Constitutive strong promoter suitable for streptomyces and application thereof

Abstract

The invention relates to a constitutive strong promoter suitable for streptomyces and application thereof. The constitutive strong promoter is: i) a nucleotide sequence as shown in any one of SEQ ID NO.1-30; or ii) a nucleotide sequence which is obtained by substituting, deleting and / or adding one or more nucleotides to any one of the nucleotide sequences shown in SEQ ID NO.1-30 and has the function of the strong promoter; or iii) a nucleotide sequence which is hybridized with any one of sequences shown in SEQ ID NO.1-30 under a highly strict condition and has the function of the strong promoter, or iv) a nucleotide sequence which has 75% or more consistency with the nucleotide sequence of i), ii) or iii) and has the function of the strong promoter. The activity of the 30 promoters disclosed by the invention is 10-88 times of that of the most common streptomycete constitutive promoter ermEp *, and a selectable promoter element is provided for gene manipulation of streptomycete.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and microbial metabolic engineering, and in particular to a constitutive strong promoter suitable for Streptomyces and its applications. Background Technology

[0002] Bacterial RNA polymerase holoenzymes are composed of α, β, β', ω subunits and a σ factor. They specifically recognize a DNA sequence upstream of a gene, known as the promoter. Promoters are key elements in gene transcription and regulation, playing an indispensable role in heterologous gene expression and the activation of silent gene clusters. Replacing the native promoter with a universal constitutive promoter or a host-specific strong promoter is an effective strategy for activating silent genes or metabolic pathways in the original strain.

[0003] Streptomyces ( Streptomyces Streptomyces (Streptomyces aquatilis) is a representative genus of Gram-positive actinomycetes, possessing a sophisticated secondary metabolic regulatory network, a strong ability to synthesize secondary metabolites, and a genetic background compatible with multiple exogenous genes. It can produce a variety of bioactive natural products and is also an ideal host for heterologous microbial expression. Commonly used Streptomyces expression strains include *Streptomyces aquatilis* (*Streptomyces cerevisiae*). Streptomyces coelicolor Streptomyces venezulatus ( Streptomyces venezuelae Streptomyces cerevisiae (S) treptomyces lividans Streptomyces albopictus () Streptomyces albus )wait.

[0004] Streptomyces genomes have a high GC content and a complex regulatory network. Streptomyces promoters exhibit significant heterogeneity; promoters available in other microorganisms are often inactive in Streptomyces. Currently, the most widely used Streptomyces promoters include those derived from… S. erythraea Erythromycin resistance gene promoter ermEp * and artificially modified promoters kasOp* Chinese patent CN113667670A discloses a strong promoter suitable for Streptomyces and its application. stnYp It can be used in commonly used Streptomyces model strains and is of great significance for protein expression and metabolite synthesis in actinomycetes.

[0005] Discovering new promoters and combining them with Streptomyces chassis strains can enable the activation of various Streptomyces silent gene clusters and the heterologous expression of natural product gene clusters. Utilizing... ermEp* The promoter regulates the jadomycin biosynthesis gene cluster, significantly increasing jadomycin yield by approximately two-fold. RNA-seq analysis of a strong promoter identified in *Streptomyces albopictus* J1074 activates the PTM gene cluster in *Bacillus oryzae*, and also... S. coelicolor The yield of the target compound in M1146 increased by 1.8 times. S. lividansThe yield of 66 target compounds increased by 1.2 times.

[0006] Replacing native promoters with universal constitutive promoters or host-specific strong promoters is an effective strategy for activating silent gene clusters and natural product biosynthetic pathways. However, universal constitutive promoters are very limited, severely restricting efficient gene initiation and heterologous expression in Streptomyces. Furthermore, while a single strong promoter can activate a gene cluster, the increase in yield is often insignificant. Moreover, transcriptional manipulation of multiple genes requires a large number of different strong promoters, and repeated use of the same promoter often carries the risk of homologous recombination.

[0007] Therefore, discovering and characterizing constitutive strong promoters that are universal, stable, and of varying strengths and applicable to Streptomyces is beneficial for the precise regulation of target genes and metabolic pathways. Summary of the Invention

[0008] The purpose of this invention is to provide a constitutive strong promoter suitable for Streptomyces and its applications. The constitutive strong promoter for Streptomyces comprises 30 promoters, the activity of which is among the most commonly used constitutive promoters for Streptomyces. ermEp* The number of promoters ranges from 10 to 88 times, enriching the natural promoter library and providing selectable promoter elements for gene manipulation in Streptomyces.

[0009] The objective of this invention can be achieved through the following technical solutions: The first objective of this invention is to provide a constitutive strong promoter suitable for Streptomyces, said constitutive strong promoter being: i) Any of the nucleotide sequences shown in SEQ ID NO. 1~30; or ii) A nucleotide sequence having the strong promoter function described above, formed by substitution, deletion, and / or addition of one or more nucleotides of any of the nucleotide sequences shown in SEQ ID NO. 1 to 30; or iii) A nucleotide sequence that hybridizes to any of the sequences shown in SEQ ID NO. 1~30 under highly stringent conditions and possesses the strong promoter function described above; or iv) A nucleotide sequence that has more than 75% identity with the nucleotide sequences of i), ii) or iii) and has the strong promoter function described above.

[0010] As a preferred technical solution, the high-rigor conditions are: treatment at 95°C for 5 minutes, incubation at 65°C for 10 minutes, and incubation at 45°C for 5 minutes in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to hybridize with the nucleotide sequences SEQ NO. 1 to 30 in the sequence listing.

