A promoter library and its preparation method and application

Abstract

The application provides a promoter library, a construction method of the promoter library and application of the promoter library. The promoter library of the application comprises promoters derived from Rhodobacter sphaeroides or modified variants thereof. The application discloses nucleic acid sequences of each promoter in the promoter library, vectors containing the nucleic acid sequences of the promoters and host cells. The application also discloses a screening method of the promoter library, and a promoter library with a gradient distribution of strength is obtained, so that high expression or low expression of a target gene can be realized and / or fine adjustment of expression thereof can be realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology; in particular, it relates to a promoter library, a vector and / or a host cell comprising the promoter, a construction method of the promoter library, a screening method of promoter activity and application thereof. BACKGROUND

[0002] Rhodobacter sphaeroides is a kind of gram-negative photosynthetic bacteria, belonging to the alpha Proteobacteria in purple nonsulfur bacteria. Rhodobacter sphaeroides has a complex metabolic pattern. It can obtain energy to maintain life through photosynthesis, and also can obtain energy to maintain life through aerobic respiration of organic matter in the absence of light. For decades, due to the fact that the bacterium contains a large amount of photosynthetic pigments, it has been used as a model organism for studying bacterial photosynthesis. In addition, Rhodobacter sphaeroides has many uses, such as synthesizing coenzyme Q 10 , which is an ideal host bacterium for it, and has high biological safety; it can also be used for biological hydrogen production, poly-β-hydroxybutyrate (PHB) production; and it is now also used for membrane protein expression systems. At present, Rhodobacter sphaeroides is widely used in food, medicine, agriculture and other fields due to the above characteristics, and has a high potential value for industrial development.

[0003] Under low-oxygen or anaerobic conditions, the cell membrane of Rhodobacter sphaeroides is easily invaginated in the form of germinating vesicles to form a large number of folded cell membranes, and differentiation produces intracytoplasmic membranes to form an intracytoplasmic membrane system (ICM). The ICM is equipped with photosynthetic devices for capturing photons for anaerobic photosynthesis, and the bacterium increases the membrane area to improve photosynthetic efficiency through this behavior. Coenzyme Q 10 is widely distributed on the biological membrane, which may also provide space for storage and function of coenzyme Q 10 . At present, the accumulation mechanism of coenzyme Q 10 is not clear, but as a model strain, the whole genome sequencing of Rhodobacter sphaeroides has been completed since 2005, and the expression and regulation mechanism of photosynthetic genes have also been studied clearly. The main light reaction center and light-harvesting complex contained in the ICM are mainly synthesized by a photosynthetic gene cluster. The expression of the genes in the photosynthetic gene cluster is regulated by multiple regulatory systems, and the promoters controlling the expression of each operon also have unique characteristics. Therefore, the photosynthetic gene cluster is important for elucidating the accumulation mechanism of coenzyme Q 10The mechanism and the potential of constructing natural promoter library have attracted the attention of the research group. Among them, the photosynthesis operon of Rhodobacter sphaeroides mainly includes pucBAC, pufQKBALMX, crt, bch, etc., which contains pucA, pucB, pufA, pufB, pufL, pufM, puhA, etc. Previous studies have shown that important regulatory proteins such as PrrA / PrrB, PpaA, AppA / PpsR, FnrL and IHF can sense oxygen concentration and light intensity to regulate the expression of photosynthesis operon.

[0004] Promoter is located on the 5' end upstream of the DNA coding strand, containing a conserved sequence that binds RNA polymerase, which plays a "control switch" role in the timing and intensity of gene expression. It mainly includes three parts of upstream element, core promoter region and response binding element. In prokaryotes, the core region of the promoter mainly includes-10 region "TATAAT" and-35 region "TTGACA". The two conservative regions can recognize the sigma factor of RNA polymerase, and it is worth noting that this binding is specific, so the promoter has species specificity. The change of the number of nucleotides in the-35 region and the-10 region conservative region can affect the activity of gene transcription. It is generally believed that the strong promoter is 16-18 bp, and when the spacing is less than 15 bp or greater than 20 bp, the activity of the promoter will be reduced.

[0005] At present, the natural promoters commonly used in Rhodobacter sphaeroides are from genes puf, pucP and aprrnB. The puf and pucP genes are from the photosynthesis operon of Rhodobacter sphaeroides, and the promoters of the two genes are affected by light and oxygen, and can be turned on or off under different culture conditions. rrnB is the promoter of ribosomal RNA in Rhodobacter sphaeroides, which is a medium-strength constitutive promoter. In addition, the σ 93 and σ 37 RNA polymerase in Rhodobacter sphaeroides can recognize the σ 70 and σ 32 dependent promoters derived from Escherichia coli compatibly, so in Rhodobacter sphaeroides, σ 70 dependent promoters derived from Escherichia coli such as Escherichia coli promoters P lacUV5 , P tac / trc , P Tn903kan , P A1 / 04 / 03 and P rrnB P1 can be recognized by Rhodobacter sphaeroides RNA polymerase. Shan Q. et al. used P J95025 , P J95026 , J95027, P tac , PrrnB Five different strengths (where P rrnB The promoters mediate the expression of CrtY gene in Rhodobacter sphaeroides, where P rrnB The yield of β-carotene is increased by 109% under the mediation of the promoter. This indicates that suitable gene expression strength is more conducive to regulating the metabolic network to achieve increased product yield, and the promoter library of Rhodobacter sphaeroides at the present stage needs to be developed. SUMMARY

[0006] In order to solve the above problems in Rhodobacter sphaeroides, the inventors of the present application analyzed the transcriptome and genome of Rhodobacter sphaeroides, and obtained a group of promoters capable of providing a series of transcription strengths in Rhodobacter sphaeroides.

[0007] Therefore, the purpose of the present application is to provide a promoter library for Rhodobacter sphaeroides and its application. Another purpose of the present application is to provide a construction method of the promoter library described in the present application.

[0008] In one aspect of the present application, a promoter library for Rhodobacter sphaeroides is provided, which comprises at least two promoters, wherein promoter A comprises a nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity with SEQ ID NO: 1, and promoter B comprises a nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence having at least 90% identity with SEQ ID NO: 2.

[0009] In a preferred embodiment, the promoter library further comprises one or more selected from the group consisting of the following promoters:

[0010] Promoter C, which comprises a nucleotide sequence shown in SEQ ID NO: 3 or a nucleotide sequence having at least 90% identity with SEQ ID NO: 3;

[0011] Promoter D, which comprises a nucleotide sequence shown in SEQ ID NO: 4 or a nucleotide sequence having at least 90% identity with SEQ ID NO: 4;

[0012] Promoter E, which comprises a nucleotide sequence shown in SEQ ID NO: 5 or a nucleotide sequence having at least 90% identity with SEQ ID NO: 5;

[0013] Promoter F, which comprises a nucleotide sequence shown in SEQ ID NO: 6 or a nucleotide sequence having at least 90% identity with SEQ ID NO: 6;

[0014] a promoter G comprising a nucleotide sequence set forth in SEQ ID NO: 7 or a nucleotide sequence having at least 90% identity to SEQ ID NO: 7;

[0015] a promoter H comprising a nucleotide sequence set forth in SEQ ID NO: 8 or a nucleotide sequence having at least 90% identity to SEQ ID NO: 8;

[0016] a promoter I comprising a nucleotide sequence set forth in SEQ ID NO: 9 or a nucleotide sequence having at least 90% identity to SEQ ID NO: 9; and

[0017] a promoter J comprising a nucleotide sequence set forth in SEQ ID NO: 10 or a nucleotide sequence having at least 90% identity to SEQ ID NO: 10.

