Method for screening target insertion sites and application thereof

Abstract

The invention relates to the field of gene engineering, and discloses a method for screening target insertion sites and application thereof, the method comprises the following steps: constructing plasmids for expressing target genes, and extracting total RNA of engineering bacteria carrying the plasmids to obtain an RNA sample; performing transcriptome sequencing on the RNA sample to obtain transcriptome sequencing data; comparing the transcriptome sequencing data with a reference genome of the engineering bacteria, and determining an FPKM value of each gene in the engineering bacteria; determining the distance between each gene in the engineering bacteria and a downstream gene; when the FPKM value of the gene is greater than 600 and the distance between the gene and a downstream gene is greater than 53 bp, the gene is a target insertion site. The method can accurately determine the target insertion site, improves the accuracy and pertinence of gRNA design, is used for optimally designing gRNA for MUCICAT gene editing, and is strong in pertinence, good in predictability, high in gene editing efficiency, good in flexibility, stable in target gene copy number, good in strain passage stability and high in target metabolite yield.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, in particular to a method for screening a target insertion site and an application thereof. Background Art

[0002] Human milk oligosaccharides (HMOs) are important nutrients in breast milk, among which 2′-fucosyllactose (2′-FL) is the third largest nutrient in breast milk after lactose and fat. HMOs are crucial for infant immunity and nervous system development. The addition of industrially produced HMOs to dairy products is an inevitable trend. The main production methods include chemical synthesis, in vitro enzymatic method and microbial fermentation. Among them, chemical synthesis cannot produce 2′-FL on a large scale due to its complex process and toxicity; enzymatic synthesis requires the addition of expensive nucleotide sugar substrates and cofactors, which limits its industrial production of 2′-FL. Therefore, the production of 2′-FL by microbial fermentation has become the mainstream of industrial production.

[0003] Currently, two technologies, Red-Et homologous recombination and CRISPR / Cas9, are used to integrate the α-1,2-fucosyltransferase gene into the genome. The Red-Et homologous recombination system is cumbersome, time-consuming, and requires the creation of a FRT scar in the genome. Furthermore, excessive scarring can destabilize the host bacterial genome and potentially lead to genomic rearrangements. The CRISPR / Cas9 system generates double-stranded breaks during editing, requiring the provision of repair fragments. However, this repair process, performed via non-homologous end joining, is highly error-prone and results in a low accuracy rate. Furthermore, due to differences in gene sequences, not all genes have suitable PAMs. Therefore, the CRISPR / Cas9 system is limited by PAM recognition sequences. Crucially, the CRISPR / Cas9 system has a high off-target efficiency, and the efficiency of site-specific gene editing decreases with the number of editing sites. The latest CRISPR-associated transposase system (MUCICAT) saves time and effort, is independent of the host homologous recombination system and DNA double-strand breaks, and its editing efficiency is unaffected by the editing site and is not restricted by PAMs. The design of sgRNA only needs to ensure the distance between the target site and the transposon integration site and not affect the expression of upstream and downstream genes. However, different genetic backgrounds and culture conditions can cause host bacteria to produce different transcriptomes to adapt to environmental changes. If this is not fully considered, gRNA design will lack specificity and predictability, and may be somewhat blind.

[0004] Therefore, MUCICAT technology faces the challenge of uncertainty in gRNA design, and there is an urgent need to develop precise and effective gRNA design methods to improve the accuracy and efficiency of gene editing. Summary of the Invention

[0005] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, the present invention provides methods and applications for screening target insertion sites. The methods of the present invention can accurately determine target insertion sites, improving the accuracy, purposefulness, flexibility, and specificity of guide RNA (gRNA) design. These methods are used to optimize the design of gRNA for MUCICAT gene editing, providing strong specificity and predictability. Furthermore, the methods can be used to construct engineered bacteria, resulting in high gene editing efficiency, good flexibility, stable target gene copy number, good strain stability, and high target metabolite production.

[0006] The present invention is based on the inventor's discovery and understanding of the following problems: Existing MUCICAT gene editing technology uses a plasmid system to regulate changes in metabolic pathways within bacteria. However, the plasmid system requires antibiotic maintenance and has limitations such as unstable copy number and easy loss. In addition, different genetic backgrounds and culture conditions can cause host bacteria to produce different transcriptomes to adapt to environmental changes, resulting in the blindness of the gRNA provided. To solve this problem, the inventors designed and developed a method for screening highly expressed gene target insertion sites. Then, based on the MUCICAT technology and the highly expressed gene target insertion sites determined by screening, at least one copy of the relevant target gene encoding the target protein in the metabolic pathway is inserted into the host bacteria to obtain a fermentation strain with a high yield of the target product. Further experimental results show that the method of constructing engineered bacteria of the present invention has strong targeting and good predictability of gRNA design, high gene editing efficiency and good accuracy. It is used to construct engineered bacteria with good target gene copy number and strain passage stability, and high target metabolite yield.