[0011] Furthermore, the constitutive strong promoter is: i) Any of the nucleotide sequences shown in SEQ ID NO. 1~30; or ii) A nucleotide sequence having the strong promoter function described above, formed by substitution, deletion, and / or addition of one or more nucleotides of any of the nucleotide sequences shown in SEQ ID NO. 1 to 30; or iii) A nucleotide sequence that hybridizes to any of the sequences shown in SEQ ID NO. 1~30 under highly stringent conditions and possesses the strong promoter function described above; or iv) A nucleotide sequence that has 85%, 90%, or 95% or more of the same nucleotide sequence as i), ii), or iii) and has the strong promoter function described above.

[0012] As a preferred technical solution, the promoter nucleotide sequence of the present invention is mutated using directed evolution and point mutation methods. Nucleotides that are artificially modified and have 75% or higher identity with the promoter nucleotide sequence isolated from the present invention, as long as they maintain the promoter activity of gene transcription, are all derived from and equivalent to the nucleotide sequence of the present invention.

[0013] As a preferred technical solution, "identity" refers to the similarity of natural nucleic acid sequences. In this invention, "identity" refers to a nucleotide sequence that has 75% or higher, 85% or higher, 90% or higher, or 95% or higher similarity to the nucleotide sequence of promoter i), ii), or iii) of this invention. Identity can be evaluated by visual inspection or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0014] A second objective of this invention is to provide a biomaterial containing a constitutive strong promoter suitable for Streptomyces, said biomaterial including recombinant DNA, expression cassette, transposon, plasmid vector, bacteriophage vector, viral vector, or engineered bacteria.

[0015] The third objective of this invention is to provide a constitutive strong promoter suitable for Streptomyces for use in constructing recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors, or engineered bacteria.

[0016] A fourth objective of this invention is to provide a recombinant DNA, which is operatively linked from the above-mentioned constitutive strong promoter to a downstream target gene.

[0017] Furthermore, the target gene is selected from nucleic acids encoding proteins, nucleic acids encoding ribozymes, and nucleic acids encoding antisense RNA.

[0018] Furthermore, the protein is an enzyme, hormone, antibody, or growth factor.

[0019] Furthermore, the enzyme is selected from oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.

[0020] Furthermore, the enzyme is the catechol 2,3-dioxygenase gene. xylE Encoded enzymes.

[0021] A fifth object of the present invention is to provide an expression vector comprising the above-described recombinant DNA.

[0022] A sixth objective of the present invention is to provide a transformant, wherein the transformant is a host bacterium carrying the above-described expression vector.

[0023] Furthermore, the host bacteria include Escherichia coli DH5α, Escherichia coli ET12567 / pUZ8002, and Streptomyces albopictus J1074.

[0024] A seventh object of the present invention is to provide a plasmid vector containing the above-described constitutive strong promoter.

[0025] Furthermore, the plasmid vector is selected from free vectors or integrative vectors. The free vector contains a replicon that can be recognized by all Gram-positive bacteria and oriT Wide host shuttle plasmid vectors for genes; The integrative vector contains a phage integrase gene. int site and oriT Genes can be widely transported across host populations via plasmid vectors.

[0026] Furthermore, the free vector includes the pBS plasmid vector, and the integrated vector includes the pDR3 plasmid vector.

[0027] An eighth object of the present invention is to provide a host cell comprising the above-described plasmid vector.

[0028] Furthermore, the host cell is a Streptomyces, including Streptomyces albopictus (Streptomyces). Streptomyces albus ).

[0029] The ninth objective of this invention is to provide an application of host cells in protein expression and heterologous synthesis of important metabolites.

[0030] Furthermore, the host cell is *Streptomyces albopictus* (…). S. albus J1074, the target gene is the catechol 2,3-dioxygenase gene. xylE The promoters are the 30 promoters mentioned above, used to measure enzyme activity at different time points.

[0031] The tenth objective of this invention is to provide a constitutive strong promoter, plasmid vector, and / or host cell suitable for Streptomyces in initiating the expression of a target gene.

[0032] Furthermore, the target gene is the catechol 2,3-dioxygenase gene.

[0033] As a preferred technical solution, in the catechol 2,3-dioxygenase gene xylE A strong promoter was inserted upstream to initiate gene transcription and expression. Enzyme activity was measured at different time points.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows: In embodiments of the present invention, 30 promoter expressions are described in detail. xylE The invention describes the construction methods of gene expression plasmids and strains. It also explains the determination and results of catechol 2,3-dioxygenase expression in Streptomyces white J1074 using 30 promoters, providing a simple and universal method for determining Streptomyces promoter activity.

[0035] This invention provides 30 constitutive promoters suitable for Streptomyces, including the sequence of the promoter, plasmid vector, and its application characterization in Streptomyces, providing a library of numerous selectable natural Streptomyces promoters.

[0036] In the catechol 2,3-dioxygenase gene xylE Inserting 30 promoters upstream of each gene enabled the expression of catechol 2,3-dioxygenase at different levels in *Streptomyces whiteus* J1074. The newly characterized constitutive promoters of varying strengths provide more options for existing *Streptomyces* promoter engineering, gene activation, heterologous expression, and metabolic remodeling. Attached Figure Description

[0037] Figure 1 In one embodiment 2 of the present invention, the plasmid vector pDR3- p69 - xylE A schematic diagram of the spectrum; Figure 2 This is a graph showing the activity of catechol 2,3-dioxygenase expressed by 30 promoters in *Streptomyces whiteus* J1074 in one embodiment 4 of the present invention. The positive control is the promoter shown. ermEp* and stnYp . Detailed Implementation

[0038] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0039] The following definitions are used in this invention: The base sequences, nucleotides, and amino acids (single letters) used in this invention are based on the nomenclature rules for biochemistry set forth by the International Union of Practical and Applied Chemistry and the International Union of Biological Sciences. Reference can be made to the abbreviations in the article "Guidelines for Preparing Instructions Containing Base and Amino Acid Sequences" published in the European Journal of Biochemistry, Volume 138, Issue 9, late 1984, as well as abbreviations commonly used in the field of biotechnology.

