CN111019873A

CN111019873A - Method for rapidly integrating large-fragment DNA on genome of clostridium acetobutylicum

Info

Publication number: CN111019873A
Application number: CN201811174829.8A
Authority: CN
Inventors: 黄鹤; 柴常升; 姜卫红; 顾阳
Original assignee: Anhui Tulugang Biotechnology Co Ltd
Current assignee: Nanjing Shiqi Biochemical Technology Co.,Ltd.
Priority date: 2018-10-09
Filing date: 2018-10-09
Publication date: 2020-04-17
Anticipated expiration: 2038-10-09
Also published as: CN111019873B

Abstract

The invention provides a method for quickly integrating large-fragment DNA on a genome of clostridium aerophagum. Specifically, the present invention comprises the steps of: (i) providing a host cell to be integrated, said host cell comprising in its genome exogenous attB sequences at the site to be integrated; (ii) introducing an integration construct comprising a foreign gene into the host cell, thereby performing targeted recombination at a site to be integrated in the genome of the host cell, thereby obtaining an integration-treated host cell; wherein the integration construct is a loop structure and comprises: an expression cassette of a phi CD27integrase, an expression cassette of a phi C31 integrase, an expression cassette of an exogenous gene, an attP sequence of phi CD27 and an attP sequence of phi C31, wherein the attP sequence of phi CD27 and the attP sequence of phi C31 are respectively positioned at two sides of the exogenous gene. The method of the invention can efficiently and stably integrate large-fragment DNA into the genome of bacillus.

Description

Method for rapidly integrating large-fragment DNA on genome of clostridium acetobutylicum

Technical Field

The invention relates to the field of biotechnology, in particular to a method for rapidly integrating large-fragment DNA on a Clostridium aerophagum (gas-benefimingcrossidia) genome.

Background

The carbon monoxide gas represented by carbon monoxide, carbon dioxide and the like is a cheap carbon resource with huge reserves, and has wide sources, including enterprise waste gas, synthesis gas prepared by gasifying carbonaceous substances such as coal, petroleum, natural gas, biomass and the like, and the like. The one-carbon resources can be converted into various bulk chemicals and liquid fuels through microbial fermentation, and the market prospect is huge. Compared with a chemical catalysis route, the biological conversion utilization route of the carbon-carbon gas has the characteristics of mild reaction conditions, low requirements on gas composition, suitability for synthesizing long-carbon-chain complex-structure compounds and the like.

Clostridium aerobica (gas-fermenting clostridium) is an anaerobic microorganism with special catabolic and anabolic pathways. Can utilize carbon monoxide gas (CO, CO)₂) Synthesizing various bulk chemicals and fuels. Thus, clostridium aerophagum has received much attention as a potentially important industrial microorganism. The domestic Bao Steel group and New Zealand Lanze company have been jointly constructed in 2012 and completed pilot-scale tests of 300 ton/year 'Steel plant Tail gas ethanol production' project based on acetogenic bacteria fermentation. The newly reported first 4.5 million tons/year industrial tail gas (CO) worldwide by the first Steel-Lanze company₂the/CO) biological fermentation method for preparing fuel ethanol is successfully debugged.

However, the current molecular manipulation techniques for clostridium aerophagum are very limited, resulting in very slow progress of the research for improving the performance of the strains through metabolic engineering.

Methods of molecular genetic manipulation for clostridium acetobutylicum have been reported to include: (1) genes of specific function are overexpressed using episomal plasmid systems. (2) By means of homologous recombination systems of Clostridium aerovorum itself, target genes on chromosomes are inactivated by means of single or double crossover, or specific DNA sequences are integrated on chromosomes. However, since the efficiency of the homologous recombination system of clostridium autogiraldii is very low, the resistance gene must be introduced as a selection marker in the above operation, and therefore, the resistance gene is inevitably introduced into the chromosome of the obtained target recombinant, thereby affecting the subsequent further genetic operation. (3) The target gene on the chromosome is subjected to insertion inactivation by using a clonon method based on a binary intron. (4) And (3) deleting or replacing genes on the chromosome by using a CRISPR/Cas9 gene editing technology.

