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Macrolactins have attracted considerable attention due to their value and application in medicine and agriculture. However, poor yields severely hinder their broader application in these fields. This study aimed to improve macrolactins production in Bacillus siamensis using a combined atmospheric and room-temperature plasma mutagenesis and a microbial microdroplet culture system. After 25 days of treatment, a desirable strain with macrolactins production 3.0-fold higher than that of the parental strain was successfully selected. The addition of 30 mg/L ZnSO 4 further increased macrolactins production to 503 ± 37.6 μg/mL, representing a 30.9 % improvement in production compared to controls. Based on transcriptome analysis, the synthesis pathways of amino acids, fengycin, and surfactin were found to be downregulated in IMD4036. Further fermentation experiments confirmed that inhibition of the comparative fengycin synthesis pathway was potentially driving the increased production of macrolactins. The strategies and possible mechanisms detailed in this study can provide insight into enhancing the production of other secondary metabolites toxic to the producer strains., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The histidine bridge is a rare and often overlooked structural motif in macrocyclic peptide natural products, yet there are several examples in nature of cyclic peptides bearing this moiety that exhibit potent biological activity. These interesting compounds have been the focus of several studies reporting their isolation, biosynthesis and chemical synthesis over the last four decades. This review summarises the findings on the structure, biological activity and, where possible, proposed biosynthesis and progress towards the synthesis of histidine-bridged cyclic peptides.

Genome analysis of Kutzneria sp. CA-103260 revealed a putative lipopeptide-encoding biosynthetic gene cluster (BGC) that was cloned into a bacterial artificial chromosome (BAC) and heterologously expressed in Streptomyces coelicolor M1152. As a result, a novel cyclic lipo-tetrapeptide containing two diaminopropionic acid residues and an exotic N , N -acetonide ring, kutzneridine A ( 1 ), was isolated and structurally characterized. Evaluation of the extraction conditions and isotope-labeling chemical modifications showed that the acetonide ring originated from acetone during isolation. The BGC was analyzed in silico and a biosynthetic pathway to 1 was proposed. Kutzneridine A displayed remarkable antibacterial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci .

