A phage-derived promoter SD13 enables strong and cross-host gene expression in Streptomyces and Escherichia coli

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In this study, the lytic bacteriophage phiSASD1 was exploited as a source of novel regulatory elements. Fifteen candidate promoter fragments were identified through bioinformatic prediction and systematically evaluated using a catechol 2,3-dioxygenase ( xylE ) reporter system in Streptomyces lividans TK54 and Escherichia coli JM109. Among these candidates, seven fragments exhibited measurable promoter activity, with SD13 showing the highest transcriptional strength. In S. lividans , SD13 displayed up to a 9.79-fold higher activity than the widely used strong promoter PermE*, while retaining robust activity in E. coli , indicating excellent cross-host compatibility. Sequence analysis combined with 5′ RACE revealed that SD13 possesses a typical σ⁷⁰-dependent promoter architecture, with its core functional region located between − 70 and + 9 bp relative to the transcription start site. Furthermore, SD13 efficiently drove the soluble expression of phage endolysin in E. coli , alleviating inclusion body formation commonly observed in T7-based systems. Collectively, these results identify SD13 as a highly efficient phage-derived promoter with broad host applicability, providing a valuable genetic tool for Streptomyces engineering and heterologous gene expression. Streptomyces bacteriophage SD13 promoter Cross-host expression Strong promoter Gene expression regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key points • Strong transcriptional promoters were systematically identified from phages. • The novel promoter SD13 shows higher activity than PermE* and works across hosts. • SD13 efficiently expresses soluble heterologous proteins, advancing synthetic biology. Introduction Streptomyces avermitilis is an important industrial microorganism responsible for the production of the widely used agricultural and medical antibiotics avermectins. Genetic engineering of this strain is critical for enhancing production yields and generating novel derivatives (Ikeda et al. 2003 ; Hao et al. 2022 ). Although Streptomyces genomes harbor numerous biosynthetic gene clusters (BGCs), many remain silent or are expressed at low levels under standard laboratory conditions, limiting their biosynthetic potential (Dong et al. 2023 ). Therefore, the development of efficient gene expression regulatory elements, particularly strong promoters, is essential for activating silent gene clusters and improving the production of target metabolites. With the rapid development of synthetic biology and metabolic engineering, the construction of efficient and controllable gene expression systems has become a key strategy for optimizing metabolic pathways in Streptomyces (Baltz et al. 2012). Promoters, as central cis-regulatory elements, directly determine transcriptional output and thereby influence the expression levels of downstream genes (Browning and Busby 2004 ). Previous studies have demonstrated that the identification or engineering of strong promoters can significantly enhance the biosynthesis of target compounds. For example, the constitutive promoter stnYp has been shown to markedly improve tylosin production (Guo et al. 2023 ). In addition, advances in understanding transcriptional regulation in Streptomyces , including the roles of SARP family regulators and global regulators such as AfsR, have provided valuable insights into promoter–regulator interactions and facilitated rational promoter design (Shi et al. 2024 ). Bacteriophages have evolved highly efficient transcriptional regulatory systems through long-term co-evolution with their hosts. Their genomes are enriched with cis-regulatory elements that can be readily recognized by host RNA polymerases, making them promising sources of novel promoters (Kronheim et al. 2023 ). Recent studies have further shown that Streptomyces can modulate phage infection through mechanisms such as siderophore secretion, thereby gaining a competitive advantage in microbial communities and revealing the complexity of phage–host regulatory networks (Zang et al. 2025 ). Previous studies have shown that promoters derived from Streptomyces phages, such as φC31 and φBT1, possess strong transcriptional activity and have been successfully applied in gene expression systems (Howe et al. 1996; Gregory et al. 2003 ; Li et al. 2020 ). However, the systematic exploration of phage-derived promoters in Streptomyces remains limited, particularly for those adapted to specific industrial hosts such as Streptomyces avermitilis , or those capable of driving efficient cross-host expression (Lu et al. 2019 ; Kronheim et al. 2023 ). In addition, differences in RNA polymerase recognition among hosts result in pronounced host dependency of promoter activity, and broadly active strong promoters that maintain high transcriptional activity across different hosts are still scarce. The bacteriophage phiSASD1 is a lytic phage infecting Streptomyces avermitilis , and its genome has been sequenced and annotated (Wang et al. 2010 ). Given that bacteriophages must rapidly hijack the host transcriptional machinery during infection, their genomes are likely to contain native promoters with high transcriptional activity. However, promoters derived from phiSASD1 have not been systematically characterized, which limits their potential application in synthetic biology. Reporter gene-based screening systems provide a reliable approach for evaluating promoter activity. By constructing promoter–reporter fusions and quantitatively measuring reporter output in different hosts, promoter strength and host compatibility can be systematically assessed (Horbal et al. 2013 ; Matsuzaki et al. 2024 ). In addition, bioinformatic prediction of core promoter elements, such as the − 10 and − 35 regions, facilitates the analysis of promoter structure–function relationships (Cheah et al. 2024 ; Nijo et al. 2017 ). In this study, we systematically screened potential promoter sequences from the phiSASD1 genome by combining bioinformatic prediction with experimental validation. The transcriptional activities of candidate promoters were evaluated in both Streptomyces and Escherichia coli , and the structural and functional characteristics of the most active promoter were further analyzed. This work aims to identify strong promoters with cross-host applicability and to provide useful genetic tools for Streptomyces metabolic engineering and heterologous gene expression. Materials and methods Strains, bacteriophage, and plasmids Streptomyces avermitilis , used as the host strain for bacteriophage phiSASD1, was maintained in our laboratory. Bacteriophage phiSASD1 is a lytic Streptomyces phage previously isolated and its genome has been sequenced and annotated (GenBank accession number: GQ379227). Escherichia coli DH5α was used for plasmid construction and propagation, whereas E. coli JM109 was used for promoter activity assays and heterologous protein expression. Streptomyces lividans TK54 was employed as the Streptomyces expression host. For promoter characterization, the E. coli – Streptomyces shuttle vector pSET152 and the reporter plasmid pIJ4083 carrying the xylE gene were used to construct the promoter probe vector pSX152. The xylE gene encodes catechol 2,3-dioxygenase (C23O), whose activity can be quantified using a catechol-based colorimetric assay. The constitutive promoter PermE*, derived from Saccharopolyspora erythraea , was used as a positive control, while the empty vector pSX152 served as a negative control. Culture conditions coli strains were cultured in LB liquid medium supplemented with appropriate antibiotics at 37°C with shaking at 200 rpm. S. avermitilis and S. lividans TK54 were cultured in YMS medium. For liquid cultivation, strains were incubated at 28°C with shaking at 200 rpm, while solid cultures were grown on agar plates containing 2% (w/v) agar. Bacteriophage phiSASD1 was propagated using the double-layer agar method, and high-titer phage lysates were prepared under optimal host growth conditions. Extraction of bacteriophage genomic DNA High-titer phage lysates were first treated with DNase I and RNase A to remove residual host nucleic acids. Subsequently, SDS and proteinase K were added to disrupt the phage capsid and release genomic DNA. Proteins were removed by phenol/chloroform extraction, followed by ethanol precipitation to recover the DNA. The integrity of the extracted DNA was verified by 1% agarose gel electrophoresis, and DNA concentration and purity were determined using a NanoDrop spectrophotometer. Bioinformatic prediction of candidate promoter sequences Potential promoter regions were predicted based on the complete genome sequence of bacteriophage phiSASD1 using BDGP, BPROM, and NNPP software. The minimum promoter score threshold in BDGP was set to 0.8 to enhance prediction reliability. Candidate promoter regions were selected from intergenic sequences upstream of annotated open reading frames (ORFs) based on GenBank annotation. A total of 15 putative promoter fragments were selected for subsequent experimental validation. Construction of promoter probe plasmids Candidate promoter fragments were amplified from phiSASD1 genomic DNA using specific primers and a high-fidelity DNA polymerase. PCR products were verified by agarose gel electrophoresis and purified prior to cloning. The purified fragments were digested with appropriate restriction enzymes and ligated into the promoterless xylE reporter vector pSX152 to generate promoter–reporter fusion constructs. The ligation mixtures were transformed into E. coli JM109 competent cells, and positive clones were selected on antibiotic-containing media. The integrity and correctness of the inserted sequences were confirmed