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Covering 2005-2024Daptomycin is a clinically important antibiotic that treats Gram-positive infections of skin and skin structure, bacteremia, and right-sided endocarditis, including those caused by methicillin-resistant Staphylococcus aureus (MRSA). Daptomycin is now generic, and many companies are involved in manufacturing and commercializing this life-saving medicine. There has been much recent interest in improving the daptomycin fermentation of Streptomyces roseosporus by mutagenesis, metabolic engineering, and synthetic biology methods. The genome sequences of two strains discovered and developed at Eli Lilly and Company, a wild-type low-producer and a high-producer induced by N -methyl- N '-nitro- N -nitrosoguanidine (MNNG) mutagenesis, are available for comparitive studies. DNA sequence analysis of the daptomycin biosynthetic gene clusters (BGCs) from these strains indicates that the high producer has two mutations in a large promoter region that drives the transcription of a giant multicistronic mRNA that includes all nine genes involved in daptomycin biosynthesis. The locations of translational start and stop codons strongly suggest that all nine genes are translationally coupled by overlapping stop and start codons or by 70S ribosome scanning. This report also reviews recent studies on this promoter region that have identified at least ten positive or negative regulatory genes suitable to manipulate by metabolic engineering, synthetic biology and focused mutagenesis for strain improvement. Improvements in daptomycin production will also enable high-level production of novel lipopeptide antibiotics identified by genome mining and combinatorial biosynthesis, and accelerate clinical and commercial development of superior lipopeptide antibiotics.

Microbial genome mining for drug discovery and development has been accelerating in recent years, driven by technical advancements in genome sequencing, bioinformatics, metabolomics/metabologenomics, and synthetic biology. Microbial genome mining is a multistep process that starts with the sequencing of microbes that encode multiple secondary metabolites and identifying new and novel secondary metabolite biosynthetic gene clusters (BGCs) to pursue. The initial steps in the process are critical for the overall success, and they encompass the most innovative new technologies to revitalize natural product discovery. As microbial genome mining has matured in recent years, unvalidated conjectures about what microbes to pursue, how to identify legitimate secondary metabolite BGCs, and how to sequence DNA to satisfactory levels of completion have been identified. The solutions to correct the misconceptions around these topics are beginning to be implemented., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

The cyclic lipopeptide antibiotics structurally related to daptomycin were first reported in the 1950s. Several have common lipopeptide initiation, elongation, and termination mechanisms. Initiation requires the use of a fatty acyl-AMP ligase (FAAL), a free-standing acyl carrier protein (ACP), and a specialized condensation (CIII) domain on the first NRPS elongation module to couple the long chain fatty acid to the first amino acid. Termination is carried out by a dimodular NRPS that contains a terminal thioesterase (Te) domain (CAT-CATTe). Lipopeptide BGCs also encode ABC transporters, apparently for export and resistance. The use of this mechanism of initiation, elongation, and termination, coupled with molecular target-agnostic resistance, has provided a unique basis for robust natural and experimental combinatorial biosynthesis to generate a large variety of structurally related compounds, some with altered or different antibacterial mechanisms of action. The FAAL, ACP, and dimodular NRPS genes were used as molecular beacons to identify phylogenetically related BGCs by BLASTp analysis of finished and draft genome sequences. These and other molecular beacons have identified: (i) known, but previously unsequenced lipopeptide BGCs in draft genomes; (ii) a new daptomycin family BGC in a draft genome of Streptomyces sedi; and (iii) novel lipopeptide BGCs in the finished genome of Streptomyces ambofaciens and the draft genome of Streptomyces zhaozhouensis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Natural product discovery from microorganisms provided important sources for antibiotics, anti-cancer agents, immune-modulators, anthelminthic agents, and insecticides during a span of 50 years starting in the 1940s, then became less productive because of rediscovery issues, low throughput, and lack of relevant new technologies to unveil less abundant or not easily detected drug-like natural products. In the early 2000s, it was observed from genome sequencing that Streptomyces species encode about ten times as many secondary metabolites as predicted from known secondary metabolomes. This gave rise to a new discovery approach-microbial genome mining. As the cost of genome sequencing dropped, the numbers of sequenced bacteria, fungi and archaea expanded dramatically, and bioinformatic methods were developed to rapidly scan whole genomes for the numbers, types, and novelty of secondary metabolite biosynthetic gene clusters. This methodology enabled the identification of microbial taxa gifted for the biosynthesis of drug-like secondary metabolites. As genome sequencing technology progressed, the realities relevant to drug discovery have emerged, the conjectures and misconceptions have been clarified, and opportunities to reinvigorate microbial drug discovery have crystallized. This perspective addresses these critical issues for drug discovery.

