Identification and utilization of two important transporters: SgvT1 and SgvT2, for griseoviridin and viridogrisein biosynthesis in Streptomyces griseoviridis

Abstract

Background: Griseoviridin (GV) and viridogrisein (VG, also referred as etamycin), both biosynthesized by a distinct 105 kb biosynthetic gene cluster (BGC) in Streptomyces griseoviridis NRRL 2427, are a pair of synergistic streptogramin antibiotics and very important in treating infections of many multi-drug resistant microorganisms. Three transporter genes, sgvT1-T3 have been discovered within the 105 kb GV/VG BGC, but the function of these efflux transporters have not been identified.

Results: In the present study, we have identified the different roles of these three transporters, SgvT1, SgvT2 and SgvT3. SgvT1 is a major facilitator superfamily (MFS) transporter whereas SgvT2 appears to serve as the sole ATP-binding cassette (ABC) transporter within the GV/VG BGC. Both proteins are necessary for efficient GV/VG biosynthesis although SgvT1 plays an especially critical role by averting undesired intracellular GV/VG accumulation during biosynthesis. SgvT3 is an alternative MFS-based transporter that appears to serve as a compensatory transporter in GV/VG biosynthesis. We also have identified the γ-butyrolactone (GBL) signaling pathway as a central regulator of sgvT1-T3 expression. Above all, overexpression of sgvT1 and sgvT2 enhances transmembrane transport leading to steady production of GV/VG in titers ≈ 3-fold greater than seen for the wild-type producer and without any notable disturbances to GV/VG biosynthetic gene expression or antibiotic control.

Conclusions: Our results shows that SgvT1-T2 are essential and useful in GV/VG biosynthesis and our effort highlight a new and effective strategy by which to better exploit streptogramin-based natural products of which GV and VG are prime examples with clinical potential.

Keywords: Antibiotic biosynthesis; Griseoviridin; Metabolite transporter; SgvT1; SgvT2; Streptogramin; Streptomyces griseoviridis; Viridogrisein.

Fig. 1

Chemical structures of griseoviridin (GV) and viridogrisein (VG)

Fig. 2

Gene organization of the GV VG gene cluster in S. griseoviridis NRRL 2427. The direction of transcription and the proposed functions of individual ORFs are indicated

Fig. 3

Comparisons of different strain growth patterns, plate-cultured morphologies, fermentation and gene expression profiles. a Growth curve of S. griseoviridis NRRL 2427 wild-type (WT) strain, ΔsgvT1/T2 mutants and WT strain complemented with sgvT1–T2; b The cultured morphology of wild-type strains, ΔsgvT1/T2 mutants and WT strain complemented with sgvT1–T2 in M-ISP4 medium plate; c HPLC analysis of GV (black diamond) and VG (black up-pointing triangle) in fermentation extract. (I) wild-type strain, (II) ΔsgvT1 mutant, (III) ΔsgvT2 mutant, (IV) ΔsgvT3 mutant; d RT-PCR analysis of sgvT1–T3 and control boundary gene orf (+2) in wild-type strain and ΔsgvD1, ΔsgvQ, ΔsgvR2, ΔsgvR3, ΔsgvA mutants and ΔsgvA::sgvA strain

Fig. 4

Comparison of GV/VG production and gene expression profiles in different strains. The GV (a) and VG (b) production curves in S. griseoviridis NRRL 2427 wild-type (WT) strain, ΔsgvT1/T2 mutants; The qPCR analysis of GV/VG biosynthesis-related gene expression during fermentation of ΔsgvT1/T2 mutants at 60 h (c) and 120 h (d) (*p < 0.05), the dash line indicate the mRNA abundance level of WT in the same conditions

Fig. 5

Proposed transfer mechanism via SgvT1–T3 in GV/VG biosynthesis in S. griseoviridis NRRL 2427. GBL signal pathway not only activates the GV/VG biosynthesis but also prompts the synchronized sgvT1–T3 expression to avoid disorder GV/VG accumulation

Fig. 6

Analysis of GV/VG high yielding recombinant strain WT::sgvT1–T2. The GV (a) and VG (b) production curves; c the HPLC analysis of GV (black diamond) and VG (black up-pointing triangle) in fermentation extract. (I) wild-type (WT) strain, (II–IV) twofold diluted sample of WT::sgvT1-T2; d the qPCR analysis of GV/VG biosynthetic gene expression profiles during fermentation of WT::sgvT1–T2 (**p < 0.01), the dash line indicate the mRNA abundance level of WT in the same conditions