by colony PCR and DNA sequencing. Construction of recombinant strains Verified recombinant plasmids were introduced into S. lividans TK54 via transformation and subsequently integrated into the host chromosome. Transformants were selected on media containing appropriate antibiotics. In parallel, recombinant plasmids carrying different promoter fragments were also transformed into E. coli JM109 to evaluate promoter activities across different hosts. Promoter activity assay Cells in the exponential growth phase were harvested by centrifugation and resuspended in an appropriate buffer. Catechol was added as the substrate, and enzymatic reactions were carried out at room temperature. The absorbance was measured at 375 nm (OD375). C23O activity was calculated using the following formula: mU (nmol·min⁻¹) = 30.03 × ΔA375 / time. One unit (mU) was defined as the amount of enzyme required to produce 1 nmol of 2-hydroxymuconic semialdehyde per minute at 30°C. Promoter activity was normalized to cell dry weight to ensure comparability across samples and expressed as mU/mg. The PermE* promoter was used as a positive control, and the empty vector pSX152 was used as a negative control. All experiments were performed with at least three independent biological replicates. Sequence feature analysis of promoters Promoter sequences exhibiting activity were subjected to sequence alignment analysis to identify conserved elements, including the − 10 and − 35 regions and spacer length. Potential transcription factor binding sites (TFBS) were predicted using the PRODORIC database and Softberry BPROM software. Sequence similarity to σ 70 -type promoter consensus motifs was analyzed to infer potential transcriptional regulatory mechanisms. Determination of transcription start site (TSS) The transcription start site of the SD13 promoter was determined using 5′ RACE (Rapid Amplification of cDNA Ends). Total RNA was extracted from S. avermitilis at different time points following phage infection and reverse-transcribed into cDNA. Nested PCR was performed using ORF13-specific primers to amplify the transcription start region. PCR products were sequenced, and the TSS was identified based on the anchor sequence and the first nucleotide immediately downstream of the poly(A) tail. Data processing and statistical analysis All experimental data were presented as mean ± standard deviation (mean ± SD). Statistical differences among groups were evaluated using one-way analysis of variance (ANOVA), and differences were considered statistically significant at P < 0.05. All statistical analyses and data visualization were performed using GraphPad Prism software. Results Identification of putative promoters in phage phiSASD1 Based on the complete genome sequence of the S. avermitilis bacteriophage phiSASD1, a total of 43 open reading frames (ORFs) were predicted. Intergenic regions between adjacent ORFs were screened for potential promoter sequences and further analyzed using the BDGP promoter prediction program. With the minimum promoter score threshold set at 0.8, nine high-scoring candidate promoter regions were identified, with prediction scores ranging from 0.82 to 0.97 (Table 1 ). Based on ORF annotation from the NCBI GenBank database, upstream intergenic sequences were further selected as candidate promoter fragments. In total, 15 putative promoter regions associated with ORF1, ORF3, ORF5, ORF8, ORF10, ORF11, ORF12, ORF13, ORF15, ORF17, ORF18, ORF19, ORF21, ORF38, and ORF40 were obtained for experimental validation (Table 2 ). These candidate fragments ranged from 92 to 703 bp in length and were associated with genes encoding proteins such as Cas4 exonuclease, DNA polymerase I, RNA polymerase subunit alpha, holin, and endolysin, suggesting potential roles in phage replication and lysis processes. Table 1 High-scoring promoter regions predicted in the phiSASD1 genome Number Start (bp) Stop (bp) Score Promoter sequence a 1 4117 4162 0.87 GTTGTGGAACTACCCGCGTGCTCCCGCT GCATCATCCTCG C CGGCCGCGA 2 12067 12112 0.94 GCCAGTTGGATGGTGCAGGTCTACAACG GTACCGTTATAT T CCGCTACAC 3 18885 18930 0.97 TATTTTTGTTTCGTCCCAGTAATAGAAAA GATAGTAACGC T CCTCGCCTT 4 19636 19681 0.88 CAGTTGGCCGCCTACCGGTACGCGGACG TGATCATTGACC C GGAGGGCAA 5 25381 25426 0.84 CTGATGGAAAACCAGGACTTCCCTCAGG ACATCTTTCCGC T GAACTACAC 6 30227 30272 0.92 CTCACTTGACCCGACTCAATTGAGTCGC CTACTATGACAA C GCGTCCACA 7 32201 32246 0.82 ACGGCTTGACTCGACTCAGTTGAGTCGG CTACTTTCGGCA T TCCAGCCGC 8 32442 32487 0.86 ACCCTTGACCTGTTCATCGTGGCCGGCG AGATCATGATGC T GGTGGCCGC 9 35074 35119 0.94 GCGGCTTGACTCGACTCAATTGAGCCGG CTAAATTGGAGC C CTCGCCAGG a Bold letters indicate the transcription start site (TSS) Table 2 Lengths of putative promoter fragments and predicted proteins encoded by the corresponding ORFs Potential promoter Length (bp) Putative product a ORF1 561 Cas4 family exonuclease ORF3 199 PAPS _reductase ORF5 142 – ORF8 703 – ORF10 204 – ORF11 106 primase/helicase ORF12 159 DNA polymerase I ORF13 265 DNA-directed RNA polymerase subunit alpha ORF15 111 – ORF17 660 HNH endonuclease ORF18 176 – ORF19 687 holin ORF21 188 Terminase small subunit ORF38 92 – ORF40 144 endolysin a “–” indicates that no homologous function was identified Promoter activity of candidate fragments in S. lividans TK54 To experimentally evaluate promoter activity, the 15 candidate fragments were individually cloned into the promoter probe vector pSX152 and integrated into the chromosome of S. lividans TK54. Promoter strength was assessed by measuring the activity of the xylE reporter gene. The constitutive promoter PermE* was used as a positive control, while the empty vector served as a negative control. Among the 15 candidates, seven promoters (SD10, SD12, SD13, SD15, SD17, SD18, and SD40) exhibited detectable transcriptional activity (Fig. 1 ). Notably, SD13 showed the highest activity, reaching 6.72-, 9.79-, and 5.06-fold that of PermE* at 12 h, 36 h, and 60 h, respectively (Fig. 1 b). SD18 also exhibited strong activity, approximately 3.38-fold higher than PermE* (Fig. 1 c), while SD15 displayed a comparable level of activity to PermE* (Fig. 1 b). Time-course analysis revealed that all active promoters reached peak C23O activity at 36 h, followed by a decline at 60 h. SD40 maintained relatively high activity at 60 h (Fig. 1 c). Interestingly, some fragments with high prediction scores (e.g., SD1 and SD38) showed no detectable promoter activity, whereas SD15, despite a relatively low prediction score, exhibited substantial transcriptional strength (Fig. 1 ). Expression activity of promoter fragments in E. coli JM109 To assess host compatibility, the seven active promoter fragments identified in S. lividans were further evaluated in E. coli JM109 using the same reporter system. The results showed that all seven promoters retained transcriptional activity in E. coli (Fig. 2 ), whereas the positive control promoter PermE* exhibited no detectable activity under the same conditions. Notably, SD13 maintained relatively high expression levels, indicating strong cross-host functionality. In addition, several promoters (e.g., SD10, SD12, and SD17) that showed relatively low activity in S. lividans exhibited enhanced activity in E. coli , highlighting the influence of host-specific transcriptional machinery on promoter performance (Fig. 2 ). Expression of endolysin driven by the SD13 promoter To evaluate the protein expression capability of the SD13 promoter, a recombinant plasmid (pIJ13-lysin) was constructed and expressed in E. coli JM109. Cells harboring the empty vector pIJ13 were used as a negative control. SDS-PAGE analysis revealed a distinct protein band at approximately 31 kDa in the recombinant strain, consistent with the predicted molecular weight of endolysin, whereas no corresponding band was observed in the control ( (Supplementary Fig. S1 ). The target protein was predominantly detected in the soluble fraction, indicating efficient soluble expression. These results demonstrate that SD13 is capable of driving effective heterologous protein expression while reducing the formation of inclusion bodies. Structural characterization of the SD13 promoter To investigate the structural features of SD13, transcription factor binding sites (TFBS) were analyzed using the PRODORIC database. The results indicated that SD13 shares high similarity with the consensus recognition sequence of the σ 70 factor in E. coli . Further analysis using Softberry BPROM revealed that SD13 contains typical prokaryotic promoter elements, including well-defined − 35 and − 10 regions (Table 3 ). In addition, a G/C-rich spacer sequence of approximately 18 bp was identified between these two regions. These structural features suggest that SD13 is a σ 70 -type promoter with canonical architecture. Table 3 Structural analysis of the SD13 promoter fragment a The sequences within the boxes represent the predicted − 10 and − 35 regions of the promoter, The bold and underlined base indicates the transcription start site, and the bold ATG denotes the start codon Determination of the Transcription Start Site of SD13 The transcription start site (TSS) of SD13 was determined using 5′ RACE. The results showed that transcripts of ORF13 could be detected as early as 20 min post-infection. Sequencing of nested PCR products revealed that the TSS is located at a guanine (G) residue positioned 107 bp upstream of the start codon (Fig. 3 ), differing by only 1 bp from the bioinformatics prediction. Effects of Conserved Regions on SD13 Promoter Activity To assess the functional importance of the conserved − 10 and − 35 regions, site-directed mutagenesis and deletion analyses were performed. Mutation of all bases in the − 10 region resulted in an approximately 3.85-fold reduction in promoter activity compared with the wild type (Fig. 4 b). In contrast, mutation of the − 35 region caused a much more