N-Deacetylation in Lincosamide Biosynthesis Is Catalyzed by a TldD/PmbA Family Protein.

Lincosamides are clinically important antibiotics originally produced as microbial specialized metabolites. The complex biosynthesis of lincosamides is coupled to the metabolism of mycothiol as a sulfur donor. Here, we elucidated the N-deacetylation of the mycothiol-derived N-acetyl-l-cysteine residue of a lincosamide intermediate, which is comprised of an amino acid and an aminooctose connected via an amide bond. We purified this intermediate from the culture broth of a deletion mutant strain and tested it as a substrate of recombinant lincosamide biosynthetic proteins in the in vitro assays that were monitored via liquid chromatography-mass spectrometry. Our findings showed that the N-deacetylation reaction is catalyzed by CcbIH/CcbQ or LmbIH/LmbQ proteins in celesticetin and lincomycin biosynthesis, respectively. These are the first N-deacetylases from the TldD/PmbA protein family, from which otherwise only several proteases and peptidases were functionally characterized. Furthermore, we present a sequence similarity network of TldD/PmbA proteins, which suggests that the lincosamide N-deacetylases are unique among these widely distributed proteins.

Biosynthesis and incorporation of an alkylproline-derivative (APD) precursor into complex natural products.

Covering: up to 2017This review covers the biosynthetic and evolutionary aspects of lincosamide antibiotics, antitumour pyrrolobenzodiazepines (PBDs) and the quorum-sensing molecule hormaomycin. These structurally and functionally diverse groups of complex natural products all incorporate rarely occurring 4-alkyl-l-proline derivatives (APDs) biosynthesized from l-tyrosine through an unusual specialized pathway catalysed by a common set of six proteins named Apd1-Apd6. We give an overview of APD formation, which involves unusual enzyme activities, and its incorporation, which is based either on nonribosomal peptide synthetase (PBDs, hormaomycin) or a unique hybrid ergothioneine-dependent condensation system followed by mycothiol-dependent sulphur atom incorporation (lincosamides). Furthermore, within the public databases, we identified 36 novel unannotated biosynthetic gene clusters that putatively encode the biosynthesis of APD compounds. Their products presumably include novel PBDs, but also novel classes of APD compounds, indicating an unprecedented potential for the diversity enhancement of these functionally versatile complex metabolites. In addition, phylogenetic analysis of known and novel gene clusters for the biosynthesis of APD compounds allowed us to infer novel evolutionary hypotheses: Apd3 methyltransferase originates from a duplication event in a hormaomycin biosynthetic gene cluster ancestor, while putative Apd5 isomerase is evolutionarily linked to PhzF protein from the biosynthesis of phenazines. Lastly, we summarize the achievements in preparing hybrid APD compounds by directing their biosynthesis, and we propose that the number of nature-like APD compounds could by multiplied by replacing l-proline residues in various groups of complex metabolites with APD, i.e. by imitating the natural process that occurs with lincosamides and PBDs, in which the replacement of l-proline for APD has proved to be an evolutionary successful concept.

Characterization of Enzymes Catalyzing Transformations of Cysteine S-Conjugated Intermediates in the Lincosamide Biosynthetic Pathway.

Lincosamides such as lincomycin A, celesticetin, and Bu-2545, constitute an important group of antibiotics. These natural products are characterized by a thiooctose linked to a l-proline residue, but they differ with regards to modifications of the thioacetal moiety, the pyrrolidine ring, and the octose core. Here we report that the pyridoxal 5'-phosphate-dependent enzyme CcbF (celesticetin biosynthetic pathway) is a decarboxylating deaminase that converts a cysteine S-conjugated intermediate into an aldehyde. In contrast, the homologous enzyme LmbF (lincomycin biosynthetic pathway) catalyzes C-S bond cleavage of the same intermediate to afford a thioglycoside. We show that Ccb4 and LmbG (downstream methyltransferases) convert the aldehyde and thiol intermediates into a variety of methylated lincosamide compounds including Bu-2545. The substrates used in these studies are the ß-anomers of the natural substrates. The findings not only provide insight into how the biosynthetic pathway of lincosamide antibiotics can bifurcate to generate different lincosamides, but also reveal the promiscuity of the enzymes involved.

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.

AIMS: To improve lincomycin A production and decrease the content of byproduct lincomycin B in an industrial lincomycin-producing strain. METHODS AND RESULTS: The in silico analysis indicated that LmbW could be involved in propylproline biosynthesis of lincomyin A. In this study, we constructed an lmbW deletion mutant and found that the mutant lost the ability to produce lincomycin A, but increased the accumulation of lincomycin B. The loss of lincomycin A production can be restored by complementing the mutant with the expression of lmbW gene. When lmbW and metK (encoding S-adenosylmethionine synthetase) was co-overexpressed, lincomycin A titre was 1744·6 mg l(-1) , a 35·83% improvement over the original strain. Meanwhile, the content of lincomycin B was reduced to 4·41%, a remarkable decrease of 34·76%, compared to that of the original strain. CONCLUSIONS: lmbW encodes a C-methyltransferase involved in the biosynthesis of lincomycin A but not lincomycin B. Co-overexpression of lmbW and metK improved lincomycin A production and decreased the content of lincomycin B. SIGNIFICANCE AND IMPACT OF THE STUDY: The engineered Streptomyces lincolnensis strain shows promising application in the fermentation production of lincomycin A, which may help cut production costs and simplify downstream separation processes.

Lincosamide synthetase--a unique condensation system combining elements of nonribosomal peptide synthetase and mycothiol metabolism.

In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.