High-efficiency breeding of Bacillus siamensis with hyper macrolactins production using physical mutagenesis and a high-throughput culture system.

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 ZnSO4 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.

Macrolactin XY, a Macrolactin Antibiotic from Marine-Derived Bacillus subtilis sp. 18.

Two new compounds, macrolactin XY (1) and (5R, 9S, 10S)-5-(hydroxymethyl)-1,3,7-decatriene-9,10-diol (2), together with nine known compounds (3-11) were isolated from the marine Bacillus subtilis sp. 18 by the OSMAC strategy. These compounds were evaluated for antibacterial activity against six tested microorganisms. Compounds 1-5 and 7-10 showed varied antibacterial activity, with the minimum inhibitory concentration (MIC) ranging from 3 to 12 µg/mL. Macrolactin XY (1) was found to possess superior antibacterial activity, especially exhibiting significant effectiveness against Enterococcus faecalis. The antibacterial activity mechanism against E. faecalis was investigated. The mechanism may disrupt bacterial cell membrane integrity and permeability, and also inhibit the expression of genes associated with bacterial energy metabolism, as established by the experiments concerning cell membrane potential, SDS-PAGE electrophoresis, cell membrane integrity, and key gene expressions. This study offers valuable insights and serves as a theoretical foundation for the future development of macrolactins as antibacterial precursors.

UV-ARTP compound mutagenesis breeding improves macrolactins production of Bacillus siamensis and reveals metabolism changes by proteomic.

Macrolactins are a type of compound with complex macrolide structure which mainly be obtained through microbiological fermentation now. They have excellent antifungal, antibacterial and antitumor activity. In order to improve macrolactins production, Bacillus siamensis YB304 was used as the research object, and a mutant Mut-K53 with stable genetic characters was selected by UV-ARTP compound mutagenesis. The yield of macrolactins was 156.46 mg/L, 3.95 times higher than original strain. The metabolic pathway changes and regulatory mechanism of macrolactins were analyzed by quantitative proteomics combined with parallel reaction monitoring. This study revealed that 1794 proteins were extracted from strain YB304 and strain Mut-K53, most of them were related to metabolism. After UV-ARTP compound mutagenesis treatment, the expression of 628 proteins were significantly changed, of which 299 proteins were significantly up-regulated. KEGG pathway analysis showed that differentially expression proteins mainly distributed in biological process, cellular component, and molecular function processing pathways. Such as utilization of carbon sources, glycolysis pathway, and amino acid metabolism pathway. Furthermore, key precursor substances such as acyl-CoA and amino acids of macrolactin biosynthesis are mostly up-regulated, which are one of the main reasons for increased production of macrolactin.This study will provide a new way to increase the yield of macrolactins through mutagenesis breeding and proteomics.

Improvement of macrolactins production by the genetic adaptation of Bacillus siamensis A72 to saline stress via adaptive laboratory evolution.

BACKGROUND: Macrolactins, a type of macrolide antibiotic, are toxic to the producer strains. As such, its level is usually maintained below the lethal concentration during the fermentation process. To improve the production of macrolactins, we applied adaptive laboratory evolution technology to engineer a saline-resistant mutant strain. The hypothesis that strains with saline resistance show improved macrolactins production was investigated. RESULTS: Using saline stress as a selective pressure, we engineered a mutant strain with saline resistance coupled with enhanced macrolactins production within 60 days using a self-made device. As compared with the parental strain, the evolved strain produced macrolactins with 11.93% improvement in non-saline stress fermentation medium containing 50 g/L glucose, when the glucose concentration increased to 70 g/L, the evolved strain produced macrolactins with 71.04% improvement. RNA sequencing and metabolomics results revealed that amino acid metabolism was involved in the production of macrolactins in the evolved strain. Furthermore, genome sequencing of the evolved strain revealed a candidate mutation, hisDD41Y, that was causal for the improved MLNs production, it was 3.42 times higher than the control in the overexpression hisDD41Y strain. Results revealed that saline resistance protected the producer strain from feedback inhibition of end-product (macrolide antibiotic), resulting in enhanced MLNs production. CONCLUSIONS: In the present work, we successfully engineered a mutant strain with enhanced macrolactins production by adaptive laboratory evolution using saline stress as a selective pressure. Based on physiological, transcriptomic and genetic analysis, amino acid metabolism was found to benefit macrolactins production improvement. Our strategy might be applicable to improve the production of other kinds of macrolide antibiotics and other toxic compounds. The identification of the hisD mutation will allow for the deduction of metabolic engineering strategies in future research.

Antagonistic Activity of Antimicrobial Metabolites Produced from Seaweed-Associated Bacillus amyloliquefaciens MTCC 10456 Against Malassezia spp.

Members of the genus Malassezia are known to be opportunistic pathogens responsible for causing skin disorders such as seborrheic dermatitis or dandruff, pityriasis versicolor, folliculitis, atopic dermatitis, and psoriasis. Due to the side effects caused by prolonged use of current topical antifungal agents, development of an alternative treatment is necessary. Fermentative production of antimicrobial metabolites from Bacillus amyloliquefaciens MTCC 10456 was carried out, and their antagonistic activity against Malassezia furfur and Malassezia globosa was evaluated. The antifungal metabolites were isolated by acid precipitation, and bioassay-guided simultaneous separation of the antimicrobial compounds was done by reversed-phase high-performance liquid chromatography (RP-HPLC). The fraction which demonstrated antifungal activity consisted of bacilysin, homologues of bacillomycin D, and members of the macrolactin family. The presence of bacilysin was detected using specific inhibitor assays and homologues of bacillomycin D, and macrolactins were identified using liquid chromatography/high-resolution electrospray ionization-mass spectrometry (LC/HRESI-MS/MS) analysis. Synergism among the identified compounds was observed which enhanced the antagonistic activity against Malassezia spp. To our knowledge, this is the first study to report the co-production and separation of members of macrolactin antibiotics, lipopeptides such as bacillomycin D and dipeptide antibiotic bacilysin, by any Bacillus strain from marine environment. Activity of individual compounds against Malassezia has been reported which may facilitate their application in the field of dermatology and in cosmetic products.