pronounced decrease, approximately 31.34-fold (Fig. 4 b). Deletion of the − 10 region led to an approximately 37.67-fold reduction, whereas deletion of the − 35 region resulted in an approximately 11-fold decrease in activity (Fig. 4 b). These results indicate that both regions are essential for SD13 function, with the − 35 region playing a particularly critical role in transcriptional strength. Identification of the Core functional Region of SD13 To define the minimal functional region of SD13, progressive truncation analyses were performed. The 5′-truncated fragment SD13-218 (− 100 to + 118) exhibited a slight increase in activity (1.13-fold of the wild type at 36 h) (Fig. 5 b). However, further truncation to SD13-167 (− 49 to + 118) resulted in a marked reduction in promoter activity (Fig. 5 b). Subsequent 3′ truncations generated SD13-110 (− 49 to + 61) and SD13-58 (− 49 to + 9) (Fig. 5 a), both of which showed gradual recovery of activity (Fig. 5 b). Notably, the fragment SD13-79 (− 70 to + 9) exhibited the highest activity, reaching approximately 1.7-fold that of the full-length promoter at 36 h (Fig. 5 b). These results indicate that the core functional region of SD13 is located between − 70 and + 9 bp, and that upstream sequences may contain elements that negatively influence transcription. Discussion This study systematically screened and identified a series of transcriptionally active promoters from the genome of the Streptomyces phage phiSASD1, among which SD13 exhibited the strongest expression capability and good cross-host applicability. Based on previous studies of the phiSASD1 genome, this phage has a genome size of approximately 37 kb, containing 43 predicted ORFs, and some gene modules share similarities in structure and organization with the Streptomyces phage φC31 (Wang et al., 2010 ). This feature suggests that phiSASD1 possesses a typical regulatory element organization pattern of actinobacteriophages and provides a theoretical basis for mining native promoters recognizable by the host from its genome. One of the important findings of this study is that SD13 shows significantly higher activity than the widely used constitutive promoter PermE* in S. lividans , reaching 9.79-fold under the tested conditions. Although PermE* has long been considered a benchmark promoter in Streptomyces , its performance has been reported to vary depending on host background and culture conditions (Zhao et al., 2024 ). In different strains, promoters such as kasOp, hrdBp, SCO5768p, SP44, and stnYp can exhibit superior performance under specific conditions (Wang et al., 2013 ; Guo et al., 2023 ). The significant superiority of SD13 over PermE* observed in this study suggests that phage-derived regulatory elements may serve as valuable supplementary resources for Streptomyces expression systems. Bacteriophages must rapidly hijack host transcriptional machinery during infection, and their genomes are therefore enriched with regulatory elements that are efficiently recognized by host RNA polymerases. Previous studies on Streptomyces phages such as φC31 have demonstrated that phage transcription largely relies on host transcriptional systems (Howe and Smith, 1996 ). More recent comparative genomics analyses further indicate that Streptomyces phages exhibit high genetic diversity and are closely associated with host metabolism and development (Kronheim et al., 2023 ). The high activity of SD13 further confirms that phage genomes are not only important sources of structural and lysis-related proteins but also valuable reservoirs of high-performance regulatory elements. Another important observation is the discrepancy between bioinformatic predictions and experimental results. Some sequences with high prediction scores showed no detectable activity, whereas others with relatively low scores exhibited strong transcriptional output. This finding highlights the limitations of current promoter prediction algorithms, particularly in high-GC organisms such as Streptomyces and in phage genomes, where sequence features may deviate from canonical models. (Lozano et al., 2021; Su et al., 2025 ). It also emphasizes the necessity of experimental validation when identifying functional regulatory elements. The cross-host functionality of SD13 is likely associated with its sequence features. Structural analysis revealed that SD13 shares high similarity with the σ 70 consensus recognition motifs of E. coli , which may facilitate its recognition by RNA polymerases from different bacterial species. This observation is consistent with previous reports indicating that promoters with conserved core elements tend to exhibit broader host compatibility (Zuo et al., 2025 ). At the same time, the differential performance of certain promoters across hosts observed in this study underscores the importance of host-specific transcriptional context in determining promoter strength (Horbal et al., 2013 ). From a structural perspective, SD13 exhibits a typical prokaryotic promoter architecture, including well-defined − 35 and − 10 regions. Functional analyses demonstrated that both regions are essential for promoter activity, with mutations in the − 35 region causing a more pronounced reduction in transcriptional strength. This is consistent with the established role of the − 35 region in RNA polymerase recognition, whereas the − 10 region is primarily involved in DNA melting and transcription initiation. Truncation analysis further defined the core functional region of SD13 as spanning from − 70 to + 9 bp relative to the transcription start site. Interestingly, the truncated variant SD13-79 exhibited even higher activity than the full-length promoter, suggesting that upstream sequences may contain regulatory elements that negatively influence transcription. Similar observations have been reported in other systems, where promoter-proximal sequences, spacer composition, and DNA structural features can modulate transcriptional efficiency (Klein et al., 2021 ). These findings provide a basis for further rational engineering of SD13 to enhance its performance. In terms of practical applications, SD13 was shown to effectively drive the soluble expression of phage endolysin in E. coli . Compared with commonly used high-expression systems such as T7, which often lead to protein aggregation and inclusion body formation, SD13 appears to provide a more balanced expression level that favors proper protein folding. Previous studies have shown that excessively strong expression often increases metabolic burden on the host and affects protein folding, thereby reducing the proportion of soluble products (Valdez-Cruz et al., 2021 ). This property may be particularly advantageous for the production of proteins that are difficult to express or prone to misfolding. Therefore, SD13 has potential applications not only in Streptomyces metabolic engineering but also in heterologous protein production platforms. Despite these promising results, several limitations should be noted. First, the interaction between SD13 and RNA polymerase has not been directly characterized at the biochemical level. Second, the performance of SD13 has not yet been validated in a broader range of industrially relevant Streptomyces strains or under fermentation conditions. In addition, its stability and performance in multi-gene expression systems remain to be investigated. Future studies addressing these aspects will further clarify its applicability in industrial biotechnology. In conclusion, this study demonstrates that Streptomyces phage genomes are valuable sources of functional regulatory elements. The promoter SD13 exhibits strong transcriptional activity, cross-host compatibility, and favorable protein expression characteristics, making it a promising tool for applications in promoter engineering, metabolic pathway optimization, and synthetic biology. Declarations Ethical approval Not applicable. Competing interests The authors declare that they have no competing interests. Funding This study was funded by the Yancheng Applied Basic Research Plan General Program (grant No. YCBK2025079). Author Contribution NL conceived and designed the research, performed the experiments, analyzed the data, and wrote the manuscript. The author read and approved the final manuscript. Acknowledgement This work was sponsored by the Yancheng Applied Basic Research Plan General Program (grant No.YCBK2025079). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability The authors declared that the article and its supplementary file provide the data that supports the study's conclusions. References Baltz RH (2012) Streptomyces temperate bacteriophage integration systems for stable genetic engineering of actinomycetes (and other organisms). J Ind Microbiol Biotechnol 39:661-672. Browning DF, Busby SJW (2004) The regulation of bacterial transcription initiation. Nat Rev Microbiol 2:57-65. 