Method for screening target insertion sites and applications thereof

Abstract

The present application relates to the field of genetic engineering, and discloses a method for screening a target insertion site and application thereof, which comprises the following steps: constructing a plasmid for expressing a target gene, extracting total RNA of an engineering bacterium carrying the plasmid to obtain an RNA sample; performing transcriptome sequencing on the RNA sample to obtain transcriptome sequencing data; performing alignment processing on the transcriptome sequencing data and a reference genome of the engineering bacterium to determine the FPKM value of each gene in the engineering bacterium; and determining the distance between each gene and a downstream gene in the engineering bacterium; when the FPKM value of a 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, improve the accuracy and pertinence of gRNA design, and be used for optimizing the design of gRNA for MUCICAT gene editing, and has the advantages of strong pertinence, good predictability, high gene editing efficiency, good flexibility, stable target gene copy number, good strain subculture stability and high target metabolite yield.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and more specifically, to a method for screening target insertion sites and its application. Background Technology

[0002] Human milk oligosaccharides (HMOs) are important nutrients in breast milk, among which 2′-fucosyllactose (2′-FL) is the third most abundant nutrient after lactose and fat. HMOs are crucial for infant immune function and nervous system development, and the industrial production of HMOs for use in dairy products is an inevitable trend. The main production methods include chemical synthesis, in vitro enzymatic methods, and microbial fermentation. Chemical synthesis, due to its complexity and toxicity, cannot be mass-produced; enzymatic synthesis requires expensive nucleotide sugar substrates and cofactors, limiting its industrial production. Therefore, microbial fermentation has become the mainstream method for producing 2′-FL in industrial settings.

[0003] Currently, the technologies used to integrate α-1,2-fucosyltransferase genes into the genome include Red-Et homologous recombination and CRISPR / Cas9. The Red-Et homologous recombination system is cumbersome, time-consuming, and leaves an FRT scar in the genome. Furthermore, excessive scarring can destabilize the host genome, potentially leading to rearrangements. The CRISPR / Cas9 system generates double-strand breaks during editing, requiring repair fragments; however, the repair method is non-homologous end joining, a highly error-prone method resulting in a low accuracy rate. In addition, due to differences in gene sequences, not all genes have suitable PAMs (penetrating amylases), thus limiting the CRISPR / Cas9 system to PAM recognition sequences. Most importantly, the CRISPR / Cas9 system has extremely high off-target efficiency, and the site-specific gene editing efficiency decreases with increasing editing sites. The latest CRISPR-associated transposases (MUCICAT) system is time-saving and labor-saving, independent of the host homologous recombination system and DNA double-strand breaks. Its editing efficiency is unaffected by the editing site and is not limited by PAM (Polymerization of Adenosine Transposons). sgRNA design only needs to meet the distance between the target site and the transposon integration site without affecting the expression of upstream and downstream genes. However, different genetic backgrounds and culture conditions can cause host bacteria to develop different transcriptomes to adapt to environmental changes. Without fully considering this, gRNA design lacks specificity and predictability, and is somewhat arbitrary.

[0004] Therefore, MUCICAT technology faces challenges 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] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides a method for screening target insertion sites and its applications. The method of this invention can accurately determine target insertion sites, improving the accuracy, purposefulness, flexibility, and specificity of gRNA design. It is used to optimize the design of gRNAs for MUCICAT gene editing, exhibiting strong specificity and good predictability. When used to construct engineered bacteria, it provides high gene editing efficiency, good flexibility, stable target gene copy number, good strain passaging stability, and high target metabolite yield.

[0006] This invention is based on the inventor's discoveries and understanding of the following problems:

[0007] Existing MUCICAT gene editing technology uses plasmid systems to regulate changes in metabolic pathways within bacteria. However, plasmid systems require antibiotics for maintenance and suffer from limitations such as copy number instability and susceptibility to loss. Furthermore, different genetic backgrounds and culture conditions lead to different transcriptomes in host bacteria to adapt to environmental changes, resulting in the blind application of gRNAs. To address this issue, the inventors designed and developed a method for screening high-expression gene target insertion sites. Then, based on MUCICAT technology and the selected high-expression gene target insertion sites, 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 that produces a high yield of the target metabolite. Further experimental results demonstrate that the method for constructing engineered bacteria in this invention exhibits highly targeted and predictive gRNA design, high gene editing efficiency and accuracy, and good target gene copy number and strain passaging stability, resulting in high yields of the target metabolite.

[0008] Therefore, in a first aspect, the present invention provides 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 engineered bacteria. The method includes: 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 a 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 a downstream gene; 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. The method according to the embodiments of the invention can accurately determine the target insertion site, improving the accuracy, purposefulness, flexibility, and specificity of gRNA design, overcoming the blindness of gRNA design, and is used to optimize the design of gRNA for MUCICAT gene editing. It has strong specificity and good predictability. When used to construct engineered bacteria, it has high gene editing efficiency and accuracy, stable target gene copy number, good strain passaging stability, and high target metabolite yield.

