CN114686990A

CN114686990A - Promoter library and preparation method and application thereof

Info

Publication number: CN114686990A
Application number: CN202011642684.7A
Authority: CN
Inventors: 张立新; 谭高翼; 石桐; 张璐; 王科峰; 梁敏东; 何昕蔚; 周立铭
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-07-01

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

Promoter library and preparation method and application thereof

Technical Field

The invention belongs to the field of biotechnology; in particular to a promoter library, a vector and/or a host cell containing the promoter, a construction method of the promoter library, a screening method of promoter activity and application thereof.

Background

Rhodobacter sphaeroides (Rhodobacter sphaeroides) is a gram-negative photosynthetic bacterium, and belongs to alpha proteobacteria among purple non-sulfur bacteria. The rhodobacter sphaeroides has a complex metabolic mode, can obtain energy through photosynthesis to maintain survival, and can also obtain energy through aerobic respiration by using organic matters under the condition of no illumination to maintain life. Since this bacterium contains a large amount of photosynthetic pigments, it has been used for decades as a model organism for studying bacterial photosynthesis. In addition, rhodobacter sphaeroides have various uses, such as the synthesis of coenzyme Q for the treatment of cardiovascular diseases₁₀The strain is an ideal host strain and has higher biological safety; can also be used for biohydrogen production and poly-beta-hydroxybutyrate (PHB) production; are also now used in membrane protein expression systems. At present, rhodobacter sphaeroides is widely applied to the fields of food, medicine, agriculture and the like due to the characteristics, and has extremely high potential value of industrial development.

Under the condition of hypoxia or no oxygen,the cell Membrane is easily invaginated in the form of a germinating vesicle to form a large number of folded cell membranes, and is differentiated to generate a cytoplasmic Membrane System (ICM). ICM is equipped with photosynthesis means for capturing photons for anaerobic photosynthesis, by which bacteria increase membrane area to increase photosynthetic efficiency due to coenzyme Q₁₀Widely distributed on biological membranes, this may also be coenzyme Q₁₀Providing multiple storage and functional spaces. The present coenzyme Q₁₀The accumulation mechanism is not clear, but rhodobacter sphaeroides is used as a model strain, whole genome sequencing is completed in 2005, and the mechanism of regulating and controlling the expression of the photosynthetic genes is well researched. The photoreaction center and the light capturing complex mainly contained in the ICM are mainly synthesized by expression of a photosynthetic gene cluster in which the expression of genes is regulated by a plurality of regulatory systems and a promoter controlling the expression of each operon has a unique characteristic, so that the photosynthetic gene cluster is used for elucidating the accumulation of coenzyme Q by rhodobacter sphaeroides₁₀The mechanisms and potential for building libraries of native promoters have attracted attention to the subject group. The photosynthetic operon of rhodobacter sphaeroides mainly comprises pucBAC, pufQKBALMX, crt, bch and the like, and genes such as pucA, pucB, pufA, pufB, pufL, pufM, puhA and the like are contained below the photosynthetic operon. The previous research also shows that PrrA/PrrB, PpaA, AppA/PpsR, FnrL, IHF and other important regulation and control proteins can sense oxygen concentration and illumination intensity and regulate the expression of photosynthesis operon.

The Promoter (Promoter) is located upstream of the 5' end of the DNA coding strand and contains a conserved sequence for RNA polymerase binding, acting as a "control switch" for the timing and strength of gene expression. The kit mainly comprises an upstream element, a core promoter region and a response binding element. In prokaryotes, the core region of the promoter consists essentially of two parts, the-10 region "TATAAT" and the-35 region "TTGACA", these two conserved regions being able to recognize each other with the sigma factor of RNA polymerase, it being noted that this binding is atopic, thus making the promoter species-specific. The activity of gene transcription can be influenced by the change of the number of nucleotides in the conserved regions of the-35 region and the-10 region, and it is generally considered that the strong promoter is 16-18bp, and when the distance is less than 15bp or more than 20bp, the activity of the promoter is reduced.

At present, the natural promoters commonly used in rhodobacter sphaeroides are derived from the genes puc, pucP and aprrnB. The genes of the puf and the pucP are both from rhodobacter sphaeroides photosynthesis operons and are adaptive to functions, and promoters of the two genes are influenced by light and oxygen and can turn on or off gene transcription under different culture conditions. P_rrnBIs the promoter of rhodobacter sphaeroides ribosomal RNA and is a constitutive promoter with medium strength. In addition, sigma in rhodobacter sphaeroides⁹³And σ³⁷RNA polymerase can compatibly recognize sigma derived from Escherichia coli⁷⁰And σ³²Dependent promoters, in rhodobacter sphaeroides, sigma derived from E.coli⁷⁰Dependent promoters such as the E.coli promoter P_lacUV5、P_tac/trc、P_Tn903kan、P_A1/04/03And P_rrnB _P1All can be recognized by RNA polymerase of rhodobacter sphaeroides. Shan Q. et al P_J95025、P_J95026、J95027、P_tac、P_rrnBFive different intensities (wherein P_rrnBPromoter not the most potent of them) mediates the expression of the CrtY gene in rhodobacter sphaeroides, where P is the gene_rrnBThe yield of the beta-carotene is improved by 109 percent under the mediation of a promoter. This indicates that the suitable gene expression strength is more favorable for regulating and controlling the metabolic network to realize the improvement of the product yield, and the promoter library of rhodobacter sphaeroides is yet to be developed at the present stage.