Fungal biosynthetic gene clusters often include genes encoding transmembrane proteins, which have been mostly thought to be transporters exporting the products. However, there is little knowledge about subcellular compartmentalization of transmembrane proteins essential for biosynthesis. Fungal mycotoxin cyclochlorotine is synthesized by non-ribosomal peptide synthetase, which is followed by modifications with three transmembrane UstYa-family proteins. Heterologous expression in Aspergillus oryzae revealed that total biosynthesis of cyclochlorotine requires additional two transporter proteins. Here, we investigated subcellular localizations of the five transmembrane proteins under heterologous expression in A. oryzae. Enhanced green fluorescent protein (EGFP) fusions to the transmembrane proteins, which were confirmed to normally function in cyclochlorotine production, were expressed together with organellar markers. All the transmembrane proteins exhibited localizations commonly in line of the trans-Golgi, endosomes, and vacuoles. This study suggests that subcellular compartmentalization of UstYa family proteins and transporters allows corporative functions of delivering intermediates and subsequent modifications, completing cyclochlorotine biosynthesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Aflatoxins (AFs) are highly toxic mycotoxins produced by the fungi, Aspergillus flavus and Aspergillus parasiticus. AFs pose severe health risks owing to their acute toxicity and carcinogenic properties. The control of AF contamination remains significantly challenging despite the extensive efforts toward controlling it. Here, we investigated the potential of mushroom extracts as a source of AF biosynthetic inhibitors. The A. parasiticus mutant strain, NFRI-95, that accumulates an AF biosynthesis intermediate, norsolorinic acid, was used in the bioassay to detect the inhibitory activity against AF biosynthesis. The screening of 195 mushroom extracts revealed that the culture filtrate extract of Chondrostereum purpureum exhibited strong inhibitory activity against AF biosynthesis. Next, large-scale culturing of C. purpureum was performed to isolate the compounds accounting for the inhibitory activity. The culture filtrate was extracted with ethyl acetate, after which the active compound was isolated by silica gel column chromatography and preparative high performance liquid chromatography (HPLC). The active compound was identified as cyclo(Val-Pro) by spectroscopic analyses. Further, four stereoisomers of cyclo(Val-Pro) were synthesized by the condensation of the N-Boc derivatives of d- and l-valine with the methyl esters of d- and l-proline. The naturally isolated compound was identified as cyclo(l-Val-l-Pro) by comparing its retention time with those of synthetic compounds by chiral HPLC analysis and CD spectra. The IC 50 value of cyclo(L-Val-L-Pro) was 2.4 mM, whereas the LD, DL, and DD isomers exhibited weaker activities, with IC 50 values of >5 mM., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: The production of surfactin, an extracellular accumulating lipopeptide produced by various Bacillus species, is a well-known representative of microbial biosurfactant. However, only limited information is available on the correlation between the growth rate of the production strain, such as B. subtilis BMV9, and surfactin production. To understand the correlation between biomass formation over time and surfactin production, the availability of glucose as carbon source was considered as main point. In fed-batch bioreactor processes, the B. subtilis BMV9 was used, a strain well-suited for high cell density fermentation. By adjusting the exponential feeding rates, the growth rate of the surfactin-producing strain, was controlled., Results: Using different growth rates in the range of 0.075 and 0.4 h -1 , highest surfactin titres of 36 g/L were reached at 0.25 h -1 with production yields Y P/S of 0.21 g/g and Y P/X of 0.7 g/g, while growth rates lower than 0.2 h -1 resulted in insufficient and slowed biomass formation as well as surfactin production (Y P/S of 0.11 g/g and Y P/X of 0.47 g/g for 0.075 h -1 ). In contrast, feeding rates higher than 0.25 h -1 led to a stimulation of overflow metabolism, resulting in increased acetate formation of up to 3 g/L and an accumulation of glucose due to insufficient conversion, leading to production yields Y P/S of 0.15 g/g and Y P/X of 0.46 g/g for 0.4 h -1 ., Conclusions: Overall, the parameter of adjusting exponential feeding rates have an important impact on the B. subtilis productivity in terms of surfactin production in fed-batch bioreactor processes. A growth rate of 0.25 h -1 allowed the highest surfactin production yield, while the total conversion of substrate to biomass remained constant at the different growth rates., (© 2024. The Author(s).)

Cyclo (Phe-Pro) (cFP), a cyclic dipeptide with notable antifungal, antibacterial, and antiviral properties, shows great promise for biological control of plant diseases. Produced as a byproduct by non-ribosomal peptide synthetases (NRPS), the regulatory mechanism of cFP biosynthesis remains unclear. In a screening test of 997 Tn5 mutants of Burkholderia seminalis strain R456, we identified eight mutants with enhanced antagonistic effects against Fusarium graminearum (Fg). Among these, mutant 88's culture filtrate contained cFP, confirmed through HPLC and LC-MS, which actively inhibited Fg. The gene disrupted in mutant 88 is part of the Dct transport system (Dct-A, -B, -D), responsible for C4-dicarboxylate transport. Knockout mutants of Dct genes exhibited higher cFP levels than the wild type, whereas complementary strains showed no significant difference. Additionally, the presence of exogenous C4-dicarboxylates reduced cFP production in wild type R456, indicating that these substrates negatively regulate cFP synthesis. Given that cFP synthesis is related to NRPS, we previously identified an NRPS cluster in R456, horizontally transferred from algae. Specifically, knocking out gene 2061 within this NRPS cluster significantly reduced cFP production. A Fur box binding site was predicted upstream of gene 2061, and yeast one-hybrid assays confirmed Fur protein binding, which increased with additional C4-dicarboxylates. Knockout of the Fur gene led to up-regulation of gene 2061 and increased cFP production, suggesting that C4-dicarboxylates suppress cFP synthesis by enhancing Fur-mediated repression of gene 2061., (© 2024. The Author(s).)

