Search

We report the successful cell-free reconstitution of two natural product biosynthetic pathways of divergent complexity and structural classes. We first constructed the teleocidin biosynthetic pathway using our BY-2 (tobacco) cell-free protein synthesis (CFPS) system. We discovered a direct interaction between TleA and MbtH, and showed that the BY-2 system is capable of producing more than 80 mg/L teleocidin B-3 with cofactor supplementation and ∼20 mg/L with no cofactors supplemented, demonstrating the high metabolic activity of the system. We then extended our methodology and report the first successful cell-free biosynthesis of UK-2 diol (precursor to the commercially valuable secondary metabolite UK-2A) from simple building blocks by refactoring a complex pathway of 10 proteins in the wheat germ CFPS system. We show that plant CFPS systems are suitable for reconstructing pathways and identifying the functions of uncharacterized genes linked to biosynthetic gene clusters and rate-limiting biosynthetic steps.

Salicylate plays a pivotal role as a pharmaceutical intermediate in drugs, such as aspirin and lamivudine. The low catalytic efficiency of key enzymes and the inherent toxicity of salicylates to cells pose significant challenges to large-scale microbial production. In this study, we introduced the salicylate synthase Irp9 into an l-phenylalanine-producing Escherichia coli , constructing the shortest salicylate biosynthetic pathway. Subsequent protein engineering increased the catalytic efficiency of Irp9 by 33.5%. Furthermore, by integrating adaptive evolution with transcriptome analysis, we elucidated the crucial mechanism of efflux proteins in salicylate tolerance. The elucidation of this mechanism guided us in the targeted modification of these transport proteins, achieving a reported maximum level of 3.72 g/L of salicylate in a shake flask. This study highlights the importance of efflux proteins for enhancing the productivity of microbial cell factories in salicylate production, which also holds potential for application in the green synthesis of other phenolic acids.

In recent decades, there has been an increase in the occurrence of fungal infections; yet, the arsenal of drugs available to fight invasive infections remains very limited. The development of new antifungal agents is hindered by the restricted number of molecular targets that can be exploited, given the shared eukaryotic nature of fungi and their hosts which often leads to host toxicity. In this paper, we examine the riboflavin biosynthetic pathway as a potential novel drug target. Riboflavin is an essential nutrient for all living organisms. Its biosynthetic pathway does not exist in humans, who obtain riboflavin through their diet. Our findings demonstrate that all enzymes in the pathway are essential for Candida albicans , Candida glabrata, and Saccharomyces cerevisiae. Auxotrophic strains, which mimic a drug targeting the biosynthesis pathway, experience rapid mortality in the absence of supplemented riboflavin. Furthermore, RIB1 is essential for virulence in both C. albicans and C. glabrata in a systemic mouse model. The fungal burden of a RIB1 deletion strain is significantly reduced in the kidneys and brain of infected mice, and this reduction becomes more pronounced over time. Nevertheless, auxotrophic cells can still take up external riboflavin when supplemented. We identified Orf19.4337 as the riboflavin importer in C. albicans and named it Rut1. We found that Rut1 only facilitates growth at external riboflavin concentrations that exceed the physiological concentrations in the human body. This suggests that riboflavin uptake is unlikely to serve as a resistance mechanism against drugs targeting the biosynthesis pathway. Interestingly, the uptake system in S. cerevisiae is more effective than in C. albicans and C. glabrata, enabling an auxotrophic S. cerevisiae strain to outcompete an auxotrophic C. albicans strain in lower riboflavin concentrations., Importance: Candida species are a common cause of invasive fungal infections. Candida albicans , in particular, poses a significant threat to immunocompromised individuals. This opportunistic pathogen typically lives as a commensal on mucosal surfaces of healthy individuals but it can also cause invasive infections associated with high morbidity and mortality. Currently, there are only three major classes of antifungal drugs available to treat these infections. In addition, the efficacy of these antifungal agents is restricted by host toxicity, suboptimal pharmacokinetics, a narrow spectrum of activity, intrinsic resistance of fungal species, such as Candida glabrata , to certain drugs, and the acquisition of resistance over time. Therefore, it is crucial to identify new antifungal drug targets with novel modes of action to add to the limited armamentarium., Competing Interests: The authors declare no conflict of interest.

