CN114686990A - Promoter library and preparation method and application thereof - Google Patents

Abstract

The invention provides a promoter library, a construction method of the promoter library and application of the promoter library. The promoter library of the present invention includes promoters derived from rhodobacter sphaeroides or modified variants thereof. The invention discloses a nucleic acid sequence of each promoter in the promoter library, a vector containing the nucleic acid sequence of the promoter and a host cell. The invention also discloses a screening method of the promoter library, which obtains the promoter library with strength gradient distribution and can realize high expression or low expression of target genes and/or fine adjustment of the expression of the target genes.

Description

A kind of promoter library and its preparation method and application

technical field

The present invention belongs to the field of biotechnology; in particular, it relates to a promoter library, a vector and/or host cell comprising the promoter, a method for constructing the promoter library, a method for screening promoter activity and applications thereof.

Background technique

Rhodobacter sphaeroides is a gram-negative photosynthetic bacterium belonging to the alpha-proteobacteria of the purple non-sulfur bacteria. The metabolic pattern of Rhodobacter sphaeroides is relatively complex. It can obtain energy through photosynthesis to maintain its existence, or it can use organic matter for aerobic respiration to obtain energy to maintain life in the absence of light. For decades, this bacterium has been used as a model organism for studying bacterial photosynthesis because it contains a large amount of photosynthetic pigments. In addition, Rhodobacter sphaeroides has various uses, such as synthesizing coenzyme Q ₁₀ for the treatment of cardiovascular diseases, which is an ideal host bacterium with high biological safety; it can also be used for biological hydrogen production , to produce poly-β-hydroxybutyrate (poly-β-hydroxybutyrate, PHB); it is now also used in membrane protein expression systems. At present, due to the above characteristics, Rhodobacter sphaeroides is widely used in food, medicine, agriculture and other fields, and has extremely high potential value for industrial development.

Under hypoxic or anaerobic conditions, the cell membrane of Rhodobacter sphaeroides is easily invaginated in the form of sprouting vesicles to form a large number of folded cell membranes, differentiated to produce an intracytoplasmic membrane, and the Intracytoplasmic Membrane System (ICM) is formed. . The ICM is equipped with a photosynthetic device for capturing photons for anaerobic photosynthesis, by which bacteria increase the membrane area to increase the photosynthetic efficiency. Since CoQ10 is widely distributed _on biofilms, this may also provide multiple storage and storage for _CoQ10 . space to function. At present, the accumulation mechanism of coenzyme Q ₁₀ is not yet clear, but as a model strain of Rhodobacter sphaeroides, the whole genome sequencing has been completed in 2005, and the regulation mechanism of photosynthetic gene expression has also been clearly studied. The photoreactive center and light-harvesting complex mainly included in the ICM are mainly expressed and 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 that control the expression of each operon also have Due to its unique characteristics, the potential of photosynthetic gene clusters in elucidating the mechanism of CoQ10 accumulation in Rhodobacter _sphaeroides and the construction of natural promoter libraries has attracted the attention of our research group. Among them, the photosynthetic operons of Rhodobacter sphaeroides mainly include pucBAC, pufQKBALMX, crt, bch, etc., which include pucA, pucB, pufA, pufB, pufL, pufM, puhA and other genes. Previous studies have also shown that important regulatory proteins such as PrrA/PrrB, PpaA, AppA/PpsR, FnrL, and IHF can sense oxygen concentration and light intensity, and regulate the expression of photosynthesis operons.

The promoter (Promoter) is located upstream of the 5' end of the DNA coding strand and contains a conserved sequence that binds RNA polymerase, and acts as a "control switch" for the timing and intensity of gene expression. It mainly includes three parts: upstream element, core promoter region and response binding element. In prokaryotes, the core region of the promoter mainly includes two parts, the -10 region "TATAAT" and the -35 region "TTGACA". These two conserved regions can recognize each other with the σ factor of RNA polymerase. It is worth noting that this kind of Binding is idiopathic, thus making the promoter species specific. Changes in the number of nucleotides in the conserved regions of -35 and -10 regions can affect the activity of gene transcription. It is generally believed that a strong promoter is 16-18 bp. When the spacing is less than 15 bp or greater than 20 bp, the activity of the promoter will be reduced. reduce.

Currently, the natural promoters commonly used in R. sphaeroides come from the genes puf, pucP and aprrnB. Both puf and pucP genes are derived from the photosynthesis operon of Rhodobacter sphaeroides, which are adapted to their functions. The promoters of these two genes are affected by light and oxygen, and can turn on or off gene transcription under different culture conditions. P _rrnB is the promoter of ribosomal RNA of Rhodobacter sphaeroides and is a moderate-strength constitutive promoter. In addition, σ ⁹³ and σ ³⁷ RNA polymerases in R. sphaeroides can compatibly recognize σ ⁷⁰ and σ ³² -dependent promoters derived from E. coli, so in R. sphaeroides, σ derived from E. coli ⁷⁰ -dependent promoters such as the E. coli promoters P _lacUV5 , P _tac/trc , P _Tn903kan , P _A1/04/03 and P _{rrnB P1} are recognized by R. sphaeroides RNA polymerase. Shan Q. et al. used promoters of P _J95025 , P _J95026 , J95027, P _tac and P _{rrnB with} five different strengths (where the P _rrnB promoter is not the strongest promoter) to mediate the CrtY gene in Rhodobacter sphaeroides expression, in which β-carotene production was increased by 109% under the _PrrnB promoter. This indicates that the appropriate gene expression intensity is more conducive to regulating the metabolic network to improve product yield, and the promoter library of Rhodobacter sphaeroides remains to be developed at this stage.

SUMMARY OF THE INVENTION

In order to solve the above problems in R. sphaeroides, the inventors of the present invention analyzed the transcriptome and genome of R. sphaeroides, and obtained a set of promoters that can provide a range of transcriptional strengths in R. sphaeroides.

Therefore, the object of the present invention is to provide a promoter library for Rhodobacter sphaeroides and its application. Another object of the present invention is to provide a method for constructing the promoter library of the present invention.

