Pseudomonas putida KT2440 endogenous PUTR library, construction method and engineering bacteria for efficiently synthesizing rhamnolipid

Abstract

The invention discloses a pseudomonas putida KT2440 endogenous PUTR library, a construction method and engineering bacteria for efficiently synthesizing rhamnolipid. KT2440 is used as host bacteria, key genes are synthesized by heterologous expression of rhamnolipid, recombinant plasmids are constructed, and the host bacteria are introduced to obtain engineering strains; carrying out fermentation culture on the wild type and the engineering strain under the same condition, and carrying out comparative transcriptomics analysis on samples in different growth stages; on the basis of transcriptome data and the key gene, acquiring a PUTR candidate element at the upstream of a starting site of a key gene sequence, connecting the PUTR candidate element to a carrier genome containing fluorescent protein, and introducing into a host bacterium competent cell to obtain a genetic engineering strain; and screening different PUTR elements to optimize key genes in a rhamnolipid synthesis route to obtain the engineering bacteria. The carbon flow distribution is accurately regulated and controlled, the synthesis efficiency of the rhamnolipid is remarkably improved, and the engineering applicability and practical value of the rhamnolipid in synthesis of the high-added-value biosurfactant are verified.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and protein engineering technology, specifically relating to an endogenous PUTR library of Pseudomonas putida KT2440, its construction method, and an engineered bacterium for efficient synthesis of rhamnolipids. Background Technology

[0002] *Pseudomonas putida* KT2440 is a Gram-negative soil bacterium widely considered a promising engineered host in synthetic biology and industrial biotechnology due to its excellent biocompatibility (no known virulence factors), strong environmental adaptability, and highly flexible metabolic network. This strain can tolerate a variety of toxic compounds, organic solvents, and a wide range of pH and temperature conditions. Its genome contains abundant metabolic enzyme systems and substance transport systems, supporting the efficient utilization of various carbon sources. Based on these advantages, *Pseudomonas putida* KT2440 has been used to synthesize various high-value-added chemicals and biomaterials, including polyhydroxyalkanoates, biosurfactants, short-chain alcohols, aromatic compound intermediates, and bioplastic precursors, showing broad application prospects in industrial fermentation, green chemistry, and environmental remediation.

[0003] Rhamnolipids are a class of glycolipid biosurfactants composed of rhamnose and fatty acids. They possess excellent surface activity, biodegradability, and biocompatibility, making them valuable for applications in petroleum extraction, environmental remediation, food, cosmetics, and pharmaceuticals. Traditionally, rhamnolipids are naturally synthesized by opportunistic pathogens such as *Pseudomonas aeruginosa*, posing biosafety risks and limiting their large-scale industrial application. In contrast, *Pseudomonas putida* KT2440 exhibits clear safety profiles and good industrial applicability. However, this strain lacks the ability to naturally synthesize rhamnolipids. Therefore, efficient and stable rhamnolipid biosynthesis requires heterologous introduction of the rhamnolipid synthesis pathway and systematic metabolic and expression regulation. Consequently, achieving precise, stable, and predictable expression regulation of multiple key genes in this host has become one of the core technical challenges hindering the efficient synthesis of rhamnolipids.

[0004] With the development of synthetic biology and metabolic engineering, precise regulation of gene expression has become a key factor in optimizing metabolic pathways and synthesizing efficient products. Researchers have developed various gene expression regulation techniques, such as CRISPR / dCas9 regulation, RNA interference, and RBS engineering, to regulate metabolic flux and balance cell growth with target product synthesis. Among these, the promoter, as a core element of transcriptional regulation, directly affects the expression intensity of downstream proteins by controlling the transcriptional level of mRNA. Currently, while synthetic promoter libraries constructed based on mutations can expand the expression intensity range to some extent, their high sequence homology leads to problems such as unexpected homologous recombination and insufficient stability. In contrast, natural promoters derived from the host genome have high sequence diversity and a wide range of transcriptional intensity, which is more conducive to achieving long-term stable and predictable expression regulation. Further research shows that relying solely on promoter regulation of transcriptional levels often fails to meet the precision requirements of complex metabolic pathways. The 5′ untranslated region (5′UTR) plays a crucial role in regulating mRNA stability, ribosome binding efficiency, and translation initiation rate, significantly influencing the final protein expression level. Therefore, designing and screening promoters and 5′UTRs as a whole to construct “promoter–5′UTR” (PUTR) combined regulatory elements can help achieve synergistic regulation at both the transcriptional and translational levels, thereby more precisely regulating metabolic flux.

[0005] Although the synthesis of promoter libraries and UTR regulation strategies have been systematically studied and applied in model microorganisms (such as Escherichia coli and Bacillus subtilis), their adaptability in Pseudomonas putida is still limited, especially the systematic exploration, characterization and engineering application of endogenous PUTR combinatorial elements in this host are still immature.

[0006] Therefore, there is an urgent need to develop a method for screening and constructing a promoter-5′UTR combinatorial element library based on the endogenous sequence of Pseudomonas putida KT2440, in order to obtain a gene regulation tool with continuously adjustable expression intensity and stable reliability, thereby providing a technical basis for the synergistic expression regulation of multiple genes related to rhamnolipid synthesis and the fine reconstruction of metabolic flux in this strain. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides an endogenous PUTR library of *Pseudomonas putida* KT2440, its construction method, and an engineered bacterium for efficient synthesis of rhamnolipids. This PUTR library is constructed based on transcriptomics data, providing a universal regulatory tool for the metabolic engineering and synthetic biology applications of this strain.

[0008] To solve the above problems, the technical solution adopted in this application is:

[0009] The first aspect of this invention is to provide a method for constructing an endogenous PUTR library of *Pseudomonas putida* KT2440, comprising the following steps:

[0010] (1) Using Pseudomonas putida KT2440 as the host strain, the key gene for rhamnolipin synthesis was heterologously expressed in the host strain, and the recombinant plasmid pBBR1MCS5-rhlAB was constructed to enable the host strain to synthesize rhamnolipin de novo.

[0011] (2) The recombinant plasmid pBBR1MCS5-rhlAB obtained in step (1) was introduced into the host bacteria to obtain the recombinant engineered strain P. putida KT2440 / pBBR1MCS5-rhlAB;

[0012] (3) Transcriptome data acquisition: Wild-type Pseudomonas putida KT2440 and recombinant engineered strain P. putidaKT2440 / pBBR1MCS5-rhlAB were fermented under the same fermentation culture conditions. Samples were taken at different growth stages and compared for transcriptome analysis.

[0013] (4) Obtaining PUTR candidate elements: Based on the transcriptome data analysis results and the selected key genes, PUTR candidate elements located upstream of the sequence start site of the key genes are obtained;

[0014] (5) Construction of recombinant expression vector: The PUTR candidate element is operatively ligated into the genome of a vector that can express fluorescent protein in competent cells of host bacteria to obtain a recombinant expression vector;

[0015] (6) Characterization of PUTR library: The recombinant expression vector obtained in step (5) is introduced into the competent cells of the host bacteria to obtain the genetically engineered strain containing the recombinant expression vector. The expression intensity of fluorescent protein at different growth stages and the cell growth of the corresponding genetically engineered strain are measured to determine the endogenous PUTR library of Pseudomonas putida KT2440.

[0016] As a preferred embodiment of this application, the PUTR candidate element is selected from at least one of the nucleotide sequences such as SEQ ID NO.1 to SEQ ID NO.80.

[0017] SEQ ID NO Sequence name SEQ ID NO Sequence name SEQ ID NO Sequence name 1 PUTR_acpP 28 PUTR_PP_0641 55 PUTR_PP_5458 2 PUTR_algQ 29 PUTR_PP_0679 56 PUTR_PP_5700 3 PUTR_amaB 30 PUTR_PP_0916 57 PUTR_ssrA 4 PUTR_atpE 31 PUTR_PP_0951 58 PUTR_PP_5730 5 PUTR_cioA 32 PUTR_PP_1111 59 PUTR_PP_2161 6 PUTR_clpA 33 PUTR_PP_1272 60 PUTR_rmf 7 PUTR_cspA 34 PUTR_PP_1753 61 PUTR_rnpB 8 PUTR_cspD 35 PUTR_PP_1910 62 PUTR_rplM 9 PUTR_csrA 36 PUTR_PP_2177 63 PUTR_rpmB 10 PUTR_flgM 37 PUTR_PP_2296 64 PUTR_rpmI 11 PUTR_fliC 38 PUTR_PP_2921 65 PUTR_rpoE 12 PUTR_fusA 39 PUTR_PP_3361 66 PUTR_rpoH 13 PUTR_gcdH 40 PUTR_PP_3580 67 PUTR_rpoS 14 PUTR_glnK 41 PUTR_PP_3595 68 PUTR_rpoZ 15 PUTR_gltI 42 PUTR_PP_3597 69 PUTR_rpsA 16 PUTR_gtsA 43 PUTR_PP_3598 70 PUTR_rpsB 17 PUTR_gtsD 44 PUTR_PP_3765 71 PUTR_rpsF 18 PUTR_infA 45 PUTR_PP_4108 72 PUTR_rpsH 19 PUTR_infC 46 PUTR_PP_4470 73 PUTR_rpsL 20 PUTR_Novel 47 PUTR_PP_4557 74 PUTR_rpsO 21 PUTR_oprD 48 PUTR_PP_4667 75 PUTR_rpsU 22 PUTR_oprF 49 PUTR_PP_4793 76 PUTR_sqR 23 PUTR_oprI 50 PUTR_PP_4975 77 PUTR_tapB 24 PUTR_phaF 51 PUTR_PP_4835 78 PUTR_yccA 25 PUTR_phhB 52 PUTR_PP_5329 79 PUTR_ydcJ 26 PUTR_PP_0354 53 PUTR_PP_5392 80 PUTR_yihS 27 PUTR_PP_0367 54 PUTR_PP_5395 .

[0018] As a preferred embodiment of this application, the key genes include the rhlA gene and / or the rhlB gene; wherein the rhlA gene and the rhlB gene are both derived from Pseudomonas aeruginosa PAO1 and are driven by the Lac promoter.

[0019] As a preferred embodiment of this application, the nucleotide sequence of the Lac promoter is shown in SEQ ID NO.81.

[0020] As a preferred embodiment of this application, the nucleotide sequence of the rhlA gene is shown in SEQ ID NO.82.

[0021] As a preferred embodiment of this application, the nucleotide sequence of the rhlB gene is shown in SEQ ID NO.83.

[0022] As a preferred embodiment of this application, the fluorescent protein is selected from green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), or superfolded fluorescent protein (sfGFP).

[0023] As a preferred embodiment of this application, the quantitative analysis index is the number of fragments per kilobase per million mapping reads of the gene (FPKM). The judgment rule is as follows: an FPKM value of 1 is used as the threshold for judging whether a gene is expressed. Eighty PUTR candidate elements with different intensities were selected from genes with an FPKM value greater than 1 to construct a PUTR library, with FPKM values ​​ranging from 823.03 to 1044427.65. The sequence approximately 500 bp upstream of the start codon of the candidate gene is extracted as the fragment of the PUTR candidate element.