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Valdez-Cruz NA, Caspeta L, Pérez NO, Ramírez OT, Trujillo-Roldán MA (2021) Challenges associated with the formation of recombinant protein inclusion bodies in Escherichia coli and strategies to address them. Front Bioeng Biotechnol 9:630551. Wang S, Qiao X, Liu X, Zhang X, Wang C, Zhao X, Chen Z, Wen Y, Song Y (2010) Complete genomic sequence analysis of the temperate bacteriophage phiSASD1 of Streptomyces avermitilis . Virology 403:78-84. Wang W, Li X, Wang J, Xiang S, Feng X, Yang K (2013) An engineered strong promoter for streptomycetes . Appl Environ Microbiol 79:4484-4492. Zang Z, Zhang C, Park KJ, Schwartz DA, Podicheti R, Lennon JT, Gerdt JP (2025) Streptomyces secretes a siderophore that sensitizes competitor bacteria to phage infection. Nat Microbiol 10(2):362-373. Zhao M, Yang Z, Li X, Zhang Y, Li Y, Wang J (2024) Development of integrated vectors with strong constitutive promoters for high-yield antibiotic production in mangrove-derived Streptomyces . Mar Drugs 22:94. Zuo W, Yin G, Zhang L, Zhang W, Xu R, Wang Y, Li J, Kang Z (2025) Engineering artificial cross-species promoters with different transcriptional strengths. Synth Syst Biotechnol 10:49-57. Additional Declarations No competing interests reported. Supplementary Files Surportingmaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9242482","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":636018919,"identity":"08c768a8-d3d5-4d98-b348-13336a279d9e","order_by":0,"name":"Nana Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYBACPhDxAcqRIEoLGxAzzoCqJl4LMw9pWiRyjz22bbOrMzjAfPA2D4NdHhFa8tKNc9uSJQwOsCVb8zAkFxOhJcdMOrftAFALj5k0D8OBxAaitFiCtfB/I0ELI8QWNiK18Lwxk+w5lyw58zCbseUcg2TCWvjZc8wkfpTZ8fMdb354402FHWEtYMAIjh0Qy4Ao9SDwh2iVo2AUjIJRMBIBACx3L1jvqovNAAAAAElFTkSuQmCC","orcid":"","institution":"Yancheng Teachers University","correspondingAuthor":true,"prefix":"","firstName":"Nana","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2026-03-27 08:54:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9242482/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9242482/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108784878,"identity":"a755c78f-9289-4911-9e92-d4aefe439480","added_by":"auto","created_at":"2026-05-08 11:00:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63735,"visible":true,"origin":"","legend":"\u003cp\u003eExpression activity of putative promoter fragments of bacteriophage phiSASD1 in \u003cem\u003eS. lividans \u003c/em\u003eTK54. (a) Expression activities of SD1, SD3, SD5, SD8, and SD10 in\u003cem\u003eS. lividans\u003c/em\u003e TK54; (b) Expression activities of SD11, SD12, SD13, SD15, and SD17 in \u003cem\u003eS. lividans\u003c/em\u003e TK54; (c) Expression activities of SD18, SD19, SD21, SD38, and SD40 in \u003cem\u003eS. lividans\u003c/em\u003e TK54. Error bars represent the standard deviation of the mean from three independent experiments. Different letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), while groups sharing the same letter show no significant difference\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9242482/v1/6aed1ffbf670c6f6550d9ef6.jpg"},{"id":108784881,"identity":"ec847602-2145-4e5b-9c59-1c7562c4e21a","added_by":"auto","created_at":"2026-05-08 11:00:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43377,"visible":true,"origin":"","legend":"\u003cp\u003eExpression activity of bacteriophage phiSASD1 promoter fragments in \u003cem\u003eE. coli\u003c/em\u003e JM109\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9242482/v1/4f6d930c395603e5351630f8.jpg"},{"id":108784879,"identity":"77baf4e5-d7bc-4533-95d8-dc8eca26a493","added_by":"auto","created_at":"2026-05-08 11:00:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":70148,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the transcription start site of the SD13 promoter by 5′ RACE. The bent arrow in the figure indicates the experimentally identified transcription start site\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9242482/v1/2cba6948e8d631d9e7e2fb37.jpg"},{"id":108807329,"identity":"08799777-dc03-41a7-a8c7-a5aacce9cb21","added_by":"auto","created_at":"2026-05-08 15:30:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81094,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of mutations in the −10 and −35 regions of the SD13 promoter (a) and the expression activities of the corresponding mutant recombinant plasmids in \u003cem\u003eS. lividans\u003c/em\u003e TK54 (b). In panel (a), red letters indicate that all bases in the −10 or −35 regions of each promoter were substituted with their complementary bases, while blank regions represent deletion mutations of the −10 or −35 regions\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9242482/v1/6d7b8f1ba7a490acdf7a0583.jpg"},{"id":108784882,"identity":"2257ea6a-74d8-4c34-a5eb-c320961963a3","added_by":"auto","created_at":"2026-05-08 11:00:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74290,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of 5′- and 3′-end deletion fragments of the SD13 promoter (a) and the expression activities of the corresponding recombinant plasmids in\u003cem\u003eS. lividans\u003c/em\u003e TK54 (b). In panel (a), blue boxes indicate promoter fragments of different lengths, and the orange arrow represents the \u003cem\u003exylE \u003c/em\u003egene. In panel (b), error bars represent the standard deviation of the mean from three independent experiments\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9242482/v1/4cbf79ea39decc06eec8b60c.jpg"},{"id":109067838,"identity":"aa3f80c9-44e7-45e7-afce-c5aaff60f82a","added_by":"auto","created_at":"2026-05-12 10:01:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":681324,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9242482/v1/092e521b-f662-4906-a4be-fef3d984a150.pdf"},{"id":108784877,"identity":"e6e74a30-0e30-409c-b6ec-1ccd32fcd022","added_by":"auto","created_at":"2026-05-08 11:00:40","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":701794,"visible":true,"origin":"","legend":"","description":"","filename":"Surportingmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9242482/v1/4c8f06f8cf24534a7a488e44.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A phage-derived promoter SD13 enables strong and cross-host gene expression in Streptomyces and Escherichia coli","fulltext":[{"header":"Key points","content":"\u003cp\u003e• Strong transcriptional promoters were systematically identified from phages.\u003c/p\u003e\u003cp\u003e• The novel promoter SD13 shows higher activity than PermE* and works across hosts.\u003c/p\u003e\u003cp\u003e• SD13 efficiently expresses soluble heterologous proteins, advancing synthetic biology.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eStreptomyces avermitilis\u003c/em\u003e is an important industrial microorganism responsible for the production of the widely used agricultural and medical antibiotics avermectins. Genetic engineering of this strain is critical for enhancing production yields and generating novel derivatives (Ikeda et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Hao et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although \u003cem\u003eStreptomyces\u003c/em\u003e genomes harbor numerous biosynthetic gene clusters (BGCs), many remain silent or are expressed at low levels under standard laboratory conditions, limiting their biosynthetic potential (Dong et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, the development of efficient gene expression regulatory elements, particularly strong promoters, is essential for activating silent gene clusters and improving the production of target metabolites. With the rapid development of synthetic biology and metabolic engineering, the construction of efficient and controllable gene expression systems has become a key strategy for optimizing metabolic pathways in \u003cem\u003eStreptomyces\u003c/em\u003e (Baltz et al. 2012). Promoters, as central cis-regulatory elements, directly determine transcriptional output and thereby influence the expression levels of downstream genes (Browning and Busby \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Previous studies have demonstrated that the identification or engineering of strong promoters can significantly enhance the biosynthesis of target compounds. For example, the constitutive promoter stnYp has been shown to markedly improve tylosin production (Guo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, advances in understanding transcriptional regulation in \u003cem\u003eStreptomyces\u003c/em\u003e, including the roles of SARP family regulators and global regulators such as AfsR, have provided valuable insights into promoter\u0026ndash;regulator interactions and facilitated rational promoter design (Shi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBacteriophages have evolved highly efficient transcriptional regulatory systems through long-term co-evolution with their hosts. Their genomes are enriched with cis-regulatory elements that can be readily recognized by host RNA polymerases, making them promising sources of novel promoters (Kronheim et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Recent studies have further shown that \u003cem\u003eStreptomyces\u003c/em\u003e can modulate phage infection through mechanisms such as siderophore secretion, thereby gaining a competitive advantage in microbial communities and revealing the complexity of phage\u0026ndash;host regulatory networks (Zang et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Previous studies have shown that promoters derived from \u003cem\u003eStreptomyces\u003c/em\u003e phages, such as φC31 and φBT1, possess strong transcriptional activity and have been successfully applied in gene expression systems (Howe et al. 1996; Gregory et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the systematic exploration of phage-derived promoters in \u003cem\u003eStreptomyces\u003c/em\u003e remains limited, particularly for those adapted to specific industrial hosts such as \u003cem\u003eStreptomyces avermitilis\u003c/em\u003e, or those capable of driving efficient cross-host expression (Lu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kronheim et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, differences in RNA polymerase recognition among hosts result in pronounced host dependency of promoter activity, and broadly active strong promoters that maintain high transcriptional activity across different hosts are still scarce.