The bacterial strains and plasmids utilized in this study are listed in Supplementary Table S1, and the corresponding primers used are listed in Supplementary Table S2. S. mobaraensis and Escherichia coli were cultured in ISP2 and LB, respectively. Phenotyping of S. mobaraensis mutants was conducted on ISP2 solid medium. Conventional TGase production utilizes an insoluble fermentation medium with the following basic fermentation formula: glycerol 2%, peptone 2%, soya flour 2%, yeast 0.5%, corn syrup 0.55%, K₂HPO₄ 0.4%, KH₂PO₄ 0.2%, MgSO₄ 0.2%, and (NH₄)₂SO₄ (Yin et al., 2021). The fermentation conditions involved maintaining the temperature at 30°C and agitating the mixture at 250 rpm. TSB soluble fermentation medium was selected for mycelia cultivation to accurately assess biomass in S. mobaraensis.

To generate a lexA deletion mutant, two fragments flanking lexA were amplified from the genomic DNA of S. mobaraensis NBRC 13819 strain. The 5′ flanking region, spanning 2,175 base pairs (positions −2,160 to +15 relative to the lexA start codon), and the 3′ flanking region, spanning 2,222 base pairs (positions +769 to +2,991), were amplified using primers SXY11/SXY12 and SXY13/SXY14, respectively (Supplementary Figure S1A).

The lexA deletion vector ΔlexA was constructed by ligating fragments amplified with SXY11/SXY14 into the EcoRI/HindIII sites of the pKC1132 plasmid (Bierman et al., 1992), and then transformed into NBRC 13819 protoplasts (Macneil and Klapko, 1987). Transformants on RM14 (Macneil and Klapko, 1987) regeneration medium were transferred to ISP2 plates for sporulation. Spores were spread on apramycin-containing ISP2 plates, which were incubated at 28°C for 2 days. pKC1132 is unable to replicate itself in Streptomyces. Therefore, only a single-crossover mutant with p∆lexA integrated into the chromosome was able to grow. Single-crossover mutants were incubated on ISP2 plates without apramycin for two rounds of sporulation, and colonies of double-crossover mutants were selected and verified by PCR analysis using the primers SXY3F/SXY3R (flanking the exchange region) and SXY4F/SXY4R (within the lexA deletion region) (Supplementary Figure S1B). Amplification with primers SXY3F/SXY3R produced a 4.5 kb band in WT DNA, while ΔlexA DNA yielded only a 3.7 kb band. With primers SXY4F/SXY4R, only WT DNA exhibited the expected 274 bp band (Supplementary Figure S1C). This process generated the lexA gene deletion mutant ΔlexA, which is characterized by the absence of lexA.

To achieve the overexpression of lexA, a 786 bp fragment of the lexA coding region and the strong promoter kasO* were amplified from DNA from S. mobaraensis NBRC 13819 and pDR4-K* (Wang et al., 2013) using the primers SXY21/SXY22 and SXY23/SXY24. The two fragments were digested with BamHI/EcoRI and HindIII/BamHI and then ligated simultaneously into EcoRI and HindIII sites of pSET152 to generate lexA overexpression vector pOlexA (Supplementary Figure S1D), which was then transformed into NBRC 13819 and TBJ3 to construct lexA overexpression strain OlexA and OlexA^TBJ3. For the complementation of ΔlexA, the amplification product containing the lexA coding region and its native promoter were ligated with the EcoRI/HindIII-digested pSET152 to create lexA-complemented vector pClexA, which was then introduced into ∆lexA to construct complemented strain ClexA (Supplementary Figure S1E).