Disclosure of Invention

In order to solve the above problems in rhodobacter sphaeroides, the inventors of the present invention analyzed the transcriptome and genome of rhodobacter sphaeroides, and obtained a set of promoters capable of providing a range of transcription intensities in rhodobacter sphaeroides.

Therefore, the present invention aims to provide a promoter library for rhodobacter sphaeroides and use thereof. The invention also aims to provide a construction method of the promoter library.

In one aspect of the invention, a promoter library for rhodobacter sphaeroides is provided, the promoter library comprising at least two promoters, wherein promoter a comprises the sequence of 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.

In a preferred embodiment, the promoter library further comprises one or more 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 corresponding to SEQ ID NO: 6 nucleotide sequences having at least 90% identity;

a promoter G comprising SEQ ID NO: 7 or a nucleotide sequence corresponding 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.

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

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

In some embodiments, the cell is a rhodobacter sphaeroides cell. For example, in the present invention, the rhodobacter sphaeroides cell may be a wild-type rhodobacter sphaeroides cell, or a modified rhodobacter sphaeroides cell (e.g., chemically mutagenized, genetically modified, or the like).

In another aspect of the present invention, there is provided a method for constructing the promoter library described herein, 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 to obtain 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.

In the present invention, the host cell is a rhodobacter sphaeroides cell. For example, the reporter gene may be selected from the group consisting of: fluorescent protein genes (e.g., green fluorescent protein gene, yellow fluorescent protein gene), luciferase (luciferase, luc) gene, and β -galactosidase gene (lacZ) and gusA gene.

In one aspect of the invention, there is provided a method of screening for a promoter, the method comprising comparing promoters in a promoter library as described herein to a reference promoter for their ability to trigger expression of a reporter gene, thereby screening for the strength of the promoter.

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. Meanwhile, the method provides a favorable tool for regulating and controlling a metabolic network in the spheroidic red cells and realizing the improvement of the product yield.

In another aspect of the invention there is provided the use of a promoter library as described herein for protein expression.

Drawings

FIG. 1: analysis of transcriptome data results of different rhodobacter sphaeroides at 24h and 48 h.

FIG. 2: schematic diagram of GUS expression plasmid assembly for promoter activity analysis.

FIG. 3: standard curve for p-nitrophenol produced was determined in GUS assay at 415nm in visible light.

FIG. 4: the promoter activity in the GUS assay was characterized by the promoter activity of the strain at three time points, 11h, 24h, and 48 h.

FIG. 5: characterization of promoter Activity in GUS assay Strain growth (OD) at three time points, 11h, 24h and 48h₇₀₀)。

Detailed Description

Definition of

The strength of the promoter is the amount of transcription of the downstream gene that it mediates. In prokaryotes, the regulatory mechanisms from transcription to translation are small, and the amount of translated protein can also be indicative of the strength of the promoter.

In the case of a nucleic acid or polypeptide, the term "isolated" or "partially purified" as used herein refers to a nucleic acid or polypeptide that is separated from at least one other component (e.g., a nucleic acid or polypeptide) with which it is found in its natural source; and/or the other component will be present with the nucleic acid or polypeptide when expressed by the cell, or secreted in the case of a secreted polypeptide. Nucleic acids or polypeptides that are chemically synthesized or synthesized using in vitro transcription/translation are considered "isolated". The term "purified" or "substantially purified" refers to an isolated nucleic acid or polypeptide that is at least 95 wt% of the target nucleic acid or polypeptide, including, for example, at least 96%, at least 97%, at least 98%, at least 99%, or more.

As used herein, the term "operably linked" refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter is placed in a specific position relative to a 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 invention is not particularly limited with respect to suitable genes of interest, including, for example, but not limited to: structural gene, gene for coding protein with specific function, enzyme, and reporter gene (such as green fluorescent protein gene, luciferase gene, galactosidase gene LazZ, GUS gene, etc.).

As used herein, "exogenous" or "heterologous" refers to a 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 is not normally found in nature. A particular sequence is "foreign" to the cell or organism into which it is inserted.

For the purposes of the present invention, "identity" is calculated by comparing two aligned sequences over a comparison window. Alignment of the sequences enables the number of positions (nucleotides or amino acids) common to both sequences to be determined over a window of comparison. The number of consensus positions was then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage homology. The determination of percent sequence identity may be done manually or using well known computer programs.