\u003c/p\u003e\u003cp\u003eThe bacteriophage phiSASD1 is a lytic phage infecting \u003cem\u003eStreptomyces avermitilis\u003c/em\u003e, and its genome has been sequenced and annotated (Wang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Given that bacteriophages must rapidly hijack the host transcriptional machinery during infection, their genomes are likely to contain native promoters with high transcriptional activity. However, promoters derived from phiSASD1 have not been systematically characterized, which limits their potential application in synthetic biology.\u003c/p\u003e\u003cp\u003eReporter gene-based screening systems provide a reliable approach for evaluating promoter activity. By constructing promoter\u0026ndash;reporter fusions and quantitatively measuring reporter output in different hosts, promoter strength and host compatibility can be systematically assessed (Horbal et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Matsuzaki et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In addition, bioinformatic prediction of core promoter elements, such as the \u0026minus;\u0026thinsp;10 and \u0026minus;\u0026thinsp;35 regions, facilitates the analysis of promoter structure\u0026ndash;function relationships (Cheah et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Nijo et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we systematically screened potential promoter sequences from the phiSASD1 genome by combining bioinformatic prediction with experimental validation. The transcriptional activities of candidate promoters were evaluated in both \u003cem\u003eStreptomyces\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e, and the structural and functional characteristics of the most active promoter were further analyzed. This work aims to identify strong promoters with cross-host applicability and to provide useful genetic tools for \u003cem\u003eStreptomyces\u003c/em\u003e metabolic engineering and heterologous gene expression.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains, bacteriophage, and plasmids\u003c/h2\u003e \u003cp\u003e \u003cem\u003eStreptomyces avermitilis\u003c/em\u003e, used as the host strain for bacteriophage phiSASD1, was maintained in our laboratory. Bacteriophage phiSASD1 is a lytic \u003cem\u003eStreptomyces\u003c/em\u003e phage previously isolated and its genome has been sequenced and annotated (GenBank accession number: GQ379227). \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α was used for plasmid construction and propagation, whereas \u003cem\u003eE. coli\u003c/em\u003e JM109 was used for promoter activity assays and heterologous protein expression. \u003cem\u003eStreptomyces lividans\u003c/em\u003e TK54 was employed as the \u003cem\u003eStreptomyces\u003c/em\u003e expression host.\u003c/p\u003e \u003cp\u003eFor promoter characterization, the \u003cem\u003eE. coli\u003c/em\u003e\u0026ndash;\u003cem\u003eStreptomyces\u003c/em\u003e shuttle vector pSET152 and the reporter plasmid pIJ4083 carrying the \u003cem\u003exylE\u003c/em\u003e gene were used to construct the promoter probe vector pSX152. The \u003cem\u003exylE\u003c/em\u003e gene encodes catechol 2,3-dioxygenase (C23O), whose activity can be quantified using a catechol-based colorimetric assay. The constitutive promoter PermE*, derived from \u003cem\u003eSaccharopolyspora erythraea\u003c/em\u003e, was used as a positive control, while the empty vector pSX152 served as a negative control.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCulture conditions\u003c/h3\u003e\n \u003cp\u003e \u003cem\u003ecoli\u003c/em\u003e strains were cultured in LB liquid medium supplemented with appropriate antibiotics at 37\u0026deg;C with shaking at 200 rpm. \u003cem\u003eS. avermitilis\u003c/em\u003e and \u003cem\u003eS. lividans\u003c/em\u003e TK54 were cultured in YMS medium. For liquid cultivation, strains were incubated at 28\u0026deg;C with shaking at 200 rpm, while solid cultures were grown on agar plates containing 2% (w/v) agar. Bacteriophage phiSASD1 was propagated using the double-layer agar method, and high-titer phage lysates were prepared under optimal host growth conditions.\u003c/p\u003e \n\u003ch3\u003eExtraction of bacteriophage genomic DNA\u003c/h3\u003e\n \u003cp\u003eHigh-titer phage lysates were first treated with DNase I and RNase A to remove residual host nucleic acids. Subsequently, SDS and proteinase K were added to disrupt the phage capsid and release genomic DNA. Proteins were removed by phenol/chloroform extraction, followed by ethanol precipitation to recover the DNA. The integrity of the extracted DNA was verified by 1% agarose gel electrophoresis, and DNA concentration and purity were determined using a NanoDrop spectrophotometer.\u003c/p\u003e \n\u003ch3\u003eBioinformatic prediction of candidate promoter sequences\u003c/h3\u003e\n \u003cp\u003ePotential promoter regions were predicted based on the complete genome sequence of bacteriophage phiSASD1 using BDGP, BPROM, and NNPP software. The minimum promoter score threshold in BDGP was set to 0.8 to enhance prediction reliability. Candidate promoter regions were selected from intergenic sequences upstream of annotated open reading frames (ORFs) based on GenBank annotation. A total of 15 putative promoter fragments were selected for subsequent experimental validation.\u003c/p\u003e \n\u003ch3\u003eConstruction of promoter probe plasmids\u003c/h3\u003e\n \u003cp\u003eCandidate promoter fragments were amplified from phiSASD1 genomic DNA using specific primers and a high-fidelity DNA polymerase. PCR products were verified by agarose gel electrophoresis and purified prior to cloning. The purified fragments were digested with appropriate restriction enzymes and ligated into the promoterless \u003cem\u003exylE\u003c/em\u003e reporter vector pSX152 to generate promoter\u0026ndash;reporter fusion constructs. The ligation mixtures were transformed into \u003cem\u003eE. coli\u003c/em\u003e JM109 competent cells, and positive clones were selected on antibiotic-containing media. The integrity and correctness of the inserted sequences were confirmed by colony PCR and DNA sequencing.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of recombinant strains\u003c/h2\u003e \u003cp\u003eVerified recombinant plasmids were introduced into \u003cem\u003eS. lividans\u003c/em\u003e TK54 via transformation and subsequently integrated into the host chromosome. Transformants were selected on media containing appropriate antibiotics. In parallel, recombinant plasmids carrying different promoter fragments were also transformed into \u003cem\u003eE. coli\u003c/em\u003e JM109 to evaluate promoter activities across different hosts.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePromoter activity assay\u003c/h3\u003e\n \u003cp\u003eCells in the exponential growth phase were harvested by centrifugation and resuspended in an appropriate buffer. Catechol was added as the substrate, and enzymatic reactions were carried out at room temperature. The absorbance was measured at 375 nm (OD375). C23O activity was calculated using the following formula: mU (nmol\u0026middot;min⁻\u0026sup1;)\u0026thinsp;=\u0026thinsp;30.03\u0026thinsp;\u0026times;\u0026thinsp;ΔA375 / time. One unit (mU) was defined as the amount of enzyme required to produce 1 nmol of 2-hydroxymuconic semialdehyde per minute at 30\u0026deg;C. Promoter activity was normalized to cell dry weight to ensure comparability across samples and expressed as mU/mg. The PermE* promoter was used as a positive control, and the empty vector pSX152 was used as a negative control. All experiments were performed with at least three independent biological replicates.\u003c/p\u003e \n\u003ch3\u003eSequence feature analysis of promoters\u003c/h3\u003e\n \u003cp\u003ePromoter sequences exhibiting activity were subjected to sequence alignment analysis to identify conserved elements, including the \u0026minus;\u0026thinsp;10 and \u0026minus;\u0026thinsp;35 regions and spacer length. Potential transcription factor binding sites (TFBS) were predicted using the PRODORIC database and Softberry BPROM software. Sequence similarity to σ\u003csup\u003e70\u003c/sup\u003e-type promoter consensus motifs was analyzed to infer potential transcriptional regulatory mechanisms.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of transcription start site (TSS)\u003c/h2\u003e \u003cp\u003eThe transcription start site of the SD13 promoter was determined using 5\u0026prime; RACE (Rapid Amplification of cDNA Ends). Total RNA was extracted from \u003cem\u003eS. avermitilis\u003c/em\u003e at different time points following phage infection and reverse-transcribed into cDNA. Nested PCR was performed using ORF13-specific primers to amplify the transcription start region. PCR products were sequenced, and the TSS was identified based on the anchor sequence and the first nucleotide immediately downstream of the poly(A) tail.