[0018] In one aspect of the present application, there is provided a vector comprising a promoter in the promoter library described herein.

[0019] In one aspect of the present application, there is provided a genetically engineered host cell, which: contains a vector comprising a promoter element in the promoter library described herein; or has integrated into its genome a promoter element in the promoter library described herein.

[0020] In some embodiments, the cell is a Rhodobacter sphaeroides cell. For example, in the present application, the Rhodobacter sphaeroides cell can be a wild-type Rhodobacter sphaeroides cell, or a modified Rhodobacter sphaeroides cell (e.g., chemically mutagenized, or genetically modified, etc.).

[0021] In another aspect of the present application, there is provided a method for constructing the promoter library described herein, comprising:

[0022] (1) obtaining a candidate promoter sequence: based on the transcriptome information of Rhodobacter sphaeroides, screening genes with expression intensity ranked at least top fifty, preferably top twenty, more preferably top ten, obtaining a candidate promoter sequence located upstream of the start site of the selected gene based on the genome information of Rhodobacter sphaeroides and the selected gene;

[0023] (2) constructing a recombinant expression vector: operably linking the candidate promoter sequence to a reporter gene sequence, and connecting to a vector capable of expressing in a host cell, thereby obtaining a recombinant expression vector; and

[0024] (3) observing the expression of the reporter gene, introducing the recombinant expression vector into the host cell and allowing the reporter gene to express, selecting the promoter capable of allowing the reporter gene to express, constituting a promoter library.

[0025] In the present application, the host cell is Rhodobacter sphaeroides cell. For example, the reporter gene can be selected from the group consisting of fluorescent protein gene (such as green fluorescent protein gene, yellow fluorescent protein gene), luciferase (luc) gene, and β-galactosidase gene (lacZ) and gusA gene.

[0026] In one aspect of the present application, a method for screening a promoter is provided, which comprises comparing the ability of a promoter in the promoter library described herein and a reference promoter to induce expression of a reporter gene, thereby screening the strength of the promoter.

[0027] The present application also discloses a screening method of the promoter library, and a promoter library with gradient distribution of strength is obtained, which can realize high expression or low expression of a target gene and / or fine-tuning of the expression thereof. Meanwhile, it provides an advantageous tool for regulating metabolic network and realizing improvement of product yield in Rhodobacter sphaeroides cell.

[0028] In another aspect of the present application, the application of the promoter library described herein in protein expression is provided. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 : Analysis of transcriptome data of different Rhodobacter sphaeroides at 24h and 48h.

[0030] Figure 2 : Schematic diagram of GUS expression plasmid assembly for promoter activity analysis.

[0031] Figure 3 : Standard curve of product p-nitrophenol in GUS test determined at visible light 415nm.

[0032] Figure 4 : Characterization of promoter activity of each strain in GUS test at 11h, 24h and 48h.

[0033] Figure 5 : Growth condition (OD 700 ) of each strain in GUS test at 11h, 24h and 48h. DETAILED DESCRIPTION

[0034] DEFINITIONS

[0035] The strength of a promoter refers to the amount of transcription of a downstream gene mediated by the promoter. In prokaryotes, there are fewer regulatory mechanisms in the process from transcription to translation, and therefore the amount of translated protein can also represent the strength of the promoter.

[0036] The terms "isolated" or "partially purified" as used herein in the context of a nucleic acid or polypeptide means a nucleic acid or polypeptide that is separated from at least one other component (e.g., nucleic acid or polypeptide) with which it is co- present in its natural source of the nucleic acid or polypeptide; and / or that would be co-present with the nucleic acid or polypeptide if expressed by a cell, or secreted if in the case of a secreted polypeptide. Chemically synthesized nucleic acids or polypeptides or nucleic acids or polypeptides synthesized using in vitro transcription / translation are considered "isolated." The terms "purified" or "substantially purified" mean an isolated nucleic acid or polypeptide that is at least 95% by weight the nucleic acid or polypeptide of interest, including, for example, at least 96%, at least 97%, at least 98%, at least 99%, or more.

[0037] As used herein, "operably linked" means a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter is placed in a particular location relative to a nucleic acid sequence of a gene of interest such that transcription of the nucleic acid sequence is directed by the promoter, whereby the promoter is "operably linked" to the nucleic acid sequence.

[0038] As used herein, "gene of interest" means a gene that can be expressed by the promoter of the present application. The present application does not have a particular limitation on a suitable gene of interest, for example, including but not limited to: a structural gene, a gene encoding a protein having a particular function, an enzyme, a reporter gene (such as a green fluorescent protein gene, a luciferase gene, a galactosidase gene LazZ, a GUS gene, and the like).

[0039] As used herein, "exogenous" or "heterologous" means the relationship between two or more nucleic acid or protein sequences from different origins. For example, a promoter is exogenous to a gene of interest if the combination of the promoter and the gene sequence is not normally found in nature. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0040] For purposes of the present application, "identity" over a comparison window is calculated by comparing two optimally aligned sequences over the comparison window, wherein the portion of the sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (for example) for optimal alignment of sequences for purposes of comparison. Optimal alignment of sequences for comparison can be conducted using known computer programs. The determination of optimal alignment is considered in context of the information available from a search of a database containing a large number of closely related sequences. The identity between two sequences can be determined using computer programs known in the art.

[0041] Promoter library

[0042] The present application provides a promoter library for Rhodobacter sphaeroides. The promoter library comprises at least two promoters, wherein promoter A comprises a nucleotide sequence set forth in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity to SEQ ID NO: 1, and promoter B comprises a nucleotide sequence set forth in SEQ ID NO: 2 or a nucleotide sequence having at least 90% identity to SEQ ID NO: 2.

[0043] In some embodiments, promoter A comprises a nucleotide sequence set forth in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity to SEQ ID NO: 1, for example, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1. In preferred embodiments, promoter A consists of a nucleotide sequence set forth in SEQ ID NO: 1.

[0044] In some embodiments, promoter B comprises a nucleotide sequence set forth in SEQ ID NO: 2 or a nucleotide sequence having at least 90% identity to SEQ ID NO: 2, for example, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 2. In preferred embodiments, promoter B consists of a nucleotide sequence set forth in SEQ ID NO: 2.

[0045] For example, the promoter library comprises promoter A consisting of a nucleotide sequence set forth in SEQ ID NO: 1 and promoter B consisting of a nucleotide sequence set forth in SEQ ID NO: 2.

