Mutasynthesis of Lincomycin Derivatives with Activity against Drug-Resistant Staphylococci

Abstract

The lincomycin biosynthetic gene lmbX was deleted in Streptomyces lincolnensis ATCC 25466, and deletion of this gene led to abolition of lincomycin production. The results of complementation experiments proved the blockage in the biosynthesis of lincomycin precursor 4-propyl-L-proline. Feeding this mutant strain with precursor derivatives resulted in production of 4-butyl-4-depropyllincomycin and 4-pentyl-4-depropyllincomycin in high titers and without lincomycin contamination. Moreover, 4-pentyl-4-depropyllincomycin was found to be more active than lincomycin against clinical Staphylococcus isolates with genes determining low-level lincosamide resistance.Lincosamides form a small yet clinically important group of antibiotics. One of the naturally occurring members of this group, lincomycin A (LIN), is active against many Gram-positive bacteria, such as staphylococci and streptococci. Its semisynthetic derivative clindamycin (CLI) is prescribed for the treatment of some infections caused by anaerobic bacteria and is also applied against the causative agent of malaria, Plasmodium falciparum (9,22).Biosynthesis of lincomycin proceeds via two separate branches from tyrosine and D-glucose to the aglycone 4-propyl-L-proline (PPL) and methylthiolincosamide (MTL), respectively. Condensation of these two precursors via an amide bond by a multimeric synthetase yields N-demethyllincomycin (NDL), which is subsequently methylated to form LIN (3) (Fig. 1A). Various LIN derivatives have been prepared chemically, and in particular, the 4Ј-alkyl-4Ј-depropyllincomycin set has been determined to be more active and have a broader antimicrobial spectrum than LIN has. Of this set, 4Ј-butyl, pentyl, and hexyl analogs have been shown to be particularly effective (12, 13). Moreover, demethylated and chlorinated 4Ј-alkyl-1Ј-demethyl-4Ј-depropylclindamycins had higher activities against Plasmodium spp. than clindamycin did (14). These more potent 4Ј-alkyl derivatives of lincomycin can be prepared by a multistep and costly chemical synthesis (13). Alternatively, feeding or genetic modifications of the natural biosynthetic pathway could be used. For example, the addition of PPL derivatives with extended alkyl residues to fermentation broths of a producer strain in a process termed precursor-directed biosynthesis has been described previously (25). This resulted in a mixture of both LIN and its more biologically active derivative being produced, which is not desirable because of the need to separate and purify the product of interest.In this study we present a new mutasynthetic approach to the preparation of the two known 4Ј-alkyl-4Ј-depropyllincomycins (Fig. 1B) by feeding a mutant strain defective in PPL biosynthesis with PPL derivatives as a practical alternative to total chemical synthesis. We tested the biological activity of these LIN derivatives on a collection of Staphylococcus strains with defined resistance profiles that have been described previously (15)(16)(17).The early reactions involved in biosynthesis of proline derivatives...

Antibiotic Resistance by Enzyme Inactivation: From Mechanisms to Solutions

“…Furthermore, a recent report by Ulanova et al has described the expansion of the chemical diversity of the lincomycins by chemical complementation of biosynthetic mutants in the producing organism to generate compounds with extended alkyl chains that retain activity against some resistant strains. [69] Rifamycin antibiotic modification by ADP-ribosylation…”

Biosynthesis, synthesis, and biological activities of pyrrolobenzodiazepines

“…Blast search of the Protein Data Bank fails to identify similar characterized proteins. However, a recent gene inactivation experiment showed the requirement of LmbX for the production of lincomycin . We have proposed that these enzymes catalyze an unusual C‐C hydrolysis of 4‐(3‐carboxy‐3‐oxo‐propenyl)‐2,3‐dihydropyrrole‐2‐carboxylic acid similarl to BphD, a C–C bond hydrolase ,…”

“…However, a recent gene inactivation experiment showed the requirement of LmbX for the production of lincomycin. 69 We have proposed that these enzymes catalyze an unusual C-C hydrolysis of 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydropyrrole-2-carboxylic acid similarl to BphD, a C-C bond hydrolase. 39,70 4-Propylidene-tetrahydropyrrole-2-carboxylic acid is the last intermediate common in the anthramycin, sibiromycin, and lincomycin A biosyntheses ( Fig.…”

Co-overexpression of lmbW and metK led to increased lincomycin A production and decreased byproduct lincomycin B content in an industrial strain of Streptomyces lincolnensis

“…2). Although depropyl butyl-or pentyl-lincomycin is produced in lmbX deletion strain by feeding butyl-or pentyl-proline (Ulanova et al 2010), the following enzymatic reactions leading to PPL forma-tion have not been elucidated. LmbB2 hydroxylates L-tyrosine to 3,4-dihydroxyphenylalanine (Neusser et al 1998;Novotna et al 2013), which is subsequently converted to 3,4-dihydropyrrole-2-carboxylic acid by LmbB1 (Neusser et al 1998;Novotna et al 2004;Colabroy et al 2008Colabroy et al , 2014.…”

“…LmbB2 hydroxylates L-tyrosine to 3,4-dihydroxyphenylalanine (Neusser et al 1998;Novotna et al 2013), which is subsequently converted to 3,4-dihydropyrrole-2-carboxylic acid by LmbB1 (Neusser et al 1998;Novotna et al 2004;Colabroy et al 2008Colabroy et al , 2014. Although depropyl butyl-or pentyl-lincomycin is produced in lmbX deletion strain by feeding butyl-or pentyl-proline (Ulanova et al 2010), the following enzymatic reactions leading to PPL forma-tion have not been elucidated. LmbR catalyses the formation of the C 8 backbone of LSM using fructose 6phosphate or sedoheptulose 7-phosphate as the C 3 donor and ribose 5-phosphate as the C 5 acceptor (Sasaki et al 2012).…”