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData processing and statistical analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). Statistical differences among groups were evaluated using one-way analysis of variance (ANOVA), and differences were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All statistical analyses and data visualization were performed using GraphPad Prism software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of putative promoters in phage phiSASD1\u003c/h2\u003e \u003cp\u003eBased on the complete genome sequence of the \u003cem\u003eS. avermitilis\u003c/em\u003e bacteriophage phiSASD1, a total of 43 open reading frames (ORFs) were predicted. Intergenic regions between adjacent ORFs were screened for potential promoter sequences and further analyzed using the BDGP promoter prediction program. With the minimum promoter score threshold set at 0.8, nine high-scoring candidate promoter regions were identified, with prediction scores ranging from 0.82 to 0.97 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Based on ORF annotation from the NCBI GenBank database, upstream intergenic sequences were further selected as candidate promoter fragments. In total, 15 putative promoter regions associated with ORF1, ORF3, ORF5, ORF8, ORF10, ORF11, ORF12, ORF13, ORF15, ORF17, ORF18, ORF19, ORF21, ORF38, and ORF40 were obtained for experimental validation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These candidate fragments ranged from 92 to 703 bp in length and were associated with genes encoding proteins such as Cas4 exonuclease, DNA polymerase I, RNA polymerase subunit alpha, holin, and endolysin, suggesting potential roles in phage replication and lysis processes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHigh-scoring promoter regions predicted in the phiSASD1 genome\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStart (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStop (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePromoter sequence\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGTTGTGGAACTACCCGCGTGCTCCCGCT\u003c/p\u003e \u003cp\u003eGCATCATCCTCG\u003cb\u003eC\u003c/b\u003eCGGCCGCGA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGCCAGTTGGATGGTGCAGGTCTACAACG\u003c/p\u003e \u003cp\u003eGTACCGTTATAT\u003cb\u003eT\u003c/b\u003eCCGCTACAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18885\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18930\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTATTTTTGTTTCGTCCCAGTAATAGAAAA\u003c/p\u003e \u003cp\u003eGATAGTAACGC\u003cb\u003eT\u003c/b\u003eCCTCGCCTT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCAGTTGGCCGCCTACCGGTACGCGGACG\u003c/p\u003e \u003cp\u003eTGATCATTGACC\u003cb\u003eC\u003c/b\u003eGGAGGGCAA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCTGATGGAAAACCAGGACTTCCCTCAGG\u003c/p\u003e \u003cp\u003eACATCTTTCCGC\u003cb\u003eT\u003c/b\u003eGAACTACAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCTCACTTGACCCGACTCAATTGAGTCGC\u003c/p\u003e \u003cp\u003eCTACTATGACAA\u003cb\u003eC\u003c/b\u003eGCGTCCACA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eACGGCTTGACTCGACTCAGTTGAGTCGG\u003c/p\u003e \u003cp\u003eCTACTTTCGGCA\u003cb\u003eT\u003c/b\u003eTCCAGCCGC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32442\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32487\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eACCCTTGACCTGTTCATCGTGGCCGGCG\u003c/p\u003e \u003cp\u003eAGATCATGATGC\u003cb\u003eT\u003c/b\u003eGGTGGCCGC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e35119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGCGGCTTGACTCGACTCAATTGAGCCGG\u003c/p\u003e \u003cp\u003eCTAAATTGGAGC\u003cb\u003eC\u003c/b\u003eCTCGCCAGG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea\u003c/sup\u003eBold letters indicate the transcription start site (TSS)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLengths of putative promoter fragments and predicted proteins encoded by the corresponding ORFs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePotential promoter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePutative product\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCas4 family exonuclease\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePAPS _reductase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eprimase/helicase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e159\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDNA polymerase I\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDNA-directed RNA polymerase subunit alpha\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHNH endonuclease\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e176\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e687\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eholin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTerminase small subunit\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORF40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eendolysin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003ea\u003c/sup\u003e \u0026ldquo;\u0026ndash;\u0026rdquo; indicates that no homologous function was identified\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePromoter activity of candidate fragments in\u003c/b\u003e \u003cb\u003eS. lividans\u003c/b\u003e \u003cb\u003eTK54\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo experimentally evaluate promoter activity, the 15 candidate fragments were individually cloned into the promoter probe vector pSX152 and integrated into the chromosome of \u003cem\u003eS. lividans\u003c/em\u003e TK54. Promoter strength was assessed by measuring the activity of the \u003cem\u003exylE\u003c/em\u003e reporter gene. The constitutive promoter PermE* was used as a positive control, while the empty vector served as a negative control. Among the 15 candidates, seven promoters (SD10, SD12, SD13, SD15, SD17, SD18, and SD40) exhibited detectable transcriptional activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, SD13 showed the highest activity, reaching 6.72-, 9.79-, and 5.06-fold that of PermE* at 12 h, 36 h, and 60 h, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). SD18 also exhibited strong activity, approximately 3.38-fold higher than PermE* (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), while SD15 displayed a comparable level of activity to PermE* (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Time-course analysis revealed that all active promoters reached peak C23O activity at 36 h, followed by a decline at 60 h. SD40 maintained relatively high activity at 60 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Interestingly, some fragments with high prediction scores (e.g., SD1 and SD38) showed no detectable promoter activity, whereas SD15, despite a relatively low prediction score, exhibited substantial transcriptional strength (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression activity of promoter fragments in\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eJM109\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess host compatibility, the seven active promoter fragments identified in \u003cem\u003eS. lividans\u003c/em\u003e were further evaluated in \u003cem\u003eE. coli\u003c/em\u003e JM109 using the same reporter system. The results showed that all seven promoters retained transcriptional activity in \u003cem\u003eE. coli\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), whereas the positive control promoter PermE* exhibited no detectable activity under the same conditions. Notably, SD13 maintained relatively high expression levels, indicating strong cross-host functionality. In addition, several promoters (e.g., SD10, SD12, and SD17) that showed relatively low activity in \u003cem\u003eS. lividans\u003c/em\u003e exhibited enhanced activity in \u003cem\u003eE. coli\u003c/em\u003e, highlighting the influence of host-specific transcriptional machinery on promoter performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eExpression of endolysin driven by the SD13 promoter\u003c/h2\u003e \u003cp\u003eTo evaluate the protein expression capability of the SD13 promoter, a recombinant plasmid (pIJ13-lysin) was constructed and expressed in \u003cem\u003eE. coli\u003c/em\u003e JM109. Cells harboring the empty vector pIJ13 were used as a negative control. SDS-PAGE analysis revealed a distinct protein band at approximately 31 kDa in the recombinant strain, consistent with the predicted molecular weight of endolysin, whereas no corresponding band was observed in the control ( (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The target protein was predominantly detected in the soluble fraction, indicating efficient soluble expression. These results demonstrate that SD13 is capable of driving effective heterologous protein expression while reducing the formation of inclusion bodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStructural characterization of the SD13 promoter\u003c/h2\u003e \u003cp\u003eTo investigate the structural features of SD13, transcription factor binding sites (TFBS) were analyzed using the PRODORIC database. The results indicated that SD13 shares high similarity with the consensus recognition sequence of the σ\u003csup\u003e70\u003c/sup\u003e factor in \u003cem\u003eE. coli\u003c/em\u003e. Further analysis using Softberry BPROM revealed that SD13 contains typical prokaryotic promoter elements, including well-defined\u0026thinsp;\u0026minus;\u0026thinsp;35 and \u0026minus;\u0026thinsp;10 regions (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, a G/C-rich spacer sequence of approximately 18 bp was identified between these two regions. These structural features suggest that SD13 is a σ\u003csup\u003e70\u003c/sup\u003e-type promoter with canonical architecture.\u003c/p\u003e \u003cp\u003eTable 3 Structural analysis of the SD13 promoter fragment\u003c/p\u003e\u003cp\u003e\u003cimg 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\" width=\"724\" height=\"90\"\u003e\u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eThe sequences within the boxes represent the predicted\u0026thinsp;\u0026minus;\u0026thinsp;10 and \u0026minus;\u0026thinsp;35 regions of the promoter, The bold and underlined base indicates the transcription start site, and the bold ATG denotes the start codon\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the Transcription Start Site of SD13\u003c/h2\u003e \u003cp\u003eThe transcription start site (TSS) of SD13 was determined using 5\u0026prime; RACE. The results showed that transcripts of ORF13 could be detected as early as 20 min post-infection. Sequencing of nested PCR products revealed that the TSS is located at a guanine (G) residue positioned 107 bp upstream of the start codon (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), differing by only 1 bp from the bioinformatics prediction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Conserved Regions on SD13 Promoter Activity\u003c/h2\u003e \u003cp\u003eTo assess the functional importance of the conserved\u0026thinsp;\u0026minus;\u0026thinsp;10 and \u0026minus;\u0026thinsp;35 regions, site-directed mutagenesis and deletion analyses were performed. Mutation of all bases in the \u0026minus;\u0026thinsp;10 region resulted in an approximately 3.85-fold reduction in promoter activity compared with the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In contrast, mutation of the \u0026minus;\u0026thinsp;35 region caused a much more pronounced decrease, approximately 31.34-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Deletion of the \u0026minus;\u0026thinsp;10 region led to an approximately 37.67-fold reduction, whereas deletion of the \u0026minus;\u0026thinsp;35 region resulted in an approximately 11-fold decrease in activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). These results indicate that both regions are essential for SD13 function, with the \u0026minus;\u0026thinsp;35 region playing a particularly critical role in transcriptional strength.