A promoter and use thereof

Abstract

The present application relates to a kind of promoter and its application, the nucleic acid sequence of the promoter is the fragment as shown in SEQ ID No.1 Or as the truncated fragment shown in SEQ ID No.1.The endogenous or exogenous target gene can be effectively expressed in its microbial cell, especially pseudomonas.For example, when prnD gene is expressed in pseudomonas using it as the promoter of prnD gene, the production of nitrapyrin can be improved.

Description

Technical Field

[0001] This invention relates to the field of nucleic acids, and in particular to a promoter and its applications. Background Technology

[0002] Pseudomonas bacteria can produce various secondary metabolites in the natural environment, such as nitropyrrolizin, phenazine, and siderophores. However, the yield of these substances is low, limiting industrial production and hindering their widespread utilization. Modern biotechnology is crucial for elucidating the functions of microbial strains and achieving targeted modification. Promoter engineering can induce efficient expression of target genes, resulting in engineered strains with high yields.

[0003] A promoter is a sequence on a DNA molecule that can be recognized, bound, and initiate transcription by RNA polymerase. Promoters are important cistropic elements in gene expression regulation, controlling the degree of gene expression. In microbial metabolic engineering, the yield can be increased by overexpressing the target gene using constitutive strong promoters. Summary of the Invention

[0004] One aspect of the present invention provides the use of a nucleic acid as a promoter, the sequence of which is shown in SEQ ID No. 1.

[0005] In one specific embodiment, the promoter is used to initiate the transcription of the prnD gene in Pseudomonas, thereby increasing the production of nitropyrrolizin by the Pseudomonas. Specifically, an efficient expression regulatory element can be formed by linking an RBS (e.g., whose sequence can be as shown in SEQ ID No. 2) to the 3' end of the promoter. The prnD gene is then linked to the 3' end of this efficient expression regulatory element to form an expression cassette. This expression cassette is ligated into a plasmid expression vector to obtain an expression vector for expressing the prnD gene. This expression vector is then transformed into Pseudomonas to increase the production of nitropyrrolizin.

[0006] In one specific embodiment, the promoter is used to initiate the overexpression of the prnD gene in Pseudomonas, thereby increasing the production of nitropyrrolizin by the Pseudomonas. Specifically, an efficient expression regulatory element can be formed by linking an RBS (e.g., whose sequence can be as shown in SEQ ID No. 2) to the 3' end of the promoter. The prnD gene is then linked to the 3' end of this efficient expression regulatory element to form an expression cassette. This expression cassette is ligated into a plasmid expression vector to obtain an expression vector for expressing the prnD gene. This expression vector is then transformed into Pseudomonas (e.g., defensive Pseudomonas, such as strain JP2-4390, engineered strain JP2ΔBGC1 / 16, engineered strain JP2ΔBGC1, or engineered strain JP2ΔBGC16) to increase the production of nitropyrrolizin.

[0007] In one specific embodiment, the sequence of the prnD gene is shown in SEQ ID No. 20.

[0008] In one specific embodiment, the wild-type promoter used to initiate the PRN BGC gene cluster in *Pseudomonas* is replaced with the promoter as described in any one of the present invention, thereby increasing the yield of nitropyrrolizin produced by *Pseudomonas*.

[0009] In one specific embodiment, the wild-type expression regulatory element used to initiate the PRN BGC gene cluster in Pseudomonas is replaced with a highly efficient expression regulatory element, thereby increasing the yield of nitropyrrolizin produced by the Pseudomonas. The highly efficient expression regulatory element consists of the promoter and RBS as described in any one of the applications of this invention, wherein the RBS is located at the 3' end of the promoter as described in any one of the applications of this invention.

[0010] In one specific embodiment, the sequence of the wild-type expression regulatory element of the PRN BGC gene cluster is shown in SEQ ID No. 23.

[0011] The second aspect of this invention provides an application of a nucleic acid as a promoter, wherein the nucleic acid sequence is truncated from the sequence shown in SEQ ID No. 1, starting from the first digit at the 3' end and extending towards the 5' end, with a truncated length greater than or equal to 32 bp and less than 1000 bp. That is, the truncated fragment from the 5' end to the 3' end of SEQ ID No. 1 includes at least the last 32 bp fragment as shown in SEQ ID No. 1; or the truncated fragment from the 3' end to the 5' end of SEQ ID No. 1 includes at least the first 32 bp fragment as shown in SEQ ID No. 1.