[0024] As a preferred embodiment of this application, the fermentation culture method is as follows:

[0025] Genetically engineered bacteria were inoculated into LB liquid medium containing gentamicin and cultured at 30°C and 200 rpm for 12 h to be used as seed culture.

[0026] The seed culture was inoculated into the fermentation medium at an inoculation rate of 2% to 5%, and fermented at a pH of 6.7 to 7.0 and a temperature of 28 to 35°C.

[0027] As a preferred embodiment of this application, the sampling nodes are the exponential period, the stable period, and the recession period.

[0028] As a preferred embodiment of this application, the expression of the fluorescent protein is obtained by transferring a genetically engineered strain cultured at 30°C and 200 rpm into a fermentation medium, fermenting at 30°C and 200 rpm, and collecting cells during the early logarithmic phase, early logarithmic phase, mid-logarithmic phase, early stationary phase, and stationary phase.

[0029] A second aspect of the present invention is to provide an endogenous PUTR library of *Pseudomonas putida* KT2440 constructed by the aforementioned construction method.

[0030] A third aspect of the present invention is to provide a recombinant genetically engineered strain constructed based on the endogenous PUTR library of the aforementioned *Pseudomonas putida* KT2440.

[0031] A fourth aspect of the present invention is to provide a method for constructing the recombinant genetically engineered strain, comprising the following steps:

[0032] (a) Using *Pseudomonas putida* KT2440 as the host bacterium, the key gene for dTDP-L-rhamnose synthesis was heterologously expressed in the host bacterium, and a recombinant expression plasmid pBBR1MCS5-Plac-rhlAB-Plac-rmlBDAC for rhamnose lipid synthesis was constructed.

[0033] (b) The recombinant expression plasmid obtained in step (a) was introduced into the *Pseudomonas putida* KT2440 to obtain the recombinant genetically engineered strain *P. putida* KT2440 / pBBR1MCS5-rhlAB-rmlBDAC for the synthesis of rhamnolipids;

[0034] (c) Based on the endogenous PUTR library of the aforementioned Pseudomonas putida KT2440, PUTR combinations with different expression intensities were screened at the plasmid level, and the expression levels of the rhlAB gene and rmlBDAC gene were optimized by combination.

[0035] (d) Using Pseudomonas putida KT2440 as the starting strain, screen for compatible PUTR elements in the PUTR library and replace the native promoters of aceE and gltA on the genome of the starting strain.

[0036] As a preferred embodiment of this application, the key genes for dTDP-L-rhamnose synthesis are derived from Pseudomonas aeruginosa PAO1, including the rmlA, rmlB, rmlC, and rmlD genes, and the genes are driven by the Lac promoter or the PUTR element.

[0037] As a preferred embodiment of this application, the nucleotide sequence of the rmlA gene is shown in SEQ ID NO.84.

[0038] As a preferred embodiment of this application, the nucleotide sequence of the rmlB gene is shown in SEQ ID NO.85.

[0039] As a preferred embodiment of this application, the nucleotide sequence of the rmlC gene is shown in SEQ ID NO.86.

[0040] As a preferred embodiment of this application, the nucleotide sequence of the rmlD gene is shown in SEQ ID NO.87.

[0041] As a preferred embodiment of this application, the nucleotide sequence of the aceE gene is shown in SEQ ID NO.88.

[0042] As a preferred embodiment of this application, the nucleotide sequence of the gltA gene is shown in SEQ ID NO.89.

[0043] A fifth aspect of the present invention is to provide the application of the recombinant genetically engineered strain in the fermentation production of rhamnolipids.

[0044] Compared with the prior art, the present invention has at least the following beneficial effects:

[0045] 1. The promoter and 5′UTR are designed as a whole functional unit to achieve synergistic regulation of transcription and translation processes;

[0046] 2. The constructed PUTR library has a wide range of expression intensity coverage and a continuously adjustable gradient distribution;

[0047] 3. The screened PUTR elements are derived from host endogenous sequences, exhibiting high genetic stability and suitability for long-term industrial fermentation;

[0048] 4. To provide a generalized tool platform for the fine regulation of complex metabolic pathways in *Pseudomonas putida* KT2440;

[0049] 5. Applying the PUTR library to the expression optimization of key genes and central metabolic nodes in the rhamnolipid synthesis pathway enables precise control of carbon flux allocation, significantly improves the synthesis efficiency of rhamnolipids, and verifies the engineering applicability and practical value of the PUTR library in the synthesis of high-value-added biosurfactants. Attached Figure Description

[0050] Figure 1 Growth curves for all PUTR-characterized recombinant strains.

[0051] Figure 2 The characterization results of the PUTR library are shown, with intensity quantified as fluorescence intensity / OD. 600 .

[0052] Figure 3 Rhamnose lipid production and OD of recombinant engineered strain KT01-KT36 600 .

[0053] Figure 4 Rhamnose lipid production and OD of recombinant engineered strain KT37-KT40 600 .

[0054] Figure 5 Rhamnose lipid production and OD of recombinant engineered strain KT41-KT44 600 . Detailed Implementation

[0055] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. This application can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application.

[0056] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0057] Furthermore, it should be understood that the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, does not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated. It should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in this invention does not preclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0058] The parental strains of this invention, *Pseudomonas putida* KT24400 and *Pseudomonas aeruginosa* PAO1, and the vector pBBR1MCS5, were purchased from Hangzhou Hongsai Biotechnology Co., Ltd.

[0059] In the following examples, the final concentration of gentamicin in the culture medium was 50 mg / L, the final concentration of tetracycline in the culture medium for Escherichia coli was 15 mg / L, and the final concentration in the culture medium for Pseudomonas putida was 25 mg / L.

[0060] The composition of LB liquid medium is: yeast 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, water as solvent, and natural pH.

[0061] LB solid plates are made by adding 20 g / L agar to LB liquid medium.

[0062] The fermentation medium consisted of 10 g / L glucose, 10 g / L glycerol, 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride, with deionized water as the solvent and natural pH.

[0063] Example 1: Determination of rhamnolipid content

[0064] (1) Take 1 mL of bacterial culture into a 2 mL EP tube, centrifuge at 12000 rpm for 3 minutes, and separate the supernatant and precipitate. The supernatant is used for the detection of rhamnolipids and other metabolites.

[0065] (2) Dilute the sample to a concentration of 0-10 g / L, mix the sample and acetonitrile in a 1:1 ratio, shake well in a constant temperature shaker, and then place at 4℃ overnight. After processing, centrifuge the sample at 12000 rpm for 3 minutes, and then put the 0.22 μm organic membrane into a liquid chromatography bottle for analysis.

[0066] (3) The instrument was a Thermo Fisher UPLC ultra-high pressure liquid chromatograph. The chromatographic column was a C18 column (4.6×150 mm, 3 μm); the electrospray detector was used; the injection volume was 2 μL; the column temperature was 40℃; the flow rate was 1 mL / min; the mobile phase used was two phases, A and B, with phase A being pure acetonitrile and phase B being 0.2% formic acid water (v / v). The gradient elution program is shown in Table 1.

[0067] Table 1: Gradient elution procedure Serial number Time (min) A（%） B（%） 1 0 70 30 2 1 70 30 3 9 100 0 4 11 100 0 5 12 70 30 6 15 70 30 .

[0068] Example 2: Construction of recombinant plasmid pBBR1MCS5-rhlAB

[0069] (1) Using pBBR1MCS5 plasmid as a template and pBB-Line-F and pBB-Line-R as primers, PCR was performed to obtain a linearized vector. The PCR products were digested with DpnI. Using the Pseudomonas aeruginosa PAO1 genome as a template and rhlAB-F / R as primers, PCR amplification was performed. All PCR products were detected by 1.0% agarose gel electrophoresis and the PCR fragments were purified.

[0070] (2) Following the instructions of the One step clone kit (Vazyme Biotech, Nanjing, China), the linearized vector was ligated with the gene fragment rhlAB and transformed into E. coli DH5α. The transformed cells were plated on gentamicin-resistant plates, and single colonies were picked for colony PCR verification using primers pBB-VF and pBB-VR. Sequencing verification yielded the pBBR1MCS5-rhlAB plasmid.

[0071] Table 2: Primers for Example 2 Primer name [[ID=9...]]Sequence (5’-3’) pBB-Line-F CCTATAGTGAGTCGTATTACGCGC pBB-Line-R CAGCTTTTGTTCCCTTTAGTGAGG rhlAB-F TAAAGGGAACAAAAGCTGATGCGGCGCGAAAGTCTG rhlAB-R GCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAATCAGGACGCAGCCTTCAGC pBB-VF GCTTCCATGTCGGCAGAATG pBB-VR CTGCGCAACTGTTGGGAAG .

[0072] Example 3: Obtaining PUTR candidate components

[0073] To provide a regulatory element library with high expression intensity, dynamic stability, and cross-product compatibility for rational metabolic engineering of *Pseudomonas putida* KT2440 chassis cells, a comparative transcriptome analysis was conducted on wild-type *Pseudomonas putida* KT2440 and engineered rhamnolipid strain *P. putida* KT2440 / pBBR1MCS5-rhlAB. The specific steps were as follows:

[0074] Single colonies of engineered rhamnolipin strain and wild-type *Pseudomonas putida* strain KT2440 / pBBR1MCS5-rhlAB were inoculated into 10 mL of LB broth and cultured overnight at 30–37 °C and 150–200 rpm. 1 mL of the pre-culture was then inoculated into a 500 mL shake flask containing 20–50 mL of fermentation medium and fermented for 48 h. Based on the growth and product accumulation curves, 50 mL of bacterial suspension was collected at the exponential, stationary, and decline phases. The suspension was centrifuged at 5500 rpm for 10 min at 4 °C, the supernatant was discarded, and the cells were resuspended in PBS buffer. This washing process was repeated three times, followed by centrifugation again, discarding the supernatant, and the precipitated cells were immediately flash-frozen in liquid nitrogen for 30 min and then stored at -80 °C. The collected samples were then sent to Novogene Biotech for comparative transcriptomics analysis.

[0075] Based on transcriptome analysis, the expression levels of genes in the six groups of samples were quantitatively analyzed. The quantitative indicator was the number of fragments per kilobase of exonper million mapped reads (FPKM). An FPKM value of 1 was used as the threshold for gene expression. Eighty PUTRs of varying intensities were selected from genes with an FPKM greater than 1 to construct a PUTR library, with FPKM values ​​ranging from 823.03 to 1044427.65. The sequence approximately 500 bp upstream of the start codon of candidate genes was extracted as the candidate PUTR element fragment.

[0076] Example 4: Construction of recombinant plasmids containing PUTR candidate elements

[0077] The expression intensity of PUTR is a combination of transcription and translation. In order to characterize the intensity of 80 endogenous PUTRs in Pseudomonas putida KT2440, this invention uses green fluorescent protein (GFP) as a reporter gene to describe and characterize the intensity of PUTRs that regulate their expression.