In one aspect of the present invention, a promoter library for Rhodobacter sphaeroides is provided, the promoter library includes at least two promoters, wherein the promoter A comprises the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence with at least 90% identity to SEQ ID NO: 1, and promoter B comprising the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence with at least 90% identity to SEQ ID NO: 2 acid sequence.

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

a promoter C comprising the nucleotide sequence shown in SEQ ID NO: 3 or a nucleotide sequence with at least 90% identity to SEQ ID NO: 3;

a promoter D comprising the nucleotide sequence shown in SEQ ID NO:4 or a nucleotide sequence having at least 90% identity with SEQ ID NO:4;

a promoter E comprising the nucleotide sequence shown in SEQ ID NO:5 or a nucleotide sequence having at least 90% identity with SEQ ID NO:5;

a promoter F comprising the nucleotide sequence shown in SEQ ID NO: 6 or a nucleotide sequence having at least 90% identity with SEQ ID NO: 6;

a promoter G comprising the nucleotide sequence shown in SEQ ID NO:7 or a nucleotide sequence having at least 90% identity with SEQ ID NO:7;

A promoter H comprising the nucleotide sequence shown in SEQ ID NO: 8 or a nucleotide sequence having at least 90% identity with SEQ ID NO: 8;

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

A promoter J comprising the nucleotide sequence set forth in SEQ ID NO:10 or a nucleotide sequence at least 90% identical to SEQ ID NO:10.

In one aspect of the invention, there is provided a vector comprising a promoter from the promoter library described herein.

In one aspect of the present invention, a genetically engineered host cell is provided, the cell: the vector comprising the promoter element in the promoter library described herein; or the promoter integrated into its genome Promoter elements in sublibraries.

In some embodiments, the cells are R. sphaeroides cells. For example, in the present invention, the R. sphaeroides cells may be wild-type R. sphaeroides cells, or modified R. sphaeroides cells (eg, chemically mutagenized, or genetically modified, etc.).

In another aspect of the present invention, there is provided a construction method of the promoter library described herein, the method comprising:

(1) Obtain candidate promoter sequences: Based on the transcriptome information of Rhodobacter sphaeroides, screen genes whose expression intensity ranks at least the top fifty, preferably the top twenty, and more preferably the top ten, based on the genome information of Rhodobacter sphaeroides And the selected gene, obtain the candidate promoter sequence that is positioned at the upstream of the starting site of the gene sequence;

(2) constructing a recombinant expression vector: the reporter gene sequence operably linked to the candidate promoter sequence is linked to a vector capable of being expressed in a host cell, thereby obtaining a recombinant expression vector; and

(3) Observing the expression of the reporter gene, introducing the recombinant expression vector into the host cell to express the reporter gene, and selecting a promoter capable of expressing the reporter gene to form a promoter library.

In the present invention, the host cells are Rhodobacter sphaeroides cells. For example, the reporter gene can be selected from the group consisting of: fluorescent protein genes (eg, green fluorescent protein gene, yellow fluorescent protein gene), luciferase (luc) gene, and beta-galactosidase gene (lacZ) and gusA gene.

In one aspect of the invention, there is provided a method of screening promoters, the method comprising comparing a promoter in a library of promoters described herein to a reference promoter for its ability to elicit expression of a reporter gene, The strength of the promoter is thus screened.

The invention also discloses a screening method for the promoter library, which obtains a promoter library with an intensity gradient distribution, which can realize high or low expression of the target gene and/or can realize the fine-tuning of its expression. At the same time, it provides a favorable tool for regulating the metabolic network in spheroid erythrocytes and achieving increased product yield.

In another aspect of the invention, there is provided the use of the promoter libraries described herein in protein expression.

Description of drawings

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

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

Figure 3: Standard curve for the determination of the resulting product p-nitrophenol at visible light 415 nm in the GUS test.

Figure 4: The promoter activity of each promoter activity characterization strain at three time points of 11h, 24h, and 48h in the GUS test.

Fig. 5: Growth conditions (OD ₇₀₀ ) of the strains used for the characterization of each promoter activity in the GUS test at three time points of 11h, 24h, and 48h.

Detailed ways

definition

The strength of a promoter is the level of transcription of the downstream gene it mediates. In prokaryotes, there are few regulatory mechanisms in the process of transcription to translation, so the amount of translated protein can also indicate the strength of the promoter.

In the context of a nucleic acid or polypeptide, the terms "isolated" or "partially purified" as used herein refer to a nucleic acid or polypeptide that is separated from at least one other component (eg, nucleic acid or polypeptide) that is The nucleic acid or polypeptide is found to co-exist with the nucleic acid or polypeptide in its natural source; and/or the other components, when expressed by a cell, or secreted in the case of a secreted polypeptide, will co-exist with the nucleic acid or polypeptide or the coexistence of polypeptides. A nucleic acid or polypeptide that is chemically synthesized or synthesized using in vitro transcription/translation is considered "isolated." The term "purified" or "substantially purified" refers to an isolated nucleic acid or polypeptide that is at least 95% by weight of the nucleic acid or polypeptide of interest, including, eg, at least 96%, at least 97%, at least 98%, at least 99%, or more.

As used herein, "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter is placed at a specific location relative to the nucleic acid sequence of a gene of interest such that transcription of the nucleic acid sequence is directed by the promoter, and thus, the promoter is "operably linked" to the nucleic acid sequence.

As used herein, "gene of interest" refers to a gene whose expression can be directed by the promoter of the present invention. The present invention has no particular limitation on suitable target genes, for example, including but not limited to: structural genes, genes encoding proteins with specific functions, enzymes, reporter genes (such as green fluorescent protein gene, luciferase gene, galactosidase gene gene LazZ, GUS gene, etc.).

As used herein, "foreign" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, a promoter is foreign to a gene of interest if the combination of the promoter and the sequence of the gene of interest does not normally occur in nature. A particular sequence is "foreign" to the cell or organism into which it is inserted.