[0046] In more preferred embodiments, the promoter library further comprises one or more promoters selected from the group consisting of:

[0047] promoter C comprising a nucleotide sequence set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 90% identity to SEQ ID NO: 3, for example, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 3;

[0048] a promoter D comprising a nucleotide sequence set forth in SEQ ID NO: 4 or a nucleotide sequence with at least 90% identity to SEQ ID NO: 4, for example, a nucleotide sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to SEQ ID NO: 4;

[0049] a promoter E comprising a nucleotide sequence set forth in SEQ ID NO: 5 or a nucleotide sequence with at least 90% identity to SEQ ID NO: 5, for example, a nucleotide sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to SEQ ID NO: 5;

[0050] a promoter F comprising a nucleotide sequence set forth in SEQ ID NO: 6 or a nucleotide sequence with at least 90% identity to SEQ ID NO: 6, for example, a nucleotide sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to SEQ ID NO: 6;

[0051] a promoter G comprising a nucleotide sequence set forth in SEQ ID NO: 7 or a nucleotide sequence with at least 90% identity to SEQ ID NO: 7, for example, a nucleotide sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to SEQ ID NO: 7;

[0052] a promoter H comprising a nucleotide sequence set forth in SEQ ID NO: 8 or a nucleotide sequence with at least 90% identity to SEQ ID NO: 8, for example, a nucleotide sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to SEQ ID NO: 8;

[0053] a promoter I comprising a nucleotide sequence set forth in SEQ ID NO: 9 or a nucleotide sequence with at least 90% identity to SEQ ID NO: 9, for example, a nucleotide sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to SEQ ID NO: 9; and

[0054] a promoter J comprising a nucleotide sequence set forth in SEQ ID NO: 10 or a nucleotide sequence having at least 90% identity to SEQ ID NO: 10, for example, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 10.

[0055] In specific embodiments, the promoter library further comprises one or more promoters selected from the group consisting of:

[0056] a promoter C consisting of a nucleotide sequence set forth in SEQ ID NO: 3;

[0057] a promoter D consisting of a nucleotide sequence set forth in SEQ ID NO: 4;

[0058] a promoter E consisting of a nucleotide sequence set forth in SEQ ID NO: 5;

[0059] a promoter F consisting of a nucleotide sequence set forth in SEQ ID NO: 6;

[0060] a promoter G consisting of a nucleotide sequence set forth in SEQ ID NO: 7;

[0061] a promoter H consisting of a nucleotide sequence set forth in SEQ ID NO: 8;

[0062] a promoter I consisting of a nucleotide sequence set forth in SEQ ID NO: 9; and

[0063] a promoter J consisting of a nucleotide sequence set forth in SEQ ID NO: 10.

[0064] For example, the promoter library can comprise at least 2 promoters, at least 3 promoters, at least 4 promoters, at least 5 promoters, at least 6 promoters, at least 7 promoters, at least 8 promoters, at least 9 promoters, or at least 10 promoters.

[0065] In the promoter library of the present application, the strength of each promoter in initiating the expression of the target gene presents a gradient distribution, which is used to direct the expression of the target gene at different strengths. For example, to express an exogenous protein in a host, or to regulate metabolic pathways in cells, etc. For example, the promoter library of the present application can be used to finely regulate metabolic pathways, to express genes in an appropriate amount, and to realize the combinatorial optimization of metabolic pathways.

[0066] The promoter of the present application can be operably linked to a gene of interest, which can be exogenous (heterologous) with respect to the promoter. The gene of interest can generally be any nucleic acid sequence (e.g., a structural nucleic acid sequence), which preferably encodes a protein having a particular function, e.g., a protein having an important property or function.

[0067] For example, when used in studies of expression strength, the gene of interest includes, but is not limited to, green fluorescent protein, yellow fluorescent protein, luciferase gene, galactosidase gene LacZ, Gus gene, etc.

[0068] As a preferred embodiment of the present application, promoters of different strengths can be selected from the promoter library of the present application, and each of them can be operably linked to a gene of interest to be studied, or the gene of interest can be operably linked to the promoter in a suitable vector, and introduced into a host cell in an appropriate manner, thereby obtaining a series of cells in which the expression level of the gene of interest is different. The function or use of the gene of interest can be known by analyzing the metabolism, phenotypic changes, protein expression or interaction, changes of various signal molecules, etc. of these cells.

[0069] As a preferred embodiment of the present application, the gene of interest can be a gene that is absent or insufficiently expressed in a certain cell, and the gene of interest can be operably linked to the promoter of the present application, or the gene of interest can be operably linked to the promoter of the present application in a suitable vector, and introduced into the cell in an appropriate manner, thereby expressing the gene of interest at a high level.

[0070] The promoter of the present application can also be operably linked to a modified gene of interest, which is exogenous (heterologous) with respect to the promoter. The gene of interest can be modified to produce various desired properties. For example, the gene of interest can be modified to increase the content of essential amino acids, improve translation of the amino acid sequence, change post-translational modification (e.g., phosphorylation sites), transport the translation product out of the cell, improve the stability of the protein, insert or delete cellular signals, etc.

[0071] In addition, the promoter and the gene of interest can be designed to down-regulate a specific gene. This is generally achieved by linking the promoter to a gene of interest sequence, which is directed in the antisense direction. Those skilled in the art are familiar with this antisense technology. Any nucleic acid sequence can be regulated in this way.

[0072] Here, in the present application, the promoter A and the promoter B can be considered as high strength promoters, the promoter C, the promoter D, the promoter E, the promoter F can be considered as medium strength promoters, and the promoter G, the promoter H, the promoter I, the promoter J can be considered as low strength promoters. Here, the high, medium, and low strength promoters are relative to the strength of the promoters tested themselves.

[0073] Vectors and host cells

[0074] The present application also provides a vector containing the promoter elements in the promoter library described herein.

[0075] Any of the promoters from the promoter library of the present application and / or the gene of interest sequence can be included in a recombinant vector.

[0076] As an embodiment, the recombinant vector comprises the promoter of the present application, and optionally comprises a multiple cloning site or at least one restriction site downstream of the promoter. When expression of the gene of interest is desired, the gene of interest is ligated into the appropriate multiple cloning site or restriction site, thereby operably linking the gene of interest to the promoter. As an embodiment, the recombinant vector comprises the promoter of the present application, and when expression of the gene of interest is desired, the gene of interest is ligated downstream of the promoter by homologous recombination, thereby operably linking the gene of interest to the promoter.

[0077] In a specific embodiment, the gene of interest is located downstream of the promoter, and is separated from the promoter by less than 2000 bp, for example, less than 1000 bp, 500 bp, 200 bp, 100 bp, 50 bp. In a preferred embodiment, there is no separation between the promoter and the gene of interest.

[0078] As an embodiment, the recombinant vector comprises (from 5' to 3' direction): a promoter that directs transcription of the gene of interest and the gene of interest. If desired, the recombinant vector can also comprise a 3' transcription terminator, a 3' polyadenylation signal, other non-translated nucleic acid sequences, transport and targeting nucleic acid sequences, resistance selection markers, enhancers or operators, etc.

[0079] In another embodiment, the gene of interest includes, but is not limited to: a structural gene, a gene encoding a protein with a specific function, a reporter gene (such as a green fluorescent protein gene, a luciferase gene, a galactosidase gene LacZ, a Gus gene).

[0080] In a preferred embodiment, the recombinant vector is an expression vector. In the present application, the term "expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, mammalian cell virus or other vector well known in the art. In general, any plasmid and vector can be used as long as it can replicate and be stably maintained in the host. Preferably, the expression vector is a Rhodobacter sphaeroides expression vector.

[0081] Methods for preparing recombinant vectors are well known to those skilled in the art. Methods well known to those skilled in the art can be used to construct an expression vector containing the promoter and / or the gene sequence of interest of the present application. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0082] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein (GFP), etc.