The species Bacillus velezensis is known for its biosynthetic potential and metabolic versatility in producing several secondary metabolites and promoting plant growth. In this study, we isolated two B. velezensis strains, WGTg-8 and WGTm-299, from the termite gut and termitarium, which exhibited antimicrobial activity against multiple clinical and phytopathogens. The whole genomes of these strains were sequenced using the Illumina platform and annotated. The genome mining of the draft genome sequences revealed 48 biological gene clusters (BGCs) responsible for synthesizing various secondary metabolites. The construction of the similarity network of the BGCs and the comparative analysis with the genetically related organisms are aided in the identification of metabolites produced by these strains. We identified biosynthetic gene clusters (BGCs) coding for macrolactin H, bacilysin, bacillibactin, amylocyclin, comX4, and LCI, found in both strains with 100% similarity. The difficidin, bacillaene, thusin_alpha, and cericidin BGCs are exclusively found in strain WGTg-8, while the colicin BGC is exclusively present in the WGTm-299 strain. The fengycin and surfactin gene clusters are present in both strains with 80% similarity. Furthermore, 28 putative NRPS BGCs, NRPS-T1PKS hybrid clusters, a T1PKS, and a bacteriocin BGC were identified with very low similarity (≤ 25%) or no similarity with known antibiotics. In addition, we found several genes coding for plant growth-promoting properties, including nitrogen metabolism, hormone synthesis, sulfur metabolism, phosphate metabolism, and a few other properties., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Boldenone (BD), a protein anabolic hormone, is commonly used to treat muscle damage, osteoporosis, and off-season muscle building in athletes. Traditional BD synthesis methods rely on chemical processes, which are costly and environmentally impactful. Therefore, developing a more sustainable and economical biosynthetic pathway is crucial for BD production. This study aimed to achieve efficient production of BD. Firstly, the catalytic performance of 17β-hydroxysteroid dehydrogenase and 3-ketosteroid-Δ 1 -dehydrogenase was improved through enzyme engineering, and their expression in the new strain of Mycobacterium neoaurum was enhanced using metabolic engineering. These improvements significantly increased BD production to 4.05 g/L, with a significant decrease in by-product generation. To further increase the yield, a multi-enzyme fusion expression system was constructed, and a key cell wall gene kasB was knocked out, resulting in a spatial-time yield of BD reaching 1.02 g/(L·d). Subsequent optimization of the transformation system further increased the BD production to 5.56 g/L, with a spatiotemporal yield of 1.39 g/(L·d). The green biosynthetic route of phytosterol one-step conversion to BD developed in this study lays the foundation for industrial production., (© 2024 Wiley‐VCH GmbH.)

β-Nicotinamide mononucleotide (NMN) is a precursor of NAD + in mammals. Research on NAD + has demonstrated its crucial role against aging and disease. Here two technical paths were established for the efficient synthesis of NMN in the yeast Pichia pastoris, enabling the production of NMN from the low-cost nicotinamide (NAM) or basic carbon sources. The yeast host was systematically modified to adapt to the biosynthesis and accumulation of NMN. To improve the semi-biosynthesis of NMN from NAM, nicotinamide phosphoribosyltransferases were expressed intracellular to evaluate their catalytic activities. The accumulation of extracellular NMN was further increased by the co-expression of an NMN transporter. Fine-tuning of gene expression level produced 72.1 mg/L NMN from NAM in flasks. To achieve de novo biosynthesis NMN, a heterologous biosynthetic pathway was reassembled in yeast cells. Fine-tuning of pathway nodes by the modification of gene expression level and enhancement of precursor generation allowed efficient NMN synthesis from glucose (36.9 mg/L) or ethanol (57.8 mg/L) in flask. Lastly, cultivations in a bioreactor in fed-batch mode achieved an NMN titre of 1004.6 mg/L at 165 h from 2 g NAM and 868 g glucose and 980.4 mg/L at 91 h from 160 g glucose and 557 g ethanol respectively. This study provides a foundation for future optimization of NMN biosynthesis by engineered yeast cell factories., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Natural products - small molecules generated by organisms to facilitate ecological interactions - are of great importance to society and are used as antibacterial, antiviral, antifungal and anticancer drugs. However, the role and evolution of these molecules and the fitness benefits they provide to their hosts in their natural habitat remain an outstanding question. In bacteria, the genes that encode the biosynthetic proteins that generate these molecules are organised into discrete loci termed biosynthetic gene clusters (BGCs). In this work, we asked the following question: How are biosynthetic gene clusters organised at the chromosomal level? We sought to answer this using publicly available high-quality assemblies of Micromonospora , an actinomycete genus with members responsible for biosynthesizing notable natural products, such as gentamicin and calicheamicin. By orienting the Micromonospora chromosome around the origin of replication, we demonstrated that Micromonospora has a conserved origin-proximal region, which becomes progressively more disordered towards the antipodes of the origin. We then demonstrated through genome mining of these organisms that the conserved origin-proximal region and the origin-distal region of Micromonospora have distinct populations of BGCs and, in this regard, parallel the organization of Streptomyces , which possesses linear chromosomes. Specifically, the origin-proximal region contains highly syntenous, conserved BGCs predicted to biosynthesize terpenes and a type III polyketide synthase. In contrast, the ori-distal region contains a highly diverse population of BGCs, with many BGCs belonging to unique gene cluster families. These data highlight that genomic plasticity in Micromonospora is locus-specific, and highlight the importance of using high-quality genome assemblies for natural product discovery and guide future natural product discovery by highlighting that biosynthetic novelty may be enriched in specific chromosomal neighbourhoods.