[0012] In one specific implementation, the truncation length is greater than or equal to 32 bp and less than or equal to 390 bp.

[0013] In one specific embodiment, the nucleic acid sequence is one of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 and SEQ ID No. 19.

[0014] In one specific embodiment, the promoter is used to initiate the transcription of the prnD gene in Pseudomonas, thereby increasing the production of nitropyrrolizin by the Pseudomonas. Specifically, an efficient expression regulatory element can be formed by linking an RBS (e.g., whose sequence can be as shown in SEQ ID No. 2) to the 3' end of the promoter. The prnD gene is then linked to the 3' end of this efficient expression regulatory element to form an expression cassette. This expression cassette is ligated into a plasmid expression vector to obtain an expression vector for expressing the prnD gene. This expression vector is then transformed into Pseudomonas to increase the production of nitropyrrolizin.

[0015] In one specific embodiment, the promoter is used to initiate the overexpression of the prnD gene in Pseudomonas, thereby increasing the production of nitropyrrolizin by the Pseudomonas. Specifically, an efficient expression regulatory element can be formed by linking an RBS (e.g., its sequence can be as shown in SEQ ID No. 2) to the 3' end of the promoter region, and an expression cassette can be formed by linking the prnD gene to the 3' end of the promoter region. The expression cassette is then linked to a plasmid expression vector to obtain an expression vector for expressing the prnD gene. This expression vector is then transformed into Pseudomonas (e.g., defensive Pseudomonas, such as strain JP2-4390, engineered strain JP2ΔBGC1 / 16, engineered strain JP2ΔBGC1, or engineered strain JP2ΔBGC16) to increase the production of nitropyrrolizin.

[0016] In one specific embodiment, the sequence of the prnD gene is shown in SEQ ID No. 20.

[0017] In one specific embodiment, the wild-type promoter used to initiate the PRN BGC gene cluster in *Pseudomonas* is replaced with the promoter as described in any one of the present invention, thereby increasing the yield of nitropyrrolizin produced by *Pseudomonas*.

[0018] In one specific embodiment, the wild-type expression regulatory element used to initiate the PRN BGC gene cluster in Pseudomonas is replaced with a highly efficient expression regulatory element, thereby increasing the yield of nitropyrrolizin produced by the Pseudomonas. The highly efficient expression regulatory element consists of the promoter and RBS as described in any one of the applications of this invention, wherein the RBS is located at the 3' end of the promoter as described in any one of the applications of this invention.

[0019] In one specific embodiment, the sequence of the wild-type expression regulatory element of the PRN BGC gene cluster is shown in SEQ ID No. 23.

[0020] The third invention provides an engineered bacterium, which is one of the following strains: JP2-Ptp199-prnD, JP2-T-Ptp199-prn, JP2-4390M1, JP2-4390M2, and JP2-4390M3.

[0021] The beneficial effects of this invention are as follows: This invention discovers that nucleic acids with sequences as shown in SEQ ID No. 1 and their truncated fragments possess strong promoter activity, which can be used to effectively express endogenous or exogenous target genes in microbial cells, especially in Pseudomonas. For example, when used as a promoter for the prnD gene, overexpression of the prnD gene in Pseudomonas can increase the yield of nitropyrrolizidine. Attached Figure Description

[0022] Figure 1 The results show that JP2-Ptp-egfp, JP2-Plac-egfp, and JP2-pBBR1 are used in OD. 600 The fluorescence expression intensity at a value of 0.6.

[0023] Figure 2 The relative transcription levels of JP2-Ptp-egfp and JP2-Plac-egfp at five different time points are shown.

[0024] Figure 3 The effects of the six truncated Ptp promoter variants on EGFP fluorescence intensity were shown.

[0025] Figure 4 The relative transcriptional levels of the six truncated Ptp promoter variants at 36 h were shown.

[0026] Figure 5 The biosynthesis of nitropyrrolizin is shown when the prnD gene is overexpressed using the Ptp199 promoter in JP2-4390.