Bacteriophage contamination and cell lysis have been recurring issues with some actinomycetes used in the pharmaceutical fermentation industry since the commercialization of streptomycin in the 1940s. In the early years, spontaneous phage-resistant mutants or lysogens were isolated to address the problem. In some cases, multiple phages were isolated from different contaminated fermentors, so strains resistant to multiple phages were isolated to stabilize the fermentation processes. With the advent of recombinant DNA technology, the early scaleup of the Escherichia coli fermentation process for the production of human insulin A and B chains encountered contamination with multiple coliphages. A genetic engineering solution was to clone and express a potent restriction/modification system in the production strains. Very recently, an E. coli fermentation of 1,3-propanediol was contaminated by a coliphage related to T1. CRISPR/Cas9 technology was applied to block future contamination by targeting seven different phage genes for double-strand cleavage. These approaches employing spontaneous mutation, genetic engineering, and synthetic biology can be applied to many current and future microorganisms used in the biotechnology industry.

Combinatorial biosynthesis of novel secondary metabolites derived from nonribosomal peptide synthetases (NRPSs) has been in slow development for about a quarter of a century. Progress has been hampered by the complexity of the giant multimodular multienzymes. More recently, advances have been made on understanding the chemical and structural biology of these complex megaenzymes, and on learning the design rules for engineering functional hybrid enzymes. In this perspective, I address what has been learned about successful engineering of complex lipopeptides related to daptomycin, and discuss how synthetic biology and microbial genome mining can converge to broaden the scope and enhance the speed and robustness of combinatorial biosynthesis of NRPS-derived natural products for drug discovery.

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Synthetic biology is a logical extension of what has been called recombinant DNA (rDNA) technology or genetic engineering since the 1970s. As rDNA technology has been the driver for the development of a thriving biotechnology industry today, starting with the commercialization of biosynthetic human insulin in the early 1980s, synthetic biology has the potential to take the industry to new heights in the coming years. Synthetic biology advances have been driven by dramatic cost reductions in DNA sequencing and DNA synthesis; by the development of sophisticated tools for genome editing, such as CRISPR/Cas9; and by advances in informatics, computational tools, and infrastructure to facilitate and scale analysis and design. Synthetic biology approaches have already been applied to the metabolic engineering of microorganisms for the production of industrially important chemicals and for the engineering of human cells to treat medical disorders. It also shows great promise to accelerate the discovery and development of novel secondary metabolites from microorganisms through traditional, engineered, and combinatorial biosynthesis. We anticipate that synthetic biology will continue to have broadening impacts on the biotechnology industry to address ongoing issues of human health, world food supply, renewable energy, and industrial chemicals and enzymes.

Actinomycetes are historically important sources for secondary metabolites (SMs) with applications in human medicine, animal health, and plant crop protection. It is now clear that actinomycetes and other microorganisms with large genomes have the capacity to produce many more SMs than was anticipated from standard fermentation studies. Indeed ~90 % of SM gene clusters (SMGCs) predicted from genome sequencing are cryptic under conventional fermentation and analytical analyses. Previous studies have suggested that among the actinomycetes with large genomes, some have the coding capacity to produce many more SMs than others, and that strains with the largest genomes tend to be the most gifted. These contentions have been evaluated more quantitatively by antiSMASH 3.0 analyses of microbial genomes, and the results indicate that many actinomycetes with large genomes are gifted for SM production, encoding 20-50 SMGCs, and devoting 0.8-3.0 Mb of coding capacity to SM production. Several Proteobacteria and Firmacutes with large genomes encode 20-30 SMGCs and devote 0.8-1.3 Mb of DNA to SM production, whereas cultured bacteria and archaea with small genomes devote insignificant coding capacity to SM production. Fully sequenced genomes of uncultured bacteria and archaea have small genomes nearly devoid of SMGCs.

Microbial genome mining is a promising technology that is revitalizing natural product discovery. It is now well documented that many bacteria with large genomes, particularly actinomycetes, encode many more secondary metabolites (SMs) than was previously known from their expressed secondary metabolomes. There are effective bioinformatics tools for counting the numbers and nature of SMs, and determining the total coding capacity from finished microbial genomes. However, these methods do not translate well to draft genomes, particularly for large SM gene clusters that contain nonribosomal peptide synthetase (NRPS) or type I polyketide synthase (PKS-I) mega-genes which are prone to fragmentation and misassembly. Small molecular beacons are required to assess the numbers and variety of NRPS, PKS-I and mixed NRPS/PKS-I pathways. In this report, I show that concatenated peptidyl carrier protein-thioesterase di-domains and acyl carrier protein-thioesterase di-domains can be used as multi-probes to survey finished or draft genomes to estimate the numbers of NRPS, PKS-I and mixed NRPS/PKS-I gene clusters to identify gifted actinomycetes.