Daptomycin (DAP), a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, is clinically important for treatment of infections caused by multidrug-resistant Gram-positive pathogens, but the low yield hampers its large-scale industrial production. Here, we describe a combination metabolic engineering strategy for constructing a DAP high-yielding strain. Initially, we enhanced aspartate (Asp) precursor supply in S. roseosporus wild-type (WT) strain by separately inhibiting Asp degradation and competitive pathway genes using CRISPRi and overexpressing Asp synthetic pathway genes using strong promoter kasOp*. The resulting strains all showed increased DAP titre. Combined inhibition of acsA4, pta, pyrB, and pyrC increased DAP titre to 167.4 μg/mL (73.5% higher than WT value). Co-overexpression of aspC, gdhA, ppc, and ecaA led to DAP titre 168 μg/mL (75.7% higher than WT value). Concurrently, we constructed a chassis strain favourable for DAP production by abolishing by-product production (i.e., deleting a 21.1 kb region of the red pigment biosynthetic gene cluster (BGC)) and engineering the DAP BGC (i.e., replacing its native dptEp with kasOp*). Titre for the resulting chassis strain reached 185.8 μg/mL. Application of our Asp precursor supply strategies to the chassis strain further increased DAP titre to 302 μg/mL (2.1-fold higher than WT value). Subsequently, we cloned the engineered DAP BGC and duplicated it in the chassis strain, leading to DAP titre 274.6 μg/mL. The above strategies, in combination, resulted in maximal DAP titre 350.7 μg/mL (2.6-fold higher than WT value), representing the highest reported DAP titre in shake-flask fermentation. These findings provide an efficient combination strategy for increasing DAP production and can also be readily applied in the overproduction of other Asp-related antibiotics., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Pneumocandin B 0 (PB 0 ) is a lipopeptide produced by the fungus Glarea lozoyensis. The existing challenges with the low-yield and the extended-fermentation cycle emphasize necessity for strain improvement. In this study, we optimized conditions to obtain high-quality protoplasts and screened effective selection markers, leading to the construction of three CRISPR/Cas9 gene editing systems. Utilizing a constitutive Cas9 expression recipient strain, combined with dual sgRNAs targeting, we achieved highly efficient editing of target genes. We successfully knocked out 10 genes within the pneumocandin putative biosynthetic gene cluster and analyzed their roles in PB 0 production. Our findings reveal that 4 of 10 genes are directly involved in PB 0 production. Specially, the deletion of gltrt or gl10050 resulted in reduced PB 0 production, while the absence of glhyp or glhtyC led to the complete loss of PB 0 biosynthesis. Notably, the deletion of glhyp caused the silencing of nearly all cluster genes, whereas overexpression of glhyp led to a 2.38-fold increase in PB 0 production. Therefore, this study provides the first comprehensive exploration of the functions of 10 genes within the pneumocandin putative biosynthetic gene cluster. Our findings provide valuable technical strategies for constructing bioengineering strains with purposefully enhanced PB 0 production., 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.)