[0027] Figure 6 The biosynthesis of nitropyrrolizin was shown after replacing the wild-type promoter Pprn with promoter Ptp199 in the defensive Pseudomonas strain. Detailed Implementation

[0028] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0029] Unless otherwise specified, the strains, plasmids, and reagents used in the embodiments of this invention can be purchased commercially.

[0030] The protective pseudomonads used in this invention ( Pseudomonas protegensThe JP2-4390 strain was first disclosed in CN202411502275.5 and is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20241968 on September 13, 2024. The GenBank accession number for the complete genome sequence of the JP2-4390 strain is CP171611. An article about the JP2-4390 strain was published in Rice Science on April 15, 2025, titled: "Efficient production of pyrrolnitrin by optimizing culture medium and blocking competitive secondary metabolic pathways in..." Pseudomonas protegens JP2-4390; Authors: Shen et al. The 16 biosynthetic gene clusters named BGC1 to BGC16 mentioned in paragraph 72 of the specification CN202411502275.5 are described in "Efficient production of pyrrolnitrin by optimizing culture medium and blocking competitive secondary metabolic pathways in..." Pseudomonas protegens The corresponding names in the article JP2-4390 are APE BGC, RIPP1BGC, MA026 BGC, HSERLACTONE BGC, OFA BGC, PLT BGC, HCN BGC, E-PCH BGC, PRN BGC (this gene cluster contains the core gene prnD for the biosynthesis of nitropyrrolizin), FEN BGC, PVD1 BGC, PVD2 BGC, NAGGN BGC, PETRICHORIN BGC, RANTHIPEPTIDE BGC, and RIPP2 BGC.

[0031] The JP2ΔBGC1 / 16 engineered strain was first disclosed in CN202411502275.5. It was obtained by knocking out the core genes of the BGC1 and BGC16 gene clusters in the wild-type strain JP2-4390. The JP2ΔBGC1 / 16 engineered strain showed a significantly higher pyrrolnitrin production compared to the wild-type strain JP2-4390. The JP2ΔBGC1 / 16 engineered strain is related to the concept of "Efficient production of pyrrolnitrin by optimizing culture medium and blocking competitive secondary metabolic pathways in..." Pseudomonas protegens The JP2Δape / ripp2 engineered bacteria in the article "JP2-4390" are the same engineered bacteria, only the strain name is different.

[0032] The JP2ΔBGC1 engineered strain was first disclosed in CN202411502275.5. It was obtained by knocking out the core gene of the BGC1 gene cluster in the wild-type strain JP2-4390. The JP2ΔBGC1 engineered strain showed a significant increase in pyrrolnitrin production compared to the wild-type strain JP2-4390. The JP2ΔBGC1 engineered strain is related to the technology of "Efficient production of pyrrolnitrin by optimizing culture medium and blocking competitive secondary metabolic pathways in..." Pseudomonas protegens The JP2Δape engineered bacteria in the article "JP2-4390" are the same engineered bacteria, only the strain name is different.

[0033] The JP2ΔBGC16 engineered strain was first disclosed in CN202411502275.5. It was obtained by knocking out the core gene of the BGC16 gene cluster in the wild-type strain JP2-4390. The JP2ΔBGC16 engineered strain showed a significant increase in pyrrolnitrin production compared to the wild-type strain JP2-4390. The JP2ΔBGC16 engineered strain is related to the technology of "Efficient production of pyrrolnitrin by optimizing culture medium and blocking competitive secondary metabolic pathways in..." Pseudomonas protegens The JP2Δripp2 engineered bacteria in the article "JP2-4390" refers to the same engineered bacteria, only the strain name is different.

[0034] The *E. coli* used in constructing the plasmids in this invention ( Escherichia coli The top 10 strains were purchased from Hangzhou EasyBio Biotechnology Co., Ltd.

[0035] The broad-host type plasmid vector pBBR1MCS-2 was purchased from Shanghai Lianmai Biotechnology Co., Ltd., and it is kanamycin resistant. Example 1

[0036] The nucleotide sequence of Ptp is shown in SEQ ID No. 1.

[0037] The nucleotide sequence of the ribosome binding site (RBS) is shown in SEQ ID No. 2.

