Regulation of daptomycin biosynthesis in Streptomyces roseosporus: new insights from genomic analysis and synthetic biology to accelerate lipopeptide discovery and commercial production.

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.

Genome mining for drug discovery: progress at the front end.

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.

Genome mining for drug discovery: cyclic lipopeptides related to daptomycin.

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.

Natural product drug discovery in the genomic era: realities, conjectures, misconceptions, and opportunities.

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-resistant industrial fermentation strains: from the cradle to CRISPR/Cas9.

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.

Synthetic biology advances and applications in the biotechnology industry: a perspective.

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.

Correction to: Synthetic biology, genome mining, and combinatorial biosynthesis of NRPS­derived antibiotics: a perspective.

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Synthetic biology, genome mining, and combinatorial biosynthesis of NRPS-derived antibiotics: a perspective.

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.

Molecular beacons to identify gifted microbes for genome mining.

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.

Gifted microbes for genome mining and natural product discovery.

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.

Natural product discovery: past, present, and future.

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.

Genetic manipulation of secondary metabolite biosynthesis for improved production in Streptomyces and other 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.