Actinomycetes continue to be important sources for the discovery of secondary metabolites for applications in human medicine, animal health, and crop protection. With the maturation of actinomycete genome mining as a robust approach to identify new and novel cryptic secondary metabolite gene clusters, it is critical to continue developing methods to activate and enhance secondary metabolite biosynthesis for discovery, development, and large-scale manufacturing. This review covers recent reports on promising new approaches and further validations or technical improvements of existing approaches to strain improvement applicable to a wide range of Streptomyces species and other actinomycetes.

Microorganisms have provided abundant sources of natural products which have been developed as commercial products for human medicine, animal health, and plant crop protection. In the early years of natural product discovery from microorganisms (The Golden Age), new antibiotics were found with relative ease from low-throughput fermentation and whole cell screening methods. Later, molecular genetic and medicinal chemistry approaches were applied to modify and improve the activities of important chemical scaffolds, and more sophisticated screening methods were directed at target disease states. In the 1990s, the pharmaceutical industry moved to high-throughput screening of synthetic chemical libraries against many potential therapeutic targets, including new targets identified from the human genome sequencing project, largely to the exclusion of natural products, and discovery rates dropped dramatically. Nonetheless, natural products continued to provide key scaffolds for drug development. In the current millennium, it was discovered from genome sequencing that microbes with large genomes have the capacity to produce about ten times as many secondary metabolites as was previously recognized. Indeed, the most gifted actinomycetes have the capacity to produce around 30-50 secondary metabolites. With the precipitous drop in cost for genome sequencing, it is now feasible to sequence thousands of actinomycete genomes to identify the "biosynthetic dark matter" as sources for the discovery of new and novel secondary metabolites. Advances in bioinformatics, mass spectrometry, proteomics, transcriptomics, metabolomics and gene expression are driving the new field of microbial genome mining for applications in natural product discovery and development.

Nonribosomal peptide synthetases (NRPSs) are giant multi-enzymes that carry out sequencial assembly line couplings of amino acids to generate linear or cyclic peptides. NRPSs are composed of repeating enzyme domains with modular organization to activate and couple specific amino acids in a particular order. From a synthetic biology perspective, they can be considered as peptide assembly machines composed of devices to couple fatty acids to l-amino acids, l-amino acids to l-amino acids, and d-amino acids to l-amino acids. The coupling devices are composed of specific parts that contain two or more enzyme domains that can be exchanged combinatorially to generate novel peptide assembly machines to produce novel peptides. The potent lipopeptide antibiotics daptomycin and A54145E have identical cyclic depsipeptide ring structures and stereochemistry but have divergent amino acid sequences. As their biosynthetic gene clusters are derived from an ancient ancestral lipopetide pathway, these lipopeptides provided an attractive model to develop combinatorial biosynthesis to generate antibiotics superior to daptomycin. These studies on combinatorial biosynthesis have helped generate guidelines for the successful assembly of NRPS parts and devices that can be used to generate novel lipopeptide structures and have established a basis for future synthetic biology studies to further develop combinatorial biosynthesis as a robust approach to natural product drug discovery.

Chemical mutagenesis continues to be an important foundational methodology for the generation of highly productive actinomycete strains for the commercial production of antibiotics and other secondary metabolites. In the past, the determination of frequencies of chemically induced resistance to rifampicin (RifR), spectinomycin (SpcR) and streptomycin (StrR) have served as surrogate markers to monitor the efficiencies and robustness of mutagenic protocols. Recent studies indicate that high level RifR, SpcR and StrR phenotypes map to specific regions of the rpoB, rpsE and rpsL genes, respectively, in actinomycetes. Moreover, mutagenesis to RifR can occur spontaneously at many different sites in rpoB, and all six types of base-pair substitutions, as well as in-frame deletions and insertions, have been observed. The RifR/rpoB system provides a robust method to rank mutagenic protocols, to evaluate mutagen specificity and to study spontaneous mutagenesis mechanisms involved in the maintenance of high G+C content in Streptomyces species and other actinomycetes.

Advances in DNA sequencing technologies have made it possible to sequence large numbers of microbial genomes rapidly and inexpensively. In recent years, genome sequencing initiatives have demonstrated that actinomycetes with large genomes generally have the genetic potential to produce many secondary metabolites, most of which remain cryptic. Since the numbers of new and novel pathways vary considerably among actinomycetes, and the correct assembly of secondary metabolite pathways containing type I polyketide synthase or nonribosomal peptide synthetase (NRPS) genes is costly and time consuming, it would be advantageous to have simple genetic predictors for the number and potential novelty of secondary metabolite pathways in targeted microorganisms. For secondary metabolite pathways that utilize NRPS mechanisms, the small chaperone-like proteins related to MbtH encoded by Mycobacterium tuberculosis offer unique probes or beacons to identify gifted microbes encoding large numbers of diverse NRPS pathways because of their unique function(s) and small size. The small size of the mbtH-homolog genes makes surveying large numbers of genomes straight-forward with less than ten-fold sequencing coverage. Multiple MbtH orthologs and paralogs have been coupled to generate a 24-mer multiprobe to assign numerical codes to individual MbtH homologs by BLASTp analysis. This multiprobe can be used to identify gifted microbes encoding new and novel secondary metabolites for further focused exploration by extensive DNA sequencing, pathway assembly and annotation, and expression studies in homologous or heterologous hosts.

