AdpA, a developmental regulator, promotes ε-poly-L-lysine biosynthesis in Streptomyces albulus

Abstract

Background: AdpA is a global regulator of morphological differentiation and secondary metabolism in Streptomyces, but the regulatory roles of the Streptomyces AdpA family on the biosynthesis of the natural product ε-poly-L-lysine (ε-PL) remain unidentified, and few studies have focused on increasing the production of ε-PL by manipulating transcription factors in Streptomyces.

Results: In this study, we revealed the regulatory roles of different AdpA homologs in ε-PL biosynthesis and morphological differentiation and effectively promoted ε-PL production and sporulation in Streptomyces albulus NK660 by heterologously expressing adpA from S. neyagawaensis NRRLB-3092 (adpA_Sn). First, we identified a novel AdpA homolog named AdpA_Sa in S. albulus NK660 and characterized its function as an activator of ε-PL biosynthesis and morphological differentiation. Subsequently, four heterologous AdpA homologs were selected to investigate their phylogenetic relationships and regulatory roles in S. albulus, and AdpA_Sn was demonstrated to have the strongest ability to promote both ε-PL production and sporulation among these five AdpA proteins. The ε-PL yield of S. albulus heterologously expressing adpA_Sn was approximately 3.6-fold higher than that of the control strain. Finally, we clarified the mechanism of AdpA_Sn in enhancing ε-PL biosynthesis and its effect on ε-PL polymerization degree using real-time quantitative PCR, microscale thermophoresis and MALDI-TOF-MS. AdpA_Sn was purified, and its seven direct targets, zwf, tal, pyk2, pta, ack, pepc and a transketolase gene (DC74_2409), were identified, suggesting that AdpA_Sn may cause the redistribution of metabolic flux in central metabolism pathways, which subsequently provides more carbon skeletons and ATP for ε-PL biosynthesis in S. albulus.

Conclusions: Here, we characterized the positive regulatory roles of Streptomyces AdpA homologs in ε-PL biosynthesis and their effects on morphological differentiation and reported for the first time that AdpA_Sn promotes ε-PL biosynthesis by affecting the transcription of its target genes in central metabolism pathways. These findings supply valuable insights into the regulatory roles of the Streptomyces AdpA family on ε-PL biosynthesis and morphological differentiation and suggest that AdpA_Sn may be an effective global regulator for enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces.

Keywords: AdpA; Morphological differentiation; Polymerization degree; Streptomyces albulus; ε-Poly-L-lysine.

Fig. 1

Amino acid alignment among AdpA_Sa, AdpA_Sd, AdpA-SH, AdpA_Sn, AdpA-C and the well-characterized AdpA_Sg. The ThiJ/PfpI/DJ-1 like domain for dimerization and two AraC/XylS family helix-turn-helix (HTH) motifs are marked. The locations of the Leu residues translated by the UUA codons are colored orange, and the locations of Arg-262 and Arg-266 residues that directly recognize the target DNA sequences of AdpA_Sg are marked with the blue stars. BAA86265.1, AdpA_Sg; AFX97763.1, AdpA_Sd; WP_018531726.1, AdpA-SH; WP_055538474.1, AdpA_Sn; WP_007264197.1, AdpA-C; and the blue font AIA03759.1 is the endogenous AdpA_Sa in S. albulus NK660

Fig. 2

Effects of AdpA_Sa on morphological development in S. albulus. A Phenotypes of S. albulus SET and S. albulus NKA grown on MSF agar plates at 30 ℃. B SEM showing morphological development of S. albulus SET and S. albulus NKA grown on MSF agar plates

Fig. 3

Effects of AdpA_Sa on ε-PL production and cell growth in S. albulus. A ε-PL yield and dry cell weight in S. albulus NKA and S. albulus SET cultured in M3G medium for 100 h. **P < 0.01 (Student's t-test). B Time course of ε-PL yield in S. albulus SET and S. albulus NKA cultured in M3G medium. C Growth curves (biomass presented by dry cell weight) of S. albulus SET and S. albulus NKA cultured in M3G medium