Lilium spp. is a world-famous bulbous flower with outstanding ornamental, edible, and medicinal value. Evaluating the taste of edible lilies and identifying important active substances and genes are necessary for germplasm improvement, new variety breeding, and industrial application. To better understand the phenylpropanoids and regalosides biosynthesis, L. davidii var. unicolor and L. lancifolium Thunb. bulbs were used for transcriptome and metabolite analysis. Results showed that the phenols and flavonoid contents in JT were lower than in LT, while the saponins and alkaloid contents in JT were higher than in LT. A total of 20,520 differentially expressed genes (DEGs) and 383 differential metabolites were searched. Integrated transcriptomics and metabolomics analysis showed that phenylpropanoid biosynthesis and flavonoid biosynthesis were differentially altered. Ninety-nine unigenes encoding ten phenolic acids and two flavonoids were identified as candidate genes involved in phenylpropanoid and flavonoid biosynthesis. WGCNA analysis showed 76 phenylpropanoid and flavonoid biosynthesis-related unigenes were verified as likely to be involved in phenylpropanoid metabolism and regalosides accumulation. Among them, 15 genes were used for qRT-PCR, and four genes were utilized for tissue-specific expression pattern analysis. Down-regulation of LdPAL2 and LdC4H1 in bulbs of L. davidii var. unicolor via virus induced gene silence (VIGS) reduced the contents of p-coumaric acid and cinnamic acid. These results contribute to understanding phenylpropanoid metabolism and identifying potential functional genes for improving the regalosides and flavonoids content in Lilium bulbs., Competing Interests: Declaration of competing interest The authors declare that there are no potential conflicts or competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

β-Caryophyllene possesses potential anticancer properties against various cancers, including breast, colon, and lung cancer. Therefore, the essential oil of Ayapana triplinervis , which is rich in β-caryophyllene, can be a potential herbal remedy for treating cancer. However, molecular and genomic studies on A. triplinervis are still sparse . In this study, we obtained 14.7 Gb of RNA-Seq data from A. triplinervis leaf RNA and assembled 137 554 transcripts with an N50 value of 1437 bp. We annotated 72 436 (52.7%) transcripts and mapped 10 640 transcripts to 156 biochemical pathways. Among them, 218 were related to terpenoid backbone biosynthesis, while 27 were linked to sesquiterpenoid and triterpenoid pathways. Ninety-four transcripts were annotated in the β-caryophyllene and lupeol pathways. From these transcripts, for the first time, we identified 25 full-length genes encoding all the 17 enzymes involved in β-caryophyllene biosynthesis and an additional five genes involved in lupeol biosynthesis. These genes will be useful for the metabolic engineering of β-caryophyllene and lupeol biosynthesis, not just in A. triplinervis but also in other species., Competing Interests: We have no conflicts of interest to disclose.

Gallic acid (GA) and β-glucogallin (BGG) are natural products with diverse uses in pharmaceutical, food, chemical and cosmetic industries. They are valued for their wide-ranging properties such as antioxidant, antibacterial, antidiabetic, and anticancer properties. Despite their significant importance, microbial production of GA and BGG faces challenges such as limited titers and yields, along with the incomplete understanding of BGG biosynthesis pathways in microorganisms. To address these challenges, we developed a recombinant Escherichia coli strain capable of efficiently producing GA. Our approach involved screening efficient pathway enzymes, integrating biosynthetic pathway genes into the genome while balancing carbon flux via adjusting expression levels, and strengthening the shikimate pathway to remove bottlenecks. The resultant strain achieved impressive results, producing 51.57 g/L of GA with a carbon yield of 0.45 g/g glucose and a productivity of 1.07 g/L/h. Furthermore, we extended this microbial platform to biosynthesize BGG by screening GA 1-O-glucosyltransferase, leading to the de novo production of 92.42 mg/L of BGG. This work establishes an efficient chassis for producing GA at an industrial level and provides a microbial platform for generating GA derivatives., (© 2024 Wiley Periodicals LLC.)

