Biology subjects

Wibowo, M.

Publications and source records attributed to Wibowo, M..

10 recordsLinked to original sources

bioRxiv2025

Sustainable biotransformation of microalgae via probiotic fermentation for enhanced functional, nutritional, and sensory properties

Microalgae represent a sustainable food source with exceptional CO{square} fixation efficiency; however, their integration into the food chain is hindered by undesirable organoleptic properties. This study establishes a green biotransformation platform using Generally Recognized As Safe (GRAS) bacterium Lactiplantibacillus plantarum to ferment Chlorella vulgaris biomass. This fermentation process operates without the use of harsh chemicals and organic solvents, enabling the full utilization of the biomass while improving sensory quality. Notably, the L. plantarum fermentation maintained dried biomass weight, in contrast to [~]15-40% loss seen with Bacillus spp., further enhancing the carbon-negative profile of microalgae. Tiered olfactory analysis and gas chromatography-mass spectrometry revealed selective reduction of polyunsaturated fatty acid-derived aldehydes and accumulation of flavor-active volatiles, including pyrazines and phenylethyl derivatives. Electronic tongue and liquid chromatography-mass spectrometry confirmed elevated umami taste, via increased glutamate and nucleotide levels. Additionally, the fermentation of microalgae with L. plantarum converted aromatic amino acids into antioxidant aromatic lactates, exemplifying catalytic, rather than stoichiometric efficiency. Overall, this renewable fermentation strategy converts photoenergy-fuelled, CO{square}-derived microalgal biomass into direct functional food ingredients under mild, organic solvent-free conditions, while bypassing conventional downstream extraction and purification steps. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/670464v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@951462org.highwire.dtl.DTLVardef@16d542forg.highwire.dtl.DTLVardef@b1da64org.highwire.dtl.DTLVardef@78f435_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Surfactin facilitates the establishment of Bacillus subtilis in synthetic communities

Soil bacteria are prolific producers of a myriad of biologically active secondary metabolites. These natural products play key roles in modern society, finding use as anti-cancer agents, as food additives, and as alternatives to chemical pesticides. As for their original role in interbacterial communication, secondary metabolites have been extensively studied under in vitro conditions, revealing a multitude of roles including antagonism, effects on motility, niche colonization, signaling, and cellular differentiation. Despite the growing body of knowledge on their mode of action, biosynthesis, and regulation, we still do not fully understand the role of secondary metabolites on the ecology of the producers and resident communities in situ. Here, we specifically examine the influence of Bacillus subtilis-produced cyclic lipopeptides (LPs) during the assembly of a bacterial synthetic community (SynCom), and simultaneously, explore the impact of LPs on B. subtilis establishment success in a SynCom propagated in an artificial soil microcosm. We found that surfactin production facilitates B. subtilis establishment success within multiple SynComs. Surprisingly, while neither a wild type nor a LP non-producer mutant had major impact on the SynCom composition over time, the B. subtilis and the SynCom metabolomes are both altered during co-cultivation. Overall, our work demonstrates the importance of surfactin production in microbial communities, suggesting a broad spectrum of action of this natural product.

microbiology↗

bioRxiv2024

Maramycin, a cytotoxic isoquinolinequinone terpenoid produced through heterologous expression of a bifunctional indole prenyltransferase /tryptophan indole-lyase in S. albidoflavus

Isoquinolinequinones represent an important family of natural alkaloids with profound biological activities. Heterologous expression of a rare bifunctional indole prenyltransferase /tryptophan indole-lyase enzyme from Streptomyces mirabilis P8-A2 in S. albidoflavus J1074 led to the activation of a putative isoquinolinequinone biosynthetic gene cluster and production of a novel isoquinolinequinone alkaloid, named maramycin (1). The structure of maramycin was determined by analysis of spectroscopic (1D/2D NMR) and MS spectrometric data. The prevalence of this bifunctional biosynthetic enzyme was explored and found to be a recent evolutionary event with only a few representatives in Nature. Maramycin exhibited moderate cytotoxicity against human prostate cancer cell lines, LNCaP and C4-2B. The discovery of maramycin (1) enriched the chemical diversity of natural isoquinolinequinones and also provided new insights into crosstalk between the host biosynthetic genes and the heterologous biosynthetic genes in generating new chemical scaffolds.