However, in general, none of the above methods can achieve specific introduction of a large fragment of a target DNA sequence into the chromosome of Clostridium perfringens (without adding a selection marker such as a resistance gene). The development of the technology is very important for clostridium acetobutylicum, and a heterologous biosynthesis pathway can be rapidly integrated in the clostridium acetobutylicum, so that the variety of target products of the clostridium acetobutylicum fermented by carbon-carbon gas can be effectively expanded.

In view of the above, there is a strong need in the art for the development of a method for efficiently and stably introducing large fragments of target DNA into the genome of clostridium acetobutylicum.

Disclosure of Invention

The invention aims to provide a method for efficiently and stably introducing large fragments of target DNA into a genome of clostridium acetobutylicum.

In a first aspect of the invention, there is provided a method for targeted genomic integration of a host cell comprising the steps of:

(i) providing a host cell to be integrated, said host cell comprising in its genome exogenous attB sequences at the site to be integrated;

(ii) introducing an integration construct comprising a foreign gene into the host cell, thereby performing targeted recombination at a site to be integrated in the genome of the host cell, thereby obtaining an integration-treated host cell;

wherein said integration construct is selected from the group consisting of:

(a) a first integration construct, said first integration construct being a loop structure and comprising: an expression cassette of phi CD27integrase, an expression cassette of exogenous genes and an attP sequence of phi CD 27;

(b) a second integration construct, said second integration construct being a loop structure and comprising: an expression cassette of the integrated enzyme of the phi C31, an expression cassette of an exogenous gene and an attP sequence of the phi C31; and

(c) a third integration construct, said third integration construct being a loop structure and comprising: an expression cassette of a phi CD27integrase, an expression cassette of a phi C31 integrase, an expression cassette of an exogenous gene, an attP sequence of phi CD27 and an attP sequence of phi C31, wherein the attP sequence of phi CD27 and the attP sequence of phi C31 are respectively positioned at two sides of the exogenous gene.

In another preferred example, the method further comprises:

(iii) introducing a selection construct into said integrated host cell, thereby obtaining a host cell which has been crossed to integrate said foreign gene into the genome;

wherein the screening construct is selected from the group consisting of:

(d1) a first screening construct, said first screening construct being a circular structure and comprising: a Cas9 expression cassette, a first sgRNA expression cassette, wherein the first sgRNA targets the coding sequence of phi CD27 integrase;

(d2) a second screening construct, said second screening construct being a circular structure and comprising: cas9 expression cassette, second sgRNA expression cassette, the coding sequence of phi C31 integrase of target of said second sgRNA.

(d3) A third selection construct, said third selection construct being a circular structure and comprising: a Cas9 expression cassette, a first sgRNA expression cassette, and a second sgRNA expression cassette; wherein the first sgRNA targets the coding sequence of Φ CD27 integrase; and the second sgRNA targets the coding sequence of Φ C31 integrase.

In another preferred example, the method further comprises: exogenous attB sequences are introduced into the host cell at the site to be integrated by gene editing.

In another preferred embodiment, the gene editing method comprises: CRISPR-Cas9 edits, TALEN edits, or ZFN edits.

In another preferred embodiment, the host cell comprises Clostridium (Clostridium Prazmowski), preferably Clostridium aerophagum (gas-transducing Clostridium), more preferably Clostridium autodahlii (Clostridium jungdahlii).

In another preferred embodiment, the site to be integrated is selected from the group consisting of: a cac08380 gene region, a cac16510 gene region, or a cac08360 gene region on the genome of clostridium autodahlii.