[0038] The nucleotide sequence of Plac is shown in SEQ ID No. 3.

[0039] The nucleotide sequence of the egfp gene is shown in SEQ ID No. 4 and was artificially synthesized.

[0040] The upstream primer used for amplifying Ptp is Ptp-F, the sequence of which is shown in SEQ ID No. 5; the downstream primer is Ptp-R, the sequence of which is shown in SEQ ID No. 6, wherein the reverse complementary sequence of RBS is located at the 5' end of the sequence shown in SEQ ID No. 6.

[0041] The upstream primer used for amplifying the egfp gene was egfp-F, the sequence of which is shown in SEQ ID No. 7; the downstream primer was egfp-R, the sequence of which is shown in SEQ ID No. 8. Using the genome of strain JP2-4390 as a template, and using primers Ptp-F and Ptp-R, a PCR product of Ptp with RBS tandem at the 3' end was amplified. Using the artificially synthesized egfp gene as a template, and using primers egfp-F and egfp-R, a PCR product of the egfp gene was amplified. The Ptp PCR product with RBS tandem at the 3' end, the PCR product of the egfp gene, and the pBBR1MCS-2 vector were ligated using the ClonExpress Ultra One StepCloning Kit (Nanjing Novizan Biotechnology Co., Ltd.) to construct the pBBR1-Ptp-egfp recombinant plasmid, in which egfp is located downstream of RBS.

[0042] The recombinant plasmid pBBR1-Ptp-egfp was transformed into JP2-4390 by electroporation, and positive transformants JP2-Ptp-egfp were selected.

[0043] Since the Plac promoter and RBS region are both relatively short, they are integrated with the upstream sequence used to amplify the egfp gene to form another upstream primer Plac-RBS-gfp-F for amplifying the egfp gene, as shown in SEQ ID No. 9, where Plac is located upstream, RBS is located downstream of Plac, and the upstream sequence of the egfp gene is located downstream of RBS.

[0044] Using the artificially synthesized egfp gene as a template, and Plac-RBS-gfp-F and egfp-R as primers, a PCR product carrying the Plac promoter and RBS region of the egfp gene was amplified. The PCR product was ligated to the pBBR1MCS-2 vector using the ClonExpress Ultra One StepCloning Kit (Nanjing Novizan Biotechnology Co., Ltd.) to construct the pBBR1-Plac-egfp recombinant plasmid. The recombinant plasmid pBBR1-Plac-egfp was transformed into JP2-4390 via electroporation, and the positive transformant JP2-Plac-egfp was selected as a positive control.

[0045] The empty vector pBBR1MCS-2 was transformed into JP2-4390 via electroporation, and the positive transformant JP2-pBBR1 was selected as a negative control.

[0046] Fresh colonies of JP2-Ptp-egfp, JP2-Plac-egfp, and JP2-pBBR1 were picked and inoculated into 5 mL of LB liquid medium, activated at 28°C and 220 rpm for 8 h. Then, 1% of each colony was inoculated into test tubes containing 5 mL of fresh LB liquid medium and incubated at 28°C and 220 rpm. After incubation until OD... 600 When the value is 0.6, fluorescence observation is performed using an LSM700 laser confocal microscope, such as... Figure 1 As shown.

[0047] Figure 1The results showed that the positive controls JP2-Plac-egfp and JP2-Ptp-egfp could produce green fluorescence, while the negative control JP2-pBBR1 recombinant strain did not produce fluorescence, indicating that the egfp gene can be expressed to produce green fluorescent protein under the action of the lac promoter Plac; the egfp gene can also be expressed to produce green fluorescent protein under the action of Ptp, that is, Ptp has a promoter function; and the green fluorescence produced by JP2-Ptp-egfp is significantly stronger than that of JP2-Plac-egfp, indicating that the green fluorescent protein production of JP2-Ptp-egfp is higher than that of JP2-Plac-egfp, further indicating that the Ptp promoter is more efficient than Plac in driving the transcription of target genes. Example 2

[0048] Fresh colonies of JP2-Ptp-egfp and JP2-Plac-egfp were picked and inoculated into 5 mL of LB liquid medium and activated at 28°C and 220 rpm for 8 h. Then, 1% of the colonies were inoculated into Erlenmeyer flasks containing 300 mL of fresh LB liquid medium and incubated at 28°C and 220 rpm for 12 h, 16 h, 22 h, 36 h, and 48 h. The bacterial cells were collected, and total RNA was extracted using the RNAprep Pure Cell / Bacteria Kit (Beijing Tiangen Biotech Co., Ltd.). Three replicates were set up for each time point.