Start sublibrary

The present invention provides a promoter library for rhodobacter sphaeroides. The promoter library comprises at least two promoters, wherein the promoter A comprises the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence corresponding to SEQ ID NO: 1, and 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.

In some embodiments, promoter a comprises SEQ ID NO: 1, or a nucleotide sequence corresponding to SEQ ID NO: 1, e.g., a nucleotide sequence having at least 90% identity to SEQ ID NO: 1, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity. In a preferred embodiment, promoter a consists of SEQ ID NO: 1, or a nucleotide sequence shown in the specification.

In some embodiments, promoter B comprises SEQ ID NO: 2, or a nucleotide sequence corresponding to SEQ ID NO: 2, e.g., a nucleotide sequence having at least 90% identity to SEQ ID NO: 2, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity. In a preferred embodiment, promoter B consists of SEQ ID NO: 2, and (b) the nucleotide sequence shown in the figure.

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

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

a promoter C comprising SEQ ID NO: 3 or a nucleotide sequence corresponding to SEQ ID NO: 3, e.g., a nucleotide sequence having at least 90% identity to SEQ ID NO: 3 a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity;

a promoter D comprising SEQ ID NO: 4 or a nucleotide sequence corresponding to SEQ ID NO: 4, e.g., a nucleotide sequence having at least 90% identity to SEQ ID NO: 4, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity;

a promoter E comprising SEQ ID NO: 5 or a nucleotide sequence corresponding to SEQ ID NO: 5, e.g., a nucleotide sequence having at least 90% identity to SEQ ID NO: 5 nucleotide sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity;

a promoter F comprising SEQ ID NO: 6 or a nucleotide sequence corresponding to SEQ ID NO: 6, e.g., a nucleotide sequence having at least 90% identity to SEQ ID NO: 6, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity;

a promoter G comprising SEQ ID NO: 7 or a nucleotide sequence corresponding to SEQ ID NO: 7, e.g., a nucleotide sequence having at least 90% identity to SEQ ID NO: 7, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity;

a promoter H comprising SEQ ID NO: 8 or a nucleotide sequence corresponding to SEQ ID NO: 8, e.g., a nucleotide sequence having at least 90% identity to SEQ ID NO: 8, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity;

a promoter I comprising SEQ ID NO: 9 or a nucleotide sequence corresponding to SEQ ID NO: 9, e.g., a nucleotide sequence having at least 90% identity to SEQ ID NO: 9, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity; and

a promoter J comprising SEQ ID NO: 10 or a nucleotide sequence corresponding to SEQ ID NO: 10, e.g., a nucleotide sequence having at least 90% identity to SEQ ID NO: 10, a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.

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

promoter C consisting of SEQ ID NO: 3;

promoter D consisting of SEQ ID NO: 4;

promoter E consisting of SEQ ID NO: 5;

a promoter F consisting of SEQ ID NO: 6;

a promoter G consisting of SEQ ID NO: 7;

a promoter H consisting of SEQ ID NO: 8;

promoter I consisting of SEQ ID NO: 9; and

a promoter J consisting of SEQ ID NO: 10, or a nucleotide sequence shown in the specification.

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.

In the promoter library of the present invention, the promoter has gradient distribution of promoter strength for directing the expression of target gene under different strength. For example, expression of foreign proteins in a host, or for modulating intracellular metabolic pathways, etc. For example, the promoter library of the present invention can be used to finely regulate metabolic pathways, so that genes are expressed in a proper amount, and the combinatorial optimization of metabolic pathways is realized.

The promoter of the present invention may be operably linked to a gene of interest, which may be foreign (heterologous) with respect to the promoter. The gene of interest can generally be any nucleic acid sequence (e.g., a structural nucleic acid sequence) that preferably encodes a protein having a particular function, e.g., certain proteins having an important property or function.

For example, when used in expression intensity studies, the genes of interest include, but are not limited to: green fluorescent protein, yellow fluorescent protein, luciferase gene, galactosidase gene LacZ, Gus gene, etc.

As a preferred mode of the present invention, promoters of different strengths can be selected from the promoter library of the present invention, and they are operably linked to a target gene to be investigated, respectively, or the target gene and the promoter are operably linked into an appropriate vector and introduced into host cells in an appropriate manner, thereby obtaining a series of cells in which the expression level of the target gene is different. The function or use of the target gene can be known by analyzing the metabolic state, phenotypic change, protein expression or interaction state, change of various signal molecules, and the like of these cells.

In a preferred embodiment of the present invention, the target gene may be a gene that is deleted or underexpressed in a cell, and the target gene may be operably linked to the promoter of the present invention, or operably linked to the promoter of the present invention in a suitable vector, and introduced into the cell in a suitable manner, thereby expressing the target gene at a high level.

