Cis-element library for thermophilic microorganism gene expression regulation and application

Abstract

The invention discloses a cis-element library for thermophilic microorganism gene expression regulation and application. The cis-element library comprises a constitutive promoter, an inducible promoter and a ribosome binding site (RBS). The invention also discloses application of the element library in gene expression of thermophilic microorganisms. The toxin-antitoxin gene is used as a new selective marker to be applied to genetic manipulation of thermophilic microorganisms by rationally controlling the strength of the promoter. Promoters of different types and strengths are used for replacing natural promoters of genes related to hydrogen production pathways, and hydrogen metabolic pathways in thermophilic microorganisms are modified, so that the yield of hydrogen is improved. And a cis-element library for accurately regulating and controlling gene expression is provided for carrying out basic research on metabolic engineering and molecular biology in thermophilic microorganisms.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a cis-element library for regulating gene expression in thermophilic microorganisms and its application. Background Art

[0002] The unique high-temperature biochemical and physiological characteristics of thermophilic microorganisms have made them a research field of great interest, but in the field of industrial biotechnology, the development of thermophilic archaea still lags behind that of bacteria and eukaryotes. The biggest limitation is the lack of genetic manipulation tools. Currently, genetic manipulation in Thermococcaceae requires the use of auxotrophic strains, such as histidine auxotrophs (his - ), uracil auxotrophic type (pyrF - ) and agmatine auxotrophs (pdaD - The hmgA gene, encoding 3-hydroxy-3-methylglutaryl-CoA reductase, is the only selectable marker in Thermococcaceae that can be used in rich culture media. Overexpression of hmgA confers resistance to simvastatin.

[0003] Thermococcus kodakarensis, a thermophilic archaeon with an optimal growth temperature of 85°C, has become an important model organism for studying mechanisms of high-temperature adaptation and the expression of thermostable enzymes. A squalene / phytoene synthase homologous gene from the thermoacidophilic archaeon Sulfolobus acidocaldarius was integrated into the T. kodakarensis chromosome under the control of a strong promoter, resulting in the successful synthesis of phytoene. Furthermore, T. kodakarensis has demonstrated potential as a platform for biohydrogen production. Although genetic manipulation systems for T. kodakarensis have been developed, auxotrophic strains can adversely affect phenotypic investigations. Therefore, researchers in this field are committed to developing a genetic toolbox for regulating gene expression in T. kodakarensis, aiming to provide more precise and flexible tools for metabolic engineering and basic biological function research. Summary of the Invention

[0004] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a cis-element library for precise regulation of gene expression for T. kodakarensis metabolic engineering and basic biological research.

[0005] To achieve the above objectives, the present invention provides a cis-element library for regulating gene expression in thermophilic microorganisms, characterized in that it includes a constitutive promoter library, and the constitutive promoter library includes the base sequences shown in SEQ ID NO.1-8.

[0006] In a preferred embodiment of the present invention, the element library further comprises an inducible promoter library, and the inducible promoter library includes a maltodextrin inducible promoter library and a pressure inducible promoter library.

[0007] In another preferred embodiment of the present invention, the maltodextrin inducible promoter library includes base sequences as shown in SEQ ID NOs. 9-13.

[0008] In another preferred embodiment of the present invention, the pressure-inducible promoter library comprises a base sequence as shown in SEQ ID NO.14.

[0009] In another preferred embodiment of the present invention, the element library further comprises a ribosome binding site RBS library, and the RBS library comprises base sequences such as those shown in SEQ ID NOs. 15, 16, and 18.

[0010] The present invention also provides use of a cis-element library for thermophilic microorganisms in gene expression in the thermophilic microorganism T. kodakarensis.

[0011] In a preferred embodiment of the present invention, a constitutive promoter is used to control the expression of the antitoxin gene, and an inducible promoter is used to control the expression of the toxin gene.