Microorganisms co-exist and co-evolve in nature, forming intricate ecological communities. The interspecies cross-talk within these communities creates and sustains their great biosynthetic potential, making them an important source of natural medicines and high-value-added chemicals. However, conventional investigations into microbial metabolites are typically carried out in pure cultures, resulting in the absence of specific activating factors and consequently causing a substantial number of biosynthetic gene clusters to remain silent. This, in turn, hampers the in-depth exploration of microbial biosynthetic potential and frequently presents researchers with the challenge of rediscovering compounds. In response to this challenge, the coculture strategy has emerged to explore microbial biosynthetic capabilities and has shed light on the study of cross-talk mechanisms. These elucidated mechanisms will contribute to a better understanding of complex biosynthetic regulations and offer valuable insights to guide the mining of secondary metabolites. This review summarizes the research advances in microbial cross-talk mechanisms, with a particular focus on the mechanisms that activate the biosynthesis of secondary metabolites. Additionally, the instructive value of these mechanisms for developing strategies to activate biosynthetic pathways is discussed. Moreover, challenges and recommendations for conducting in-depth studies on the cross-talk mechanisms are presented., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chinese cabbage is rich in vitamins, fibre, and nutrients and is one of the primary vegetables consumed in autumn and winter in South Asia. 'Purple pak choi' sprouts are particularly rich in anthocyanins and are favoured by consumers. However, reports on the regulation of anthocyanin synthesis in purple pak choi sprouts do not exist. In this study, we examined the phenotypic development of purple pak choi sprouts after germination. The total anthocyanin content increased from 0.02 to 0.52 mg/g FW from days 0 to 6. RNA-seq data analysis revealed an increase in differentially expressed genes corresponding to the development of purple pak choi sprouts. Expression pattern analysis of genes associated with the anthocyanin biosynthesis pathway revealed a significant upregulation of structural genes during the purple phase, suggesting that the transcription factors PAP2 and MYBL2 may play crucial regulatory roles. BraPAP2.A03 , BraTT8.A09 , and BraMYBL2.A07 exhibited strong interactions with key genes in the anthocyanin biosynthesis pathway, specifically BraDFR.A09 . Furthermore, the expression of BraPAP2.A03 aligned with the expression patterns of most anthocyanin biosynthesis-related genes, whereas those of BraTT8.A09 and BraMYBL2.A07 corresponded with the expression pattern of BraDFR.A09 . These results provide valuable insights into regulatory mechanisms underlying anthocyanin synthesis in purple pak choi sprouts.

Soil microorganisms mediate several biological processes through the secretion of natural products synthesized in specialized metabolic pathways, yet functional characterization in ecological contexts remains challenging. Using culture-independent metagenomic analyses of microbial DNA derived directly from soil samples, we examined the potential of biosynthetic gene clusters (BGCs) from six bacterial communities distributed along an altitudinal gradient of the Andes Mountains in the Atacama Desert. We mined 38 metagenome-assembled genomes (MAGs) and identified 168 BGCs. Results indicated that most predicted BGCs were classified as non-ribosomal-peptides (NRP), post-translational modified peptides (RiPP), and terpenes, which were mainly identified in genomes of species from Acidobacteriota and Proteobacteria phyla. Based on BGC composition according to types of core biosynthetic genes, six clusters of MAGs were observed, three of them with predominance for a single phylum, of which two also showed specificity to a single sampling site. Comparative analyses of accessory genes in BGCs showed associations between membrane transporters and other protein domains involved in specialized metabolism with classes of biosynthetic cores, such as resistance-nodulation-cell division (RND) multidrug efflux pumps with RiPPs and the iron-dependent transporter TonB with terpenes. Our findings increase knowledge regarding the biosynthetic potential of uncultured bacteria inhabiting pristine locations from one of the oldest and driest nonpolar deserts on Earth.IMPORTANCEMuch of what we know about specialized metabolites in the Atacama Desert, including Andean ecosystems, comes from isolated microorganisms intended for drug development and natural product discovery. To complement research on the metabolic potential of microbes in extreme environments, comparative analyses on functional annotations of biosynthetic gene clusters (BGCs) from uncultivated bacterial genomes were carried out. Results indicated that in general, BGCs encode for structurally unique metabolites and that metagenome-assembled genomes did not show an obvious relationship between the composition of their core biosynthetic potential and taxonomy or geographic distribution. Nevertheless, some members of Acidobacteriota showed a phylogenetic relationship with specific metabolic traits and a few members of Proteobacteria and Desulfobacterota exhibited niche adaptations. Our results emphasize that studying specialized metabolism in environmental samples may significantly contribute to the elucidation of structures, activities, and ecological roles of microbial molecules., Competing Interests: The authors declare no conflict of interest.