For purposes of the present invention, "identity" is calculated by comparing two aligned sequences in a comparison window. Alignment of the sequences enables determination of the number of positions (nucleotides or amino acids) shared by the two sequences in the comparison window. Then, divide the number of shared positions by the total number of positions in the comparison window and multiply by 100 to obtain percent homology. Determination of percent sequence identity can be accomplished manually or using well-known computer programs.

promoter library

The present invention provides a promoter library for Rhodobacter sphaeroides. The promoter library includes at least two promoters, wherein the promoter A comprises the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence with at least 90% identity with SEQ ID NO: 1, and the promoter Sub-B comprises the nucleotide sequence set forth in SEQ ID NO:2 or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2.

In some embodiments, promoter A comprises the nucleotide sequence set forth in SEQ ID NO: 1, or a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1, eg, has SEQ ID NO: 1 Nucleotide sequences of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity. In a preferred embodiment, promoter A consists of the nucleotide sequence shown in SEQ ID NO:1.

In some embodiments, promoter B comprises the nucleotide sequence set forth in SEQ ID NO:2, or a nucleotide sequence that is at least 90% identical to SEQ ID NO:2, eg, has SEQ ID NO:2 Nucleotide sequences of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity. In a preferred embodiment, promoter B consists of the nucleotide sequence shown in SEQ ID NO:2.

For example, the promoter library comprises: promoter A consisting of the nucleotide sequence shown in SEQ ID NO: 1 and promoter B consisting of the nucleotide sequence shown in SEQ ID NO: 2.

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

A promoter C comprising the nucleotide sequence set forth in SEQ ID NO:3 or a nucleotide sequence at least 90% identical to SEQ ID NO:3, eg, having SEQ ID NO:3 Nucleotide sequences of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity;

A promoter D comprising the nucleotide sequence set forth in SEQ ID NO:4 or a nucleotide sequence at least 90% identical to SEQ ID NO:4, eg, with SEQ ID NO:4 Nucleotide sequences of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity;

A promoter E comprising the nucleotide sequence set forth in SEQ ID NO:5 or a nucleotide sequence at least 90% identical to SEQ ID NO:5, eg, having SEQ ID NO:5 Nucleotide sequences of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity;

A promoter F comprising the nucleotide sequence set forth in SEQ ID NO:6 or a nucleotide sequence at least 90% identical to SEQ ID NO:6, eg, having SEQ ID NO:6 Nucleotide sequences of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity;

A promoter G comprising the nucleotide sequence set forth in SEQ ID NO:7 or a nucleotide sequence at least 90% identical to SEQ ID NO:7, eg, having SEQ ID NO:7 Nucleotide sequences of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity;

A promoter H comprising the nucleotide sequence set forth in SEQ ID NO:8 or a nucleotide sequence at least 90% identical to SEQ ID NO:8, eg, having SEQ ID NO:8 Nucleotide sequences of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity;

A promoter I comprising the nucleotide sequence set forth in SEQ ID NO:9 or a nucleotide sequence at least 90% identical to SEQ ID NO:9, eg, having SEQ ID NO:9 Nucleotide sequences of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity; and

A promoter J comprising the nucleotide sequence set forth in SEQ ID NO: 10 or a nucleotide sequence at least 90% identical to SEQ ID NO: 10, eg, having SEQ ID NO: 10 Nucleotide sequences of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity.

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

Promoter C, which is composed of the nucleotide sequence shown in SEQ ID NO: 3;

Promoter D, the promoter D consists of the nucleotide sequence shown in SEQ ID NO: 4;

Promoter E, the promoter E consists of the nucleotide sequence shown in SEQ ID NO: 5;

Promoter F, said promoter F consists of the nucleotide sequence shown in SEQ ID NO: 6;

Promoter G, said promoter G consists of the nucleotide sequence shown in SEQ ID NO: 7;

Promoter H, described promoter H consists of the nucleotide sequence shown in SEQ ID NO: 8;

a promoter I consisting of the nucleotide sequence shown in SEQ ID NO: 9; and

Promoter J consisting of the nucleotide sequence shown in SEQ ID NO:10.

For example, the promoter library may 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 promoter, at least 9 promoters, or at least 10 promoters.

In the promoter library of the present invention, the intensity of each promoter to promote the expression of the target gene presents a gradient distribution, which is used to guide the expression of the target gene under different intensities. For example, the expression of exogenous proteins in the host, or the regulation of intracellular metabolic pathways, etc. For example, the promoter library of the present invention can be used to finely regulate the metabolic pathway, so that the gene can be expressed in an appropriate amount, and the combinatorial optimization of the metabolic pathway can be realized.

The promoter of the present invention may be operably linked to a gene of interest, which may be foreign (heterologous) to the promoter. The gene of interest can generally be any nucleic acid sequence (eg, a structural nucleic acid sequence), and the gene of interest preferably encodes a protein with a specific function, such as certain proteins with important properties or functions.

For example, when used for the study of expression intensity, the target genes include but are not limited to: green fluorescent protein, yellow fluorescent protein, luciferase gene, galactosidase gene LacZ, Gus gene and the like.

As a preferred mode of the present invention, promoters with different strengths can be selected from the promoter library of the present invention, and they are respectively operably linked to the target gene to be studied, or the target gene is operably linked to the promoter. It is ligated into a suitable vector and introduced into a host cell in an appropriate manner, thereby obtaining a series of cells with different expression levels of the target gene. The function or use of the target gene can be known by analyzing the metabolism, phenotypic changes, protein expression or interaction, and changes of various signal molecules in these cells.

As a preferred mode of the present invention, the target gene can be a gene that is missing or insufficiently expressed in a certain cell, and the target gene can be operably linked to the promoter of the present invention, or the target gene can be linked with the promoter of the present invention. The promoter of the present invention is operably linked into a suitable vector and introduced into the cell in an appropriate manner, thereby expressing the target gene at a high level.

The promoters of the present invention may also be operably linked to an improved sequence of a gene of interest that is foreign (heterologous) 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 amino acid sequences, alter post-translational modifications (such as phosphorylation sites), transport translation products out of the cell, improve protein stability, insertion or deletion cell signaling, etc.