[0083] In addition to the promoter of the present application, the recombinant vector can also contain one or more other promoters. The other promoters are, for example, tissue-specific, constitutive or inducible.

[0084] The vector containing the appropriate promoter and the gene of interest described above can be used to transform an appropriate host cell to enable it to express the protein.

[0085] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include yeast, E. coli, animal tissue cells, plant cells, etc. Those skilled in the art are well aware of how to select an appropriate vector and host cell. As a preferred embodiment of the present application, the host cell is a Rhodobacter sphaeroides cell. For example, in the present application, the Rhodobacter sphaeroides cell can be a wild-type Rhodobacter sphaeroides cell, or a modified Rhodobacter sphaeroides cell (e.g., chemically mutagenized, or genetically modified, etc.). For example, the Rhodobacter sphaeroides cell can be a Rhodobacter sphaeroides cell that has been chemically mutagenized or genetically modified to produce coenzyme Q 10 at a high yield.

[0086] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known in the art. When the host is a prokaryote, such as E. coli, the transformation of the host cell can be effected by the use of techniques such as calcium chloride precipitation. If necessary, the transformation can be performed by electroporation. When the host is a eukaryote, the transformation can be effected by the use of techniques such as calcium phosphate precipitation, conventional mechanical procedures such as microinjection, electroporation, or liposome-mediated transfection.

[0087] For example, the transformation of Rhodobacter sphaeroides with the recombinant vector can be performed by electroporation, conjugation, or the like.

[0088] The present application also provides a genetically engineered host cell, which:

[0089] has integrated into its genome a promoter element of the promoter library described herein.

[0090] In a preferred embodiment, the cell is an E. coli or Rhodobacter sphaeroides cell.

[0091] Method of constructing a promoter library

[0092] The method for constructing the promoter library described herein comprises:

[0093] (1) obtaining a candidate promoter sequence: based on the transcriptome information of Rhodobacter sphaeroides, screening genes with expression intensity ranked at least top 50, preferably top 20, more preferably top 10, based on the genome information of Rhodobacter sphaeroides and the selected genes, obtaining a candidate promoter sequence located upstream of the start site of the gene sequence;

[0094] (2) constructing a recombinant expression vector: operably linking the candidate promoter sequence to a reporter gene sequence, and connecting to a vector capable of expressing in a host cell, thereby obtaining a recombinant expression vector; and

[0095] (3) observing the expression of the reporter gene, introducing the recombinant expression vector into the host cell and allowing the reporter gene to express, selecting a promoter capable of allowing the reporter gene to express, thereby constituting a promoter library.

[0096] The transcriptome and genome information can be obtained using information disclosed in databases in the art (e.g., KEGG, NCBI), or by sequencing.

[0097] The candidate promoter sequence is usually selected from a 100-500 bp fragment upstream of the start codon, which contains the core promoter part. In a preferred embodiment, the fragment can be further modified, for example, by deleting a part that inhibits the activity of the promoter, for example, a ppsR binding site. For determination of the ppsR binding site, see Patrice Bruscella et al., The Use of Chromatin Immunoprecipitation to Define PpsR Binding Activity in Rhodobacter sphaeroides 2.4.1, JOURNAL OF BACTERIOLOGY, Oct. 2008, p. 6817-6828, which is hereby incorporated by reference in its entirety.

[0098] As a reporter gene, a green fluorescent protein gene, a luciferase gene, a galactosidase gene LacZ, a Gus gene, etc. can be used.

[0099] Fluorescent protein gene: Green fluorescent protein (GFP) is a typical endogenous fluorescent protein, which is safe to the host, and the fluorescence intensity directly reflects the expression amount, without the need for additional substrate, with high sensitivity, which is conducive to non-destructive detection of the host.

[0100] Yellow fluorescent protein (YFP) can be regarded as a mutant of green fluorescent protein, which has a red spectrum shift relative to green fluorescent protein, and shares the GFP fluorescent protein, with longer wavelength excitation and emission light, and most importantly, it has a low background when imaging in cells.

[0101] Luciferase (luc) gene: Luc is an enzyme that catalyzes the production of luciferin or fatty aldehyde in an aerobic environment, and luc catalyzes the substrate ATP, inorganic luciferin and oxygen to produce a luminescent reaction, and the enzyme and the substrate are highly specific, with minimal impact on the host, high sensitivity, easy to detect, no excitation light interference, etc.

[0102] GusA gene: derived from Escherichia coli, encoding β-D-glucuronidase, monomer molecular weight 68200 Da, resistant to various detergents, and has very high activity in the presence of thiol reducing agents such as β-mercaptoethanol or DTT. This enzyme can catalyze the hydrolysis of many β-glucoside ester substances without coenzyme factors or cations, and some substances will change color after hydrolysis. A small number of divalent metal ions such as Zn 2+ , Cu 2+GUS activity can be inhibited. Its optimum pH is 5.2-8.0, and the anti-heat half-life is 50°C for 2h, and the activity is linear for a long time. The GUS substrate is soluble, and the most commonly used catalytic substrate is 5-bromo-4-chloro-3-indole glucuronide (X-gluc), and the product is a blue compound. In the early years, 4-methyl umbelliferone glucuronide (MUG) and 9-hydroxy-3- isoxazolone glucuronide (ReG) were also commonly used. ReG has high fluorescence activity, and its product is excited at 560nm and emitted at 590nm. In biological research, the gene is often placed downstream of the promoter to be studied, and the activity of the promoter can be analyzed by detecting the enzyme. This is the most widely used report gene, which plays the role of "reporting" the activity of the promoter. In one embodiment, the GUSA sequence is derived from Enterobacteriaceae, LOCUS: WP_000945901.

[0103] The GUS test is an enzymatic reaction detection experiment based on colorimetric determination. For example, the color development of the product produced by the Gus enzyme catalytic substrate contained in the sample is collected by an enzyme marker, and the amount of catalytic substrate is calculated from the concentration of the product, and then the strength of the used promoter is reflected. For example, when using p-nitrophenyl-β-D-glucuronide as a substrate to generate a product, p-nitrophenol, the color reaction is detected at 415nm for 20-30min (the color reaction tends to be stable at this time), and then the corresponding consumed substrate in the sample is calculated by the standard curve of the product relative to the absorbance at 415nm (for example Figure 3 ), and then the strength of the tested promoter is reflected by the slope of the substrate and the reaction time. For GUS testing, see Siegl, Theresa, et al. "Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes." Metabolic Engineering 19. Complete (2013): 98-106, which is hereby incorporated by reference in its entirety.

[0104] In some embodiments, the candidate promoter sequence can also be modified. The modification includes substitution, deletion, and / or insertion. In preferred embodiments, the candidate promoter sequence is modified by deletion of a repressor site. For example, the ppsR binding site in the candidate promoter sequence is deleted. For determination of the ppsR binding site, see Patrice Bruscella et al., The Use of Chromatin Immunoprecipitation to Define PpsR Binding Activity in Rhodobacter sphaeroides 2.4.1, JOURNAL OF BACTERIOLOGY, Oct. 2008, p. 6817-6828, which is incorporated herein by reference in its entirety.