The promoters of the present invention may also be operably linked to improved sequences of genes of interest that are foreign (heterologous) to the promoter. The gene of interest can be modified to produce various desired characteristics. For example, the gene of interest may be modified to increase the content of essential amino acids, to enhance the translation of amino acid sequences, to alter post-translational modifications (e.g., phosphorylation sites), to transport the translated product outside the cell, to improve the stability of the protein, to insert or delete cellular signals, and the like.

In addition, the promoter and gene of interest can be designed to down-regulate a particular gene. This is generally achieved by linking the promoter to the gene sequence of interest, which is directed in the antisense orientation. Those of ordinary skill in the art are familiar with such antisense technology. Any nucleic acid sequence may be modulated in this manner.

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

Vectors and host cells

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

Any promoter and/or gene sequence of interest from the promoter library of the present invention may be contained in a recombinant vector.

As an embodiment, said recombinant vector comprises a promoter of the invention, optionally comprising a multiple cloning site or at least one cleavage site downstream of said promoter. When the target gene is required to be expressed, the target gene is ligated into a suitable multiple cloning site or enzyme cleavage site, thereby operably linking the target gene with the promoter. As an embodiment, the recombinant vector includes the promoter of the present invention, and when it is desired to express the target gene, the target gene is operably linked to the promoter by ligating the target gene downstream of the promoter by homologous recombination.

In a specific embodiment, the gene of interest is located downstream of a promoter and is separated from the promoter by less than 2000bp, such as less than 1000bp, 500bp, 200bp, 100bp, 50 bp. In a preferred embodiment, the promoter is not spaced from the gene of interest.

As an embodiment, the recombinant vector comprises (in the 5 'to 3' direction): a promoter for directing the transcription of a target gene and the target gene. If desired, the recombinant vector may also include a 3 'transcription terminator, a 3' polyadenylation signal, other untranslated nucleic acid sequences, transport and targeting nucleic acid sequences, resistance selection markers, enhancers or operators, and the like.

In another embodiment, the gene of interest includes, but is not limited to: structural genes, genes encoding proteins having specific functions, reporter genes (e.g., 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 general, any plasmid and vector may be used as long as they are capable of replication and stability 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 gene sequence of interest described herein. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably comprises 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 Fluorescent Protein (GFP), among others.

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

Vectors comprising the appropriate promoters and genes of interest described above may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, e.g. yeast cells(ii) a Or higher eukaryotic cells, such as plant cells. Representative examples are: yeast, Escherichia coli, tissue cells of animals, plant cells, and the like. It will be clear to one of ordinary skill in the art how to select an appropriate vector and host cell. As a preferred mode of the present invention, the host cell is rhodobacter sphaeroides. For example, in the present invention, the rhodobacter sphaeroides cell may be a wild-type rhodobacter sphaeroides cell, or a modified rhodobacter sphaeroides cell (e.g., chemically mutagenized, genetically modified, or the like). For example, rhodobacter sphaeroides cells can be chemically mutagenized or genetically modified to produce high levels of coenzyme Q₁₀Rhodobacter sphaeroides of (4).

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

For example, the recombinant vector transformation of rhodobacter sphaeroides can be performed by means of electrotransformation, conjugative transfer, and the like.

The invention also provides genetically engineered host cells that: a vector containing a promoter having promoter elements in a promoter library described herein; or

The genome of which incorporates the promoter elements of the promoter libraries described herein.

In a preferred embodiment, the cell is an E.coli or rhodobacter sphaeroides cell.

Method for constructing starter library

The method for constructing the promoter library comprises the following steps:

Transcriptome and genomic information may be obtained using information disclosed in various databases in the art (e.g., KEGG, NCBI), or by sequencing.

Candidate promoter sequences are typically selected from 100-500bp upstream of the initiation codon, including the core promoter portion. In a preferred embodiment, the fragment may be further modified, for example by deleting a portion in which promoter activity is inhibited, for example, the ppsR binding site. For The determination OF The ppsR Binding site, see Patrice Bruscella et al, The Use OF chromatography in immunopropraction to Define PpsR Binding Activity in Rhodobacter sphaeroides 2.4.1, JOURNAL OF BACTERIOLOGY, Oct.2008, p.6817-6828, herein incorporated by reference in their entirety.

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 (GFP) is a typical endogenous fluorescent protein, GFP is safe to a host, the fluorescence intensity can directly reflect the expression amount, no substrate is required to be added, the sensitivity is high, and nondestructive detection of the host is facilitated.

Yellow Fluorescent Protein (YFP) can be regarded as a mutant of green fluorescent protein, whose fluorescence is shifted in the red spectrum relative to green fluorescent protein, and which, in common with GFP fluorescent protein, has excitation and emission light of longer wavelength, most importantly with low background when imaged intracellularly.