[0007] Therefore, in the first aspect of the present invention, a method for screening target insertion sites is proposed. According to an embodiment of the present invention, the target insertion site is used to insert a target gene into an engineered bacterium. The method comprises: constructing a plasmid carrying the target gene, extracting total RNA from the engineered bacterium carrying the plasmid to obtain an RNA sample; performing transcriptome sequencing on the RNA sample to obtain transcriptome sequencing data; comparing the transcriptome sequencing data with the reference genome of the engineered bacterium to determine the FPKM value of each gene in the engineered bacterium; and determining the distance between each gene in the engineered bacterium and a downstream gene; when the FPKM value of a gene is greater than 600 and the distance from the downstream gene is greater than 53 bp, the gene is a target insertion site. The method according to the embodiment of the invention can accurately determine the target insertion site, improve the accuracy, purposefulness, flexibility and pertinence of gRNA design, overcome the blindness of gRNA design, and is used to optimize the design of gRNA for MUCICAT gene editing, with strong pertinence and good predictability. It is used to construct engineered bacteria with high gene editing efficiency and accuracy, stable target gene copy number, good strain passage stability, and high target metabolite production.

[0008] In a second aspect of the present invention, a method for designing a gRNA is provided. According to an embodiment of the present invention, the gRNA is used to insert a target gene into the genome of an engineered bacterium, and the method comprises: determining a target insertion site for the gRNA corresponding to the genome of the engineered bacterium according to the aforementioned method; and determining the nucleotide sequence of the gRNA based on the target insertion site. According to the method of an embodiment of the present invention, the gRNA for MUCICAT gene editing obtained by screening based on the aforementioned target insertion site has strong targeting, good predictability, high gene editing efficiency, and good accuracy.

[0009] In a third aspect of the present invention, the present invention provides a gRNA. According to an embodiment of the present invention, the gRNA is designed according to the aforementioned method.

[0010] Those skilled in the art will understand that the features and advantages described above for the method of screening target insertion sites and the method of designing gRNA are also applicable to this gRNA and will not be repeated here.

[0011] In a fourth aspect, the present invention provides a method for constructing an engineered bacterium. According to an embodiment of the present invention, the method comprises: obtaining a gRNA based on the aforementioned method, and inserting at least one copy of the target gene into the genome of the engineered bacterium using the MUCICAT gene editing system and the gRNA; wherein the target gene encodes a target protein, and the target protein is used to produce a target product. The method according to an embodiment of the present invention has high gene editing efficiency and good accuracy, and the constructed engineered bacterium has a stable copy number of the target gene, good strain stability, and high yield of the target metabolite.

[0012] In a fifth aspect, the present invention provides an engineered bacterium. According to an embodiment of the present invention, the engineered bacterium is constructed according to the aforementioned method.

[0013] Those skilled in the art will understand that the features and advantages described above for the method of screening target insertion sites, the method of designing gRNA, and the method of constructing engineered bacteria are also applicable to the engineered bacteria and will not be repeated here.

[0014] In a sixth aspect of the present invention, the present invention provides the use of the aforementioned engineered bacteria in producing a target product, wherein the target product is 2'-fucosyllactose.

[0015] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which: Figure 1 Schematic diagram of the 2′-fucosyllactose metabolic pathway according to an embodiment of the present invention; Figure 2 For use in Example 2 of the present invention GFP The expression level of the insertion site was verified and investigated. (a) shows the expression level of the insertion site using the original gRNA as a guide in 4 ( ydgA-uidc ) and No. 8 ( yiM-cpxA )have GFP The random insertion strain is called strain 1; (b) is the result of using the designed gRNA as a guide in strain 2 ( ldtE-lpp )have GFP The randomly inserted strain is called bacteria 2; (c) is the result of bacteria 1 and bacteria 2 GFP Results of emission light detection; (d) bacteria 1 and bacteria 2 GFP Excitation light detection investigation results diagram; Figure 3In Example 3 of the present invention BKHT-SUMO Copy number detection and plasmid elimination results, where (a) is 3 copies ( fur-chiQ , ldtE-lpp and yecH-ftnA ) strain colony PCR test results; (b) 4 copies ( fur-chiQ , ldtE-lpp , yecH-ftnA and yjgM-rraB ) strain colony PCR test results; (c) is 5 copies ( fur-chiQ , ydhP-sodB , ldtE-lpp , yecH-ftnA and yjgM-rraB ) BKHT-SUMO Colony PCR test results of the strain; (d) 3 copies, 4 copies and 5 copies BKHT-SUMO Results of the pQCasTns(Ptr) plasmid elimination study of the strain; (e) 3 copies, 4 copies, and 5 copies BKHT-SUMO Results of the pDonor plasmid elimination study of the strain; (f) 3 copies, 4 copies, and 5 copies BKHT-SUMO Results of pCutamp plasmid elimination investigation of strains; (g) 3 copies, 4 copies, and 5 copies BKHT-SUMO Figure 1 shows the results of the pCutamp plasmid elimination verification investigation of the strain; Figure 4 This is a graph showing the results of investigating the content of the target metabolite 2′-FL after fermentation with the multi-copy strain in Example 4 of the present invention. DETAILED DESCRIPTION

[0017] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0018] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0019] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0020] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0021] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0022] Terms and Definitions Herein, the term "FutC" refers to a fucosyltransferase derived from Helicobacter pylori; italics indicate the gene encoding the protein.

