A library of genome insertion sites of *Pseudomonas putida* and its construction method

Abstract

This invention belongs to the field of biotechnology, specifically relating to a *Pseudomonas putida* genome insertion site library and its construction method. This invention, for the first time, systematically screened and obtained 17 specific genome insertion sites suitable for *Pseudomonas putida*. The constructed insertion site library covers different expression intensities, filling the gap in existing technologies lacking efficient integration sites for *Pseudomonas putida*, and providing diverse options for personalized expression of target genes. The screened insertion sites are all located in the regions of opposing transcription genes, avoiding interference with the host's basal metabolic network. The recombinant strain exhibits stable growth performance without significant growth inhibition, making it suitable for large-scale industrial applications. The integration method, based on homologous recombination, achieves stable integration of the target gene without plasmid maintenance, avoiding expression instability caused by plasmid loss and copy number fluctuations. It also reduces the continuous use of antibiotics, lowering industrialization costs and environmental risks, and the construction method is simple and efficient.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a genome insertion site library suitable for *Pseudomonas putida*, including the specific location of the genome insertion site, the method for constructing the insertion site library, and the method for characterizing the expression intensity of the insertion site. Background Technology

[0002] In heterologous gene expression and the construction of microbial cell factories, stable and efficient expression of the target gene is the core objective, and the selection of the gene integration site directly determines the expression effect and strain stability. Compared with plasmid-mediated gene expression systems, integrating the target gene into the microbial genome can avoid problems such as unstable expression levels and plasmid loss caused by copy number fluctuations during plasmid replication, while also reducing the continuous use of antibiotic selection markers, making it more suitable for large-scale applications such as industrial fermentation. In addition, different genomic integration sites exhibit significant differences in transcriptional activity and chromosomal structural compatibility. Screening for specific sites that have high transcriptional efficiency, do not interfere with the function of essential host genes, and can achieve continuous and stable overexpression of the target gene is a key prerequisite for improving heterologous gene expression efficiency and promoting the industrialization of microbial synthetic biology technology.

[0003] *Pseudomonas putida*, a Gram-negative bacterium with broad-spectrum metabolic capabilities, possesses unique advantages in biodegradation, biocatalysis, and bio-based product synthesis, making it an important foundational strain for genetic engineering. However, current research on gene overexpression in *P. putida* still faces significant site-related technical bottlenecks: on the one hand, existing research on *P. putida* integration sites is limited, lacking systematically screened high-fit sites, resulting in low target gene expression levels, poor stability, and susceptibility to interference with the host metabolic network due to improper insertion sites, leading to strain growth inhibition; on the other hand, the *P. putida* genome contains complex regulatory elements and gene clusters, and random insertion or improper site selection can easily lead to transcriptional inhibition and low translation efficiency of the target gene. Furthermore, existing sites are insufficient to meet the personalized needs of different expression intensities (such as high expression and medium expression), severely limiting its potential for modification and application as a cell factory.

[0004] In light of the current technological status quo, there is an urgent need to develop a specific and efficient method for screening genome integration sites and adaptor sites for *Pseudomonas putida*, to address issues such as unstable expression, poor adaptability, and interference with host growth associated with existing sites. Systematic screening to obtain genome integration sites with excellent transcriptional activity and strong compatibility can not only achieve efficient and stable overexpression of the target gene but also avoid disrupting the metabolic functions of *Pseudomonas putida*, thereby improving the growth performance and industrial adaptability of engineered strains. Furthermore, the development of such sites can fill the technological gap in *Pseudomonas putida*-specific high-expression integration sites, providing key technical support for its engineering applications in fields such as biocatalysis and environmental remediation, and possessing significant practical value and industrialization prospects. Summary of the Invention

[0005] The present invention aims to overcome the defects of existing technologies, such as unstable expression, poor adaptability, and interference with host growth, in the integration site of *Pseudomonas putida*. It provides a *Pseudomonas putida* genome insertion site library and its construction method.

[0006] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: A library of insertion sites for the genome of *Pseudomonas putida*, wherein the insertion sites are located in the intergenic region of the genome of *Pseudomonas putida* KT2440, the intergenic region being formed by adjacent genes with opposite transcription directions, and the insertion sites being suitable for the integration of exogenous genes and enabling stable expression.

[0007] Because regions between genes with opposite transcription directions have a more open chromosomal spatial structure, their transcriptional activity is usually higher, and they can effectively avoid negative effects such as transcriptional readthrough or promoter interference with exogenous gene insertion fragments, thereby minimizing interference with the host's original metabolic network. This invention, by screening specific regions in the genome of *Pseudomonas putida* KT2440—namely, intergenic regions separated by adjacent genes with opposite transcription directions—as insertion sites, significantly improves the accuracy and scientific rigor of insertion site selection, ensuring that the selected insertion sites inherently possess good expression compatibility and host-friendliness.

[0008] Based on a site selection strategy that emphasizes the relative direction of transcription, this invention successfully obtained 17 specific insertion sites and constructed a library, achieving stable and efficient expression of exogenous genes and effectively avoiding the instability problem of plasmid systems. Simultaneously, the library exhibits gradient differences in expression intensity, providing diverse options to meet the personalized needs of gene expression levels in different application scenarios. Users can select the most suitable integration site according to the characteristics of the target product, achieving "programmable" and "tunable" expression levels.

[0009] Furthermore, because the selected insertion sites strictly avoid essential genes and key regulatory regions, the integration of exogenous genes hardly interferes with the host's normal life activities, effectively improving the growth compatibility with the host strain. This strongly demonstrates the friendliness of the selected insertion sites to the host metabolic network, helping to ensure that the engineered strain maintains vigorous growth during scale-up culture. In addition, the genome integration-based strategy eliminates the need for continuous antibiotic addition during fermentation for screening, significantly reducing production costs and mitigating the environmental risks and safety concerns associated with antibiotic use. This makes the technology more industrially viable and has greater potential for sustainable development.

[0010] Preferably, the number of insertion sites is 17.

[0011] Preferably, the sequence of genomic coordinates corresponding to the insertion site is shown in SEQ ID NO.1~17.