In the second aspect of the present invention, a genetically engineered bacterium is provided, wherein the genetically engineered bacterium is clostridium, and a nucleic acid sequence from 5 'to 3' of the structure of formula I is inserted into the genome of the genetically engineered bacterium:

Z1-W-Z2 (I)

in the formula (I), the compound is shown in the specification,

z1 is an attL sequence which is an attL27 sequence of phi CD27 formed by integrating an attB sequence and an attP sequence of phi CD 27; or attL31 sequence of phi C31 formed by integrating attB sequence and attP sequence of phi C31;

w is a foreign gene integrated into the genome;

z2 is an attR sequence; the attR sequence is an attB sequence of the phi CD27 and an attP sequence which are integrated to form an attR27 sequence of the phi CD 27; or attB sequences and attP sequences of phi C31 are integrated to form attR31 sequences of phi C31.

In another preferred embodiment, Z1 is an attL31 sequence and Z2 is an attR27 sequence.

In another preferred embodiment, Z1 is an attL27 sequence and Z2 is an attR31 sequence.

In another preferred embodiment, "-" is a linker sequence or a residual sequence formed by recombination of DNA.

In another preferred embodiment, the foreign gene is a butyrate synthesis pathway (BAPP) gene.

In another preferred embodiment, the genetically engineered bacterium comprises Clostridium, preferably Clostridium aerophagum, more preferably Clostridium alterniformis.

In a third aspect of the invention, there is provided a construct for site-directed integration into a genome, said construct comprising an integration construct selected from the group consisting of:

In another preferred embodiment, the construct further comprises: a screening construct selected from the group consisting of:

In another preferred embodiment, the construct comprises a third integration construct and a screening construct.

In another preferred embodiment, the exogenous gene comprises a single gene or a plurality of genes.

In another preferred embodiment, the exogenous gene comprises a gene cluster.

In another preferred embodiment, the exogenous gene is located on a large fragment of DNA.

In another preferred embodiment, the large fragment of DNA is 1-1000kb, preferably 5-500kb in length.

In another preferred embodiment, the genome comprises the genome of clostridium.

In another preferred embodiment, the genome comprises the genome of clostridium aerophagum.

In another preferred embodiment, the Clostridium acetobutylicum comprises Clostridium autodahlii (Clostridium ljungdahlii).

In another preferred embodiment, the length of the foreign gene or nucleic acid sequence to be integrated is greater than or equal to 1kb, preferably greater than or equal to 2kb, more preferably greater than or equal to 2.5kb, greater than or equal to 3kb, greater than or equal to 3.5kb, greater than or equal to 4kb, greater than or equal to 5kb, greater than or equal to 10kb, greater than or equal to 15kb, greater than or equal to 20kb, greater than or equal to 25kb, greater than or equal to 30kb, greater than or equal to 35kb, greater than or equal to 40kb, greater than or equal to 45kb, greater than or equal to 50kb, greater than or equal to 100kb, greater than or equal to 1000 kb.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a schematic representation of the integration system of phage Φ CD27 from Clostridium difficile (Clostridium difficile) into Clostridium immortal. Wherein the content of the first and second substances,

FIG. 1A: the cac08380 gene on the genome of clostridium Yongdalensis is replaced by an attB site of Phage Phage CD27 derived from clostridium difficile by a CRISPR-Cas9 genome editing method. The pHGcas-08380 plasmid carries the flanking homology arms of the cas9, sgRNA, attP and cac08380 genes.

FIG. 1B: the integrase (integrase) gene of Phage CD27 was codon optimized and cloned into plasmid pHG-CD27int along with the attP site. The recombination between attP and attB sites was achieved by introduction into a host bacterium having integrated attB sites on the chromosome as shown in FIG. 1A.

FIG. 1C: the entire plasmid pHG-CD27int plasmid is integrated into the genome of Clostridium Yondall by recombination between attP and attB sites.

FIG. 1D: the sequencing results demonstrate recombination between the attP and attB sites, forming new sites attL and attR on the chromosome.

FIG. 2 shows a schematic representation of the integration of the phage Φ C31 integration system derived from Streptomyces (Streptomyces) into Clostridium immortal.

FIG. 3 shows the PCR amplification results of the attL and attR sites and flanking sequences formed in Clostridium Yondall with integrated system of Φ C31.