[0023] Herein, the term "FucT" refers to a fucosyltransferase derived from Helicobacter pylori; italics indicate the gene encoding the protein.

[0024] Herein, the term "BKHT" refers to a fucosyltransferase derived from Helicobacter pylori; italics indicate the gene encoding the protein.

[0025] As used herein, the term "SUMO protein" refers to small ubiquitin-like modifiers, which affect the function and localization of target proteins through reversible post-translational modifications.

[0026] Herein, the term "2'-fucosyllactose" is equivalent to "2'-FL".

[0027] In this article, the term "PAM" refers to a short DNA sequence that is used by the CRISPR-Cas system to distinguish "self" from "invaders."

[0028] In this article, the term "GFP" refers to green fluorescent protein, which can form a chromophore by self-catalysis and emit green fluorescence under the excitation of blue light or ultraviolet light.

[0029] In this article, the term "pQCasTns plasmid" refers to a vector for expressing a CRISPR-associated transposase, which can target specific genomic regions under the guidance of the CRISPR system to achieve the insertion or deletion of DNA fragments.

[0030] In this article, the term "pDonor plasmid" refers to a vector used for gene knock-in experiments, which is usually used to provide homology arms or homology sequences to promote homologous recombination at the target site.

[0031] As used herein, the term "pCutamp plasmid" refers to a plasmid deletion system for use in E. coli.

[0032] Exemplarily, the "pCutamp plasmid" is used to eliminate the dual plasmids pQcasTns and pDonor.

[0033] The present invention provides a method for screening target insertion sites, a method for designing gRNA and gRNA, a method for constructing engineered bacteria and engineered bacteria and their uses, which will be described in detail below.

[0034] Method for screening target insertion sites The present invention proposes a method for screening target insertion sites. According to an embodiment of the present invention, the target insertion site is used to insert a target gene into an engineered bacterium. The method comprises: constructing a plasmid carrying the target gene, extracting total RNA from the engineered bacterium carrying the plasmid to obtain an RNA sample; performing transcriptome sequencing on the RNA sample to obtain transcriptome sequencing data; comparing the transcriptome sequencing data with a reference genome of the engineered bacterium to determine the FPKM value of each gene in the engineered bacterium; and determining the distance between each gene in the engineered bacterium and a downstream gene; when the FPKM value of a gene is greater than 600 and the distance from the downstream gene is greater than 53 bp, the gene is a target insertion site. The method according to the embodiment of the invention can accurately determine the target insertion site, improve the accuracy, purposefulness, flexibility, and pertinence of gRNA design, overcome the blindness of gRNA design, and is used to optimize the design of gRNA for MUCICAT gene editing, with strong pertinence and good predictability. It is used to construct engineered bacteria with high gene editing efficiency and accuracy, stable target gene copy number, good strain passage stability, and high target metabolite production.

[0035] The method according to the embodiment of the invention is through transcriptome sequencing and data analysis, and highly expressed target sites are excavated, and these sites are directly related to the design of gRNA, thereby improving the accuracy and purposefulness of gRNA design. The method according to the embodiment of the invention utilizes the transcriptome data design gRNA of different strains, and carries out personalized design according to the transcriptome background specific to strain, thereby improving the flexibility and pertinence of gRNA design. Compared with the gRNA provided by existing MUCICAT technology, the method according to the embodiment of the invention overcomes the blindness of gRNA design, overcomes the technical defect that different genetic backgrounds and culture conditions make host bacteria produce different transcriptomes, and improves the production performance of engineering bacteria.

[0036] In this article, the term "FPKM value" represents the number of fragments per kilobase of exon model per million mapped reads, which is a metric used to measure gene expression levels.

[0037] In this article, the term "MUCICAT gene editing" refers to the multi-copy chromosome integration technology of the CRISPR-associated transposase system (CRISPR-associated transposases). It is a new type of gene editing tool that uses transposases associated with the CRISPR-Cas system to insert DNA fragments into specific sites of the genome through RNA guidance without relying on the homologous recombination mechanism of the host cell.

[0038] In this article, the term "gRNA" is equivalent to "guide RNA", which is one of the key components of the CRISPR-Cas9 system. It is composed of CRISPR RNA (crRNA) and transcription-activating CRISPR RNA (tracrRNA). It can specifically recognize and complementarily pair with the target DNA sequence, guiding the Cas9 nuclease to cut the target DNA at a specific location, achieving editing operations such as gene knockout, insertion or site-directed mutagenesis.

[0039] According to some specific embodiments of the present invention, the transcriptome sequencing data is filtered, and the data volume is no less than 6 Gb. The filtering process includes removing adapter sequences, removing sequences with low sequencing quality scores, and removing sequences with a base uncertainty ratio greater than 5%. Low sequencing quality scores are sequences in which bases with a quality score less than 15 account for greater than 20% of the total base count in the sequence. This results in high-quality sequencing data (clean data), further improving the accuracy of screening results.

[0040] According to a specific embodiment of the present invention, the comparison software used in the comparison process is Bowtie2.