[0105] For example, the modification of the candidate promoter sequence can be performed before step (2). Alternatively, the modification can be performed after step (3) and then return to step (2). In some embodiments, the modification can be performed based on the isolated candidate promoter sequence or based on the recombinant expression vector obtained in step (2).

[0106] Screening method

[0107] A method of screening a promoter, the method comprising comparing and ranking the promoters in the promoter library according to their ability to initiate expression of a gene of interest, thereby screening the strength of the promoters.

[0108] In preferred embodiments, the gene of interest can be selected from a reporter gene. For definition of a reporter gene, see the description above herein.

[0109] In the present application, it is preferred to use a reference promoter for comparison with the candidate promoter. For selection of a reference promoter, it is able to give more intuitive and direct feedback on the ability of the candidate promoter to initiate expression of a reporter gene. Therefore, generally the reference promoter is selected from a well-characterized promoter in the host cell. In preferred embodiments, two or more reference promoters with different strengths are selected.

[0110] In preferred embodiments, the screening method is performed by comparing the strength of the promoters described herein relative to a reference promoter in initiating expression of a gene of interest, including higher than the reference or lower than the reference, the method comprising: culturing host cells with the gene of interest initiated by the promoters of the present application and host cells with the gene of interest initiated by the reference promoter, respectively, under the same culture conditions, and comparing the expression strength (or expression level) of the gene of interest in the two types of cells.

[0111] In the above screening method, the preferred host cell is Rhodobacter sphaeroides cell. The conditions for culturing Rhodobacter sphaeroides cell are well known to those skilled in the art, and are not particularly limited in the present application. Preferably, the conditions for culturing Rhodobacter sphaeroides cell are 31 ± 1°C, 210 ± 100 rpm.

[0112] The method for determining the expression of the gene of interest (particularly the reporter gene) in the culture system is a well-known technique to those skilled in the art, and can be adjusted according to the gene of interest. As a preferred mode of the present application, the reporter gene is Gus protein, and the method for determining the expression of the reporter gene comprises determining the activity of the enzyme in the culture system. In some embodiments, the reporter gene is a fluorescent protein (e.g., green fluorescent protein), and the method for determining the expression of the reporter gene comprises determining the fluorescence intensity in the culture system.

[0113] In addition, the present application also provides the use of the promoter library described herein in protein expression.

[0114] Examples

[0115] The present application is further described below in conjunction with examples and drawings, but the present application is not limited by the following examples. Those skilled in the art should understand that equivalent substitutions or corresponding improvements of the technical features of the present application still fall within the scope of protection of the present application. Unless otherwise specified, the reagents used in the following examples are commercially available products, and the preparation of solutions can use conventional techniques in the art.

[0116] Example 1 Obtaining of candidate promoters

[0117] The promoter starts transcription of mRNA after binding to RNA polymerase, and the transcription initiation site is generally located downstream of the promoter, or there is overlap. The mRNA expression level is semi-quantitatively determined by transcriptome information, and the position of the corresponding gene in the genome of the strain can be found by the sequence of the expressed mRNA, to determine the corresponding coding sequence on the genome and the sequence containing the promoter upstream of the coding sequence. In this experiment, the experimental strain was subjected to transcriptomic analysis, and the relevant photosynthetic gene cluster was located on the single-stranded circular genome of Rhodobacter sphaeroides. The adjacent sequence of the relevant gene in the genome located by mRNA was regarded as the existence region of the candidate promoter, and thus the candidate promoter sequence information was obtained for characterization of the experiment.

[0118] Specifically, Rhodobacter sphaeroides HY01 (hereinafter referred to as R. sp HY01, obtained by screening Rhodobacter sphaeroides 2.4.1 by nitrosoguanidine mutagenesis, high-yield coenzyme Q 10 , in Figure 1 ind) wild-type Rhodobacter sphaeroides 2.4.1 (in Figure 1The transcriptional information of the Rhodopseudomonas palustris ind24, ind48, wt24, and wt48 in 24h and 48h under the condition of 32℃, 220rpm with fermentation medium is shown in FIG. 1 and FIG. 2, respectively. Figure 1 Figure 1 FIG. 3 shows the transcriptional information of the Rhodopseudomonas palustris ind24, ind48, wt24, and wt48 in 24h and 48h under the condition of 32℃, 220rpm with fermentation medium, wherein the horizontal axis represents the transcriptional intensity in different Rhodopseudomonas palustris strains (including Rhodopseudomonas palustris ind24, ind48, wt24, and wt48), the vertical axis represents the corresponding operon / gene name, and the right side is the color scale.

[0119] The gene sequences of the top thirteen genes in transcriptional intensity were selected, and the genomic information of these genes was obtained based on the information in the KEGG database (the genomic information here is from the wild-type Rhodopseudomonas palustris 2.4.1).

[0120] Specifically, the top thirteen genes in transcriptional intensity are from the following operons / genes (from top to bottom in FIG. 4): Figure 1 rnpB (KEGG number RSP_4342, this gene is not shown together with the transcriptional information of other genes in FIG. 1 due to its excessively high transcriptional intensity), RSP_1185, RSP_6124, RSP_7571, RSP_2718, puhA (KEGG number RSP_0291), puc2B (KEGG number RSP_1556), crtA (KEGG number RSP_0272), rpsM (KEGG number RSP_1737), pucBAC (KEGG number RSP_0314, shown as pucB in FIG. 1), bchEJ (KEGG number RSP_0280, shown as bchJ in FIG. 1), pufQ (KEGG number RSP_0259), and crtEF (KEGG number RSP_0264, shown as crtF in FIG. 1). Figure 1 Figure 1 Figure 1 Figure 1

[0121] ​​​​​PufQ, puc2B, puhA, crtEF, pucBAC, crtA, bchEJ are all from photosynthetic gene cluster. rpsM is 30S ribosomal protein S13 gene; rnpB is RNAase P gene. RSP_7571, RSP_6124, RSP_2718, RSP_1185 are genes whose expression proteins' functions are not clear, and their promoters also have no clear sequence information.

[0122] The fragments containing core promoters upstream of the start codon of genes RSP_7571, RSP_6124, RSP_2718, RSP_1185, puhA and rnpB were cut, to obtain candidate promoters P RSP-7571 (SEQ ID NO: 2, 500bp), P RSP-6124 (SEQ ID NO: 1, 500bp), P RSP_2718 (SEQ ID NO: 10, 500bp), P RSP-1185 (the information of this promoter is not shown), P puhA (SEQ ID NO: 9, 500bp), P rnpb (SEQ ID NO: 4, 500bp).

[0123] The fragments containing core promoters upstream of the start codon of genes pufQ, crtEF, pucBAC, crtA, bchEJ were cut and the ppsR binding sites in them were deleted (see Patrice Bruscella et al., The Use of Chromatin Immunoprecipitation to Define PpsR Binding Activity in Rhodobacter sphaeroides 2.4.1, JOURNAL OF BACTERIOLOGY, Oct. 2008, p. 6817-6828), to obtain the following candidate promoters: P pufQ (SEQ ID NO: 7, 152bp), P crtEF (SEQ ID NO: 5, 132bp), P pucBAC (SEQ ID NO: 8, 203bp), P crtA (SEQ ID NO: 6, 157bp), P bchEJ (SEQ ID NO: 3, 147bp).