FIG. 4 shows a schematic diagram of the synthetic route of integrating large fragment butyric acid specifically on the chromosome of Clostridium Youdaiensis by using two sets of integration systems of PhiCD 27 and PhiC 31 and the PCR amplification identification result thereof.

FIG. 4A: and the scheme of specifically integrating large fragment butyric acid synthesis paths on the chromosome of clostridium Yondall is realized by utilizing two integration systems of PhiCD 27 and PhiC 31. BAPP: butyl acid production pathway.

FIG. 4B: the pHGcas-CD27 plasmid (containing CRISPR-Cas9 system) is introduced into the cells. The plasmid pHGcas-CD27 carries on it a sgRNA recognizing the gene coding for the gene, [ phi ] CD27 integrin, and therefore targets the plasmid pHG-BA02, either free in cells of Clostridium ljungdahlii or single-crossover integrated on the chromosome, and cleaves it. Thus, colonies that can grow on solid plates are either wild-type or mutant strains that integrate the BAPP pathway on the chromosome by double crossover.

FIG. 4C: colonies on the plates were identified by PCR. The DNA bands of 1.2kb and 9.1kb represent the genotypes of the wild type and BAPP integrated mutant, respectively.

Detailed Description

The inventor of the invention develops two sets of phage PhCD 27 and PhiC 31 phage site specificity recombination systems for the first time through extensive and intensive research and a large amount of screening, wherein the phage PhiCD 27 and the PhiC 31 can carry out efficient large-fragment DNA recombination in clostridium aerovorans (particularly clostridium aerovorax). On the basis, the inventor establishes a clostridium aerophagum genome editing method based on the two systems, so that a large-fragment DNA sequence can be efficiently and stably integrated on a chromosome. The present invention has been completed based on this finding.

Term(s) for

As used herein, the terms "nucleic acid sequence to be integrated", "exogenous gene to be integrated" are used interchangeably to refer to any nucleic acid sequence of interest, including (but not limited to): a functional gene, a CDS sequence, a resistance gene coding sequence, an antibiotic biosynthesis gene cluster, a promoter sequence, a terminator sequence, a replicon sequence, or a combination thereof.

Phage integrase

Site-specific recombination (site-specific recombination) mediated by phage integrase is a category of genetic recombination. Unlike conventional homologous recombination methods, this type of recombination does not depend on long stretches of homologous DNA sequences, only requires the combination of integrase catalysis and several nucleotide homologous sequences, and recombination occurs only within these short homologous sequences.

The inventor surprisingly finds that large-fragment DNA can be efficiently introduced into the genome of clostridium aerophagum based on the phi CD27 and phi C31 integrase through a large amount of screening.

The method of the present invention can be applied to clostridium aerovorans and other clostridia (particularly various industrial clostridia) so as to carry out site-specific recombination.

CRISPR/Cas9 mediated gene editing method

CRISPR/Cas9 is an adaptive immune defense formed during long-term evolution of bacteria and archaea, and can be used to fight invading viruses and foreign DNA. The CRISPR/Cas9 system provides immunity by integrating fragments of invading phage and plasmid DNA into the CRISPR and using the corresponding CRISPR RNAs (sgRNAs) to direct degradation of the homologous sequences.

The working principle of this system is that crRNA (CRISPR-derived RNA) is bound to tracrRNA (trans-activating RNA) by base pairing to form a tracrRNA/crRNA complex, which directs the nuclease Cas9 protein to cleave double-stranded DNA at the sequence target site paired with the crRNA. By artificially designing the two RNAs, sgRNA (single-guide RNA) with guiding function can be transformed, and the single-guide RNA can guide the site-specific cleavage of the DNA by Cas 9.

