Mario Wibowo

Cocoa products are widely appreciated worldwide due to their unique properties, including their distinctive color. The characteristic color of cocoa mainly comes after the various processes of cocoa processing, including fermentation, roasting, and alkalization, which also aim to improve its taste and solubility. Polyphenols are believed to be responsible for the formation of color in cocoa. However, the understanding of specific polyphenols causing a color change after processing is still quite limited. This review presents current findings about the role of polyphenolic compounds in color development in cocoa. The identification of key cocoa polyphenols in color formation and vital chromophoric compounds in cocoa products will provide more understanding of chemical markers to predict the color potential of both unprocessed and processed cocoa beans, which will be useful in quality control and industrial applications. Finally, future perspectives on polyphenols and cocoa sensory quality are also discussed.

Lactic acid bacteria (LAB) are important part of the complex microbiome of many Southeast Asian fermented foods, contributing to the health‐promoting effects in these foods. In this study, 16 LAB isolates were obtained from tempeh and oncom , and amongst those, four novel strains ( Lacticaseibacillus paracasei Tem A13 and Tem B18, Lactiplantibacillus plantarum Onc A8, Pediococcus pentosaceus Onc A1) exhibited desired probiotic attributes (tolerance to stomach and intestinal conditions), comparable to the commercial strain Lacticaseibacillus rhamnosus GG. Submerged fermentation utilizing the probiotic strains were subsequently performed on mung bean water extract, and the fermented samples were subjected to metabolomic profiling. Bioactive metabolites with anti‐inflammatory and antioxidative properties, such as indole lactic acid, phenyl lactic acid, and hydroxyphenyl lactic acid were identified in Tem A13‐, Tem B18‐, Onc A8‐ and GG‐fermented, but not in Onc A1‐fermented and unfermented mung beans. Furthermore, N‐lactoyl phenylalanine, a metabolite associated with appetite suppression was identified in Tem A13‐ and Tem B18‐fermented samples. Undesired metabolites associated with bitter taste perception (dipeptides Phe‐Ile, Phe‐Val, and Val‐Ile) were present in GG‐fermented mung bean. Altogether, this study provides insights into the use of specific probiotic strain to produce functional beverage enriched in desired bioactives associated with health‐promoting benefits.

Basidiomycetous fungi are prolific producers of terpenoids, yet many biosynthetic pathways remain unexplored. Here, we report the discovery of two novel terpene synthases: PoTS6 from the oyster mushroom (Pleurotus ostreatus) and AgTS2 from the bulbous honey fungus (Armillaria gallica). Both produce a unique terpene scaffold, pleostene (1), whose structure was elucidated by using NMR spectroscopy and GC-MS analysis. Despite sharing only 44% sequence identity, PoTS6 and AgTS2 exhibit highly similar three-dimensional conformations, including conserved active-site residues and their spatial arrangement within the substrate-binding pockets. Our finding highlights the importance of structural conservation over sequence similarity in product specificity. Phylogenetic analysis reveals two distinct clades of homologous enzymes, suggesting pleostene biosynthesis may be widespread among Agaricales fungi. Functional analysis demonstrates that the C-terminal region of PoTS6 (residues 331-407) is dispensable for catalytic activity. Genomic context analysis predicts that PoTS6 is not part of a gene cluster, whereas AgTS2 resides within a putative biosynthetic gene cluster containing 3 cytochrome P450 enzymes. These P450s may oxidize pleostene into bioactive derivatives such as clitocybulols and nebucanes, known for their cytotoxic and antimicrobial activities. These findings expand our understanding of fungal terpenoid biosynthesis and offer new opportunities for natural product biosynthesis.

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.

Bacterial-fungal interactions influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Here, we use a co-culture experimental evolution approach to investigate bacterial adaptation to the presence of a fungus, using a simple model of bacterial-fungal interactions encompassing the bacterium Bacillus subtilis and the fungus Aspergillus niger. We find in one evolving population that B. subtilis was 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 possibly provoking an RlmA-dependent cell wall stress. Thus, our results indicate that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates the competitive success of the bacterium.

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.

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.

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 knockout 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 an azo compound followed by further oxygenation. Enzymes involved in modifications of the precursor could be postulated based on their predicted function and the intermediates identified in the knockout strains. 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.

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 less clear. Here, we use a four-species synthetic community (SynCom) in which individual members are sensitive towards key P. protegens antimicrobial metabolites (DAPG, pyoluteorin, and orfamide A) to determine how antibiotic production contributes to P. protegens community invasion and to identify community traits that counteract the antimicrobial effects. We show that P. protegens readily invades and alters the SynCom composition over time, and that P. protegens establishment requires production of DAPG and pyoluteorin. An orfamide A-deficient mutant of P. protegens invades the community as efficiently as wildtype, and both cause similar perturbations to community composition. Here, we identify the microbial interactions underlying the absence of an orfamide A mediated impact on the otherwise antibiotic-sensitive SynCom member, and show that the cyclic lipopeptide is inactivated and degraded by the combined action of 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.