[0049] Total RNA from each sample was used as a template and reverse transcribed into cDNA using the reverse transcription reagent HiScript III All-in-one RTSuperMixPerfect for qPCR (Nanjing Novizan Biotechnology Co., Ltd.).

[0050] The upstream primer for qPCR of the egfp gene is qRT-egfp-F, the sequence of which is shown in SEQ ID No. 10, and the downstream primer is qRT-egfp-R, the sequence of which is shown in SEQ ID No. 11.

[0051] Using the rfl-5122 gene as an internal reference gene, the upstream primer for qPCR was qRT-rfl-5122-F, the sequence of which is shown in SEQ ID No. 12, and the downstream primer was qRT-rfl-5122-R, the sequence of which is shown in SEQ ID No. 13.

[0052] Quantitative real-time PCR was performed on the egfp transcription levels of the two strains, JP2-Ptp-egfp and JP2-Plac-egfp, at five time points using Taq Pro Universal SYBR qPCR Master Mix (Nanjing Novizan Biotechnology Co., Ltd.). The reaction conditions were set as follows: denaturation at 95°C for 30 seconds, 95°C for 10 seconds, and 60°C for 30 seconds, for a total of 40 cycles. The relative transcription levels of egfp compared to the internal reference gene were calculated using the ΔΔCt method. Data analysis was performed using GraphPadPrism 8.0 software. Significant differences were analyzed using one-way ANOVA with a two-tailed t-test. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Results are shown in [Figure number missing]. Figure 2 .

[0053] Figure 2 The results showed that the egfp gene in both JP2-Plac-egfp and JP2-Ptp-egfp was transcribed, indicating that Ptp has a promoter function; and the transcription level of JP2-Ptp-egfp was higher than that of JP2-Plac-egfp at all stages, indicating that the Ptp promoter is more efficient at driving the transcription of target genes than Plac. Example 3

[0054] Ptp was truncated from the 3' end to the 5' end to 390 bp, 348 bp, 199 bp, 109 bp, 52 bp, and 32 bp, respectively. These truncated fragments were named Ptp390, Ptp348, Ptp199, Ptp109, Ptp52, and Ptp32, respectively. Their nucleotide sequences are shown in SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, and SEQ ID No. 19, respectively.

[0055] Using the same procedures as in Example 1 for constructing the recombinant plasmid pBBR1-Ptp-egfp, the Ptp values ​​on the pBBR1-Ptp-egfp recombinant plasmid were replaced with Ptp390, Ptp348, Ptp199, Ptp109, Ptp52, and Ptp32, respectively, to obtain the recombinant plasmids pBBR1-Ptp390-egfp, pBBR1-Ptp348-egfp, pBBR1-Ptp199-egfp, pBBR1-Ptp109-egfp, pBBR1-Ptp52-egfp, and pBBR1-Ptp32-egfp.

[0056] pBBR1-Ptp390-egfp, pBBR1-Ptp348-egfp, pBBR1-Ptp199-egfp, pBBR1-Ptp109-egfp, pBBR1-Ptp52-egfp, and pBBR1-Ptp32-egfp were electroporated into strain JP2-4390, thereby obtaining recombinant strains JP2-Ptp390-egfp, JP2-Ptp348-egfp, JP2-Ptp199-egfp, JP2-Ptp109-egfp, JP2-Ptp52-egfp, and JP2-Ptp32-egfp.

[0057] The recombinant strains JP2-Ptp390-egfp, JP2-Ptp348-egfp, JP2-Ptp199-egfp, JP2-Ptp109-egfp, JP2-Ptp52-egfp, and JP2-Ptp32-egfp were observed at OD using the same procedures as in Example 1. 600 Fluorescence at 0.6. JP2-Ptp-egfp was used as a positive control-1; JP2-Plac-egfp as a positive control-2; and JP2-pBBR1 as a negative control. Results are shown below. Figure 3 .