As an RNA-guided dsDNA binding protein, Cas9 effector nuclease is the first known unifying factor (unification factor) that is able to co-localize RNA, DNA and proteins. Fusion of the protein with nuclease-free Cas9(Cas9nuclease-null) and expression of the appropriate sgRNA can target any dsDNA sequence, while the ends of the sgRNA can be ligated to the target DNA without affecting binding of Cas 9. Thus, Cas9 can bring about any fusion protein and RNA at any dsDNA sequence. This technique is called CRISPR/Cas9 gene editing system.

Vectors and host cells

The invention also provides a vector comprising a DNA construct of the invention. Preferably, the carrier is selected from: bacterial plasmids, bacteriophages, yeast plasmids, or animal cell vectors, shuttle vectors; the vector is a transposon vector. Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. Any plasmid and vector may be used as long as it can replicate and is stable in the host.

The term "expression cassette" as used herein refers to a polynucleotide sequence comprising the sequence components of the gene to be expressed and the elements required for expression. For example, in the present invention, the term "selectable marker expression cassette" refers to a polynucleotide sequence comprising a sequence encoding a selectable marker and a sequence module for expressing a desired element. Components required for expression include a promoter and polyadenylation signal sequence. In addition, the selectable marker expression cassette may or may not contain other sequences, including (but not limited to): enhancers, secretory signal peptide sequences, and the like.

In the present invention, the promoter suitable for the foreign gene expression cassette and the selection marker gene expression cassette may be any one of common promoters, which may be a constitutive promoter or an inducible promoter. Preferably, the promoter is a strong promoter that is constitutive.

As used herein, "operably connected to" refers to a condition: i.e. certain parts of a linear DNA sequence can influence the activity of other parts of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a precursor and is involved in secretion of the polypeptide, the signal peptide (secretory leader) DNA is operably linked to the polypeptide DNA; a promoter is operably linked to a coding sequence if it controls the transcription of that sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, "operably linked" means adjacent, and for secretory leaders means adjacent in reading frame.

The various elements used in the constructs of the invention are known in the art, and thus the corresponding elements can be obtained by conventional methods, such as PCR, total artificial chemical synthesis, enzymatic digestion, and then ligated together by well-known DNA ligation techniques to form the constructs of the invention.

One of ordinary skill in the art can use well-known methods to construct expression vectors containing the promoter and/or gene sequences of interest described herein. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

The invention also provides a host cell comprising said construct or vector, or said host cell having said construct or vector chromosomally integrated therein. In another preferred embodiment, the host cell further comprises a vector comprising a gene encoding a transposase or having a transposase gene integrated into its chromosome.

Preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

In another preferred embodiment, the host cell comprises Escherichia coli and Clostridium aerovorum.

In another preferred embodiment, the prokaryotic cell includes (but is not limited to): escherichia coli and the like.

The constructs or vectors of the invention may be used to transform appropriate host cells. The host cell may be a prokaryotic cell, such as E.coli, Streptomyces, Clostridium aerovorans: or lower eukaryotic cells, such as yeast cells. It will be clear to one of ordinary skill in the art how to select an appropriate vector and host cell. Transformation of host cells with recombinant DNA Using routine techniques well known to those skilled in the artThe operation is performed. When the host is prokaryote (such as Escherichia coli or Clostridium aerophagum), CaCl can be used₂The treatment 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 (e.g., microinjection, electroporation, liposome encapsulation, etc.).

Integration method

The invention also provides a method for site-specific integration of a genome into a host cell, comprising the steps of:

wherein the integration construct is as described above.

In another preferred example, the method further comprises:

(iii) introducing a selection construct into said integration-treated host cell, thereby obtaining a host cell which has been crossed to integrate said foreign gene into the genome, wherein said selection construct is as described above.

In one embodiment, the present inventors integrated large fragment of DNA of butyrate synthesis pathway into clostridium Yondall by the integration method to obtain an engineered bacterium integrated with butyrate synthesis pathway. The engineering bacteria are cultured in a medium containing CO/CO₂The fermentation is carried out in the gas of (2). After 96 hours of culture, the engineering bacteria can synthesize 1g/L of butyric acid, and the butyric acid pathway integrated into the chromosome is proved to catalyze the reaction of butyric acid synthesis.