Additionally, promoters and genes of interest can be designed to downregulate specific genes. This is typically accomplished by ligating the promoter to the gene sequence of interest, which is directed in the antisense reverse direction. One of ordinary skill in the art is familiar with such antisense technology. Any nucleic acid sequence can be modulated in this manner.

Here, in the present invention, promoter A and promoter B can be regarded as high-strength promoters, promoter C, promoter D, promoter E, and promoter F can be regarded as medium-strength promoters, and promoter G , Promoter H, Promoter I, and Promoter J can be regarded as low-strength promoters. Here, high, medium and low strength promoters are relative to the strength of the tested promoter itself.

Vectors and Host Cells

The present invention also provides vectors containing promoter elements from the promoter libraries described herein.

Any one of the promoter and/or gene sequences of interest from the promoter library of the present invention can be included in the recombinant vector.

As an embodiment, the recombinant vector includes the promoter of the present invention, and optionally contains multiple cloning sites or at least one restriction enzyme site downstream of the promoter. When the target gene needs to be expressed, the target gene is ligated into a suitable multiple cloning site or restriction enzyme site, thereby operably linking the target gene to the promoter. As an embodiment, the recombinant vector includes the promoter of the present invention, and when the target gene needs to be expressed, the target gene is connected to the downstream of the promoter by means of homologous recombination, so that the target gene and the promoter are operably connected connect.

In a specific embodiment, the target gene is located downstream of a promoter, and the distance from the promoter is 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 spacer between the promoter and the gene of interest.

As an embodiment, the recombinant vector includes (from 5' to 3' direction): a promoter for directing the transcription of the target gene and the target gene. If desired, the recombinant vector may also include 3' transcription terminators, 3' polynucleotideization signals, other untranslated nucleic acid sequences, transport and targeting nucleic acid sequences, resistance selectable markers, enhancers or operators, and the like.

In another embodiment, the target genes include but are not limited to: structural genes, genes encoding proteins with specific functions, reporter genes (eg, green fluorescent protein gene, luciferase gene, galactosidase gene LacZ, Gus gene).

In a preferred embodiment, the recombinant vector is an expression vector. In the present invention, the term "expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, mammalian cell viruses or other vectors well known in the art. In conclusion, any plasmids and vectors can be used as long as they are replicable and stable in the host. Preferably, the expression vector is an expression vector suitable for Rhodobacter sphaeroides.

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 expression vectors containing the promoter and/or target gene sequences of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like. Expression vectors also include a ribosome binding site for translation initiation and a transcription terminator.

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

In addition to the promoter of the present invention, the recombinant vector may contain one or more other promoters. Said other promoters are, for example: tissue-specific, constitutive or inducible.

A vector comprising the appropriate promoter and gene of interest described above can be used to transform an appropriate host cell so that it can express the protein.

Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: yeast, Escherichia coli, animal tissue cells, plant cells, etc. It will be clear to those of ordinary skill in the art how to select appropriate vectors and host cells. As a preferred embodiment of the present invention, the host cell is Rhodobacter sphaeroides. For example, in the present invention, the R. sphaeroides cells may be wild-type R. sphaeroides cells, or modified R. sphaeroides cells (eg, chemically mutagenized, or genetically modified, etc.). For example, R. sphaeroides cells can be R. sphaeroides that have been chemically mutagenized or genetically modified to produce high levels of coenzyme Q ₁₀ .

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use MgCl ₂ . If desired, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

For example, transformation of Rhodobacter sphaeroides by a recombinant vector can be carried out by means of electroporation, conjugation transfer and the like.

The present invention also provides a genetically engineered host cell that: contains a vector having a promoter element from a promoter library described herein; or

The promoter elements of the promoter library described herein are integrated into its genome.

In a preferred embodiment, the cells are Escherichia coli or Rhodobacter sphaeroides cells.

Construction method of promoter library

The construction method of the promoter library of the present invention includes:

(1) Obtain candidate promoter sequences: Based on the transcriptome information of Rhodobacter sphaeroides, screen genes whose expression intensity ranks at least the top fifty, preferably the top twenty, and more preferably the top ten, based on the genome information of Rhodobacter sphaeroides And the selected gene, obtain the candidate promoter sequence that is positioned at the upstream of the starting site of the gene sequence;

(2) constructing a recombinant expression vector: the reporter gene sequence operably linked to the candidate promoter sequence is linked to a vector capable of being expressed in a host cell, thereby obtaining a recombinant expression vector; and

(3) Observing the expression of the reporter gene, introducing the recombinant expression vector into the host cell to express the reporter gene, and selecting a promoter capable of expressing the reporter gene to form a promoter library.

Transcriptomic and genomic information can be obtained using information published in various databases in the art (eg, KEGG, NCBI), or by sequencing.

Candidate promoter sequences are usually chosen to be 100-500 bp upstream of the start codon that contains the core promoter portion. In preferred embodiments, the fragment may be further modified, eg, to delete a portion thereof that inhibits promoter activity, eg, the ppsR binding site. For the determination of ppsR binding sites, see Patrice Bruscella et al., The Use of Chromatin Immunoprecipitation to Define PpsR Binding Activity in Rhodobactersphaeroides 2.4.1, JOURNAL OF BACTERIOLOGY, Oct. 2008, p. 6817-6828, incorporated herein by reference Its entirety is incorporated herein.

As the reporter gene, a green fluorescent protein gene, a luciferase gene, a galactosidase gene LacZ, a Gus gene, or the like can be used.

Fluorescent protein gene: Green fluorescent protein (green flourscent protein, GFP) is a typical endogenous fluorescent protein, GFP is safe for the host, and the fluorescence intensity can directly reflect the expression level, no external substrate is required, and the sensitivity is high, which is conducive to Non-destructive testing of the host.

Yellow fluorescent protein (YFP) can be regarded as a mutant of green fluorescent protein. Compared with green fluorescent protein, its fluorescence image is shifted to the red spectrum. It is shared with GFP fluorescent protein and has longer wavelength excitation and emission light. The most important thing is Because of its low background when imaged inside cells.