Microbial genome mining is a rapidly developing approach to discover new and novel secondary metabolites for drug discovery. Many advances have been made in the past decade to facilitate genome mining, and these are reviewed in this Special Issue of the Journal of Industrial Microbiology and Biotechnology. In this Introductory Review, we discuss the concept of genome mining and why it is important for the revitalization of natural product discovery; what microbes show the most promise for focused genome mining; how microbial genomes can be mined; how genome mining can be leveraged with other technologies; how progress on genome mining can be accelerated; and who should fund future progress in this promising field. We direct interested readers to more focused reviews on the individual topics in this Special Issue for more detailed summaries on the current state-of-the-art.

ϕC31, ϕBT1, R4, and TG1 are temperate bacteriophages with broad host specificity for species of the genus Streptomyces. They form lysogens by integrating site-specifically into diverse attB sites located within individual structural genes that map to the conserved core region of streptomycete linear chromosomes. The target genes containing the ϕC31, ϕBT1, R4, and TG1 attB sites encode a pirin-like protein, an integral membrane protein, an acyl-CoA synthetase, and an aminotransferase, respectively. These genes are highly conserved within the genus Streptomyces, and somewhat conserved within other actinomycetes. In each case, integration is mediated by a large serine recombinase that catalyzes unidirectional recombination between the bacteriophage attP and chromosomal attB sites. The unidirectional nature of the integration mechanism has been exploited in genetic engineering to produce stable recombinants of streptomycetes, other actinomycetes, eucaryotes, and archaea. The ϕC31 attachment/integration (Att/Int) system has been the most widely used, and it has been coupled with the ϕBT1 Att/Int system to facilitate combinatorial biosynthesis of novel lipopeptide antibiotics in Streptomyces fradiae.

Mycobacterium tuberculosis encodes mycobactin, a peptide siderophore that is biosynthesized by a nonribosomal peptide synthetase (NRPS) mechanism. Within the mycobactin biosynthetic gene cluster is a gene that encodes a 71-amino-acid protein MbtH. Many other NRPS gene clusters harbor mbtH homologs, and recent genetic, biochemical, and structural studies have begun to shed light on the function(s) of these proteins. In some cases, MbtH-like proteins are required for biosynthesis of their cognate peptides, and non-cognate MbtH-like proteins have been shown to be partially complementary. Biochemical studies revealed that certain MbtH-like proteins participate in tight binding to NRPS proteins containing adenylation (A) domains where they stimulate adenylation reactions. Expression of MbtH-like proteins is important for a number of applications, including optimal production of native and genetically engineered secondary metabolites produced by mechanisms that employ NRPS enzymes. They also may serve as beacons to identify gifted actinomycetes and possibly other bacteria that encode multiple functional NRPS pathways for discovery of novel secondary metabolites by genome mining.

With the recent advances in DNA sequencing technologies, it is now feasible to sequence multiple actinomycete genomes rapidly and inexpensively. An important observation that emerged from early Streptomyces genome sequencing projects was that each strain contains genes that encode 20 or more potential secondary metabolites, only a fraction of which are expressed during fermentation. More recently, this observation has been extended to many other actinomycetes with large genomes. The discovery of a wealth of orphan or cryptic secondary metabolite biosynthetic gene clusters has suggested that sequencing large numbers of actinomycete genomes may provide the starting materials for a productive new approach to discover novel secondary metabolites. The key issue for this approach to be successful is to find ways to turn on or turn up the expression of cryptic or poorly expressed pathways to provide material for structure elucidation and biological testing. In this review, I discuss several genetic approaches that are potentially applicable to many actinomycetes for this application.

A54145 is a complex of lipopeptide antibiotics produced by Streptomyces fradiae. A54145 factors are structurally related to daptomycin, with four modified amino acids, only one of which is present in daptomycin. We generated three mutants defective in lptJ, lptK or lptL, whose gene products are involved in the formation of hydroxy-Asn(3) (hAsn(3)) and methoxy-Asp(9) (moAsp(9)). Each of the mutants produced novel lipopeptides related to A54145 and the profiles allowed assignment of functions for those genes. We constructed strains carrying different combinations of these genes coupled with a mutation in the lptI gene involved in the biosynthesis of 3-methyl-Glu(12) (3mGlu(12)), and all recombinants produced novel lipopeptides. One of the compounds displayed very good antibacterial activity in the presence of bovine surfactant, which interacts with daptomycin or A54145E to inhibit their antibacterial activities.