[0012] In another preferred embodiment of the present invention, the constitutive promoter P1431M1 is combined with the 5'UTR element RhmtB to control the expression of the antitoxin gene PYCH_17730, and the pressure-inducible promoter P9950-M3 is combined with the 5'UTR element RhmtB to control the expression of the toxin gene PYCH_17720, wherein the P1431M1 sequence is shown as SEQ ID NO.2, the P9950-M3 sequence is shown as SEQ ID NO.14, the RhmtB sequence is shown as SEQ ID NO.17, the PYCH_17730 gene sequence is shown as SEQ ID NO.19, and the PYCH_17720 gene sequence is shown as SEQ ID NO.20.

[0013] In another preferred embodiment of the present invention, the cloning vector used for induced expression uses plasmid pUC18 as a backbone.

[0014] The present invention also provides the use of the above-mentioned cis-element library for thermophilic microorganisms in regulating hydrogen production, characterized in that the inducible promoter in the element library is used to replace the natural promoter of the gene related to the hydrogen production pathway in the thermophilic microorganism T. kodakarensis.

[0015] Technical Effects

[0016] 1. This application established a cis-element library for precise regulation of gene expression in T. kodakarensis, including constitutive promoters, inducible promoters, and ribosome binding sites (RBS). Based on the primary transcript sequencing data of Thermococcus kodakarensis, promoters and ribosome binding sites were mined. The effects of the two types of cis-elements on transcriptional activity were evaluated by using thermophilic glycosidase as a reporter gene, and the natural promoters and ribosome binding sites were optimized and modified. The element library has 76 constitutive promoters with expression intensities ranging from approximately 8×10 3 The researchers also identified 22 inducible promoters, including 15 maltodextrin-inducible promoters and 7 pressure-inducible promoters, with a maximum induction strength of approximately 8-fold. A genetic system has been established in T. kodakarensis that differs from traditional genetic manipulation (which relies on auxotrophic strains).

[0017] 2. Utilizing endogenous toxins of thermophilic archaea as genetic selection markers overcomes the limitations of these microorganisms, which rely solely on auxotrophic markers and simvastatin resistance selection. Specifically, a constitutive promoter controls the expression of the antitoxin gene, while an inducible promoter controls the expression of the toxin gene. By adjusting the strength of both promoters, they successfully achieved scarless gene knockout in the parent strain, T. kodakarensis. By combining promoters of varying strengths and types, they induced high expression of the endogenous toxin under specific conditions, inhibiting cell growth and mediating scarless gene knockout in thermophilic archaea.

[0018] 3. Knockout of the cytoplasmic hydrogenase system in Thermococcus kodakarensis has led to the efficient construction of a new gene knockout technology system. By replacing the native promoters of membrane-bound hydrogenase (MBH) and ferredoxin with promoters of different types and strengths, the expression of multiple genes in the hydrogen production metabolic pathway of T. kodakarensis was increased, resulting in a 2.68-fold increase in hydrogen production.

[0019] The present application provides a new tool for controlling gene expression for the genetic manipulation and metabolic engineering of such thermophilic microorganisms from the perspective of synthetic biotechnology.

[0020] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the expression intensity of the constitutive promoter in T. kodakarensis of a preferred embodiment of the present invention;

[0022] Figure 2It is a preferred embodiment of the present invention rational mutation constitutive promoter P0560 and P2065 core region;

[0023] Figure 3 It is a preferred embodiment of the present invention based on rational design and modification of the T. kodakarensis constitutive promoter;

[0024] Figure 4 It is a preferred embodiment of the present invention to construct a constitutive promoter library in T. kodakarensis;

[0025] Figure 5 It is a preferred embodiment of the present invention to evaluate the induction strength of 17 maltodextrin inducible promoters by β-glucosidase activity;

[0026] Figure 6 It is a core region of a rationally optimized maltodextrin-inducible promoter in a preferred embodiment of the present invention;