[0009] In a second aspect, the present invention provides a method for designing gRNAs. According to an embodiment of the invention, the gRNA is used to insert a target gene into the genome of an engineered bacterium, and the method includes: determining the target insertion site of the gRNA corresponding to the engineered bacterium genome according to the aforementioned method; and determining the nucleotide sequence of the gRNA based on the target insertion site. According to the method of the present invention, the gRNA for MUCICAT gene editing obtained based on the aforementioned target insertion site is highly targeted, predictive, and has high gene editing efficiency and accuracy.

[0010] In a third aspect, the present invention provides a gRNA. According to embodiments of the present invention, the gRNA is designed according to the foregoing method.

[0011] Those skilled in the art will understand that the features and advantages described above regarding the methods for screening target insertion sites and designing gRNAs also apply to this gRNA, and will not be repeated here.

[0012] In a fourth aspect, the present invention provides a method for constructing engineered bacteria. According to an embodiment of the present invention, the method includes: obtaining gRNA based on the foregoing method; inserting at least one copy number 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 generate a target product. The method according to the embodiments of the present invention has high gene editing efficiency and accuracy, and the constructed engineered bacteria exhibit stable target gene copy numbers, good strain passaging stability, and high yield of target metabolites.

[0013] 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.

[0014] Those skilled in the art will understand that the features and advantages described above regarding the methods for screening target insertion sites, designing gRNAs, and constructing engineered bacteria are also applicable to this engineered bacteria, and will not be repeated here.

[0015] In a sixth aspect, the present invention provides the use of the aforementioned engineered bacteria in the production of a target product, wherein the target product is 2′-fucosylated lactose.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0017] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0018] Figure 1 This is a schematic diagram of the 2′-fucosylation-lactose metabolic pathway according to an embodiment of the present invention;

[0019] Figure 2 Used in Embodiment 2 of the present invention GFP The results of the validation study on the expression level at the insertion site are shown in the figure. Among them, (a) shows the expression level at 4 ( ) using the original gRNA as a guide. ydgA-uidc ) and No. 8 ( yiM-cpxA )have GFP (a) Results of randomly inserted strains, referred to as strain 1; (b) Results of using a designed gRNA as a guide in strain 2. ldtE-lpp )have GFP The randomly inserted strains are referred to as strain 2 in the result observation diagram; (c) shows strains 1 and 2. GFP The results of the emission light detection are shown in Figure 1; (d) shows bacteria 1 and bacteria 2. GFP Figure showing the results of the excitation light detection.

[0020] Figure 3 As in Embodiment 3 of the present invention BKHT-SUMO Figure 1 shows the results of copy number detection and plasmid elimination investigation. In figure (a), 3 copies are considered (…). fur-chiQ , ldtE-lpp and yecH-ftnA (a) Colony PCR detection results of strain (b) are shown; (c) shows 4 copies of (a) fur-chiQ , ldtE-lpp , yecH-ftnA and yjgM-rraB (c) shows the colony PCR detection results of strain 5; fur-chiQ , ydhP-sodB , ldtE-lpp , yecH-ftnA and yjgM-rraB ) BKHT-SUMO Image showing colony PCR results of the strain; (d) shows 3, 4, and 5 copies. BKHT-SUMO Figure 1 shows the results of the pQCasTns(Ptr) plasmid elimination study of the strain; (e) shows 3, 4, and 5 copies. BKHT-SUMO Figure 1 shows the results of the pDonor plasmid elimination study of the strain; (f) shows 3, 4, and 5 copies. BKHT-SUMO Figure 1 shows the results of the pCutamp plasmid elimination study of the strain; (g) represents 3, 4, and 5 copies. BKHT-SUMO Figure showing the results of the pCutamp plasmid elimination verification study of the strain;

[0021] [[ID=二十九]]Figure 4 This is a graph showing the results of investigating the content of the target metabolite 2′-FL after fermentation by the multi-copy strain in Example 4 of the present invention. Detailed Implementation

[0022] 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 construed as limiting the present invention.

[0023] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0024] The endpoints and any values ​​of the ranges 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 endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0026] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0027] Terms and Definitions

[0028] In this article, the term "FutC" refers to fucosyltransferase derived from Helicobacter pylori; italics indicate the gene encoding this protein.

[0029] In this article, the term "FucT" refers to fucosyltransferase derived from Helicobacter pylori; italics indicate the gene encoding the protein.

[0030] In this article, the term "BKHT" refers to fucosyltransferase derived from Helicobacter pylori; italics indicate the gene encoding this protein.

[0031] In this paper, the term "SUMO protein" refers to a small ubiquitin-like modifier that affects the function and localization of a target protein through reversible post-translational modifications.

[0032] In this paper, the term “2′-fucosylated lactose” is equivalent to “2′-FL”.

[0033] In this paper, the term “PAM” refers to a short DNA sequence used by the CRISPR-Cas system as a marker to distinguish “self” from “invader”.

