Promoter and application thereof

Abstract

The invention relates to a promoter and application thereof. The nucleotide sequence of the promoter is a fragment as shown in SEQ ID No. 1 or a truncated fragment as shown in SEQ ID No. 1. Endogenous or exogenous target genes can be effectively expressed in microbial cells, especially in pseudomonas. For example, when a prnD gene is expressed in a Pseudomonas using the same as a promoter of the prnD gene, the yield of nitropyrrole can be increased.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acids, and particularly to a promoter and its application. Background Art

[0002] Pseudomonas can produce various secondary metabolites such as pyrrolnitrin, phenazine, and siderophore in the natural environment. However, the yields of these substances are relatively low, and industrial production is limited, making it impossible to promote and utilize them. Rational improvement of microbial strains by modern biotechnology is an important means to clarify their functions and achieve directional transformation of strains. Promoter engineering can induce the high expression of target genes and obtain engineering strains with high-yield performance.

[0003] A promoter refers to a segment of DNA molecule that can be recognized, bound, and initiate transcription by RNA polymerase. A promoter is an important cis-acting element in the process of gene expression regulation, and it can control the degree of gene expression. In microbial metabolic engineering, the yield can be increased by overexpressing the target gene using some constitutive strong promoters. Summary of the Invention

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

[0005] In a specific embodiment, the promoter is used to initiate the transcription of the prnD gene in Pseudomonas, thereby increasing the yield of pyrrolnitrin produced by the Pseudomonas. Specifically, an efficient expression regulatory element can be formed by connecting the 3'-end of the promoter with an RBS (for example, its sequence can be as shown in SEQ ID No. 2), an expression cassette can be formed by connecting the 3'-end of the efficient expression regulatory element with the prnD gene, the expression cassette can be connected to a plasmid expression vector to obtain an expression vector for expressing the prnD gene, and the expression vector can be transformed into Pseudomonas to achieve an increase in the yield of pyrrolnitrin.

[0006] In a specific embodiment, the promoter is used to overexpress the prnD gene in Pseudomonas, thereby increasing the yield of pyrrolnitrin produced by the Pseudomonas. Specifically, an efficient expression regulatory element can be formed by connecting the 3'-end of the promoter with an RBS (for example, its sequence can be as shown in SEQ ID No. 2), an expression cassette can be formed by connecting the 3'-end of the efficient expression regulatory element with the prnD gene, the expression cassette can be connected to a plasmid expression vector to obtain an expression vector for expressing the prnD gene, and the expression vector can be transformed into Pseudomonas (such as Pseudomonas protegens, such as strain JP2-4390, JP2ΔBGC1 / 16 engineering strain, JP2ΔBGC1 engineering strain, or JP2ΔBGC16 engineering strain) to achieve an increase in the yield of pyrrolnitrin.

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

[0008] In a specific embodiment, the wild-type promoter for initiating the PRN BGC gene cluster in Pseudomonas is replaced with the promoter in any one of the applications described in the present invention, thereby increasing the yield of pyrrolnitrin produced by the Pseudomonas.

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

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

[0011] The second aspect of the present invention provides an application of a nucleic acid as a promoter, and the sequence of the nucleic acid is intercepted from the sequence shown in SEQ ID No. 1, starting from the first position at the 3'-end and intercepting towards the 5'-end, and the intercepted length is greater than or equal to 32 bp and less than 1000 bp. That is to say, from the 5'-end to the 3'-end of SEQ ID No. 1, the intercepted fragment contains at least the last 32 bp fragment shown in SEQ ID No. 1; or from the 3'-end to the 5'-end of SEQ ID No. 1, the intercepted fragment contains at least the first 32 bp fragment shown in SEQ ID No. 1.

[0012] In a specific embodiment, the intercepted length is greater than or equal to 32 bp and less than or equal to 390 bp.

[0013] In a specific embodiment, the sequence of the nucleic acid is one of those 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.