[0027] Figure 7 It is a preferred embodiment of the present invention to screen, characterize and rationally design a pressure-inducible promoter;

[0028] Figure 8 It is a characterization of the T. kodakarensis 5′UTR of a preferred embodiment of the present invention;

[0029] Figure 9 It is a rationally optimized T. kodakarensis 5′UTR sequence of a preferred embodiment of the present invention;

[0030] Figure 10 It is a preferred embodiment of the present invention to screen the HHP-induced toxin-antitoxin (TA) system in T. kodakarensis TS559;

[0031] Figure 11 It is a preferred embodiment of the present invention to design a reverse toxin selection marker regulated by antitoxin in T. kodakarensis to achieve scarless gene deletion;

[0032] Figure 12 It is a preferred embodiment of the present invention to improve H2 production in T.kodakarensis; DETAILED DESCRIPTION

[0033] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0034] Example 1. Expression intensity of constitutive promoter in T. kodakarensis

[0035] To evaluate the strength of the constitutive promoter in T. kodakarensis, a β-glucosidase reporter system was used for testing. The sequence 45 bp upstream of the TSS (containing the core elements of the promoter, BRE and TATA-box) was selected for evaluating the strength of the promoter. The β-glucosidase reporter gene was fused with the selected promoter elements and inserted into the expression plasmid pTE1. The control plasmid used the most commonly used constitutive promoter and RBS (PhmtB and RhmtB) in T. kodakarensis and was named pTE1-PhmtB-RhmtB-glu. All promoter activity evaluation plasmids were derived from pTE1-PhmtB-RhmtB-glu, in which the RBS remained unchanged and only the upstream promoter sequence was changed. Using the T. kodakarensis genome as a template, the promoter fragment was cloned and connected to the linearized pTE1-PhmtB-RhmtB-glu vector using the One Step Cloning Kit. The constructed promoter plasmid was transformed into E. coli. After verification by PCR, the plasmid was extracted and sequenced. The correctly sequenced plasmid was then transformed into T. kodakarensis TS559 using the calcium chloride method to obtain a strain expressing the promoter to be tested. The promoter strength in T. kodakarensis TS559 was determined by enzyme activity assay.

[0036] The enzyme hydrolyzes the corresponding monosaccharide p-nitrophenyl glycoside to release p-nitrophenol, which exhibits a yellow color under alkaline conditions. Enzyme activity is measured spectrophotometrically at 405 nm using p-nitrophenyl-β-D-glucoside as the substrate. The recombinant strain harboring the glycosidase reporter gene was cultured at 85°C for approximately 12 hours, normalized to normalized cell density, and harvested by centrifugation at 10,000 rpm for 5 minutes at 4°C. Cells were then disrupted by repeated freeze-thaw cycles using liquid nitrogen. The cell suspension was centrifuged at 12,000 rpm for 10 minutes at 4°C, resulting in the crude enzyme supernatant. The glycosidase assay was performed in a 500 μL aliquot containing a final concentration of 2.8 mM substrate, an appropriate amount of crude enzyme, and 50 mM sodium phosphate buffer (pH 6.5). The reaction mixture was transferred to an 85°C water bath and incubated for 30 minutes. Then, an equal volume of 0.25 M sodium carbonate solution was added to terminate the reaction, and the absorbance was measured at a wavelength of 405 nm using a spectrophotometer. Figure 1 shown.

[0037] Example 2: Rational mutation of the constitutive promoter P0560 and P2065 core regions

[0038] The base pairs in the core region of the T. kodakarensis promoter were scanned for mutations. By rationally designing the promoter sequence, a series of constitutive promoters with different strengths were obtained, and the key sequence patterns that affect the strength of the T. kodakarensis promoter were summarized. Among the promoters characterized above, promoters P0560 (DNA binding protein Alba, TK0560) and P2065 (TRAM protein, TK2065) were selected as the initial promoters, and rational design was mainly carried out on the BRE and TATA-box regions of the promoters, such as Figure 2 shown.