[0012] A recombinant *Pseudomonas putida* comprising a foreign gene integrated into an insertion site in a *Pseudomonas putida* genome insertion site library as described above.

[0013] Preferably, the exogenous gene is a reporter gene or a functional gene.

[0014] As a further preferred embodiment, the reporter gene is the green fluorescent protein gene (eGFP), the sequence of which is shown in SEQ ID NO. 18.

[0015] Preferably, the recombinant *Pseudomonas putida* uses *Pseudomonas putida* KT2440 as the chassis strain.

[0016] As a further preferred option, the exogenous gene is integrated into the insertion site through gene editing.

[0017] The above settings enable stable expression of exogenous genes.

[0018] A genome integration vector for constructing recombinant *Pseudomonas putida* as described above, the genome integration vector comprising recombinant sequences homologous to the insertion site upstream and downstream, a target gene expression cassette, and an sgRNA sequence for guiding integration.

[0019] Preferably, the genome integration vector is based on the CRISPR / Cas9 system and includes pCas vector and pSD vector.

[0020] As a further preferred embodiment, the pCas vector contains the Cas9 gene for DNA cleavage.

[0021] As a further preferred embodiment, the pSD vector comprises upstream and downstream homologous arm sequences of the insertion site, a target gene expression cassette, and sgRNA that guides the Cas9 protein to the insertion site.

[0022] A method for constructing a Pseudomonas putida genome insertion site library as described above includes the following steps: S1: Screening candidate insertion sites: Based on the genome annotation information of Pseudomonas putida KT2440, intergenic regions with opposite transcription directions were selected as candidate insertion sites; S2: Constructing a genome integration vector: Designing homologous arms and sgRNAs for candidate insertion sites, and assembling the target gene expression cassette; S3: Integration and Validation: The target gene was integrated into the corresponding insertion site in the genome of *Pseudomonas putida*, and recombinant *Pseudomonas putida* was obtained through resistance screening and PCR validation.

[0023] As a further preferred embodiment, a method for constructing a library of *Pseudomonas putida* genome insertion sites as described above includes the following steps: S1: Screening for candidate insertion sites: Based on the genome annotation information of Pseudomonas putida KT2440, intergenic regions with opposite transcription directions were selected as candidate insertion sites, and 17 candidate insertion sites were finally identified. S2: Constructing a genome integration vector: For each candidate insertion site, primers containing upstream and downstream homologous arms of the insertion site were designed and synthesized. Homologous arm fragments, Ptac promoter fragments, and reporter gene eGFP fragments were amplified by PCR. The above fragments were simultaneously inserted into the vector pSEVA using a one-step cloning technique to obtain 17 pSEVA-Donor vectors (pSD). The first 20 bp of sgRNA sequence on pSD was replaced with the corresponding target sequence (the first 20 bp of Pam site) by PCR to construct 17 specific genome integration vectors. S3: Integration and Validation (Construction of Recombinant Pseudomonas putida): The pCAS9 vector and pSD genome integration vector were transformed into Pseudomonas putida KT2440 competent cells. The genes were integrated into the corresponding insertion sites of the host genome using CRISPR gene editing technology. Positive recombinant strains were obtained through resistance screening and PCR verification, thus completing the construction of the Pseudomonas putida genome insertion site library. S4: Library characterizing insertion sites: The recombinant strain was cultured, and the expression level of the reporter gene at different culture stages was measured to determine the expression intensity level of each insertion site.

[0024] Preferably, the length of the homologous arm in step S2 is 500~800bp.

[0025] The above settings ensure the efficiency of homologous recombination.

[0026] Preferably, the target gene expression cassette in step S2 is driven by the Ptac promoter.

[0027] The above settings ensure the inducible expression of the target gene.

[0028] Preferably, the integration process in step S3 uses an electrical conversion method with conversion parameters of 2.5kV, 25μF, and 200Ω.

[0029] Preferably, in step S3, the resistance screening uses LB solid medium containing tetracycline and gentamicin.

[0030] Preferably, the method also includes a step of characterizing the expression intensity of the insertion site: culturing the recombinant *Pseudomonas putida* strain and measuring the relative expression level by reporter gene fluorescence intensity.

[0031] As a further preferred option, the culture conditions are 30°C and 220 rpm.

[0032] Preferably, the expression level of the reporter gene is determined by measuring the eGFP fluorescence intensity using a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 520 nm, and the relative fluorescence intensity is calculated to characterize the expression intensity.

[0033] The formula for calculating relative fluorescence intensity (RFI) is as follows: Relative fluorescence intensity (RFI) = Sample fluorescence intensity / Cell OD 600 .

[0034] Therefore, the present invention has the following beneficial effects: (1) This invention systematically screened and obtained 17 specific genomic insertion sites suitable for Pseudomonas putida for the first time. The constructed insertion site library covers different expression intensities, which fills the gap in existing technologies that lack efficient integration sites for Pseudomonas putida and provides diversified options for personalized expression of target genes. (2) The selected insertion sites are all located in the region of the opposite transcription gene, which avoids interference with the host's basic metabolic network. The recombinant strain has stable growth performance and no obvious growth inhibition, making it suitable for large-scale industrial application. (3) The integration of the target gene is achieved by homologous recombination, which does not require plasmid maintenance, avoids the expression instability caused by plasmid loss and copy number fluctuation, and reduces the continuous use of antibiotics, thus reducing industrialization costs and environmental risks. (4) The construction method is simple and efficient. It adopts CRISPR gene editing technology, which has high editing efficiency and provides a clear basis for the industrial application of insertion sites. Attached Figure Description

[0035] Figure 1 The growth curve of recombinant *Pseudomonas putida* over time.