The lexA coding region (261 amino acids) was amplified from S. mobaraensis NBRC 13819 using the primers SXY31/SXY32 and cloned and inserted into the pET-28a (+) plasmid to generate pET-lexA. The expression plasmids were introduced into E. coli Rosetta (DE3), and the recombinant E. coli were cultured in Luria-Bertani medium supplemented with 100 μg/mL kanamycin and 100 μg/mL chloramphenicol overnight at 37°C with shaking at 250 rpm. Subsequently, the seed culture was transferred to 1 L of LB medium containing equivalent antibiotic concentrations, and the inoculum size was adjusted to 1%. The culture was continued at 37°C for 3 h. At an OD600 of 0.8, the inducer isopropyl β-D-1 thiogalactolate was added at a final concentration of 100 μM, and induction was carried out at 16°C for 19 h. Bacteria containing recombinant protein were harvested, disrupted in lysis buffer (300 mM NaCl, 50 mM NaH₂PO₄, 10 mM, Imidazole) by sonication on ice, and centrifuged. His₆-tagged recombinant protein in supernatant was purified on a column packed with Ni²⁺-NTA agarose beads (Bio-Works; Sweden) and eluted from resin by lysis buffer plus 200 mM imidazole. The purified His₆-LexA were dialyzed against dialysis buffer (10 mM Tris-HCl, 1 mM EDTA, 80 mM NaCl, 4% glycerol, 20 mM β-mercaptoethanol, pH 7.5) to eliminate imidazole, and stored at −80°C until use.

PCR was used to amplify the DNA sequence of the promoter region of the target gene (primers are listed in Supplementary Table S2), and the obtained product was used to prepare the electrophoretic mobility shift assay (EMSA) probe. 0 ng, 100 ng, 200 ng His₆-LexA were coincubated with the 25 nM EMSA probe in a 20 μL binding system (binding buffer: 30 mM KCl, 10 mM Tris-HCl, 0.4 mM DTT, 2 mM MgCl₂, 0.2 mM EDTA, 2% glycerol) for 30 min at 30°C. This was followed by electrophoresis on an 8% native polyacrylamide gel at 0°C. After 40 min of constant voltage electrophoresis at 100 V, 1% GoldenView was added to pure water, mix in a horizontal shaker for 15 min with the gel, and then DNA is detected using gel imaging (Tanon-5200; China).

For RNA isolation, S. mobaraensis strains were grown on ISP2 solid medium or in liquid TSB medium. Samples were collected from three distinct Streptomyces cultures at different time intervals and subsequently pulverized in liquid nitrogen. Total RNAs were extracted utilizing Trizol reagent (Tiangen; China). Following extraction, crude RNA samples underwent treatment with DNase I (TaKaRa; Japan) to eliminate any potential genomic DNA contaminants. Reverse transcription of total RNA and subsequent reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) analysis were performed as described previously (Yan et al., 2020). Primer pairs listed in Supplementary Table S2 were utilized for RT-qPCR amplification. DNase I-treated RNA samples, which did not undergo reverse transcription, served as negative controls to validate the absence of DNA contamination. The transcription levels of each gene were normalized relative to the internal control gene 16S rRNA (LOCUS_r00030) using the comparative Ct method. The expression value of each gene at the initial time point was set as 1. Gene expression analyses were performed in three biological replicates.

The activity of TGase was measured by hydroxamate assay (Oteng-Pabi and Keillor, 2013). The quantification of hydroxamate was determined by measuring the absorbance at 525 nm of the red complex formed with FeCl₃-TCA (trichloroacetic acid) (Tao et al., 2024). Twenty microliters of fermentation broth supernatant was transferred into the 1.5 mL eppendorf tube. For the blank control, first add 200 μL of reagent B, react at 37°C for 10 min, and then add 200 μL reagent A. Pipette 200 μL of the reaction solution into a 96-well plate, and use a multifunctional microplate reader (EN 60825-1:2007; PerkinElmer, United States) to measure the absorbance of the supernatant at 525 nm. For the tested sample, 200 μL reagent A was added, and the reaction was carried out at 37°C for 10 min. After adding 200 μL reagent B to terminate the reaction, the absorbance of the supernatant was measured at 525 nm. Reagent A: 12.114 g trishydroxylaminomethane (Tris), 3.474 g hydroxylamine hydrochloride, 1.537 g reduced glutathione (GSH), 1.687 g substrate (Nα-Cbz-Gln-Gly), and adjust the pH to 6.0, add water to make the volume to 500 mL. Reagent B: 3 mol/L HCl, 12% trichloroacetic acid, 5% FeCl₃ dissolved in 0.1 mol/L HCl, mix the three solutions in equal amounts and mix evenly with a magnetic stirrer.