Luciferase (luciferase, luc) gene: the luc is an enzyme for catalyzing fluorescein or lipase to generate fluorescence in an aerobic environment of an organism, catalyzes ATP (adenosine triphosphate) substrates, inorganic fluorescein and oxygen to generate a luminous reaction, has strong specificity with the substrates, has little influence on a host, and has the characteristics of high sensitivity, easy detection, no excitation light interference and the like.

gusA gene: is derived from Escherichia coli, encodes beta-D-glucuronidase, has monomer molecular weight of 68200Da, is resistant to various detergents, and has extremely high activity in the presence of thiol reducing agent beta-mercaptoethanol or DTT. The enzyme can catalyze the hydrolysis of a plurality of beta-glucoside ester substances without coenzyme factors or cations, and the hydrolysis of some substances can generate color change. A small number of divalent metal ions such as Zn²⁺、Cu²⁺Can inhibit its activity. The optimum pH value is 5.2-8.0, the heat-resistant half-life period is 50 ℃ for 2h, and the activity is stored in a linear relation for a long time. GUS substrates are soluble, the most commonly used catalytic substrate is 5-bromo-4-chloro-3-indole glucuronide (X-gluc), the product is a blue compound, 4-methylumbelliferone glucuronide (MUG) and 9-hydroxy-3-isophenoxazolone glucuronide (ReG) are commonly used in the early years, ReG has high fluorescence activity, and the product is excited at 560nm and emits at 590 nm. In biological studies, the gene is often placed downstream of the promoter to be investigated, and the activity of the promoter can be analyzed by detecting the enzyme. This is also the most widespread use of reporter genes, with the aim of "reporting" the promoter activity. In one embodiment, the GUSA sequence is derived from: enterobacteriaceae, LOCUS: WP _ 000945901.

The GUS assay is an enzymatic reaction detection assay based on a colorimetric assay. For example, the color development of a product produced by catalyzing a substrate with Gus enzymes contained in a sample is collected by a microplate reader, the amount of the catalyzed substrate is calculated from the concentration of the product, and the intensity of the promoter used is reflected thereby. For example, when p-nitrophenyl- β -D-glucuronic acid is used as a substrate to produce p-nitrophenol as a product, a color reaction is detected at 415nm for 20-30min (during which time the color reaction tends to stabilize), then the intensity of the promoter to be tested is reflected by the slope of the substrate versus reaction time curve corresponding to consumed substrate in the sample, as deduced from a standard curve of the product versus absorbance at 415nm (e.g., FIG. 3). With respect to GUS testing, reference may be made to Siegl, Theresa et al, "Design, description and characterization of a synthetic promoter library for fine-tuned gene expression in microorganisms," metabolism Engineering19. compact (2013):98-106, which is hereby incorporated by reference in its entirety.

In some embodiments, the candidate promoter sequence may also be modified. Such modifications include substitutions, deletions, and/or insertions. In a preferred embodiment, the suppression site in the candidate promoter sequence is subject to deletion modification. For example, the ppsR binding site in the candidate promoter sequence is deleted. For The determination OF The ppsR Binding site, see Patrice Bruscella et al, The Use OF chromatographic immunological prediction to Define PpsR Binding Activity in Rhodobacter sphaeroides 2.4.1, JOURNAL OF BACTERIOLOGY, Oct.2008, p.6817-6828, herein incorporated by reference in its entirety.

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

Screening method

A method of screening for promoters, the method comprising comparing and ranking promoters in a library of promoter sequences according to the present invention with respect to their ability to trigger expression of a gene of interest, thereby screening for the strength of the promoters.

In a preferred embodiment, the gene of interest may be selected from a reporter gene. For the definition of the reporter gene, see the description herein above.

In the present invention, it is preferred to use a reference promoter in comparison with a candidate promoter. With regard to the selection of the reference promoter, it is possible to more intuitively and directly feed back the ability of the candidate promoter to trigger the expression of the reporter gene. Thus, in general, a reference promoter is chosen for a promoter that has good characteristics in the host cell. In a preferred embodiment, two or more reference promoters are selected that differ in promoter strength.

In a preferred embodiment, the screening method comprises comparing the promoter described herein with a reference promoter at an intensity level that promotes expression of a gene of interest (including a level of promotion above or below the reference level), the method comprising: under the same culture conditions, the host cell in which the expression of the target gene is promoted by the promoter of the present invention and the host cell in which the expression of the target gene is promoted by the reference promoter are separately cultured, and the expression intensity (or expression amount) of the target gene in the two cells is compared.

In the above screening method, the preferred host cell is a rhodobacter sphaeroides cell. The conditions for culturing the rhodobacter sphaeroides cells are well known to those skilled in the art, and there is no particular limitation in the present invention. Preferably, the conditions for culturing the rhodobacter sphaeroides cells are 31 + -1 deg.C, 210 + -100 rpm.