[0014] In a specific embodiment, the promoter is used to initiate the transcription of the prnD gene in Pseudomonas, thereby increasing the yield of pyrrolnitrin produced by the Pseudomonas. Specifically, an efficient expression regulatory element can be formed by connecting the 3'-end of the promoter with an RBS (for example, its sequence can be as shown in SEQ ID No. 2), and an expression cassette is formed by connecting the 3'-end of this efficient expression regulatory element with the prnD gene. The expression cassette is connected to a plasmid expression vector to obtain an expression vector for expressing the prnD gene, and this expression vector is transformed into Pseudomonas to achieve an increase in the yield of pyrrolnitrin.

[0015] In a specific embodiment, the promoter is used to overexpress the prnD gene in Pseudomonas, thereby increasing the yield of pyrrolnitrin produced by the Pseudomonas. Specifically, an efficient expression regulatory element can be formed by connecting the 3'-end of the promoter with an RBS (for example, its sequence can be as shown in SEQ ID No. 2), and an expression cassette is formed by connecting the 3'-end of this promoter region with the prnD gene. The expression cassette is connected to a plasmid expression vector to obtain an expression vector for expressing the prnD gene, and this expression vector is transformed into Pseudomonas (such as Pseudomonas protegens, such as strain JP2-4390, engineering strain JP2ΔBGC1 / 16, engineering strain JP2ΔBGC1, or engineering strain JP2ΔBGC16) to achieve an increase in the yield of pyrrolnitrin.

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

[0017] In a specific embodiment, the wild-type promoter in Pseudomonas that initiates the PRN BGC gene cluster is replaced with the promoter in any one of the applications described in the present invention, thereby increasing the yield of pyrrolnitrin produced by the Pseudomonas.

[0018] In a specific embodiment, the wild-type expression regulatory element in Pseudomonas that initiates the PRN BGC gene cluster is replaced with an efficient expression regulatory element, thereby increasing the yield of pyrrolnitrin produced by the Pseudomonas. Among them, the efficient expression regulatory element is composed of the promoter and an RBS in any one of the applications described in the second aspect of the present invention, and the RBS is located at the 3'-end of the promoter in any one of the applications described in the second aspect of the present invention.

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

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

[0021] Advantages of the present invention: The present invention discovers that the nucleic acid shown in SEQ ID No. 1 and its truncated fragments have strong promoter activity, and can effectively express endogenous or exogenous target genes in microbial cells, especially in Pseudomonas. For example, when it is used as the promoter of the prnD gene to overexpress the prnD gene in Pseudomonas, the production of pyrrolnitrin can be increased. Description of the Drawings

[0022] Figure 1 Shows the fluorescence expression intensities of JP2-Ptp-egfp, JP2-Plac-egfp, and JP2-pBBR1 at OD 600 = 0.6.

[0023] Figure 2 Shows the relative transcriptional levels of JP2-Ptp-egfp and JP2-Plac-egfp at five different times.

[0024] Figure 3 Shows the effects of six truncated forms of the Ptp promoter on the EGFP fluorescence intensity.

[0025] Figure 4 Shows the relative transcriptional levels of six truncated forms of the Ptp promoter at 36 h.

[0026] Figure 5 Shows the biosynthesis amount of pyrrolnitrin when overexpressing the prnD gene using the Ptp199 promoter in JP2-4390.

[0027] Figure 6 Shows the biosynthesis amount of pyrrolnitrin after replacing the wild-type promoter Pprn in the Pseudomonas protegens strain with the promoter Ptp199. Detailed Embodiments

[0028] The above content of the present invention will be further described in detail below in the form of preferred embodiments, but it does not constitute a limitation to the present invention.

[0029] Unless otherwise specified, the strains, plasmids, and reagents in the embodiments of the present invention can be purchased through commercial channels.

[0030] Pseudomonas protegens used in the present invention ( Pseudomonas protegens)The strain JP2-4390 was first disclosed in CN202411502275.5. It is deposited in the China Center for Type Culture Collection with the deposit number CCTCC M20241968, and the deposit date is September 13, 2024. The GenBank accession number of the complete genome sequence of the strain JP2-4390 is CP171611. An article about the strain JP2-4390 was published in Rice Science on April 15, 2025. Article name: 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 described in paragraph 72 of the specification of CN202411502275.5 have the corresponding names in the article "Efficient production of pyrrolnitrin by optimizing culture medium and blocking competitive secondary metabolic pathways in Pseudomonas protegens JP2-4390" as APE BGC, RIPP1 BGC, MA026 BGC, HSERLACTONE BGC, OFA BGC, PLT BGC, HCN BGC, E-PCH BGC, PRN BGC (the core gene prnD for the biosynthesis of pyrrolnitrin is included in this gene cluster), FEN BGC, PVD1 BGC, PVD2 BGC, NAGGN BGC, PETRICHORIN BGC, RANTHIPEPTIDE BGC, and RIPP2 BGC.