One outstanding advantage of the integration method of the invention is that the integrated engineering bacteria have excellent genetic stability. In one embodiment of the present invention, the butyric acid pathway-integrated engineered bacteria are continuously subcultured (24 hours for fresh medium and 10 times for total) in a medium without any selection pressure factor (e.g., antibiotic). Subsequently, the 10 th generation and the 1 st generation of the engineering bacteria were subjected to fermentation comparison. The results show that the growth and butyric acid synthesis of the two engineering bacteria are not obviously different, which proves that the engineering bacteria prepared by the integration method of the invention have excellent genetic stability.

The main advantages of the invention are:

(1) the method can efficiently integrate the exogenous gene (especially large fragment DNA) at a specific position of the genome of clostridium aerophagum, and avoid the defects of undefined integration site and transgenic expression interference caused by random integration of the exogenous gene;

(2) according to the method, the plasmids containing the exogenous genes are directly transfected into the clostridium aerovorans, so that the steps are simple and convenient, and the operability is greatly improved;

(3) in the cells with the transgene positioned and integrated, which are obtained by the method, the gene integration position is clear, and the gene integration detection is simple;

(4) the method has high integration efficiency and good stability. The foreign gene integrated into the genome can stably exist for a long period of time even in the absence of selective pressure.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1: it was verified that the phage Φ CD27 integration system derived from Clostridium difficile (Clostridium difficile) is functional in Clostridium immortal (Clostridium ljungdahlii)

1. Experimental procedure

1) The elements contained in several of the reported phage integration systems were collected, including integrase (integrase), attachment site (attachment site) attP and attB sequences.

2) And (3) replacing the Clju _08380 gene on the chromosome of the clostridium Youdaiensis by an attB sequence of the phi CD27 through a CRISPR-Cas9 gene editing system.

3) The codon optimized gene for Φ CD27integrase (integrase) and the attP sequence were cloned on an expression plasmid suitable for clostridium andraeanum. Expression of the integrase gene on the plasmid was driven by the bulk ptb promoter from c. The plasmid pHG-27int obtained was introduced into the above-mentioned chassis cells of Clostridium alterniforme containing attB by electrotransformation.

4) The competent cells after electrotransformation were plated on solid medium containing thiamphenicol (5. mu.g/mL).

5) After 2-3 days of culture, colonies on the solid medium were picked for PCR identification and sequencing.

6) The results indicated that attP × attB site-specific recombination occurred in the identified colonies.

2. Results of the experiment

The results of the experiment are shown in FIG. 1.

As shown in fig. 1A, the cac08380 gene on the clostridium andreanum genome was replaced by attB site of Phage CD27 derived from clostridium difficile (clostridium difficile) by CRISPR-Cas9 genome editing method. The pHGcas-08380 plasmid carries the flanking homology arms of the cas9, sgRNA, attP and cac08380 genes.

As shown in FIG. 1B, the integrase (integrase) gene of Phage Phage CD27 was codon optimized and cloned into plasmid pHG-CD27int together with the attP site. The recombination between attP and attB sites was achieved by introduction into a host bacterium having integrated attB sites on the chromosome as shown in FIG. 1A.

The entire plasmid pHG-CD27int plasmid is integrated into the genome of C.perpendicularis by recombination between attP and attB sites, as shown in FIG. 1C.

The sequencing results, as shown in FIG. 1D, demonstrate recombination between the attP and attB sites, forming new sites attL and attR on the chromosome.

Example 2: proves that the integrated system derived from Streptomyces phage phi C31 has function in Clostridium immortal (Clostridium ljungdahlii)

1. Experimental procedure

The procedure is as in example 1, except that the integrase and attP/attB sites are replaced by the φ C31 integration system.

2. Results of the experiment

The results of the experiment are shown in fig. 2 and 3. Wherein FIG. 2 is a flow chart; as shown in FIG. 3, the attL and attR sites and the sequences on both sides are amplified by PCR, and the size of the PCR band obtained by electrophoresis is in accordance with the expectation, which indicates that the integrated system of the PhiC 31 can realize the recombination between the attP and attB sites in Clostridium Youdaier.

