Modular metabolic engineering of Bacillus amyloliquefaciens for high-level production of green biosurfactant iturin A.

As a kind of biosurfactants, iturin A has attracted people's wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens. Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5'-UTRs of downstream genes (ituA, ituB, and ituC) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH, serC, and introducing ocD. Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. KEY POINTS: • Optimizing 5'-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far.

Cyclic Peptides from Graspetide Biosynthesis and Native Chemical Ligation.

The ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily of natural products includes many examples of cyclic peptides with diverse macrocyclization chemistries. The graspetides, one family of macrocyclized RiPPs, harbor side chain-side chain ester or amide linkages. We recently reported the structure and biosynthesis of the graspetide pre-fuscimiditide, a 22-amino-acid (aa) peptide with two ester cross-links forming a stem-loop structure. These cross-links are introduced by a single graspetide synthetase, the ATP-grasp enzyme ThfB. Here we show that ThfB can also catalyze the formation of amide or thioester cross-links in prefuscimiditide, with thioester formation being especially efficient. We further show that upon proteolysis to reveal an N-terminal cysteine residue, the thioester-linked peptide rapidly and quantitatively rearranges via native chemical ligation into an isopeptide-bonded head-to-tail cyclic peptide. The solution structure of this rearranged peptide was determined by using 2D NMR spectroscopy experiments. Our methodology offers a straightforward recombinant route to head-to-tail cyclic peptides.

Possible Functional Roles of Patellamides in the Ascidian-Prochloron Symbiosis.

Patellamides are highly bioactive compounds found along with other cyanobactins in the symbiosis between didemnid ascidians and the enigmatic cyanobacterium Prochloron. The biosynthetic pathway of patellamide synthesis is well understood, the relevant operons have been identified in the Prochloron genome and genes involved in patellamide synthesis are among the most highly transcribed cyanobacterial genes in hospite. However, a more detailed study of the in vivo dynamics of patellamides and their function in the ascidian-Prochloron symbiosis is complicated by the fact that Prochloron remains uncultivated despite numerous attempts since its discovery in 1975. A major challenge is to account for the highly dynamic microenvironmental conditions experienced by Prochloron in hospite, where light-dark cycles drive rapid shifts between hyperoxia and anoxia as well as pH variations from pH ~6 to ~10. Recently, work on patellamide analogues has pointed out a range of different catalytic functions of patellamide that could prove essential for the ascidian-Prochloron symbiosis and could be modulated by the strong microenvironmental dynamics. Here, we review fundamental properties of patellamides and their occurrence and dynamics in vitro and in vivo. We discuss possible functions of patellamides in the ascidian-Prochloron symbiosis and identify important knowledge gaps and needs for further experimental studies.

Biosynthesis of Tasikamides via Pathway Coupling and Diazonium-Mediated Hydrazone Formation.

Naturally occurring hydrazones are rare despite the ubiquitous usage of synthetic hydrazones in the preparation of organic compounds and functional materials. In this study, we discovered a family of novel microbial metabolites (tasikamides) that share a unique cyclic pentapeptide scaffold. Surprisingly, tasikamides A-C (1-3) contain a hydrazone group (C═N─N) that joins the cyclic peptide scaffold to an alkyl 5-hydroxylanthranilate (AHA) moiety. We discovered that the biosynthesis of 1-3 requires two discrete gene clusters, with one encoding a nonribosomal peptide synthetase (NRPS) pathway for assembling the cyclic peptide scaffold and another encoding the AHA-synthesizing pathway. The AHA gene cluster encodes three ancillary enzymes that catalyze the diazotization of AHA to yield an aryl diazonium species (diazo-AHA). The electrophilic diazo-AHA undergoes nonenzymatic Japp-Klingemann coupling with a ß-keto aldehyde-containing cyclic peptide precursor to furnish the hydrazone group and yield 1-3. The studies together unraveled a novel mechanism whereby specialized metabolites are formed by the coupling of two biosynthetic pathways via an unprecedented in vivo Japp-Klingemann reaction. The findings raise the prospect of exploiting the arylamine-diazotizing enzymes (AAD) for the in vivo synthesis of aryl compounds and modification of biological macromolecules.

