Adaptive Optimization Boosted the Production of Moenomycin A in the Microbial Chassis Streptomyces albus J1074.

Great efforts have been made to improve Streptomyces chassis for efficient production of targeted natural products. Moenomycin family antibiotics, represented by moenomycin (Moe) and nosokomycin, are phosphoglycolipid antibiotics that display extraordinary inhibition against Gram-positive bacteria. Herein, we assembled a completed 34 kb hybrid biosynthetic gene cluster (BGC) of moenomycin A (moe-BGC) based on a 24 kb nosokomycin analogue biosynthetic gene cluster (noso-BGC). The heterologous expression of the hybrid moe-BGC in Streptomyces albus J1074 achieved the production of moenomycin A in the recombinant strain LX01 with a yield of 12.1 ± 2 mg/L. Further strong promoter refactoring to improve the transcriptional levels of all of the functional genes in strain LX02 enhanced the production of moenomycin A by 58%. However, the yield improvement of moenomycin A resulted in a dramatic 38% decrease in the chassis biomass compared with the control strain. To improve the weak physiological tolerance to moenomycin A of the chassis, another copy of the gene salb-PBP2 (P238N&F200D), encoding peptidoglycan biosynthetic protein PBP2, was introduced into the chassis strain, producing strain LX03. Cell growth was restored, and the fermentation titer of moenomycin A was 130% higher than that of LX01. Additionally, the production of moenomycin A in strain LX03 was further elevated by 45% to 40.0 ± 3 mg/L after media optimization. These results suggested that the adaptive optimization strategy of strong promoter refactoring in the BGC plus physiological tolerance in the chassis was an efficient approach for obtaining the desired natural products with high titers.

Cyclic di-GMP cyclase SSFG_02181 from Streptomyces ghanaensis ATCC14672 regulates antibiotic biosynthesis and morphological differentiation in streptomycetes.

Streptomycetes are filamentous bacteria famous for their ability to produce a vast majority of clinically important secondary metabolites. Both complex morphogenesis and onset of antibiotic biosynthesis are tightly linked in streptomycetes and require series of specific signals for initiation. Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, one of the well-known bacterial second messengers, has been recently shown to govern morphogenesis and natural product synthesis in Streptomyces by altering the activity of the pleiotropic regulator BldD. Here we report a role of the heme-binding diguanylate cyclase SSFG_02181 from Streptomyces ghanaensis in the regulation of the peptidoglycan glycosyltransferase inhibitor moenomycin A biosynthesis. Deletion of ssfg_02181 reduced the moenomycin A accumulation and led to a precocious sporulation, while the overexpression of the gene blocked sporogenesis and remarkably improved antibiotic titer. We also demonstrate that BldD negatively controls the expression of ssfg_02181, which stems from direct binding of BldD to the ssfg_02181 promoter. Notably, the heterologous expression of ssfg_02181 in model Streptomyces spp. arrested morphological progression at aerial mycelium level and strongly altered the production of secondary metabolites. Altogether, our work underscores the significance of c-di-GMP-mediated signaling in natural product biosynthesis and pointed to extensively applicable approach to increase antibiotic production levels in streptomycetes.

Secondary nucleotide messenger c-di-GMP exerts a global control on natural product biosynthesis in streptomycetes.

Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria. In streptomycetes, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating an activity of the pleiotropic regulator BldD. Here we report that c-di-GMP plays a key role in regulating secondary metabolite production in streptomycetes by altering the expression levels of bldD. Deletion of cdgB encoding a diguanylate cyclase in Streptomycesghanaensis reduced c-di-GMP levels and the production of the peptidoglycan glycosyltransferase inhibitor moenomycin A. In contrast to the cdgB mutant, inactivation of rmdB, encoding a phosphodiesterase for the c-di-GMP hydrolysis, positively correlated with the c-di-GMP and moenomycin A accumulation. Deletion of bldD adversely affected the synthesis of secondary metabolites in S. ghanaensis, including the production of moenomycin A. The bldD-deficient phenotype is partly mediated by an increase in expression of the pleiotropic regulatory gene wblA. Genetic and biochemical analyses demonstrate that a complex of c-di-GMP and BldD effectively represses transcription of wblA, thus preventing sporogenesis and sustaining antibiotic synthesis. These results show that manipulation of the expression of genes controlling c-di-GMP pool has the potential to improve antibiotic production as well as activate the expression of silent gene clusters.

A gene cluster for the biosynthesis of moenomycin family antibiotics in the genome of teicoplanin producer Actinoplanes teichomyceticus.

Moenomycins are phosphoglycolipid antibiotics notable for their extreme potency, unique mode of action, and proven record of use in animal nutrition without selection for resistant microflora. There is a keen interest in manipulation of structures of moenomycins in order to better understand their structure-activity relationships and to generate improved analogs. Only two almost identical moenomycin biosynthetic gene clusters are known, limiting our knowledge of the evolution of moenomycin pathways and our ability to genetically diversify them. Here, we report a novel gene cluster (tchm) that directs production of the phosphoglycolipid teichomycin in Actinoplanes teichomyceticus. Its overall genetic architecture is significantly different from that of the moenomycin biosynthesis (moe) gene clusters of Streptomyces ghanaensis and Streptomyces clavuligerus, featuring multiple gene rearrangements and two novel structural genes. Involvement of the tchm cluster in teichomycin biosynthesis was confirmed via heterologous co-expression of amidotransferase tchmH5 and moe genes. Our work sets the background for further engineering of moenomycins and for deeper inquiries into the evolution of this fascinating biosynthetic pathway.