Methods for determining the expression of a desired gene (particularly a reporter gene) in a culture system are well known to those skilled in the art, and may be adjusted depending on the desired gene. In a preferred embodiment of the present invention, the reporter gene is a Gus protein, and the method for determining the expression of the reporter gene comprises: the activity of the enzyme in the culture system was measured. 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: the fluorescence intensity in the culture system was measured.

In addition, the invention also provides the application of the promoter library in protein expression.

Examples

The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples. It should be understood by those skilled in the art that equivalent substitutions and corresponding modifications of the technical features of the present application are also within the protective scope of the present application. The reagents used in the following examples are commercially available products, and the solutions can be prepared by techniques conventional in the art, except where otherwise specified.

EXAMPLE 1 acquisition of candidate promoters

The transcription of mRNA from a promoter begins after binding of RNA polymerase, and typically the transcription initiation site is located downstream of the promoter and also coincides therewith. The mRNA expression level is semi-quantitatively measured through transcriptome information, the position of a corresponding gene in a strain genome can be found through the sequence of the expressed mRNA, and a corresponding coding sequence on the genome and a sequence containing a promoter at the upstream of the coding sequence are determined. In the experiment, transcriptomics analysis is carried out on the experimental strain, related photosynthetic gene clusters are positioned on the rhodobacter sphaeroides single-chain circular genome, and the adjacent sequences of related genes in the genome positioned by mRNA are regarded as the existence regions of candidate promoters, so that the candidate promoter sequence information is obtained and used for characterization experiments.

Specifically, rhodobacter sphaeroides HY01 (hereinafter referred to as R.spHY01, obtained by screening rhodobacter sphaeroides 2.4.1 by nitrosoguanidine mutagenesis and having high coenzyme Q yield is obtained₁₀Referred to as ind in FIG. 1) wild-type rhodobacter sphaeroides 2.4.1 (also referred to as wt in FIG. 1) was cultured with the fermentation medium at 32 ℃ and 220rpm for 24h and 48h (ind 24, ind48, wt24, wt48 in FIG. 1, respectively). The transcriptome information obtained was subjected to transcript intensity analysis (see FIG. 1 for analytical results, where the abscissa is transcript intensity in different rhodobacter sphaeroides strains (including rhodobacter sphaeroides ind24, ind48, wt24, and wt48), the ordinate is the corresponding operon/gene name, and the right side is the color scale). Wherein the culture medium is 5g/L of ammonium sulfate, 5g/L of monosodium glutamate, 7g/L of corn starch, 20g/L of glucose, 0.3g/L of monopotassium phosphate, 7g/L of magnesium sulfate, 3g/L of sodium chloride, 1g/L of ferrous sulfate, 0.4g/L of manganese sulfate, 0.008g/L of cobalt chloride, 4g/L of calcium carbonate, adjusted to pH 6.5, and sterilized at 121 ℃ for 25 min.

The gene sequences with transcription intensities ranked top thirteen were selected and genomic information for these genes was obtained based on the information in the KEGG database (here genomic information is genomic information from wild type rhodobacter sphaeroides 2.4.1).

Specifically, the top thirteen genes ranked in transcription intensity were from the following operons/genes, respectively (top to bottom in fig. 1): rnpB (KEGG No. RSP _4342, which is not shown in FIG. 1 together with the transcription information of other genes because the transcription intensity is too high), RSP _1185, RSP _6124, RSP _7571, RSP _2718, puhA (KEGG No. RSP _0291), puc2B (KEGG No. RSP _1556), crtA (KEGG No. RSP _0272), rpsM (KEGG No. RSP _1737), pucBAC (KEGG No. RSP _0314, shown as pucB in FIG. 1), bchEJ (KEGG No. RSP _0280, shown as bchJ in FIG. 1), pufQ (KEGG No. RSP _0259), crtEF (KEGG No. RSP _0264, shown as crtF in FIG. 1).

Wherein, the pufQ, puc2B, puhA, crtEF, pucBAC, crtA and bchEJ are all from the photosynthetic gene cluster. rpsM is 30S ribosomal protein S13 gene; rnpB is the RNase P gene. RSP _7571, RSP _6124, RSP _2718 and RSP _1185 show the functions of the expressed protein, and the promoter has no clear sequence information.

Intercepting the segment containing the core promoter 500bp upstream of the initiation codon of the genes RSP _7571, RSP _6124, RSP _2718, RSP _1185, puhA and rnpB to obtain a candidate promoter 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(information of the promoter is not shown), P_puhA(SEQ ID NO：9，500bp)、P_rnpb(SEQ ID NO：4，500bp)。

The fragment containing The core promoter upstream OF The initiation codon OF The genes pfQ, crtEF, pucBAC, crtA, bchEJ was excised and The ppsR Binding site therein was deleted (see Patrices Bruscella et al, The Use OF chromatography immunization to Define PpsR Binding Activity in Rhodobacter sphaeroides 2.4.1, JOURNAL OF BACTERIOLOGY, Oct.2008, p.6817-6828), thereby obtaining 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)。

Example 2 construction of strains for promoter Activity characterization

The promoters Pkana (SEQ ID NO: 13, 121bp) and Ptac (SEQ ID NO: 12, 79bp) were synthesized. Both Pkana and Ptac are commonly used promoters in E.coli plasmids.