[0039] Using plasmid pTE1-P0560-RhmtB-glu as a PCR template, and centered around the base corresponding to the desired mutation site, using primers 10-20 bp upstream and downstream, a linearized mutant plasmid was amplified by whole-plasmid PCR. Using the plasmid template from the restriction nuclease Dpn I digestion product, the digested PCR product was transformed into E. coli DH5α. The linearized plasmid was circularized and replicated using the E. coli autorepair system. Single clones were selected for sequencing verification, and the mutant plasmid with the correct sequence was transformed into T. kodakarensis TS559 to test promoter strength.

[0040] Example 3: Modification of the T. kodakarensis constitutive promoter based on rational design

[0041] According to the base composition regularity of the core region of the T. kodakarensis promoter, mutations were made in the BRE and TATA-box regions, and rational mutations were made in some non-standard promoters to improve their expression levels ( Figure 3 ).

[0042] Example 4: Construction of a constitutive promoter library in T. kodakarensis

[0043] Characterization and rational modification of constitutive promoters in T. kodakarensis resulted in a library of 76 constitutive promoters ( Figure 4 ), the intensity fluctuation range reaches 8×10 3 .

[0044] Example 5: Evaluation of the induction strength of 17 maltodextrin-inducible promoters by β-glucosidase activity

[0045] ChIP-seq was used to map the binding sites of the TrmBL1 transcription factor genome-wide in P. furiosus under pyruvate growth conditions, identifying 28 TrmBL1 binding sites within the organism. To identify inducible promoters regulated by TrmBL1 in T. kodakarensis, the presence of the TrmBL1 protein was first determined. Blast sequence alignment revealed that the TrmBL1 protein is encoded by gene TK1769 in T. kodakarensis. Therefore, the promoters of these TrmBL1-regulated genes are promising candidates for inducible promoters in Thermococcus.

[0046] The sequence 200bp upstream of TSS (including the core elements of the promoter, BRE and TATA-box) was selected to evaluate the strength of the maltodextrin-inducible promoter, and the RBS sequence itself was selected. The vector construction method was referred to Example 1. The constructed promoter plasmid was transformed into Escherichia coli. After verification by PCR of the bacterial solution, the plasmid was extracted for sequencing. The correctly sequenced plasmid was transferred into T. kodakarensis TS559 by the calcium chloride transformation method to obtain a strain expressing the promoter to be tested. The strain was grown under gluconeogenic conditions (added with 0.5% sodium pyruvate) with an OD of 0. 600 When the concentration reached 0.2, maltodextrin was added to induce expression. The strength of the promoter in T. kodakarensis TS559 was determined by enzyme activity assay. Figure 5 shown.

[0047] Example 6: Rational Optimization of the Core Region of the Maltodextrin-Inducible Promoter

[0048] Using the above-established base composition rules of the promoter core region, five natural maltodextrin-inducible promoters were rationally mutated ( Figure 6 ), and finally obtained 15 maltodextrin-inducible promoters with different induction strengths.

[0049] Example 7: Screening, characterization, and rational design of pressure-inducible promoters

[0050] Two promoters were selected, named P1255 (promoter of ABC transporter encoding gene PAP_RS01255) and P9950 (promoter of SPASM domain protein encoding gene PAP_RS09950) for the next characterization experiment. The sequence 60bp upstream of TSS (containing the core elements BRE and TATA-box of the promoter) was selected to evaluate the strength of the promoter, and the RBS was RhmtB. The vector construction method and promoter strength test refer to Example 1. The screened promoters are as follows. Figure 7The expression level of the promoter was tested after the strain was cultured at 0.1 MPa and 30 MPa for 12 h.