A potent new lipopeptide antibiotic, A54145E(Asn(3)Asp(9)), was isolated from the fermentation broth of Streptomyces fradiae DA1489 engineered to delete genes encoding enzymes involved in hydroxylation of Asn(3) and methoxylation of Asp(9). The chemical structure predicted from the genetic changes in the biosynthetic pathway was determined by analyses of chemical transformations, D, L-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS and 2D NMR techniques. These studies confirmed the primary amino acid sequence of A54145E(Asn(3)Asp(9)) predicted from the genetic engineering strategy, and also confirmed the structure and locations of three D-amino acids predicted from bioinformatic studies.

A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the φC31 or φBT1 site-specific integration system allowed manipulation of A54145 biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu₁₂), at the expense of factors containing 3-methyl-glutamic acid (3mGlu₁₂). This provided a practical route to produce high levels of pure Glu₁₂-containing lipopeptides. A suite of mutant strains and plasmids was created for combinatorial biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.

Natural products discovery from actinomycetes has been on the decline in recent years, and has suffered from a lack of innovative ways to discover new secondary metabolites within a background of the thousands of known compounds. Recent advances in whole genome sequencing have revealed that actinomycetes with large genomes encode multiple secondary metabolite pathways, most of which remain cryptic. One approach to address the expression of cryptic pathways is to first identify novel pathways by bioinformatics, then clone and express them in well-characterized hosts with known secondary metabolomes. This process should eliminate the tedious dereplication process that has hampered natural products discovery. Several laboratory and industrial production strains have been used for heterologous production of secondary metabolite pathways. This review discusses the results of these studies, and the pros and cons of using various Streptomyces and one Saccharopolyspora strain for heterologous expression. This information should provide an experimental basis to help researchers choose hosts for current application and future development to express heterologous secondary metabolite pathways in yields sufficient for rapid scale-up, biological testing, and commercial production.

Daptomycin is a clinically useful lipopeptide antibiotic produced by Streptomyces roseosporus. The antibiotic is assembled by a nonribosomal peptide synthetase (NRPS) mechanism, and the cyclized tridecapeptide contains three non-proteinogenic-amino acids: ornithine (Orn), 3-methyl-glutamic acid (3mGlu) and kynurinine (Kyn). Daptomycin also has three D-amino acids. The two genes that encode proteins involved in the formation of 3mGlu and Kyn have no known orthologs, and hence they are good probes to search for daptomycin-like gene clusters among newly sequenced actinomycete genomes. A recent search with these genes revealed a daptomycin-like gene cluster in Saccharomonospora viridis, a causative agent for farmer's lung disease. S. viridis has a genome of 4.3 Mb, which is about one half the size of Streptomyces genomes. Searches for other NRPS and polyketide synthase (PKS) genes revealed only one other NRPS gene and no PKS genes in S. viridis. The orthologous lipopeptide biosynthetic genes and gene products in S. viridis and S. roseosporus have diverged extensively from a last common ancestor dating back to a pathway that had evolved over 1 billion years ago.

Daptomycin is a cyclic lipopeptide antibiotic approved for the treatment of skin and skin structure infections caused by Gram-positive pathogens and for that of bacteremia and right-sided endocarditis caused by Staphylococcus aureus. Daptomycin failed to meet noninferiority criteria for the treatment of community-acquired pneumonia, likely due to sequestration in pulmonary surfactant. Many analogues of daptomycin have been generated by combinatorial biosynthesis, but only two displayed improved activity in the presence of bovine surfactant, and neither was as active as daptomycin in vitro. In the present study, we generated hybrid molecules of the structurally related lipopeptide A54145 in Streptomyces fradiae and tested them for antibacterial activity in the presence of bovine surfactant. Hybrid A54145 nonribosomal peptide synthetase (NRPS) biosynthetic genes were constructed by genetic engineering and were expressed in combination with a deletion of the lptI methyltransferase gene, which is involved in the formation of the 3-methyl-glutamic acid (3mGlu) residue at position 12. Some of the compounds were very active against S. aureus and other Gram-positive pathogens; one compound was also highly active in the presence of bovine surfactant, had low acute toxicity, and showed some efficacy against Streptococcus pneumoniae in a mouse model of pulmonary infection.

Daptomycin has been approved and successfully launched for the treatment of complicated skin and skin-structure infections caused by gram-positive pathogens [1] and bacteremia and right-sided endocarditis due to Staphylococcus aureus, including strains that are resistant to methicillin or other antibiotics [2]. The development of the drug, however, was not straightforward; it involved a cast of characters, including scientists at Eli Lilly and at Cubist Pharmaceuticals. Of most importance, the development of daptomycin involved the tenacious leadership of Dr. Francis Tally. As a tribute to Dr. Tally, we attempt to reconstruct the path of daptomycin from the mountain to the clinic.