To measure the dry weight of the cells, the strain was initially cultured in seed medium for 22 h and then transferred to fermentation medium. Seed medium (w/v): 2% glycerol, 2% peptone, 0.5% yeast powder, 0.2% K₂HPO₄ and 0.2% MgSO₄. Adjust pH to 7.0. Fermentation medium (w/v): 2% glycerol, 2% peptone, 0.5% yeast powder, 2% soy protein powder, 0.2% KH₂PO₄, 0.4% K₂HPO₄, 0.2% CaCO₃ and 0.2% MgSO₄ adjusted to pH 7.0. Mycelium samples were collected at 8 h intervals and dried overnight at 80°C until complete evaporation of water. The dry weight was determined using an electronic scale (dry weight = sample tube weight − empty tube weight; n = 3) (Yin et al., 2021).

Mycelial phenotypes were examined using scanning electron microscopy (Hitachi SU8010; Japan). The samples were cut into 1 cm*1 cm pieces by the inoculation shovel and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH = 7.2) for 8 h. Subsequently, the samples were washed three times with ultrapure water (8 min per immersion). Dehydration was achieved by treating the samples with ethanol solutions of increasing concentrations (30, 50, 70, 85, 95, and 100%), with each immersion lasting 15 min. Finally, critical point drying was performed, followed by gold spraying and observation using a Hitachi Cold-Field Emission Scanning Electron Microscope (Xie et al., 2023).

To analyze the differential proteins expressed in S. mobaraensis NBRC 13819 and TBJ3 strains, fermentation was conducted in the 7.5 L fermenter (New Brunswick BioFlo^®/CelliGen^® 115, Germany) at 30°C. The rotation speed was related to dissolved oxygen and set to 300–600 rpm. The dissolved oxygen threshold was 1.8 mg/L. One millilitre of bacterial cells were collected at 12, 27 and 42 h for differential protein expression analysis. Sample processing, liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and data standardization was performed by the Novogene company (Beijing, China) as described previously (Qiu et al., 2024). Differentially expressed proteins were listed in Supplementary Table S3.

The genome-wide scan of S. mobaraensis NBRC 13819 used the PREDetector 3.1 website.¹ The sequences used to calculate LexA conserved motifs are listed in Supplementary Table S4. The S. mobaraensis NBRC 13819 genome information of the strain was downloaded from NCBI. The score of the target genes is cut at 9. The predicted target genes are listed in the Supplementary Table S5.

Using the original strain (S. mobaraensis NBRC 13819) and the TGase industrial strain TBJ3 for fermentation, the culture broth samples were collected every 3 h to analyze the accumulation of TGase. The results showed that TGase production was significantly greater in the TBJ3 strain than NBRC 13819 in the supernatant (Figure 1A). Concurrently, growth curves, determined by measuring cell dry weight, indicated that the growth cycles of WT and TBJ3 were essentially identical, suggesting that the observed difference in TGase production was not attributed to variations in biomass (Supplementary Figure S2A). Further analysis of the TGase activity profiles of the WT and TBJ3 strains revealed that the TGase activity of TBJ3 was 3-fold greater than that of the WT at the 40 h maturation point, when all Pro-TGase forms TGase (Figure 1B).

In addition, to investigate the functional genes influencing TGase synthesis, the bacterial cells were selected at 12 h (logarithmic or log phase), 27 h (stationary phase), and 42 h (lag phase) (Supplementary Figure S2B) for the differential protein expression analysis. The results revealed significant alterations in protein expression between the TBJ3 and WT strains at all three examined time points, with 59 proteins upregulated and 82 downregulated (Figure 1C and Supplementary Table S3). Notably, among the proteins exhibiting substantial upregulation, LexA—an essential transcription factor associated with the regulation of DNA damage repair stress—displayed remarkable fold increases of 4.73, 5.43, and 5.78, respectively, in TBJ3. Based on the significant up-regulation of LexA in TBJ3, we hypothesize that LexA is involved in regulating TGase production.