[0078] (1) Using pTrc99a-gfp plasmid as a template and pB-gfp-F and pB-gfp-R as primers, the gfp fragment was amplified by PCR. Using pBBR1MCS5 plasmid as a template and pB-line-F and pB-line-R as primers, the linearized vector was amplified by PCR. The PCR products were digested with DpnI. All PCR products were detected and purified by 1.0% agarose gel electrophoresis. The linearized vector was ligated to the gfp gene fragment according to the instructions of the One step clone kit (Vazyme Biotech, Nanjing, China). The cloning product was transformed into E. coli DH5α and plated on gentamicin-resistant plates. The transformation conditions were: incubation on ice for 30 min, heat shock at 42℃ for 90 s, incubation on ice for 2 min, addition of 700 μL of liquid LB, and incubation on a shaker at 37℃ for 60 min. Single colonies were selected and colony PCR was performed using primers pBB-VF and pBB-VR for verification. Positive transformants were obtained through sequencing. The pBB-Plac-gfp plasmid was extracted from the positive transformants.

[0079] (2) Using plasmid pBB-Plc-gfp as a template and pBB-gfp-line-F1 and pBB-gfp-line-R1 as primers, PCR was performed to obtain the linearized vector pBB-gfp-Line with the original promoter deleted. The PCR products were digested with DpnI. Using the genome of *Pseudomonas putida* KT2440 as a template and primers “gene name”-F and “gene name”-R, 80 PUTR fragments were amplified. All PCR products were detected by 1.0% agarose gel electrophoresis and the PCR fragments were purified. Following the instructions of the One-Step Cloning Kit (Vazyme Biotech, Nanjing, China), the linearized vector was ligated with the PUTR fragment and transformed into E. coli DH5α. The transformed cells were plated on gentamicin-resistant plates under the following conditions: incubation on ice for 30 min, heat shock at 42°C for 90 s, incubation on ice for 2 min, addition of 700 μL of LB liquid, and incubation on a shaker at 37°C for 60 min. Single colonies were picked and colony PCR was performed using primers pBB-VF and pBB-VR for verification. Sequencing confirmed the presence of positive transformants. The pBB-PUTR (gene name)-gfp plasmid was extracted from the positive transformants. The reaction system and procedure for colony PCR were the same as above. This yielded 80 recombinant plasmids showing expression of fluorescent protein genes regulated by different intensities of PUTR.

[0080] (3) Using pTrc99a-gfp plasmid as a template and pV-gfp-F and pV-gfp-R as primers, the gfp fragment was amplified by PCR. Using pVLT33 plasmid as a template and pV-line-F and pV-line-R as primers, the linearized vector was amplified by PCR. The PCR products were digested with DpnI. All PCR products were detected and purified by 1.0% agarose gel electrophoresis. The linearized vector was ligated to the gfp gene fragment according to the instructions of the One step clone kit (Vazyme Biotech, Nanjing, China). The cloning product was transformed into E. coli DH5α and plated on gentamicin-resistant plates. The transformation conditions were: incubation on ice for 30 min, heat shock at 42℃ for 90 s, incubation on ice for 2 min, addition of 700 μL of liquid LB, and incubation on a shaker at 37℃ for 60 min. Single colonies were selected and colony PCR was performed using primers pV-VF ​​and pV-VR for verification. Positive transformants were obtained through sequencing. The pV-gfp plasmid was extracted from the positive transformants.

[0081] (4) Using plasmid pV-gfp as a template and primers Ptac-gfp-F and gfp-R as primers, PCR was performed to amplify the Ptac-gfp fragment. The PCR product was detected by 1.0% agarose gel electrophoresis and the PCR fragment was purified. The Ptac-gfp fragment and the linearized vector pBB-gfp-Line were ligated according to the instructions of the One Step Cloning Kit (Vazyme Biotech, Nanjing, China). The cloning product was transformed into E. coli DH5α, plated on gentamicin-resistant plates, and single colonies were picked for colony PCR verification using primers pBB-VF and pBB-VR. Sequencing verification yielded positive transformants. The pBB-Ptac-gfp plasmid was extracted from the positive transformants.