In addition, based on SEQ ID NO: 11 to synthesize GUS gene.

According to the schematic diagram of FIG. 2, each of the above candidate promoters and GUS genes was assembled into a pBBR1MCSK vector (purchased from Novagen) by Ezmax (purchased from Takara Bio Inc.), and a plasmid having each of the candidate promoters and GUS genes was constructed. After sequencing verification, transformation into E.coli DH10b competent cells and selection of positive transformants by colony PCR. Transformation conditions are as follows: incubate on ice for 30min, heat at 42 ℃ for 90s, incubate on ice for 2min, add 700uL LB, incubate at 37 ℃ for 45min with a shaker.

Plasmids were extracted from the positive transformants, and a single clone of wild type rhodobacter sphaeroides 2.4.1 (strain No. ATCC17023) was electrically transformed to obtain a recombinant strain. The prepared strains are shown in table 1 below.

The electrotransformation conditions were as follows:

1. adding 2mL of thallus into 2mL sterile EP tube (culturing thallus with TSB culture medium until OD is 1-3), and centrifuging at 9000rpm for 1 min;

2. removing supernatant, adding 1mL of deionized water, blowing, mixing uniformly, centrifuging at 9000rpm for 1min, and pouring out supernatant.

3. Repeating the step 2 to wash for three times;

4. adding 80 μ L of 10% glycerol into each EP tube from which the supernatant is poured, resuspending the thalli, adding the plasmid (500ng), and mixing uniformly;

5. the mixture was taken out, and after all was poured into a 2mm electric rotor and incubated on ice for 20min, electric rotation was performed.

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

7. after the electric transfer is finished, 600 mu L of the antibiotic-free TSB culture medium is added, the liquid is completely taken out after the air suction and the uniform mixing, the liquid is placed in a 2mL EP tube, and the incubation is carried out for 2h at 32 ℃ and 200 rpm;

8.12000 g, centrifuging for 1min, pouring out part of supernatant, mixing the rest bacteria solution, spreading on Carna resistant plate, and culturing in 32 deg.C incubator for 6-7 days.

TSB medium: 30g/L Tryptone Soya Broth. The solid medium was supplemented with 2% agar and sterilized at 115 ℃ for 20 min.

TABLE 1 Strain for promoter Activity characterization constructed in this example

Example 3 Activity analysis of candidate promoters

In this example, GUS assays were used to analyze candidate priming activities. For information, see Siegl, Theresa et al, "Design, construction and characterization of a synthetic promoter library for fine-tuned gene expression in microorganisms", Metabolic Engineering19.Complete (2013): 98-106.

GUS experimental principle:

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

The specific experimental steps are as follows:

1. after 1-2 colonies of each strain prepared in example 2 were picked and inoculated into 5mL of TSB broth and cultured at 220rpm in 50mL centrifuge tube 32 ℃ for 24 hours, 5mL of the culture broth was transferred to 45mL of fermentation medium (the same as that in example 1) and cultured at 220rpm at 32 ℃ for a plateau (48 hours) (the fermentation medium contained 50ng/mL kanamycin in this step).

2. Taking OD₇₀₀Same bacterial liquid (OD)₇₀₀Value in the range of 10-25), the supernatant was removed by centrifugation at 12000rpm × 1 min.

3. The cells were resuspended in 900. mu.L of Gus buffer 2, and then incubated in a water bath at 37 ℃ for 20min to obtain lysates of the respective strains.

4. The lysate was then diluted with 900. mu.L of Gus buffer 1 and centrifuged at 14000rpm for 10min at 4 ℃.

5. mu.L of the supernatant was added to 500. mu.L of Gus buffer 3.

6. The enzyme-linked immunosorbent assay measures the absorbance at 415nm, 200 mu L of mixed liquid is added into a 96-well plate, the measurement time is 30min, and 100 mu L of Gus buffer 3 is mixed with 100 mu L of Gus buffer 2 and Gus buffer 1 in the same proportion to serve as a control group.

7. Collecting data of the color reaction process by an enzyme-labeling instrument, and detecting for 30min on a 96-well plate; simultaneously, a standard curve of the product with respect to the absorbance at 415nm was prepared (see FIG. 3); converting light absorption values corresponding to all time points on a reaction curve into product concentrations according to a standard curve so as to calculate the amount of the catalyzed substrate, then drawing a curve graph of each strain until the product color is stable within time by taking the reaction time as an abscissa and the catalyzed substrate concentrations as an ordinate, fitting the reaction progress through office software, calculating the slope of the curve, expressing the activity of the enzyme by the slope, and determining the formula according to the enzymatic reaction principle to calculate the specific activity of the enzyme. Each group of strains was replicated three times.