[0058] Figure 3 The results showed that all truncated fragments of Ptp could initiate the transcription of the egfp gene and subsequent expression of EGFP protein, and the fluorescence intensity of their expression products was higher than that of Plac. This indicates that these truncated fragments all possess promoter activity, and that their promoter activity is stronger than that of Plac. Furthermore, based on fluorescence intensity, Ptp199 showed the strongest activity, followed by Ptp390, and then Ptp348, with the promoter activity of these truncated fragments all being stronger than that of Ptp. Example 4

[0059] The recombinant strains JP2-Ptp390-egfp, JP2-Ptp348-egfp, JP2-Ptp199-egfp, JP2-Ptp109-egfp, JP2-Ptp52-egfp, and JP2-Ptp32-egfp were used in Example 2 to determine the relative transcription levels of the egfp genes driven by Ptp390, Ptp348, Ptp199, Ptp109, Ptp52, and Ptp32 at 36 h. JP2-Ptp-egfp was used as a positive control-1; JP2-Plac-egfp was used as a positive control-2. Results are shown below. Figure 4 .according to Figure 4 The results show that it is related to Figure 3The results were consistent: Ptp199 showed the strongest activity, followed by Ptp390, and then Ptp348. Furthermore, Ptp and its truncated fragments showed stronger activity than Plac. Example 5

[0060] The core gene for the biosynthesis of nitropyrrolizin is the prnD gene, the nucleic acid sequence of which is shown in SEQ ID No. 20. The upstream primer used to amplify the full length of the prnD gene is prnD-F, the sequence of which is shown in SEQ ID No. 21; the downstream primer is prnD-R, the sequence of which is shown in SEQ ID No. 22.

[0061] Using the genome of strain JP2-4390 as a template, a PCR product of Ptp199 with RBS tandemly at the 3' end was amplified, namely the expression regulatory element Ptp199-RBS. The reverse complementary sequence of RBS is located at the 5' end of the downstream primer.

[0062] Using the genome of strain JP2-4390 as a template, and prnD-F and prnD-R as primers, the PCR product of the prnD gene was amplified.

[0063] The PCR products of Ptp199 and prnD gene were ligated with the pBBR1MCS-2 vector using the ClonExpress Ultra One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.) to construct the pBBR1-Ptp199-prnD recombinant plasmid, in which prnD is located downstream of RBS.

[0064] The pBBR1-Ptp199-prnD recombinant plasmid was electroporated into strain JP2-4390 to obtain the JP2-Ptp199-prnD recombinant strain.

[0065] Fresh colonies of JP2-Ptp199-prnD were inoculated into 5 mL of LB liquid medium and activated at 28°C and 220 rpm for 8 h. Then, 1% of the colonies were inoculated into 100 mL of fresh KIDO-4390 liquid medium in a 500 mL Erlenmeyer flask and incubated at 28°C and 220 rpm for 120 h. The supernatant was collected by centrifugation at 12000×g for 15 min, and 50 g of ammonium sulfate was added to the supernatant and stirred until completely dissolved. The mixture was then incubated overnight at 4°C. The precipitate was extracted with methanol, and the organic phase was collected by centrifugation at 12000×g for 15 min and dried using a rotary evaporator to obtain the crude extract of the prnD overexpression metabolites. The crude extract of the metabolites extracted using the wild-type strain JP2-4390 as a control was the crude extract of the prnD overexpression metabolites.

[0066] The two crude extracts were analyzed using Agilent Technologies 1260 Infinity liquid chromatography to determine the amount of nitropyrrolidone synthesized. The mobile phase was acetonitrile-water solution (acetonitrile was chromatographic grade); the mobile phase flow rate was 1.0 mL / min, and the elution time was 30 min; the detection wavelength was 210 nm, and the injection volume was 5 μL. The procedure was followed according to the instrument manual, and the results are as follows: Figure 5 As shown, the yield of nitropyrrolizin in the recombinant strain JP2-Ptp199-prnD after overexpression of the prnD gene was increased by approximately 81.3% compared to the wild-type strain. Example 6

[0067] The sequence of the expression regulatory element of the PRN BGC gene cluster (i.e., the BGC9 gene cluster in patent CN202411502275.5) is shown in SEQ ID No. 23. The upstream sequence Pprn-up of the PRN BGC gene cluster expression regulatory element is shown in SEQ ID No. 24. The sequence of the upstream primer Pprn-upF used to amplify Pprn-up is shown in SEQ ID No. 25, and the sequence of the downstream primer Pprn-upR is shown in SEQ ID No. 26.

