly inhibited the induction activity. We also observed significant inhibition of the growth of Thermoanaerifungin cells, indicating that high concentrations of d-mannose have certain cytotoxicity to Thermoanaerifungin, which in turn affects the induction activity. In summary, this example demonstrates that l-arabinose is a specific inducer of ThermoARAl and is not affected by most other sugars.

[0063] Example 5: l-arabinose-dependent whole-cell saccharification of cellulosic substrates

[0064] This example evaluates the utility of the ThermoARAl system for secretory expression. It is well known that BGL is a rate-limiting enzyme in the process of cellulose degradation, as it can convert cellobiose to glucose. In this paper, a ThermoARAl -controlled BGL excretion strain (ThermoARAl -BGL) with an N-terminal signal peptide (from Thermoanaerifungin cellulase Cel48S) was constructed. Since the expressed BGL protein is secreted outside the cell, the supernatant of the Thermoanaerifungin culture solution is directly used for BGL activity determination with p-nitrophenyl- -d-glucopyranoside (pNPG) as the substrate. The specific operation is as follows: the supernatant sample is mixed with a reaction buffer (50 mM sodium acetate, pH 5.5) containing 5 mM pNPG, and the total volume is 100 μL. After the reaction is carried out at 55 °C for 10 min, 40 μL of the reaction solution is added to 200 μL of 1 M Na2CO3 to terminate the reaction, and the absorbance at 405 nm is immediately measured. The BGL enzyme activity unit (U) is defined as the amount of β-glucosidase required to produce 1 μmol of p-nitrophenol (pNP) per minute. The BGL enzyme activity is normalized by the protein concentration of the Thermoanaerifungin culture supernatant.

[0065] After determining that BGL is successfully expressed and excreted, whole-cell saccharification applications of cellulosic substrates are carried out, as follows:

[0066] (1) Thermoanaerifungin strains are cultured in GS-2 medium with 5 g / L Avicel as the carbon source to the mid-log phase. Then, Thermoanaerifungin is inoculated into a saccharification medium containing 4% (w / v) pretreated cellulosic substrate at a ratio of 10% (v / v), and Tm and L-arabinose are added as needed.

[0067] (2) Using ThermoARAi-BGL strain for whole-cell saccharification with 4% (w / v) pretreated xylose residues as substrate. The xylose residues (Shandong Futian Science and Technology Group Co., Ltd, China) were pretreated with 10% (w / w) KOH at 90 °C for 2 h. The resulting reaction liquid was transferred to a 300-mesh nylon bag, and the waste liquid was filtered to separate the solid substrate. Then, the solid substrate was washed to neutral with deionized water and stored at 4 °C for subsequent saccharification experiments. Whole-cell saccharification was carried out at 60 °C, 200 rpm, with an anaerobic culture medium volume of 50 mL. The saccharification process was carried out for 10 days, and about 0.5 mL of culture was sampled every 1-2 days to analyze the supernatant components obtained by whole-cell saccharification using high-performance liquid chromatography for monitoring the production of reducing sugars. The high-performance liquid chromatography system (Agilent 1260 series, Agilent Technologies) was equipped with an Aminex HPX-87H chromatographic column (Bio-Rad, Hercules, CA, USA) and a parallax detector (Agilent 1260 infinity RID; Agilent, Santa Clara, CA, USA). The mobile phase was 5 mM sulfuric acid, the flow rate was 0.6 mL / min, and the temperature was 55 °C.

[0068] The results are shown in detail in Figure 8 . Among them, Figure 8 a is the extracellular BGL activity of the ThermoARAi-BGL construct under the induction and non-induction of 4 g / L l-arabinose. As shown in Figure 8 a, (1) the strain was cultured with cellobiose as carbon source, and the BGL activity of the supernatant protein of the strain was 0.1 U / mg without the addition of l-arabinose induction, which was similar to that of Clostridium thermocellum DSM1313; (2) after induction of 4 g / L l-arabinose, the BGL activity of the supernatant protein of the strain reached 2.83 U / mg, confirming that it had been successfully induced and excreted. Figure 8 b is the result of whole-cell saccharification with 4% (w / v) pretreated xylose residues containing 84.0% glucan as substrate. Among them, 0, 1, 2 and 4 g / L of l-arabinose were added as inducers to induce BGL expression, and the concentrations of glucose and cellobiose in the culture supernatant were determined by HPLC. As shown in Figure 8As shown in Figure b, the cellobiose production of the uninduced group increased first and then decreased gradually after the third day, with a final concentration of 1.2 g / L. The cellobiose produced by the induced groups was degraded by the expressed BGL, and the degradation rate was related to the concentration of the inducer. The glucose production of the uninduced group was 10.9 g / L, which was lower than that of the induced groups (13.3-13.5 g / L), possibly due to the inhibitory effect of cellobiose accumulation. Correspondingly, the glucose concentration of the 1 g / L l-arabinose induced group was slightly lower than that of the other induced groups initially, but at the end of saccharification, the glucose concentrations of all the induced groups reached the same level (13.4 ± 0.1 g / L). In summary, the ThermoARAi system can be successfully used for the expression of BGL in lignocellulose saccharification.