Specifically, the enzyme activity can be calculated by the method disclosed in Siegl, Theresa et al, "Design, description and characterization of a synthetic promoter library for fine-tuned gene expression in microorganisms", Metabolic engineering19.complete (2013):98-106, wherein the standard curve used is the standard curve in FIG. 3.

Solution:

gus buffer 1: 30mL of 50mM phosphate buffer (pH 7.0), 150. mu.L of 5mM DTT, and 30. mu.L of 0.1% Triton X-100 were used.

Gus buffer 2: 30mL of 50mM phosphate buffer (pH 7.0), 150. mu.L of 5mM DTT, 30. mu.L of 0.1% Triton X-100, and 30mg of lysozyme (final concentration: about 1mg/mL) were used.

Gus buffer 3: 30mL of 50mM phosphate buffer (pH 7.0), 150. mu.L of 5mM DTT, 30. mu.L of 0.1% Triton X-100, and a final concentration of p-nitrophenyl-beta-D-glucuronic acid of 2 mM.

Wherein: phosphate buffer: 1.14g K₂HPO₄·3H₂The O is prepared to 100mL by deionized water, and 0.6g NaH is used₂PO₄Deionized water is added to 100mL, the deionized water is used as main liquid, the deionized water is used as auxiliary liquid to adjust the pH, and when the pH is 7.0, K is added₂HPO₄·3H₂O：NaH₂PO₄＝5：4。

2mM p-nitrophenyl-beta-D-glucuronic acid: 0.63g p-nitrophenyl-beta-D-glucuronic acid was added to 1L dH₂O, when 30mL of the solution was prepared, 18.9mg was added.

And (4) analyzing results:

FIG. 5 shows the growth conditions of each strain at 11h, 24h, and 48h in culture, as OD, during the period from culture to plateau₇₀₀Shown.

The method comprises the steps of firstly calculating the slope of an absorption curve of each promoter, converting the slope into substrate concentration according to a standard curve, wherein the concentration is positively correlated with the enzyme activity of a reporter gene expression product, and the enzyme activity is positively correlated with the strength of the promoter, so that the screened promoter activities are sequenced, the ordinate is the enzyme activity (activity) in fig. 4, the abscissa is each promoter, and the strength of the promoter is increased sequentially from left to right. The first of these was a blank control and the second was a gusA reporter control without promoter. Among the promoters, P is weaker_kana、P₂₇₁₈、P_puhA、P_pucBAC、P_PufQOf moderate intensity is P_CrtA、P_crtEF、P_tac、P_rnpb、P_bchEJOf high strength is P₇₅₇₁And P₆₁₂₄。

The experiment successfully obtains a series of promoters with 5 magnitude order strength differences, proves that the method can effectively quantify and characterize the promoter strength, and can be continuously used for expanding the rhodobacter sphaeroides promoter element library. Meanwhile, experimental results show that the extremely strong promoter influences the growth rate and the growth amount of the thalli, and the metabolic burden born by the strain is displayed to a certain extent. According to P_rnpBAs a result, it is presumed that an up-regulation mechanism may exist. Common promoter P_tac、P_kanaThe introduction of contrast also makes these results easier to apply.

The accumulation of expressed protein is not determined by the transient translational strength of the gene for a single independent variable, and different protein products will also have different degradation rates. Therefore, the promoter strength characterization in the experiment should be regarded as the horizontal reference data at the same time point. In the future, if the expression degradation time sequence of the reporter protein can be controlled, the nutrition level can be regulated and controlled by a constant fermentation tank, the degradation and accumulation time can be distinguished, and the clear explanation of the strength of the promoter and the nutrition limiting factors can be facilitated.

Furthermore, promoter engineering is rapidly advancing and many researchers have established artificial mutation-engineered promoter libraries for the cells under study, and the GUS method used is also from studies that characterize artificial promoter libraries. The artificial promoter has higher possibility of being not regulated by a cell metabolism network, and is beneficial to limiting the range of uncontrollable variables by various single gene modification works. In addition, it is possible for the artificial promoter to fill in the intensity region where the native promoter is not present, and in this experiment a promoter from the top ten of the transcriptome data intensities was selected, as in P₆₁₂₄、P₇₅₇₁Thus the region near the strong promoter still has a large gradient filling the space. If application needs to be carried out in the future, the two sequences can be analyzed, and the site influencing the promoter activity can be found out for mutation, so that the library is further enriched.

Sequence listing

<110> university of east China's college of science

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cttctgctcg cggcggttcg cctgcggttt aggccctggg cgcgggggcc gctcacgggc 420

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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

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<213> Artificial Sequence (Artificial Sequence)

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<210> 13

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 60

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t 121

Claims

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 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;

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:

(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

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.