Background: Light is a critical environmental factor in plants, encompassing two vital aspects: intensity and quality. To assess the influence of different light intensities on Origanum majorana L., pots containing the herb were subjected to four levels of light intensity: 20, 50, 70, and 100% natural light. After a 60-day treatment period, the plants were evaluated for metabolite production, including total sugar content, protein, dry weight, antioxidant indices, expression of monoterpenes biosynthesis genes, and essential oil compounds. The experimental design followed a randomized complete blocks format, and statistical analysis of variance was conducted., Results: The results indicated a correlation between increased light intensity and elevated total sugar and protein content, which contributed to improved plant dry weight. The highest levels of hydrogen peroxide and malondialdehyde (MDA) were observed under 100% light intensity. Catalase and superoxide dismutase enzymes exhibited increased activity, with a 4.23-fold and 2.14-fold increase, respectively, under full light. In contrast, peroxidase and polyphenol oxidase enzyme activities decreased by 3.29-fold and 3.24-fold, respectively. As light intensity increases, the expression level of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) gene increases. However, beyond a light intensity of 70%, the DXR gene expression level decreased. Furthermore, the expression levels of the cytochrome P450 genes CYP71D178 and CYP71D179 exhibited an increasing trend in response to elevated light intensity. Essential oil content increased from 0.02 to 0.5% until reaching 70% light intensity. However, with further increases in light intensity, the essential oil content decreased by 54 to 0.23%., Conclusions: These findings emphasize the importance of balancing plant growth promotion and stress management under different light conditions. The research suggests that sweet marjoram plants thrive best in unshaded open spaces, resulting in maximum biomass. However, essential oil production decreases under the same conditions. For farmers in areas with an average light intensity of approximately 1700 µmol m -2 s -1 , it is recommended to cultivate sweet marjoram in shade-free fields to optimize biomass and essential oil production. Towards the end of the growth cycle, it is advisable to use shades that allow 70% of light to pass through. The specific duration of shade implementation can be further explored in future research., (© 2024. The Author(s).)

Levan biopolymer and levan-type fructooligosaccharides (L-FOSs) are β-2,6-linked fructans that have been used as non-digestible dietary fiber and prebiotic oligosaccharides in food and cosmeceutical applications. In this study, we explore the operon responsible for levan and L-FOSs production in Priestia koreensis HL12. Presented is the first genomic perspective on sucrose utilization and the levan biosynthesis pathway in this bacterium. Regarding sequence annotation, the putative levansucrase operon responsible for β-2,6-linked fructan was identified in the genome of strain HL12, and comprises sacB levansucrase gene belonging to GH68, located adjacent to levB endo-levanase gene, which belongs to GH32. Importantly, sugars related with the levan biosynthesis pathway are proposed to be transported via 3 types of transportation systems, including multiple ABC Sugar and glucose/H + transporters, as well as glucose- and fructose-specific PTS systems. Based on product profile analysis, the HL12 strain exhibited high efficiency in levan production from high sucrose concentration (300 g/l), achieving the highest yield of 127 g/l (equivalent to 55% conversion based on sucrose consumption), together with short-chain L-FOSs (DP3-5) and long-chain L-FOSs with respective size larger than DP6 after 48 h incubation. These findings highlight the potential of P. koreensis HL12 as a whole-cell biocatalyst for producing levan and L-FOSs, and underscore its novelty in converting sugars into high-value-added products for diverse commercial and industrial applications.