[0124] Construction of strains for characterization of promoter activity in Example 2

[0125] Synthetic promoters Pkana (SEQ ID NO: 13, 121 bp) and Ptac (SEQ ID NO: 12, 79 bp). Both Pkana and Ptac are commonly used promoters in E. coli plasmids.

[0126] In addition, a GUS gene was synthesized based on the sequence shown in SEQ ID NO: 11.

[0127] According to the schematic diagram of Figure 2 The above-mentioned each candidate promoter and GUS gene were assembled into a pBBR1MCSK vector (purchased from Novagen Company) by Ezmax (purchased from Tu Lu Gang Biological Company) to construct a plasmid with each candidate promoter and GUS gene. After sequencing verification, it was transformed into E. coli DH10b competent cells, and positive transformants were screened by colony PCR. The transformation conditions were as follows: incubation on ice for 30 min, heat shock at 42°C for 90 s, incubation on ice for 2 min, addition of 700 uL LB, and incubation at 37°C for 45 min.

[0128] Plasmids were extracted from the positive transformants, and recombinant strains were obtained by electroporation of wild-type Rhodobacter sphaeroides 2.4.1 monoclonal (strain number ATCC17023). The prepared strains are shown in Table 1 below.

[0129] The electroporation conditions are as follows:

[0130] 1. Add 2 mL of bacterial cells (culture the bacterial cells in TSB medium to an OD of 1-3) to each 2 mL sterile EP tube, and centrifuge at 9000 rpm for 1 min;

[0131] 2. Remove the supernatant, add 1 mL of deionized water, mix by blowing, centrifuge at 9000 rpm for 1 min, and discard the supernatant.

[0132] 3. Repeat step 2 for three times of water washing;

[0133] 4. Add 80 uL of 10% glycerol to each EP tube after discarding the supernatant, resuspend the bacterial cells and add the plasmid (500 ng) and mix;

[0134] 5. Take out the mixture, inject it into a 2 mm electroporation cup, and incubate on ice for 20 min, then perform electroporation.

[0135] 6. Electroporation conditions: 2000V / 200Ω / 2mm;

[0136] 7. After electroporation, add 600 uL of antibiotic-free TSB medium, mix by blowing and sucking, then take out the liquid and place it in a 2 mL EP tube, and incubate at 32°C, 200 rpm for 2 h;

[0137] 8. 12000g centrifuge for 1 min, pour off part of the supernatant, mix the remaining bacterial liquid, and spread on kanamycin-resistant plates and place in a 32°C incubator for 6-7 days.

[0138] TSB medium: 30 g / L Tryptone Soya Broth. Solid medium is added with 2% agar, and the sterilization condition is 115°C x 20 min.

[0139] Table 1 Strains used for characterization of promoter activity in this example

[0140]

[0141] Example 3 Activity analysis of candidate promoters

[0142] In this example, GUS assay was used to analyze the activity of the candidate promoters. For more information, see Siegl, Theresa, et al. "Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes." Metabolic Engineering 19. Complete (2013): 98-106.

[0143] Principle of GUS experiment:

[0144] The enzyme activity determination reaction substrate is p-nitrophenyl-β-D-glucuronide, and the highly sensitive β-D-glucuronidase (GUS reporter protein) is the catalyst to generate p-nitrophenol. The reaction system is measured by spectrophotometry at 415 nm for 30 minutes, and the slope of the final absorbance curve is used to calculate the enzyme activity.

[0145] The specific experimental steps are as follows:

[0146] 1. Pick 1-2 colonies of each strain prepared in Example 2 and inoculate in 5 mL TSB liquid medium in a 50 mL centrifuge tube at 32°C, 220 rpm for 24 h, then take 5 mL and transfer to 45 mL fermentation medium (the fermentation medium is the same as in Example 1) and incubate at 32°C, 220 rpm until the stationary phase (48 h) (in this step, the fermentation medium contains 50 ng / ml of kanamycin).

[0147] 2. Take OD 700 The same bacterial liquid (OD 700 in the range of 10-25), centrifuge at 12000 rpm x 1 min to remove the supernatant.

[0148] 3. Resuspend the bacterial pellet in 900 μL Gus buffer 2 and incubate in a water bath at 37 °C for 20 min to obtain the lysis solution of each strain.

[0149] 4. Dilute the lysis solution with 900 μL Gus buffer 1 and centrifuge at 14000 rpm at 4 °C for 10 min.

[0150] 5. Take 500 μL supernatant and add 500 μL Gus buffer 3.

[0151] 6. Measure the absorbance at 415 nm with a microplate reader. Add 200 μL of the mixed solution to a 96-well plate and measure for 30 min. Take 100 μL Gus buffer 3, 100 μL Gus buffer 2 and 100 μL Gus buffer 1 as the control group.

[0152] 7. Collect the data of the color development reaction process with a microplate reader for 30 min in a 96-well plate. Meanwhile, prepare a standard curve of the product relative to the absorbance at 415 nm (see Figure 3 ). According to the standard curve, convert the absorbance values at each time point on the reaction curve to the product concentration to calculate the amount of catalyzed substrate. Then, take the reaction time as the horizontal coordinate and the catalyzed substrate concentration as the vertical coordinate to draw a curve graph of each strain within the time for the product color to stabilize, fit the reaction progress with office software, calculate the slope of the curve, and express the enzyme activity with the slope. According to the principle of enzyme reaction, determine the formula to calculate the specific enzyme activity. Each group of strains has three repeats.

[0153] The enzyme activity can be calculated by the method disclosed in Siegl, Theresa et al. “Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes.” Metabolic Engineering 19. Complete (2013): 98-106, wherein the standard curve used is the standard curve in Figure 3 .

[0154] Solution:

[0155] Gus buffer 1: 30 mL of 50 mM phosphate buffer (pH 7.0), 150 μL of 5 mM DTT, and 30 μL of 0.1% Triton X-100.

[0156] Gus buffer 2: 30 mL of 50 mM phosphate buffer (pH 7.0), 150 μL of 5 mM DTT, 30 μL of 0.1% Triton X-100, 30 mg of lysozyme (final concentration about 1 mg / mL).

[0157] Gus buffer 3: 30 mL of 50 mM phosphate buffer (pH 7.0), 150 μL of 5 mM DTT, 30 μL of 0.1% Triton X-100, final concentration of 2 mM of p-nitrophenyl-β-D-glucuronide.

[0158] Phosphate buffer: 1.14 g of K2HPO4·3H2O was dissolved in 100 mL of deionized water, 0.6 g of NaH2PO4 was dissolved in 100 mL of deionized water, and the pH was adjusted to 7.0 by using the former as the main solution and the latter as the auxiliary solution, K2HPO4·3H2O:NaH2PO4=5:4.

[0159] 2 mM of p-nitrophenyl-β-D-glucuronide: 0.63 g of p-nitrophenyl-β-D-glucuronide was added to 1 L of dH2O, and 18.9 mg was added when preparing a 30 mL solution.

[0160] Result analysis:

[0161] Figure 5 The growth of each strain at 11 h, 24 h, and 48 h during the cultivation to the plateau phase is shown as OD 700 is shown.