[0051] Example 8. Characterization of T. kodakarensis 5′UTR

[0052] The expression intensity of 11 natural RBSs was evaluated using the β-glucosidase reporter system ( Figure 8 ). The selected RBS is located within the 5′UTR with a length of 25-46nt, and it is predicted by RNA Folding Form V2.3 that it will not form a complex structure at high temperature. To identify 5′UTR elements of different strengths in T.kodakarensis, it is necessary to control the 5′UTR to be tested in the transcription unit regulated by the same promoter. In this study, the above 11 5′UTR elements were characterized under the regulation of the promoter P0560. Using pTE1-PhmtB-RhmtB-glu as the starting plasmid, the promoter was kept as P0560, and only the 5′UTR sequence downstream was replaced. Using the T.kodakarensis genome as a template, the 5′UTR fragment was cloned and connected to the linearized pTE1-PhmtB-RhmtB-glu vector using the OneStep Cloning Kit. The constructed 5′UTR characterization plasmid was transformed into Escherichia coli. After bacterial liquid PCR verification, the plasmid was extracted and sequenced. The correctly sequenced plasmid was transformed into T. kodakarensis TS559 by calcium chloride transformation to obtain a test strain with the corresponding 5′UTR, and its β-glucosidase activity was tested.

[0053] Example 9: Rational Optimization of T. kodakarensis 5′UTR Sequence

[0054] UTR2289 was selected for further mutation. The secondary structure of UTR2289 predicted by mFold showed that the RNA sequence was simple and relatively stable at 85°C ( Figure 9 ). Base mutations were designed in the RBS or spacer region of the 5′UTR of UTR2289, and their effects on the expression intensity of UTR2289 were detected by β-glucosidase.

[0055] Example 10: Screening for HHP-induced toxin-antitoxin (TA) system in T. kodakarensis TS559

[0056] Functional toxin-antitoxin systems were screened in T.kodakarensis. A constitutive promoter was used to control the expression of the antitoxin gene, while an inducible promoter was used to control the expression of the toxin gene, to screen a heterologous type II toxin-antitoxin system (TA system) in T.kodakarensis. In order for the toxin protein to play a screening role under induction conditions, it is necessary to match different constitutive promoters, inducible promoters and TA systems. Referring to the strength of the constitutive promoter and 5′UTR characterized above, it was finally determined to use the two element combinations of P1431M1+RhmtB and P2289+R2289M1 to control the expression of the antitoxin gene, respectively, while the toxin gene selection pressure inducible promoter P9950-M3 controlled its expression. As Figure 10 As shown, the toxin protein PYCH_17720 and the antitoxin protein PYCH_17730 have a screening effect on T. kodakarensis.

[0057] Example 11: Design of a reverse toxin selection marker regulated by antitoxin to achieve scarless gene deletion in T. kodakarensis

[0058] First, the parent strain T. kodakarensis was used as the host and simvastatin (Sim) was used as the positive selection marker for the first homologous recombination. Second, under the induction of 30 MPa, the second homologous recombination was performed. The genome of the strain T. kodakarensis was used as a template to amplify the upstream fragment TK1827-Up, the downstream fragment TK1827-Dw and the gene TK1827 itself. The obtained fragments were 1000 bp, 1000 bp and 1582 bp in length, respectively, and inserted into Figure 11The knockout plasmid pTA-TK1827 was obtained by amplifying pTA-TK1827 using primers Primer F / Primer R. The linearized fragment was transformed into the strain T.kodakarensis and screened in a roller tube containing 4 μM Sim. The strain was cultured at 85°C for about 12 hours until a single colony grew out. A single colony was picked in an anaerobic operation box and placed in 5 mL of liquid culture medium containing 10 μM Sim. The genome of each transformant was extracted with phenol-chloroform, and the upstream and downstream primers of gene TK1827 were used to amplify and detect whether the first homologous recombination was successful. The strain in which the linear gene deletion fragment was successfully inserted into the genomic DNA of strain T.kodakarensis was named TK1827i. The strain TK1827i was inoculated into a liquid culture medium without Sim and transferred to a syringe. After culturing for about 14 hours at 30 MPa and 85°C (to complete the second homologous recombination), dilute the bacterial solution and pipette 1 mL into a roller tube without Sim, then place it in an 85°C incubator and culture until a single colony grows. Pick a single colony and culture it in a normal liquid medium. Identify it by PCR. Figure 11 shown.