Daptomycin is a lipopeptide antibiotic used clinically to treat infections caused by Gram-positive bacteria. Laboratory studies have shown that Staphylococcus aureus resistance to daptomycin occurs stepwise and slowly. Mutations associated with decreased susceptibility were mapped in mprF, yycG, rpoB, and rpoC, each giving about twofold increases in the minimal inhibitory concentration (MIC) and combinations giving higher MICs. The mprF gene encodes a dual functional enzyme that couples lysine to phosphatidylglycerol (PG) and transfers the lysyl-PG (LPG) to the outer leaflet of the membrane. LPG is less acidic than PG, and thus reduces the binding of Ca(++)-bound daptomycin to bacterial membranes. The mprF mutants have higher LPG/PG ratios in the membrane outer leaflet and bind less daptomycin than the wild-type strain. YycG is a sensor histidine kinase of a two component signal transduction system required for viability in many low G+C Gram-positive bacteria. The observation of DapR mutations in yycG suggests that YycG may be a target for daptomycin antibacterial activity. Daptomycin inserts into PG rich membrane at the cell division septum, but also inserts into lung surfactant, explaining why it failed to meet non-inferiority criteria in clinical trials for community acquired pneumonia (CAP). Recent advances in biosynthetic engineering have provided new tools to generate novel lipopeptides with modifications in the core peptide: several were very potent antibiotics against Gram-positive pathogens, and some were active in the presence of surfactant.

Daptomycin is an acidic cyclic lipopeptide antibiotic approved for treatment of infections caused by Gram-positive pathogens, including Staphylococcus aureus strains resistant to other antibiotics. Daptomycin biosynthesis is carried out by a giant multisubunit, multienzyme nonribosomal peptide synthetase (NRPS). The daptomycin (dpt) biosynthetic genes have been cloned in a bacterial artificial chromosome (BAC) vector, sequenced, and expressed in Streptomyces lividans. Several of the dpt genes, including the three NRPS genes, are transcribed as a lengthy polycistronic message. The daptomycin-producing strain, Streptomyces roseosporus, can be genetically manipulated, and a number of deletion mutants encompassing one or more of the dpt genes have been constructed. Several of the dpt genes have been expressed from ectopic chromosomal loci (varphiC31 or IS117 attB sites) under the transcriptional control of the strong constitutive ermEp* promoter, and recombinant strains produced high levels of lipopeptides, thus establishing a trans-complementation system for combinatorial biosynthesis. A number of hybrid NRPS subunits have been generated by lambda-Red-mediated recombination, and combinatorial libraries of lipopeptides have been generated by NRPS subunit exchanges, module exchanges, multidomain exchanges, deletion mutagenesis, and multiple natural lipidations, using the ectopic trans-complementation system in S. roseosporus.

The soil actinomycetes have been important sources of antibiotics, but were nearly abandoned in recent years in favor of high-throughput target-based screening of chemical libraries. The latter approach has not been productive, so it is time to reinvigorate the discovery of new antibiotics from a proven source. Recent progress has been made on antibiotic discovery from actinomycetes by using high-throughput fermentation, isolation of marine actinomycetes, mining genomes for cryptic pathways, and combinatorial biosynthesis to generate new secondary metabolites related to existing pharmacophores.

Genetic engineering has been applied to reprogramme non-ribosomal peptide synthetases (NRPSs) to produce novel antibiotics, but little is known about what determines the efficiency of production. We explored module exchanges at nucleotide sequences encoding interpeptide linkers in dptD, a gene encoding a di-modular NRPS subunit that incorporates 3-methylglutamic acid (3mGlu(12)) and kynurenine (Kyn(13)) into daptomycin. Mutations causing amino acid substitutions, deletions or insertions in the inter-module linker had no negative effects on lipopeptide yields. Hybrid DptD subunits were generated by fusing the 3mGlu(12) module to terminal modules from calcium-dependent antibiotic (CDA) or A54145 NRPSs, and recombinants produced daptomycin analogues with Trp(13) or Ile(13) at high efficiencies. A recombinant expressing DptD with a hybrid Kyn(13) module containing a di-domain from a d-Asn module caused the production of a new daptomycin analogue containing Asn(13).

Daptomycin is a clinically important antibiotic approved for the treatment of complicated skin and skin structure infections caused by Gram-positive pathogens, and for the treatment of bacteremia and endocarditis caused by Staphylococcus aureus. Daptomycin and related acidic cyclic lipopeptide antibiotics have ten amino acids in the ring, and exocyclic tails containing one or three amino acids. The N-termini of the exocyclic amino acids are generally coupled to long chain fatty acids. Biosynthesis is initiated by the coupling of fatty acids to the N-terminal amino acids, followed by the coupling of the remaining amino acids by nonribosomal peptide synthetase (NRPS) mechanisms, then cyclization and release of the lipopeptides. The biosynthetic genes for daptomycin, calcium dependent antibiotic (CDA), A54145 and friulimicin have been cloned, sequenced, analyzed bioinformatically, and in some cases genetically or biochemically. The information on the organization and expression of the NRPS and other genes has been exploited to generate combinatorial libraries of hybrid lipopeptide antibiotics related to daptomycin, including several compounds with very good antibacterial activities.