Luciferase (luciferase, luc) gene: luc is an enzyme that catalyzes luciferin or fatty aldolase to produce fluorescence in an organism in an aerobic environment. luc catalyzes the luminescence reaction of substrate ATP, inorganic luciferin and oxygen, and the enzyme and It has strong substrate specificity, minimal impact on the host, high sensitivity, easy detection, and no excitation light interference.

gusA gene: derived from Escherichia coli, encoding β-D-glucuronidase, with a monomer molecular weight of 68200Da, resistant to a variety of detergents, and extremely active in the presence of mercapto alcohol reducing agents β-mercaptoethanol or DTT. This enzyme can catalyze the hydrolysis of many β-glucoside esters without coenzyme factors or cations, and some substances will change color after hydrolysis. A few divalent metal ions such as Zn ²⁺ and Cu ²⁺ can inhibit its activity. Its optimum pH is 5.2-8.0, the half-life of heat resistance is 50 ℃ 2h, and the activity has a linear relationship in a long time. GUS substrates are all soluble, the most commonly used catalytic substrate is 5-bromo-4-chloro-3-indole glucuronide (X-gluc), the product is a blue compound, and 4-methyl umbrella was also commonly used in the early years. Form ketone glucuronide (MUG), 9-hydroxy-3-isophenoxazolone glucuronide (ReG), ReG has high fluorescence activity, its product excitation at 560nm, emission 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 also the most widely used reporter gene, which serves the purpose of "reporting" the activation activity. In one embodiment, the GUSA sequence source: Enterobacteriaceae, LOCUS: WP_000945901.

The GUS assay is an enzymatic reaction detection assay based on a colorimetric assay. For example, the color of the product produced by the Gus enzyme catalyzed substrate contained in the sample is collected by a microplate reader, and the amount of the catalyzed substrate is calculated from the concentration of the product, which reflects the strength of the promoter used. For example, when using p-nitrophenyl-β-D-glucuronic acid as the substrate to generate the product p-nitrophenol, the color reaction is detected at 415nm for 20-30min (the color reaction tends to be stable during this time), The corresponding substrate consumed in the sample was then extrapolated from a standard curve of product versus absorbance at 415 nm (eg Figure 3), followed by the slope of the substrate versus reaction time curve to reflect the strength of the promoter tested. For the GUS test, please refer to Siegl, Theresa et al., "Design, construction and characterisation of asynthetic promoter library for fine-tuned gene expression in actinomycetes.", Metabolic Engineering 19.Complete(2013):98-106, incorporated herein by reference The manner in which it is incorporated herein in its entirety.

In some embodiments, the candidate promoter sequence may also be modified. The modifications include substitutions, deletions, and/or insertions. In a preferred embodiment, the repression site in the candidate promoter sequence is modified by deletion. For example, delete the ppsR binding site in the candidate promoter sequence. For the determination of the ppsR binding site, see Patrice Bruscella et al., The Use of Chromatin Immunoprecipitation to Define PpsR BindingActivity in Rhodobacter sphaeroides 2.4.1, JOURNAL OF BACTERIOLOGY, Oct. 2008, p. 6817-6828, which is incorporated herein by reference Its entirety is incorporated herein.

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

Screening method

A method for screening promoters, the method comprising comparing and ranking the promoters in the promoter library according to the present invention for their ability to induce the expression of a target gene, thereby screening the strength of the promoters.

In a preferred embodiment, the gene of interest can be selected from reporter genes. For the definition of reporter gene, see description above in this article.

In the present invention, a reference promoter is preferably used for comparison with a candidate promoter. Regarding the selection of the reference promoter, it can more intuitively and directly feed back the ability of the candidate promoter to induce the expression of the reporter gene. Therefore, a promoter that is well characterized in the host cell is usually selected in reference to the promoter. In a preferred embodiment, two or more reference promoters with different activation strengths are selected.

In a preferred embodiment, the screening method is performed by comparing the level of strength of the promoters described herein relative to a reference promoter in promoting the expression of a gene of interest (including a level above the reference or a level below the reference) ), the method comprises: under identical culturing conditions, cultivate respectively the host cell that the promoter of the present invention starts the target gene expression and the host cell that the reference promoter starts the target gene expression, compares the expression intensity of the target gene in two kinds of cells (or expression).

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

The method for measuring the expression of the target gene (especially the reporter gene) in the culture system is a technique well known to those skilled in the art, and it can also be adjusted according to the difference of the target gene. As a preferred mode of the present invention, the reporter gene is Gus protein, and the method for measuring the expression of the reporter gene includes: measuring the activity of the enzyme in the culture system. In some embodiments, the reporter gene is a fluorescent protein (eg, green fluorescent protein), and the method for determining the expression of the reporter gene includes: determining the fluorescence intensity in the culture system.

In addition, the present invention also provides the use of the promoter library described herein in protein expression.

Example

The present invention will be further described below with reference to the embodiments and the accompanying drawings, but the present invention is not limited by the following embodiments. Those skilled in the art should understand that equivalent replacements or corresponding improvements made to the technical features of the present application still fall within the protection scope of the present application. Unless otherwise specified, the reagents used in the following examples are all commercially available products, and conventional techniques in the art can be used for the preparation of the solution.

Example 1 Acquisition of Candidate Promoters

After the promoter binds to RNA polymerase, mRNA starts to be transcribed. Generally, the transcription initiation site is located downstream of the promoter, and there are also overlaps. The mRNA expression level can be measured semi-quantitatively through the transcriptome information, and the position of the corresponding gene in the strain genome can be found through the sequence of the expressed mRNA, and the corresponding coding sequence on the genome and the sequence including the promoter upstream of the coding sequence can be determined. In this experiment, a transcriptomic analysis of the experimental strain was performed to locate the relevant photosynthetic gene clusters on the single-stranded circular genome of Rhodobacter sphaeroides, and the adjacent sequences of the relevant genes in the genome mapped by mRNA were regarded as candidate promoters The presence of the promoter region, thereby obtaining candidate promoter sequence information, used for characterization experiments.