Macrolactins: biological activity and biosynthesis.

Marine microorganisms have proven to be a rich source of natural products with unique structures and novel activities, due to their special living conditions. Macrolactins (MLNs), mostly produced by marine-derived microorganisms, are a group of 24-membered lactone natural products, which exhibit potent antibacterial, antifungal, antiviral, anticancer, anti-inflammatory, anti-angiogenic and other activities. Their extensive biological activities make them potential compounds for drug development. MLNs are biosynthesized via a type I polyketide synthase (PKS) pathway with different tailoring steps, such as epoxidation, glycosylation and acylation. These modification steps provide opportunities to diversify their structures by combinatorial biosynthesis strategies. This review mainly focuses on the newly discovered MLNs in the past five years, including their biological activities and relevant biosynthetic studies.

Rapid improvement in the macrolactins production of Bacillus sp. combining atmospheric room temperature plasma with the specific growth rate index.

Macrolactins (MLNs) have attracted considerable attention due to their antibacterial and antiviral properties. Here, the MLN production of Bacillus sp. strain IMDGX0108 was improved using a breeding strategy of atmospheric room temperature plasma (ARTP) technique. Combining with a selection procedure based on the colony morphology and specific growth rate index (SGRI), two genetically stable mutants A29 and A72 were identified. The MLN production of A29 and A72 was 35.2% and 52.8% greater than that of the parent strain, respectively. The best-performing mutant A72 was subjected to RNA-sequence analysis. Five pathways were significantly enriched, and fatty acid bioprocesses might play an important role in improving the production of MLNs. The combined strategy developed herein (i.e., ARTP mutation plus an efficient screening procedure) might be an appropriate method by which to obtain strains overproducing MLNs.

Marine bacteria as source of antimicrobial compounds.

The marine environment encompasses a huge biological diversity and can be considered as an underexplored location for prospecting bioactive molecules. In this review, the current state of art about antimicrobial molecules from marine bacteria has been summarized considering the main phylum and sources evolved in a marine environment. Considering the last two decades, we have found as most studied group of bacteria producers of substances with antimicrobial activity is the Firmicutes phylum, in particular strains of the Bacillus genus. The reason for that can be attributed to the difficult cultivation of typical Actinobacteria from a marine sediment, whose members are the major producers of antimicrobial substances in land environments. However, a reversed trend has been observed in recent years with an increasing number of reports settling on Actinobacteria. Great diversity of chemical structures have been identified, such as fijimicyns and lynamicyns from Actinomycetes and macrolactins produced by Bacillus.

Macrolactin Antibiotics: Amazing Natural Products.

The resistance among various microbial species (infectious agents) to different antimicrobial drugs has emerged as a cause of serious public health problem all over the world. In this sense, natural products have been a rich source of compounds for drug discovery with antibiotic activity. Macrolactins are amazing structures which have antibiotic activity against some clinically relevant pathogens. In addition, they have anti-inflammatory, antifungal, antimicrobial, and antitumor activities. They are macrolides containing 24-membered lactone ring with some differences in their chemical structures. The synthesis of these compounds is a difficult task which has attracted attention of researchers; however few syntheses have been reported. In this review, the isolation of all reported macrolactins, their syntheses and biological activities are revisited.

Macrolactins from Marine-Derived Bacillus subtilis B5 Bacteria as Inhibitors of Inducible Nitric Oxide and Cytokines Expression.

In order to find new natural products with anti-inflammatory activity, chemical investigation of a 3000-meter deep-sea sediment derived bacteria Bacillus subtilis B5 was carried out. A new macrolactin derivative was isolated and identified as 7,13-epoxyl-macrolactin A (1). Owing to the existence of the epoxy ring, 1 exhibited a significant inhibitory effect on the expression of inducible nitric oxide and cytokines, compared with previously isolated known macrolactins (2-5). Real-time Polymerase Chain Reaction (PCR) analysis showed that the new compound significantly inhibited the mRNA expressions of inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Reverse transcription-PCR analysis demonstrated that the new compound reduced the mRNA expression level of IL-1ß in a concentration-dependent manner.

Uncovering a glycosyltransferase provides insights into the glycosylation step during macrolactin and bacillaene biosynthesis.

Macrolactins (MLNs) have unique structural patterns containing a 24-membered ring lactone and diverse bioactivities. The MLN skeleton is biosynthesized via a trans-acyl transferase (AT) type I polyketide synthase (PKS) pathway, but the tailoring steps are still unknown. Herein, we report the identification of a glycosyltransferase (GT) gene bmmGT1, which is located at different locus from the MLN gene cluster in the genome of marine-derived Bacillus marinus B-9987, and its functional characterization as an MLN GT, thus affording five novel MLNs analogues. Surprisingly, this GT is also capable of catalyzing the glycosylation of bacillaenes (BAEs), which are the prototypes of trans-AT polyketides, thus suggesting broad substrate flexibility. These results provide the first significant insights into the glycosylation step in MLN and BAE biosynthetic pathways.