[0082] Table 3: Primers for Example 4 Primer Name Sequence (5'-3') pB-gfp-F CTAAAGGGAACAAAAGCTGATGGTGAGCAAAGGCGAG pB-gfp-R CTATAGGGCGAATTGTCACTTGTACAGTTCGTCCATAC pB-line-F CAATTCGCCCTATAGTGAGTCGTATTAC pB-line-R CAGCTTTTGTTCCCTTTAGTGAGG pBB-VF GCTTCCATGTCGGCAGAATG pBB-VR CTGCGCAACTGTTGGGAAG pBB-gfp-line-F1 ATGGTGAGCAAAGGCGAGGAAC pBB-gfp-line-R1 CGTGCCAGCTGCATTAATGAATCG pV-gfp-F CAATTTCACACAGGAAACAATGGTGAGCAAAGGCGAG pV-gfp-R CATCCGCCAAAACAGCCTCACTTGTACAGTTCGTCCATAC pV-line-F GGCTGTTTTGGCGGATGAG pV-line-R TGTTTCCTGTGTGAAATTGTTATCCGC pV-VF TTTTTCCCGCGTTTTCGCAGAAACGT Pv-VR ATCATGAAAGGCTGGCTTTTTCTTGTT Ptac-gfp-F TTAATGCAGCTGGCACGTTGACAATTAATCATCGGCTCGTATAATGTGTGG ssrA-F TCATTAATGCAGCTGGCACGGTGCCATCCCTACTTCAATATCG ssrA-R CCTCGCCTTTGCTCACCATACGCTAGCGTTACGCTAC rpnB-F TTAATGCAGCTGGCACGCGAAGGCTTCCTGCCGG rnpB-R CCTCGCCTTTGCTCACCATCGCCAAGGTTACCGACAC oprI-F ATTAATGCAGCTGGCACGCCTCGCAGGTCTTCTCGG oprI-R CCTCGCCTTTGCTCACCATCGTGTTCCCCTTCAAGGAC PP_RS2785-F ATTAATGCAGCTGGCACGTCACTGCCAACCTGGTG PP_RS27855-R CCTCGCCTTTGCTCACCATTGCCTGTCTCCCCAGATAG glnK-F TTAATGCAGCTGGCACGGGAGAAATGGCCGCCAG glnK-R CCTCGCCTTTGCTCACCATGAAACTCTCTCCCGATTTGG gltI-F CATTAATGCAGCTGGCACGCAAGAAGGGATGGCCTG gltI-R CTCGCCTTTGCTCACCATATTTTCCTCGACGTGTTTG csrA-F ATTAATGCAGCTGGCACGGAGAGTGAAGCCAAACGC csrA-R CTCGCCTTTGCTCACCATGTAGTTCTCCTTGGTGATGTA oprF-F ATTAATGCAGCTGGCACGTGATGCTGAAAGTGCTGTAC oprF-R CTCGCCTTTGCTCACCATCCGTTAAATCCCCATCTGG atpE-F TTAATGCAGCTGGCACGAGGTCAAGGGACTGGGC atpE-R CTCGCCTTTGCTCACCATCTTTCCTCCCGACTTTTACG yccA-F TTAATGCAGCTGGCACGCCCATTGCAAGCACTCC yccA-R CCTCGCCTTTGCTCACCATGGCGACACTCCTTGGTAA PP_5329-F TTAATGCAGCTGGCACGTCCCGCTGTATGCCGTG PP_5329-R CTCGCCTTTGCTCACCATGCCTTGCTCCTAGCAGG acpP-F ATTAATGCAGCTGGCACGGCAGGTCAGGCCAACTAC acpP-R CTCGCCTTTGCTCACCATACCTAGTTTTCACTCCTAATGG rpsA-F TTAATGCAGCTGGCACGTGAGGACGTCAGCAACG rpsA-R CTCGCCTTTGCTCACCATTGTGGGTTCGTTTTAAGTAAAG tapB-F ATTAATGCAGCTGGCACGGCCATGGGTTATATCGATAG tapB-R CTCGCCTTTGCTCACCATTGTCTTATTTCCTATGAAACTT PP_4470-F TTAATGCAGCTGGCACGCGAACGGGTGATGACC PP_4470-R CTCGCCTTTGCTCACCATATAAATTGACCAGAGTTTGGT cspD-F ATTAATGCAGCTGGCACGGGTAAACAATCCGCACACTG cspD-R CCTCGCCTTTGCTCACCATCCCTATGTCCCTCTGCAAAG PP_0367-F TAATGCAGCTGGCACGACCTTGATAGAGGGGGCC PP_0367-R CGCCTTTGCTCACCATTTAAAACAGGCTAGGGGCG PP_5700-F TTAATGCAGCTGGCACGGAACAAAGGCAACCAGG PP_5700-R CTCGCCTTTGCTCACCATGATTCAATCCTTATTCAGCTTG algQ-F TAATGCAGCTGGCACGTTTGCTGGCAGCACGG algQ-R CTCGCCTTTGCTCACCATGGCGATCTCTTCCCTTCTG PP_3580-F TAATGCAGCTGGCACGCGCCGGTTCCTGGCTGC PP_3580-R CGCCTTTGCTCACCATATCGATGCGGCCGCC PP_4793-F TAATGCAGCTGGCACGCATCGCCGCGTTTTCC PP_4793-R CTCGCCTTTGCTCACCATCCGTTCACCTCATTGAAGG PP_0641-F TTAATGCAGCTGGCACGTTTGTGCCGTACACCCC PP_0641-R CTCGCCTTTGCTCACCATGTTTGACTCCTGACGTCTTC rmf-F ATTAATGCAGCTGGCACGCCGTACACCTTTCCAGCAG rmf-R CTCGCCTTTGCTCACCATAGGGTGTTGCCCTCAC rpsO-F TTAATGCAGCTGGCACGGCGTAGGATGCAGCAAA rpsO-R CGCCTTTGCTCACCATATCCCTGTATTAATAAAAAAGGTG rpoH-F tAATGCAGCTGGCACGATCCTCAAGCTGGGTG rpoH-R CGCCTTTGCTCACCATTCAAGAACCTCCGACTTAC rpsB-F ATTAATGCAGCTGGCACGCGTTGACGATATAATCGTGGC rpsB-R CTCGCCTTTGCTCACCATGATAGTTCCTTGATAAGTCGGG PP_0679 -F TAATGCAGCTGGCACGTCTGCTGGCGCTTGCC PP_0679 -R CGCCTTTGCTCACCATGGGTCAGGGTTTCTGGC rpsA-F TAATGCAGCTGGCACGTGAGGACGTCAGCAAC rpsA-R CTCGCCTTTGCTCACCATTGTGGGTTCGTTTTAAGTAAAG PP_5392-F TTAATGCAGCTGGCACGGCGAGAGAAGCCGTACC PP_5392-R CGCCTTTGCTCACCATGGTGAATGAGCTCCAACG oprF-F TTAATGCAGCTGGCACGGAACAAGTCGCGGTAACAC oprD-R CGCCTTTGCTCACCATCGTGATTTGCTCCTTTGG rpoZ-F TTAATGCAGCTGGCACGTACCAGTGCAGCAGCAT rpoZ-R CTCGCCTTTGCTCACCATGGTCTTCCTCAGTAGCAATTG clpSA-F TTAATGCAGCTGGCACGCAGTCAGGCTTTTGCAG clpSA-R CTCGCCTTTGCTCACCATGGAAAGAATTTCATCATGAGC PP_1910-F TAATGCAGCTGGCACGACCTGCTCCCTGCTCAG PP_1910-R CTCGCCTTTGCTCACCATAGGCGCAGCATTCTAGGG infC-F TTAATGCAGCTGGCACGTTTTTTCGCGGTGGCAGC infC-R CTCGCCTTTGCTCACCATGGCGCCTCCTCTCCTAG PP_0951-F TTAATGCAGCTGGCACGAACCCTGATGAAGGTTGCC PP_0951-R CTCGCCTTTGCTCACCATACAGCTTCTCCTTTGTTGCC fliC-F TTAATGCAGCTGGCACGCGCTGCTGCTGCAGAAG fliC-R CGCCTTTGCTCACCATGACGAATTCCTCGTTGTATGG PP_2161-F TTAATGCAGCTGGCACGCTCAATACCGCGCAAAGC PP_2161-R CTCGCCTTTGCTCACCATTGGCTATTCTCCATCAATGG cspA-F TTAATGCAGCTGGCACGGGGGGTCAGGTAGAAGAAC cspA-R CTCGCCTTTGCTCACCATCTGAACTACTCCGGATTTT rpsH-F TTAATGCAGCTGGCACGCATGAAAAACCGCGAGC rpsH-R CTCGCCTTTGCTCACCATGGGCTAGATGCTCCTG rplM-F TAATGCAGCTGGCACGCATGGTCACGGAACCAG rplM-R CTCGCCTTTGCTCACCATTCTCTAAAGCCTCAGAGGC rpmI-F TAATGCAGCTGGCACGAGGTTGGCATCGTCTC rpmI-R GCCTTTGCTCACCATTCGATAAAATTACACATAATCATCAG rpsF-F TAATGCAGCTGGCACGCGCGCCTGTTTTTTTCG rpsF-R CGCCTTTGCTCACCATGAATGCTCCTTACGGG infA-F TAATGCAGCTGGCACGGCGCGTTCGTTAATTC infA-R CTCGCCTTTGCTCACCATGTACAGACTCCAGATACG fusA-F AATGCAGCTGGCACGAGACAATCAGTGGAGCTAG fusA-R CTCGCCTTTGCTCACCATGATAAGCCCTCAAACGG rpmB-F TAATGCAGCTGGCACGTGGTGAGCGAATTGTACTG rpmB-R CGCCTTTGCTCACCATGACTCAGCCCTCTAAAACC flgM-F TAATGCAGCTGGCACGCGCCATGCCAGACTCC flgM-R CGCCTTTGCTCACCATCGTGTTGTCGCGCTTG rpsL-F TAATGCAGCTGGCACGAGTGCTGCTGGGTATC rpsL-R CGCCTTTGCTCACCATCTACTAGCTCCACTGATTG PP_3765-F ATTAATGCAGCTGGCACGGGTTGAAATAGACATTGCG PP_3765-R CGCCTTTGCTCACCATTGTAATCACTCCTGTACGTC rpsU-F TAATGCAGCTGGCACGATGTCCCAGGCAAAGG rpsU-R CTCGCCTTTGCTCACCATCCAGGGCTACCTTAATTCA PP_1111-F TAATGCAGCTGGCACGAGCTTGGTGAAGTGCTGG PP_1111-R CGCCTTTGCTCACCATTGGGTGCATGGGCTTG rpoS-F TAATGCAGCTGGCACGTAGGTGGGTGGACCTG rpoS-R TCGCCTTTGCTCACCATTGTTATAATCCTTTGCTGAGTTC PP_5395-F ATTAATGCAGCTGGCACGAGTGGTCATGGTCGTGCTG PP_5395-R CGCCTTTGCTCACCATCCTCCATGTCAGCGGC phaF-F ATTAATGCAGCTGGCACGTGAAACACATGGGGTGGG phaF-R CTCGCCTTTGCTCACCATGCTGTGTACCTCATGCTC gcdH-F TTAATGCAGCTGGCACGTGAGCCGTGGTAGCAGC gcdH-R CTCGCCTTTGCTCACCATGAGCGAATCCTCGAAATAGGG PP_4667-F TTAATGCAGCTGGCACGTGGCTTAGCTGCGGCG PP_4667-R CTCGCCTTTGCTCACCATCTGCGTTCTCCTTGGAATTG amaB-F TTAATGCAGCTGGCACGTGCCGTTTGCCAAACCC amaB-R CTCGCCTTTGCTCACCATTTGTGCATCTCCTACATCGG PP_2296-F ATTAATGCAGCTGGCACGCTTTAACTGGATCGACTGGC PP_2296-R CTCGCCTTTGCTCACCATGTTTCTTATCTACCACCTTGT cioA-F ATTAATGCAGCTGGCACGTCAATTGGTAGAGGTGCC cioA-R CTCGCCTTTGCTCACCATGGTGCTTCCTCTTCAGAT gtsA-F ATTAATGCAGCTGGCACGGCATTGTTCGACACAGCCTG gtsA-R CTCGCCTTTGCTCACCATCGGAGCACCTTTCTTGTTG ydcj-F ATTAATGCAGCTGGCACGGCTGGTGCTGCTGCAAAG ydcJ-R CTCGCCTTTGCTCACCATCGCTAGCTTCCTGGTTGG PP_5458-F TTAATGCAGCTGGCACGGCAACTGGTCGATGTGG PP_5458-R CTCGCCTTTGCTCACCATTTGAAACTACCTTCTTGCGG PP_4108-F CATTAATGCAGCTGGCACGTTGCAATGCGTGTCGCGGC PP_4108-R TCGCCTTTGCTCACCATGGGGCACGCGGACCTTG phhB-F TAATGCAGCTGGCACGCGTTCGCTGGGCATCAG phhB-R CTCGCCTTTGCTCACCATGAGGCTCTCCTGGTGAGG yhiS-F AATGCAGCTGGCACGCAAGTACGGCGAACGGG yihS-R CGCCTTTGCTCACCATGGGCCAGCTTGATCCTTG PP_3598-F TAATGCAGCTGGCACGCGCAGGTGTTCTTCAACC PP_3598-R CGCCTTTGCTCACCATATGTCACCTTGGGACAGG PP_4975-F TTAATGCAGCTGGCACGCGGTCTGGATGGTCATG PP_4975-R TCGCCTTTGCTCACCATCTGTACTCCTGACCTGC PP_0916-F TTAATGCAGCTGGCACGACGCATGCGTTTCAGG PP_0916-R CTCGCCTTTGCTCACCATAAAGGGTCACGAAAGATGC PP_2177-F TAATGCAGCTGGCACGTGAGACGCCTGTGGCC PP_2177-R CGCCTTTGCTCACCATCGTTTCGGACTGCCGTG PP_4835-F TAATGCAGCTGGCACGGAAGGGGCCAAGCAGG PP_4835-R CGCCTTTGCTCACCATGGAGAGATCCGGTCAGG PP_4557-F TAATGCAGCTGGCACGAGCCACCCCTTGGTTG PP_4557-R CGCCTTTGCTCACCATGTGGTTTTGCCTTTTGAGC PP_0354-F TTAATGCAGCTGGCACGACGAATCGTCCTGATCAAG PP_0354-R TCGCCTTTGCTCACCATGAAACTGACCGTATCGC PP_1753-F TAATGCAGCTGGCACGAGTTGGTGGATGCCGG PP_1753-R CTCGCCTTTGCTCACCATAAAATCCTCGATCAAGCCC PP_3361-F ATTAATGCAGCTGGCACGACGTACCTGGTTGAGGTCG PP_3361-R CGCCTTTGCTCACCATATCATCAGGTTCCGAGCG sqr-F TAATGCAGCTGGCACGGGTGTGTGATGAATGTGC sqr-R CTCGCCTTTGCTCACCATCACGTTCTCCAATTTATCCG PP_2921-F TAATGCAGCTGGCACGGCTCCAGTGTCACGCCC PP_2921-R CGCCTTTGCTCACCATGACCGCGTCTCCTGGC PP_1272-F TAATGCAGCTGGCACGGGGCCACCCTATCTTC PP_1272-R CTCGCCTTTGCTCACCATGAGAATACATCTCCAGCAAC gtsD-F TTAATGCAGCTGGCACGGTTGGTGGTGTTCAAGC gtsD-R CTCGCCTTTGCTCACCATGTATTGGATCCCGAGGTAG PP_3595-F TTAATGCAGCTGGCACGATCGACAGCGCCCAGACC PP_3595-R CTCGCCTTTGCTCACCATGACGAACCTCTTGGGCAGG PP_3597-F TAATGCAGCTGGCACGCCTGTTCAGCGGCCTG PP_3597-R TCGCCTTTGCTCACCATCTTGTTGTTCTCCCGCG Novel-F AATGCAGCTGGCACGAAGACCTGTCGCTGGAAG Novel-R CTCGCCTTTGCTCACCATCAGTGCTTGTTATTGTTCTTG rpoE-F TAATGCAGCTGGCACGGGTTAAGTGCCAGGCTGAG rpoE-R CCTCGCCTTTGCTCACCATGAACACTCCTCAGTGAACTCG .

[0083] Example 5: Construction of recombinant characterization strains of *Pseudomonas putida*

[0084] (1) Preparation of competent cells of *Pseudomonas putida* KT2440: The *Pseudomonas putida* KT2440 strain was taken out from the -80℃ freezer and streaked onto LB solid medium. It was cultured overnight at 30℃. A single colony was picked and inoculated into 10 mL of liquid LB medium. After culturing at 30℃ and 200 rpm for 12 h, 1% of the inoculum was added to 50 mL of liquid LB medium and cultured at 30℃ and 200 rpm until OD. 600 =0.8~1.2, immediately incubate on ice for 20 min. Transfer the bacterial culture to a pre-chilled 50 mL sterile centrifuge tube, centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend in 20 mL pre-chilled HEPES buffer, centrifuge at 5000 rpm for 10 min, repeat the washing three times, and finally resuspend in 1 mL 15% glycerol and aliquot into sterile 1.5 mL EP tubes, 100 μL per tube, and store at -80℃.

[0085] (2) The correctly sequenced recombinant plasmid pBB-PUTR (specific gene)-gfp was electroporated into competent Pseudomonas putida KT2440 cells at 2.5 kV. 1-2 mL of LB liquid medium was added, and the cells were incubated for 2 h at 30℃ and 200 rpm in a shaker. The bacterial culture was then spread on LB plates containing gentamicin and cultured until single colonies grew to obtain recombinant bacteria containing the recombinant plasmid. The plasmid pBBR1MCS5-gfp without the promoter was introduced into KT2440 cells as a negative control. The plasmids pBBR1MCS5-Plac-gfp and pBBR1MCS5-Ptac-gfp containing the commonly used promoters Plac and Ptac were introduced into KT2440 cells as the other two control groups.