bioengineering↗

bioRxiv2023

Biosynthesis of the azoxy compound azodyrecin from Streptomyces mirabilis P8-A2

Azoxy compounds are a distinctive group of bioactive secondary metabolites, characterized by a unique RN=N+(O-)R moiety. The azoxy moiety is present in various classes of metabolites that exhibit various biological activities. The enzymatic mechanisms underlying azoxy bond formation remain enigmatic. Azodyrecins are cytotoxic azoxy metabolites produced by Streptomyces mirabilis P8-A2. Here we cloned and confirmed the putative azd biosynthetic gene cluster through CATCH cloning followed by expression and production of azodyrecins in two heterologous hosts, S. albidoflavus J1074 and S. coelicolor M1146, respectively. We explored the function of 14 enzymes in azodyrecin biosynthesis through gene knock-out using CRISPR-Cas9 base editing in the native producer, S. mirabilis P8-A2. The key intermediates were analyzed in the mutants through MS/MS fragmentation studies, revealing azoxy bond formation via the conversion of hydrazine to azo compound; followed by further oxygenation. Additionally, N-oxygenase and dehydrogenase activities were confirmed among 8 core biosynthetic genes and five helper genes. Moreover, the distribution of the azoxy biosynthetic gene clusters across Streptomyces spp. genomes is explored, highlighting the presence of these clusters in over 20% of the Streptomyces spp. genomes and revealing that azoxymycin and valanimycin are scarce, while azodyrecin and KA57A like clusters are widely distributed across the phylogenetic tree.

microbiology↗

bioRxiv2023

Resistance towards and biotransformation of Pseudomonas-produced secondary metabolites during community invasion

The role of antagonistic secondary metabolites produced by Pseudomonas protegens in suppression of soil-borne phytopathogens has been clearly documented. However, their contribution to the ability of P. protegens to establish in soil and rhizosphere microbiomes remains ambiguous. Here, we use a four-species synthetic community to determine how antibiotic production contributes to P. protegens community invasion and identify community traits that alter the abundance of key P. protegens antimicrobial metabolites (DAPG, pyoluteorin and orfamide A). Surprisingly, mutants deficient in antimicrobial production caused similar perturbations in community composition compared to invasion by wildtype P. protegens. Intriguingly, while pyoluteorin and orfamide A are secreted at levels toxic to individual bacterial strains, community-level resistance circumvents toxicity. Here, we identify the underlying mechanism by which the cyclic lipopeptide, orfamide A, is inactivated and degraded by Rhodococcus globerulus D757 and Stenotrophomonas indicatrix D763. Altogether, the demonstration that the synthetic community constrains P. protegens invasion by detoxifying its antibiotics may provide a mechanistic explanation to inconsistencies in biocontrol effectiveness in situ.

microbiology↗

bioRxiv2023

Enhanced niche colonisation and competition during bacterial adaptation to a fungus

Bacterial-fungal interactions (BFIs) influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Using a simple BFI system encompassing the Gram-positive bacterium Bacillus subtilis and the black mould fungus Aspergillus niger, we established a co-culture experimental evolution method to investigate bacterial adaptation to the presence of a fungus. In the evolving populations, B. subtilis was rapidly selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles and RlmA-dependent cell wall stress induction. Increased surfactin production typically enhances the competitive success of bacteria against fungi, which likely explains the primary adaption path in the presence of A. niger. Significance statementExperimental evolution and co-cultivation of different microbes are important and useful techniques for discovering new traits and unravelling cryptic regulatory connections. We combined these methods by evolving the Gram-positive bacterium Bacillus subtilis in the presence of the black mould fungus Aspergillus niger that were previously shown to engage in an intricate and physical interaction. Both are ubiquitous, environmentally and industrially relevant model microbes in the colonisation of rhizo- and endosphere and in the production of enzymes. Our results demonstrate how laboratory adaptation can be exploited to improve biocontrol properties of bacteria.

microbiology↗

bioRxiv2022

Establishment of a transparent soil system to study Bacillus subtilis chemical ecology

Bacterial secondary metabolites are structurally diverse molecules that drive microbial interaction by altering growth, cell differentiation, and signaling. Bacillus subtilis, a Gram-positive soil-dwelling bacterium, produces a wealth of secondary metabolites, among them, lipopeptides have been vastly studied by their antimicrobial, antitumor, and surfactant activities. However, the natural functions of secondary metabolites in the lifestyles of the producing organism remain less explored under natural conditions, i.e. in soil. Here, we describe a hydrogel-based transparent soil system to investigate B. subtilis chemical ecology under controllable soil-like conditions. The transparent soil matrix allows the growth of B. subtilis and other isolates gnotobiotically and under nutrient-controlled conditions. Additionally, we show that transparent soil allows the detection of lipopeptides production and dynamics by HPLC-MS and MALDI-MS imaging, along with fluorescence imaging of 3-dimensional bacterial assemblages. We anticipate that this affordable and highly controllable system will promote bacterial chemical ecology research and help to elucidate microbial interactions driven by secondary metabolites.