[0059] Example 12: Improving H2 production in T. kodakarensis

[0060] T. kodakarensis can grow in a medium containing pyruvate. In the absence of sulfur, electrons released by substrate oxidation are accepted by protons, ultimately producing hydrogen (H2). Increasing microbial H2 production through genetic engineering primarily involves two strategies: overexpressing enzymes involved in H2 production and deleting enzymes involved in H2-consuming pathways. These two enzymes in T. kodakarensis correspond to membrane-bound hydrogenases MBH (TK2080-TK2093) and cytosolic hydrogenases Hyh (TK2069-TK2072), respectively. The specific process for improving H2 production in T. kodakarensis is as follows: 1) First, the gene cluster TK2069-TK2072 is seamlessly deleted using the gene deletion method established in T. kodakarensis as described above; 2) In the Hyh deletion strain, the native promoter of MBH is replaced with a strong constitutive promoter (P2289+R2289M1) or a strong inducible promoter (P1136M1 and P2129M1), and the native promoter of the ferredoxin encoding gene TK2012 is replaced with a constitutive promoter (P0615M2) or an inducible promoter (P1771M1). Figure 12 It was shown that replacing the natural promoters of key genes in the H2 metabolic pathway with strong promoters can significantly enhance the H2 production capacity of T. kodakarensis.

[0061] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A cis-element library for regulating gene expression in thermophilic microorganisms, characterized in that: It comprises a constitutive promoter library, which comprises the base sequences shown in SEQ ID NO. 1-8.

2. The cis-element library according to claim 1, wherein The element library further comprises an inducible promoter library, which includes a maltodextrin inducible promoter library and a pressure inducible promoter library.

3. The cis-element library according to claim 2, wherein The maltodextrin inducible promoter library includes base sequences as shown in SEQ ID NOs. 9-13.

4. The cis-element library according to claim 2, wherein The pressure-inducible promoter library includes a base sequence as shown in SEQ ID NO.

14.

5. The cis-element library according to claim 1, wherein The element library also includes a ribosome binding site RBS library, and the RBS library includes base sequences shown as SEQ ID NOs. 15, 16, and 18.

6. Use of the cis-element library for thermophilic microorganisms according to any one of claims 1 to 5 in regulating gene expression in the thermophilic microorganism T. kodakarensis.

7. The use according to claim 6, characterized in that A constitutive promoter is used to control the expression of the antitoxin gene, and an inducible promoter is used to control the expression of the toxin gene.

8. The use according to claim 7, characterized in that The constitutive promoter P1431M1 and the 5'UTR element RhmtB control the expression of the antitoxin gene PYCH_17730, and the pressure-inducible promoter P9950-M3 and the 5'UTR element RhmtB control the expression of the toxin gene PYCH_17720, wherein the P1431M1 sequence is shown in SEQ ID NO.2, the P9950-M3 sequence is shown in SEQ ID NO.14, the RhmtB sequence is shown in SEQ ID NO.17, the PYCH_17730 gene sequence is shown in SEQ ID NO.19, and the PYCH_17720 gene sequence is shown in SEQ ID NO.

20.

9. The use according to claim 8, characterized in that The cloning vector used in the inducible expression was based on the plasmid pUC18.

10. Use of the cis-element library for thermophilic microorganisms according to any one of claims 1 to 5 in regulating hydrogen production, characterized in that: The inducible promoter in the element library was used to replace the natural promoter of the gene related to the hydrogen production pathway in the thermophilic microorganism T. kodakarensis.

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