The roseoflavin producer Streptomyces davaonensis has a high catalytic capacity and specific genetic adaptations with regard to the biosynthesis of riboflavin

Abstract

Summary The bacterium Streptomyces davaonensis synthesizes the antibiotic roseoflavin in the stationary phase of growth. The starting point for roseoflavin biosynthesis is riboflavin (vitamin B2) and four enzymes (RibCF, RosB, RosA and RosC) are necessary to convert a vitamin (riboflavin) into a potent, broad‐spectrum antibiotic (roseoflavin). In S. davaonensis, seven enzymatic functions are required to synthesize the roseoflavin precursor riboflavin from the central building blocks GTP and ribulose 5‐phosphate. When compared with other bacterial and in particular Streptomyces genomes the S. davaonensis genome contains an unusual high number (21) of putative riboflavin biosynthetic genes (rib genes), including a rib gene encoding an additional riboflavin synthase originating from an Archaeon. We show by complementation analyses and enzyme assays that 17 out of these 21 putative rib genes indeed encode for riboflavin biosynthetic enzymes. Biochemical analyses of selected enzymes support this finding. Transcriptome analyses show that all of the rib genes are expressed either in the exponential or in the stationary phase of growth and thus do not represent silent genes. We conclude that the Rib enzymes produced in the stationary phase represent a physiological adaptation to support roseoflavin biosynthesis.

The novel phosphatase RosC catalyzes the last unknown step of roseoflavin biosynthesis in Streptomyces davaonensis

“…The second cluster comprises 10 genes including rosA whose gene product synthesizes RoF from AF. Both clusters are expressed in the stationary phase of growth (Kissling et al ., 2020). We anticipated that the missing AFP phosphatase gene would be part or close to the two known ros gene clusters.…”

“…A RoRP sensitive RP riboswitch (RFN) is located upstream of ribB9 (BN159_8007) encoding a monofunctional 3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase (Pedrolli et al ., 2012). The gene ribB9 strongly is expressed in the stationary phase of growth and RibB9 is thought to support roseoflavin biosynthesis by enhancing the levels of riboflavin (the precursor of RoF) (Kissling et al ., 2020). To generate the strains S. davaonensis Δclus_ rosA and S. davaonensis Δclus_ rosB the corresponding gene clusters were replaced by an apramycin resistance cassette (Apr R ) (Schwarz et al ., 2016) [Colour figure can be viewed at wileyonlinelibrary.com]…”

“…A recent transcriptome analysis of S. davaonensis suggested that RosA (>32‐fold induction), RosB (>128‐fold induction) and RosC (>32‐fold induction) were present in comparably high amounts in the stationary growth phase when RoF production occurred being in line with their role in secondary metabolite synthesis (Kissling et al ., 2020). Genome‐wide mapping of transcriptional start sites revealed that rosB is part of a gene cluster ( rosGE2XYB ) comprising 5 genes.…”

“…The gene rosG encodes a putative γ‐glutamyl‐transferase and possibly is involved in generation of glutamate (a substrate for RosB) from cytoplasmic glutathione. The gene ribE2 encodes an additional riboflavin synthase (Kissling et al ., 2020). The genes rosXY encode two proteins which represent a highly efficient riboflavin uptake system (C.S.…”

“…The functions of the remaining genes are unknown. A candidate for a regulator controlling RoF biosynthesis (i.e., both ros gene clusters) is a MarR type protein encoded by the S. davaonensis gene (BN159_43085 or marR ) located immediately downstream of rosB (but transcribed from the opposite direction) (Kissling et al ., 2020).…”