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of the Core functional Region of SD13\u003c/h2\u003e \u003cp\u003eTo define the minimal functional region of SD13, progressive truncation analyses were performed. The 5\u0026prime;-truncated fragment SD13-218 (\u0026minus;\u0026thinsp;100 to +\u0026thinsp;118) exhibited a slight increase in activity (1.13-fold of the wild type at 36 h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). However, further truncation to SD13-167 (\u0026minus;\u0026thinsp;49 to +\u0026thinsp;118) resulted in a marked reduction in promoter activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Subsequent 3\u0026prime; truncations generated SD13-110 (\u0026minus;\u0026thinsp;49 to +\u0026thinsp;61) and SD13-58 (\u0026minus;\u0026thinsp;49 to +\u0026thinsp;9) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), both of which showed gradual recovery of activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Notably, the fragment SD13-79 (\u0026minus;\u0026thinsp;70 to +\u0026thinsp;9) exhibited the highest activity, reaching approximately 1.7-fold that of the full-length promoter at 36 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). These results indicate that the core functional region of SD13 is located between \u0026minus;\u0026thinsp;70 and +\u0026thinsp;9 bp, and that upstream sequences may contain elements that negatively influence transcription.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study systematically screened and identified a series of transcriptionally active promoters from the genome of the \u003cem\u003eStreptomyces\u003c/em\u003e phage phiSASD1, among which SD13 exhibited the strongest expression capability and good cross-host applicability. Based on previous studies of the phiSASD1 genome, this phage has a genome size of approximately 37 kb, containing 43 predicted ORFs, and some gene modules share similarities in structure and organization with the \u003cem\u003eStreptomyces\u003c/em\u003e phage φC31 (Wang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This feature suggests that phiSASD1 possesses a typical regulatory element organization pattern of actinobacteriophages and provides a theoretical basis for mining native promoters recognizable by the host from its genome.\u003c/p\u003e \u003cp\u003eOne of the important findings of this study is that SD13 shows significantly higher activity than the widely used constitutive promoter PermE* in \u003cem\u003eS. lividans\u003c/em\u003e, reaching 9.79-fold under the tested conditions. Although PermE* has long been considered a benchmark promoter in \u003cem\u003eStreptomyces\u003c/em\u003e, its performance has been reported to vary depending on host background and culture conditions (Zhao et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In different strains, promoters such as kasOp, hrdBp, SCO5768p, SP44, and stnYp can exhibit superior performance under specific conditions (Wang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The significant superiority of SD13 over PermE* observed in this study suggests that phage-derived regulatory elements may serve as valuable supplementary resources for \u003cem\u003eStreptomyces\u003c/em\u003e expression systems.\u003c/p\u003e \u003cp\u003eBacteriophages must rapidly hijack host transcriptional machinery during infection, and their genomes are therefore enriched with regulatory elements that are efficiently recognized by host RNA polymerases. Previous studies on \u003cem\u003eStreptomyces\u003c/em\u003e phages such as φC31 have demonstrated that phage transcription largely relies on host transcriptional systems (Howe and Smith, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). More recent comparative genomics analyses further indicate that \u003cem\u003eStreptomyces\u003c/em\u003e phages exhibit high genetic diversity and are closely associated with host metabolism and development (Kronheim et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The high activity of SD13 further confirms that phage genomes are not only important sources of structural and lysis-related proteins but also valuable reservoirs of high-performance regulatory elements.\u003c/p\u003e \u003cp\u003eAnother important observation is the discrepancy between bioinformatic predictions and experimental results. Some sequences with high prediction scores showed no detectable activity, whereas others with relatively low scores exhibited strong transcriptional output. This finding highlights the limitations of current promoter prediction algorithms, particularly in high-GC organisms such as \u003cem\u003eStreptomyces\u003c/em\u003e and in phage genomes, where sequence features may deviate from canonical models. (Lozano et al., 2021; Su et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). It also emphasizes the necessity of experimental validation when identifying functional regulatory elements.\u003c/p\u003e \u003cp\u003eThe cross-host functionality of SD13 is likely associated with its sequence features. Structural analysis revealed that SD13 shares high similarity with the σ\u003csup\u003e70\u003c/sup\u003e consensus recognition motifs of \u003cem\u003eE. coli\u003c/em\u003e, which may facilitate its recognition by RNA polymerases from different bacterial species. This observation is consistent with previous reports indicating that promoters with conserved core elements tend to exhibit broader host compatibility (Zuo et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). At the same time, the differential performance of certain promoters across hosts observed in this study underscores the importance of host-specific transcriptional context in determining promoter strength (Horbal et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFrom a structural perspective, SD13 exhibits a typical prokaryotic promoter architecture, including well-defined\u0026thinsp;\u0026minus;\u0026thinsp;35 and \u0026minus;\u0026thinsp;10 regions. Functional analyses demonstrated that both regions are essential for promoter activity, with mutations in the \u0026minus;\u0026thinsp;35 region causing a more pronounced reduction in transcriptional strength. This is consistent with the established role of the \u0026minus;\u0026thinsp;35 region in RNA polymerase recognition, whereas the \u0026minus;\u0026thinsp;10 region is primarily involved in DNA melting and transcription initiation.\u003c/p\u003e \u003cp\u003eTruncation analysis further defined the core functional region of SD13 as spanning from \u0026minus;\u0026thinsp;70 to +\u0026thinsp;9 bp relative to the transcription start site. Interestingly, the truncated variant SD13-79 exhibited even higher activity than the full-length promoter, suggesting that upstream sequences may contain regulatory elements that negatively influence transcription. Similar observations have been reported in other systems, where promoter-proximal sequences, spacer composition, and DNA structural features can modulate transcriptional efficiency (Klein et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These findings provide a basis for further rational engineering of SD13 to enhance its performance.\u003c/p\u003e \u003cp\u003eIn terms of practical applications, SD13 was shown to effectively drive the soluble expression of phage endolysin in \u003cem\u003eE. coli\u003c/em\u003e. Compared with commonly used high-expression systems such as T7, which often lead to protein aggregation and inclusion body formation, SD13 appears to provide a more balanced expression level that favors proper protein folding. Previous studies have shown that excessively strong expression often increases metabolic burden on the host and affects protein folding, thereby reducing the proportion of soluble products (Valdez-Cruz et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This property may be particularly advantageous for the production of proteins that are difficult to express or prone to misfolding. Therefore, SD13 has potential applications not only in \u003cem\u003eStreptomyces\u003c/em\u003e metabolic engineering but also in heterologous protein production platforms.\u003c/p\u003e \u003cp\u003eDespite these promising results, several limitations should be noted. First, the interaction between SD13 and RNA polymerase has not been directly characterized at the biochemical level. Second, the performance of SD13 has not yet been validated in a broader range of industrially relevant \u003cem\u003eStreptomyces\u003c/em\u003e strains or under fermentation conditions. In addition, its stability and performance in multi-gene expression systems remain to be investigated. Future studies addressing these aspects will further clarify its applicability in industrial biotechnology.