[0034] In this paper, the term "GFP" refers to green fluorescent protein, which can form a chromophore through self-catalysis and emit green fluorescence when excited by blue light or ultraviolet light.

[0035] In this paper, the term "pQCasTns plasmid" refers to a vector for expressing CRISPR-associated transposases, which can target specific genomic regions under the guidance of the CRISPR system to achieve the insertion or deletion of DNA fragments.

[0036] In this paper, the term "pDonor plasmid" refers to a vector used in gene knock-in experiments, typically to provide homologous arms or homologous sequences to facilitate homologous recombination at the target site.

[0037] In this paper, the term "pCutamp plasmid" refers to a plasmid knockout system used in Escherichia coli.

[0038] For example, the "pCutamp plasmid" is used to eliminate the dual plasmids pQcasTns and pDonor.

[0039] This invention proposes 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.

[0040] Methods for screening target insertion sites

[0041] This invention proposes a method for screening target insertion sites. According to an embodiment of the invention, the target insertion site is used to insert a target gene into engineered bacteria. The method includes: 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 a 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 a downstream gene. 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 considered a target insertion site. The method according to the embodiments of the invention can accurately determine the target insertion site, improving the accuracy, purposefulness, flexibility, and specificity of gRNA design, overcoming the blindness of gRNA design, and is used to optimize the design of gRNA for MUCICAT gene editing. It has strong specificity and good predictability. When used to construct engineered bacteria, it has high gene editing efficiency and accuracy, stable target gene copy number, good strain passaging stability, and high target metabolite yield.

[0042] The method according to the embodiments of the invention identifies highly expressed target sites through transcriptome sequencing and data analysis. These sites are directly related to gRNA design, thereby improving the accuracy and purposefulness of gRNA design. The method also utilizes transcriptome data from different strains to design gRNAs, allowing for personalized design based on the specific transcriptome background of each strain, thus enhancing the flexibility and targeting of gRNA design. Compared to gRNAs provided by existing MUCICAT technology, the method according to the embodiments of the invention overcomes the blindness of gRNA design and the technical deficiency of different genetic backgrounds and culture conditions leading to different transcriptomes in host bacteria, thereby improving the production performance of engineered bacteria.

[0043] In this paper, the term "FPKM value" refers to the number of fragments read per million maps per thousand exons, and is an indicator used to measure gene expression levels.

[0044] In this paper, the term "MUCICAT gene editing" refers to the multicopy chromosome integration technology of CRISPR-associated transposases, a novel gene editing tool that utilizes CRISPR-Cas system-associated transposases to insert DNA fragments into specific sites of the genome via RNA guidance, without relying on the host cell's homologous recombination mechanism.

[0045] In this article, the term "gRNA" is equivalent to "guide RNA," a key component of the CRISPR-Cas9 system. It consists of CRISPR RNA (crRNA) and transcription-activating CRISPR RNA (tracrRNA), which can specifically recognize and pair with the target DNA sequence to guide the Cas9 endonuclease to cut the target DNA at a specific location, thereby enabling gene knockout, insertion, or site-directed mutation editing operations.

[0046] According to some specific embodiments of the present invention, the transcriptome sequencing data is filtered, and the transcriptome sequencing data is no less than 6 Gb. The filtering process includes: removing adapter sequences, removing sequences with low sequencing quality values, and removing sequences with an uncertainty base ratio higher than 5%. Sequences with low sequencing quality values ​​are those where the proportion of bases with a quality value lower than 15 is greater than 20% of the total number of bases in the sequence. This obtains high-quality sequencing data (cleandata), further improving the accuracy of the screening results.

[0047] According to a specific embodiment of the present invention, the comparison software for the comparison processing is Bowtie2.

[0048] According to embodiments 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*, lactic acid bacteria, *Streptomyces*, *Xanthomonas*, and *Pseudomonas*. The method according to embodiments of the present invention can be used to screen for high-expression target insertion sites in the genomes of various strains producing secondary metabolites, and based on this, optimize the design of gRNAs, thereby improving the efficiency of gene editing and the yield of target metabolites, overcoming the influence of different genetic backgrounds and culture conditions on the effectiveness of gRNAs, and enhancing the production performance of the strains.

[0049] According to embodiments of the present invention, the target gene encodes a target protein, the target protein comprising: fucosyltransferase and / or SUMO protein. Figure 1As shown, the production of 2′-FL involves a key gene, namely the α-1,2-fucosyltransferase gene. According to the method of this embodiment, suitable insertion sites for high expression of this gene are screened and identified. Based on this, gRNAs for MUCICAT gene editing are optimized and screened, resulting in high gene editing efficiency and accuracy. The constructed engineered bacteria have stable copy numbers, good strain passaging stability, and high yield of the target metabolite 2′-FL. According to embodiments of this invention, the target proteins include fucosyltransferase and SUMO protein.

[0050] In this document, the term "SUMO protein" is equivalent to "Small Ubiquitin-like Modifier". According to the method of embodiments of the present invention, fucosyltransferase and SUMO protein are fused and expressed to further improve the stability, expression level, and protease activity of fucosyltransferase.