[0086] Example 6: Characterization of PUTR library

[0087] Single colonies of the recombinant strain containing the candidate PUTR recombinant plasmid and the control recombinant strain were inoculated into 10 mL of LB medium and cultured overnight at 30–37°C and 150–200 rpm. 1% of the pre-culture was inoculated into 48-well plates containing 1–2 mL of fermentation medium. To prevent the inoculation amount from affecting the nutrient concentration in the medium, the medium concentration was scaled up proportionally. Each strain was subcultured three times, and each subculturation was performed on five 48-well plates. Appropriate concentrations of antibiotics were added, and the plates were cultured at 30–37°C and 150–200 rpm. Samples were taken at the early logarithmic, prologial, mid-logarithmic, late logarithmic, and early stationary phases. One plate was taken at each sampling point, and 1 mL of bacterial culture was collected from each well. The samples were centrifuged at 12,000 rpm at 4°C for 1 min, the supernatant was discarded, and the samples were washed three times with PBS buffer (pH 7.4) before fluorescence intensity and OD were measured. 600 The fluorescence intensity of GFP was measured at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Relative fluorescence intensity was expressed as per OD of the whole cell. 600 The fluorescence intensity is used to characterize it, i.e., GFP / OD 600 .

[0088] PUTR characterization of recombinant strain growth Figure 1 As shown. The characterization results are as follows. Figure / / As shown, the results indicate that each PUTR exhibits either very stable expression levels or a gradually increasing expression level with cell growth at various growth stages. Furthermore, among the 80 PUTR candidate elements screened, 28 are PUTR elements (PUTR... PP_5700 PUTR cspD PUTR PP_4470 PUTR tapB PUTR acpP PUTR csrAPUTR oprI PUTR PP_0951 PUTR rpoH PUTR rpsB PUTR PP_2126 PUTR rmf PUTR fliC PUTR PP_0641 PUTR rpmB PUTR rpsH PUTR rpsU PUTR rpsF PUTR rpsL PUTR rplM PUTR rpoS PUTR PP_3765 PUTR PP_4835 PUTR rpoE PUTR cioA PUTR PP_2296 PUTR PP_5458 PUTR phaF PUTR exhibits higher fluorescence intensity than Plac and Ptac. These high-intensity expression elements provide strong driving components for high-yield metabolic pathways. Among them, PUTR... PP_2296 PUTR rpsL PUTR rplM PUTR PP_4470 PUTR csrA PUTR oprI It exhibits stable expression levels across all growth stages, unaffected by fluctuations in growth metabolism, making it suitable for genes requiring continuous expression. Of the remaining PUTRs, 14 showed fluorescence intensity similar to Plac, while the fluorescence intensity of the remaining PUTRs gradually decreased to levels similar to the negative control, equivalent to no or very weak fluorescence intensity.

[0089] The intensity range of the PUTR library in the early logarithmic, mid-logarithmic, late logarithmic, and early stationary phases was 47.38%-790.51%, 15.14%-948.31%, 17.12%-1168.1%, and 19.13%-1468.22% of that of the Plac promoter, respectively. Overall, the PUTR library exhibited a continuous and broad gradient at the transcriptional and translational levels. Furthermore, the library contains PUTRs with different characteristics, including stable expression at different growth stages, high expression at the logarithmic stage, or high expression at the stationary phase. These PUTRs could provide an effective tool for regulating gene expression in metabolic engineering of *Pseudomonas putida* KT2440.

[0090] Example 7: Construction of recombinant plasmid pBBR1MCS5-Plac-rhlAB-Plac-rmlBDAC

[0091] (1) Using pBBR1MCS5-rhlAB plasmid as a template and pBAB-Line-F and pBAB-Lac-Line-R as primers, PCR was performed to obtain a linearized vector. The PCR products were digested with DpnI. Using the Pseudomonas aeruginosa PAO1 genome as a template and Lac-rmlBDAC-F / R as primers, PCR amplification was performed. All PCR products were detected by 1.0% agarose gel electrophoresis and the PCR fragments were purified.

[0092] (2) Following the instructions of the One step clone kit (Vazyme Biotech, Nanjing, China), the linearized vector was ligated with the gene fragment rmlBDAC, transformed into E. coli DH5α, plated on gentamicin-resistant plates, and single colonies were picked for colony PCR verification using primers pBB-VF and pBB-VR. Sequencing verification yielded the plasmid pBBR1MCS5-Plac-rhlAB-Plac-rmlBDAC.

[0093] Table 4: Primers for Example 7 Primer Name Sequence (5'-3') pBAB-Line-F CCTATAGTGAGTCGTATTACGCGCGCT pBAB-Lac-Line-R GCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAATCAGGACGCAGCCTTCAGC Lac-rmlBDAC-F TTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTGCTTGAACAAGGTATGACTC rmlBDAC-R TAATACGACTCACTATAGGTCAGGGGAAGCAGTCG pBB-VF GCTTCCATGTCGGCAGAATG pBB-VR CTGCGCAACTGTTGGGAAG .

[0094] Example 8: Combination optimization of rhlAB and rmlBDAC

[0095] (1) Using pBBR1MCS5-Plac-rhlAB-Plac-rmlBDAC plasmid as a template, and rmlBDAC-PUTR-Line-F and rhlAB-PUTR-Line-R as primers, PCR amplification was performed to obtain a linearized vector. The PCR product was digested with DpnI. Using the genome of *Pseudomonas putida* KT2440 as a template, and AB-0951-PA-F / R as primers, PCR amplification was performed to obtain PUTR. PP_0951 Fragments. All PCR products were detected and purified by 1.0% agarose gel electrophoresis. The linearized vector and gene fragment PUTR were then cloned according to the instructions of the One-Step Cloning Kit (Vazyme Biotech, Nanjing, China). PP_0951 The ligation was performed, and the cells were transformed into E. coli DH5α. The cells were plated on gentamicin-resistant plates, and single colonies were picked for colony PCR verification using primers pBB-VF and pBB-VR. Sequencing confirmed the presence of pBBR1MCS5-Plac-rhlAB-PUTR. PP_0951-rmlBDAC plasmid was obtained according to the method described. rmf -rmlBDAC, pBBR1MCS5-Plac-rhlAB-PUTR PP_3765 -rmlBDAC, pBBR1MCS5-Plac-rhlAB-PUTR PP_1111 -rmlBDAC, pBBR1MCS5-Plac-rhlAB-PUTR PP_3595 -rmlBDAC plasmid.

[0096] (2) Using pBBR1MCS5-Plac-rhlAB-Plac-rmlBDAC plasmid as a template, and AB-PUTR-Line-F and pBB-gfp-line-R1 as primers, PCR amplification was performed to obtain a linearized vector. The PCR product was digested with DpnI. Using the genome of *Pseudomonas putida* KT2440 as a template, and AB-0951-F / R as primers, PCR amplification was performed to obtain PUTR. PP_0951 Fragments. All PCR products were detected and purified by 1.0% agarose gel electrophoresis. The linearized vector and gene fragment PUTR were then cloned according to the instructions of the One-Step Cloning Kit (Vazyme Biotech, Nanjing, China). PP_0951 The ligation was performed, and the cells were transformed into E. coli DH5α. The cells were plated on gentamicin-resistant plates, and single colonies were picked for colony PCR verification using primers pBB-VF and pBB-VR. Sequencing confirmed the presence of pBBR1MCS5-PUTR. PP_0951 -rhlAB-Plac-rmlBDAC plasmid was obtained according to the method described. rmf -rhlAB-Plac-rmlBDAC, pBBR1MCS5-PUTR PP_3765 -rhlAB-Plac-rmlBDAC, pBBR1MCS5-PUTR PP_1111 -rhlAB-Plac-rmlBDAC, pBBR1MCS5-PUTR PP_3595 -rhlAB-Plac-rmlBDAC plasmid.

[0097] (3) Using pBBR1MCS5-PUTR PP_0951Using the plasmid rhlAB-Plac-rmlBDAC as a template, and with primers rmlBDAC-PUTR-Line-F and rhlAB-PUTR-Line-R, PCR amplification was performed to obtain a linearized vector. The PCR product was digested with DpnI. Using the genome of *Pseudomonas putida* KT2440 as a template, and with primers AB-0951-PA-F / R, PCR amplification was performed to obtain the PUTR vector. PP_0951 Fragments. All PCR products were detected and purified by 1.0% agarose gel electrophoresis. The linearized vector and gene fragment PUTR were then cloned according to the instructions of the One-Step Cloning Kit (Vazyme Biotech, Nanjing, China). PP_0951 The ligation was performed, and the cells were transformed into E. coli DH5α. The cells were plated on gentamicin-resistant plates, and single colonies were picked for colony PCR verification using primers pBB-VF and pBB-VR. Sequencing confirmed the presence of pBBR1MCS5-PUTR. PP_0951 -rhlAB-PUTR PP_0951 -rmlBDAC plasmid. pBBR1MCS5-PUTR was obtained according to the method described. PP_0951 -rhlAB-PUTR rmf -rmlBDAC, pBBR1MCS5-PUTR PP_0951 -rhlAB-PUTR PP_3765 -rmlBDAC, pBBR1MCS5-PUTR PP_0951 -rhlAB-PUTR PP_1111 -rmlBDAC, pBBR1MCS5-PUTR PP_0951 -rhlAB-PUTR PP_3595 -rmlBDAC plasmid.

[0098] (4) Using pBBR1MCS5-PUTR rmf Using the plasmid rhlAB-Plac-rmlBDAC as a template, and primers rmlBDAC-PUTR-Line-F and rhlAB-PUTR-Line-R as primers, PCR amplification was performed to obtain a linearized vector. The PCR product was digested with DpnI. Using the genome of *Pseudomonas putida* KT2440 as a template, and primers AB-rmf-PA-F / R as primers, PCR amplification was performed to obtain PUTR. rmfFragments. All PCR products were detected and purified by 1.0% agarose gel electrophoresis. The linearized vector and gene fragment PUTR were then cloned according to the instructions of the One-Step Cloning Kit (Vazyme Biotech, Nanjing, China). rmf The cells were ligated and transformed into E. coli DH5α, plated on gentamicin-resistant plates, and single colonies were picked for colony PCR verification using primers pBB-VF and pBB-VR. Sequencing confirmed the presence of pBBR1MCS5-PUTR. rmf -rhlAB-PUTR rmf -rmlBDAC plasmid. pBBR1MCS5-PUTR was obtained according to the method described. rmf -rhlAB-PUTR PP_0951 -rmlBDAC, pBBR1MCS5-PUTR rmf -rhlAB-PUTR PP_3765 -rmlBDAC, pBBR1MCS5-PUTR rmf -rhlAB-PUTR PP_1111 -rmlBDAC, pBBR1MCS5-PUTR rmf -rhlAB-PUTR PP_3595 -rmlBDAC plasmid.

[0099] (5) pBBR1MCS5-PUTR PP_3765 Using the plasmid -rhlAB-Plac-rmlBDAC as a template, and with primers rmlBDAC-PUTR-Line-F and rhlAB-PUTR-Line-R, PCR amplification was performed to obtain a linearized vector. The PCR product was digested with DpnI. Using the genome of *Pseudomonas putida* KT2440 as a template, and with primers AB-PP_3765-PA-F / R, PCR amplification was performed to obtain PUTR. PP_3765 Fragments. All PCR products were detected and purified by 1.0% agarose gel electrophoresis. The linearized vector and gene fragment PUTR were then cloned according to the instructions of the One-Step Cloning Kit (Vazyme Biotech, Nanjing, China). PP_3765 The ligation was performed, and the cells were transformed into E. coli DH5α. The cells were plated on gentamicin-resistant plates, and single colonies were picked for colony PCR verification using primers pBB-VF and pBB-VR. Sequencing confirmed the presence of pBBR1MCS5-PUTR. PP_3765 -rhlAB-PUTR PP_3765-rmlBDAC plasmid. pBBR1MCS5-PUTR was obtained according to the method described. PP_3765 -rhlAB-PUTR PP_0951 -rmlBDAC, pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR rmf -rmlBDAC, pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_1111 -rmlBDAC, pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_3595 -rmlBDAC plasmid.