[0036] Figure 2 The graph shows the expression curve of green fluorescent protein (eGFP) in recombinant Pseudomonas putida over time. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0038] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0039] Example 1: Screening and Determination of Candidate Genomic Insertion Sites (1) Acquisition and preliminary screening of genomic data: The genome sequence of Pseudomonas putida KT2440 (GenBank: CP169744.1) and complete genome annotation information were found from the NCBI database. Adjacent genes with opposite transcription directions were screened, that is, two adjacent non-essential genes with opposite transcription directions. The middle position of the intergenic region between such gene pairs was determined as the initial candidate integration site. At the same time, the length of the intergenic region where the candidate site is located was limited to ≥80bp to avoid the influence of insufficient space on the insertion and expression of foreign genes.

[0040] (2) Screening of highly specific Pam sites: Subsequently, based on the genome sequence of this strain in the NCBI database, the Protospacer adjacent motif (PAM) sites in the core region (integration region of the inserted fragment) of the selected site were systematically screened (NGG-type PAM sites were preferentially screened for commonly used CRISPR / Cas9-mediated homologous recombination systems); the specificity of the screened PAM sites was verified by the NCBIBLAST tool to ensure that the site sequence exists only uniquely in the genome of Pseudomonas putida KT2440. Finally, 17 preferred genome insertion sites were screened, and their core sequences are shown in SEQ ID NO.1~SEQ ID NO.17 (see Table 1 below).

[0041] The eGFP gene sequence is shown below: ATGGTGAGCAAAGGCGAGGAACTGTTCACCGGTGTGGTTCCGATCCTGGTTGAGCTGGACGGCGATGTGAACGGTCACAAGTTTAGCGTTAGCGGCGAGGGCGAAGGTGACGCGACCTACGGCAAGCTGACCCTGAAATTCATTTGCACCACCGGTAAACTGCCGGTTCCGTGGCCGACCCTGGTGACCACCCTGACCTACGGTGTGCAGTGCTTTAGCCGTTATCCGGACCACATGAAGCAACACGATTTCTTTAAAAGCGCGATGCCGGAGGGCTACGTTCAGGAACGTACCATCTTCTTTAAGGACGATGGTAACTATAAAACCCGTGCGGAAGTGAAGTTCGAAGGCGACACCCTGGTGAACCGTATCGAGCTGAAGGGTATTGACTTTAAAGAAGATGGCAACATTCTGGGTCACAAGCTGGAGTACAACTATAACAGCCACAACGTTTATATCATGGCGGATAAGCAGAAAAACGGCATTAAGGTGAACTTTAAAATCCGTCACAACATTGAAGACGGTAGCGTTCAACTGGCGGATCACTACCAGCAAAACACCCCGATTGGTGATGGTCCGGTGCTGCTGCCGGATAACCACTATCTGAGCACCCAGAGCGCGCTGAGCAAGGACCCGAACGAGAAACGTGATCACATGGTTCTGCTGGAATTCGTGACCGCGGCGGGTATCACCCTGGGTATGGACGAACTGTACAAGTGA。

[0042] Table 1: Core sequence of genomic integration site GS1 GCAGCGGAGGCGACCGCTTTCACGCCTTGAATCCTAGGCGATCCGGTCAT GS2 TGAGGTTTGCGCCTGCATCGCCGTCGGATTCCCGCTCACTGGGCGTCGGTGGCTTCAGGCGCA GS3 GGGCATGACACCGTTAACACAGGTAGTTCACTGGCCAGCAGGGCCGCCCTTTCGCGGGTGAACCCGCTCCTACGGGCGAGCGCGCAGGCGGT GS4 TATTGAATCGGGTTGGTCGAAAGTGTGTGTGTGGGCTTTGGGTCATGTACAAGCGCATCG GS5 AAAGATGAAACGGCTGGGTGCAGGCGTCCGAACGCCTGGTGCAATTCAGGCGGAAAACAGCGAACCCTTCGATTGGGAAT GS6 GCGGCCCCTGTGGGAGCGGCCTTGTGTCGCGATCGGGCCGCACAGCGGCCCCAGCAGTCTTTGCCGCGATACAGACATCGAGGGGCCGCTTCGCGGCCCGATCGCGACACAAGGCCGCTCCCACAAAAA GS7 GCGCGCTTTGCGGCCCTATCACGACACAAGGCCGCTCCTGTAGAGATCAAGAAATTCTCTGCAGGAGCGGCCTTGTGTCGTGATAGGGCTGCGAAG GS8 CCGCTTCGCAGCAAAAGGCTTCTACGGTGCTGTGCGGGCAGGGCTGAAGAAAAACGCCGACACAAGGTGTCGGCGTTGGCACAT GS9 CGATTCGCGGGCAAGCCCGCTCCCACAGGTACTCCACCGCCTTGGTAAACGGTGCAATATCTGTGGGAGTGGGC GS10 GAGCTTCAAGCGACTACAAAAACCAGAGCGGATCGCGCATTGCCCT GS11 CGCGGGCAAGCCCGCCCCCGCAGAGAAATCACAAGCCCAAGGGCTGTGGTATTCGCTGTGGAAGCGGGAGGCACGCGGCCCGGGTGTCAGCAGCAGGGCACAAGATTGCT GS12 GGGTTTACCTGCGAATACGGTAGCGGCGGAACGGTGAACGGCGGGTGGAGATTGGTCGGCAGGGCCGGCCCTTTCGCGGGTAAACCCGC GS13 TCGATTGCGCGAAATCAGACCTGCCTTGTGTGCCCGCTATGTGAAGCAACCTTCGGTTGCGTTACATAGCGGACACACAGCGTCGTGACTTGCCGGGGAAACTACTTGGTCGCGGCGTGTGGAGCAGAGACTGTAGGGGAGCGTTGA GS14 CTCAATCTATGGCACTTTTTTGATTTATGCGTGAGTAACTCCTTAGCGTTTAAGAATTAGGCCACATGAGACTTACATTGAAACAACAGCCGCCTTAGTTCCGGGGTAGCAAAGGCCTCCCTCCAAATTTCCGTGCCGATTAGTG GS15 TGCAGAAGACGTACTCACCTTCGTTGAGGATTGGGCTCATACCGCGTAATCCAAGTGCTCACGCAAAAGGTGGAGGAGGGTAGTCGAGGGGTTCGCCTGCAGTATTCGATGTC GS16 AATTTCTAACAGCTGGCGCGATTTTGGACTGTGGGAGCGGGCTTGCCCGCGAACACCGGCGAAGCCGGTGCCATGCACCGCGTCGCTTGTTTCGCGGGCACGCCCGCTCCCACAGGTAAAGCGTCATCATCCCCGTCGGGCT GS17 CTACAAGGGATGCGCGTTGTGAGGTGCGGGTGAGTCCGGCACAAAAAAAGGGGAGCATCCGCTCCCCAGAGGTTAA 。