[0051] According to an embodiment of the present invention, the target gene includes FucT-SUMO , FutC-SUMO , BKHT-SUMO At least one of them.

[0052] According to the method of this invention, after mining high-expression gene target insertion sites based on MUCICAT technology and transcriptome data, key genes are... BKHT-SUMO Multiple copies are integrated into the genome, constructing different copy numbers. BKHT-SUMO The engineered E. coli can effectively enhance the synthesis of 2′-fucosylated lactose (2′-FL) and achieve efficient synthesis of 2′-FL.

[0053] Methods for designing gRNA

[0054] This invention proposes a method for designing gRNA. According to an embodiment of the invention, the gRNA is used to insert a target gene into the genome of an engineered bacterium, and the method includes: determining the target insertion site of the gRNA corresponding to the genome of the engineered bacterium according to the foregoing method; and determining the nucleotide sequence of the gRNA based on the target insertion site.

[0055] According to the method of the present invention, the gRNA for MUCICAT gene editing obtained by screening based on the aforementioned target insertion site is highly targeted, predictive, and has high gene editing efficiency and accuracy.

[0056] gRNA

[0057] This invention proposes a gRNA. According to embodiments of the invention, the gRNA is designed according to the aforementioned method.

[0058] Those skilled in the art will understand that the features and advantages described above regarding the methods for screening target insertion sites and designing gRNAs also apply to this gRNA, and will not be repeated here.

[0059] According to an embodiment of the present invention, the target insertion site of the gRNA corresponding to the engineered bacterial genome includes: [[ID=三十六]]ychH , fur , sodB , ftnA , lpp , ytfK , glpD and rraB At least one of them. According to the method of embodiments of the present invention, the stability, expression level, and biological activity of the target protein are further improved.

[0060] According to an embodiment of the present invention, the gRNA has a nucleotide sequence as shown in at least one of SEQ ID NO: 25-32.

[0061] According to some specific embodiments of the present invention, the engineered bacteria is BL21Star™ (DE3). ΔlacZ ΔwcaJ ΔnudD ΔnudK .

[0062] It should be noted that the above can be achieved in BL21Star™ (DE3). " In Escherichia coli, the accurate and efficient insertion of the nucleotide sequence of the gRNA of the target gene using MUCICAT editing technology is an example.

[0063] Methods for constructing engineered bacteria

[0064] This invention proposes a method for constructing engineered bacteria. According to an embodiment of the invention, the method includes: obtaining gRNA based on the aforementioned method; inserting at least one copy number 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 generate a target product. The method according to the embodiment of the invention has high gene editing efficiency and accuracy, and the constructed engineered bacteria exhibit stable target gene copy numbers, good strain passaging stability, and high yield of target metabolites.

[0065] According to embodiments 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, lactic acid bacteria, Streptomyces, Xanthomonas, and Pseudomonas.

[0066] According to an embodiment of the present invention, the target protein includes: fucosyltransferase and / or SUMO protein; the target product is 2′-fucosyllactose.

[0067] According to an embodiment of the present invention, the target gene includes ΔlacZ ΔwcaJ ΔnudD ΔnudK , FucT-SUMO , FutC-SUMO At least one of them.

[0068] According to embodiments of the present invention, 1 to 8 copies of the target gene are inserted into the genome of the engineered bacteria. The method according to embodiments of the present invention is used to construct bacteria with different copy numbers. BKHT-SUMO Engineered E. coli genes enable efficient synthesis of 2′-FL.

[0069] According to embodiments of the present invention, the copy number is 2 to 6. The method according to embodiments of the present invention further improves the synthesis efficiency of 2′-FL.

[0070] For example, the number of copies can be 2, 3, 4, 5, or 6.

[0071] engineered bacteria

[0072] This invention proposes an engineered bacterium. According to an embodiment of the invention, the engineered bacterium is constructed according to the aforementioned method.

[0073] Those skilled in the art will understand that the features and advantages described above regarding the methods for screening target insertion sites, designing gRNAs, and constructing engineered bacteria are also applicable to this engineered bacteria, and will not be repeated here.

[0074] use

[0075] This invention proposes the use of the aforementioned engineered bacteria in the production of the target product.

[0076] According to an embodiment of the present invention, the target product is 2′-fucosylated lactose.

[0077] Those skilled in the art will understand that the features and advantages described above regarding the methods for screening target insertion sites, designing gRNAs, and constructing engineered bacteria are also applicable to this purpose, and will not be repeated here.