Example 3: realizing the specific integration of large fragment butyric acid synthesis path on the chromosome of Clostridium Yongdahli (Clostridium jungdahlii) by utilizing two sets of integrated systems of Phi CD27 and Phi C31

1. Experimental procedure

1) The attB sequences of the phi CD27 and the phi C31 are respectively replaced by Clju _08380 and Clju _08360 genes on a Clju-Cas 9 gene editing system to obtain Chassis bacteria CLJU_B27/31。

2) Codon optimized genes of Φ CD27 and Φ C31 integrase (integrase) and the corresponding attP sequences were cloned on expression plasmids suitable for clostridium andraeanum. Expression of the integrase gene on the plasmid was driven by the bulk ptb promoter from c. The obtained plasmid pHG-BA02 was introduced into the above-mentioned attB-containing ClJU of Clostridium Yondall Chassis cell by electrotransformation_B27/31。

3) The competent cells after electrotransformation were plated on solid medium containing thiamphenicol (5. mu.g/mL).

4) After 2-3 days of culture, collecting colonies growing on the solid culture medium, and transferring the colonies into a fresh liquid culture medium for culture. When the bacterial suspension concentration reached OD600 of 0.3-0.5, competent cells were prepared, and then the second plasmid pHGcas-CD27 was introduced into the competent cells by electroporation. The bacterial liquid after electric shock was spread on a solid medium containing erythromycins (10. mu.g/mL).

5) After 2-3 days of culture, after a certain number of colonies grow on the solid medium, several bacteria are selected for PCR verification, and colonies (9.1 kb DNA band in FIG. 4C) capable of amplifying butyric acid synthesis pathway from chromosome are found.

6) In order to further isolate and purify the mutant colonies (excluding wild-type individuals mixed therein) having butyric acid pathway integrated into the genome, colonies of the colonies (FIG. 4C) on the solid medium were re-inoculated into a liquid medium, subcultured for 2 times in a continuous manner, and dipped into a bacterial solution to perform streaking to obtain pure mutants of interest. The obtained mutant strain was further confirmed for genotype by sequencing.

2. Results of the experiment

The results of the experiment are shown in FIG. 4.

In which, as shown in FIG. 4A, a scheme (BAPP) for specifically integrating large fragment butyric acid synthesis pathway on chromosome of Clostridium immortal (Clostridium jungdahlii) was implemented using two integration systems of Φ CD27 and Φ C31. As shown in FIG. 4B, pHGcas-CD27 plasmid (containing CRISPR-Cas9 system) was introduced into the cells. The plasmid pHGcas-CD27 carries sgRNA capable of recognizing the gene for realizing the gene encoding the phi CD27 integrin, so that the plasmid pHG-BA02 which is free in cells of the clostridium Youdalium or integrated on a chromosome in a single exchange mode can be targeted and cut. Thus, colonies that can grow on solid plates are either wild-type or mutant strains that integrate the BAPP pathway on the chromosome by double crossover. As in fig. 4C, colonies on the plate were identified by PCR. The DNA bands of 1.2kb and 9.1kb represent the genotypes of the wild type and BAPP integrated mutant, respectively.

In conclusion, the present example succeeded in obtaining pure desired mutants.

Example 4: stability detection

For the butyric acid pathway-integrated engineered bacteria prepared in example 3, serial subculture was performed in a medium without any addition of a selection pressure factor (e.g., antibiotic) (fresh medium was inoculated for 24 hours, and 10 times total inoculation) to the culture medium. Subsequently, the 10 th generation and the 1 st generation of the engineering bacteria were subjected to fermentation comparison.

The results show that no detectable sequence difference occurs in growth and butyric acid synthesis of the two engineering bacteria, and the BAPP sequence integrated into the genome is not lost. The engineering bacteria prepared by the integration method of the invention have excellent genetic stability.