[0041] According to an embodiment of the present invention, the starting bacteria of the engineered bacteria include, but are not limited to, at least one of: Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Bacillus subtilis, Corynebacterium glutamicum, Lactobacillus, Streptomyces, Xanthomonas, and Pseudomonas. The method according to an embodiment of the present invention can be used to screen for highly expressed target insertion sites in the genomes of a variety of strains producing secondary metabolites, and based on this, optimize the design of gRNA, thereby improving the efficiency of gene editing and the yield of target metabolites, overcoming the effects of different genetic backgrounds and culture conditions on the effect of gRNA, and improving the production performance of the strain.

[0042] According to an embodiment of the present invention, the target gene encodes a target protein, and the target protein includes: fucosyltransferase and / or SUMO protein. Figure 1As shown, the production process of 2′-FL involves a key gene, the α-1,2-fucosyltransferase gene. According to the methods of the present invention, a target insertion site suitable for high expression of this gene was screened and determined. The gRNA for MUCICAT gene editing, thus optimized and designed, was screened and obtained, with high gene editing efficiency and good accuracy. The resulting engineered bacteria had a stable copy number, good strain stability, and high yield of the target metabolite 2′-FL. According to the embodiments of the present invention, the target proteins include: fucosyltransferase and SUMO protein.

[0043] As used herein, the term "SUMO protein" is equivalent to "Small Ubiquitin-like Modifier." According to the methods of the present invention, fusion expression of a fucosyltransferase and a SUMO protein further improves the stability and expression level of the fucosyltransferase and enhances protease activity.

[0044] According to an embodiment of the present invention, the target gene includes FucT-SUMO 、 FutC-SUMO 、 BKHT-SUMO At least one of .

[0045] According to the method of the embodiment of the present invention, after mining the target insertion sites of highly expressed genes based on MUCICAT technology combined with transcriptome data, the key genes are inserted into the target sites. BKHT-SUMO Multiple copies are integrated into the genome to construct different copy numbers BKHT-SUMO Genetically engineered Escherichia coli can effectively enhance the synthesis of 2′-fucosyllactose (2′-FL) and achieve efficient synthesis of 2′-FL.

[0046] Methods for designing gRNA The present invention provides a method for designing a gRNA. According to an embodiment of the present invention, the gRNA is used to insert a target gene into the genome of an engineered bacterium, and the method comprises: determining a target insertion site of the gRNA corresponding to the genome of the engineered bacterium according to the aforementioned method; and determining the nucleotide sequence of the gRNA based on the target insertion site.

[0047] According to the method of the embodiment of the present invention, the gRNA for MUCICAT gene editing obtained based on the aforementioned target insertion site screening has strong targeting, good predictability, high gene editing efficiency and good accuracy.

[0048] gRNA The present invention provides a gRNA. According to an embodiment of the present invention, the gRNA is designed according to the aforementioned method.

[0049] Those skilled in the art will understand that the features and advantages described above for the method of screening target insertion sites and the method of designing gRNA are also applicable to this gRNA and will not be repeated here.

[0050] According to an embodiment of the present invention, the target insertion site of the gRNA corresponding to the engineered bacterial genome includes: ychH , fur , sodB , ftnA , lpp , ytfK , glpD and rraB According to the method of the embodiment of the present invention, the stability, expression level and biological activity of the target protein are further improved.

[0051] According to an embodiment of the present invention, the gRNA has a nucleotide sequence as shown in at least one of SEQ ID NOs: 25 to 32.

[0052] According to some specific embodiments of the present invention, the engineered bacteria is BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK .

[0053] It should be noted that the above can be realized on BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK In Escherichia coli, the nucleotide sequence of the gRNA for accurate and efficient insertion into the target gene using MUCICAT editing technology is exemplified.

[0054] Methods for constructing engineered bacteria The present invention proposes a method for constructing an engineered bacterium. According to an embodiment of the present invention, the method comprises: obtaining a gRNA based on the aforementioned method, and inserting at least one copy of the target gene into the genome of the engineered bacterium using the MUCICAT gene editing system and the gRNA; wherein the target gene encodes a target protein, and the target protein is used to produce a target product. The method according to the embodiment of the present invention has high gene editing efficiency and good accuracy. The constructed engineered bacterium has a stable copy number of the target gene, good strain stability, and high yield of the target metabolite.

[0055] According to an embodiment of the present invention, the starting bacteria of the engineered bacteria include but are not limited to: at least one of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Bacillus subtilis, Corynebacterium glutamicum, Lactobacillus, Streptomyces, Xanthomonas, and Pseudomonas.

[0056] According to an embodiment of the present invention, the target protein includes: fucosyltransferase and / or SUMO protein; and the target product is 2′-fucosyllactose.

[0057] According to an embodiment of the present invention, the target gene includes FucT-SUMO 、 FutC-SUMO 、 BKHT-SUMO At least one of .

[0058] According to an embodiment of the present invention, 1 to 8 copies of the target gene are inserted into the genome of the engineered bacteria. BKHT-SUMO Genetically engineered Escherichia coli achieves efficient synthesis of 2′-FL.

[0059] According to an embodiment of the present invention, the copy number is 2 to 6. The method according to an embodiment of the present invention further improves the synthesis efficiency of 2'-FL.

[0060] Illustratively, the copy number can be 2, 3, 4, 5, or 6.

[0061] Engineered bacteria The present invention provides an engineered bacterium. According to an embodiment of the present invention, the engineered bacterium is constructed according to the aforementioned method.