[0043] Example 2: Construction of genomic integration vector (1) Primer design and synthesis: For 17 candidate insertion sites, amplification primers for upstream and downstream homologous arms were designed. The 5' ends of the F-terminal primer of the upstream homologous arm and the R-terminal primer of the downstream homologous arm were introduced with homologous sequences to the vector. Ptac gene amplification primers were designed, with the 5' end of the upstream primer introducing a homologous sequence to the upstream homologous arm and the 5' end of the downstream primer introducing a homologous sequence to the eGFP gene. eGFP gene amplification primers were designed, with the 5' end of the upstream primer introducing a homologous sequence to the Ptac promoter and the 5' end of the downstream primer introducing a homologous sequence to the downstream homologous arm. All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the specific primer sequences are shown in Table 2 below.

[0044] Table 2: Primer sequences GS1 Up F tgcaggcatgcaagcttgcg ACCAGCCTGCTGATGAGC GS1 Up R agccgatgattaattgtcaaTGCCCGGGCCATGCAG GS1 Ptac F TCAACTGCATGGCCCGGGCAttgacaattaatcatcggctc Ptac R TCCTCGCCTTTGCTCACCATtgtttcctgtgtgaaattgttatc eGFP F gataacaatttcacacaggaaacaATGGTGAGCAAAGGCGAGGA GS1 eGFP R TGAATAATTACGGCCGCTGGTCACTTGTACAGTTCGTCCATAC GS1 Down F GTATGGACGAACTGTACAAGTGACCAGCGGCCGTAATTATTC GS1 Down R cagggttttcccagtcacgaCAGTTCAGTTGGATACTTTACTGC GS1 pS MF ataatgctagcTAGGATTCAAGGCGTGAAAGgttttagagctagaaatagcaag GS1 pS MR gctctaaaacCTTTCACGCCTTGAATCCTAgctagcattatacctaggactgagc pS LF tcgtgactgggaaaaccctg pS LR cgcaagcttgcatgcctg pS VF tcacccttggcgtccaacca pS VR gtgagcgcatacgctacttg GS1 GVF CTGCCCATCGTCGGGTGAC GS1 GVR CCAGCACCGAGTATTGTTTTGG GS2 Up F tgcaggcatgcaagcttgcgAGACGCCAACCGTTCTCC GS2 Up R agccgatgattaattgtcaaTGCACAGATATTACTCATACCTGACTG GS2 Ptac F GTATGAGTAATATCTGTGCAttgacaattaatcatcggctc GS2 eGFP R TCGAGCGCATTGATCCGCCTCACTTGTACAGTTCGTCC GS2 Down F GGACGAACTGTACAAGTGAGGCGGATCAATGCGCTCG GS2 Down R cagggttttcccagtcacgaTCGCTTGTCAGGCACCGC GS2 pS MF CAGTGAGCGGGAATCCGAgttttagagctagaaatagcaagttaaaataag GS2 pS MR TCGGATTCCCGCTCACTGGGgctagcattatacctaggactgagctag GS2 GVF CGGGAGCAGATCATTTTCGC GS2 GVR TTCCATGGACGGTGTGGAGC GS3 Up F tgcaggcatgcaagcttgcgCATCCTCATTCTCGCCGG GS3 Up R agccgatgattaattgtcaaTCAGTCCTGCAGCCGCTC GS3 Ptac F GAGCGGCTGCAGGACTGAttgacaattaatcatcggct GS3 eGFP R CGGCCTTGCCCAAGATTTGATCACTTGTACAGTTCGTCC GS3 Down F TGGACGAACTGTACAAGTGATCAAATCTTGGGCAAGGCCG GS3 Down R cagggttttcccagtcacgaCCGGCTGGAGAGCAAGG GS3 pS MF GCATGACACCGTTAACACgttttagagctagaaatagcaagttaaaataag GS3 pS MR GTGTTAACGGTGTCATGCCCgctagcattatacctaggactgagctag GS3 GVF ATGCCTGACGCACTCCCC GS3 GVR CGAGATGCACATGGCCGAAG GS4 Up F tgcaggcatgcaagcttgcgCAAGCTTATCGCCAACCT GS4 Up R gagccgatgattaattgtcaaTCAGGCAACCCGTCGGATAC GS4 Ptac F GTATCCGACGGGTTGCCTGAttgacaattaatcatcggctcg GS4 eGFP R GTGGGATCTGACGATCTGTGGTTCACTTGTACAGTTCGTCCATAC GS4 Down F GTATGGACGAACTGTACAAGTGAACCACAGATCGTCAGATCCCAC GS4 Down R cagggttttcccagtcacgaCCACCAATGCACTGTCG GS4 pS MF GGTCGAAAGTGTGTGTGTgttttagagctagaaatagcaagttaaaataag GS4 pS MR ACACACACACTTTCGACCAAgctagcattatacctaggactgagctag GS4 GVF CGAACGCGCGCAAGATG GS4 GVR CTTGCTCGGCTATGCCCGG GS5 Up F tgcaggcatgcaagcttgcgCCGATCATGCTGGTGCTGTTC