The emergence of multidrug-resistant fungi Candida auris is a worldwide health crisis connected with high rates of mortality. There is a critical need to find novel and unique antifungal compounds for treating infections of multidrug-resistant fungi such as C. auris . This study aimed to illustrate that biosynthetic gene clusters in native bacterial isolates are able to produce antifungal compounds against the multidrug-resistant fungus C. auris. It was successfully achieved using large-scale antifungal activity screening, cytotoxicity analysis, and whole genome sequencing integrated with genome mining-guided analysis and liquid chromatography-mass spectrometry (LC/MS). A list of possible gene candidates was initially identified with genome mining methods to predict secondary metabolite gene clusters of antifungal-compound-producing bacteria. Then, gene clusters present in the antifungal-compound-producing bacteria were identified and aligned with the reference genome using comparative genomic approaches. Bacillus halotolerans AQ11M9 was identified through large-scale antifungal activity screening as a natural compound-producer against multidrug-resistant C. auris , while it was nontoxic to normal human skin fibroblast cells (confirmed using a cell viability assay). The genome (4,197,347 bp) of B. halotolerans AQ11M9 with 2931 predicted genes was first mined for detecting and characterizing biosynthetic gene clusters, which revealed 10 candidate regions with antifungal activity. Clusters of AQ11M9 encoded non-ribosomal peptide synthase (NRPS) (bacilysin, bacillibactin, paenibactin, surfactin, plipastin, and fengycin) and polyketide (macrobrevin). The presence of gene clusters with anti- C. auris activity, and surfactin identified through LC/MS, from AQ11M9 suggests the potential of utilizing it as a source for a novel and powerful anti- C. auris compound.

Cyclic peptides, with remarkable stability, cellular permeability, and proteolysis resistance, display promising potential in pharmaceutical applications. Labionin (Lab), a unique bicyclic cross-link containing both C-C and C-S bonds, provides high rigidity and better control of conformation compared to monocyclic cross-links. To discover more Lab-containing scaffolds with highly rigid conformation for cyclic peptide drug development, herein, a cryptic class III lanthipeptide biosynthetic gene cluster (BGC) (i.e., rcs ) was identified in the sponge-associated Streptomyces rochei MB037 and expressed in Escherichia coli , incorporating an N-terminal SUMO-tag on the RcsA precursor peptide to prevent proteolysis. Subsequently, a novel class III lanthipeptide, i.e., rochsin A, exhibiting a highly rigid conformation with coupled Lab cross-links crowded by bulky aromatic amino acids, was produced. Three AplP-like proteases outside the rcs BGC were proven to remove the leader peptide of rochsin A through their dual endo- and aminopeptidase activities, resulting in mature rochsin A in vitro . Ala mutation experiments revealed the C to N cyclization direction, like most class III lanthipeptides. However, RcsKC displays a high substrate breadth, enabling various ring topologies that are rarely observed in other class III lanthipeptides. Overall, the established expression system broadens the chemical diversity of cyclic peptides with unique Lab cross-links and offers a highly rigid scaffold for cyclic peptide drug development.