[0062] Those skilled in the art will understand that the features and advantages described above for the method of screening target insertion sites, the method of designing gRNA, and the method of constructing engineered bacteria are also applicable to the engineered bacteria and will not be repeated here.

[0063] use The present invention proposes the use of the aforementioned engineered bacteria in producing a target product.

[0064] According to an embodiment of the present invention, the target product is 2′-fucosyllactose.

[0065] Those skilled in the art will understand that the features and advantages described above for the method of screening target insertion sites, the method of designing gRNA, and the method of constructing engineered bacteria are also applicable to this use and will not be repeated here.

[0066] In order to achieve the above object, the present invention is to perform a series of experiments on five strains BL21Star™ (DE3), including BL21Star™ (DE3) carrying the dual plasmids pCDFduet-BCGW and pETDuet-FucT-SUMO (no exogenous gene expression is induced), referred to as BL21Star™ (DE3)-FucT-SUMO (-), BL21Star™ (DE3) carrying the dual plasmids pCDFduet-BCGW and pETDuet-FucT-SUMO (exogenous gene expression is induced), referred to as BL21Star™ (DE3)- FucT-SUMO (+), and BL21Star™ (DE3) carrying the dual plasmids pCDFduet-BCGW and pETDuet-FutC-SUMO (no exogenous gene expression is induced), referred to as BL21Star™ (DE3)- FucT-SUMO ( (-) and BL21Star™ (DE3) carrying the dual plasmids pCDFduet-BCGW and pETDuet-FutC-SUMO (inducing exogenous gene expression) (abbreviated as BL21Star™ (DE3) - Fut C-SUMO (+) strains were fermented and then transcriptome sequencing was performed. Comprehensive data analysis was conducted to mine 8 highly expressed target sites, and gRNA was designed. At the same time, the gRNA given in the literature [Yang S, Zhang Y, Xu J, et al. Orthogonal CRISPR-associated transposasesfor parallel and multiplexed chromosomal integration[J]. Nucleic Acids Research, 2021, 49(17): 10192-10202.] was used. GFP was used as a probe and GFP was inserted into the genome of the host bacteria for fermentation. The highly expressed target sites were verified by microplate reader detection. Therefore, the gRNA designed according to the different transcriptome data of the strains in the present invention has flexibility compared with the gRNA given in the literature, and the yield of the target product produced is higher. Afterwards, the key gene α-1,2-fucosyltransferase gene was used to replace GFP and multiple copies were inserted into Escherichia coli for fermentation. Mass spectrometry detection verified that the copy number of the key enzyme gene was proportional to the 2′-FL production.

[0067] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0068] Example 1: Culture and sequencing of strains 1: Strain culture Bacterial strain: Escherichia coli BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK Experiments have already been done.

[0069] Culture media: SOB liquid (20 g / L tryptone, 5 g / L yeast extract, 1.16 g / L sodium chloride, and 0.19 g / L potassium chloride, pH = 7.4-7.5), LB liquid (10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride), and solid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 20 g / L agar).

[0070] The strain BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK , BL21Star™ (DE3) Δ lacZ ΔwcaJ ΔnudD ΔnudK - FutC-SUMO and BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD Δ nudK - FucT-SUMO In 3 mL LB liquid medium in a 12 mL shaking tube, culture at 37°C with constant temperature shaking at 220 rpm until the OD600 reaches 0.6–0.8. ΔlacZ ΔwcaJ ΔnudD ΔnudK - FutC-SUMO and BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FucT-SUMO 0.5 mM IPTG was added and all strains were cultured at 220 rpm and 25°C for 72 h. RNA was then extracted from strain BL21Star™ (DE3) using the Tiangen RNA extraction kit. ΔlacZ ΔwcaJ ΔnudD ΔnudK , BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FutC-SUMO (-), BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FutC-SUMO (+), BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FucT-SUMO (-) and BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FucT -SUMO (+) RNA was sent to Hainan BGI Life Science Technology Co., Ltd. for transcriptome sequencing.

[0071] 2: Sequencing and gene screening Transcriptome sequencing was performed on the samples using the BGI high-throughput sequencing platform T20, generating no less than 6Gb of raw data for each sample. The filtering software SOAPnuke (version 1.5.6) was used to filter the raw data, removing adapters, sequences with low sequencing quality values, and sequences with high N ratios to obtain high-quality sequencing data (clean data). The clean data was then compared with the Escherichia coli reference genome ( Escherichia coli K-12) were aligned using Bowtie2 (version 2.4.5) software. The expression level (FPKM value) of each gene was calculated based on the alignment results, and gene regions with significantly high expression and adjacent transcription directions (FPRM value greater than 600) were identified.

[0072] After genetic screening, BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK , BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FucT-SUMO (-), BL21Star™ (DE3) ΔlacZ ΔwcaJ Δ nudD ΔnudK - FucT-SUMO (+), BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FutC-SUMO (-) and BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FutC-SUMO (+) Transcriptome data analysis of 5 strains screened genes with relatively high expression levels, and genes with a distance of more than 53 bp from downstream genes were used as target insertion sites, and the target gene was inserted between the upstream and downstream genes.