[0078] To achieve the above objectives, this invention relates to five strains of BL21Star™(DE3): BL21Star™(DE3) carrying the dual plasmids pCDFduet-BCGW and pETDuet-FucT-SUMO (without inducing exogenous gene expression), abbreviated as BL21Star™(DE3)-FucT-SUMO (-); BL21Star™(DE3) carrying the dual plasmids pCDFduet-BCGW and pETDuet-FucT-SUMO (with inducing exogenous gene expression), abbreviated as BL21Star™(DE3)-FucT-SUMO (+); and BL21Star™(DE3) carrying the dual plasmids pCDFduet-BCGW and pETDuet-FutC-SUMO (without inducing exogenous gene expression), abbreviated as BL21Star™(DE3)-FutC-SUMO. The transcriptomes of BL21Star™ (DE3) (inducing exogenous gene expression) and BL21Star™ (DE3)-Fut C-SUMO (+) strains carrying the dual plasmids pCDFduet-BCGW and pETDuet-FutC-SUMO were fermented and then sequenced. Comprehensive data analysis identified eight highly expressed target sites, and gRNAs were designed. Using the gRNAs described in the literature [Yang S, Zhang Y, Xu J, et al. Orthogonal CRISPR-associated transposases for parallel and multiplexed chromosomal integration[J]. Nucleic AcidsResearch, 2021, 49(17): 10192-10202.], and with GFP as a probe, GFP was inserted into the host bacterial genome for fermentation. The highly expressed target sites were verified by microplate reader detection. Therefore, the gRNAs designed based on different transcriptome data of the strains in this invention are more flexible than the gRNAs described in the literature, and the yield of the target products is higher. Subsequently, the key gene α-1,2-fucosyltransferase was used to replace GFP and multiple copies were inserted into E. coli for fermentation. Mass spectrometry was used to verify that the copy number of the key enzyme gene was directly proportional to the yield of 2′-FL.

[0079] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0080] Example 1: Culture and sequencing of the strain

[0081] 1: Strain culture

[0082] Strain: Bacillus BL21Star™ (DE3) BKHT-SUMO The experiment has already been conducted.

[0083] 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 culture media (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 20 g / L agar).

[0084] strain BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK Δ - lacZ ΔwcaJ ΔnudD ΔnudK And BL21Star™ (DE3) FutC-SUMO ΔlacZ ΔwcaJ ΔnudD Δ - nudK In 12 mL shake tubes containing 3 mL of LB liquid medium, the culture was incubated at 220 rpm and 37°C until the OD600 reached 0.6–0.8. The culture was then transferred to BL21Star™ (DE3). FucT-SUMO - ΔlacZ ΔwcaJ ΔnudD ΔnudK And BL21Star™ (DE3) FutC-SUMO - ΔlacZ ΔwcaJ ΔnudD ΔnudK 0.5 mM IPTG was added, and all strains were incubated at 220 rpm and 25°C with shaking for 72 h. Then, strain BL21Star™(DE3) was extracted using the Tiangen RNA extraction kit. FucT-SUMO BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - ΔlacZ ΔwcaJ ΔnudD ΔnudK (-), BL21Star™ (DE3) FutC-SUMO - ΔlacZ ΔwcaJ ΔnudD ΔnudK (+), BL21Star™ (DE3) FutC-SUMO - ΔlacZ ΔwcaJ ΔnudD ΔnudK (-) and BL21Star™ (DE3) [[ID=六十九]]FucT-SUMO ΔlacZ ΔwcaJ - ΔnudD ΔnudK The (+) RNA was sent to BGI Genomics Co., Ltd. in Hainan for transcriptome sequencing.

[0085] 2: Sequencing and gene screening

[0086] Transcriptome sequencing was performed on the samples using the BGI Genomics T20 high-throughput sequencing platform, yielding at least 6Gb of raw data per sample. The raw data was then filtered using SOAPnuke (version 1.5.6) to remove adapters, sequences with excessively low sequencing quality values, and sequences with excessively high N-ratios, resulting in high-quality clean data. This clean data was then compared with the *E. coli* reference genome (…). FucT -SUMO The K-12 gene was compared and analyzed using Bowtie2 (version 2.4.5). The expression level (FPKM value) of each gene was calculated based on the comparison results. Gene regions with significantly high expression and adjacent transcription directions (FPRM value greater than 600) were identified.

[0087] Genetic screening was conducted to select BL21Star™ (DE3). Escherichia coli BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - ΔlacZ ΔwcaJ ΔnudD ΔnudK (-), BL21Star™ (DE3) FucT-SUMO ΔlacZ ΔwcaJ Δ - nudD ΔnudK (+), BL21Star™ (DE3) FucT-SUMO - ΔlacZ ΔwcaJ ΔnudD ΔnudK (-) and BL21Star™ (DE3) FutC-SUMO - ΔlacZ ΔwcaJ ΔnudD ΔnudK (+) Transcriptome data analysis of 5 strains screened for genes with relatively high expression levels and genes that were more than 53 bp away from downstream genes as target insertion sites, and the target gene was inserted between upstream and downstream genes.

[0088] The highly expressed genes selected include FutC-SUMO , ychH , fur , sodB , ftnA , lpp , ytfK and glpD .

[0089] In this embodiment, the five Escherichia coli strains were sequenced, and the expression levels of the high-expression genes screened are shown in Table 1 below.