Specifically, Rhodobacter sphaeroides HY01 (hereinafter referred to as R.sphHY01, obtained from Rhodobacter sphaeroides 2.4.1 through nitrosoguanidine mutagenesis screening, high-yield coenzyme Q ₁₀ , referred to as ind in Figure 1) wild The transcriptome information of Rhodobacter sphaeroides 2.4.1 (also referred to as wt in Figure 1) was cultured with fermentation medium at 32°C and 220rpm for 24h and 48h (respectively ind24, ind48, wt24, wt48). Transcription intensity analysis was performed on the obtained transcriptome information (the analysis results are shown in Figure 1, where the abscissa is the transcription intensity, vertical axis in different Rhodobacter sphaeroides strains (including R. Coordinates are the corresponding operon/gene name, color scale on the right). Among them, the medium is ammonium sulfate 5g/L, monosodium glutamate 5g/L, corn steep liquor 7g/L, glucose 20g/L, potassium dihydrogen phosphate 0.3g/L, magnesium sulfate 7g/L, sodium chloride 3g/L, Ferrous sulfate 1g/L, manganese sulfate 0.4g/L, cobalt chloride 0.008g/L, calcium carbonate 4g/L, pH adjusted to 6.5, sterilized at 121°C for 25min.

The top thirteen gene sequences with transcription intensity were selected, and the genome information of these genes was obtained based on the information in the KEGG database (the genome information here is the genome information from wild-type R. sphaeroides 2.4.1).

Specifically, the top thirteen genes with transcription intensity are from the following operons/genes (from top to bottom in Figure 1): rnpB (KEGG number RSP_4342, this gene is not transcribed with other genes due to its high transcription intensity) information is shown together in Figure 1), 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 Figure 1), bchEJ (KEGG code RSP_0280, shown as bchJ in Figure 1), pufQ (KEGG code RSP_0259), crtEF (KEGG code RSP_0264, shown as crtF in Figure 1).

Among them, pufQ, puc2B, puhA, crtEF, pucBAC, crtA, bchEJ are all from photosynthetic gene clusters. rpsM is 30S ribosomal protein S13 gene; rnpB is RNase P gene. The functions of RSP_7571, RSP_6124, RSP_2718, and RSP_1185 have not yet been clarified, and the promoter sequence information has not yet been clarified.

The fragment containing the core promoter 500bp upstream of the initiation codon of the genes RSP_7571, RSP_6124, RSP_2718, RSP_1185, puhA, and rnpB was cut to obtain candidate promoters P _RSP-7571 (SEQ ID NO: 2, 500bp), P _RSP-6124 (SEQ ID NO: 1,500bp), _{PRSP_2718} (SEQ ID NO:10,500bp), PRSP _-1185 (information on this promoter not shown), _PpuhA (SEQ ID NO:9,500bp), P _rnpb (SEQ ID NO: 4, 500bp).

Cut out the fragment containing the core promoter upstream of the start codon of the genes pufQ, crtEF, pucBAC, crtA, bchEJ and delete the ppsR binding site (please refer to Patrice Bruscella et al., The Use of Chromatin Immunoprecipitation to Define PpsR Binding Activity in Rhodobactersphaeroides 2.4 .1, JOURNAL OF BACTERIOLOGY, Oct. 2008, p. 6817-6828), to obtain the following candidate promoters: P _pufQ (SEQ ID NO: 7, 152 bp), P _crtEF (SEQ ID NO: 5, 132 bp), P _pucBAC (SEQ ID NO: 8, 203 bp), P _crtA (SEQ ID NO: 6, 157 bp), P _bchEJ (SEQ ID NO: 3, 147 bp).

Example 2 Construction of strains for characterizing promoter activity

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.

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

According to the schematic diagram in Figure 2, the above-mentioned candidate promoters and GUS genes were assembled into pBBR1MCSK vector (purchased from Novagen) through Ezmax (purchased from Tolo Biotechnology Co., Ltd.) to construct plasmids with each candidate promoter and GUS gene. After verification by sequencing, it was transformed into E. coli DH10b competent, and positive transformants were screened by colony PCR. Transformation conditions: incubate on ice for 30 min, heat at 42 °C for 90 s, incubate on ice for 2 min, add 700 uL LB, and incubate at 37 °C for 45 min on a shaker.

The plasmid was extracted from the positive transformant, and the wild-type Rhodobacter sphaeroides 2.4.1 single clone (strain number ATCC17023) was electroporated to obtain a recombinant strain. The prepared strains are shown in Table 1 below.

The electroconversion conditions are as follows:

1. Add 2 mL of bacterial cells to each tube of 2 mL sterile EP tubes (cultivate the bacterial cells with TSB medium to an OD of 1-3), and centrifuge at 9000 rpm for 1 min;

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

3. Repeat step 2 to wash with water three times;

4. Add 80 μL of 10% glycerol to each EP tube from which the supernatant was discarded, resuspend the cells and add plasmid (500 ng) to mix well;

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

6. Electric transfer conditions: 2000V/200Ω/2mm;

7. After electroporation, add 600 μL of antibiotic-free TSB medium, blow and suck and mix evenly, remove all the liquid, put it in a 2 mL EP tube, and incubate at 32°C and 200 rpm for 2 hours;

8. Centrifuge at 12000g for 1 min, pour off part of the supernatant, mix the remaining bacterial liquid, spread it on a kana-resistant plate, and place it in a 32°C incubator for 6-7 days.

TSB medium: 30g/L Tryptone Soya Broth. 2% agar was added to the solid medium, and the sterilization conditions were 115°C × 20min.

Table 1 The strains for characterizing promoter activity constructed in this example

Example 3 Activity analysis of candidate promoters

In this example, the activity of candidate priming was analyzed using the GUS assay. For related information, see Siegl, Theresa et al., "Design, construction and characterisation of a syntheticpromoter library for fine-tuned gene expression in actinomycetes.", Metabolic Engineering 19.Complete(2013):98-106.