[0040] In the following examples, the specific steps and conditions for PCR amplification, plasmid extraction, enzyme digestion, ligation of digestion products, and transformation were all performed according to the instructions of the purchased enzymes and reagents. The DNA polymerase used for PCR amplification was purchased from Nanjing Novizan Biopharmaceutical Technology Co., Ltd., and the restriction endonucleases used for enzyme digestion and the ligases used for ligation of digestion products were purchased from Thermo Fisher Scientific. The plasmid extraction kit, DNA gel recovery kit, and PCR purification kit were purchased from Wuhan HanHai New Enzyme Biotechnology Co., Ltd., and the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. and Platinum Biotech (Shanghai) Co., Ltd.

[0041] In the following embodiments, the sources of the strains are as follows: Streptomyces velutipes ( S. flocculus CGMCC 4.1223 and Streptomyces albopictus ( S. albus ) J1074 has been reported in the article (Guo W, Xiao Z, Huang T, Zhang K, Pan H, Tang G, Peng W, Deng Z, Liang R, Lin S. Identification and characterization of a strong constitutive promoterstnYp for activating biosynthetic genes and producing natural products instreptomyces. Microbial Cell Factories. 2023, 22(1):127. doi: 10.1186 / s12934-023-02136-9).

[0042] Escherichia coli ET12567 / pUZ8002 was purchased from Beyotime Biotechnology.

[0043] Unless otherwise specified, the experimental techniques and methods used in this embodiment are all conventional techniques and methods.