GS5 Up R agccgatgattaattgtcaaTGCAACGAAAGGCCAGTAC GS5 Ptac F TACTGGCCTTTCGTTGCAttgacaattaatcatcggctcgtataatg GS5 eGFP R CGGCCCATTTTTCGCCATTTTCACTTGTACAGTTCGTCCATAC GS5 Down F GTATGGACGAACTGTACAAGTGAAAATGGCGAAAAATGGGCCG GS5 Down R cagggttttcccagtcacgaGTGGGAAGGCAATGTGATC GS5 pS MF AACGCCTGGTGCAATTCAGGgttttagagctagaaatagcaagttaaaataag GS5 pS MR tgaattgcaccaggcgttGCTAGCATTATACCTAGGACTGAGCTAG GS5 GVF GGCTCAAGCCTGTCGTTCATC GS5 GVR CCTGGCATTTGACTTCAAGCC GS6 Up F tgcaggcatgcaagcttgcgAAACGCGGGGCTTGCCG GS6 Up R cgagccgatgattaattgtcaaGTCAGCTGTTCAGGCGGTGA GS6 Ptac F TCACCGCCTGAACAGCTGACttgacaattaatcatcggctcg GS6 eGFP R CATTCCAGGTGGACGTGGTCTCACTTGTACAGTTCGTCCATAC GS6 Down F GTATGGACGAACTGTACAAGTGAGACCACGTCCACCTGGAATG GS6 Down R ccagggttttcccagtcacgaTCCATGGGCGATATCGCCGAGC GS6 pS MF TCGCGGCAAAGACTGCTGgttttagagctagaaatagcaagttaaaataag GS6 pS MR CAGCAGTCTTTGCCGCGATAgctagcattatacctaggactgagctag GS6 GVF CCGATCGTCGGAGACCAGAG GS6 GVR CAAGCCTGATGGCCAATCAC GS7 Up F tgcaggcatgcaagcttgcgGGTACCGCTCTGCTCTCTGCTg GS7 Up R agccgatgattaattgtcaaCCTCGGTTTTCAGCTGGCAG GS7 Ptac F TTCTGCCAGCTGAAAACCGAGGttgacaattaatcatcggctcg GS7 eGFP R TAGAAGTACCGAGGGGGCTGTCACTTGTACAGTTCGTCCATAC GS7 Down F GTATGGACGAACTGTACAAGTGACAGCCCCCTCGGTACTTCTATC GS7 Down R cagggttttcccagtcacgaGCCGGACGACAAGGAAAAG GS7 pS MF GATCAAGAAATTCTCTGCgttttagagctagaaatagcaagttaaaataag GS7 pS MR gcagagaatttcttgatctcGCTAGCATTATACCTAGGACTGAGCTAG GS7 GVF CTCAATTACACAGGAAGGAACAGG GS7 GVR AGGTAGAAGCCGAGAAGCCC GS8 Up F tgcaggcatgcaagcttgcgTATGCCGCGTTGCATGAC GS8 Up R agccgatgattaattgtcaaCCGCAAAGCAGCCTCTTAC GS8 Ptac F AGTAAGAGGCTGCTTTGCGGttgacaattaatcatcggctcg GS8 eGFP R TTTCGCTAGCAACGCCTGATTCACTTGTACAGTTCGTCCATAC GS8 Down F GTATGGACGAACTGTACAAGTGAATCAGGCGTTGCTAGCGAAA GS8 Down R cagggttttcccagtcacgaCCAGAAGCGTCTGGACGC GS8 pS MF aggcttctacggtgctgtgGTTTTAGAGCTAGAAATAGCAAGTTAAAATAag GS8 pS MR acagcaccgtagaagccttGCTAGCATTATACCTAGGACTGAGCTAG GS8 GVF CGAGGAAGAGGAACTGGTCG GS8 GVR ACGCCGAAATCCTCGAATTG GS9 Up F tgcaggcatgcaagcttgcgACTGGACGAAGACCCAAAGC GS9 Up R agccgatgattaattgtcaaCCTTCAGCGCCCCATCATTTC GS9 Ptac F AAATGATGGGGCGCTGAAGGttgacaattaatcatcggctcg GS9 eGFP R GTCATACTCTTCGCGGGCTCTCACTTGTACAGTTCGTCC GS9 Down F GGACGAACTGTACAAGTGAGAGCCCGCGAAGAGTATGAC GS9 Down R cagggttttcccagtcacgaCTGTGCCCTGGAAATTGTCG GS9 pS MF CACCGTTTACCAAGGCGGgttttagagctagaaatagcaagttaaaataag GS9 pS MR CCGCCTTGGTAAACGGTGCAgctagcattatacctaggactgagctag GS9 GVF AGGGTGGCATTGAAGACAGC GS9 GVR TGCTAATGCCGACACTGGAG GS10 Up F tgcaggcatgcaagcttgcgCTGGCGCTTCTTCAACAGC GS10 Up R agccgatgattaattgtcaaGAAACCTGCCGCTCTGGAAG GS10 Ptac F CTTCCAGAGCGGCAGGTTTCttgacaattaatcatcggctcg GS10 eGFP R TCAGGCGGCAAGGAAAATTCTCACTTGTACAGTTCGTCCATAC GS10 Down F GGACGAACTGTACAAGTGAGAATTTTCCTTGCCGCCTGAAG GS10 Down R cagggttttcccagtcacgaCCATCGCCATGATCAAGCAG GS10 pS MF CGACTACAAAAACCAGAGgttttagagctagaaatagcaagttaaaataag GS10 pS MR CTCTGGTTTTTGTAGTCGCTgctagcattatacctaggactgagctag GS10 GVF ACAACTTGCTGCAGCGTTAC GS10 GVR CATTACCGACGCCATGATCC GS11 Up F tgcaggcatgcaagcttgcgCGTGCAGGTGAACCTGGTAC GS11 Up R agccgatgattaattgtcaaAAGAGGCCGCTACAGGTCAAG GS11 Ptac F TTGACCTGTAGCGGCCTCTTttgacaattaatcatcggctcg GS11 eGFP R CCTGGCCATTTGGGGATATCTCACTTGTACAGTTCGTCCATAC GS11 Down F GGACGAACTGTACAAGTGAGATATCCCCAAATGGCCAGGG GS11 Down R cagggttttcccagtcacgaCCAGCCTCAAGGACAAC