[0090] Table 1. Expression levels of highly expressed genes

[0091]

[0092] in,

[0093] BL21star_F_S indicates strain BL21Star™ (DE3). rraB - ΔlacZ ΔwcaJ ΔnudD ΔnudKFutC- (-) exogenous genes SUMO Uninduced expression; BL21star_F indicates strain BL21Star™ (DE3). FutC-SUMO ΔlacZ - ΔwcaJ ΔnudD ΔnudK (+) exogenous genes FutC-SUMO Induced expression, BL21star represents strain BL21Star™ (DE3). FutC-SUMO ΔlacZ ΔwcaJ ΔnudD ΔnudK , BL21star_T_S indicates strain BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FucT-SUMO (-) exogenous genes FucT-SUMO Uninduced expression; BL21star_T indicates strain BL21Star™ (DE3). ΔlacZ ΔwcaJ ΔnudD ΔnudK - FucT-SUMO (+) exogenous genes FucT-SUMO Induced expression.

[0094] Example 2: Discovering insertion sites of highly expressed gene targets

[0095] 1: gRNA and primer design

[0096] Highly expressed genes were screened using transcriptome data from five different strains. Eight highly expressed genes were selected, and eight gRNA sequences were designed downstream of them according to the MUCICAT gRNA design principle. The publicly available "original gRNA sequences," the eight gRNA sequences designed in this example, and the validation primers are shown in Tables 2 and 3.

[0097] 2: Plasmid Construction

[0098] pQCasTns(Ptr)-array 8 (carrying the "original gRNA") and pCutamp were purchased from GenScript, while pQCasTns(Ptr)-array 8 (carrying the "designed gRNA") was synthesized by GenScript. The cargo gene was ligated via Gibson-Assembly. FutC-SUMO and GFP pDonor, construct pDonor-RE- FutC-SUMO -LE and pDonor- RE- GFP -LE.

[0099] In this paper, the nucleotide sequence of the pQCasTns(Ptr)-array 8 carrying the "original gRNA" is determined by reference: Yang S, Zhang Y, Xu J, et al. Orthogonal CRISPR-associated transposases for parallel and multiplexed chromosomal integration[J]. Nucleic AcidsResearch, 2021, 49(17): 10192-10202. This is referred to simply as the "original gRNA". Detailed sequence information is shown in Table 2.

[0100] In this paper, pQCasTns(Ptr)-array 8 carrying the "designed gRNA" has its gRNA nucleotide sequence determined according to the target insertion site identified in Example 1. ychH , fur , sodB , ftnA , lpp , ytfK , glpD and rraB The design was obtained. See Table 3 for detailed sequence information.

[0101] Table 2. Original gRNA and primer sequences

[0102]

[0103] Table 3. Designed gRNA and primer sequences

[0104]

[0105] Note: NR indicates an unnamed gene.

[0106] 3: Production of electrocompetent cells

[0107] First, the strain BL21Star™ (DE3) stored at -80℃ was... ΔlacZ ΔwcaJ ΔnudD ΔnudKActivate the cells by streaking on LB agar plates, and then incubate single colonies overnight. Next, inoculate 1% of the culture into 250 mL SOB liquid medium in a 500 mL Erlenmeyer flask and incubate at 37°C with shaking at 220 rpm until the OD600 reaches 0.4–0.5. After incubation, let the culture stand on ice for 15 min, then centrifuge at 5000g for 30 min at 4°C. Collect the cells and gently shake them on a decolorizing shaker with 10% pre-chilled glycerol until the cells disperse. Centrifuge at 5000g for 30 min at 4°C. Repeat this washing process three times. For the final centrifugation, dispersing the cells with approximately 1 mL of 10% glycerol, aliquot 50 μL into 1.5 mL centrifuge tubes. Finally, flash-freeze the competent cells in liquid nitrogen and store at -80°C until use.

[0108] 4: GFP transposition experiment, transposition efficiency analysis, and sequencing

[0109] CAST's transposition experiments involve the transformation and induction of pDonor and pQCasTns(Ptr)-array 8. First, pQCasTns(Ptr)-array 8 (the original gRNA) and pDonor-RE- GFP -LE, pQCasTns(Ptr)-array 8 (designed gRNA) and pDonor-RE- GFP -LE is converted to BL21Star™ (DE3) via common electrostatic conversion. ΔlacZ ΔwcaJ ΔnudD ΔnudK Competent cells were added to 800 μL of liquid SOB medium and incubated at 37°C and 220 rpm for 1 hour. The cells were then collected by centrifugation at 5000g for 1 min. 100 μL of liquid LB medium was used to dilute the cells and spread them onto LB agar plates containing 100 μg / ml ampicillin and 100 μg / ml kanamycin. The cells were incubated overnight at 37°C for 16 hours. The next day, hundreds of colonies were scraped from the plates, resuspended in fresh LB liquid medium, and then spread onto LB agar plates containing 100 ng / mL acyclovir (aTC) and the two antibiotics. The cells were 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 redistributed and then re-coated onto plates containing 1000 ng / ml aTC and the two antibiotics. The colonies were incubated at 25°C for 48 hours. Finally, the colonies were streaked and cultured at 25°C to form single clones, and colony PCR was performed to analyze transposition efficiency and screen positive clones for sequencing.