Scalable and Efficient In Planta Biosynthesis of Sunflower Trypsin Inhibitor-1 (SFTI) Peptide Therapeutics.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14 amino acid cyclic peptide which has been effectively employed as a scaffold for engineering a range of peptide therapeutic candidates. Typically, synthesis of SFTI-1-based therapeutics is performed via solid-phase peptide synthesis and native chemical ligation, with significant financial and environmental costs associated. In planta synthesis of SFTI-1 based therapeutics serves as a greener approach for environmentally sustainable production. Here, we detail the methods for the transient expression, production, and purification of SFTI-1-based therapeutic peptides in Nicotiana benthamiana using a scalable and high-throughput approach. We demonstrate that a prerequisite for this is the co-expression of specialized asparaginyl endopeptidases (AEPs) that perform the backbone cyclization of SFTI-1. In our founding study, we were able to achieve in planta yields of a plasmin inhibitor SFTI-1 peptide at yields of ~60 µg/g of dried plant material.

Discovery and biosynthesis of cyclic plant peptides via autocatalytic cyclases.

Many bioactive plant cyclic peptides form side-chain-derived macrocycles. Lyciumins, cyclic plant peptides with tryptophan macrocyclizations, are ribosomal peptides (RiPPs) originating from repetitive core peptide motifs in precursor peptides with plant-specific BURP (BNM2, USP, RD22 and PG1beta) domains, but the biosynthetic mechanism for their formation has remained unknown. Here, we characterize precursor-peptide BURP domains as copper-dependent autocatalytic peptide cyclases and use a combination of tandem mass spectrometry-based metabolomics and plant genomics to systematically discover five BURP-domain-derived plant RiPP classes, with mono- and bicyclic structures formed via tryptophans and tyrosines, from botanical collections. As BURP-domain cyclases are scaffold-generating enzymes in plant specialized metabolism that are physically connected to their substrates in the same polypeptide, we introduce a bioinformatic method to mine plant genomes for precursor-peptide-encoding genes by detection of repetitive substrate domains and known core peptide features. Our study sets the stage for chemical, biosynthetic and biological exploration of plant RiPP natural products from BURP-domain cyclases.

Simple and rapid pipeline for the production of cyclic and linear small-sized peptides in E. coli.

Small and medium-sized peptides are gaining popularity in biomedical applications, including therapeutic target development. As an alternative to chemical synthesis, we describe a complete pipeline for the production of linear as well as structurally constrained cyclic peptides in an E. coli expression system in this study. A plasmid vector containing a novel N terminal HOE tag (28 amino acids in length) that fuses with the peptide was created. The HOE tag contains sites for both chemical (CNBr) and enzymatic (enterokinase) cleavage, making it easy to isolate the peptide after production. A total of 21 peptides (17 cyclic and 4 linear) were synthesized, and the HOE tag was successfully removed using either CNBr (9 peptides) or enterokinase (12 peptides). The presence of a disulfide bond was confirmed in six representative cyclic peptides. In this study we have provided detailed instructions on primers design strategy, overexpression and purification of HOE tagged peptides, chemical and enzymatic cleavage, and confirmation of the cyclic form of peptides. We are confident that this pipeline will assist researchers in producing multiple recombinant peptides in a cost-effective and time-efficient manner.

Gene editing enables rapid engineering of complex antibiotic assembly lines.

Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.

In vitro elucidation of the crucial but complex oxidative tailoring steps in rufomycin biosynthesis enables one pot conversion of rufomycin B to rufomycin C.

The antimycobacterial peptides, rufomycins, have their antibiotic activity conferred by oxidative tailoring of the cyclic peptide. Here we elucidate the roles of cytochrome P450s RufS and RufM in regioselective epoxidation and alkyl oxidation respectively and demonstrate how RufM and RufS create a complex product profile dependent on redox partner availability. Finally, we report the in vitro one pot conversion of rufomycin B to rufomycin C.