[0100] (6) Using pBBR1MCS5-PUTR PP_1111 Using the plasmid -rhlAB-Plac-rmlBDAC as a template, and with primers rmlBDAC-PUTR-Line-F and rhlAB-PUTR-Line-R, PCR amplification was performed to obtain a linearized vector. The PCR product was digested with DpnI. Using the genome of *Pseudomonas putida* KT2440 as a template, and with primers AB-PP_1111-PA-F / R, PCR amplification was performed to obtain PUTR. PP_1111 Fragments. All PCR products were detected and purified by 1.0% agarose gel electrophoresis. The linearized vector and gene fragment PUTR were then cloned according to the instructions of the One-Step Cloning Kit (Vazyme Biotech, Nanjing, China). rmf The ligation was performed, and the cells were transformed into E. coli DH5α. The cells were plated on gentamicin-resistant plates, and single colonies were picked for colony PCR verification using primers pBB-VF and pBB-VR. Sequencing confirmed the presence of pBBR1MCS5-PUTR. PP_1111 -rhlAB-PUTR PP_1111 -rmlBDAC plasmid. pBBR1MCS5-PUTR was obtained according to the method described. PP_1111 -rhlAB-PUTR PP_0951 -rmlBDAC, pBBR1MCS5-PUTR PP_1111 -rhlAB-PUTR rmf -rmlBDAC, pBBR1MCS5-PUTR PP_1111 -rhlAB-PUTR PP_3765 -rmlBDAC, pBBR1MCS5-PUTR PP_1111 -rhlAB-PUTR PP_3595 -rmlBDAC plasmid.

[0101] (7) pBBR1MCS5-PUTR PP_3595 Using the plasmid -rhlAB-Plac-rmlBDAC as a template, and with primers rmlBDAC-PUTR-Line-F and rhlAB-PUTR-Line-R, PCR amplification was performed to obtain a linearized vector. The PCR product was digested with DpnI. Using the genome of *Pseudomonas putida* KT2440 as a template, and with primers AB-PP_3595-PA-F / R, PCR amplification was performed to obtain PUTR. PP_3595 Fragments. All PCR products were detected and purified by 1.0% agarose gel electrophoresis. The linearized vector and gene fragment PUTR were then cloned according to the instructions of the One-Step Cloning Kit (Vazyme Biotech, Nanjing, China). PP_3595 The ligation was performed, and the cells were transformed into E. coli DH5α. The cells were plated on gentamicin-resistant plates, and single colonies were picked for colony PCR verification using primers pBB-VF and pBB-VR. Sequencing confirmed the presence of pBBR1MCS5-PUTR. PP_3595 -rhlAB-PUTR PP_3595 -rmlBDAC plasmid. pBBR1MCS5-PUTR was obtained according to the method described. PP_3595 -rhlAB-PUTR PP_0951 -rmlBDAC, pBBR1MCS5-PUTR PP_3595 -rhlAB-PUTR rmf -rmlBDAC, pBBR1MCS5-PUTR PP_3595 -rhlAB-PUTR PP_3765 -rmlBDAC, pBBR1MCS5-PUTR PP_3595 -rhlAB-PUTR PP_1111 -rmlBDAC plasmid.

[0102] (8) Prepare competent cells of Pseudomonas putida KT2440, and insert the plasmid pBBR1MCS5-Plac-rhlAB-PUTR into the cells. PP_0951 -rmlBDAC, pBBR1MCS5-Plac-rhlAB-PUTR rmf -rmlBDAC, pBBR1MCS5-Plac-rhlAB-PUTR PP_3765 -rmlBDAC, pBBR1MCS5-Plac-rhlAB-PUTR PP_1111 -rmlBDAC, pBBR1MCS5-Plac-rhlAB-PUTRPP_3595 -rmlBDAC;pBBR1MCS5-PUTR PP _0951 -rhlAB-Plac-rmlBDAC、pBBR1MCS5-PUTR rmf -rhlAB-Plac-rmlBDAC、pBBR1MCS5-PUTR PP_3765 -rhlAB-Plac-rmlBDAC、pBBR1MCS5-PUTR PP_1111 -rhlAB-Plac-rmlBDAC、pBBR1MCS5-PUTR PP_3595 -rhlAB-Plac-rmlBDAC;pBBR1MCS5-PUTR PP_0951 -rhlAB-PUTR PP_0951 - rmlBDAC、pBBR1MCS5-PUTR PP_0951 -rhlAB-PUTR rmf -rmlBDAC、pBBR1MCS5-PUTR PP_0951 -rhlAB-PUTR PP_3765 -rmlBDAC、pBBR1MCS5-PUTR PP_0951 -rhlAB-PUTR PP_1111 -rmlBDAC、pBBR1MCS5-PUTR PP_0951 -rhlAB-PUTR PP_3595 -rmlBDAC;pBBR1MCS5-PUTR rmf -rhlAB-PUTR rmf -rmlBDAC、pBBR1MCS5-PUTR rmf -rhlAB-PUTR PP_0951 -rmlBDAC、pBBR1MCS5-PUTR rmf -rhlAB-PUTR PP_3765 -rmlBDAC、pBBR1MCS5-PUTR rmf -rhlAB-PUTR PP_1111 -rmlBDAC、pBBR1MCS5-PUTR rmf -rhlAB-PUTR PP_3595 -rmlBDAC;pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_3765 -rmlBDAC、pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_0951 -rmlBDAC、pBBR1MCS5-PUTR PP_3765-rhlAB-PUTR rmf -rmlBDAC, pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_1111 -rmlBDAC, pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_3595 -rmlBDAC; pBBR1MCS5-PUTR PP_1111 -rhlAB-PUTR PP_1111 -rmlBDAC, pBBR1MCS5-PUTR PP_1111 -rhlAB-PUTR PP_0951 -rmlBDAC, pBBR1MCS5-PUTR PP_1111 -rhlAB-PUTR rmf -rmlBDAC, pBBR1MCS5-PUTR PP_1111 -rhlAB-PUTR PP_3765 -rmlBDAC, pBBR1MCS5-PUTR PP_1111 -rhlAB-PUTR PP_3595 -rmlBDAC; pBBR1MCS5-PUTR PP_3595 -rhlAB-PUTR PP_3595 -rmlBDAC, pBBR1MCS5-PUTR PP_3595 -rhlAB-PUTR PP_0951 -rmlBDAC, pBBR1MCS5-PUTR PP_3595 -rhlAB-PUTR rmf -rmlBDAC, pBBR1MCS5-PUTR PP_3595 -rhlAB-PUTR PP_3765 -rmlBDAC, pBBR1MCS5-PUTR PP_3595 -rhlAB-PUTR PP_1111 -rmlBDAC; and the control plasmid pBBR1MCS5-Plac-rhlAB-Plac-rmlBDAC were transformed into the host cells by electroporation to obtain the recombinant genetically engineered strain KT01-KT36.

[0103] (9) The recombinant genetically engineered strain KT01-KT36 was inoculated into 10 mL of LB medium and cultured overnight at 30-37°C and 150-200 rpm. 1 mL of the pre-culture was then inoculated into a 500 mL shake flask containing 50 mL of fermentation medium and fermented for 2-4 days. The fermentation broth was tested according to the method in Example 2, and the OD... 600and the rhamnolipin content in the fermentation broth supernatant, such as Figure 3 As shown.

[0104] like Figure 3 As shown, under the condition that the rhlAB gene is expressed driven by the Plac promoter, gradually increasing the strength of the PUTR element driving rmlBDAC expression can promote the production of rhamnolipids to a certain extent. However, when the rmlBDAC expression level reaches its maximum intensity, the production of rhamnolipids actually decreases slightly. These results indicate that excessive accumulation of dTDP-L-rhamnose precursors may have a feedback inhibition effect on the rml enzyme system or induce intracellular metabolic stress, thus hindering the continuous synthesis of the target product. Furthermore, the experimental results also show that the production of rhamnolipids does not show a linear increasing trend with increasing rhlAB expression. Overexpression of rhlAB significantly increases the metabolic burden on cells and reduces the utilization efficiency of activated rhamnose donors; while insufficient rhlAB expression makes it the rate-limiting step in the rhamnolipid synthesis pathway. Therefore, efficient biosynthesis of rhamnolipids depends on a precise match between rhlAB expression level and the supply capacity of dTDP-L-rhamnose precursors, rather than simply increasing the expression intensity of the synthase. Among all tested combinations, the production of rhamnolipids decreased slightly when the PUTR element was at its maximum intensity. PP_3765 rhlAB is expressed under promoter control and in PUTR PP_1111 The strain expressing rmlBDAC under promoter control (KT24) achieved the highest rhamnolipid production at 72 hours, at 2.68 g / L, which was 37.6% higher than the control group. This expression configuration effectively balanced the metabolic flux between precursor supply (rmlBDAC driven by a relatively weak PUTR) and rhamnolipid synthesis (rhlAB driven by a moderate PUTR).

[0105] Table 5: Primers for Example 8 Primer Name Sequence (5’-3’) rmlBDAC-PUTR-Line-F ATGACGATTCTCGTGACCGGC rhlAB-PUTR-Line-R TCAGGACGCAGCCTTCAGC AB-0951-PA-F GCTGAAGGCTGCGTCCTGAAACCCTGATGAAGGTTGCCAC AB-0951-PA-R GTCACGAGAATCGTCATACAGCTTCTCCTTTGTTGCCC AB-rmf-PA-F GCTGAAGGCTGCGTCCTGACCGTACACCTTTCCAGCAGG AB-rmf-PA-R GCCGGTCACGAGAATCGTCATAGGGTGTTGCCCTCACTTG AB-3765-PA-F GCTGAAGGCTGCGTCCTGAGGTTGAAATAGACATTGCGTGC AB-3765-PA-R GCCGGTCACGAGAATCGTCATTGTAATCACTCCTGTACGTCGG AB-1111-PA-F GCTGAAGGCTGCGTCCTGAAGCTTGGTGAAGTGCTGGC AB-1111-PA-R GCCGGTCACGAGAATCGTCATTGGGTGCATGGGCTTG AB-3595-PA-F GCTGAAGGCTGCGTCCTGAATCGACAGCGCCCAGACC AB-3595-PA-R GCCGGTCACGAGAATCGTCATGACGAACCTCTTGGGCAGG AB-PUTR-Line-F ATGCGGCGCGAAAGTCTG AB-0951-F ATTAATGCAGCTGGCACGAACCCTGATGAAGGTTGCCAC AB-0951-R CAGACTTTCGCGCCGCATACAGCTTCTCCTTTGTTGCCC AB-rmf-F ATTAATGCAGCTGGCACGCCGTACACCTTTCCAGCAG AB-rmf-R CAGACTTTCGCGCCGCATAGGGTGTTGCCCTCAC AB-3765-F ATTAATGCAGCTGGCACGGGTTGAAATAGACATTGCG AB-3765-R CAGACTTTCGCGCCGCATTGTAATCACTCCTGTACGTC AB-1111-F TAATGCAGCTGGCACGAGCTTGGTGAAGTGCTGG AB-1111-R CAGACTTTCGCGCCGCATTGGGTGCATGGGCTTG AB-3595-F TTAATGCAGCTGGCACGATCGACAGCGCCCAGACC AB-3595-R CAGACTTTCGCGCCGCATGACGAACCTCTTGGGCAGG .