[0044] In the following embodiments, the sequence of the promoters is as follows: The promoter p69 The sequence is shown in SEQ ID NO.1: ACGTCCCTGAGGAACAGGTCGTAGTAGAACGGGATCGCGACGGCCGCCGCGGCGAAGAGCGCGAGCTCGTTGAGCGCGGACCACACGAAACCAGGACGGCCGTTGGACACCAGCGCCAGGCTCAGCCCGCCGAGGAAGACACACAGCGCG AGAGCACCACGGAGCCTGCGCAGCAGACCACGGCTCCCCGCTATATCGCCGTTCATCGACCACCCCCGGTGTCAGGGTGTCTTTCCCCTCTCGGCAGGCCGCCAAGCCTTGCCCCGGCAACTAGCTTGCTAGGATGCTAGCATCGGCAC; The promoter p07 The sequence is shown in SEQ ID NO.2: GGGTCGGTGTAACTCGCTGCGGCGCCGACTCGCCTACGGCATACGGTGGTTGTACGCGCTATTCACGGCGCCTTCGCATTCTCGCGCAGCACGCCCATCGCGCCCATGGTGAATGCCGGTGGCGGGCCGAGGCGGCGAATACGGGGCGGT CGCCGCCGGCCGCCGGCCGTGGCGGGGCAGGGAGCGGCGGGGGACGGCATCGTCCGCATCCGGTCCGCGAAGGATGGCCGGAACCTTCTCCATGAGGTCGCCGCGGCGGGCATGCTTGGCGTGCGACGGCTAGCCTGCTAGCATGCTCAC; The promoter p73 The sequence is shown in SEQ ID NO.3: ATGCGCCTATCAACGACGGTTGTTGGCGGTGCCCCGCGCGCACCGGCTGCACGGCGGCGCCGCGCACGCACGCGGGGCGGCGCGAGCCGGGTGATGTGGGAAGGAGTCCGGGCCGGCGCGGCGGAGCTCGCGGCCGGCGGCAGTCGGCCCTGGAGTCTCCCGTGCCGTCGCGGGGACTTCAACAGGGTGCTACGCGGACGTACTGGTGCCGACGAGAGAGGGGCCCTGGTCCGTGAGGGCCCTCGCGGAGCCACCCCGTACGCGGTGGTTGGCAACCTGTCCGTGTGCTAGCATGCTAGC; The promoter pV0 has the sequence shown in SEQ ID NO.4: CCGCGGAAGCCGTAGCGGCCCACGACGATCAGGTCGGCGTGGGAGAGCCGGGACACGTCCTCGGCCAGGACGTCGTGCTCGCGCTGGTCGGCGGGCAGGGCCCGCAGGGTGGTCACGACGGCGTCCAGGTAGCGGGTGGTCCACAGCGCGGCGGCGTTCAGCACTGGTCCGAGCGCGCCGAGCTGGTCCTCCTCGCCCGTCCGGTACGCCTGCATGATCTGCCCGCGCCTGCCGGGGCAGATGTCGCGGGCGAGCTGTTGCGATCGACATCTAGCGATTTAGCTTGCTAGCGTGCTTGCC; The promoter pW9 has the sequence shown in SEQ ID NO.5: CGATGACGAACAGGAGCCGCCTGCCGTACAGGTCGGCCAGCGAGCCTGAGGTAAGCAGGAGCGCTGCGAGGCTCAACGCGTATGCGTCCGTCGTCCATTGCAGGCCACTGAAGTCGGCGTCCAGCGAGTGCTGGATGTCGGGCAGCGCGACCGCGACGATCGTGACGTCCAGCAGCAACATGAATGTGGCGGCGCACACGACTAACAGCGTCCACCATTGACGTTTCATCGTTCGAGCTCCTTCTCGATGCAAAGCTCTTGCGCGTCATGCCCGATCGCGGCGCATGCTAGTATGCTAGC; The promoter pC8 has the sequence shown in SEQ ID NO.6: AACAGGCGCCGCCTGCCGTACAGGTCGGCCAGGGAGCCCGAGGTGAGCAGGAGCGCCGCCAGGCTTAACGCGTAGGCATCCGTCGTCCATTGCAGGCCGCTGAAGCTCGCGTCCAGCGAGTGCTGGATGTCGGGGAGCGCGACCGCGACGATCGTGACGTCCAGCAGCAGCATGAACGTGGCGGCGCACACCACCAGCAGCGTCCACCATTGGCGTTTCATTGTTGGGATTCCTCCTCGGTTCGTAACTCTTGTTCGATCGGCCCTAGTTGGACGAGCGGACGGATGCTAGCATCCTAGT; The promoter pH1 has the sequence shown in SEQ ID NO.7: CGGCCAAGTCGCCCCGGCGCCGCCACGCCGAGGCGCGCGGCGGAATGTTGCCCTTTCCCTCGCTGAACCCCGCCGCGCCATGGGTGAGTTCCCGGGATGAAGACGGCGTGCCGGTGTGTATCGGCGAGCACATGCACTGCGGCGTGCTGCAGCGCCGCCTCGTTCTTGTCGCCGAGGCAGCCCGGTTCCACGCCGGCCGGCTGCTGCACCAGCCCCGGGGCGGGCGGGAGGACCGAAAGAACAGACAGAAGGAATTCTGTGGGCTAGCATGCTAGCAATCTAGCGAGCTAGTATCCGTGT; The promoter pW3 has the sequence shown in SEQ ID NO.8: CCTCCGCGACCGGCCCGGACCACAACACGCACGCGCCTGCCGCACCCGCCACTGCGGTCAGTGCGGTTGCCCTGTTCAACGCACGACGCCATCTGAACCACATGACTCTCACGTTCCCCACCCTTCATGTGCACACGAAAGCAACACGCGCCCAACTACCCACGCTGGTAACGAACATGCGCCGTTCTGCTGAGGGAGGTCGCCGAGTTGTTGAGCCGGTCCACGTGGCCCCCGGTCGCGAGTGGGTGCTGCGCGGCAGGGCTTGTCAGGCTAGGCGTCTAGCATGCTAGCATCGCCCTC; The promoter pY7 has the sequence shown in SEQ ID NO.9: GAGGGAGATGCTAGCATCCTAGATACCTAGCCAGATAGTCCATCGACAAGGACCCGCTCATGTCTGCTTCTGCGGCCGCCCCGCACGACCCGGTTGTCGTCCGCGCGGGCGATGCCGAGCAGTTGCCCGAGATCGGCCACTACCTGCTGGCTGACGCCGGCGCCAGCAACGGCGCGCTCAGCGCCCACCGCATCCAGCTCGCCCGCGGCGCCGACGGCGCCGTCCCGCACCGGCACGACCACGCGTCCGAACTGTTCTTCGTCGTCGACGGAACGCTGGAGGTGCTCATGGGATCGAAGA; The promoter pG4 has the sequence shown in SEQ ID NO.10: ACTAGGATGCTAGCATCCGTCCGCTCGTCCAACTAGGGCCGATCGAACAAGAGTTACGAACCGAGGAGGACTCCCAACAATGAAACGCCAATGGTGGACGCTGCTGGTGGTGTGCGCCGCCACGTTCATGCTGCTGCTGGACGTCACGATCGTCGCGGTCGCGCTCCCCGACATCCAGCACTCGCTGGACGCGAGCTTCAGCGGCCTGCAATGGACGACGGATGCCTACGCGTTAAGCCTGGCGGCGCTCCTGCTCACCTCGGGCTCCCTGGCCGACCTGTACGGCAGGCGGCGCCTGTT; The promoter p51 has the sequence shown in SEQ ID NO.11: TGATGACCTTCGGTCCCACAAGCACCTGGAGCGTTCCGTCGACGACGAAGAACAGTTCGGAGGAGTGGTCGTGCCGGTGCGGGACCGCGCCGTCGGCGCCGCGGGCGAGTCGGATGCGGTGGGCGCTGAGCGCCCCGTTGCTGGCACTGGCGTCAGCCAGTAGGCAGTGGCCGATCTCGGGCAGTTGCTCGGCGTCGCCTGCGCGGACGACAACCGGGTCGTGTGGATCAGCTGCAAAAACAGACACGAGAGACCCCCTGGGACTAGTATGCTAGCAATCTAGCGAGCTAGTATCGCCCT; The promoter pE6 has the sequence shown in SEQ ID NO.12: GCCCATGGCGGAAGAGGCCATGCGTCGGCCAAGTCGCCCCGGCGCCGCCACGCCGAGGCGCGCGGCGGAATGTTGCCCTTTCCCTCGCTGAACCCCGCCGCGCCATGGGTGAGTTCCCGGGATGAAGACGGCGTGCCGGTGTGTATCGGCGAGCACATGCACTGCGGCGTGCTGCAGCGCCGCCTCGTTCTTGTCGCCGAGGCAGCCCGGTTCCACGCCGGCCGGCTGCTGCACCAGCCCCGGGGCGGGCGGGAGGACCGAAAGAACAGACAGAAGGAATTCTGTGGGCTAGCATGCTAG; The promoter pN3 has the sequence shown in SEQ ID NO.13: GAACAGCCGCCGCCTGCCGTACAGGTCGGCGAGGGACCCTGAGGTCAGCAGGAGCGCCGCCAGGCTTAACGCGTAGGCGTCCGTGGTCCATTGCAGACCGCTGAAGCTCGCGTGCAGTGACTGCTGGATGTCGGGCAGCGCGACCGCGACGATCGTGACGTCGAGCAGCAGCATGAACGTGGCGGCGCACACCACGATCAGCGTCCACCATTGGCGTTTCATTGTTCGGAGCCCTCCTCGGCTCGTAACTCGCGTTCTTTCACGCGTGGTTCAACAAATCGGCGCATGCTAGCATGCTAGC; The promoter pN4 has the sequence shown in SEQ ID NO.14: AACAGGCGCCGCCTGCCGTACAGGTCGGCCAGGGAGCCCGAGGTGAGCAGGAGCGCCGCCAGGCTTAACGCGTAGGCATCCGTCGTCCATTGCAGGCCGCTGAAGCTCGCGTCCAGCGAGTGCTGGATGTCGGGGAGCGCGACCGCGACGATCGTGACGTCCAGCAGCAGCATGAACGTGGCGGCGCACACCACCAGCAGCGTCCACCATTGGCGTTTCATTGTTGGGATTCCTCCTCGGTTCGTAACTCTTGTTCGATCGGCCCTAGTTGGACGAGCGGACGGATGCTAGCATCCTAGT; The promoter pA7 has the sequence shown in SEQ ID NO.15: GCGCGGCGCGGTCGAGTACGGACTGGAGGTCGGCGTTCTCGGTCACCGGGGCGTCCCTTTCGCAGGCGGGCATCACGAACTGAACCAGCGTACGTCAGGGGTCCGACAGCCACGCCGCCGGTCAGCGAGCACCCCACGTCAGCGCACCCGCGCCCACACCACCGACCGCCCGGCACCCCCGCACCAGGGACCCGCCCCTCGCACCTCCCGCCGGGGACGCGCCCCCGCGCCGTACTGCCCCTGCCCTCTCCTGGCGCCGACCCCTCGACACTAGATTGCTAGCATGCTAGCTTCCTTCTC; The promoter p86 has the sequence shown in SEQ ID NO.16: GCTGGTGCTCCAGGCGCGCGGGGACGGCTGAGCGTCGTACCGCACGACGGTCTGAGTGCCCTCGCCCGGTGTTTCATGTCCGGTTCCCGCGCGCAGTCGGCCGGTGCTGTCCGGTCCTCGTGCGGGACGGTCGCCCTCGGCCGGCTTCGCCGGGGGTGCCCCCGGTTGCGGCGCTGGGGGCGGGCTGTGTGACGGCGGCCGGGACCTTCACGTGCGGTTGAGTGGCCCCTGCTGGTAGGCCGGTTTCCGATCACCGCGCGGATCCTGCATCGCTGGGGCTATGTTGCTAGCATCCTAACC; [[ID=?]]The promoter pI4 has the sequence shown in SEQ ID NO.17: It should be noted that there seems to be a typo in the original text where "所述启动子" in line 8 should probably be something more specific or correct in the context. Here I've translated it as "The promoter" as it's the most common way to start the relevant description in English for this kind of patent text.AGGCCATCGTTCTGCCGGAGCACGAGGGCCCGGGGAGCTGCCGCGTCGAGGTGGTCTACCGCCACTACACCGGCGTGGACCGGGTGACGGGGACCGAACGGGTCGCCCTCGTCCTCAAGGGGGCGGGCCGCAAGGGCAGGCCCTGCGAACTGGCCACCGAGCTCGCCGGTTCGGCCGCCGCCGCGCTGCCGCCCGGCTGACGGGCTGATTCCTCTTCTATCGACCCAGTTTGTGAACTGGGTCGAGGAGCAGCCCTGTCTCGAGGTGTGGCTAGCTTGCTAGGATGCTAGCATCGCACTC; The promoter pF3 has the sequence shown in SEQ ID NO.18: GCGCATGGACGACCTTCACCGCGACGGTGCGCCCGCCCGCGCTGCGCCCGAGGTAGACCCGGCCCATCCCGCCCGCGCCGAGCCGGCCCAGCAGCCGGTACCCGCCCAGGCTTCGTGGATCATCCGCCGCCAACGACTCCATGGCGCCTCCCCCTTGATACGGCGCCCCCAGCTTATGACCCGGCCCTGCCGGCCCCCCAGAACTCCCCCTCGCCCCGTCGCCCCTCCCCCACCTGCCTGCCCGCCCCGGAGGACCCCCGACCCCTCGACGCCAGGTTGCTAGCATGCTAGCTTCCTACC; The promoter pX4 has the sequence shown in SEQ ID NO.19: GTACGGCGGGATCACCCCCGTCGGCCTGCCGGGGGGATGGCCCCTGCTCGTCGACGAGGCCGTCGCCGCCACTCCGTTCGTCCTCATCGGCAGCGGCAAGCGCCGGAGCAAGCTGATCCTCCCCGGCGCGCTCCTGGCGCGGTTGCCGAACGCCGAGGTCGTCGCCGGTCTGGCCTGCTGAGCACGGCCGGCCGCGACTACGGGGCCGGAACCGGGGCGCCGCCGCCGGTCCCGGCCCGTCTCCCGCAGATGCCGTTAACAACTACCCCGGCACGTCTCTAGATTGCTAGCATCCTAACC; The promoter pU5 has the sequence shown in SEQ ID NO.20: GCTCGTCACCCTGCGCCCCGACGGCTTCGGGCTCGCCCCGGCCGGCCGTACGGCGGACGTCACGGTGGACGCGACCGTCGAGGCGACCGCGGCCGACCTCCTCCTGCTGCTCTACGGTCGTCGGCGCCACGACGCCGAGGTCGTCGCGGTCGTAGGCGATCAGGATCTGCTCCTGCGTTGGTTCGCCAACTCCGCTTTCTAGAACGCGTTCGGTGGGTCGCACCGCCGCGGCCGGAGCATGCCCGCGGCGGTGTGACGCCGATCACGCGCGGGCATATCTAGAGTGCTAGCATTCTAGAT; The promoter p08 has the sequence shown in SEQ ID NO.21: GTACGTGTCCGTCCTTCCGTGGGGCCGTCCGTCCTTCTGTGCGGCCAAACGTCCAGTGTGCCTCATCCGCCGGGGGCGGCGGGGGGTGGCGGGAGAGGCCCCGGACCCCGCTTGTCGCGGCTTCGCCGCTCGTCCTCAAACGCCGGACGGGCTTGATTTCGCGCCCGGGCGCACGATTCAGCCCGTCCGGCGTTTGAGGACAACCGCGCGGAGCGCGGTTTCGGGGGTGCGGGGGCTTGCCCCCGCAAGAAACGGTGAATGGGGGTGCCCCCTCTGGGGGAGGGACTGGGGCACCCTTCCCCCACCCTGCCTAGATTGCTAGCATCCTAACC; The promoter pB6 has the sequence shown in SEQ ID NO.22: GTACGTGTCCGTCCTTCCGTGGGGCCGTCCGTCCTTCTGTGCGGCCAAACGTCCAGTGTGCCTCATCCGCCGGGGGCGGCGGGGGGTGGCGGGAGAGGCCCCGGACCCCGCTTGTCGCGGCTTCGCCGCTCGTCCTCAAACGCCGGACGGGCTTGATTTCGCGCCCGGGCGCACGATTCAGCCCGTCCGGCGTTTGAGGACAACCGCGCGGAGCGCGGTTTCGGGGGTGCGGGGGCTTGCCCCCGCAAGAAACGGAGAATGGGGGTGCCCCCTCTGGGGGAGGGGCTGGGGCACCCTTCCCCCACCATCCCTAGCTTGCTAGCATCCTAGCC; The promoter p49 has the sequence shown in SEQ ID NO.23: CAACGGCCGAAAGTATGGTCATGGCGTCATTGTGGATGGATCGCGCCGGGGCCGGGCGGCTGGGGTGCGCGGTCCGGGCACCACGGCCGCGGGCCCTGCCGACACGACACGCGCGCGTTCCTGGCCGGCGCGCACCGGCGACGGTTGGCATCCGGCGCGCACCCGCCGAGGGGTGCGCGTACCGAAGCCCGGCCCTCACCGGCCGACCGGCCTCGCCCGGCGGCGGCCCGCCGCCCCGTCACGACTGTTCGTCCACCGGAGACCAGGACATCTAGGTTGCTAGCATGCTAGCCTCCCAGC; The promoter pA4 has the sequence shown in SEQ ID NO.24: GGACCAGGACCTGCTCTCGCGTTGGTTCGCCAACTCCGCCTTCTAGGGCGCGTCCGGTGGGTCACGCGGTGACCTGCCAGGGCTCGCGGTGACCCATCAGGGTTCGCGGTGACGTGGCGGGCGGCCTGGGGATGGCTGGTGAGTTTGCCGGTGTACAGGGTGGCGTCGCGCCACTGCTGAGCCTGTGAGCGACCCCGTCCAGCCATTCCGGGCCGCCGTTCTCCTCCGAGCGGGCCGGCAAAGCCGTTTCGTGTGACGTCGATCTCACACCCTCGCGCTTAGAATGCTAGCATTCTAAGT; The promoter pU7 has the sequence shown in SEQ ID NO.25: TGCTCCTCACCGCCGTCTTCACCAAGACCCTCATCGACATGTGGAACCCGGCCTACGGCTCCGGCAGCTCCGTCCTCGGCATCGGCTCGGTCTTCGTCATCGGCGTCGGCCTGCTGCTGCTCGGCTTCGTGCTGATGCTGGTGATGCGGCGGCGCAGCCCGGCGTTCTTCCGGGGCGAGGTGCTGGGGCGGGGGACGCCGGCGCTGGTGGTGGAGGAGTAGAGCCCGCGCGAGCCGTCCCGCCCTCCCGTCTCGGCCAGATTGCTAGCATCCTAGTTGGCTAGGGTGCTAGCCTCCCATC; The promoter pH2 has the sequence shown in SEQ ID NO.26: GGTGTACTTGGCCTTGTCGTACGCGTTGTGCGTCCCGATCGCCGTCGTCTCGGAGAACTTCGGCGCCGCCGCGTTCGCGTTGCCCGGCCCCACCATCAGCAGAGCACCGGCACCCACGAGTGCCGCCAGTCGTCTCACCGCGCCCATTGCTCAACTTCCCTTCGCTGTCGCCGAGTTGGCGGCAGCGTAGGAGCGAGCGGTTACTGCGCGGTAGCCCCTGAAGGAACATCAGCCACTCTCCGGCCGTCGGGCGACCTGTCACCACTGGTGGCTAGATTGCTAGCATGCTAGCCTCAAGGT; The promoter pS1 has the sequence shown in SEQ ID NO.27: GAGCTGTCCCGTAAGTGATCTTGCTGTGGGATGGCCGTGGCCGTGGCTGGTGTGATCACTGCATCGGAGCCTTCTTGGATGGCCCCCTTCACCGGGCTGAGCCCGCGCACCTTCGCCAAGTTGGTGACGATGCTGCGGCGCGAAGGTGCCGATGCGCCGGGGCGAGGCGGCCACGGAAGATGCCGCTGGAGGACCGCGTGCCGCTGGCGGCTGCGTACTGACGCACCAATTTGACGCAACGTCAGTTTGGCTCGCTGCTTGGTGGGTTAATTACATTGCTAGCATGCTAGCATCGCACTC; The promoter p The sequence of G8 is shown in SEQ ID NO.28: CGCATCGTGCACCCGATGGTGGGTGAGCTGGCCCTCCGCTACGAGACCTTCACGCTCCCCGACGACGACCAGGAGCAGTCGCTGAGCAGCTACCACGCGGAGCCCGGCTCGCCCTCCGAGGAGGCGCTGCGCCTGCTGGCGAGCTGGGGAGCGGACGCCTCCCGGGACTTCTCGGCCGACGCCGGTACGGACGCCGACACGGACACCGGTACGGAGTCCTGACCCCGGGCCCGGCTCGCCACCTCAGCCTGCCGCGCAACCCCGGCTCAGGTCCTTTCTTAGAATGCTAGCATCCTAATT; The promoter p33 The sequence of which is shown in SEQ ID NO.29: CCGACTCGCCGTCTTCTTCGGGGAGGACGGCAAGATCCGCCGGGCTGACGCGGACGGCAAGGACGTGGAGGAGGTCATCCGGGAGGCCGTAGCCCGAGATCTGGAAATGGCGTAAGCGCGCCTGAGCGCCCGTCGCGGCTTGCTACTGGG ATTCCCCCCCCGGCGGCGGGCCGTTCGGCGTTTGAGGACGAGCGGCGCAGCCGCGAAAAGGGGGTCCGGGGGCTTGCCCCCAGGAAACGGAGAAAGGGCGGGTCCGGGGCACCCTTCCCACCCCCACTCTAGATTGCTAGCATCCTAGCC; The promoter pF6 The sequence is shown in SEQ ID NO.30: ACGTGTACTTCGCCTTGTCGTACGCGTTGTGCGTGCCGATCGAAGTCGTCTCGGAGAGCCTCGGCGCCGCCGCGTGCGCGTTGCCCGGCCCCGCCATCAGCACGCCGGCCGCTCCCAGAGTGCCGCCGCTCGTCTCACTGCGCCCATCGC CCAACTCTCCTTCGTTTCCGTCGAGTTGACGGAAGCGTAGGAGCAAGCGGTTACTGCACGGTAGCTCCCGGAGGAACATCAGTCACTCCCGGTCCGGCGCGGCATCTCACGGCTGGCGGCTAGATTGCTAGCATGCTAGCCTCGCAAGC.