Introduction: Optimal exploitation of the huge amounts of agro-industrial residuals that are produced annually, which endangers the ecosystem and ultimately contributes to climate change, is one of the solutions available to produce value-added compounds., Aim and Objectives: This study aimed at the economic production and optimization of surfactin. Therefore, the production was carried out by the microbial conversion of Potato Peel Waste (PPW) and Frying Oil Waste (FOW) utilizing locally isolated Bacillus halotolerans. Also, investigating its potential application as an antimicrobial agent towards some pathogenic strains., Results: Screening the bacterial isolates for surfactin production revealed that the strain with the highest yield (49 g/100 g substrate) and efficient oil displacement activity was genetically identified as B. halotolerans. The production process was then optimized utilizing Central Composite Design (CCD) resulting in the amelioration of yield by 11.4% (from 49 to 55.3 g/100 g substrate) and surface tension (ST) by 8.3% (from 36 to 33 mN/m) with a constant level of the critical micelle concentration (CMC) at 125 mg/L. Moreover, the physiochemical characterization studies of the produced surfactin by FTIR, 1 H NMR, and LC-MS/MS proved the existence of a cyclic lipopeptide (surfactin). The investigations further showed a strong emulsification affinity for soybean and motor oil (E24 = 50%), as well as the ability to maintain the emulsion stable over a wide pH (4-10) and temperature (10-100 °C) range. Interestingly, surfactin had a broad-spectrum range of inhibition activity against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, klebsiella pneumonia, and Candida albicans., Conclusion: Subsequently, the screening of the isolates and the utilized food-processing wastes along with the extraction technique resulted in a high yield of surfactin characterized by acceptable ST and CMC levels. However, optimization of the cultural conditions to improve the activity and productivity was achieved using Factor-At-A-Time (OFAT) and Central Composite Design (CCD). In contrast, surface activity recorded a maximum level of (33 mN/n) and productivity of 55.3 g/100 g substrate. The optimized surfactin had also the ability to maintain the stability of emulsions over a wide range of pH and temperature. Otherwise, the obtained results proved the promising efficiency of the surfactin against bacterial and fungal pathogens., (© 2024. The Author(s).)

Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes ( rapC , rapF , and rapH ) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens .

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.

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., (© 2024. The Author(s).)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 β-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.

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.

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., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2021. The Author(s).)

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.

Covering: 1950s up to the end of 2020Bottromycins are a class of macrocyclic peptide natural products that are produced by several Streptomyces species and possess promising antibacterial activity against clinically relevant multidrug-resistant pathogens. They belong to the ribosomally synthesised and post-translationally modified peptide (RiPP) superfamily of natural products. The structure contains a unique four-amino acid macrocycle formed via a rare amidine linkage, C-methylation and a D-amino acid. This review covers all aspects of bottromycin research with a focus on recent years (2009-2020), in which major advances in total synthesis and understanding of bottromycin biosynthesis were achieved.

We conducted global genome mining of 162,672 bacterial genomes and identified 829 cyclodipeptide (CDP) biosynthesis gene clusters (BGC) containing a cytochrome P450 gene. Heterologous expression of BGC from Saccharopolyspora hirsuta DSM 44795 led to the identification of two novel crownlike CDPs, cyctetryptomycin A ( 4 ) and B ( 5 ), which possess unprecedented complex macrocycle and show neuroprotective activity. The two cytochrome P450s found in the BGC catalyze sequential reactions leading to the cyclization of diketopiperazine dimers.

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., (Copyright © 2021 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

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.

Proline-rich macrocyclic peptides (PRMPs) are natural products present in geographically and phylogenetically dispersed marine sponges. The large diversity and low abundance of PRMPs in sponge metabolomes precludes isolation and structure elucidation of each individual PRMP congener. Here, using standards developed via biomimetic enzymatic synthesis of PRMPs, a mass spectrometry-based workflow to sequence PRMPs was developed and validated to reveal that the diversity of PRMPs in marine sponges is much greater than that has been realized by natural product isolation-based strategies. Findings are placed in the context of diversity-oriented transamidative macrocyclization of peptide substrates in sponge holobionts., (© 2021 Wiley-VCH GmbH.)

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 mg Jagaricin ∙g CDW -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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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·4SiO 2 ·H 2 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., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

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.

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.

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.

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., (© 2021 Wiley-VCH GmbH.)