MoeH5: a natural glycorandomizer from the moenomycin biosynthetic pathway.

The biosynthesis of the phosphoglycolipid antibiotic moenomycin A attracts the attention of researchers hoping to develop new moenomycin-based antibiotics against multidrug resistant Gram-positive infections. There is detailed understanding of most steps of this biosynthetic pathway in Streptomyces ghanaensis (ATCC14672), except for the ultimate stage, where a single pentasaccharide intermediate is converted into a set of unusually modified final products. Here we report that only one gene, moeH5, encoding a homologue of the glutamine amidotransferase (GAT) enzyme superfamily, is responsible for the observed diversity of terminally decorated moenomycins. Genetic and biochemical evidence support the idea that MoeH5 is a novel member of the GAT superfamily, whose homologues are involved in the synthesis of various secondary metabolites as well as K and O antigens of bacterial lipopolysaccharide. Our results provide insights into the mechanism of MoeH5 and its counterparts, and give us a new tool for the diversification of phosphoglycolipid antibiotics.

Tuning the moenomycin pharmacophore to enable discovery of bacterial cell wall synthesis inhibitors.

New antibiotic drugs need to be identified to address rapidly developing resistance of bacterial pathogens to common antibiotics. The natural antibiotic moenomycin A is the prototype for compounds that bind to bacterial peptidoglycan glycosyltransferases (PGTs) and inhibit cell wall biosynthesis, but it cannot be used as a drug. Here we report the chemoenzymatic synthesis of a fluorescently labeled, truncated analogue of moenomycin based on the minimal pharmacophore. This probe, which has optimized enzyme binding properties compared to moenomycin, was designed to identify low-micromolar inhibitors that bind to conserved features in PGT active sites. We demonstrate its use in displacement assays using PGTs from S. aureus, E. faecalis, and E. coli. 110,000 compounds were screened against S. aureus SgtB, and we identified a non-carbohydrate based compound that binds to all PGTs tested. We also show that the compound inhibits in vitro formation of peptidoglycan chains by several different PGTs. Thus, this assay enables the identification of small molecules that target PGT active sites, and may provide lead compounds for development of new antibiotics.

The molecular biology of moenomycins: towards novel antibiotics based on inhibition of bacterial peptidoglycan glycosyltransferases.

Moenomycins are phosphoglycolipid antibiotics and the only known natural product inhibitors of peptidoglycan glycosytransferases (PGTs). Techniques that would allow facile diversification of the moenomycin structure would facilitate the development of novel antibiotics, which are urgently needed in the wake of multidrug resistant bacterial infections. The cloning and initial characterization of the moenomycin biosynthetic genes has already redefined the minimal moenomycin pharmacophore and now opens the door for the biocombinatorial generation of bioactive moenomycin fragments. Here, we highlight the importance of research on the genetic mechanisms that regulate moenomycin biosynthesis and that confer moenomycin resistance to bacteria in the development of novel anti-infectives based on PGT inhibition.

Genetic factors that influence moenomycin production in streptomycetes.

Moenomycin, a natural phosphoglycolipid product that has a long history of use in animal nutrition, is currently considered an attractive starting point for the development of novel antibiotics. We recently reconstituted the biosynthesis of this natural product in a heterologous host, Streptomyces lividans TK24, but production levels were too low to be useful. We have examined several other streptomycetes strains as hosts and have also explored the overexpression of two pleiotropic regulatory genes, afsS and relA, on moenomycin production. A moenomycin-resistant derivative of S. albus J1074 was found to give the highest titers of moenomycin, and production was improved by overexpressing relA. Partial duplication of the moe cluster 1 in S. ghanaensis also increased average moenomycin production. The results reported here suggest that rational manipulation of global regulators combined with increased moe gene dosage could be a useful technique for improvement of moenomycin biosynthesis.

Complete characterization of the seventeen step moenomycin biosynthetic pathway.

The moenomycins are phosphoglycolipid antibiotics produced by Streptomyces ghanaensis and related organisms. The phosphoglycolipids are the only known active site inhibitors of the peptidoglycan glycosyltransferases, an important family of enzymes involved in the biosynthesis of the bacterial cell wall. Although these natural products have exceptionally potent antibiotic activity, pharmacokinetic limitations have precluded their clinical use. We previously identified the moenomycin biosynthetic gene cluster in order to facilitate biosynthetic approaches to new derivatives. Here, we report a comprehensive set of genetic and enzymatic experiments that establish functions for the 17 moenomycin biosynthetic genes involved in the synthesis of moenomycin and variants. These studies reveal the order of assembly of the full molecular scaffold and define a subset of seven genes involved in the synthesis of bioactive analogues. This work will enable both in vitro and fermentation-based reconstitution of phosphoglycolipid scaffolds so that chemoenzymatic approaches to novel analogues can be explored.