Discussion of the related Art

The inventor firstly tests the effectiveness of site-specific recombination systems from various phages in Clostridium Yondall, and firstly discovers that two sets of phage site-specific recombination systems, namely, phage CD27 from Clostridium difficile (Clostridium difficile) and phageC31 from streptomycete, can realize integration of exogenous genes on chromosomes in Clostridium Yondall, and the efficiency reaches 100%.

However, the disadvantage is that the integration of the whole plasmid sequence containing the target gene on the chromosome can only be realized by using any one set of the phage site-specific recombination system. The chromosomally integrated plasmid is unstable and may be self-sheared again, resulting in a loss of phenotype of the resulting mutant strain.

Therefore, the present inventors further improved the above technology by introducing two sets of phage site-specific recombination systems, phage CD27 and phage C31, into clostridium alterniformis simultaneously, and using their orthogonality, integrating the DNA fragment of interest specifically into the chromosome of clostridium alterniformis by a double crossover event without introducing any additional sequences. In this process, in order to eliminate the interference of transformants which do not undergo recombination and only undergo single crossover recombination, the present inventors introduced the CRISPR-Cas9 system into the above transformants and specifically cut a plasmid (episomal or integrated into the chromosome) containing the phage recombination system, thereby trying to ensure that the surviving transformants are all recombinants which undergo double crossover (the possibility of the existence of a wild-type strain with a small probability). By this strategy, the inventors successfully integrated a short DNA sequence MCS followed by a 8.5kb butyrate synthesis pathway from C.acetobutylicum at a specific position on the chromosome of C.immortal (without introducing any additional selection marker).

In order to verify whether Clostridium Yondall integrated with butyric acid synthesis pathway has functions, the inventor puts engineering bacteria into the state of containing CO/CO₂The fermentation is carried out in the gas of (2). The results indicate that the butyrate pathway integrated into the chromosome can catalyze the reaction of butyrate synthesis.

In addition, the engineered bacteria integrated with the butyric acid pathway are subjected to continuous subculture (10 times of transfer of fresh culture medium for 24 hours) in a culture medium without adding any screening pressure factor (such as antibiotic). Subsequently, the 10 th generation and the 1 st generation of the engineering bacteria were subjected to fermentation comparison. The results show that the growth and butyric acid synthesis of the two engineering bacteria are not obviously different, and the engineering bacteria are proved to have good genetic stability.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A method for site-directed genomic integration of a host cell, comprising the steps of:

wherein said integration construct is selected from the group consisting of:

2. The method of claim 1, wherein the method further comprises:

wherein the screening construct is selected from the group consisting of:

(d2) a second screening construct, said second screening construct being a circular structure and comprising: a Cas9 expression cassette, a second sgRNA expression cassette, wherein the second sgRNA targets the coding sequence of the phi C31 integrase; and

3. The method of claim 1, wherein the method further comprises: exogenous attB sequences are introduced into the host cell at the site to be integrated by gene editing.

4. The method according to claim 1, wherein the host cell comprises Clostridium (Clostridium prazmowski), preferably Clostridium aerophagum (gas-transducing Clostridium), more preferably Clostridium autodahlii (Clostridium ljungdahlii).

5. A genetically engineered bacterium is clostridium, and a nucleic acid sequence from 5 'to 3' of a structure shown in a formula I is inserted into a genome of the genetically engineered bacterium:

Z1-W-Z2 (I)

in the formula (I), the compound is shown in the specification,

w is a foreign gene integrated into the genome;

6. The genetically engineered bacterium of claim 5, wherein said foreign gene is a butyrate synthesis pathway (BAPP) gene.

7. A construct for site-directed integration into a genome, said construct comprising an integration construct selected from the group consisting of:

8. The construct of claim 7, further comprising: a screening construct selected from the group consisting of:

9. The construct of claim 7, wherein said construct comprises a third integration construct and a screening construct.

10. The construct of claim 7, wherein the genome comprises the genome of C.