Three daptomycin-related lipopeptides, A21978C1-3(d-Asn11) (2-4), were purified from the fermentation broth of a recombinant Streptomyces roseosporus strain. Their chemical structures were determined by analyses of the biosynthetic pathway, chemical transformations, d,l-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS, and 2D-NMR techniques. Compounds 2-4 exhibited potent antibacterial activity against Staphylococcus aureus with MIC values of 0.6, 0.3, and 0.15 microM, respectively, well correlated to the acyl tail chain length.

Molecular engineering approaches to producing new antibiotics have been in development for about 25 years. Advances in cloning and analysis of antibiotic gene clusters, engineering biosynthetic pathways in Escherichia coli, transfer of engineered pathways from E. coli into Streptomyces expression hosts, and stable maintenance and expression of cloned genes have streamlined the process in recent years. Advances in understanding mechanisms and substrate specificities during assembly by polyketide synthases, nonribosomal peptide synthetases, glycosyltransferases and other enzymes have made molecular engineering design and outcomes more predictable. Complex molecular scaffolds not amenable to synthesis by medicinal chemistry (for example, vancomycin (Vancocin), daptomycin (Cubicin) and erythromycin) are now tractable by molecular engineering. Medicinal chemistry can further embellish the properties of engineered antibiotics, making the two disciplines complementary.

Daptomycin, a cyclic lipopeptide produced by Streptomyces roseosporus, is the active ingredient of Cubicin (daptomycin-for-injection), a first-in-class antibiotic approved for treatment of skin and skin-structure infections caused by Gram-positive pathogens and bacteremia and endocarditis caused by Staphylococcus aureus, including methicillin-resistant strains. Genetic engineering of the nonribosomal peptide synthetase (NRPS) in the daptomycin biosynthetic pathway was exploited for the biosynthesis of novel active antibiotics. lambda-Red-mediated recombination was used to exchange single or multiple modules in the DptBC subunit of the NRPS to modify the daptomycin cyclic peptide core. We combined module exchanges, NRPS subunit exchanges, inactivation of the tailoring enzyme glutamic acid 3-methyltransferase, and natural variations of the lipid tail to generate a library of novel lipopeptides, some of which were as active as daptomycin against Gram-positive bacteria. One compound was more potent against an Escherichia coli imp mutant that has increased outer membrane permeability. This study established a robust combinatorial biosynthesis platform to produce novel peptide antibiotics in sufficient quantities for antimicrobial screening and drug development.

Daptomycin is a lipopeptide antibiotic produced by Streptomyces roseosporus and recently commercialized as Cubicin (daptomycin-for-injection) for treatment of skin and skin-structure infections caused by Gram-positive pathogens. Daptomycin is synthesized by a non-ribosomal peptide synthetase (NRPS) encoded by three overlapping genes, dptA, dptBC and dptD. The dptE and dptF genes, immediately upstream of dptA, are likely to be involved in the initiation of daptomycin biosynthesis by coupling decanoic acid to the N-terminal Trp. Analysis of RT-PCR data suggests that dptE, dptF, dptA, dptBC, dptD and possibly other dpt genes are transcribed as one large message; however, it has been demonstrated that sequential translation of these genes from a long transcript is not essential for robust daptomycin production. The dptA and the dptD genes were deleted from the dpt gene cluster, and expressed from ectopic positions in the chromosome under the control of the strong constitutive ermEp* promoter to produce high levels of lipopeptides. This three-locus trans-complementation system was used to produce hybrid lipopeptide antibiotics by introducing the heterologous lptD and cdaPS3 genes from Streptomyces fradiae and Streptomyces coelicolor, respectively, to complement the DeltadptD mutation.

In many peptide antibiotics, modified amino acids are important for biological activity. The amino acid 3-methyl-glutamic acid (3mGlu) has been found only in three cyclic lipopeptide antibiotics: daptomycin and the A21978C family produced by Streptomyces roseosporus, calcium-dependent antibiotic produced by Streptomyces coelicolor and A54145 produced by Streptomyces fradiae. We studied the non-ribosomal peptide synthetase genes involved in A21978C biosynthesis and the downstream genes, dptG, dptH, dptI and dptJ predicted to encode a conserved protein of unknown function, a thioesterase, a methyltransferase (MTase) and a tryptophan 2,3-dioxygenase respectively. Deletion of dptGHIJ reduced overall lipopeptide yield and led to production of a series of novel A21978C analogues containing Glu12 instead of 3mGlu12. Complementation by only dptI, or its S. coelicolor homologue, glmT, restored the biosynthesis of the 3mGlu-containing compounds in the mutant. Compared with A21978C, the Glu12-containing derivatives were less active against Staphylococcus aureus. Further genetic analyses showed that members of the dptGHIJ locus cooperatively contributed to optimal A21978C production; deletion of dptH, dptI or dptJ genes reduced the yield significantly, while expression of dptIJ or dptGHIJ from the strong ermEp* promoter substantially increased lipopeptide production. The results indicate that these genes play important roles in the biosynthesis of daptomycin, and that dptI encodes a Glu MTase.