Thirtieth Anniversary of the Discovery of Laxaphycins. Intriguing Peptides Keeping a Part of Their Mystery.

Lipopeptides are a class of compounds generally produced by microorganisms through hybrid biosynthetic pathways involving non-ribosomal peptide synthase and a polyketyl synthase. Cyanobacterial-produced laxaphycins are examples of this family of compounds that have expanded over the past three decades. These compounds benefit from technological advances helping in their synthesis and characterization, as well as in deciphering their biosynthesis. The present article attempts to summarize most of the articles that have been published on laxaphycins. The current knowledge on the ecological role of these complex sets of compounds will also be examined.

Anabaenopeptins: What We Know So Far.

Cyanobacteria are microorganisms with photosynthetic mechanisms capable of colonizing several distinct environments worldwide. They can produce a vast spectrum of bioactive compounds with different properties, resulting in an improved adaptative capacity. Their richness in secondary metabolites is related to their unique and diverse metabolic apparatus, such as Non-Ribosomal Peptide Synthetases (NRPSs). One important class of peptides produced by the non-ribosomal pathway is anabaenopeptins. These cyclic hexapeptides demonstrated inhibitory activity towards phosphatases and proteases, which could be related to their toxicity and adaptiveness against zooplankters and crustaceans. Thus, this review aims to identify key features related to anabaenopeptins, including the diversity of their structure, occurrence, the biosynthetic steps for their production, ecological roles, and biotechnological applications.

Biosynthetic Strategies for Macrocyclic Peptides.

Macrocyclic peptides are predominantly peptide structures bearing one or more rings and spanning multiple amino acid residues. Macrocyclization has become a common approach for improving the pharmacological properties and bioactivity of peptides. A variety of ribosomal-derived and non-ribosomal synthesized cyclization approaches have been established. The biosynthesis of backbone macrocyclic peptides using seven new emerging methodologies will be discussed with regard to the features and strengths of each platform rather than medicinal chemistry tools. The mRNA display variant, known as the random nonstandard peptide integrated discovery (RaPID) platform, utilizes flexible in vitro translation (FIT) to access macrocyclic peptides containing nonproteinogenic amino acids (NAAs). As a new discovery approach, the ribosomally synthesized and post-translationally modified peptides (RiPPs) method involves the combination of ribosomal synthesis and the phage screening platform together with macrocyclization chemistries to generate libraries of macrocyclic peptides. Meanwhile, the split-intein circular ligation of peptides and proteins (SICLOPPS) approach relies on the in vivo production of macrocyclic peptides. In vitro and in vivo peptide library screening is discussed as an advanced strategy for cyclic peptide selection. Specifically, biosynthetic bicyclic peptides are highlighted as versatile and attractive modalities. Bicyclic peptides represent another type of promising therapeutics that allow for building blocks with a heterotrimeric conjugate to address intractable challenges and enable multimer complexes via linkers. Additionally, we discuss the cell-free chemoenzymatic synthesis of macrocyclic peptides with a non-ribosomal catalase known as the non-ribosomal synthetase (NRPS) and chemo-enzymatic approach, with recombinant thioesterase (TE) domains. Novel insights into the use of peptide library tools, activity-based two-hybrid screening, structure diversification, inclusion of NAAs, combinatorial libraries, expanding the toolbox for macrocyclic peptides, bicyclic peptides, chemoenzymatic strategies, and future perspectives are presented. This review highlights the broad spectrum of strategy classes, novel platforms, structure diversity, chemical space, and functionalities of macrocyclic peptides enabled by emerging biosynthetic platforms to achieve bioactivity and for therapeutic purposes.

Stepwise genetic engineering of Pseudomonas putida enables robust heterologous production of prodigiosin and glidobactin A.

Polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) comprise biosynthetic pathways that provide access to diverse, often bioactive natural products. Metabolic engineering can improve production metrics to support characterization and drug-development studies, but often native hosts are difficult to genetically manipulate and/or culture. For this reason, heterologous expression is a common strategy for natural product discovery and characterization. Many bacteria have been developed to express heterologous biosynthetic gene clusters (BGCs) for producing polyketides and nonribosomal peptides. In this article, we describe tools for using Pseudomonas putida, a Gram-negative soil bacterium, as a heterologous host for producing natural products. Pseudomonads are known to produce many natural products, but P. putida production titers have been inconsistent in the literature and often low compared to other hosts. In recent years, synthetic biology tools for engineering P. putida have greatly improved, but their application towards production of natural products is limited. To demonstrate the potential of P. putida as a heterologous host, we introduced BGCs encoding the synthesis of prodigiosin and glidobactin A, two bioactive natural products synthesized from a combination of PKS and NRPS enzymology. Engineered strains exhibited robust production of both compounds after a single chromosomal integration of the corresponding BGC. Next, we took advantage of a set of genome-editing tools to increase titers by modifying transcription and translation of the BGCs and increasing the availability of auxiliary proteins required for PKS and NRPS activity. Lastly, we discovered genetic modifications to P. putida that affect natural product synthesis, including a strategy for removing a carbon sink that improves product titers. These efforts resulted in production strains capable of producing 1.1 g/L prodigiosin and 470 mg/L glidobactin A.

Residual biomass from surfactin production is a source of arginase and adsorbed surfactin that is useful for environmental remediation.

Lipopeptides are important secondary metabolites produced by microbes. They find applications in environmental decontamination and in the chemical, pharmaceutical and food industries. However, their production is expensive. In the present work we propose three strategies to lower the production costs of surfactin. First, the coproduction of surfactin and arginase in a single growth. Second, extract the fraction of surfactin that adsorbs to the biomass and is removed from the growth medium through centrifugation. Third, use microbial biomass for the remediation of organic and inorganic contaminants. The coproduction of surfactin and arginase was evaluated by factorial design experiments using the LB medium supplemented with arginine. The best conditions for surfactin production were 22 h of growth at 37 °C using LB supplemented with arginine 7.3 g/L. Almost similar conditions were found to produce highest levels of arginase, 24 h and 6.45 g/L arginine. Decontamination of phenol and copper from artificial samples was attained by treatment with residues from lipopeptide production. Thus, cell suspensions and wash-waters used to extract surfactin from the biomass. Cell suspensions were used to successfully remove hydroquinone. Cell suspensions and wash-waters containing surfactin were successfully used to recover copper from solution. Specific monitoring methods were used for phenol and metal solutions, respectively a biosensor based on tyrosinase and either atomic absorption flame ionization spectrometry or absorbance coupled to the Arduino™ platform. Therefore, we report three alternative strategies to lower the production costs in lipopeptide production, which include the effective recovery of copper and phenol from contaminated waters using residues from surfactin production. Sustainable and profitable production of surfactin can be achieved by a coproduction strategy of lipopeptides and enzymes. Lipopeptides are collected in the supernatant and enzymes in the biomass. In addition, lipopeptides that coprecipitate with biomass can be recovered by washing. Lipopeptide wash-waters find applications in remediation and cells can also be used for environmental decontamination.

Host nutrition-based approach for biotechnological production of the antifungal cyclic lipopeptide jagaricin.

In today's, society multi-resistant pathogens have become an emerging threat, which makes the search for novel anti-infectives more urgent than ever. A promising class of substances are cyclic lipopeptides like the antifungal jagaricin. Jagaricin is formed by the bacterial mushroom pathogen Janthinobacterium agaricidamnosum. It has shown antifungal activity against human pathogenic fungi like Candida albicans and Aspergillus fumigatus. In addition, jagaricin is nearly non-toxic for plants, which makes it a promising agent for agricultural applications. Cyclic lipopeptides formed by microorganisms originate from their secondary metabolism. This makes it very challenging to determine the inducing factor for product formation, especially for unknown microbial systems like J. agaricidamnosum. In the presented study, a biotechnological process for jagaricin formation was developed, investigating impact factors like the medium, oxygen availability, and phosphate. For this reason, experiments were conducted on microtiter plate, shake flask, and stirred tank bioreactor level. Ultimately, a final maximum jagaricin concentration of 251 mg L-1 (15.5 mgJagaricin∙gCDW-1) could be achieved, which is an increase of approximately 458 % in comparison to previous results in standard glucose medium. This concentration allows the production of significantly higher amounts of jagaricin and enables further experiments to investigate the potential of this substance.