[0031] The engineered strain JP2ΔBGC1 / 16 was first disclosed in CN202411502275.5 and was obtained by knocking out the core genes of two gene clusters, BGC1 and BGC16, in the wild-type strain JP2-4390. The pyrrolnitrin production of the engineered strain JP2ΔBGC1 / 16 was significantly increased compared with that of the wild-type strain JP2-4390. The engineered strain JP2ΔBGC1 / 16 is the same engineered strain as the JP2Δape / ripp2 engineered strain in the article " Pseudomonas protegens Efficient production of pyrrolnitrin by optimizing culture medium and blocking competitive secondary metabolic pathways in JP2-4390", only with different strain names.

[0032] The engineered strain JP2ΔBGC1 was first disclosed in CN202411502275.5 and was obtained by knocking out the core gene of the BGC1 gene cluster in the wild-type strain JP2-4390. The pyrrolnitrin production of the engineered strain JP2ΔBGC1 was significantly increased compared with that of the wild-type strain JP2-4390. The engineered strain JP2ΔBGC1 is the same engineered strain as the JP2Δape engineered strain in the article " Pseudomonas protegens Efficient production of pyrrolnitrin by optimizing culture medium and blocking competitive secondary metabolic pathways in JP2-4390", only with different strain names.

[0033] The engineered strain JP2ΔBGC16 was first disclosed in CN202411502275.5 and was obtained by knocking out the core gene of the BGC16 gene cluster in the wild-type strain JP2-4390. The pyrrolnitrin production of the engineered strain JP2ΔBGC16 was significantly increased compared with that of the wild-type strain JP2-4390. The engineered strain JP2ΔBGC16 is the same engineered strain as the JP2Δripp2 engineered strain in the article " Pseudomonas protegens Efficient production of pyrrolnitrin by optimizing culture medium and blocking competitive secondary metabolic pathways in JP2-4390", only with different strain names.

[0034] The Escherichia coli ( Escherichia coli ) TOP 10 strain used in constructing the plasmid of the present invention was purchased from Hangzhou Eastep Biotech Co., Ltd.

[0035] The broad-host plasmid vector pBBR1MCS-2 was purchased from Shanghai Lianmai Bioengineering Co., Ltd. and has kanamycin resistance. 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 for amplifying Ptp is Ptp-F, and its sequence is shown in SEQ ID No. 5; the downstream primer is Ptp-R, and its sequence is shown in SEQ ID No. 6. Among them, 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 for amplifying the egfp gene is egfp-F, and its sequence is shown in SEQ ID No. 7; the downstream primer is egfp-R, and its sequence is shown in SEQ ID No. 8. Using the genomic DNA of strain JP2-4390 as a template and Ptp-F and Ptp-R as primers, a PCR product of Ptp with RBS tandemly linked at the 3' end was amplified; using the artificially synthesized egfp gene as a template and egfp-F and egfp-R as primers, a PCR product of the egfp gene was amplified. The ClonExpress Ultra One Step Cloning Kit (Nanjing Novoprotein Scientific Inc.) was used to ligate the PCR product of Ptp with RBS tandemly linked at the 3' end, the PCR product of the egfp gene, and the pBBR1MCS-2 vector to construct the pBBR1-Ptp-egfp recombinant plasmid, where egfp is located downstream of RBS.

[0042] The recombinant plasmid pBBR1-Ptp-egfp was transferred into JP2-4390 by electroporation, and the positive transformant JP2-Ptp-egfp was screened out.