[0106] Example 9: PUTR Element-Mediated Regulation of aceE Expression Levels

[0107] (1) Using pSEVA-gRic6T plasmid (Addgene Plasmid#106401) as a template and aceE-PSTB-F / R as primers, PCR amplification was performed, and site-directed mutagenesis of the gRNA was carried out. The PCR product was digested with DpnI. The digested product was transferred into E. coli DH5α, plated on gentamicin plates, and single colonies were picked for sequencing verification (primers pS-VF and pS-VR) to screen for successfully mutated pSEVA plasmids. The successfully mutated pSEVA plasmids were linearized using linearization primers PSL-F / R, and the product was detected by 1.0% agarose gel electrophoresis. The PCR product was digested and purified with DpnI to obtain the linearized vector.

[0108] (2) Using the genome of *Pseudomonas putida* KT2440 as a template, and with aceE-Up-F / R and aceE-Down-F / R primers, PCR amplification was performed to obtain the upstream and downstream homologous arms of the aceE gene promoter (500 bp). Using PUTR... amaB aceE-F / R, PUTR atpE aceE-F / R, PUTR PP_0641 aceE-F / R, PUTR PP_5700 Using aceE-F / R primers, PCR amplification was performed to obtain the PUTR fragment replacing the native aceE promoter. The DNA fragments from the upper and lower homologous arms were then fused with the PUTR fragment using fusion PCR to obtain the Donor-PUTR fragment. amaB aceE, Donor-PUTR atpE aceE, Donor-PUTR PP_0641 aceE, Donor-PUTR PP_ 5700 aceE. All PCR products were detected by 1.0% agarose gel electrophoresis and the PCR fragments were purified.

[0109] (3) Following the instructions of the One-Step Cloning Kit (Vazyme Biotech, Nanjing, China), the linearized vector was ligated with the gene fragment Donor and transformed into E. coli DH5α. The transformed cells were plated on gentamicin-resistant plates, and single colonies were picked for colony PCR verification using primers pS-VF and pS-VR. Sequencing confirmed the presence of positive transformants. The plasmid PSD-PUTR was extracted from the positive transformants. amaB aceE, PSD-PUTR atpE aceE, PSD-PUTR PP_0641 aceE, PSD-PUTR PP_5700 aceE.

[0110] (4) Prepare competent cells of Pseudomonas putida KT2440. Electroporate plasmid pCAS-RK2T (Addgene Plasmid#106400) into competent cells at 2.5 kV. Add 1-2 mL LB and revive in a shaker at 30℃ and 200 rpm for 2 h. Spread the bacterial solution on LB plates containing 25 mg / L tetracycline and culture until a single colony grows to obtain recombinant bacteria KT2440 / pCas containing recombinant plasmid.

[0111] (5) Single colonies of recombinant KT2440 / pCas were picked and inoculated into 10 mL of liquid LB medium. Tetracycline with a final concentration of 25 mg / L and 40 mM arabinose were added to induce pCas expression. KT2440 / pCas were prepared into electrocompetent cells, and plasmid PSD-PUTR was transferred to them. amaB aceE, PSD-PUTR atpE aceE, PSD-PUTR PP_0641 aceE, PSD-PUTR PP_5700 aceE was electroporated into KT2440 / pCas competent cells at 2.5 kV. 1-2 mL of LB broth was added, and the cells were incubated for 2 h at 30℃ and 200 rpm in a shaker. The bacterial culture was then plated onto LB agar plates containing 50 mg / L gentamicin and 25 mg / L tetracycline and cultured until single colonies appeared, yielding recombinant bacteria containing the recombinant plasmid. Single colonies were selected for colony PCR verification (primers aceE-GVF / R). PCR products with the correct bands were selected for sequencing verification, and successfully edited strains were screened.

[0112] (6) Select a positive single colony and inoculate it into a 10 mL LB tube containing 10 mM L-Rha and 25 mg / L tetracycline. Incubate overnight at 30°C. Stir the colony on an LB plate containing 25 mg / L tetracycline. Incubate at 30°C for 24 h. Select a single colony and spot it on an LB plate containing 25 mg / L tetracycline. Stir the colony on an LB plate containing 50 mg / L gentamicin. If a single colony cannot be found on an LB plate containing gentamicin, its PSD plasmid has been successfully eliminated. Single colonies successfully eliminated from the PSD plasmid were picked and placed in 10 mL LB tubes containing 5 g / L glucose and incubated overnight at 30°C. The next day, the bacterial culture was streaked onto LB agar plates containing 5 g / L glucose and 10 g / L sucrose and incubated at 30°C for 24 h. Single colonies were then picked and streaked onto LB agar plates containing 25 mg / L tetracycline. Single colonies that could not be streaked onto LB agar plates containing tetracycline had their pCas plasmids successfully eliminated, resulting in the plasmid-free strain KT2440ΔaceE::PUTR. amaB -aceE、KT2440ΔaceE::PUTRatpE -aceE、KT2440ΔaceE::PUTR PP_0641 -aceE、KT2440ΔaceE::PUTR PP_5700 - aceE.

[0113] (7) Plasmid-free strain KT2440ΔaceE::PUTR amaB -aceE、KT2440ΔaceE::PUTR atpE -aceE、KT2440ΔaceE::PUTR PP_0641 -aceE、KT2440ΔaceE::PUTR PP_5700 -aceE was used to prepare electrocompetent cells, and pBBR1MCS5-PUTR constructed in Example 8 was used to prepare the cells. PP_3765 -rhlAB-PUTR PP_1111 The -rmlBDAC plasmid was transformed into the above competent cells using electroporation to obtain strain KT2440ΔaceE::PUTR amaB -aceE / pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_1111 -rmlBDAC (KT37), KT2440ΔaceE::PUTR atpE -aceE / pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_1111 -rmlBDAC (KT38), KT2440ΔaceE::PUTR PP_0641 -aceE / pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_1111 -rmlBDAC (KT39), KT2440ΔaceE::PUTR PP_5700 -aceE / pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_1111 -rmlBDAC(KT40).

[0114] (8) The recombinant engineered strain KT37-KT40 and the control strain KT30 were inoculated into 10 mL of LB medium and cultured overnight at 30-37°C and 150-200 rpm. 1 mL of the pre-culture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium and fermented for 2-4 days. The fermentation broth was tested according to the method in Example 1, and the OD... 600 and the rhamnolipin content in the fermentation broth supernatant, such as Figure 4 As shown.

[0115] like Figure 4 As shown, based on *Pseudomonas putida* KT2440, PUTR elements with progressively increasing expression intensity (PUTR) were used. amaB PUTR atpE PUTR PP_0641 and PUTR PP_5700 By replacing the endogenous aceE promoter in the genome, the transcriptional input intensity at the pyruvate-to-acetyl-CoA conversion node was systematically regulated. Experimental results showed that as aceE expression levels gradually increased, rhamnolipin production in the engineered strains exhibited a trend of first increasing and then decreasing. Specifically, in the engineered strain (KT38) where aceE expression was driven by the PUTRatpE promoter, rhamnolipin production reached its highest value of 2.82 g / L; however, when aceE expression was driven by a higher-intensity PUTR element, rhamnolipin production decreased. These results indicate that there is an optimal regulatory range for transcriptional input in the acetyl-CoA synthesis pathway at the pyruvate metabolism node, and excessively low or high aceE expression levels are detrimental to the efficient allocation of carbon to the rhamnolipin synthesis pathway.

[0116] Table 6: Primers for Example 9 Primer Name Sequence (5'-3') aceE-PSTB-F taatgctagcGTAGTAAAACTACAACGCGCgttttagagctagaaatagc aceE-PSTB-R gctctaaaacGCGCGTTGTAGTTTTACTACgctagcattatacctaggac aceE-Up-F gctcggtacccggggatccAGCTCGTCCTCGCTCTG aceE-Up-R TGCGACGTTTGGTCGCA aceE-Down-F ATGCAAGACCTCGATCCAATC aceE-Down-R ggttttcccagtcacgacgcggTTGCCGTCCACTTCCTGG PUTRamaBaceE-F TGCGACCAAACGTCGCATGCCGTTTGCCAAACCC PUTRamaBaceE-R TGGATCGAGGTCTTGCATTTGTGCATCTCCTACATCGGTG PUTRatpEaceE-F TGCGACCAAACGTCGCAAGGTCAAGGGACTGGGC PUTRatpEaceE-R GGATCGAGGTCTTGCATCTTTCCTCCCGACTTTTACG PUTRPP_0641aceE-F GTGCGACCAAACGTCGCATTTGTGCCGTACACCCC PUTRPP_0641aceE-R GATTGGATCGAGGTCTTGCATGTTTGACTCCTGACGTCTTCTG PUTRPP_5700aceE-F GGTGCGACCAAACGTCGCAGAACAAAGGCAACCAGGTG PUTRPP_5700aceE-R GATTGGATCGAGGTCTTGCATGATTCAATCCTTATTCAGCTTGC pS-VF agggcggcggatttgtcc pS-VR gcggcaaccgagcgttc PSL-F gccgcgtcgtgactgggaaaacc PSL-R ggatccccgggtaccgagctcctcaa aceE-GVF CCAAGTTCCGCCTGACG aceE-GVR [[ID= .

[0117] Example 10: PUTR Element-Mediated Regulation of gltA Expression Levels

[0118] (1) Using pSEVA-gRic6T plasmid (Addgene Plasmid#106401) as a template and gltA-PSTB-F / R as primers, PCR amplification was performed, and site-directed mutagenesis of the gRNA was carried out. The PCR product was digested with DpnI. The digested product was transferred into E. coli DH5α, plated on gentamicin plates, and single colonies were picked for sequencing verification (primers pS-VF and pS-VR) to screen for successfully mutated pSEVA plasmids. The successfully mutated pSEVA plasmids were linearized using linearization primers PSL-F / R, and the product was detected by 1.0% agarose gel electrophoresis. The PCR product was digested and purified with DpnI to obtain the linearized vector.