[0073] The highly expressed genes screened out include ychH , fur , sodB , ftnA , lpp , ytfK , glpD and rraB .

[0074] In this example, the above five E. coli strains were sequenced, and the expression levels of the screened highly expressed genes are shown in Table 1 below.

[0075] Table 1. Expression levels of highly expressed genes

[0076] in, BL21star_F_S indicates strain BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FutC- SUMO (-) foreign geneFutC-SUMO Uninduced expression, BL21star_F indicates strain BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FutC-SUMO (+) foreign gene FutC-SUMO Inducible expression, BL21star refers to the strain BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK , BL21star_T_S indicates strain BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FucT-SUMO (-) foreign gene FucT-SUMO Uninduced expression, BL21star_T indicates strain BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FucT-SUMO (+) foreign gene FucT-SUMO Inducible expression.

[0077] Example 2: Mining highly expressed gene target insertion sites 1: gRNA and primer design Combining transcriptome data from five different strains to screen for highly expressed genes, eight gRNA sequences were designed downstream of these eight highly expressed genes according to the MUCICAT gRNA design principles. The "original gRNA sequences" disclosed in the prior art and the eight gRNA sequences designed in this example, along with the validation primers, are shown in Tables 2 and 3.

[0078] 2: Plasmid construction pQCasTns(Ptr)-array 8 (carrying the original gRNA) and pCutamp were purchased from GenScript, and pQCasTns(Ptr)-array 8 (carrying the designed gRNA) was synthesized by GenScript. FutC-SUMO and GFP ) of pDonor, construct pDonor-RE- FutC-SUMO -LE and pDonor- RE- GFP -LE.

[0079] In this article, the pQCasTns(Ptr)-array 8 carrying the “original gRNA” was determined based on the gRNA nucleotide sequence from Yang S, Zhang Y, Xu J, et al. Orthogonal CRISPR-associated transposasesfor parallel and multiplexed chromosomal integration[J]. Nucleic Acids Research, 2021, 49(17): 10192-10202. The specific sequence information is shown in Table 2.

[0080] In this article, pQCasTns(Ptr)-array 8 carrying the "designed gRNA" has a gRNA nucleotide sequence according to the target insertion site determined by screening in Example 1. ychH , fur , sodB , ftnA , lpp , ytfK , glpD and rraB The specific sequence information is shown in Table 3.

[0081] Table 2. Original gRNA and primer sequences

[0082] Table 3. Designed gRNA and primer sequences

[0083] Note: NR indicates unnamed gene 3: Preparation of electroporated competent cells First, the strain BL21Star™ (DE3) stored at -80℃ ΔlacZ ΔwcaJ ΔnudD ΔnudK Streak activation onto LB solid plates, and the next day, isolate a single colony and culture overnight. Then, inoculate 1% of the culture into 250 mL of SOB liquid medium in a 500 mL Erlenmeyer flask and shake at 37°C at 220 rpm until the OD600 reaches 0.4–0.5. Allow the culture to rest on ice for 15 minutes, then centrifuge at 5000 g for 30 minutes at 4°C to harvest the cells. Gently shake with 10% pre-cooled glycerol on a decolorizing shaker until the cells are dissociated. Centrifuge at 5000 g for 30 minutes at 4°C. Repeat this washing process three times, finally dissolving the cells in approximately 1 mL of 10% glycerol and aliquoting 50 μL of the cells into 1.5 mL centrifuge tubes. The resulting competent culture was snap-frozen in liquid nitrogen and stored at -80°C until ready for use.

[0084] 4: GFP transposition experiment and transposition efficiency analysis and sequencing The CAST transposition experiment includes the transformation and induction of pDonor and pQCasTns(Ptr)-array 8. First, pQCasTns(Ptr)-array 8 (original gRNA) and pDonor- RE- GFP -LE, pQCasTns(Ptr)-array 8 (designed gRNA) and pDonor- RE- GFP -LE were co-electroporated into BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK Competent cells were added to 800 μL of liquid SOB medium and revived at 37°C, 220 rpm for 1 hour. The cells were then centrifuged at ×5000g for 1 minute to collect the cells. 100 μL of liquid LB medium was diluted and plated onto double-antibiotic LB agar plates (100 μg / mL ampicillin and 100 μg / mL kanamycin). The cells were grown overnight at 37°C for 16 hours. The next day, several hundred colonies were scraped from the plates, resuspended in fresh LB medium, and then plated onto double-antibiotic LB agar plates containing 100 ng / mL anhydrotetracycline (aTC) and incubated at 25°C for 48 hours to induce protein expression. The resulting film was scraped off and resuspended in LB medium. The cells were rediluted and re-plated onto plates containing 1000 ng / mL aTC and double-antibiotic LB. The colonies were grown at 25°C for 48 hours. Finally, the cells were streaked and diluted and cultured at 25°C to generate single clones. Colony PCR was performed to analyze the transposition efficiency, and positive clones were selected for sequencing.