[0043] Due to the short lengths of both the Plac promoter and the RBS region, they were integrated with the upstream sequence for amplifying the egfp gene to form another upstream primer, Plac-RBS-gfp-F, for amplifying the egfp gene. Its sequence is 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 of the egfp gene carrying the Plac promoter and the RBS region was amplified. The above PCR product was ligated to the pBBR1MCS-2 vector using the ClonExpress Ultra One Step Cloning Kit (Nanjing Novoprotein Scientific Inc.) to construct the pBBR1-Plac-egfp recombinant plasmid. The recombinant plasmid pBBR1-Plac-egfp was transferred into JP2-4390 by electroporation, and positive transformants JP2-Plac-egfp were screened out as the positive control.

[0045] The empty plasmid pBBR1MCS-2 was transferred into JP2-4390 by electroporation, and positive transformants JP2-pBBR1 were screened out as the 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 at 28 °C and 220 rpm for 8 h of activation. Then, they were inoculated into test tubes containing 5 mL of fresh LB liquid medium at an inoculation amount of 1% and cultured at 28 °C and 220 rpm. When the OD 600 reached 0.6, fluorescence observation was performed using the laser confocal microscope LSM700, as Figure 1 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 could express to produce green fluorescent protein under the action of the lac promoter Plac; the egfp gene could also express to produce green fluorescent protein under the action of Ptp, that is, Ptp had promoter function; and the green fluorescence produced by JP2-Ptp-egfp was significantly stronger than that of JP2-Plac-egfp, indicating that the yield of green fluorescent protein expressed by JP2-Ptp-egfp was higher than that of JP2-Plac-egfp, further indicating that the efficiency of the Ptp promoter driving the transcription of the target gene was stronger than that of Plac. Example 2

[0048] Pick fresh colonies of JP2-Ptp-egfp and JP2-Plac-egfp and inoculate them into 5 mL of LB liquid medium at 28 °C and 220 rpm for 8 h of activation. Then inoculate them into a triangular flask containing 300 mL of fresh LB liquid medium at an inoculation amount of 1%, and sample and collect the bacteria at 12 h, 16 h, 22 h, 36 h, and 48 h of culture at 28 °C and 220 rpm. Use the RNAprep Pure Cell / Bacteria Kit (Tiangen Biochemical Technology Co., Ltd., Beijing) to extract the total RNA of the bacteria. Set 3 replicates for the samples at each time point.

[0049] Using the total RNA of each sample as a template, reverse transcribe it into cDNA using the reverse transcription reagent HiScript Ⅲ All-in-one RT SuperMix Perfect for qPCR (Nanjing Novoprotein Science and Technology Co., Ltd.).

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

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

[0052] The fluorescence quantitative PCR was used to detect the transcriptional levels of egfp in the above-mentioned JP2-Ptp-egfp and JP2-Plac-egfp strains at five time points using Taq Pro Universal SYBR qPCR Master Mix (Nanjing Novoprotein Scientific Inc.). The reaction conditions were set as follows: denaturation at 95 °C for 30 s, 95 °C for 10 s, and 60 °C for 30 s, for a total of 40 cycles. The relative transcriptional level of egfp relative to the internal reference gene was calculated using the ΔΔCt method, and the data statistics were completed by GraphPad Prism 8.0 software; the analysis of significant differences in the data was obtained using the one-way ANOVA and two-tailed t test procedures, where * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and the results are shown in Figure 2 .

[0053] Figure 2 The results in show that the egfp gene is transcribed in both JP2-Plac-egfp and JP2-Ptp-egfp, indicating that Ptp has promoter function; and the transcriptional level of JP2-Ptp-egfp at each stage is higher than that of JP2-Plac-egfp, indicating that the efficiency of the Ptp promoter in driving the transcription of target genes is stronger than that of Plac. Example 3

[0054] Ptp was truncated from the first position at the 3' end to the 5' end to 390 bp, 348 bp, 199 bp, 109 bp, 52 bp and 32 bp. These truncated fragments were named Ptp390, Ptp348, Ptp199, Ptp109, Ptp52 and Ptp32 in turn, and 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 in turn.