Background: Surfactin, a representative biosurfactant of lipopeptide mainly produced by Bacillus subtilis, consists of a cyclic heptapeptide linked to a β-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 nC 14 -surfactin increased 6.4 times compared to the control strain. A comparison of further properties revealed that the product with more nC 14 -surfactin had higher surface activity and better performance in oil-washing. Finally, the product with more nC 14 -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 nC 14 -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.

The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881-1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Clostridia coordinate many important processes such as toxin production, infection, and survival by density-dependent communication (quorum sensing) using autoinducing peptides (AIPs). Although clostridial AIPs have been proposed to be (thio)lactone-containing peptides, their true structures remain elusive. Here, we report the genome-guided discovery of an AIP that controls endospore formation in Ruminiclostridium cellulolyticum. Through a combination of chemical synthesis and chemical complementation assays with a mutant strain, we reveal that the genuine chemical mediator is a homodetic cyclopeptide (cAIP). Kinetic analyses indicate that the mature cAIP is produced via a cryptic thiolactone intermediate that undergoes a rapid S→N acyl shift, in a manner similar to intramolecular native chemical ligation (NCL). Finally, by implementing a chemical probe in a targeted screen, we show that this novel enzyme-primed, intramolecular NCL is a widespread feature of clostridial AIP biosynthesis., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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.

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 (Δ empC , Δ empD , and Δ empE ) could not produce empedopeptin, while dioxygenase gene null-mutant strains (Δ empA and Δ empB ) produced several unique empedopeptin analogs. However, the antibiotic activity of Δ empA and Δ empB 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.

Mycotoxin cyclochlorotine ( 1 ) and structurally related astins are cyclic pentapeptides containing unique nonproteinogenic amino acids, such as β-phenylalanine, l- allo -threonine, and 3,4-dichloroproline. Herein, we report the biosynthetic pathway for 1 , which involves intriguing tailoring processes mediated by DUF3328 proteins, including stereo- and regiospecific chlorination and hydroxylation and intramolecular O , N -transacylation. Our findings demonstrate that DUF3328 proteins, which are known to be involved in oxidative cyclization of fungal ribosomal peptides, have much higher functional diversity than previously expected.

Coprisamides C and D ( 1 and 2 ) were isolated from a gut bacterium, Micromonospora sp. UTJ3, of the carrion beetle Silpha perforata . Based on the combined analysis of UV, MS, and NMR spectral data, the planar structures of 1 and 2 were elucidated to be unreported derivatives of coprisamides A and B, cyclic depsipeptides bearing a 2-alkenylcinnamic acid unit and the unusual amino acids β-methylaspartic acid and 2,3-diaminopropanoic acid. The absolute configuration of 1 was determined using the advanced Marfey's method, phenylglycine methyl ester derivatization, and J -based configuration analysis. The biosynthetic gene clusters for the coprisamides were investigated based on genomic data from coprisamide-producing strains Micromonospora sp. UTJ3 and Streptomyces sp. SNU533. Coprisamide C ( 1 ) was active against the Mycobacterium tuberculosis mc 2 6230 strain.

Novel natural products are urgently needed to address the worldwide incidence of bacterial resistance to antibiotics. Extreme environments are a major source of novel compounds with unusual chemical structures. Pedobacter lusitanus NL19 is a new bacterial species that was isolated from one such environment and which produces compounds with potent activity against relevant microorganisms in the clinical, food, veterinary and aquaculture areas. The production of antimicrobials by P. lusitanus NL19 was identified in tryptic soy agar (TSA), but not in its equivalent broth (TSB). It was observed that in TSB medium a high concentration of casein peptone (PC) repressed the production of antibacterial compounds. HPLC, MS and MS/MS spectra with de novo sequencing revealed that the bioactivity of P. lusitanus NL19 was due to the production of pedopeptins. Hence, biosynthesis of pedopeptins is inhibited by high concentrations of PC in the broth medium. Furthermore, a nonribosomal peptide synthetase (NRPS) gene cluster was identified in the genome of NL19 encoding the biosynthesis of the peptides. qPCR analysis confirmed that the transcription of these genes is repressed in cells cultivated in high concentrations of PC. It is shown that pedopeptins are nonribosomal peptides with a broad-spectrum activity, including against Gram-positive and Gram-negative bacteria and yeasts., (Copyright © 2020. Published by Elsevier B.V.)