Genetic engineering of the precursor supply pathway for the overproduction of the nC14-surfactin isoform with promising MEOR applications.

BACKGROUND: Surfactin, a representative biosurfactant of lipopeptide mainly produced by Bacillus subtilis, consists of a cyclic heptapeptide linked to a ß-hydroxy fatty acid chain. The functional activity of surfactin is closely related to the length and isomerism of the fatty acid chain. RESULTS: In this study, the fatty acid precursor supply pathway in Bacillus subtilis 168 for surfactin production was strengthened through two steps. Firstly, pathways competing for the precursors were eliminated with inactivation of pps and pks. Secondly, the plant medium-chain acyl-carrier protein (ACP) thioesterase (BTE) from Umbellularia californica was overexpressed. As a result, the surfactin titer after 24 h of cultivation improved by 34%, and the production rate increased from 0.112 to 0.177 g/L/h. The isoforms identified by RP-HPLC and GC-MS showed that the proportion of nC14-surfactin increased 6.4 times compared to the control strain. A comparison of further properties revealed that the product with more nC14-surfactin had higher surface activity and better performance in oil-washing. Finally, the product with more nC14-surfactin isoform had a higher hydrocarbon-emulsification index, and it increased the water-wettability of the oil-saturated silicate surface. CONCLUSION: The obtained results identified that enhancing the supply of fatty acid precursor is very essential for the synthesis of surfactin. At the same time, this study also proved that thioesterase BTE can promote the production of nC14-surfactin and experimentally demonstrated its higher surface activity and better performance in oil-washing. These results are of great significance for the MEOR application of surfactin.

Microparticles enhance the formation of seven major classes of natural products in native and metabolically engineered actinobacteria through accelerated morphological development.

Actinobacteria provide a rich spectrum of bioactive natural products and therefore display an invaluable source towards commercially valuable pharmaceuticals and agrochemicals. Here, we studied the use of inorganic talc microparticles (hydrous magnesium silicate, 3MgO·4SiO2 ·H2 O, 10 µm) as a general supplement to enhance natural product formation in this important class of bacteria. Added to cultures of recombinant Streptomyces lividans, talc enhanced production of the macrocyclic peptide antibiotic bottromycin A2 and its methylated derivative Met-bottromycin A2 up to 109 mg L-1 , the highest titer reported so far. Hereby, the microparticles fundamentally affected metabolism. With 10 g L-1 talc, S. lividans grew to 40% smaller pellets and, using RNA sequencing, revealed accelerated morphogenesis and aging, indicated by early upregulation of developmental regulator genes such as ssgA, ssgB, wblA, sigN, and bldN. Furthermore, the microparticles re-balanced the expression of individual bottromycin cluster genes, resulting in a higher macrocyclization efficiency at the level of BotAH and correspondingly lower levels of non-cyclized shunt by-products, driving the production of mature bottromycin. Testing a variety of Streptomyces species, talc addition resulted in up to 13-fold higher titers for the RiPPs bottromycin and cinnamycin, the alkaloid undecylprodigiosin, the polyketide pamamycin, the tetracycline-type oxytetracycline, and the anthramycin-analogs usabamycins. Moreover, talc addition boosted production in other actinobacteria, outside of the genus of Streptomyces: vancomycin (Amycolatopsis japonicum DSM 44213), teicoplanin (Actinoplanes teichomyceticus ATCC 31121), and the angucyclinone-type antibiotic simocyclinone (Kitasatospora sp.). For teicoplanin, the microparticles were even crucial to activate production. Taken together, the use of talc was beneficial in 75% of all tested cases and optimized natural and heterologous hosts forming the substance of interest with clusters under native and synthetic control. Given its simplicity and broad benefits, microparticle-supplementation appears as an enabling technology in natural product research of these most important microbes.