[0045] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0046] Example 1 This embodiment provides a method for constructing a constitutive strong promoter suitable for Streptomyces, the specific steps of which are as follows: The EFI-GNT (Enzyme Function Initiative-Genome Neighborhood Tool) online server aims to find neighboring genes with conserved gene arrangements in genomes that are geographically distant. It utilizes *Streptomyces velutipes* (…). S. flocculusThe amino acid sequence of StnY from CGMCC 4.1223 was obtained. The genome information of StnY homologous proteins was mined and retrieved using the EFI-GNT online server. Further analysis was performed on the genome of Streptomyces, and the promoter sequences of StnY homologous proteins in the genome were retrieved, resulting in the sequences of 30 promoters, as shown in SEQ ID NO.1~30, which were obtained by gene synthesis by Platinum Biotech (Shanghai) Co., Ltd. and Jiutian Gene Technology (Tianjin) Co., Ltd.

[0047] Example 2 This embodiment provides a promoter with the sequence shown in SEQ ID NO.1. p69 Express xylE Genes were obtained from the plasmid vector pDR3- p69 - xylE The construction method and specific steps are as follows: The plasmid vector pDR3 contains the phage integrase gene. int site and oriT The Streptomyces integration vector for the gene, a pSET152-derived plasmid, contains the reporter gene. xylE - neo April R The reference for the construction method of plasmid vector pDR3 is Wang W, Li X, Wang J, Xiang S, Feng X, Yang K. An engineered strong promoter for streptomycetes. Appl Environ Microbiol. 2013 Jul;79(14):4484-92. doi: 10.1128 / AEM.00985-13. Epub 2013 May 17. PMID: 23686264; PMCID:PMC3697493. catechol 2,3-dioxygenase gene xylE Derived from *Pseudomonas putida*, this enzyme catalyzes the formation of yellow 2-hydroxymucosanol semialdehyde from colorless catechol, which can be quantitatively detected at a wavelength of 375 nm.