A streamlined metabolic pathway for the biosynthesis of moenomycin A.

Moenomycin A (MmA) is a member of the phosphoglycolipid family of antibiotics, which are the only natural products known to directly target the extracellular peptidoglycan glycosyltransferases involved in bacterial cell wall biosynthesis. The structural and biological uniqueness of MmA make it an attractive starting point for the development of new antibacterial drugs. In order both to elucidate the biosynthesis of this unusual compound and to develop tools to manipulate its structure, we have identified the MmA biosynthetic genes in Streptomyces ghanaensis (ATCC14672). We show via heterologous expression of a subset of moe genes that the economy of the MmA pathway is enabled through the use of sugar-nucleotide and isoprenoid building blocks derived from primary metabolism. The work reported lays the foundation for genetic engineering of MmA biosynthesis to produce novel derivatives.

Aptamers that recognize the lipid moiety of the antibiotic moenomycin A.

Moenomycin A is an amphiphilic phosphoglycolipid antibiotic that interferes with the transglycosylation step in peptidoglycan biosynthesis. The antibiotic consists of a branched pentasaccharide moiety, connected to the moenocinol lipid via a glycerophosphate linker. We have previously described the selection of aptamers that require the lipid group and the disaccharide epitopes of the oligosaccharide moiety for moenomycin binding. Here we report that the enriched moenomycin-binding library contains sequences that evolved for specific recognition of the unpolar lipid group of the antibiotic. These results suggest that the evolution of hydrophobic binding pockets in RNA molecules may be much more common than previously assumed.

Isolation and analysis of moenomycin and its biosynthetic intermediates from Streptomyces ghanaensis (ATCC 14672) wildtype and selected mutants.

Streptomyces ghanaensis (ATCC 14672) produces the phosphoglycolipid antibiotic moenomycin consisting of several components. A solid phase extraction procedure was developed which allowed a rapid isolation of both moenomycin and its biosynthetic intermediates from culture filtrates. Semi-preparative high performance liquid chromatography followed by high performance liquid chromatography-mass spectrometry provided structural data on the different moenomycin components. In order to obtain initial information on the biosynthetic pathway, moenomycin non-producing mutants were isolated. They were shown to release intermediates with shorter lipid chains suggesting that the lipid chain synthesis probably takes place at a later stage of the moenomycin biosynthesis. Based on the biological activity and the analytical data, we assume that a modification and in particular a shorter lipid portion drastically influences the inhibitory activity of this antibiotic.

[Functioning of integrative vectors, based on phage phi C 31, in the strain Streptomyces bambergiensis 712]. / Funktsionirovanie integrativnykh vektorov, sozdannykh na osnove faga phiC31, v shtamme Streptomyces bambergiensis 712.

Actinomycete integrative vectors were constructed. The vectors contain the Escherichia coli plasmid ColE1 replicon, the thiostrepton resistance gene used for selection in Streptomyces and a fragment of the phi C31 actinophage genome with integrative functions. The pS133 and PS135 vector DNAs transformed Streptomyces bambergiensis 712, a strain producing the phosphoglycolipid antibiotic moenomycin. Two types of the transformants were detected. The first type was not affected in the ability to produce moenomycin and the vector pS135 DNA was shown to integrate into the S. bambergiensis 712 genome by the site-specific pattern with the psi C31 phage DNA fragment. The second type of the transformants lost the ability to produce moenomycin. The Southern analysis and cloning of the inserted DNA indicated that in this case the vector pS135 and pS133 DNAs also integrated specifically into the genome but the integration took place not within the phage DNA fragment. Its realization was suggested to proceed via homologous recombination.

[Effect of plasmid pIJ350 on genetic stability of antibiotic production by Streptomyces bambergiensis]. / Vliianie plazmidy pIJ350 na geneticheskuiu stabil'nost' priznaka antibiotikoobrazovaniia u Streptomyces bambergiensis.

It was shown that S. bambergiensis S800 was genetically instable with respect to the property of the antibiotic production (Ant) while in strain S712 of S. bambergiensis this property was stable. Transformation of S. bambergiensis protoplasts with pIJ350 plasmid DNA and analysis of the transformants screening revealed induction of the Ant instability in both the strains. In case of plasmids pVG101 and pIJ943 this effect was not shown. Analysis of the S800 (pIJ350) transformant screening revealed five groups of mutants differing in the antibiotic production level and the presence of pIJ350 plasmid. Restriction analysis of the total DNA of the mutants showed that there were large deletions in the genome of two of them. Retransformation of the mutants with pIJ350 plasmid DNA showed that in all the cases induction of the instability was lacking. The behaviour of the spontaneous mutants Ant- of strain S800 with respect to pIJ350 plasmid was analogous to that of the mutants Ant- from the transformant S800 (pIJ350) screening. A hypothetic model for the determinant incompatibility with pIJ350 plasmid genetically linked to the Ant property in the genome of S. bambergiensis and unstable in strain S800 was proposed.