GS11 pS MF CTTGTGATTTCTCTGCGGgttttagagctagaaatagcaagttaaaataag GS11 pS MR CCGCAGAGAAATCACAAGCCgctagcattatacctaggactgagctag GS11 GVF GAAGGTCGAGCAGCTGTACG GS11 GVR GAGCGCATCGACCTGCAAAG GS12 Up F tgcaggcatgcaagcttgcgCGTTCATCCGTGGCATGAATG GS12 Up R agccgatgattaattgtcaaGACTTGTGGGAACGAACAGG GS12 Ptac F CCTGTTCGTTCCCACAAGTCttgacaattaatcatcggctcg GS12 eGFP R AAGGGGGATAGAGCGTAGGATCACTTGTACAGTTCGTCCATAC GS12 Down F TGGACGAACTGTACAAGTGATCCTACGCTCTATCCCCCTTTG GS12 Down R cagggttttcccagtcacgaGACCTTCGCCTGGTCAA GS12 pS MF CGGCGGAACGGTGAACGGgttttagagctagaaatagcaagttaaaataag GS12 pS MR CCGTTCACCGTTCCGCCGCTgctagcattatacctaggactgagctag GS12 GVF GACCTGGACCACCAGCGTAG GS12 GVR CCAGCGCGATACGTATGAAG GS13 Up F tgcaggcatgcaagcttgcgTTCCTGAGAGAACGTCTGAAGC GS13 Up R agccgatgattaattgtcaaGTAGGAGGCGGATTCACACC GS13 Ptac F GGTGTGAATCCGCCTCCTACttgacaattaatcatcggctcg GS13 eGFP R CTAGCTTGAAACCGCCACTGTCACTTGTACAGTTCGTCCATAC GS13 Down F GTATGGACGAACTGTACAAGTGACAGTGGCGGTTTCAAGCTAG GS13 Down R cagggttttcccagtcacgaGAGCAGTGAAGGTCAAACCAGG GS13 pS MF GCGACCAAGTAGTTTCCCgttttagagctagaaatagcaagttaaaataag GS13 pS MR GGGAAACTACTTGGTCGCGGgctagcattatacctaggactgagctag GS13 GVF GCCATCGCTTCGTACGTTATG GS13 GVR TGGACGCAGCTATTTTGTCC GS14 Up F tgcaggcatgcaagcttgcgGGAGAAAATCCTCCGCGAAC GS14 Up R agccgatgattaattgtcaaGGCCTTTTCCCAAGCTACAG GS14 Ptac F CTGTAGCTTGGGAAAAGGCCttgacaattaatcatcggctcg GS14 eGFP R CACTATCAGGCAGTTTGGCATCACTTGTACAGTTCGTCCATAC GS14 Down F GTATGGACGAACTGTACAAGTGATGCCAAACTGCCTGATAGTG GS14 Down R cagggttttcccagtcacgaGACCACTACAAAGCACACTCAC GS14 pS MF GCTACCCCGGAACTAAGGgttttagagctagaaatagcaagttaaaataag GS14 pS MR CCTTAGTTCCGGGGTAGCAAgctagcattatacctaggactgagctag GS14 GVF CAGCAGCAACTTCCAGTGAC GS14 GVR GATTGACTCTTGTTGGAGCACTTATC GS15 Up F tgcaggcatgcaagcttgcgGGCAAACCTTCCGATCTGC GS15 Up R agccgatgattaattgtcaaCCAAGGCATTTACGCAGTCC GS15 Ptac F GGACTGCGTAAATGCCTTGGttgacaattaatcatcggctcg GS15 eGFP R CAGGCAAGAAGGCGACTTTCTCACTTGTACAGTTCGTCCA GS15 Down F TGGACGAACTGTACAAGTGAGAAAGTCGCCTTCTTGCCTG GS15 Down R cagggttttcccagtcacgaCGCTAAACCGGTGAACCATTTAC GS15 pS MF GAGCCCAATCCTCAACGAgttttagagctagaaatagcaagttaaaataag GS15 pS MR TCGTTGAGGATTGGGCTCATgctagcattatacctaggactgagctag GS15 GVF CAACGCCTTGGATGTCATGC GS15 GVR TGAGGTGGCTGCCAATGATC GS16 Up F tgcaggcatgcaagcttgcgCCCTACTGGCAACAATCGATCTAC GS16 Up R agccgatgattaattgtcaaGAGCAAGACAGTTGCTCCCG GS16 Ptac F CGGGAGCAACTGTCTTGCTCttgacaattaatcatcggctcg GS16 eGFP R AACCAAAGAAGAAAGGCTGATCACTTGTACAGTTCGTCCATAC GS16 Down F TGGACGAACTGTACAAGTGATCAGCCTTTCTTCTTTGGTTTCAG GS16 Down R cagggttttcccagtcacgaCCTTCAAGCCTGGCAAGAAC GS16 pS MF AAGCGTCATCATCCCCGTgttttagagctagaaatagcaagttaaaataag GS16 pS MR ACGGGGATGATGACGCTTTAgctagcattatacctaggactgagctag GS16 GVF CAAGATCCGCTCGGTGGAAG GS16 GVR CTGGACAAGGACACCGTGAC GS17 Up F tgcaggcatgcaagcttgcgTCATCCGCCTGGAGAAGCTC GS17 Up R agccgatgattaattgtcaaGAGCTGATTCAGCACACCGC GS17 Ptac F CGGTGTGCTGAATCAGCTCttgacaattaatcatcggctcg GS17 eGFP R GATTTAAGCCCTCATCTACAAGTGGTCACTTGTACAGTTcgtccatac GS17 Down F TGGACGAACTGTACAAGTGACCACTTGTAGATGAGGGCTTAaatc GS17 Down R cagggttttcccagtcacgaCCAAGTTGCGTAGCGACATC GS17 pS MF CAAGGGATGCGCGTTGTGgttttagagctagaaatagcaagttaaaataag GS17 pS MR CACAACGCGCATCCCTTGTAgctagcattatacctaggactgagctag GS17 GVF ATGACAGACGCATTGAACTGG GS17 GVR GTACCTGCAAGGTGGCTATG .

