Identification and characterization of two Streptomyces davawensis riboflavin biosynthesis gene clusters.
In Streptomyces davawensis roseoflavin is synthesized from GTP and ribulose-5-phosphate through riboflavin. As a first step towards the molecular analysis of flavin metabolism in S. davawensis the genes involved in riboflavin biosynthesis were cloned by hybridization of heterologous probes to a genomic library on a high-density colony-array. The genes ribB (riboflavin synthase, alpha-chain; EC 2.5.1.9), ribM (putative membrane protein), ribA (bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase; EC 3.5.4.25) and ribH (lumazine synthase; EC 2.5.1.9) are organized in an operon-like cluster. Northern blot analysis of this cluster revealed two transcripts of 1.7 and 3.1 kb, respectively. The gene ribB was overexpressed in Escherichia coli. The specific riboflavin synthase activity in a cell-free extract of a recombinant strain was 0.246 nmol mg(-1 )min(-1). Overexpression of ribM enhanced the transport of riboflavin in the corresponding recombinant E. coli strain. Furthermore, overexpression of ribM increased roseoflavin sensitivity of E. coli. On another subgenomic fragment a putative S. davawensis ribG gene coding for the missing pyrimidine deaminase/reductase (EC 3.5.4.26 and EC 1.1.1.193) of the riboflavin biosynthetic pathway and ribY coding for a second (monofunctional) GTP cyclohydrolase II were identified.
Grill
Simon
S
Institute for Technical Microbiology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany.
Yamaguchi
Hiroyuki
H
Wagner
Helen
H
Zwahlen
Laure
L
Kusch
Ute
U
Mack
Matthias
M
D001483:Base Sequence; D053898:Biosynthetic Pathways / Q000235:genetics*; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D023061:Gene Order; D015698:Genomic Library; D008969:Molecular Sequence Data; D005810:Multigene Family; D009693:Nucleic Acid Hybridization; D009876:Operon; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012256:Riboflavin / Q000235:genetics*; D012258:Riboflavin Synthase / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012258:Riboflavin Synthase; D012256:Riboflavin