[0055] Using the same operation as in Example 1 to construct the recombinant plasmid pBBR1-Ptp-egfp, Ptp on the pBBR1-Ptp-egfp recombinant plasmid was 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 respectively electrotransformed into strain JP2-4390 to obtain recombinant strains JP2-Ptp390-egfp, JP2-Ptp348-egfp, JP2-Ptp199-egfp, JP2-Ptp109-egfp, JP2-Ptp52-egfp and JP2-Ptp32-egfp accordingly.

[0057] Using the same operation as in Example 1, the fluorescence of recombinant strains JP2-Ptp390-egfp, JP2-Ptp348-egfp, JP2-Ptp199-egfp, JP2-Ptp109-egfp, JP2-Ptp52-egfp and JP2-Ptp32-egfp was observed at an OD 600 of 0.6. JP2-Ptp-egfp was used as positive control -1; JP2-Plac-egfp was used as positive control -2; JP2-pBBR1 was used as negative control. The results are shown in Figure 3 .

[0058] Figure 3 The results showed that the truncated fragments of Ptp could all initiate the transcription of the egfp gene and the subsequent expression of the EGFP protein, and the fluorescence intensity of their expression products was higher than that of Plac. On the one hand, this indicated that these truncated fragments all had promoter activity; on the other hand, it indicated that the promoter activity of these truncated fragments was stronger than that of Plac. In addition, based on the fluorescence intensity, it was preliminarily judged that Ptp199 had the strongest activity, followed by Ptp390, and then Ptp348, and the promoter activity of these several truncated fragments was stronger than that of Ptp. Example 4

[0059] Based on the same operation as in Example 2, the relative transcription levels of the egfp gene driven by Ptp390, Ptp348, Ptp199, Ptp109, Ptp52 and Ptp32 in recombinant strains JP2-Ptp390-egfp, JP2-Ptp348-egfp, JP2-Ptp199-egfp, JP2-Ptp109-egfp, JP2-Ptp52-egfp and JP2-Ptp32-egfp were measured at 36 h. JP2-Ptp-egfp was used as positive control -1; JP2-Plac-egfp was used as positive control -2. The results are shown in Figure 4 . According to Figure 4 the results, it can be known that it is the same as Figure 3The results are consistent. The activity of Ptp199 is the strongest, followed by Ptp390, then Ptp348, and the activities of Ptp and its truncated fragments are stronger than those of Plac. Example 5

[0060] The core gene for the biosynthesis of pyrrolnitrin is the prnD gene, and its nucleic acid sequence is shown in SEQ ID No. 20. The upstream primer for amplifying the full length of the prnD gene is prnD-F, and its sequence is shown in SEQ ID No. 21; the downstream primer is prnD-R, and its sequence is shown in SEQ ID No. 22.

[0061] Using the genome of strain JP2-4390 as a template, a PCR product of Ptp199 with an RBS tandem at the 3' end was amplified, namely the expression regulatory element Ptp199-RBS. Among them, the reverse complementary sequence of the 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, a PCR product of the prnD gene was amplified.

[0063] Using the ClonExpress Ultra One Step Cloning Kit (Nanjing Novoprotein Scientific Co., Ltd.), the PCR product of Ptp199, the PCR product of the prnD gene, and the pBBR1MCS-2 vector were ligated to construct the pBBR1-Ptp199-prnD recombinant plasmid, where prnD is located downstream of the RBS.

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

[0065] Fresh colonies of JP2-Ptp199-prnD were picked and inoculated into 5 mL of LB liquid medium at 28 °C and 220 rpm for 8 h of activation. Then, they were inoculated into a 500 mL Erlenmeyer flask containing 100 mL of fresh KIDO-4390 liquid medium at an inoculation amount of 1%, and cultured at 28 °C and 220 rpm for 120 h. The supernatant was collected by centrifugation at 12,000×g for 15 min. Then, 50 g of ammonium sulfate was added to the supernatant and stirred until completely dissolved, and it was kept overnight at 4 °C. The precipitate was extracted with methanol, and then the organic phase was collected by centrifugation at 12,000×g for 15 min and dried with a rotary evaporator to obtain a crude extract of the metabolite after overexpressing prnD. The crude extract of the metabolite extracted with the wild-type strain JP2-4390 as a control is the crude extract of the metabolite after normal expression of prnD.