GUS experiment principle:

Using p-nitrophenyl-β-D-glucuronic acid as the reaction substrate for enzyme activity assay, and highly sensitive β-D-glucuronidase (GUS reporter protein) as the catalyst, p-nitrophenol is generated. The reaction system was spectrophotometrically measured for light absorption at 415 nm for 30 minutes, and the slope of the final absorbance curve was used to calculate the enzymatic activity.

The specific experimental steps are as follows:

1. Pick 1-2 colonies of each bacterial strain prepared in Example 2 and inoculate it in 5mL TSB liquid medium, after culturing 24h at 32°C and 220rpm in a 50mL centrifuge tube, get 5mL and transfer it to 45mL fermentation medium (fermentation culture medium). The medium was the same as that in Example 1), and cultured to the plateau phase (48h) at 32°C and 220rpm (in this step, the fermentation medium contained 50ng/ml of kanamycin).

2. Take the same bacterial solution with OD ₇₀₀ (in the range of OD ₇₀₀ value of 10-25), centrifuge at 12000rpm×1min to remove the supernatant.

3. Resuspend the bacteria with 900 μL of Gus buffer 2, and then incubate in a water bath at 37°C for 20 minutes to obtain the lysate of each strain.

4. Then dilute the lysate with 900 μL of Gus buffer 1 and centrifuge at 14000 rpm for 10 min at 4°C.

5. Add 500 μL of the supernatant to 500 μL of Gus buffer 3.

6. The microplate reader was used to measure the absorbance at 415 nm. Add 200 μL of mixed liquid to the 96-well plate, and the measurement time was 30 min. Mix 100 μL of Gus buffer 3 with 100 μL of Gus buffer 2 and Gus buffer 1 in the same proportion as a control group.

7. The data of the color reaction process was collected by a microplate reader and detected on a 96-well plate for 30 minutes; at the same time, a standard curve of the product relative to the absorbance at 415 nm was prepared (see Figure 3); The absorbance value corresponding to the time point is converted into the concentration of the product to calculate the amount of the catalyzed substrate, and then take the reaction time as the abscissa and the concentration of the catalyzed substrate as the ordinate, draw the time from each strain to the product color is stable, with time Change the curve graph, fit the reaction progress through the office software, calculate the slope of the curve, express the activity of the enzyme by the slope, and calculate the specific activity of the enzyme according to the principle of the enzymatic reaction. Each group of strains was replicated in triplicate.

Specifically, the enzymatic 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 inactinomycetes.", Metabolic Engineering 19. Complete (2013): 98-106, wherein the The standard curve used is the one in Figure 3.

Solution:

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% TritonX-100.

Gus buffer 2: 30 mL of 50 mM phosphate buffer (pH 7.0), 150 μL of 5 mM DTT, 30 μL of 0.1% TritonX-100, and 30 mg of lysozyme (the final concentration is about 1 mg/mL).

Gus buffer 3: 30 mL of 50 mM phosphate buffer (pH 7.0), 150 μL of 5 mM DTT, 30 μL of 0.1% TritonX-100, p-nitrophenyl-β-D-glucuronic acid final concentration 2 mM.

Among them: Phosphate buffer: 1.14g K ₂ HPO ₄ · 3H ₂ O was made up to 100mL with deionized water, and 0.6g NaH ₂ PO ₄ was made up to 100mL with deionized water. The former was the main solution and the latter was the auxiliary solution. pH, K ₂ HPO ₄ ·3H ₂ O:NaH ₂ PO ₄ =5:4 at pH 7.0.

2 mM p-nitrophenyl-β-D-glucuronic acid: 0.63 g p-nitrophenyl-β-D-glucuronic acid was added with 1 LdH ₂ O, and 18.9 mg was added to prepare a 30 mL solution.

Result analysis:

Figure 5 shows the growth status of each strain at 11h, 24h, and 48h during cultivation to plateau, shown as _OD700 .

First, the slope of the absorption curve of each promoter is calculated, and the slope is converted into the substrate concentration according to the standard curve. The concentration is positively correlated with the enzyme activity of the reporter gene expression product, and the enzyme activity is positively correlated with the strength of the promoter. In Figure 4, the ordinate is the enzyme activity, the abscissa is each promoter, and the promoter strength increases sequentially from left to right. The first is a blank control, and the second is a gusA reporter gene control without a promoter. Among the promoters, the weak ones are P _kana , P ₂₇₁₈ , P _puhA , P _pucBAC , and P _PufQ , the medium ones are P _CrtA , P _crtEF , P _tac , P _rnpb , P _bchEJ , and the high ones are P ₇₅₇₁ and P ₆₁₂₄ .

This experiment successfully obtained a series of promoters with differences in strength across 5 orders of magnitude, proving that this method can effectively quantitatively characterize promoter strength, and can continue to use this method to expand the library of R. sphaeroides promoter elements. At the same time, the experimental results show the effect of extremely strong promoters on the growth rate and amount of bacteria, and to a certain extent, it shows the metabolic burden that the strain can carry. According to the results of _PrnpB , it is speculated that there may be an up-regulation mechanism. The introduction of the commonly used promoters P _tac , P _kana also makes these results easier to apply.

The accumulation of expressed proteins is not determined by the instantaneous gene translation strength of a single independent variable, and different protein products will also have different degradation rates. Therefore, the characterization of promoter strength in this experiment should be regarded as horizontal reference data at the same time point. In the future, if the timing of reporter protein expression and degradation can be controlled, and the nutrient level can be controlled by a chemostat fermenter to achieve the distinction between degradation and accumulation time, it will be more conducive to clearly clarify the strength of the promoter and the nutrient limiting factors.