[0085] 5: GFP enzyme marker detection Take the double plasmid pQCasTns(Ptr)-array 8 (original gRNA) and pDonor- RE- GFP -LE positive clone BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK , containing double plasmids pQCasTns(Ptr)-array 8 (designed gRNA) and pDonor- RE- GFP -LE positive clone BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK and the control group BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK Cultured in LB liquid medium for 3 h, adjusted to OD 0.25, and 100 μL of bacterial solution was taken and analyzed by microplate reader (λ ex = 460 nm, λ em = 500-750 nm; λ em=550,λ ex =300-500nm) for detection. Based on the MUCICAT system, the original gRNA and the designed gRNA were used as guides. GFP Multi-copy insertion into E. coli BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK Sequencing, fermentation and microplate reader detection were performed in the genome.

[0086] The results are as follows Figure 2 shown.

[0087] The results showed that the double plasmids pQCasTns(Ptr)-array 8 (original gRNA) and pDonor- RE- GFP -LE ， pQCasTns(Ptr)-array 8 (designed gRNA) and pDonor- RE- GFP -LE was randomly inserted into 2 and 1 sites respectively after 1 transposition experiment. GFP From the fluorescence intensity of the GFP protein, it can be seen that the fluorescence intensity value of the GFP protein inserted at one site by the designed gRNA is close to the fluorescence intensity value of the GFP protein inserted at two sites by the original gRNA.

[0088] The above results show that combining transcriptome data to mine highly expressed gene sites has a significant effect on promoting the protein expression of gene insertion. Therefore, the designed gRNA was selected for the target gene (fucosyltransferase gene) BKHT-SUMO of transposition insertions.

[0089] Example 3: BKHT-SUMO Copy number detection and plasmid elimination 1: BKHT-SUMO Transposition experiments and next-generation sequencing First, the double plasmid pQCasTns(Ptr)-array 8 (designed gRNA) and pDonor- RE- GFP -LE co-electroporation to BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudKCompetent cells were added with 800 μL of liquid SOB medium and revived at 37°C, 220 rpm for 1 hour. The cells were then centrifuged at ×5000g for 1 minute, harvested, diluted with 100 μL of liquid LB medium, and plated onto solid LB plates containing double antibiotics (100 μg / mL ampicillin and 100 μg / mL kanamycin). The plates were grown overnight at 37°C for 16 hours. The next day, colonies were scraped from the plates and partially resuspended in fresh liquid LB medium. The plates were then plated onto solid LB plates containing 100 ng / mL anhydrotetracycline (aTC) and double antibiotics and incubated at 25°C for 48 hours to induce protein expression. The resulting film was scraped off and partially resuspended in LB liquid medium. The plate was then re-plated onto plates containing 1000 ng / ml aTC and double-antibiotic LB. The colonies were grown at 25°C for 48 hours. Afterwards, the colonies were plated onto solid medium containing 1000 ng / ml aTC and double-antibiotic LB. This process was repeated eight times. Each replicate was streaked and cultured at 25°C to isolate single colonies. Colony PCR was performed to analyze transposition efficiency. Positive clones were selected for sequencing to confirm that the transposon had successfully inserted into the target genomic locus, in the intended orientation, and without unexpected mutations or recombination events.

[0090] 2: Plasmid elimination The pCutamp plasmid contains sgRNAs targeting the pQcasTns and pDonor plasmids. The sgRNA spacer is a consensus sequence, 5′-TGCTTCAATAATATTGAAAAAGG-3′, within the AmpR promoters of both plasmids. Rhamnose induces transcription of the sgRNA, directing the Streptococcus pyogenes Cas9 protein to target and cleave the consensus sequence. Because no homologous template is provided, repair of the targeted plasmids is impossible. First, the positive clones were made electrocompetent. pCutamp was electroporated into the electrocompetent cells of the positive clones. 800 μL of liquid SOB medium was added and the cells were revived at 37°C, 220 rpm for 1 hour. To improve the efficiency of plasmid elimination, the revived cells were transferred to 4 mL of liquid LB medium containing 50 μg / mL apramycin and 10 mM rhamnose and shaken at 37°C for 6 hours. 100 μL of the culture was spread onto solid LB plates containing 50 μg / mL apramycin and 10 mM rhamnose and incubated at 37°C overnight. To eliminate the pCutamp plasmid, the overnight-grown strains were re-spread onto three different LB plates containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 10 g / L sucrose. Colonies that failed to grow on plates containing ampicillin and kanamycin were considered to have eliminated pDonor and pQCasTns. Then, the corresponding colonies grown on the sucrose plates were re-plated on non-selective LB agar plates and plates containing apramycin to verify the elimination of the pCutamp plasmid. Colonies that cannot grow on the plates containing apramycin are plasmid-free multicopy strains.

[0091] The results of copy number colony PCR detection and plasmid elimination investigation are as follows Figure 3 shown.

[0092] The results showed that: in BL21Star™ (DE3) of pQCasTns(Ptr)-array 8 (designed gRNA) and pDonor-RE-BKHT-SUMO-LE ΔlacZ ΔwcaJ ΔnudD ΔnudK The fucosyltransferase gene BKHT-SUMO was used to replace the GFP gene in the strain for transposition experiments, and BL21Star™ (DE3) containing 3 copies, 4 copies, and 5 copies of BKHT-SUMO were obtained. ΔlacZ ΔwcaJ ΔnudD ΔnudK strains (the strains were named strain 3, strain 4 and strain 5).