[0110] 5: GFP microplate reader detection

[0111] Take samples containing the dual plasmids pQCasTns(Ptr)-array 8 (original gRNA) and pDonor-RE- GFP -LE positive clone BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK Contains dual plasmids pQCasTns(Ptr)-array 8 (designed gRNA) and pDonor-RE- GFP -LE positive clone BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK and control group BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK Incubate in LB liquid medium for 3 hours, adjust OD to 0.25, and then take 100 μL of bacterial culture and transfer it to a microplate reader (λ). ex = 460 nm, λ em = 500-750 nm; λ em =550, λ ex Detection was performed at 300-500 nm. Based on the MUCICAT system, using the original gRNA and the designed gRNA as guides, [the following was observed / detected]. GFP Multiple copies inserted into E. coli BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK The genome is sequenced, fermented, and analyzed using an enzyme-linked immunosorbent assay (ELISA) reader.

[0112] The results are as follows Figure 2 As shown.

[0113] The results showed that using the dual 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 in one transposition experiment, and 2 and 1 locations were detected respectively. GFP As can be seen from the fluorescence intensity of the GFP protein, the fluorescence intensity value of the GFP protein inserted at one location using the designed gRNA is close to the fluorescence intensity value of the GFP protein inserted at two locations using the original gRNA.

[0114] The above results indicate that combining transcriptome data mining of highly expressed gene sites significantly promotes protein expression levels after gene insertion. Therefore, a designed gRNA was selected for the target gene (fucosyltransferase gene). BKHT-SUMO Insert the rotatable connector.

[0115] Example 3: BKHT-SUMO Copy number detection and plasmid elimination

[0116] 1: BKHT-SUMOTransposon experiments and first-generation sequencing

[0117] First, the dual plasmid pQCasTns(Ptr)-array 8 (designed gRNA) and pDonor-RE- GFP -LE common-electric conversion to BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK Competent cells were added to 800 μL of liquid SOB medium and incubated at 37°C and 220 rpm for 1 hour. The cells were then collected by centrifugation at 5000g for 1 min and diluted with 100 μL of liquid LB medium. The cells were then spread onto LB agar plates containing 100 μg / ml ampicillin and 100 μg / ml kanamycin and incubated overnight at 37°C for 16 hours. The next day, colonies were scraped off the plates and partially resuspended in fresh LB liquid medium. These colonies were then spread onto LB agar plates containing 100 ng / mL acyclovir (aTC) and the two antibiotics and incubated at 25°C for 48 hours to induce protein expression. The resulting membrane was scraped off and partially resuspended in LB liquid medium, then re-coated onto plates containing 1000 ng / ml aTC and LB broth containing two antibiotics. Colonies were grown at 25°C for 48 h. Subsequent operations were performed on LB solid culture containing 1000 ng / ml aTC and two antibiotics, and this step was repeated 8 times. For each replicate, a streaking dilution was performed at 25°C to create single colonies, which were then subjected to colony PCR to analyze transposon efficiency. Positive clones were screened for sequencing to confirm successful insertion of the transposon into the specific site of the target genome, following the designed transposon direction, without any unexpected mutations or recombination events.

[0118] 2: Plasmid elimination

[0119] The pCutamp plasmid contains sgRNAs targeting the pQcasTns and pDonor plasmids. The spacer region of the sgRNAs is the shared sequence 5′-TGCTTCAATAATATTGAAAAAGG-3′ of the AmpR promoter in both plasmids. Rhamnose induces sgRNA transcription, guiding the Streptococcus pyogenes Cas9 protein to target and cleave the shared sequence. Because no homologous template was provided, the target plasmid could not be repaired. First, the positive clones were converted into electrocompetent cells. pCutamp was electroporated into these cells, and 800 μL of liquid SOB medium was added. The cells were incubated at 37°C and 220 rpm for 1 hour. To improve plasmid elimination efficiency, the incubated culture was transferred to 4 mL of liquid LB medium containing 50 μg / mL apramycin and 10 mM rhamnose, and incubated at 37°C with shaking for 6 hours. 100 μL of the incubated culture was then spread onto LB agar plates containing 50 μg / mL apramycin and 10 mM rhamnose and incubated overnight at 37°C. To eliminate the pCutamp plasmid, the overnight-grown strains were re-spread onto LB agar plates containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 10 g / L sucrose, respectively. Colonies that could not grow on plates containing ampicillin and kanamycin were considered to have eliminated pDonor and pQCasTns. The corresponding colonies grown on sucrose plates were then re-inoculated onto non-selective LB agar plates and plates containing apramycin to verify the elimination of the pCutamp plasmid. Colonies that could not grow on plates containing apramycin were plasmid-free, multi-copy strains.

[0120] The results of copy number colony PCR detection and plasmid elimination investigation are as follows: Figure 3 As shown.