[0119] (2) Using the genome of *Pseudomonas putida* KT2440 as a template, and with gltA-Up-F / R and gltA-Down-F / R as primers, PCR amplification was performed to obtain the upstream and downstream homologous arms of the gltA promoter (500 bp). Using PUTR... rpoZ gltA-F / R, PUTR acpPgltA-F / R, PUTR csrA gltA-F / R, PUTR rpmB Using gltA-F / R as primers, PCR amplification was performed to obtain the PUTR fragment replacing the native gltA promoter. The DNA fragments from the upper and lower homologous arms were then fused with the PUTR fragment using fusion PCR to obtain the Donor-PUTR fragment. rpoZ gltA, Donor-PUTR acpP gltA, Donor-PUTR csrA gltA, Donor-PUTR rpmB gltA. All PCR products were detected by 1.0% agarose gel electrophoresis and the PCR fragments were purified.

[0120] (3) Following the instructions of the One-Step Cloning Kit (Vazyme Biotech, Nanjing, China), the linearized vector was ligated with the gene fragment Donor and transformed into E. coli DH5α. The transformed cells were plated on gentamicin-resistant plates, and single colonies were picked for colony PCR verification using primers pS-VF and pS-VR. Sequencing confirmed the presence of positive transformants. The plasmid PSD-PUTR was extracted from the positive transformants. rpoZ gltA, PSD-PUTR acpP gltA, PSD-PUTR csrA gltA, PSD-PUTR rpmB gltA.

[0121] (4) Pick recombinant bacteria KT2440ΔaceE::PUTR atpE Single colonies of -aceE / pCas were inoculated into 10 mL of liquid LB medium, and tetracycline (final concentration 25 mg / L) and arabinose (40 mM) were added to induce pCas expression. KT2440 / pCas were then used to prepare electrotransfer competent cells, and plasmid PSD-PUTR was applied. rpoZ gltA, PSD-PUTR acpP gltA, PSD-PUTR csrA gltA, PSD-PUTR rpmB gltA is electrically transferred to KT2440ΔaceE::PUTR at 2.5 kV. atpEIn aceE / pCas competent cells, add 1-2 mL LB broth and incubate at 30℃ and 200 rpm for 2 h in a shaker. Then, spread the bacterial culture onto LB agar plates containing 50 mg / L gentamicin and 25 mg / L tetracycline, and culture until single colonies appear to obtain recombinant bacteria containing the recombinant plasmid. Select single colonies for colony PCR verification (primers gltA-GVF / R), and sequence the PCR products with the correct bands to screen for successfully edited strains.

[0122] (5) Select a positive single colony and inoculate it into a 10 mL LB tube containing 10 mM L-Rha and 25 mg / L tetracycline. Incubate overnight at 30°C. Stir the colony on an LB plate containing 25 mg / L tetracycline. Incubate at 30°C for 24 h. Select a single colony and spot it on an LB plate containing 25 mg / L tetracycline. Stir the colony on an LB plate containing 50 mg / L gentamicin. If a single colony cannot be found on an LB plate containing gentamicin, its PSD plasmid has been successfully eliminated. Single colonies successfully eliminated from the PSD plasmid were picked and placed in 10 mL LB tubes containing 5 g / L glucose and incubated overnight at 30°C. The next day, the bacterial culture was streaked onto LB agar plates containing 5 g / L glucose and 10 g / L sucrose and incubated at 30°C for 24 h. Single colonies were then picked and streaked onto LB agar plates containing 25 mg / L tetracycline. Single colonies that could not be streaked onto LB agar plates containing tetracycline had their pCas plasmids successfully eliminated, resulting in the plasmid-free strain KT2440ΔaceE::PUTR. atpE -aceEΔgltA::PUTR rpoZ -gltA、KT2440ΔaceE::PUTR atpE -aceEΔgltA::PUTR acpP -gltA、KT2440ΔaceE::PUTR atpE -aceEΔgltA::PUTR csrA -gltA、KT2440ΔaceE::PUTR atpE -aceEΔgltA::PUTR rpmB -gltA.

[0123] (6) Prepare electrocompetent cells from the plasmid-free strain, and use the pBBR1MCS5-PUTR strain constructed in Example 8. PP_3765 -rhlAB-PUTR PP_1111 The -rmlBDAC plasmid was transformed into the above competent cells using electroporation to obtain strain KT2440ΔaceE::PUTR atpE -aceEΔgltA::PUTR rpoZ -gltA / pBBR1MCS5-PUTR PP_3765-rhlAB-PUTR PP_1111 -rmlBDAC (KT41), KT2440ΔaceE::PUTR atpE -aceEΔgltA::PUTR acpP -gltA / pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_1111 -rmlBDAC (KT42), KT2440ΔaceE::PUTR atpE -aceEΔgltA::PUTR csrA -gltA / pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_1111 -rmlBDAC (KT43), KT244ΔaceE::PUTR atpE -aceE 0ΔgltA::PUTR rpmB -gltA / pBBR1MCS5-PUTR PP_3765 -rhlAB-PUTR PP_1111 -rmlBDAC(KT44).

[0124] (7) The recombinant engineered strains KT41-KT44 and the control strain KT30 were inoculated into 10 mL of LB medium and cultured overnight at 30-37°C and 150-200 rpm. 1 mL of the pre-culture was then inoculated into a 500 mL shake flask containing 50 mL of fermentation medium and fermented for 2-4 days. The fermentation broth was tested according to the method in Example 1, and the OD... 600 and the rhamnolipin content in the fermentation broth supernatant, such as ​ As shown.

[0125] like ​ As shown, in an engineered strain with optimized aceE expression, the transcriptional level of the key tricarboxylic acid cycle gene gltA was further regulated using a PUTR gradient. The results indicated that using a medium to medium-high intensity PUTR element (PUTR... rpoZ PUTR acpP PUTR csrA and PUTR rpmB When gltA expression was driven, the engineered strains showed a significant increase in rhamnolipid production while maintaining normal growth. Among them, the engineered strain (KT42) with the PUTRAcpP promoter replacing the endogenous gltA promoter achieved the best balance between growth performance and product synthesis, with a rhamnolipid production of 2.95 g / L.

[0126] Table 7: Primers for Example 10 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ <![CDATA[PUTR rpoZ gltA-F]]> ​ <![CDATA[PUTR rpoZ gltA-R]]> ​ <![CDATA[PUTR acpP gltA-F]]> ​ <![CDATA[PUTR acpP gltA-R]]> ​ <![CDATA[PUTR csrA gltA-F]]> ​ <![CDATA[PUTR csrA gltA-R]]> ​ <![CDATA[PUTR rpmB gltA-F]]> ​ <![CDATA[PUTR rpmB gltA-R]]> ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ .

[0127] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for constructing an endogenous PUTR library of *Pseudomonas putida* KT2440, characterized in that, Includes the following steps: (1) Using Pseudomonas putida KT2440 as the host bacterium, the key gene for rhamnolipid synthesis was heterologously expressed in the host bacterium, and the recombinant plasmid pBBR1MCS5-rhlAB was constructed. (2) The recombinant plasmid pBBR1MCS5-rhlAB obtained in step (1) was introduced into the host bacteria to obtain the recombinant engineered strain P. putida KT2440 / pBBR1MCS5-rhlAB; (3) Transcriptome data acquisition: Wild-type Pseudomonas putida KT2440 and recombinant engineered strain P. putidaKT2440 / pBBR1MCS5-rhlAB were fermented under the same fermentation culture conditions. Samples were taken at different growth stages and compared for transcriptome analysis. (4) Obtaining PUTR candidate elements: Based on the transcriptome data analysis results and the selected key genes, PUTR candidate elements located upstream of the sequence start site of the key genes are obtained; (5) Construction of recombinant expression vector: The candidate PUTR element is operatively ligated into the genome of a vector carrying a fluorescent protein that can express the protein in the competent cells of the host bacterium to obtain a recombinant expression vector; (6) Characterization of PUTR library: The recombinant expression vector obtained in step (5) is introduced into the competent cells of the host bacteria to obtain the genetically engineered strain containing the recombinant expression vector. The expression intensity of fluorescent protein at different growth stages and the cell growth of the corresponding genetically engineered strain are measured to determine the endogenous PUTR library of Pseudomonas putida KT2440.

2. The method for constructing an endogenous PUTR library of *Pseudomonas putida* KT2440 according to claim 1, characterized in that, The PUTR candidate element is selected from at least one of the nucleotide sequences such as SEQ ID NO.1 to SEQ ID NO.

80.

3. The method for constructing an endogenous PUTR library of *Pseudomonas putida* KT2440 according to claim 1, characterized in that, The key genes include the rhlA gene and / or the rhlB gene; wherein the rhlA gene and the rhlB gene are both derived from Pseudomonas aeruginosa PAO1 and are driven by the Lac promoter.

4. The method for constructing an endogenous PUTR library of *Pseudomonas putida* KT2440 according to claim 3, characterized in that, The nucleotide sequence of the Lac promoter is shown in SEQ ID NO.81, the nucleotide sequence of the rhlA gene is shown in SEQ ID NO.82, and the nucleotide sequence of the rhlB gene is shown in SEQ ID NO.

83.

5. The method for constructing an endogenous PUTR library of *Pseudomonas putida* KT2440 according to claim 1, characterized in that, The fluorescent protein is selected from green fluorescent protein, enhanced green fluorescent protein, or superfolded fluorescent protein.

6. An endogenous PUTR library of Pseudomonas putida KT2440 constructed by the construction method according to any one of claims 1-5.

7. A recombinant genetically engineered strain constructed based on the endogenous PUTR library of *Pseudomonas putida* KT2440 as described in claim 6.

8. The method for constructing the recombinant genetically engineered strain according to claim 8, characterized in that, Includes the following steps: (a) Using *Pseudomonas putida* KT2440 as the host bacterium, the key gene for dTDP-L-rhamnose synthesis was heterologously expressed in the host bacterium, and a recombinant expression plasmid pBBR1MCS5-Plac-rhlAB-Plac-rmlBDAC for rhamnose lipid synthesis was constructed. (b) The recombinant expression plasmid obtained in step (a) was introduced into the *Pseudomonas putida* KT2440 to obtain the recombinant genetically engineered strain *P. putida* KT2440 / pBBR1MCS5-rhlAB for the synthesis of rhamnolipids; (c) Based on the endogenous PUTR library of the aforementioned Pseudomonas putida KT2440, PUTR combinations with different expression intensities were screened at the plasmid level, and the expression levels of the rhlAB gene and rmlBDAC gene were optimized by combination. (d) Using Pseudomonas putida KT2440 as the starting strain, suitable PUTR elements were screened and replaced with native promoters of aceE and gltA on the genome.

9. The method for constructing recombinant genetically engineered strains according to claim 8, characterized in that, The key genes for dTDP-L-rhamnose synthesis are derived from Pseudomonas aeruginosa PAO1, including the rmlA, rmlB, rmlC, and rmlD genes, and these genes are driven by the Lac promoter or the PUTR element.

10. The method for constructing recombinant genetically engineered strains according to claim 9, characterized in that, The nucleotide sequence of the rmlA gene is shown in SEQ ID NO. 84, the nucleotide sequence of the rmlB gene is shown in SEQ ID NO. 85, the sequence of the rmlC gene is shown in SEQ ID NO. 86, the nucleotide sequence of the rmlD gene is shown in SEQ ID NO. 87, the nucleotide sequence of the aceE gene is shown in SEQ ID NO. 88, and the nucleotide sequence of the gltA gene is shown in SEQ ID NO. 89.

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