[0093] The results of this experiment showed that the designed gRNA was used for the target gene BKHT-SUMO The transposition insertion is efficient and specific.

[0094] Furthermore, these three strains were subjected to post-plasmid elimination fermentation and mass spectrometry detection of the target metabolite 2′-FL.

[0095] Example 4: Multi-copy strain fermentation and mass spectrometry detection of target metabolites The plasmid-cured strain was streaked and revived, and a single colony was cultured overnight. A 1% inoculum was then used to inoculate 3 mL of LB liquid medium containing 30 g / L glycerol in a 12 mL shake tube. The culture was incubated at 37°C and 220 rpm. When the OD reached 0.6-0.8, 0.5 mM IPTG was added. After incubation at 25°C for 2 h, 10 g / L lactose was added and fermentation continued for 72 h to verify 2′-FL synthesis. A 100-200 μl sample of the fermentation broth was transferred to a 1.5 mL centrifuge tube, incubated in a boiling water bath at 100°C for 10 min, cooled to room temperature, and centrifuged at 15,000 rcf for 10 min. 10 μl of the supernatant was added to 990 μl of the diluent, and diluted 100-fold with 990 μL of 90% acetonitrile for analysis by LC-MS / MS.

[0096] The results are as follows Figure 4 shown.

[0097] The results showed that: BKHT-SUMO With the increase of copy number, the 2′-FL production gradually increased, reaching 112.45 mg / L (OD=6.49), 121.68 mg / L (OD=6.45), and 131.81 mg / L (OD=6.43), respectively.

[0098] The above results show that the target gene of the engineered bacteria constructed in Example 3 BKHT-SUMO The copy number is stable and the target metabolite production is high, which can effectively enhance the synthesis of 2′-fucosyllactose (2′-FL) and achieve efficient synthesis of 2′-FL.

[0099] In the description of this specification, the reference terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0100] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for screening target insertion sites, characterized in that, The target insertion site is used to insert a target gene into the genome of an engineered bacterium, and the method comprises: Constructing a plasmid carrying the target gene, extracting total RNA from the engineered bacteria carrying the plasmid to obtain an RNA sample; performing transcriptome sequencing on the RNA sample to obtain transcriptome sequencing data; Comparing the transcriptome sequencing data with the reference genome of the engineered bacteria to determine the FPKM value of each gene in the engineered bacteria; and determining the distance between each gene in the engineered bacteria and downstream genes; When the FPKM value of a gene is greater than 600 and the distance to the downstream gene is greater than 53 bp, the gene is a target insertion site.

2. The method according to claim 1, characterized in that The starting bacteria of the engineering bacteria include but are not limited to: at least one of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Bacillus subtilis, Corynebacterium glutamicum, Lactobacillus, Streptomyces, Xanthomonas, and Pseudomonas.

3. The method according to claim 1, characterized in that The target gene encodes a target protein, and the target protein includes: fucosyltransferase and / or SUMO protein.

4. The method according to claim 3, characterized in that The target genes include FucT-SUMO 、 FutC-SUMO 、 BKHT-SUMO At least one of .

5. A method for designing gRNA, characterized in that, The gRNA is used to insert a target gene into the genome of an engineered bacterium, and the method comprises: Determining the target insertion site of the gRNA corresponding to the engineered bacterial genome according to the method according to any one of claims 1 to 4; Based on the target insertion site, the nucleotide sequence of the gRNA is determined.

6. A gRNA, characterized in that The gRNA is designed according to the method of claim 5.

7. The gRNA according to claim 6, characterized in that The target insertion site of the gRNA corresponding to the engineered bacterial genome includes: yhH , fur , sodB , ftnA , lpp , ytfK , glpD and rB At least one of .

8. The gRNA according to claim 7, wherein The gRNA has a nucleotide sequence as shown in at least one of SEQ ID NOs: 25 to 32.

9. A method for constructing an engineered bacterium, characterized in that: The method comprises: obtaining gRNA based on the method of claim 5, and inserting at least one copy of the target gene into the genome of the engineered bacteria based on the MUCICAT gene editing system and the gRNA; Wherein, the target gene encodes a target protein, and the target protein is used to achieve the production of a target product.

10. The method according to claim 9, characterized in that The starting bacteria of the engineering bacteria include but are not limited to: at least one of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Bacillus subtilis, Corynebacterium glutamicum, Lactobacillus, Streptomyces, Xanthomonas, and Pseudomonas.

11. The method according to claim 9, characterized in that The target protein includes: fucosyltransferase and / or SUMO protein; the target product is 2'-fucosyllactose.

12. The method according to claim 9, characterized in that The target genes include FucT-SUMO 、 FutC- SUMO 、 BKHT-SUMO At least one of .

13. The method according to claim 11 or 12, characterized in that 1 to 8 copies of the target gene are inserted into the genome of the engineered bacteria.

14. The method according to claim 13, characterized in that The copy number is 2 to 6.

15. An engineered bacterium, characterized in that: The engineered bacteria are constructed according to the method according to any one of claims 9 to 14.

16. Use of the engineered bacteria according to claim 15 in producing a target product.

17. The use according to claim 16, characterized in that The target product is 2'-fucosyllactose.

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