[0121] The results showed that BL21Star™ (DE3) in pQCasTns(Ptr)-array 8 (designed gRNA) and pDonor-RE-BKHT-SUMO-LE... ΔlacZ ΔwcaJ ΔnudD ΔnudK In transposition experiments, the GFP gene was replaced with the fucosyltransferase gene BKHT-SUMO in the strain, and BL21Star™ (DE3) containing 3, 4, and 5 copies of BKHT-SUMO were obtained. ΔlacZ ΔwcaJ ΔnudD ΔnudK The strains (the strains are named strain 3, strain 4 and strain 5 respectively).

[0122] The experimental results show that the designed gRNA was used for the target gene. BKHT-SUMO The transposable insertion method is highly efficient and specific.

[0123] Furthermore, the three strains were subjected to plasmid elimination post-fermentation and mass spectrometry analysis of the target metabolite 2′-FL.

[0124] Example 4: Fermentation of multi-copy strains and mass spectrometry detection of target metabolites

[0125] The plasmid-free strain was streaked and revived. Single clones were cultured overnight, then inoculated at a 1% inoculation rate into 3 mL LB liquid medium containing 30 g / L glycerol in 12 mL shake tubes. The culture was carried out at 37°C and 220 rpm. When the OD reached 0.6–0.8, 0.5 mM IPTG was added, and the culture was continued at 25°C for 2 h. Then, 10 g / L lactose was added, and fermentation was continued for 72 h to verify the synthesis of 2′-FL. 100–200 μl of the fermentation broth sample was placed in a 1.5 mL centrifuge tube, boiled at 100°C for 10 min, cooled to room temperature, and centrifuged at 15000 rcf for 10 min. 10 μl of the supernatant was taken, diluted 100-fold with 990 μl of diluent and analyzed by LC-MS / MS.

[0126] The results are as follows Figure 4 As shown.

[0127] The results showed that: with the genome BKHT-SUMO With increasing copy number, 2′-FL production gradually increased to 112.45 mg / L (OD=6.49), 121.68 mg / L (OD=6.45), and 131.81 mg / L (OD=6.43), respectively.

[0128] The above results show that the target gene of the engineered bacteria constructed using Example 3 is effective. BKHT-SUMO With stable copy number and high yield of target metabolites, it can effectively enhance the synthesis of 2′-fucosylated lactose (2′-FL) and achieve efficient synthesis of 2′-FL.

[0129] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0130] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to 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 includes: A plasmid carrying the target gene was constructed, and total RNA was extracted from the engineered bacteria carrying the plasmid to obtain an RNA sample. The RNA sample was subjected to transcriptome sequencing to obtain transcriptome sequencing data; The transcriptome sequencing data is compared with the reference genome of the engineered bacteria to determine the FPKM value of each gene in the engineered bacteria; and the distance between each gene in the engineered bacteria and downstream genes is determined. 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 engineered bacteria were selected from BL21Star™ (DE3). ΔlacZ ΔwcaJ ΔnudD ΔnudK BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FutC-SUMO Or BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FucT-SUMO ; The target insertion site includes: ychH , fur , sodB , ftnA , lpp , ytfK , glpD and rraB At least one of them.

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

3. The method according to claim 2, characterized in that, The target gene includes FucT-SUMO , FutC-SUMO , BKHT-SUMO At least one of them.

4. 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 includes: The method according to any one of claims 1 to 3 determines the target insertion site of the gRNA corresponding to the genome of the engineered bacteria; The nucleotide sequence of the gRNA is determined based on the target insertion site.

5. A method for constructing engineered bacteria, characterized in that, The method includes: obtaining gRNA based on the method of claim 4, 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; The target gene encodes a target protein, which is used to generate the target product. The target insertion sites of the gRNA corresponding to the engineered bacterial genome include: ychH , fur , sodB , ftnA , lpp , ytfK , glpD and rraB At least one of them; The engineered bacteria were selected from BL21Star™ (DE3). ΔlacZ ΔwcaJ ΔnudD ΔnudK BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FutC-SUMO Or BL21Star™ (DE3) ΔlacZ ΔwcaJ ΔnudD ΔnudK - FucT-SUMO ; The target protein includes: fucosyltransferase and / or SUMO protein; the target product is 2′-fucosyllactose.

6. The method according to claim 5, characterized in that, The gRNA has a nucleotide sequence as shown in at least one of SEQ ID NO: 25-32.

7. The method according to claim 5, characterized in that, The target gene includes FucT-SUMO , FutC-SUMO , BKHT-SUMO At least one of them.

8. The method according to claim 5 or 7, characterized in that, Insert 1 to 8 copies of the target gene into the genome of the engineered bacteria.

9. The method according to claim 8, characterized in that, The number of copies is 2 to 6.

10. An engineered bacterium, characterized in that, The engineered bacteria are constructed according to any one of claims 5 to 9.

11. Use of the engineered bacteria of claim 10 in the production of the target product.

12. The use according to claim 11, characterized in that, The target product is 2′-fucosylated lactose.

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