LexA, an SOS response repressor, activates TGase synthesis in Streptomyces mobaraensis

Abstract

Transglutaminase (EC 2.3.2.13, TGase), an enzyme that catalyzes the formation of covalent cross-links between protein or peptide molecules, plays a critical role in commercial food processing, medicine, and textiles. TGase from Streptomyces is the sole commercial enzyme preparation for cross-linking proteins. In this study, we revealed that the SOS response repressor protein LexA in Streptomyces mobaraensis not only triggers morphological development but also enhances TGase synthesis. The absence of lexA significantly diminished TGase production and sporulation. Although LexA does not bind directly to the promoter region of the TGase gene, it indirectly stimulates transcription of the tga gene, which encodes TGase. Furthermore, LexA directly enhances the expression of genes associated with protein synthesis and transcription factors, thus favorably influencing TGase synthesis at both the transcriptional and posttranscriptional levels. Moreover, LexA activates four crucial genes involved in morphological differentiation, promoting spore maturation. Overall, our findings suggest that LexA plays a dual role as a master regulator of the SOS response and a significant contributor to TGase regulation and certain aspects of secondary metabolism, offering insights into the cellular functions of LexA and facilitating the strategic engineering of TGase overproducers.

Keywords: LexA; TGase; regulation; streptomyces; transcription factor.

Figure 1

Differential protein expression analysis of type and industrial Streptomyces mobaraensis. (A) SDS-PAGE analysis of Sm-TGase. Protein Marker: M. Left panel: changes in TGase protein expression (yield) in the fermentation broth of the departure strain (WT) at different time points. The panels represent samples of the fermentation broth of the departure strain at different fermentation times. Right panel: changes in TGase protein expression (yield) in the fermentation broth of the high-yielding strain (TBJ3) at different time points. The numbers represent fermentation samples of high-yielding strains at different fermentation times. The time points marked in red represent the samples were used for differential protein expression analysis. The green arrow indicates the bands of Pro-TGase and the red arrow indicates the bands of TGase. (B) Dry cell weight growth curves and enzyme activity curves of the WT and TBJ3 strains. (C) Volcano plots comparing the comparative proteomics of the departure strain and the high-yielding strain at 27 h. The red dots indicate upregulated genes, and blue dots indicate downregulated genes. Data in (B) are shown as the mean ± SD (n = 3 biological replicates).

Figure 2

Analysis of enzyme production in lexA-related strains. (A) SDS–PAGE analysis of the WT, ∆lexA, OlexA, and ClexA strains after 40 h of culture. Numbers marked in red are samples sent for differential protein expression analysis. (B) Grayscale quantitative analysis of WT, ∆lexA, OlexA, and ClexA after 45 h of culture. The precise data of protein quantification was acquired via grayscale analysis utilizing ImageJ (National Institutes of Health, USA) software. (C) Enzyme activity curves of the WT, ∆lexA, and OlexA strains. (D) Dry cell weight growth curves of the WT, ∆lexA, and OlexA strains. (E) Enzyme activity curves of the TBJ3 and OlexA^TBJ3 strains. Data in (C, D, E) are shown as the mean ± SD (n=3 biological replicates). Two-tailed Student’s t test was used in (B) to analyze the statistical significance (n.s. p  > 0.05; ***p  < 0.001).

Figure 3

Effect of the lexA gene on the phenotype of S. mobaraensis. (A) Phenotypes of the WT, LexA deletion mutant (ΔlexA), complementation (ClexA), and overexpression (OlexA) strains grown on ISP2 agar media at 30°C. (B) Scanning electron microscopy images of the WT and ΔlexA strains.

Figure 4

Identification of LexA Target Genes Associated with Development. (A) RT-qPCR analysis of the transcript levels of TGase-encoding genes in the WT, ΔlexA, and OlexA strains after 16, 32, and 48 h of culture. (B) Electrophoretic mobility shift assays (EMSAs) to detect the interaction of His₆-LexA with TGase-encoding genes. (C) WebLogo analysis of LexA binding sequences. Data in (A) are shown as the mean ± SD (n = 3 biological replicates). Two-tailed student’s t-test was used in (A) to analyze the statistical significance (^**p < 0.01 and ^***p < 0.001). Two-tailed Student’s t-test was used in (B) to analyze the statistical significance (n.s. p  > 0.05; ***p  < 0.001).

Figure 5

Target identification and regulatory validation of LexA. (A) EMSAs to detect the interaction of His₆-LexA with protein synthesis-related probes. Probe marR1 (112 bp), Probe araC1 (110 bp), Probe rplJ (118 bp), Probe sti (111 bp). (B) RT-qPCR analysis of the WT and ΔlexA strains with protein synthesis-related genes after 16, 32, and 48 h of culture. (C) EMSAs to detect the interaction of His₆-LexA with probes related to influencing morphology. Probe whiB (123 bp), Probe ssgA (106 bp), Probe divIVA (101 bp), Probe ftsH (117 bp). (D) RT-qPCR analysis of the morphology-related genes in the WT and ΔlexA strains after 16 h, 32 h, and 48 h of culture. Data in (B,D) are shown as the mean ± SD (n = 3 biological replicates). Two-tailed student’s t-test was used in (B,D) to analyze the statistical significance (^*p < 0.05, ^**p < 0.01, and ^***p < 0.001).

Figure 6

Conceptual model of regulatory role of LexA in control of TGase production and S. mobaraensis development.