[0069] The stability of the inducer during cell growth is a key factor affecting the performance of the induction system. To evaluate the stability of l-arabinose in ThermoARAi, the change in inducer concentration during the 10-day saccharification process was monitored using high-performance liquid chromatography for the 4 g / L l-arabinose induced group, and the results are shown in Figure Figure 9 . As shown in Figure Figure 9 , the l-arabinose concentration remained relatively constant. Thus, l-arabinose is a stable inducer for Clostridium thermocellum ThermoARAi, and it is also confirmed that it can be used for long-term fermentation experiments.

[0070] Example 6: l-arabinose-dependent amorphous PET flake degradation

[0071] Due to the great potential of Clostridium thermocellum as a whole-cell catalyst for PET degradation, in order to further demonstrate the usability of the ThermoARAi system, this example uses the system to exogenously express the LCC that degrades PET in Clostridium thermocellum; and after 2 days of induction, the ester hydrolysis activity of the extracellular LCC on bis-hydroxyethylterephthalate (BHET) is determined. Using a high-performance liquid chromatography system equipped with a variable wavelength detector (Agilent 1260 infinity VWD) and an Agilent Eclipse XDB-C18 chromatographic column, the hydrolysis products terephthalic acid (TPA) and mono-hydroxyethylterephthalate (MHET) released from BHET into the culture supernatant are determined. Standard curves are drawn using the corresponding commercial reagents. The ester hydrolysis activity unit (U) is defined as the amount of enzyme required to hydrolyze 1 mmol of BHET per hour. The exogenous expression of LCC is determined according to the esterase activity. Subsequently, amorphous PET flake degradation applications are carried out.

[0072] Specifically, the amorphous PET flake with a thickness of 250 pm was purchased from Goodfellow Ltd. (Bad Nauheim, Germany; product number ES301445). The PET flake was cut into pieces with a size of about 2 cm x 0.8 cm, and each piece weighed about 48 mg, which was used for whole-cell degradation. After sterilization by soaking in 75% ethanol overnight and air-drying under sterile airflow, each small flake was sequentially added to 10 mL of GS-2 medium containing 5 g / L cellobiose, and the process was carried out under anaerobic conditions. The Clostridium thermocellum seed liquid was inoculated into the culture system at a ratio of 1% (v / v) to start the degradation process, and the culture was continuously incubated at 60°C and 170 rpm for 10 days. Different concentrations of L-arabinose (0-4 g / L) were added as inducers. After the incubation, the residual small flake was washed and dried, and the weight loss of the small flake before and after degradation was calculated. The composition of the culture supernatant was analyzed by high-performance liquid chromatography (HPLC), and the results are shown in Figure 10 .

[0073] wherein, Figure 10 a is the extracellular esterase activity with BHET as the substrate. As shown in Figure 10 a, the extracellular esterase activity of the 0.5-4 g / L L-arabinose induction group was higher than that of the control group, indicating that significant extracellular LCC esterase activity was detected in all induction groups. However, unlike the application of the ThermoARAi system in Example 5, high inducer concentration seemed to have a negative impact on the expression of LCC, and the highest enzyme activity was obtained at 0.7 or 1 g / L inducer.