[0048] Will xylE The gene sequence, synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd., is shown in the promoter of SEQ ID NO.1. p69 Downstream of the fragment ( xylE 5' end and promoter p (69' 3' direct connection), to obtain p69 - xylE The fragment will p69 -xylE Fragments via restriction enzyme sites BamH I and Spe I. Inserted into plasmid pDR3, resulting in plasmid pDR3- p69 - xylE The plasmid pDR3- was synthesized by a commissioned company. p69 - xylE The diagram is as follows Figure 1 As shown. The construction of other promoter-containing plasmid vectors and plasmid pDR3- p69 - xylE resemblance.

[0049] Example 3 This embodiment provides a promoter with the sequence shown in SEQ ID NO.1. p69 The strain expressing catechol 2,3-dioxygenase (XylE strain) was obtained Streptomyces albus J1074 / pDR3- p69 - xylE The construction method and specific steps are as follows: (1) Inoculate Streptomyces white onto YEME medium (10 g / L glucose, 3 g / L malt extract, 5 g / L peptone, 3 g / L yeast extract, 103 g / L sucrose, pH 7.2) and culture at 30°C until the logarithmic phase.

[0050] (2) The recombinant plasmid pDR3- obtained in Example 2 was used to... p69 - xylE Transformed into Escherichia coli ET12567 / pUZ8002 and plated on LB agar plates containing 30 mg / L apopramycin, 50 mg / L kanamycin and 25 mg / L chloramphenicol.