Influence of Culture Substrates on Morphology and Function of Pulmonary Alveolar Cells In Vitro.

Cell's microenvironment has been shown to exert influence on cell behavior. In particular, matrix-cell interactions strongly impact cell morphology and function. The purpose of this study was to analyze the influence of different culture substrate materials on phenotype and functional properties of lung epithelial adenocarcinoma (A549) cells. A549 cells were seeded onto two different biocompatible, commercially available substrates: a polyester coverslip (Thermanox™ Coverslips), that was used as cell culture plate control, and a polydimethylsiloxane membrane (PDMS, Elastosil® Film) investigated in this study as alternative material for A549 cells culture. The two substrates influenced cell morphology and the actin cytoskeleton organization. Further, the Yes-associated protein (YAP) and its transcriptional coactivator PDZ-binding motif (TAZ) were translocated to the nucleus in A549 cells cultured on polyester substrate, yet it remained mostly cytosolic in cells on PDMS substrate. By SEM analysis, we observed that cells grown on Elastosil® Film maintained an alveolar Type II cell morphology. Immunofluorescence staining for surfactant-C revealing a high expression of surfactant-C in cells cultured on Elastosil® Film, but not in cells cultured on Thermanox™ Coverslips. A549 cells grown onto Elastosil® Film exhibited morphology and functionality that suggest retainment of alveolar epithelial Type II phenotype, while A549 cells grown onto conventional plastic substrates acquired an alveolar Type I phenotype.

An Atypical Arginine Dihydrolase Involved in the Biosynthesis of Cyclic Hexapeptide Longicatenamides.

The incorporation of non-proteinogenic amino acids (NPAAs) enriches the structural diversity of nonribosomal peptides. Recently, four NPAA-containing cyclic hexapeptides, longicatenamides A-D, were isolated using a combined-culture strategy. Based on in silico analysis, we discovered their putative biosynthetic gene cluster (lon) and proposed a possible biosynthetic mechanism. Surprisingly, the lon22 gene encodes an atypical arginine dihydrolase, which can also catalyze the hydrolysis of citrulline to ornithine. Phylogenetic analysis showed that Lon22-like proteins form a novel clade that is separated from other guanidine-modifying enzymes. After rational design, the catalytic efficiencies of a Lon22 Y80F mutant for arginine and citrulline substrates were 2.31- and 4.70-fold that of the wild-type (WT), respectively. In addition, characterization of the Lon20-A4 adenylation domain suggested that it can incorporate both ornithine and lysine into the final products.

Integrated Omics Strategy Reveals Cyclic Lipopeptides Empedopeptins from Massilia sp. YMA4 and Their Biosynthetic Pathway.

Empedopeptins-eight amino acid cyclic lipopeptides-are calcium-dependent antibiotics that act against Gram-positive bacteria such as Staphylococcus aureus by inhibiting cell wall biosynthesis. However, to date, the biosynthetic mechanism of the empedopeptins has not been well identified. Through comparative genomics and metabolomics analysis, we identified empedopeptin and its new analogs from a marine bacterium, Massilia sp. YMA4. We then unveiled the empedopeptin biosynthetic gene cluster. The core nonribosomal peptide gene null-mutant strains (ΔempC, ΔempD, and ΔempE) could not produce empedopeptin, while dioxygenase gene null-mutant strains (ΔempA and ΔempB) produced several unique empedopeptin analogs. However, the antibiotic activity of ΔempA and ΔempB was significantly reduced compared with the wild-type, demonstrating that the hydroxylated amino acid residues of empedopeptin and its analogs are important to their antibiotic activity. Furthermore, we found seven bacterial strains that could produce empedopeptin-like cyclic lipopeptides using a genome mining approach. In summary, this study demonstrated that an integrated omics strategy can facilitate the discovery of potential bioactive metabolites from microbial sources without further isolation and purification.