[0162] First, the slope of the absorption curve of each promoter was calculated, and the slope was converted into the substrate concentration according to the standard curve, which was positively correlated with the enzyme activity of the reporter gene expression product, and the enzyme activity was positively correlated with the strength of the promoter, thereby sorting the activity of the screened promoters, Figure 4 The middle ordinate is the enzyme activity (activity), and the abscissa is each promoter, and the promoter strength increases from left to right. The first one is a blank control, and the second one is a gusA reporter gene control without a promoter. The weaker ones among the promoters are P kana , P 2718 , P puhA , P pucBAC , P PufQ , the medium-strength ones are P CrtA , P crtEF , P tac , P rnpb , P bchEJ , and the high-strength ones are P 7571 and P 6124 .

[0163] The experiment successfully obtained a series of promoters spanning five orders of magnitude in strength, demonstrating that the method can effectively quantify promoter strength and can continue to be used to expand the Rhodobacter sphaeroides promoter element library. At the same time, the experimental results show the strong influence of very strong promoters on the growth rate and amount of bacteria, to some extent, showing the metabolic burden that the strain can bear. According to P rnpB As a result, it is speculated that there may be an up-regulation mechanism. The commonly used promoters P tac , P kana The introduction of these results also makes these results more easily applied.

[0164] The accumulation of expressed proteins is not determined by the instantaneous gene translation strength of a single independent variable, and different protein products will have different degradation rates. Therefore, the characterization of promoter strength in this experiment should be considered as a horizontal reference at the same time point. In the future, if the expression and degradation timing of the reporter protein can be controlled, the nutrient level can be regulated in a chemostat fermenter to distinguish between degradation and accumulation time, which will be more conducive to clearly elucidating the strength of the promoter and the nutrient limiting factor.

[0165] In addition, promoter engineering is developing rapidly, and many researchers have established artificial mutant promoter libraries for the cells they study, and the GUS method used is also from the study of characterizing artificial promoter libraries. Artificial promoters have a higher possibility of being unregulated by the cell's metabolic network, which helps to limit the scope of uncontrolled variables in various single-gene modification work. In addition, artificial promoters have the potential to fill in the intensity regions that natural promoters do not have. In this experiment, promoters from the top ten in the transcriptome data have been selected, while there is still a large amount of gradient filling space in the vicinity of such strong promoters as P 6124 , P 7571 In the future, if there is a need for application, the sequences of both can be analyzed to find the sites that affect promoter activity for mutation, further enriching this library. SEQUENCE LISTING <110> East China University of Technology <120> A Promoter Library and Its Preparation Method and Application <130> 1 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 500 <212> DNA <213> Artificial Sequence <400> 1 gtctcgacct ccagaaggtc gtcgaagacc gccgggcgca gatagtcggc ctcgacgcgg 60 cggacggcga agacgatccc gctctcggcg cgcagccgcg cctgatcgac gccgagcccg 120 cgcacccatt cgctgcgggc ccgctcgatg aacttcagat agttggcgta atagacgatt 180 cccgcgagat cggtgtcctc gtagtagacg cgcagggcga aacggtgcgg catgggcatc 240 tcccggcagg atggcgcttt ccgcaggggc tagctcgaag ctgccggcgc cgcaaggaag 300 gcgcggaatt tccttctgaa attgagcctc cgggcgcccg tttccatgcg gtgcgagtgt 360 atgccgcacc ttcccgcgcg cgtttgtgcg tgaggaagcc gcggcacatg ctaacttcgg 420 ctccgcaggt gcatccgcat catcccgagg cgtgttgcgc gtctgatcat gagttctaat 480 gcccgttaga ggagagcact 500 <210> 2 <211> 500 <212> DNA <213> Artificial Sequence <400> 2 cgccgcagcc gatggtccag acctcggccc cggcgagggc gggcgtggcc cggatccgct 60 cggcgagcct ctgcgccagc gccgcctcgc cgggcgagcc cgtgaccgac cgttgccgcg 120 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 CCTTCTCCCT GCGGCGGCGG CGGCGGCGGC GCGGCGGCGG CGGCGGCGGC GCGGCGGCGG 180 CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG 240 CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG 300 CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG 360 CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG 420 CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG 480 TTGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 540 <211> 147 <212> DNA <213> Artificial Sequence <400> 3 CCTTCTCCCT GCGGCGGCGG CGGCGGCGGC GCGGCGGCGG CGGCGGCGGC GCGGCGGCGG 60 CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG 120 CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG CGCGCGCGCG 180 <210> 4 <211> 500 <212> DNA <213> Artificial Sequence <400> 4 gagctgcgcg cctggcacgc gggggcggga tcctgggggg gcgtgacgga tgtcaactcg 60 cgctcgatcg ggatcgagct cgccaatccc ggcgacaggc ccttttccga gccgcagatg 120 gcggcgctgg agcggctgct ggcggggatc ctcgcacggt ggcggatccc gcccgcgcgg 180 gtaatcggcc attccgacat ggcgccggag cggaaatgcg atccgggacc gcgcttcgac 240 tggcgccgtc tggcccgcgg cgggctctcg gtctggccct cggacggctg tccgcctgcg 300 gaggcggaga cttttgccgc ttcggcccgc gccttcggct atcccgccgt ggaggcggag 360 cttctgctcg cggcggttcg cctgcggttt aggccctggg cgcgggggcc gctcacgggc 420 gaggatgcag ggatgatggc cgatcttgcg gcgcgctatc cggttgacgg acggacgccc 480 caagcgtaag ccaccgccgt 500 <210> 5 <211> 112 <212> DNA <213> Artificial Sequence <400> 5 gcccgggatt gttgacaccc ccgtgcgggc tgtccagtat caaagaccag aaggtgtgga 60 gtcggcggca cggtggccgc cccggggcgc agcgatgcgc cggagctgtc ct 112 <210> 6 <211> 150 <212> DNA <213> Artificial Sequence <400> 6 ggcgcgaact cctgcagtgc ttgtcaacat gccccataca atagacctag tcaggttttg 60 cggcttgagg gcggtgcgac ttcgcgacta atgtccgccc gcttcggttc ggtgtttgat 120 cgcgcccacc ccatctgcag gaggaatgac 150 <210> 7 <211> 152 <212> DNA <213> Artificial Sequence <400> 7 cgcgcgggcg cagcgcaatc ggacgcaggg tgacacaacg taagcgctcg tcaggctcac 60 gatgtcccgt taatgttaca cctgaagcgt cgatataggc cgacccgagt cccgcagggc 120 aggtcggccg gaagagcgcg ggggaaaacg ct 152 <210> 8 <211> 183 <212> DNA <213> Artificial Sequence <400> 8 tccggcggcc aataagtcgc acccaaaacg gtcttgtcag ccaacactga cattgaatcc 60 ataatgcgag ccggggcgga tcagaaatcg ccgacaaggt gatccaggtc tctccggtct 120 GAGAATAGTA ATAGTAATAG TAATAGTAAT AGTAATAGTA ATAGTAATAG TAATAGTATG TATGTAATAG TAATAGTA 60 GTAATAGTA 183 <210> 9 GTAATAGTA 500 <212> DNA <213> Artificial Sequence <400> 9 TCGACCCCCGC CCATGGCGGG ACGGGGCATC CTGATCGGCC TTGCCGCCTT CCTGCTCG 60 TGCTTTTCGC CTGGCCTGCC GCgtcgaagg cgatgttctt cgccggtgcg ggcctgatcg 120 GGATGGGCGG CGGGCTCTTT TCCGTCGCCA CCCTCACGAT GGCgatggcc atcccggtgg 180 Cgggtctggc cggccgcggc ctcgcgctcg gcgcctgggg ggctgcgcag gcgaccgccg 240 Cgggcctcgc catcctcatg ggcggcgcgc tgcgcgacgt catcggccac tgggccaagg 300 Cggggcatct cggtgccgcg ctgcaggacg cggccatcgg ctacagctcc gtgtacctcc 360 TCGAGATCGG GCTGCTGTTC GCCACACTGA TCgtgctggg gcctctggtc Cgaaccacga 420 TCCTCTCATC TGAACGACCG GCCGGACCCC GCgtgggact cgccgacttc CcCacCt 480 GACACCGGAG GACCCCTTAA 500 <210> 10 <211> 500 <212> DNA <213> Artificial Sequence <400> 10 gagccttcgc gcgaggcccc gaaggccgcg ccgaagcccg aggcgcgcaa gctctcggaa 60 ggcctcacct tcaccgagcg caagcgtctc gacgcgcttc cgggcctcat cgagcggctc 120 gaggccgaga tcgcgaagct cggggaattc ctcgcggccg acgacctctt cacccgcgag 180 ccggtcaaat tccagaaggc cagcgaggcg atggcggagc gtcaggccct tttgtcgcag 240 gccgaagagg agtggctgac gctcgaggac aaggccagca aaggttagcc gatgtcctgt 300 ccgccttgcg ctggaattgg cgcatggcac tctccgcata tcacctcctc gtgagcggat 360 ctggcgaacc ccccttccga gaaataggac atgtgcacgg aaggcgtcgt catccccggc 420 gacgcgagac cgtcaacgag agatccggca tccccgcaaa agcagcagtg cggacgtcgg 480 aagcttaatt ggagacagag 500 <210> 11 <211> 1812 <212> DNA <213> Escherichia coli <400> 11 atgttacgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg cctgtgggca 60 ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag cgcgttacaa 120 gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga tgcagatatt 180 cgtaattatg tgggcaacgt ctggtatcag cgcgaagtct ttataccgaa aggttgggca 240 ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt gtgggtcaat 300 aatcaggaag tgatggagca tcagggcggc tatacgccat ttgaagccga tgtcacgccg 360 tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga actgaactgg 420 cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa gcagtcttac 480 ttccatgatt tctttaacta cgccggcatc catcgcagcg taatgctcta caccacgccg 540 aacacctggg tggacgatat caccgtggtg acgcatgtcg cgcaagcctg taaccacgcg 600 tctgttgact ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg tgatgcggat 660 caacaggtgg ttgcaactgg acaaggcacc agcgggactt tgcaagtggt gaatccgcac 720 ctctggcaat cgggtgaagg ttatctctat gaactgtgcg tcacagccaa aagccagaca 780 gagtgtgata tctacccgct gcgcgtcggc atccggtcag tggcagtgaa gggcgaacag 840 ttcctgatca accacaaacc gttctacttt actggctttg gccgtcatga agatgcggat 900 ttgcgcggca aaggattcga taacgtgctg atggtgcacg atcacgcatt aatggactgg 960 attggggcca actcctaccg tacctcgcat tacccttacg ctgaagagat gctcgactgg 1020 gcagatgaac atggcatcgt ggtgattgat gaaactgcag ctgtcggctt taacctctct 1080 ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga agaggcagtc 1140 aacggggaaa ctcagcaggc gcacttacag gcgattaaag agctgatagc gcgtgacaaa 1200 aaccacccaa gcgtggtgat gtggagtatt gccaacgaac cggatacccg tccgcaaggt 1260 gcacgggaat atttcgcgcc actggcggaa gcaacgcgta aactcgaccc gacgcgtccg 1320 atcacctgcg tcaatgtaat gttctgcgac gctcacaccg ataccatcag cgatctcttt 1380 gatgtgctgt gcctgaaccg ttattacgga tggtatgtcc aaagcggcga tttggaaacg 1440 gcagagaagg tactggaaaa agaacttctg gcctggcagg agaaactgca tcagccgatt 1500 atcatcaccg aatacggcgt ggatacgtta gccgggctgc actcaatgta caccgacatg 1560 tggagtgaag agtatcagtg tgcatggctg gatatgtatc accgcgtctt tgatcgcgtc 1620 agcgccgtcg tcggtgaaca ggtatggaat ttcgccgatt ttgcgacctc gcaaggcata 1680 ttgcgcgttg gcggtaacaa gaagggcatc ttcacccgcg accgcaaacc gaagtcggcg 1740 gcttttctgc tgcaaaaacg ctggactggc atgaacttcg gtgaaaaacc gcagcaggga 1800 ggcaaacaat ga 1812 <210> 12 <211> 79 <212> DNA <213> Artificial Sequence <400> 12 ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc 60 acacaggaaa cagtattcg 79 <210> 13 <211> 121 <212> DNA <213> Artificial Sequence <400> 13 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 60 gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt 120 t 121