Bicyclic peptides, marinostatins, are protease inhibitors derived from the marine bacterium Algicola sagamiensis. The biosynthetic gene cluster of marinostatin was previously identified, although no heterologous production was reported. In this report, the biosynthetic gene cluster of marinostatin (mstA and mstB) was cloned into the expression vector pET-41a(+). As a result of the coexpression experiment, a new analogous peptide named marinostatin E was successfully produced using Escherichia coli BL21(DE3). The structure of marinostatin E was determined by a combination of chemical treatments and tandem mass spectrometry experiments. Marinostatin E exhibited inhibitory activities against chymotrypsin and subtilisin with an IC50 of 4.0 and 39.6 μm, respectively., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Bottromycins are ribosomally synthesized and post-translationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillin-resistant Staphylococcus aureus . The total synthesis of bottromycins involves at least 17 steps, with a poor overall yield. Here, we report the characterization of the cytochrome P450 enzyme BotCYP from a bottromycin biosynthetic gene cluster. We determined the structure of a close BotCYP homolog and used our data to conduct the first large-scale survey of P450 enzymes associated with RiPP biosynthetic gene clusters. We demonstrate that BotCYP converts a C-terminal thiazoline to a thiazole via an oxidative decarboxylation reaction and provides stereochemical resolution for the pathway. Our data enable the two-pot in vitro production of the bottromycin core scaffold and may allow the rapid generation of bottromycin analogues for compound development.

Background: Genome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories. Surfactin, a lipopeptide-type biosurfactant with broad spectrum antibiotic activity, has wide application prospects in anticancer therapy, biocontrol and bioremediation. Bacillus amyloliquefaciens LL3, previously isolated by our lab, contains an intact srfA operon in the genome for surfactin biosynthesis., Results: In this study, a genome-reduced strain GR167 lacking ~ 4.18% of the B. amyloliquefaciens LL3 genome was constructed by deleting some unnecessary genomic regions. Compared with the strain NK-1 (LL3 derivative, ΔuppΔpMC1), GR167 exhibited faster growth rate, higher transformation efficiency, increased intracellular reducing power level and higher heterologous protein expression capacity. Furthermore, the chassis strain GR167 was engineered for enhanced surfactin production. Firstly, the iturin and fengycin biosynthetic gene clusters were deleted from GR167 to generate GR167ID. Subsequently, two promoters PR suc and PR tpxi from LL3 were obtained by RNA-seq and promoter strength characterization, and then they were individually substituted for the native srfA promoter in GR167ID to generate GR167IDS and GR167IDT. The best mutant GR167IDS showed a 678-fold improvement in the transcriptional level of the srfA operon relative to GR167ID, and it produced 311.35 mg/L surfactin, with a 10.4-fold increase relative to GR167., Conclusions: The genome-reduced strain GR167 was advantageous over the parental strain in several industrially relevant physiological traits assessed and it was highlighted as a chassis strain for further genetic modification. In future studies, further reduction of the LL3 genome can be expected to create high-performance chassis for synthetic biology applications.