[0045] (2) Amplification of the target fragment: Homologous arm fragments were amplified using genomic DNA of *Pseudomonas putida* KT2440 as a template, and PCR reactions were performed using 17 sets of primers (34 pairs) for both upper and lower homologous arms. The reaction system (50 μL) consisted of: 25 μL of 2×Phanta Max Master Mix, 1 μL each of forward and reverse primers (10 μM), 1 μL of template DNA (100 ng / μL), and 22 μL of sterile water. Reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 45 s, 30 cycles; 72℃ final extension for 5 min; and incubation at 4℃. The Ptac promoter was amplified using the pTrc99a vector as a template, and PCR reactions were performed using 17 pairs of primers, with the same reaction system and conditions as for homologous arm amplification. eGFP gene expression amplification was performed using plasmid pUC19-eGFP provided by Beijing Qingke Biotechnology Co., Ltd. as a template. PCR reactions were conducted using 17 pairs of primers, with the reaction system and conditions identical to those for homologous arm amplification. All amplification products were verified by 1% agarose gel electrophoresis. The target fragment was recovered using a DNA gel extraction kit, and after concentration determination, it was stored at -20℃.

[0046] (3) sgRNA construction and vector linearization: Using pSEVA vector as a template, PCR was performed using 17 pairs of sgRNA mutant primers, with the reaction system being the same as the homologous arm amplification. Reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 3 min, 30 cycles; 72℃ final extension for 5 min; 4℃ incubation. The cells were chemically transformed into commercial DH5α competent cells, plated on LB plates containing GM antibiotics, and cultured overnight. Single colonies were picked and added to the colony PCR reaction system (using a colony PCR kit purchased from Nanjing Novizan Biotechnology Co., Ltd.). PCR verification was performed using primers pS VF and pS VR. Positive clones were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Seventeen single clones of recombinant plasmids containing the correct sequences were inoculated into LB liquid medium containing 50 μg / mL GM and cultured at 37°C and 220 rpm for 12 h. Plasmids were extracted (using a plasmid miniprep kit, Beijing Qingke Biotechnology Co., Ltd.) to obtain 17 pSEVA vectors containing the corresponding sgRNAs. After concentration determination, the vectors were stored at -20°C. Using the plasmids as templates, PCR linearization was performed using primers pS LF and pS LR. The reaction system was the same as that for homologous arm amplification. The reaction conditions were: 95°C pre-denaturation for 3 min; 95°C denaturation for 3 min, 62°C annealing for 15 s, 72°C extension for 3 min, 35 cycles; and a final extension at 72°C for 5 min. The linearized products were separated by 1% agarose gel electrophoresis and recovered for later use.

[0047] (4) One-step cloning and ligation: The pSEVA linearized vector, its corresponding upper and lower homologous arms, Ptac promoter, and eGFP gene fragment were cloned using a one-step cloning kit (Nanjing Novizan Biotechnology Co., Ltd.). After the reaction, the sample was immediately placed on ice and cooled for 5 min.

[0048] (5) Transformation and positive verification: The 17 ligation products were transformed into Escherichia coli DH5α competent cells (Beijing Qingke Biotechnology Co., Ltd.), and incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, and incubated on ice for 2 min. 800 μL of LB liquid medium was added and cultured at 37℃ and 180 rpm for 1 h with shaking. 100 μL of bacterial culture was spread on LB solid medium containing 50 μg / mL GM antibiotic and cultured upside down at 37℃ for 12 h.

[0049] (6) Verification of positive clones: Single colonies were picked and added to the colony PCR reaction system (colony PCR kit, Nanjing Novizan Biotechnology Co., Ltd.). PCR verification was performed using primers pS VF and pS VR. Positive clones were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Single clones (a total of 17) containing the correct recombinant plasmid sequence were inoculated into LB liquid medium containing 50 μg / mL GM (gentamicin) antibiotic and cultured at 37℃ and 220 rpm for 12 h. Plasmids were extracted (using a plasmid mini-prep kit, purchased from Beijing Qingke Biotechnology Co., Ltd.), and the concentration was determined before storage at -20℃.

[0050] Example 3: Construction of recombinant *Pseudomonas putida* (1) Preparation of competent cells of *Pseudomonas putida* KT2440: *Pseudomonas putida* KT2440 containing pCas plasmid was inoculated into 10 mL of LB liquid medium, and 25 μg / mL tetracycline and 6 g / L arabinose were added. The culture was carried out at 30 °C and 220 rpm for 12 h. 1 mL of the bacterial culture was transferred to 100 mL of LB liquid medium and cultured at 30 °C and 220 rpm until OD200. 600 =0.6; ice bath for 30 min, centrifuge at 4℃ and 5000 rpm for 10 min, discard the supernatant; resuspend the bacterial cells in 50 mL of pre-cooled sterile water, centrifuge at 4℃ and 5000 rpm for 10 min, discard the supernatant; repeat washing twice, resuspend in 2 mL of pre-cooled sterile water, add 2 mL of 30% glycerol, aliquot into 1.5 mL centrifuge tubes, and store at -80℃ for later use.

[0051] (2) Electroporation and screening: Take 17 100 μL competent cells and add 10 μL of the 17 genome integration vectors constructed in Example 2 (1 μg / μL); transfer to a pre-cooled electroporation cup (0.2 cm) and perform electroporation using an electroporator (Bio-Rad) with the following parameters: 2.5 kV, 25 μF, 200 Ω; immediately add 1 mL of LB liquid medium and culture at 30 °C and 220 rpm for 2 h with shaking; take 200 μL of bacterial culture and spread it on LB solid medium containing 50 μg / mL and incubate upside down at 30 °C for 24 h.