There are few new antibiotics in the pipeline today. The reasons may include starvation at the front of the pipeline due to inadequate sources of suitable compounds to screen coupled with poorly validated discovery methodologies. A successful antibiotic discovery approach in the past, based upon whole cell antibiotic screening of natural products from actinomycetes and fungi, eventually suffered from constipation in the middle of the pipeline due to rediscovery of known compounds, even though low throughput methodology was employed at the front end. The current lack of productivity may be attributed to the poor choice of strategies to address the discovery of new antibiotics. Recent applications of high throughput in vitro screening of individual antibacterial targets to identify lead compounds from combinatorial chemical libraries, traditional chemical libraries, and partially purified natural product extracts has not produced any significant clinical candidates. The solution to the current dilemma may be to return to natural product whole cell screening. For this approach to work in the current millennium, the process needs to be miniaturized to increase the throughput by orders of magnitude over traditional screening, and the rediscovery of known antibiotics needs to be minimized by methods that can be readily monitored and improved over time.

Daptomycin is a lipopeptide antibiotic produced by a nonribosomal peptide synthetase (NRPS) in Streptomyces roseosporus. The holoenzyme is composed of three subunits, encoded by the dptA, dptBC, and dptD genes, each responsible for incorporating particular amino acids into the peptide. We introduced expression plasmids carrying dptD or NRPS genes encoding subunits from two related lipopeptide biosynthetic pathways into a daptomycin nonproducing strain of S. roseosporus harboring a deletion of dptD. All constructs successfully complemented the deletion in trans, generating three peptide cores related to daptomycin. When these were coupled with incomplete methylation of 1 amino acid and natural variation in the lipid side chain, 18 lipopeptides were generated. Substantial amounts of nine of these compounds were readily obtained by fermentation, and all displayed antibacterial activity against gram-positive pathogens.

Daptomycin and the A21978C antibiotic complex are lipopeptides produced by Streptomyces roseosporus and also in recombinant Streptomyces lividans TK23 and TK64 strains, when a 128 kbp region of cloned S. roseosporus DNA containing the daptomycin gene cluster is inserted site-specifically in the phiC31 attB site. A21978C fermentation yields were initially much lower in S. lividans than in S. roseosporus, and detection was complicated by the production of host metabolites. However A21978C production in S. lividans was improved by deletion of genes encoding the production of actinorhodin and by medium optimization to control the chemical form of the calcium dependent antibiotic (CDA). This latter compound has not previously been chemically characterized as a S. lividans product. Adding phosphate to a defined fermentation medium resulted in formation of only the phosphorylated forms of CDA, which were well separated from A21978C on chromatographic analysis. Adjusting the level of phosphate in the medium led to an improvement in A21978C yield from 20 to 55 mg/l.

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus. Cubicin (daptomycin-for-injection) was approved in 2003 by the FDA to treat skin and skin structure infections caused by Gram-positive pathogens. Daptomycin is particularly significant in that it represents the first new natural product antibacterial structural class approved for clinical use in three decades. The daptomycin gene cluster contains three very large genes (dptA, dptBC, and dptD) that encode the nonribosomal peptide synthetase (NRPS). The related cyclic lipopeptide A54145 has four NRPS genes (lptA, lptB, lptC, and lptD), and calcium dependent antibiotic (CDA) has three (cdaPS1, cdaPS2, and cdaPS3). Mutants of S. roseosporus containing deletions of one or more of the NRPS genes have been trans-complemented with dptA, dptBC, and dptD by inserting these genes under the control of the ermEp* promoter into separate conjugal cloning vectors containing phiC31 or IS117 attachment (attP int) sites; delivering the plasmids into S. roseosporus by conjugation from Escherichia coli; and inserting the plasmids site-specifically into the chromosome at the corresponding attB sites. This trans-complementation system was used to generate subunit exchanges with lptD and cdaPS3 and the recombinants produced novel hybrid molecules. Module exchanges at positions D: -Ala(8) and D: -Ser(11) in the peptide have produced additional novel derivatives of daptomycin. The approaches of subunit exchanges and module exchanges were combined with amino acid modifications of Glu at position 12 and natural variations in lipid side chain starter units to generate a combinatorial library of antibiotics related to daptomycin. Many of the engineered strains produced levels of novel molecules amenable to isolation and antimicrobial testing, and most of the compounds displayed antibacterial activities.