[0074] Figure 10 b is the yield of TPA and MHET in the culture supernatant and the weight loss of the PET flake after 10 days of degradation. As shown in Figure 10 b, by degrading the PET flake in 10 mL of GS-2 medium with 0-4 g / L L-arabinose for 10 days, the degradation of PET by the ThermoARAi-LCC strain was demonstrated. Among them, the weight loss of the PET flake was significantly induced by 0.7 g / L L-arabinose, and the highest degradation rate reached 62.9%. The PET flake degradation rates corresponding to other 0.5, 1 and 4 g / L L-arabinose induction concentrations were 51.4%, 44.1% and 44.7%, respectively. In addition, the hydrolysis products TPA and MHET in the culture supernatant were detected by HPLC, and their concentrations were related to the weight loss of PET. These results show that the ThermoARAi system can be used to induce the expression of LCC in the PET degradation system, but the optimal induction concentration is different from other applications tested in this study. When using the ThermoARAi system to express different proteins, it is necessary to optimize the induction conditions.

[0075] Example 7: Induction of expression of ThermoARAi system in Escherichia coli

[0076] To verify whether the ThermoARAi system constructed in *Clostridium thermophilum* can function in the mesophilic bacterium *Escherichia coli*, the AraR system from Example 2 was used... T2 P C -P e GusB construct plasmid was transformed into Escherichia coli BL21(DE3) competent cells according to the chemical transformation protocol.

[0077] The specific method is as follows: competent cells are thawed naturally on ice (5-10 min), and the plasmid to be transformed, pHKm-AraR, is then placed on ice. T2 P C -P e GusB was pre-cooled on ice. Centrifuge tubes containing 1 mL of LB medium were removed from the refrigerator and preheated in a 37°C incubator. 3 µL of the plasmid to be transformed was added to 50 µL of competent cells, gently mixed, and incubated on ice for 30 min. After heat shock at 42°C for 90 s, the cells were immediately incubated on ice for 2-3 min. 900 µL of preheated LB liquid medium (without antibiotics) was added to a clean bench. The cells were incubated at 37°C with shaking for 1 h (~200 rpm). An appropriate amount of the incubated culture was then spread onto an ampicillin-resistant plate. After verifying positive clones by colony PCR and sequencing, single colonies were picked and cultured. β-glucuronidase activity was measured with or without 4 g / L of L-arabinose in the culture medium. The specific procedure was the same as the activity assay in Example 1. The induction effect was observed based on GusB activity; the results are detailed in [link to example]. Figure 11 .

[0078] like Figure 11 As shown, the baseline GusB activity of BL21 is close to 0, similar to that of Clostridium thermocellum DSM1313. The construct BL21::AraR T2 P C -P e The inducible activity of GusB was 94477 U / mg, exhibiting a 21.2-fold increase in activated expression compared to the uninduced state. These results demonstrate that the ThermoARAi system can induce the expression of the target protein in *E. coli*, although its dynamic range is lower than that of *Clostridium thermocellum*, it still possesses practical application value.

[0079] Example 8: Induction of expression of the ThermoARAi system in Clostridium difficile

[0080] To verify whether the ThermoARAi system constructed in T. thermophilus can work in mesophilic bacteria E. coli, the AraR T2 P C -P e The GusB construct plasmid was transformed into C. cellulolyticum H10.

[0081] The specific method is as follows: 1 mL of seed culture was inoculated into 100 mL of GS-2 liquid medium, and incubated at 34°C for 17-24 h. When the cells grew to the late logarithmic growth phase (OD 600 =0.5-0.8), they were taken out and placed in an ice-water mixture for 20 min to stop cell metabolism. The bacterial solution was dispensed into anaerobic centrifuge tubes in an anaerobic box, and centrifuged at 3000 g at 4°C for 10 min. The supernatant was discarded. The cells were washed twice with pre-cooled electroporation buffer, centrifuged at 3000 g at 4°C for 10 min, and the cells were taken out. The washed cells were suspended in 2 mL of pre-cooled electroporation buffer and placed on ice for standby. 200 μL of competent cells were added to about 1 μg of methylated plasmid, mixed, and then added to a 0.2 cm electroporation cup and placed on ice for 5 min. The cells were quickly transferred to a preheated anaerobic tube containing 4 mL of GS-2 medium after electroporation, and incubated at 34°C for 6 h. The bacterial solution after recovery was centrifuged, and the cells were spread on GS-2 solid medium containing erythromycin. After the bacterial solution was completely absorbed, it was inverted in a 2.5 L anaerobic barrel and incubated at 34°C. After 4-6 days of culture, the colonies were grown, and positive clones were verified by colony PCR and sequencing. Single colonies were picked and cultured. The β-glucuronidase activity was determined by adding or not adding 4 g / L of l-arabinose to the culture medium. The specific operation method was consistent with the activity determination method of Example 1. The induction effect was observed according to the GusB activity, and the results are shown in Figure 12 .