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that \u003cem\u003eStreptomyces\u003c/em\u003e phage genomes are valuable sources of functional regulatory elements. The promoter SD13 exhibits strong transcriptional activity, cross-host compatibility, and favorable protein expression characteristics, making it a promising tool for applications in promoter engineering, metabolic pathway optimization, and synthetic biology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthical approval\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was funded by the Yancheng Applied Basic Research Plan General Program (grant No. YCBK2025079).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eNL conceived and designed the research, performed the experiments, analyzed the data, and wrote the manuscript. The author read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was sponsored by the Yancheng Applied Basic Research Plan General Program (grant No.YCBK2025079). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe authors declared that the article and its supplementary file provide the data that supports the study's conclusions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBaltz RH (2012) \u003cem\u003eStreptomyces\u003c/em\u003e temperate bacteriophage integration systems for stable genetic engineering of actinomycetes (and other organisms). J Ind Microbiol Biotechnol 39:661-672.\u003c/li\u003e\n\u003cli\u003eBrowning DF, Busby SJW (2004) The regulation of bacterial transcription initiation. Nat Rev Microbiol 2:57-65.\u003c/li\u003e\n\u003cli\u003eCheah HL, Citartan M, Lee LP, Ahmed SA, Salleh MZ, Teh LK, Tang TH (2024) Exploring transcription start sites and genomic features facilitates identification of small RNAs in \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e. Funct Integr Genomics 24:160.\u003c/li\u003e\n\u003cli\u003eDeal C, De Wannemaeker L, De Mey M (2024) Towards a rational approach to promoter engineering: understanding the complexity of transcription initiation in prokaryotes. FEMS Microbiol Rev 48(2):fuae004. \u003c/li\u003e\n\u003cli\u003eDong JX, Zhou Q, Luo YZ (2023) Recent advances and applications of \u003cem\u003eStreptomyces \u003c/em\u003epromoters. Microbiol China 50:3588-3605.\u003c/li\u003e\n\u003cli\u003eGregory MA, Till R, Smith MCM (2003) Integration site for\u003cem\u003e Streptomyces\u003c/em\u003e phage \u0026phi;BT1 and development of site-specific integrating vectors. J Bacteriol 185:5320-5323.\u003c/li\u003e\n\u003cli\u003eGuo W, Xiao Z, Huang T, Zhang K, Pan HX, Tang GL, Deng Z, Liang R, Lin S (2023) Identification and characterization of a strong constitutive promoter \u003cem\u003estnYp \u003c/em\u003efor activating biosynthetic genes and producing natural products in\u003cem\u003e streptomyces\u003c/em\u003e. Microb Cell Fact 22:127.\u003c/li\u003e\n\u003cli\u003eHao Y, You Y, Chen Z, Li J, Liu G, Wen Y (2022) Avermectin B1a production in \u003cem\u003eStreptomyces avermitilis\u003c/em\u003e is enhanced by engineering \u003cem\u003eaveC\u003c/em\u003e and precursor supply genes. Appl Microbiol Biotechnol 106: 2191-2205.\u003c/li\u003e\n\u003cli\u003eHorbal L, Kobylyanskyy A, Yushchuk O, Zaburannyi N, Luzhetskyy A, Ostash B, Marinelli F, Fedorenko V (2013) Evaluation of heterologous promoters for genetic analysis of Actinoplanes teichomyceticus\u0026mdash;producer of teicoplanin, drug of last defense. J Biotechnol 168:367-372.\u003c/li\u003e\n\u003cli\u003eHowe CW, Smith MCM (1996) Characterization of a late promoter from the \u003cem\u003eStreptomyces \u003c/em\u003etemperate phage \u0026phi;C31. J Bacteriol 178:2127-2130.\u003c/li\u003e\n\u003cli\u003eIkeda H, Ishikawa J, Hanamoto A, Shinose M, Kikuchi H, Shiba T, Sakaki Y, Hattori M, Omura S (2003) Complete genome sequence and comparative analysis of the industrial microorganism \u003cem\u003eStreptomyces avermitilis\u003c/em\u003e. Nat Biotechnol 21:526-531.\u003c/li\u003e\n\u003cli\u003eKlein CA, Teufel M, Weile CJ, Sobetzko P (2021) The bacterial promoter spacer modulates promoter strength and timing by length, TG-motifs and DNA supercoiling sensitivity. Sci Rep 11:24399.\u003c/li\u003e\n\u003cli\u003eKronheim S, Solomon E, Ho L, Davidson AR, Maxwell KL (2023) Complete genomes and comparative analyses of \u003cem\u003eStreptomyces\u003c/em\u003e phages that influence secondary metabolism and sporulation. Sci Rep 13:9820.\u003c/li\u003e\n\u003cli\u003eLi Y, Zhang J, Zheng J, Guan H, Liu W, Tan H (2020) Co-expression of a SARP Family Activator ChlF2 and a Type II Thioesterase ChlK Led to High Production of Chlorothricin in \u003cem\u003eStreptomyces\u003c/em\u003e antibioticus DSM 40725. Front Bioeng Biotechnol 8:1013.\u003c/li\u003e\n\u003cli\u003eLozano Terol G, Gallego-Jara J, Sola Mart\u0026iacute;nez RA, Mart\u0026iacute;nez Vivancos A, C\u0026aacute;novas D\u0026iacute;az M, de Diego Puente T (2021) Impact of the Expression System on Recombinant Protein Production in \u003cem\u003eEscherichia coli\u003c/em\u003e BL21. Front Microbiol 12:682001. \u003c/li\u003e\n\u003cli\u003eLu N, Kim C, Chen Z, Wen Y, Wei Q, Qiu Y, Wang S, Song Y (2019) Characterization and genome analysis of the temperate bacteriophage \u0026phi;SAJS1 from \u003cem\u003eStreptomyces avermitilis\u003c/em\u003e. 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Genome Biol 26:353.\u003c/li\u003e\n\u003cli\u003eValdez-Cruz NA, Caspeta L, P\u0026eacute;rez NO, Ram\u0026iacute;rez OT, Trujillo-Rold\u0026aacute;n MA (2021) Challenges associated with the formation of recombinant protein inclusion bodies in\u003cem\u003e Escherichia coli \u003c/em\u003eand strategies to address them. Front Bioeng Biotechnol 9:630551.\u003c/li\u003e\n\u003cli\u003eWang S, Qiao X, Liu X, Zhang X, Wang C, Zhao X, Chen Z, Wen Y, Song Y (2010) Complete genomic sequence analysis of the temperate bacteriophage phiSASD1 of \u003cem\u003eStreptomyces avermitilis\u003c/em\u003e. Virology 403:78-84.\u003c/li\u003e\n\u003cli\u003eWang W, Li X, Wang J, Xiang S, Feng X, Yang K (2013) An engineered strong promoter for \u003cem\u003estreptomycetes\u003c/em\u003e. Appl Environ Microbiol 79:4484-4492.\u003c/li\u003e\n\u003cli\u003eZang Z, Zhang C, Park KJ, Schwartz DA, Podicheti R, Lennon JT, Gerdt JP (2025) \u003cem\u003eStreptomyces\u003c/em\u003e secretes a siderophore that sensitizes competitor bacteria to phage infection. Nat Microbiol 10(2):362-373.\u003c/li\u003e\n\u003cli\u003eZhao M, Yang Z, Li X, Zhang Y, Li Y, Wang J (2024) Development of integrated vectors with strong constitutive promoters for high-yield antibiotic production in mangrove-derived \u003cem\u003eStreptomyces\u003c/em\u003e. Mar Drugs 22:94.\u003c/li\u003e\n\u003cli\u003eZuo W, Yin G, Zhang L, Zhang W, Xu R, Wang Y, Li J, Kang Z (2025) Engineering artificial cross-species promoters with different transcriptional strengths. Synth Syst Biotechnol 10:49-57.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Streptomyces bacteriophage, SD13 promoter, Cross-host expression, Strong promoter, Gene expression regulation","lastPublishedDoi":"10.21203/rs.3.rs-9242482/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9242482/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEfficient and tunable promoters are essential tools for metabolic engineering and synthetic biology in \u003cem\u003eStreptomyces\u003c/em\u003e. In this study, the lytic bacteriophage phiSASD1 was exploited as a source of novel regulatory elements. Fifteen candidate promoter fragments were identified through bioinformatic prediction and systematically evaluated using a catechol 2,3-dioxygenase (\u003cem\u003exylE\u003c/em\u003e) reporter system in \u003cem\u003eStreptomyces lividans\u003c/em\u003e TK54 and \u003cem\u003eEscherichia coli\u003c/em\u003e JM109. Among these candidates, seven fragments exhibited measurable promoter activity, with SD13 showing the highest transcriptional strength. In \u003cem\u003eS. lividans\u003c/em\u003e, SD13 displayed up to a 9.79-fold higher activity than the widely used strong promoter PermE*, while retaining robust activity in \u003cem\u003eE. coli\u003c/em\u003e, indicating excellent cross-host compatibility. Sequence analysis combined with 5\u0026prime; RACE revealed that SD13 possesses a typical σ⁷⁰-dependent promoter architecture, with its core functional region located between \u0026minus;\u0026thinsp;70 and +\u0026thinsp;9 bp relative to the transcription start site. Furthermore, SD13 efficiently drove the soluble expression of phage endolysin in \u003cem\u003eE. coli\u003c/em\u003e, alleviating inclusion body formation commonly observed in T7-based systems. Collectively, these results identify SD13 as a highly efficient phage-derived promoter with broad host applicability, providing a valuable genetic tool for \u003cem\u003eStreptomyces\u003c/em\u003e engineering and heterologous gene expression.\u003c/p\u003e","manuscriptTitle":"A phage-derived promoter SD13 enables strong and cross-host gene expression in Streptomyces and Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-08 11:00:31","doi":"10.21203/rs.3.rs-9242482/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"694c6bf1-5f01-40d0-9822-0aaf07386a56","owner":[],"postedDate":"May 8th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-07T08:21:25+00:00","index":26,"fulltext":""},{"type":"reviewerAgreed","content":"166838723468007501468225453466791754002","date":"2026-05-06T01:31:19+00:00","index":25,"fulltext":""},{"type":"reviewerAgreed","content":"18634906557034146053939653132058306010","date":"2026-05-05T10:10:22+00:00","index":24,"fulltext":""},{"type":"reviewersInvited","content":"9","date":"2026-04-30T04:55:16+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T11:00:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-08 11:00:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9242482","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9242482","identity":"rs-9242482","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}