Fig. 4

Effects of AdpA_Sd, AdpA-SH, AdpA_Sn and AdpA-C on morphological development in S. albulus. A Phenotypes of four heterologous adpA genes expression mutants and S. albulus SET grown on MSF agar plates at 30 ℃. B SEM showing morphological development of four heterologous adpA genes expression mutants, S. albulus NKA and S. albulus SET grown on MSF agar plates for 3 days

Fig. 5

The ε-PL yield and dry cell weight in four heterologous adpA genes expression mutants and S. albulus SET cultured in M3G medium. **P < 0.01 (Student's t-test)

Fig. 6

Simplified metabolic network and transcription levels of key genes for ε-PL biosynthesis in S. albulus. A Simplified metabolic network of ε-PL production using glucose as the carbon source in S. albulus. The key genes for ε-PL biosynthesis are indicated, and the green, red and blue fonts or arrows stand for decreased transcription level, increased transcription level and no significant change in transcription level of these genes caused by the expression of adpA_Sn, respectively. The dotted arrow means the transcription level of pls is regulated by the sigma factor HrdD. B Transcription levels of the key genes for ε-PL biosynthesis in S. albulus SNA and S. albulus NK660

Fig. 7

Schematic diagram and purified AdpA_Sn protein for MST analysis. A Schematic diagram of the MST analysis. B SDS-PAGE of the purified AdpA_Sn protein for MST analysis. Lane M, protein marker; Lane 1, bovine serum albumin (BSA); Lane 2, purified His-tagged AdpA_Sn protein

Fig. 8

Interaction between promoter regions of targets and AdpA_Sn. A Interaction between promoter region of zwf and AdpA_Sn. The upstream genes of zwf, intergenic distances among these three genes and their common promoter region are displayed. RT-PCR results confirmed that genes DC74_2409, tal and zwf shared common promoter: Lane M, DNA marker III; Lane 1,4,7,10, amplification products using primers p4/p5 with gDNA, cDNA, RNA from S. albulus SNA and ddH₂O as templates, respectively; Lane 2,5,8,11, amplification products using primers p2/p3 with gDNA, cDNA, RNA from S. albulus SNA and ddH₂O as templates, respectively; Lane 3,6,9,12, amplification products using primers p1/p3 with gDNA, cDNA, RNA from S. albulus SNA and ddH₂O as templates, respectively. The binding curve for interaction between promoter region of zwf and AdpA_Sn is indicated. B Interaction between promoter region of pyk2 and AdpA_Sn. The upstream genes of pyk2, intergenic distances among these three genes and their common promoter region are displayed. RT-PCR results confirmed that genes pta, ack and pyk2 shared common promoter: Lane M, DNA marker III; Lane 1,4,7,10, amplification products using primers p9/p10 with gDNA, cDNA, RNA from S. albulus SNA and ddH₂O as templates, respectively; Lane 2,5,8,11, amplification products using primers p7/p8 with gDNA, cDNA, RNA from S. albulus SNA and ddH₂O as templates, respectively; Lane 3,6,9,12, amplification products using primers p6/p8 with gDNA, cDNA, RNA from S. albulus SNA and ddH₂O as templates, respectively. The binding curve for interaction between promoter region of pyk2 and AdpA_Sn is indicated. C The binding curve for interaction between promoter region of pepc and AdpA_Sn. D Comparison of the binding affinities between AdpA_Sn and the promoter regions of three target genes

Fig. 9

Polymerization degree of the purified ε-PL products produced by S. albulus NK660 and S. albulus SNA, respectively

Fig. 10

The proposed model showing the regulation of AdpA_Sn in central metabolism and acetate metabolism pathways. A Proposed model of the AdpA_Sn-mediated regulatory network in S. albulus SNA. Solid lines indicate the direct regulation confirmed experimentally in this paper. Dashed lines indicate that bldA could affect the translation level of the UUA codon-containing gene adpA_Sn. B The effects of AdpA_Sn on metabolic network and ε-PL biosynthesis are displayed. The seven target genes of AdpA_Sn are indicated in the simplified metabolic network, and the green, red and blue fonts or arrows stand for negative regulation, positive regulation and no regulation of AdpA_Sn, respectively. The green dotted arrows mean these metabolic pathways may be negatively regulated because the transcription level change of the gene (DC74_334) encoding the isozyme of DC74_2409 is unknown