[0052] (3) Validation of recombinant strains: single colonies were picked and added to the colony PCR reaction system (using a colony PCR kit purchased from Nanjing Novizan Biotechnology Co., Ltd.). PCR validation was performed using primers on the outer side of the homologous arm. Positive clones were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Single colonies containing the correct sequence were inoculated into 10 mL LB medium containing 25 μg / mL tetracycline and 10 mM rhamnose, and cultured at 30°C with shaking at 220 rpm for 12 h. The colonies were then streaked onto plates containing 25 μg / mL tetracycline and cultured at 30°C for 24 h. Single colonies were then picked and streaked onto plates containing 50 μg / mL gentamicin. Strains that did not grow were considered strains that had successfully eliminated the pSEVA vector. The same strains were then inoculated into 10 mL LB medium containing 5 g / L glucose and cultured at 30°C with shaking at 220 rpm for 12 h. The colonies were then streaked onto plates containing 5 g / L glucose and 10 g / L sucrose and cultured at 30°C for 24 h. Single colonies were then picked and streaked onto plates containing 25 μg / mL tetracycline. Strains that did not grow were considered strains that had successfully eliminated the pCAS vector. Using this method, 17 recombinant *Pseudomonas putridae* strains that successfully inserted the Ptac-eGFP expression cassette into different genomic integration sites were obtained.

[0053] Example 4: Characterization of growth levels at different insertion sites (1) Culture of strains: 17 recombinant strains and control strain KT2440 were inoculated into 10 mL LB medium and cultured at 30℃ and 220 rpm for 12 h to obtain seed liquid; the inoculum was transferred to fresh liquid medium containing 50 mL LB at an inoculation rate of 1% (v / v) (3 replicates per strain), and cultured at 30℃ and 220 rpm with shaking. Samples were taken at 6 h and 12 h.

[0054] (2) Growth curve determination: Take 1 mL of bacterial culture at each time point, centrifuge at 12000 rpm for 1 min, discard the supernatant, resuspend the bacterial cells in sterile water and dilute 10 times, transfer to a 96-well clear microplate; use a microplate reader to measure OD. 600 Values, record data and plot growth curves (see...) Figure 1 ). Figure 1 The growth curve of recombinant *Pseudomonas putida* over time.

[0055] Figure 1 The results showed that the growth curves of the 17 recombinant strains were not significantly different from those of wild-type Pseudomonas putida KT2440 (P>0.05), indicating that the choice of insertion site did not interfere with the host's growth and metabolism.

[0056] Example 5: Characterization of expression intensity at different insertion sites (1) Culture of strains: 17 recombinant strains and control strain KT2440 were inoculated into LB liquid medium and cultured at 30℃ and 220rpm for 12h to obtain seed liquid; the inoculum was transferred to fresh LB liquid medium containing 50mL at a 1% (v / v) inoculation rate (3 replicates per strain) and cultured at 30℃ and 220rpm with shaking. Samples were taken at 6h and 12h.

[0057] (2) Growth assay: Take 1 mL of bacterial culture at each time point, centrifuge at 12000 rpm for 1 min, discard the supernatant, resuspend the bacterial cells in sterile water and dilute 10 times, transfer to a 96-well clear microplate; measure OD using a microplate reader. 600 Values, record data and plot growth curves.

[0058] (3) Expression intensity determination: Take 1 mL of bacterial culture at each time point, centrifuge at 12000 rpm for 1 min, discard the supernatant, resuspend the bacterial cells in sterile water and dilute 10 times, transfer to a 96-well black ELISA plate; use an ELISA reader to measure the fluorescence intensity, with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Calculate the relative fluorescence intensity (RFI) of each recombinant strain (RFI = sample fluorescence intensity / cell OD). 600 Record the data and plot the eGFP expression level curve (see...). Figure 2 ). Figure 2 The graph shows the expression curve of green fluorescent protein (eGFP) in recombinant Pseudomonas putida over time.

[0059] Figure 2 The results showed that all 17 obtained genomic integration sites achieved green fluorescent protein expression, with significant differences in expression intensity and broad coverage, which can meet the personalized expression needs of different functional genes. These results indicate that the genomic integration sites screened in this invention possess both good expression activity and host compatibility, have minimal impact on host growth, and exhibit stable expression levels, providing a powerful tool for the synthetic biology and systems metabolic engineering of *Pseudomonas putida*.

[0060] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant Pseudomonas putida, characterized in that, The recombinant *Pseudomonas putida* contains a foreign gene, which is integrated into an insertion site in a *Pseudomonas putida* genome insertion site library. The insertion site is located in the intergenic region of the *Pseudomonas putida* KT2440 genome, and the sequence of the genomic coordinates corresponding to the insertion site is shown in SEQ ID NO. 1~17.

2. The recombinant *Pseudomonas putida* according to claim 1, characterized in that, The exogenous gene is a reporter gene or a functional gene.

3. The recombinant *Pseudomonas putida* according to claim 2, characterized in that, The reporter gene is the green fluorescent protein gene eGFP, the sequence of which is shown in SEQ ID NO.

18.

4. A genome integration vector for constructing the recombinant *Pseudomonas putida* as described in any one of claims 1 to 3, characterized in that, The genome integration vector contains recombinant sequences homologous to the upstream and downstream of the insertion site, a target gene expression cassette, and an sgRNA sequence that guides integration.

5. A method for constructing a library of *Pseudomonas putida* genome insertion sites, characterized in that, Includes the following steps: S1: Selection of insertion sites: Based on the genome annotation information of Pseudomonas putida KT2440, intergenic regions with opposite transcription directions are selected as insertion sites, and the sequence of the genome coordinates corresponding to the insertion sites is shown in SEQ ID NO.1~17; S2: Constructing a genome integration vector: Designing homologous arms and sgRNAs for the insertion site, and assembling the target gene expression cassette; S3: Integration and verification: The target gene is integrated into the corresponding insertion site in the genome of *Pseudomonas putida*, and recombinant *Pseudomonas putida* as described in any one of claims 1 to 3 is obtained through resistance screening and PCR verification. S4: Characterization of insertion site library: The recombinant strain was cultured, and the expression level of the reporter gene at different culture stages was measured to determine the expression intensity level of each insertion site.

6. The method for constructing a Pseudomonas putida genome insertion site library according to claim 5, characterized in that, It also includes a step of characterizing the expression intensity of the insertion site: culturing the recombinant *Pseudomonas putida* strain and measuring the relative expression level by reporter gene fluorescence intensity.

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