A urinary tract infection (UTI) is a multi-factorial disease including cystitis, pyelonephritis, and pyelitis. After Escherichia coli, Proteus mirabilis is the most common UTI-associated opportunistic pathogen. Antibiotic resistance of bacteria and infection recurrence can be connected to biofilm formation by P. mirabilis. In this study, human and sheep isolates of P. mirabilis were investigated for antibiotic sensitivity using an antibiotic disk test. Co-aggregation of the tested potential probiotic bacilli, Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933, with the isolated pathogen was also evaluated. Then, the anti-biofilm activity of naturally derived metabolites, such as subtilin and subtilosin, in the bacilli-free supernatants was assessed against biofilms of P. mirabilis isolates. The isolated pathogens were sensitive to 30 μg of amikacin and 5 μg of ciprofloxacin but resistant to other tested antibiotics. After 24 h, auto-aggregation of B. amyloliquefaciens B-1895 was at 89.5% and higher than auto-aggregation of B. subtilis KATMIRA1933 (59.5%). B. amyloliquefaciens B-1895 strongly co-aggregated with P. mirabilis isolates from human UTIs. Cell-free supernatants of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 showed higher antimicrobial activity against biofilms of P. mirabilis isolated from humans as compared with biofilms of sheep isolates. According to our knowledge, this is the first report evaluating the anti-biofilm activity of probiotic spore-forming bacilli against clinical and animal UTI isolates of P. mirabilis. Further studies are recommended to investigate the anti-biofilm activity and the mode of action for the antimicrobial substances produced by these bacilli, subtilosin and subtilin.

The potential of sponge-associated bacteria for the biosynthesis of natural products with antibacterial activity was evaluated. In a preliminary screening 108 of 835 axenic isolates showed antibacterial activity. Active isolates were identified by 16S rRNA gene sequencing and selection of the most promising strains was done in a championship like approach, which can be done in every lab and field station without expensive equipment. In a competition assay, strains that inhibited most of the other strains were selected. In a second round, the strongest competitors from each host sponge competed against each other. To rule out that the best competitors selected in that way represent similar strains with the same metabolic profile, BOX PCR experiments were performed, and extracts of these strains were analysed using metabolic fingerprinting. This proved that the strains are different and have various metabolic profiles, even though belonging to the same genus, i.e. Bacillus. Furthermore, it was shown that co-culture experiments triggered the production of compounds with antibiotic activity, i.e. surfactins and macrolactin A. Since many members of the genus Bacillus possess the genetic equipment for the biosynthesis of these compounds, a potential synergism was analysed, showing synergistic effects between C14-surfactin and macrolactin A against methicillin-resistant Staphylococcus aureus (MRSA).

Background: Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production., Results: The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10 T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (P pps ) against the strong constitutive promoter P veg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (P pps ::P veg ) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition., Conclusions: This study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

CRISPR/Cas9 is one of the robust and effective gene manipulation tools which has been widely applied in various organisms. In this study, the plipastatin gene cluster was successfully expressed in genome-modified Bacillus subtilis 1A751 by disrupting the surfactin operon ( srf ) through CRISPR/Cas9 technology. The presumed plipastatin biosynthetic pathway was proposed based on the analysis of its biosynthetic gene cluster. Two new plipastatins were identified by a combination of ultra-high performance liquid chromatography-coupled electron spray ionization-tandem mass spectrometry and gas chromatography-mass spectrometry analyses, together with nine known plipastatins or their derivatives. The yield of plipastatin was as high as 1600 mg/L which is the highest reported to date. Antimicrobial experiments revealed that its methanolic extracts exhibited powerful inhibitory effects on the growth of the tested pathogens and fungi. The results from this investigation highlight the remarkable utility of CRISPR/Cas9 in mining new plipastatins and increasing the total plipastatin yield, providing a new pipeline for the industrial application of plipastatin.

Ustiloxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) first reported in Ascomycetes. Originally identified as metabolites of the rice pathogenic fungus Ustilaginoidea virens, they were recently identified among the metabolites of the mold Aspergillus flavus, along with their corresponding biosynthetic gene cluster. Ustilaginoidea virens produces ustiloxins A and B, whereas A. flavus produces only ustiloxin B. Correspondingly, in U. virens, the ustiloxin precursor peptide, from which the compound backbone is cleaved and cyclized, contains the core peptides Tyr(Y)-Val(V)-Ile(I)-Gly(G) and Tyr(Y)-Ala(A)-Ile(I)-Gly(G) for ustiloxins A and B, respectively, whereas that of A. flavus contains only the YAIG motif for ustiloxin B. In this study, the gene that encodes the precursor peptide in A. flavus, ustA, was replaced with synthetic genes encoding the core peptides YVIG or FAIG, to investigate their compatibility with the ustiloxin biosynthetic machinery. We also examined the importance of the hydroxyl group on the aromatic ring of Tyr for cyclization of the YAIG core peptide. Against our expectation, the ustA variant possessing YVIG core peptides did not produce a detectable amount of ustiloxin A, even though the ustiloxin biosynthetic gene clusters of A. flavus and U. virens both contain 13 homologous genes. We confirmed that the lack of ustiloxin A production was not due to lack or insufficient expression of the substituted synthetic gene. This result, along with the differences between the primary sequences of UstYa and UstYb in A. flavus and U. virens, suggests that the ustiloxin biosynthetic machinery is optimized for the native core peptide sequences. The synthetic FAIG-encoding ustA did not yield any compounds specific to the FAIG core peptide, suggesting that the hydroxyl group on the aromatic ring of Tyr in the core peptide is indispensable for cyclization of the core peptide, even though it is not structurally involved in the cyclization., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Strain S150 was isolated from the tobacco rhizosphere as a plant growth-promoting rhizobacterium. It increased plant fresh weight significantly and lateral root development, and it antagonized plant pathogenic fungi but not phytobacteria. Further tests showed that strain S150 solubilized organic phosphate and produced ammonia, siderophore, protease, amylase, and cellulase, but it did not produce indole-3-acetic acid. Using morphology, physiological characteristics, and multi-locus sequence analysis, strain S150 was identified as Pseudomonas koreensis. The complete genome of strain S150 was sequenced, and it showed a single circular chromosome of 6,304,843 bp with a 61.09% G + C content. The bacterial genome contained 5,454 predicted genes that occupied 87.7% of the genome. Venn diagrams of the identified orthologous clusters of P. koreensis S150 with the other three sequenced P. koreensis strains revealed up to 4,167 homologous gene clusters that were shared among them, and 21 orthologous clusters were only present in the genome of strain S150. Genome mining of the bacterium P. koreensis S150 showed that the strain possessed 10 biosynthetic gene clusters for secondary metabolites, which included four clusters of non-ribosomal peptide synthetases (NRPSs) involved in the biosynthesis of cyclic lipopeptides (CLPs). One of the NRPSs possibly encoded lokisin, a cyclic lipopeptide produced by fluorescent Pseudomonas. Genomic mutation of the lokA gene, which is one of the three structural NRPS genes for lokisin in strain S150, led to a deficiency in fungal antagonism that could be restored fully by gene complementation. The results suggested that P. koreensis S150 is a novel plant growth-promoting agent with specific cyclic lipopeptides and contains a lokisin-encoding gene cluster that is dominant against plant fungal pathogens., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

This study examined the storage instability of cyclic lipopeptides (CLs) extracted from Bacillus subtilis culture; CLs were easily oxidized and therefore quickly lost antifungal activity during storage. Solid lipid nanoparticle (SLN) encapsulation effectively suppressed the oxidation and prolonged and enhanced the antifungal efficacy of CLs through controlled release. Thermal gravimetric analysis, X-ray diffraction, and Fourier transform infrared spectroscopy revealed that hydrophobic interactions between the fatty acid moieties of CLs and SLNs fortified the crystal structure of the CL-SLN complex, thereby improving the storage stability of the encapsulated CLs. Furthermore, the encapsulated CLs also demonstrated more potent antifungal activity than free CLs in damaging fungal cellular membranes, affecting the growth of hyphae and spores, and decreasing ochratoxin A levels. SLN encapsulation is an effective method to protect and improve the function of CLs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH-VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)