To elucidate the role of LexA in S. mobaraensis, we generated a lexA deletion mutant strain (ΔlexA) through homologous double exchange. Additionally, an OlexA overexpression strain of lexA was constructed using the integrative vector pSET152 and the potent kasO*p promoter. To restore the lexA gene, the complementation strain ClexA was generated based on the integrating vector pSET152.

In shake flask fermentation experiments, we analyzed the fermentation broth collected at 40 h using SDS-PAGE. The results revealed a significant reduction in TGase production in ΔlexA, a notable increase in OlexA, and a substantial return to wild-type levels in ClexA following lexA complementation (Figures 2A,B). These findings strongly suggest the involvement of LexA in regulating TGase synthesis. Moreover, the overexpression of lexA not only compensates for the reduced TGase production observed in ΔlexA but also significantly surpasses the wild-type levels. This finding underscores the potential effectiveness of lexA overexpression as a strategy to enhance TGase production in S. mobaraensis.

To determine the positive regulation of TGase production by LexA without impacting bacterial growth, we conducted enzyme activity and growth profile analyses on the WT, ΔlexA, and OlexA strains. At 40 h of TGase maturation, OlexA exhibited a 38% increase in TGase enzyme activity compared to that of the WT, while the TGase activity of the ΔlexA mutant decreased by 62% (Figure 2C) and the TGase activity of the ClexA mutant basically can be restored to the WT (Supplementary Figure S3A). The growth curves, determined by measuring the cell dry weight of lexA-associated strains, indicated that OlexA, ΔlexA and ClexA do not influence the growth of the bacterium (Figure 2D; Supplementary Figure S3B). These findings confirm that LexA regulates TGase expression without affecting the growth of S. mobaraensis. To further increase the TGase production in industrial strain TBJ3, we overexpressed lexA in TBJ3 to obtain strain OlexA^TBJ3. By measuring the enzyme activity curve, we found that overexpressing lexA, the TGase activity increased by nearly 25% in 40 h (Figure 2E), indicating that regulating the lexA expression level is an effective strategy to increase TGase production.

In order to investigate the effect of LexA on morphological differentiation of S. mobaraensis, OlexA, ClexA, and ΔlexA were incubated on ISP2 solid media for 5 days at 30°C, with WT serving as the control strain. Bacterial growth was observed to assess morphological changes. The results demonstrated that OlexA exhibited earlier sporulation, initiating on the first day, while the WT formed mature spores by the second day. In contrast, ΔlexA produced fewer spores even by the fifth day, indicating a bald phenotype. Remarkably, the ClexA phenotype was restored to that of the wild type (Figure 3A). Further scanning electron microscopy revealed that by day 2, the WT exhibited a substantial number of filamentous features on the sprocket, whereas the ΔlexA strain presented a relatively cleaner appearance, with only a few filamentous filaments at the initial stage (Figure 3B). These findings strongly suggest that LexA plays a regulatory role in the morphological differentiation of S. mobaraensis.

To further understand the regulatory mechanism of LexA, mycelia from the WT, ΔlexA, and OlexA strains fermented for 16 h (logarithmic or log phase), 32 h (stationary phase), or 48 h (death phase) were harvested in shake flasks. RNA extraction was performed, and changes in the transcript levels of tga, the gene encoding TGase, were assessed through RT-qPCR. The results revealed a significant downregulation of the tga transcript in the ΔlexA strain compared to that in the WT, while a substantial upregulation was observed in the OlexA strain relative to that in the WT (Figure 4A). This indicates that LexA positively regulates TGase expression at the transcriptional level.

To explore the direct regulatory role of LexA in TGase expression, the recombinant protein His₆-LexA, which was tagged with His₆ at the N-terminus, was heterologously expressed and purified in E. coli (Supplementary Figure S4A). Electrophoretic mobility shift assays (EMSAs) using His₆-LexA with a sequence probe of the tga promoter region demonstrated that LexA could not bind to the tga promoter (Figure 4B). This finding suggested that LexA does not directly regulate TGase expression.