[0068] The downstream sequence Pprn-dw of the PRN BGC gene cluster expression regulatory element is shown in SEQ ID No. 27. The upstream primer Pprn-dwF sequence used to amplify Pprn-dw is shown in SEQ ID No. 28; the downstream primer Pprn-dwR sequence is shown in SEQ ID No. 29.

[0069] Using the genome of strain JP2-4390 as a template, a PCR product of Ptp199 with RBS tandemly at the 3' end was amplified, namely the expression regulatory element Ptp199-RBS. The reverse complementary sequence of RBS is located at the 5' end of the downstream primer.

[0070] Using the genome of strain JP2-4390 as a template, the PCR product of Pprn-up was amplified using Pprn-upF and Pprn-upR as primers; the PCR product of Pprn-dw was amplified using Pprn-dwF and Pprn-dwR as primers.

[0071] The PCR products of Ptp199, Pprn-up, and Pprn-dw were ligated with the pK18mobsacB vector (Shanghai Zeye Biotechnology Co., Ltd.) using the ClonExpress Ultra One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.) to construct the pK18-Ptp199-prn recombinant plasmid, in which Pprn-up is located upstream of Ptp199 and Pprn-dw is located downstream of RBS.

[0072] Referring to CN202411502275.5, the pK18-Ptp199-prn recombinant plasmid was electroporated into strain JP2-4390. Through homologous recombination, the wild-type expression regulatory element of the PRN BGC gene cluster was replaced with the expression regulatory element Ptp199-RBS to obtain the engineered strain JP2-T-Ptp199-prn. The pK18-Ptp199-prn recombinant plasmid was electroporated into the engineered strain JP2ΔBGC1 / 16. Through homologous recombination, the wild-type expression regulatory element of the PRN BGC gene cluster was replaced with the expression regulatory element Ptp199-RBS to obtain the engineered strain JP2-4390M1.

[0073] Strains JP2-T-Ptp199-prn and JP2-4390M1 were cultured, and crude extracts of metabolites were extracted. The synthesis of nipyrrolizin was analyzed using Agilent Technologies 1260 Infinity liquid chromatography, as in Example 5. The nipyrrolizin yield was then calculated by comparing the peak area of ​​the crude metabolite extract of the corresponding strain with that of the nipyrrolizin standard. The nipyrrolizin yield of JP2-T-Ptp199-prn and JP2-4390M1 strains was analyzed using the starting strains JP2-4390 and JP2ΔBGC1 / 16 as controls. The results are as follows: Figure 6 As shown, the engineered strain JP2-T-Ptp199-prn has a 3.16 times higher nitropyrrolizin production capacity than the wild type; the engineered strain JP2-4390M1 has a 4.17 times higher nitropyrrolizin production capacity than the wild type strain and a 2.59 times higher nitropyrrolizin production capacity than the JP2ΔBGC1 / 16 strain.

[0074] The above results indicate that the Ptp199-RBS expression regulatory element has stronger activity than the wild-type expression regulatory element of the PRN BGC gene cluster, and the Ptp199 promoter has stronger activity than the wild-type promoter of the PRN BGC gene cluster, and can effectively increase the yield of nitropyrrolizin. Example 7

[0075] Referring to CN202411502275.5, the pK18-Ptp199-prn recombinant plasmid was electroporated into the engineered strain JP2ΔBGC1. The wild-type expression regulatory element of the PRN BGC gene cluster was replaced with the expression regulatory element Ptp199-RBS through homologous recombination to obtain the engineered strain JP2-4390M2.

[0076] Referring to CN202411502275.5, the pK18-Ptp199-prn recombinant plasmid was electroporated into the engineered strain JP2ΔBGC19. The wild-type expression regulatory element of the PRN BGC gene cluster was replaced with the expression regulatory element Ptp199-RBS through homologous recombination to obtain the engineered strain JP2-4390M3.