microbiology↗

bioRxiv2020

Genomic and chemical diversity of Bacillus subtilis secondary metabolites against plant pathogenic fungi

Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant-growth-promoting and biocontrol abilities. These secondary metabolites include non-ribosomal peptides (NRPs) with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates and therefore, a comparative overview of B. subtilis secondary metabolites is missing. In this study, we have isolated 23 B. subtilis strains from eleven sampling sites, compared the fungal inhibition profiles of wild types and their NRPs mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that non-ribosomal peptide production varied among B. subtilis strains co-isolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium sp., a combination of plipastatin and surfactin is required to hinder the growth of Botrytis cinerea. Detailed genomic analysis revealed that altered NRP production profiles in certain isolates is due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical NRPs detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis. IMPORTANCESecondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which non-ribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack production of certain secondary metabolites.

microbiology↗

bioRxiv2020

Development of a whole cell biosensor for detection of 2, 4-diacetylphloroglucinol (DAPG) producing bacteria from grassland soil

Fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (DAPG) are ecologically important in the rhizosphere as they can control phytopathogens and contribute to disease suppressiveness. While studies of DAPG-producing Pseudomonas have predominantly focused on rhizosphere niches, the ecological role of DAPG as well as the distribution and dynamics of DAPG-producing bacteria remains less well understood for other environments such as bulk soil and grassland, where the level of DAPG producers are predicted to be low. Here, we construct a whole cell biosensor for detection of DAPG and DAPG-producing bacteria from environmental samples. We show that the sensor is highly specific towards DAPG, with a sensitivity in the low nanomolar range (<20 nm). This sensitivity is comparable to the DAPG levels identified in rhizosphere samples by chemical analysis. The biosensor enables guided isolation of DAPG-producing Pseudomonas. Using the biosensor, we probed the same grassland soil sampling site to isolate genetically related DAPG-producing Pseudomonas kilonensis strains over a period of 12 months. Next, we used the biosensor to determine the frequency of DAPG-producing Pseudomonads within three different grassland soil sites and show that DAPG producers can constitute part of the Pseudomonas population in the range of 0.35-17% at these sites. Finally, we show that the biosensor enables detection of DAPG produced by non-Pseudomonas species. Our studies show that a whole-cell biosensor for DAPG detection can facilitate isolation of bacteria that produce this important secondary metabolite and provide insight into the population dynamics of DAPG producers in natural grassland soil. IMPORTANCEThe interest has grown for bacterial biocontrol agents as biosustainable alternatives to pesticides to increase crop yields. Currently, we have a broad knowledge of antimicrobial compounds, such as DAPG, produced by bacteria growing in the rhizosphere surrounding plant roots. However, compared to the rhizosphere niches, the ecological role of DAPG as well as the distribution and dynamics of DAPG-producing bacteria remains less well understood for other environments such as bulk and grassland soil. Currently, we are restricted to chemical methods with detection limits and time-consuming PCR-based and probe-hybridization approaches to detect DAPG and its respective producer. In this study, we have developed a whole-cell biosensor, which can circumvent the labor-intensive screening process, as well as increase the sensitivity at which DAPG is detected. This enables quantification of relative amounts of DAPG-producers, which in turn increases our understanding of the dynamics and ecology of these producers in natural soil environments.

microbiology↗

bioRxiv2019

Surfactin production is not essential for pellicle and root-associated biofilm development of Bacillus subtilis

Secondary metabolites have an important impact on the biocontrol potential of soil-derived microbes. In addition, various microbe-produced chemicals have been suggested to impact the development and phenotypic differentiation of bacteria, including biofilms. The non-ribosomal synthesized lipopeptide of Bacillus subtilis, surfactin, has been described to impact the plant promoting capacity of the bacterium. Here, we investigated the impact of surfactin production on biofilm formation of B. subtilis using the laboratory model systems; pellicle formation at the air-medium interface and architecturally complex colony development, in addition to plant root-associated biofilms. We found that the production of surfactin by B. subtilis is not essential for pellicle biofilm formation neither in the well-studied strain, NCIB 3610, nor in the newly isolated environmental strains, but lack of surfactin reduces colony expansion. Further, plant root colonization was comparable both in the presence or absence of surfactin synthesis. Our results suggest that surfactin-related biocontrol and plant promotion in B. subtilis strains are independent of biofilm formation.

microbiology↗