[0066] The above two crude extracts were respectively analyzed by Agilent Technologies 1260 infinity liquid chromatography to detect the synthesis amount of pyrrolnitrin. The mobile phase used was an acetonitrile - aqueous solution, and the acetonitrile was a chromatographic grade solution; the flow rate of the mobile phase was 1.0 mL / min, and the elution time was 30 min; the detection wavelength was 210 nm, and the injection volume was 5 microliters. Run the program according to the instrument instructions, and the results are as Figure 5 shown. The production of pyrrolnitrin in the recombinant strain JP2 - Ptp199 - prnD overexpressing the gene prnD increased by approximately 81.3% compared with 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 upstream primer Pprn - upF sequence for amplifying Pprn - up is shown in SEQ ID No. 25, and the downstream primer Pprn - upR sequence 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 for amplifying 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 the JP2 - 4390 strain as a template, a PCR product of Ptp199 with RBS tandemly linked at the 3' end was amplified, that is, the expression regulatory element Ptp199 - RBS. Among them, the reverse complementary sequence of RBS is located at the 5' end of the downstream primer.

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

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

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

[0073] The JP2-T-Ptp199-prn and JP2-4390M1 strains were cultured, and the crude extracts of the metabolites were extracted. The synthesis amount of pyrrolnitrin was detected by Agilent Technologies 1260 infinity liquid chromatography analysis, the same as in Example 5. Then, the pyrrolnitrin yield was calculated by comparing the peak areas of the crude extracts of the metabolites of the corresponding strains with the pyrrolnitrin standard. Using the starting strains JP2-4390 and JP2ΔBGC1 / 16 as controls, the pyrrolnitrin yields of JP2-T-Ptp199-prn and JP2-4390M1 were analyzed. The results are as Figure 6 shown. The ability of the engineered strain JP2-T-Ptp199-prn to produce pyrrolnitrin is 3.16 times that of the wild type; the pyrrolnitrin yield of the engineered strain JP2-4390M1 is 4.17 times that of the wild-type strain and 2.59 times that of the JP2ΔBGC1 / 16 strain.

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

[0075] Referring to CN202411502275.5, the pK18-Ptp199-prn recombinant plasmid was electrotransformed into the engineered bacterium JP2ΔBGC1, and the wild-type expression regulatory elements of the PRN BGC gene cluster were replaced with the expression regulatory element Ptp199-RBS by homologous recombination to obtain the engineered bacterium JP2-4390M2.

[0076] Referring to CN202411502275.5, the pK18-Ptp199-prn recombinant plasmid was electrotransformed into the engineered bacterium JP2ΔBGC19, and the wild-type expression regulatory elements of the PRN BGC gene cluster were replaced with the expression regulatory element Ptp199-RBS by homologous recombination to obtain the engineered bacterium JP2-4390M3.

Claims

1. Use of a nucleic acid as a promoter, wherein the sequence of the nucleic acid is as shown in SEQ ID No.

1.

2. Use of a nucleic acid as a promoter, wherein the sequence of the nucleic acid is truncated from the sequence as shown in SEQ ID No. 1, starting from the first position at the 3'-end and truncating towards the 5'-end, and the truncation length is 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, wherein The sequence of the nucleic acid is one of the sequences as 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.

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 yield of pyrrolnitrin produced by the Pseudomonas.

6. The application according to claim 5, characterized in that, The promoter is used to overexpress the prnD gene in Pseudomonas, thereby increasing the yield of pyrrolnitrin produced by the Pseudomonas.

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

20.

8. The application according to claim 5, characterized in that, Replacing the wild-type expression regulatory element for initiating the PRN BGC gene cluster in Pseudomonas with a highly efficient expression regulatory element, thereby increasing the yield of pyrrolnitrin produced by the Pseudomonas, wherein the highly efficient expression regulatory element consists of the promoter and RBS in the use according to any one of claims 1 to 4, and the RBS is located at the 3'-end of the promoter in the use according to any one of claims 1 to 4.

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

23.

10. An engineered bacterium, which is one of the strains JP2-Ptp199-prnD, JP2-T-Ptp199-prn and JP2-4390M1, JP2-4390M2 and JP2-4390M3.

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