In S. venezuelae, the conserved binding motif for LexA is a 16 bp incomplete palindrome sequence (Stratton et al., 2022). The lexA gene in S. mobaraensis is 786 base pairs in length and encodes 281 amino acids. By analyzing 268 Streptomyces species, we found that LexA is present in more than 99% of them. Protein homology analysis revealed that the LexA protein in S. mobaraensis shares high homology (94.8, 96.3, 94.2, and 96.8%) with LexA proteins in S. venezuelae, S. coelicolor, Streptomyces griseus, and Streptomyces avermitilis (Supplementary Figure S5). These genes exhibit complete agreement in their DNA-binding HTH domains, suggesting that LexA has a highly conserved regulatory function across Streptomyces species.

Based on the identified LexA binding sequence in S. venezuelae, we predicted a conserved binding motif for LexA, represented by a 16-nucleotide binding sequence: 5′-TCGAACRNNGNNCGA-3′ (S=C/G; R = G/A; W = A/T; N = A/T/C/G; D = G/A/T) (Figure 4C). Additionally, a genome-wide scan of S. mobaraensis using the PREDetector 3.1 website (see text footnote 1) predicted a total of 157 target genes for LexA with a score greater than 9.0 (Supplementary Table S5). Among the predicted target genes, we identified 32 transcription factors and 5 target genes associated with protein synthesis. For the EMSAs, we randomly selected two transcription factors: marR1 (LOCUS_20350; MarR family transcriptional regulator) and araC1 (LOCUS_63640; AraC family transcriptional regulator). The results demonstrated that LexA effectively binds to the promoter regions of these two genes (Figure 5A). Further analysis of the transcript levels of marR1 and araC1 in the ΔlexA and WT strains by RT-qPCR revealed significant downregulation in the ΔlexA strain compared to the WT strain (Figure 5B). Therefore, LexA directly upregulates the transcript levels of marR1 and araC1, which influences their expression.

We found that the tga gene transcription level was significantly down-regulated in lexA deletion strain, but the EMSA experimental results showed that LexA was not directly involved in regulating the transcription of the tga gene. This shows that LexA regulates the tga transcription indirectly by regulating other transcription factors. Among our predicted target genes, 32 genes exert regulatory functions, indicating that LexA may indirectly regulate the tga transcription by regulating the expression of these genes. Additionally, we focused on two target genes associated with protein synthesis, namely, rplJ (Gomez-Escribano et al., 2006) (LOCUS_22270; 50S ribosomal protein L10) and sti (LOCUS_52980; membrane protein). The encoded protein, 50S-subunit ribosomal protein L10, plays a vital role in the process of protein translation, while sti encodes a protease inhibitory protein involved in the process of protein degradation. The EMSA results demonstrated that LexA directly interacts with the promoter regions of rplJ and sti (Figure 5A). RT-qPCR experiments confirmed that the transcript levels of rplJ and sti were significantly lower in the ΔlexA mutant than in the WT (Figure 5B). These findings indicate that LexA directly activates the expression of rplJ and sti. Thus, LexA can also affect the TGase production by regulating the processes involved in protein synthesis and degradation.

Plate phenotyping and scanning electron microscopy experiments were conducted to investigate the role of LexA in the maturation of S. mobaraensis spores. From our analysis of predicted target genes, we identified four key genes that are crucial for morphological differentiation: whiB (Davis and Chater, 1992), ssgA (Traag and van Wezel, 2008), divIVA (Passot et al., 2022) and ftsH (Xu et al., 2022) (Supplementary Table S5). To confirm the regulatory impact of LexA on these genes, probes were generated through PCR amplification of their promoter regions (Probe whiBp, ssgAp, divIVAp, and ftsHp). Subsequent EMSA experiments using purified His₆-LexA demonstrated specific binding of LexA to the promoter regions of these genes (Figure 5C), further validating their predicted regulation.

To assess the regulatory effects of LexA on the aforementioned four target genes, colonies from both the ∆lexA and WT strains were harvested at 16, 32, and 48 h time points on ISP2 solid media. RNA extraction and RT-qPCR were performed to measure the transcript levels of these genes. Our results consistently showed a significant downregulation of the transcript levels in ∆lexA compared to those in WT at all time points (Figure 5D). These findings strongly indicate that LexA plays a positive role in the developmental differentiation of S. mobaraensis by activating the expression of these key target genes.