[0082] As Figure 12 shown, the background GusB activity of H10 was low, which was comparable to that of T. thermophilus DSM1313. The construct H10::AraR T2 P C -P e GusB exhibited a 73.3-fold activated expression, and the GusB activity after induction was 67044 U / mg. The leaky expression without induction was also low, between E. coli and T. thermophilus. These results show that the ThermoARAi system can work in C. cellulolyticum, has a high dynamic range, and has practical application value.

[0083] In summary, the present application successfully established a thermo-ARai system in Clostridium thermocellum. By optimizing the expression promoter of AraR inhibitor protein and further optimizing the induction conditions, the Thermo-ARai showed a dynamic range of 175-fold, low leakage level and good inducer specificity. Moreover, the Thermo-ARai system has the advantages of high host orthogonality, sustainable expression, and no toxicity to the host. In addition, the Thermo-ARai can induce the excretion expression of BGL and LCC in Clostridium thermocellum, and construct whole-cell catalysts for the saccharification of cellulose substrate and the degradation of amorphous PET sheet, respectively. In mesophilic bacteria Escherichia coli and Clostridium cellulolyticum, the Thermo-ARai system can also play an inducing role, with an induction efficiency of 21-fold and 73-fold, respectively. This shows that the Thermo-ARai system provided by the present application has application prospects in various biotechnologies, and is expected to become a new tool for metabolic engineering and synthetic biology applications of Clostridium thermocellum, and has important practical application value.

[0084] Table 1. List of strains and plasmids

[0085]

[0086]

[0087] Table 2. List of primers

[0088]

[0089]

Claims

1. A promoter for regulating the expression of an isolated AraR repressor protein, characterized in that: The promoter is P. CbpB - araRT1 Or P CbpB - araRT2 The P CbpB - araRT1 The nucleotide sequence is shown in SEQ ID NO:1; the promoter P CbpB - araRT2 The nucleotide sequence is shown in SEQ ID NO:

2.

2. An AraR inhibitory protein, characterized in that: Its expression is controlled by the expression promoter as described in claim 1; the amino acid sequence of the AraR repressor protein is shown in SEQ ID NO:

3.

3. The AraR inhibitory protein according to claim 2, characterized in that: The AraR repressor protein and promoter P araD or P abnE Specific binding; the promoter P araD The nucleotide sequence is shown in SEQ ID NO:4, and the promoter P abnE The nucleotide sequence is shown in SEQ ID NO:

5.

4. The AraR inhibitory protein according to claim 2 or 3, characterized in that: The codon-optimized nucleotide sequence of the AraR repressor protein is shown in SEQ ID NO:

23.

5. An AraR expression cassette, characterized in that: Includes the expression promoter P as described in claim 1 CbpB-araRT1 Or P CbpB-araRT2 And the gene encoding the AraR repressor protein as described in claim 2.

6. A thermophilic inducible gene expression regulation system, characterized in that: It comprises (a) the AraR expression cassette as described in claim 5, (b) an inducible promoter regulated by an AraR repressor protein, and (c) a reporter gene or a target gene induced to be expressed in a regulated manner and operably linked to said inducible promoter; said inducible promoter is P araD or P abnE .

7. An engineered strain carrying an inducible gene expression regulation system, characterized in that: The strain integrates or is transformed with the thermophilic inducible gene expression regulation system as described in claim 6; the strain is a thermophilic strain or a mesophilic strain.

8. The engineered strain according to claim 7, characterized in that: The thermophilic strain is *Clostridium thermophilum* or bacteria with a growth temperature of 45-65°C; the mesophilic strain is *Escherichia coli*, *Clostridium fibrinolyticum*, or bacteria with a growth temperature of 25-45°C.

9. The engineered strain according to claim 7 or 8, characterized in that: The inducer is L-arabinose, the concentration of the inducer is 2~4 g / L, and the induction time is 6-8 h.

10. A biosynthesis or degradation system based on the engineered strain according to any one of claims 7-9, characterized in that: This is used to induce the production of a target protein, which is a recombinant protein as a target product, an enzyme in a biosynthetic system for synthesizing a target compound, or an enzyme in a biodegradation system for degrading a specific compound.

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