Claims

1. A promoter A, the nucleotide sequence of which is the nucleotide sequence set forth in SEQ ID NO:

1.

2. A library of promoters comprising at least two promoters, wherein, The at least two promoters comprise the promoter A of claim 1, and a promoter B, the nucleotide sequence of which is the nucleotide sequence set forth in SEQ ID NO:

2.

3. The promoter library of claim 2, wherein, The promoter library further comprises one or more selected from the group consisting of: a promoter C, the nucleotide sequence of which is the nucleotide sequence set forth in SEQ ID NO: 3; a promoter D, the nucleotide sequence of which is the nucleotide sequence set forth in SEQ ID NO: 4; a promoter E, the nucleotide sequence of which is the nucleotide sequence set forth in SEQ ID NO: 5; a promoter F, the nucleotide sequence of which is the nucleotide sequence set forth in SEQ ID NO: 6; a promoter G, the nucleotide sequence of which is the nucleotide sequence set forth in SEQ ID NO: 7; a promoter H, the nucleotide sequence of which is the nucleotide sequence set forth in SEQ ID NO: 8; a promoter I, the nucleotide sequence of which is the nucleotide sequence set forth in SEQ ID NO: 9; and a promoter J, the nucleotide sequence of which is the nucleotide sequence set forth in SEQ ID NO:

10.

4. A vector comprising the promoter of claim 1.

5. The vector of claim 4, wherein, The vector further comprises one or more of the promoters B, C, D, E, F, G, H, I and J in the promoter library of claim 2 or 3.

6. A genetically engineered host cell, the cell: comprising the vector of claim 4 or 5; or having integrated into its genome the promoter of claim 1.

7. The host cell of claim 6, wherein, further having integrated into its genome one or more of the promoters B, C, D, E, F, G, H, I and J in the promoter library of claim 2 or 3.

8. The host cell of claim 6, wherein, The cell is a Rhodobacter sphaeroides cell.

9. The host cell of claim 6, wherein, The cell is selected from a wild-type Rhodobacter sphaeroides cell or a modified Rhodobacter sphaeroides cell.

10. Use of a promoter according to claim 1 or of a library of promoters according to claim 2 or 3 for protein expression, wherein, The protein expression is performed in a Rhodobacter sphaeroides cell.

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