In addition, promoter engineering has developed rapidly, and many researchers have established artificially mutated promoter libraries for the cells under study. Artificial promoters have a higher probability of being unregulated by cellular metabolic networks, and help to limit the range of uncontrollable variables for various single-gene modification efforts. In addition, artificial promoters may fill the strength regions that do not exist in natural promoters. In this experiment, the top ten promoters from the transcriptome data have been selected, but they still have strong regions near strong promoters such as P ₆₁₂₄ and P ₇₅₇₁ . Lots of gradients to fill the space. If there are application needs in the future, the sequences of the two can be analyzed to find out the sites that affect the promoter activity and mutate to further enrich the library.

sequence listing

<110> East China University of Science and Technology

<120> A promoter library and its preparation method and application

<130> 1

<160> 13

<170> PatentIn version 3.5

<210> 1

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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

gccccgttaga ggagagcact 500

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<213> Artificial Sequence

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cgccgcagcc gatggtccag acctcggccc cggcgagggc gggcgtggcc cggatccgct 60

cggcgagcct ctgcgccagc gccgcctcgc cgggcgagcc cgtgaccgac cgttgccgcg 120

tcagccacag cgcccaatcc cgggcgcggt cacgataatg ggccatgtct ctctcctgct 180

cgtccgccgg atcgcctgcc cggctgtgtc ctgtccgccc gcacgggtcc ccgtgcgcag 240

cgccccggcc catcctgcgc cgaaggccgg ggcgaccgga agccccgcgc agagtgggca 300

gtccgaaggc cggctttgcc tgcgcgggct atgcccgcag ctattcctgc gcccctccgc 360

cgggaagcgc ccgccacgaa agcgtgagag ggggcggcgt gatgtcgccc atgggccgca 420

gcgtcgccct tcggcttgac gcaccccctg ccgggcggca acggtgaggg ctggcccagc 480

ttgcatctga aggaggcacc 500

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cccctgcgcc ccgctgccgc agggcatttc ccccgccttt gggaaagggt gggcccgtcc 60

gcgcgggtcg aatggtcgct ttgacatgca tcaatttgcc cgccacagaa gtaaacgtaa 120

gcgctcgtca ggcgcgggag tccacca 147

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<212> DNA

<213> Artificial Sequence

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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

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<213> Artificial Sequence

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gcccgggatt gttgacaccc ccgtgcgggc tgtccagtat caaagaccag aaggtgtgga 60

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<213> Artificial Sequence

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ggcgcgaact cctgcagtgc ttgtcaacat gccccataca atagacctag tcaggttttg 60

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<213> Artificial Sequence

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cgcgcgggcg cagcgcaatc ggacgcaggg tgacacaacg taagcgctcg tcaggctcac 60

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aggtcggccg gaagagcgcg ggggaaaacg ct 152

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<212> DNA

<213> Artificial Sequence

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tccggcggcc aataagtcgc acccaaaacg gtcttgtcag ccaacactga cattgaatcc 60

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cgtcgaagcc cgcgtgcagg ccctacacgc aaaccgtcga tttaccagtt gggagacgac 180

aca 183

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<212> DNA

<213> Artificial Sequence

<400> 9

tcgaccccgc ccgcatggcg ggacggggca tcctgatcgg ccttgccgcc ttcctgctcg 60

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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 gccggcggga cccgcgtggg actcgccgac ttccccacct 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

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<212> DNA

<213> Escherichia coli

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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 (10)

1. A promoter library comprising at least two promoters, wherein promoter a comprises SEQ ID NO: 1 or a nucleotide sequence corresponding to SEQ ID NO: 1, promoter B comprises a nucleotide sequence of at least 90% identity to SEQ ID NO: 2 or a nucleotide sequence corresponding to SEQ ID NO: 2 a nucleotide sequence having at least 90% identity.

2. The promoter library of claim 1, wherein the promoter library further comprises one or more promoters selected from the group consisting of:

a promoter C comprising SEQ ID NO: 3 or a nucleotide sequence corresponding to SEQ ID NO: 3 a nucleotide sequence having at least 90% identity;

a promoter D comprising SEQ ID NO: 4 or a nucleotide sequence corresponding to SEQ ID NO: 4 nucleotide sequences having at least 90% identity;

a promoter E comprising SEQ ID NO: 5 or a nucleotide sequence corresponding to SEQ ID NO: 5 nucleotide sequences having at least 90% identity;

a promoter F comprising SEQ ID NO: 6 or a nucleotide sequence substantially identical to SEQ ID NO: 6 nucleotide sequences having at least 90% identity;

a promoter G comprising SEQ ID NO: 7 or a nucleotide sequence substantially identical to SEQ ID NO: 7 nucleotide sequences having at least 90% identity;

a promoter H comprising SEQ ID NO: 8 or a nucleotide sequence corresponding to SEQ ID NO: 8 nucleotide sequences having at least 90% identity;

a promoter I comprising SEQ ID NO: 9 or a nucleotide sequence corresponding to SEQ ID NO: 9 a nucleotide sequence having at least 90% identity; and

a promoter J comprising SEQ ID NO: 10 or a nucleotide sequence corresponding to SEQ ID NO: 10 nucleotide sequences having at least 90% identity.

3. A vector comprising a promoter from the promoter library of claim 1 or 2.

4. A genetically engineered host cell, said cell:

comprising the vector of claim 4; or

A promoter integrated in its genome in the promoter library according to claim 1 or 2,

preferably, the cells are rhodobacter sphaeroides cells.

5. The host cell of claim 4, wherein the cell is selected from the group consisting of: wild-type rhodobacter sphaeroides cells and modified rhodobacter sphaeroides cells.

6. A method of building a promoter library according to claim 1 or 2, the method comprising:

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

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

(3) observing the expression of a reporter gene, introducing the recombinant expression vector into the host cell and expressing the reporter gene, and selecting a promoter capable of expressing the reporter gene to form a promoter library.

7. The method of claim 6, wherein the reporter gene is selected from the group consisting of: green fluorescent protein gene, luciferase gene, galactosidase gene LacZ and Gus gene.

8. The method of claim 6 or 7, wherein the host cell is selected from rhodobacter sphaeroides cells.

9. A method of screening for a promoter, the method comprising comparing the ability of promoters in the promoter library of claim 1 or 2 to trigger expression of a reporter gene, and determining the strength of the promoter based on the comparison, thereby screening for the promoter.

10. Use of a promoter library according to claim 1 or 2 for protein expression.

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