Claims

1. The use of a nucleic acid as a promoter, the sequence of which is shown in SEQ ID No.

1.

2. The application of a nucleic acid as a promoter, wherein the sequence of the nucleic acid is truncated from the sequence shown in SEQ ID No. 1, starting from the first digit at the 3' end and extending to the 5' end, with a truncated length greater than or equal to 32 bp and less than 1000 bp.

3. The application according to claim 2, characterized in that, The truncation length is greater than or equal to 32 bp and less than or equal to 390 bp.

4. The application according to claim 2, characterized in that, The nucleic acid sequence is one of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 and SEQ ID No.

19.

5. The application according to any one of claims 1 to 4, characterized in that, The promoter is used to initiate the transcription of the prnD gene in Pseudomonas, thereby increasing the production of nitropyrrolizin by the Pseudomonas.

6. The application according to claim 5, characterized in that, The promoter is used to initiate the overexpression of the prnD gene in Pseudomonas, thereby increasing the production of nitropyrrolizin by the Pseudomonas.

7. The application according to claim 6, characterized in that, The sequence of the prnD gene is shown in SEQ ID No.

20.

8. The application according to claim 5, characterized in that, The wild-type expression regulatory element used to initiate the PRN BGC gene cluster in Pseudomonas is replaced with a highly efficient expression regulatory element, thereby increasing the production of nitropyrrolizin by the Pseudomonas, wherein the highly efficient expression regulatory element consists of the promoter and RBS as described in any one of claims 1 to 4, and the RBS is located at the 3' end of the promoter as described in any one of claims 1 to 4.

9. The application according to claim 8, characterized in that, The sequence of the wild-type expression regulatory element of the PRN BGC gene cluster is shown in SEQ ID No.

23.

10. An engineered bacterium, wherein the engineered bacterium is one of strains JP2-Ptp199-prnD, JP2-T-Ptp199-prn, JP2-4390M1, JP2-4390M2, and JP2-4390M3; wherein, The JP2-Ptp199-prnD strain was obtained by electroporation transformation of the pBBR1-Ptp199-prnD recombinant plasmid into the JP2-4390 strain; wherein, the pBBR1-Ptp199-prnD recombinant plasmid was obtained by ligating the expression regulatory elements Ptp199-RBS and the prnD gene into the pBBR1MCS-2 vector; The JP2-T-Ptp199-prn strain was obtained by replacing the wild-type expression regulatory element of the PRN BGC gene cluster in the JP2-4390 strain with the expression regulatory element Ptp199-RBS through homologous recombination. The JP2-4390M1 was obtained by replacing the wild-type expression regulatory element of the PRN BGC gene cluster in the JP2ΔBGC1 / 16 strain with the expression regulatory element Ptp199-RBS through homologous recombination. The JP2-4390M2 strain was obtained by replacing the wild-type expression regulatory element of the PRN BGC gene cluster in the JP2ΔBGC1 strain with the expression regulatory element Ptp199-RBS through homologous recombination. The JP2-4390M3 strain was obtained by replacing the wild-type expression regulatory element of the PRN BGC gene cluster in the JP2ΔBGC16 strain with the expression regulatory element Ptp199-RBS through homologous recombination. The sequence of the RBS is shown in SEQ ID No. 2; the sequence of the Ptp199 is shown in SEQ ID No. 16; the nucleic acid sequence of the prnD gene is shown in SEQ ID No. 20; and the sequence of the wild-type expression regulatory element of the PRN BGC gene cluster is shown in SEQ ID No.

23. The JP2-4390 strain is deposited at the China Center for Type Culture Collection, with accession number CCTCC M 20241968; The JP2ΔBGC1 / 16 strain was obtained by knocking out the core genes of the two gene clusters BGC1 and BGC16 in the wild-type strain JP2-4390. The JP2ΔBGC1 strain was obtained by knocking out the core gene of the BGC1 gene cluster in the wild-type strain JP2-4390. The JP2ΔBGC16 strain was obtained by knocking out the core gene of the BGC16 gene cluster in the wild-type strain JP2-4390.

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