Isolation and identification of 3-propylidene-.DELTA.1-pyrroline-5-carboxylic acid, a biosynthetic precursor of lincomycin.

Abstract

An accumulated lincomycin intermediate in UC8292, a lincomycin nonproducing strain of Streptomyces lincolnensis, has been isolated and purified by employing an assay system based on complementation of UC11066, another lincomycin nonproducing strain of S. lincolnensis. The structure of the purified intermediate is shown to be 3-propylidene-z1 1-pyrroline-5-carboxylic acid, or 1, 2, 3, 6-tetradehydro-propylproline by mass spectrometry and NMRspectroscopic studies. Based on the structure of this newly found intermediate, a biosynthetic pathway for propylproline is proposed as tyrosine-»L-3-hydroxytyrosine (Dopa) -^-> -^->3-propylidene-zl 1-pyrroline-5-carboxylic acid -åº 3-propyl-zl 2-pyrroline-5-carboxylic acid ->>propylproline. 1773It has been shown previously that Streptomyces lincolnensis strain UC8292 (originally designated NTG-3), a lincomycin nonproducer, is incapable of synthesizing lincomycin cosynthetic factor (LCF), a deazariboflavin1'2^Lacking this deazariboflavin, UC 8292 cannot synthesize propylproline and consequently lincomycin. In this paper, we report that a previously unidentified lincomycin intermediate in UC 8292 has been isolated and purified by employing an assay system based on complementation of S. lincolnensis strain UC 1 1066 (originally designated IIc-2-3-3)3), another lincomycin nonproducer. Materials and MethodsBacterial Strains Two mutants of S. lincolnensis blocked in the biosynthesis of lincomycin were used in this study, UC 8292, a riboflavin auxotroph which does not produce LCFnecessary for the biosynthesis of lincomycin1'2), and UC1 1066, a mutant which does not produce propylproline, an intermediate in the lincomycin biosynthetic pathway. The latter strain was obtained from a derivative of S. lincolnensis strain UC5124 by transposon mutagenesis with the Streptomycesfradiae transposon Tn45603\Fermentation Media and Conditions The media and fermentation conditions for shake flask fermentations were described previously2*. Twenty liter fermentations were carried out in the same media at 29°C with an agitation speed of 700 rpm and an aeration rate of 20liters per minute. Both flasks and tanks were harvested at 4 days. Assay for Lincomycin Biosynthetic Intermediates (LBI)In an effort to find a nonproducing strain of S. lincolnensis capable of complementing UC8292, several other strains, including UC1 1066, were tested in cross-feeding experiments. Fermentations of all strains were carried out in 500ml shake flasks and supernatants and mycelia mixed in all combinations at 96 hours. Mixed test flasks were incubated for an additional 24 hours and assayed for lincomycin activity. Addition ofUC 8292 supernatant to mycelia of UC1 1066 also resulted in lincomycin production indicating that the genetic lesion in UC11066 precedes the block in UC8292. Based on these results, S. lincolnensis

Biosynthesis of Sibiromycin, a Potent Antitumor Antibiotic

“…6). The six enzymes (SibS, SibT, SibU, SibV, SibY, and SibZ) involved in this multistep conversion were assigned based on the comparative analysis of the anthramycin, sibiromycin, and lincomycin A gene clusters and on the accumulation of the 4-propylidene-3,4-dihydropyrrole-2-carboxylic acid in an F420-deficient Streptomyces lincolnensis strain (23). The first two steps are catalyzed by SibU and SibV, highly similar to LmbB2 and LmbB1, respectively, whose tyrosine hydroxylase and L-DOPA 2,3-dioxygenase activities have been previously shown in vitro (Table 1) PBDs and lincomycin gene clusters strongly supports a role for SibS in the formation of 4-vinyl-2,3-dihydropyrrole-2-carboxylic acid.…”

New carbasugars from Streptomyces lincolnensis

“…[6,7] Other metabolites isolated from the same source were a [1] -pyrroline derivative 7 (a biosynthetic precursor of lincomycin), [8] γ -aminobutyric acid, [9] 3-methylthioacrylic acid, [10] 3,4-dihydroxybenzaldehyde, [7] and 7,8-didemethyl-8-hydroxy-5-deazariboflavin. [11] We studied a low-producing strain of S. lincolnensis DSM 40 355 looking for possible carbohydrate precursors of lincomycin.…”

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions

“…Encoded by the oxytetracycline (oxy), chlorotetracycline (ctc), and dactylocyclinone (dac) gene clusters (29), these enzymes are members of the flavin/deazaflavin oxidoreductase (FDOR) superfamily (30) and utilize F 420 H 2 reduced through the action of Fno (387). F 420 is also required for the synthesis of lincosamide antibiotics by Streptomyces lincolnensis strains (146,388,389), including lincomycin, the precursor of the clinical semisynthetic antibiotic clindamycin (390). On the basis of the accumulation of 4-propylidene-3,4-dihydropyrrole-2-carboxylic acid by strains unable to biosynthesize F 420 , it is proposed that an F 420 H 2 -dependent reductase catalyzes the reduction of the imine moiety of the dihydropyrrole to tetrapyrrole (Fig.…”