[0051] (3) Pick the single clones on the LB plate obtained in step (2) and put them into LB liquid medium containing 30 mg / L apopramycin, 50 mg / L kanamycin and 25 mg / L, and culture at 37°C until the logarithmic phase.

[0052] (4) Take 10 mL of Escherichia coli ET12567 / pUZ8002 cultured in step (1), wash it 3 times, and mix it with 1 mL of Streptomyces albopictus J1074 obtained in the logarithmic phase obtained in step (3). Spread the mixture on MS plates containing 40 mM MgCl2 (20 g / L soybean meal, 20 g / L mannitol, pH 7.2), and incubate at 30°C for 12 h. Cover the plates with sterile water containing apopramine and trimethoprim (900 µL sterile water with 25 µL of 50 mg / L apopramine and 50 µL of 100 mg / L trimethoprim), and continue incubation at 30°C until zygotes grow. After picking and rescreening the zygotes, the positive bacteria are named S. albus J1074 / pDR3- p69 - xylE The remaining tasks included the construction of promoter-containing plasmid vectors and the development of bacterial strains. Streptomyces albus J1074 / pDR3- p69 - xylE resemblance.

[0053] Example 4 This embodiment provides a method for detecting XylE enzyme activity in a catechol 2,3-dioxygenase strain (XylE strain) using a promoter with sequences shown in SEQ ID NO.1~30. The specific steps are as follows: (1) Single clones of Streptomyces leuciscinus expressing XylE using the 30 promoters obtained in Example 3 were inoculated into YEME medium and cultured at 30°C until the stationary phase. After transfer, 1 mL samples were taken at 24 hours and 48 hours of culture.

[0054] (2) After ultrasonic disruption of the bacterial cells, centrifuge at 12,000 rpm for 15 minutes at 4°C and collect the supernatant. The total protein concentration in the cell disruption supernatant was determined using the Bradford method.

[0055] (3) Prepare reaction buffer (10 mM PBS, 0.2 mM catechol, pH 7.5). Add 200 μL of reaction buffer and 4 μL of cell lysis supernatant to the reaction wells of a 96-well plate. Use a multi-functional microplate reader to measure the enzyme kinetic curve of each well at 30℃. The total measurement time is 15 minutes. Measure the absorbance at 375 nm every minute.

[0056] According to the literature "Practical" StreptomycesGenetics. Norwich: The John Innes Foundation. The formula for calculating the enzyme activity of catechol-2,3-dioxygenase XylE is: mU [nmol / min]≈(30.03×ΔA375) / (time [min]); the formula for calculating the normalized enzyme activity is: XylE activity=(XylE enzyme activity [nmol / min]) / (total amount of added protein [mg]).

[0057] In the formula: 30.03 is derived from Tobias K, Bibb MJ, Mark JB. Practical Streptomyces Genetics. Norwich: The John Innes Foundation, 2000. The activity assay of catechol 2,3-dioxygenase expressed by the 30 promoters in Streptomyces white J1074 is shown in the figure below. Figure 2 As shown, the positive control is the promoter. ermEp* and stnYp The 30 promoters exhibited varying degrees of activity, with their 24-hour activity being the highest among the promoters. ermEp* It is 0.8 to 50 times more potent than the promoter, and its activity over 48 hours is [missing information]. ermEp* The activity levels of these 30 promoters ranged from 0.24 to 98 times, significantly enriching the natural promoter library of Streptomyces. Although some of the activity of these 30 promoters was found in the positive control promoters... stnYp Despite its activity, but compared to the most widely used Streptomyces strong constitutive promoter today... ermEp* It exhibits excellent activity advantages. In research and actual production of Streptomyces natural products, promoters of different strengths can be selected for overexpression based on the actual required expression regulation level of the gene, thereby achieving precise gene regulation.

[0058] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A constitutive strong promoter suitable for Streptomyces, characterized in that, The constitutive strong promoter is: i) Any of the nucleotide sequences shown in SEQ ID NO. 1~30; or ii) A nucleotide sequence having the strong promoter function described above, formed by substitution, deletion, and / or addition of one or more nucleotides of any of the nucleotide sequences shown in SEQ ID NO. 1 to 30; or iii) A nucleotide sequence that hybridizes to any of the sequences shown in SEQ ID NO. 1~30 under highly stringent conditions and has the strong promoter function described above, or iv) A nucleotide sequence that has more than 75% identity with the nucleotide sequences of i), ii) or iii) and has the strong promoter function described above.

2. A biomaterial containing the constitutive strong promoter of Streptomyces as described in claim 1, characterized in that, The biomaterials include recombinant DNA, expression cassettes, transposons, plasmid vectors, bacteriophage vectors, viral vectors, or engineered bacteria.

3. The application of the constitutive strong promoter for Streptomyces as described in claim 1 in the construction of recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors or engineered bacteria.

4. A recombinant DNA, characterized in that, It is formed by operatively linking the constitutive strong promoter as described in claim 1 with the downstream target gene.

5. An expression carrier, characterized in that, The expression vector comprises the recombinant DNA of claim 4.

6. A transformant, characterized in that, The transformant is a host bacterium carrying the expression vector of claim 5.

7. A plasmid vector, characterized in that, The plasmid vector contains the constitutive strong promoter as described in claim 1.

8. A host cell, characterized in that, The host cell comprises the plasmid vector of claim 7.

9. The use of a host cell as described in claim 8 in protein expression and heterologous synthesis of important metabolites.

10. The application of a constitutive strong promoter for Streptomyces as described in claim 1, a plasmid vector as described in claim 7, and / or a host cell as described in claim 8 in initiating the expression of a target gene.

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