Methionine (Met), a sulfur-containing amino acid, is essential for the underlying biological processes in living organisms. In addition to its importance as a starting building block for peptide chain elongation in protein biosynthesis, Met is a direct precursor of S -adenosyl-l-methionine, an indispensable methyl donor molecule in primary and secondary metabolism. Streptomyces bacteria are well known to produce diverse secondary metabolites, but many strains lack canonical Met pathway genes for l-homocysteine, a direct precursor of Met in bacteria, plants, and archaea. Here, we report the identification of a novel gene ( metM ) responsible for the Met biosynthesis in Streptomyces strains and demonstrate the catalytic function of the gene product, MetM. We further identified the metO gene, a downstream gene of metM , and showed that it encodes a sulfur-carrier protein (SCP). In in vitro analysis, MetO was found to play an important role in a sulfur donor by forming a thiocarboxylated SCP. Together with MetO (thiocarboxylate), MetM directly converted O -phospho-l-homoserine to l-homocysteine. O -Phospho-l-homoserine is also known as an intermediate for threonine biosynthesis in bacteria and plants, and MetM shares sequence homology with threonine synthase. Our findings thus revealed that MetM seizes O -phospho-l-homoserine from the threonine biosynthetic pathway and uses it as an intermediate of the Met biosynthesis to generate the sulfur-containing amino acid. Importantly, this MetM/MetO pathway is highly conserved in Streptomyces bacteria and distributed in other bacteria and archaea.IMPORTANCEMethionine (Met) is a sulfur-containing proteinogenic amino acid. Moreover, Met is a direct precursor of S -adenosyl-l-methionine, an indispensable molecule for expanding the structural diversity of natural products. Because Met and its derivatives benefit humans, the knowledge of Met biosynthesis is important as a basis for improving their fermentation. Streptomyces bacteria are well known to produce diverse and valuable natural products, but many strains lack canonical Met pathway genes. Here, we identified a novel l-homocysteine synthase (MetM) in Streptomyces and demonstrated that it converts O -phospho-L-homoserine to l-homocysteine using a thiocarboxylated sulfur-carrier protein as a sulfur donor. Since the metM is distributed in other bacteria and archaea, our pioneering study contributes to understanding Met biosynthesis in these organisms., Competing Interests: The authors declare no conflict of interest.

The human gut microbiota possesses the capacity to synthesize vitamins, especially B group vitamins, which are recognized as indispensable for various biological processes both among members of these bacterial communities and host cells. Accordingly, vitamin production by intestinal commensals has attracted significant interest. Nevertheless, our current understanding of bacterial vitamin synthesis is primarily based on individual genomic and monoculture investigations, therefore not providing an overall view of the biosynthetic potential of complex microbial communities. In the current study, we utilized over 100 bacterial genes known to be involved in the biosynthesis of B group and K vitamins to assess the corresponding vitamin biosynthetic potential of approximately 8,000 human gut microbiomes. Our analyses reveal that host-associated factors, such as age and geographical origin, appear to influence the diversity and abundance of vitamin biosynthetic pathways. Furthermore, we identify gut microbiota members that substantially contribute to these biosynthetic functions at each stage of human life. Interestingly, inference of microbial co-associations and network relationships uncovered the apparent key role played by folate and cobalamin in equilibrium establishment of the infant and adult gut microbial communities, respectively.IMPORTANCEOverall, this study expands our understanding of microbe-mediated vitamin biosynthesis in the human gut and may provide potential novel targets to improve availability of these essential micronutrients in the host., Competing Interests: The authors declare no conflict of interest.

Ginsenoside Ro, as one of the few oleanane-type ginsenosides, is well known for its unique molecular structure and biological activities. Currently, research on the biosynthesis of ginsenoside Ro is still in its early stages. Therefore, the establishment of a new ginsenoside Ro cell factory is of great significance for the in-depth development and utilization of genes related to ginsenoside Ro synthesis, as well as for the exploration of pathways to obtain ginsenoside Ro. In this study, we cloned endogenous constitutive promoters, terminators, and other genetic elements from S. cerevisiae BY4741. These elements were then sequentially assembled with the uridine diphosphate glucuronic acid transferase gene identified in our previously study ( PgUGAT252645 ) and several other reported key enzyme genes, to construct DNA fragments used for integration into the genome of S. cerevisiae BY4741. By sequentially transferring these DNA fragments into chemically competent cells of engineering strains and conducting screening and target product detection, we successfully constructed an engineered S. cerevisiae strain (BY-Ro) for ginsenoside Ro biosynthesis using S. cerevisiae BY4741 as the host cell. Strain BY-Ro produced 253.32 μg/L of ginsenoside Ro under optimal fermentation conditions. According to subsequent measurements and calculations, this equates to 0.033 mg/g DCW, corresponding to approximately 31% of the ginsenoside Ro content found in plant samples. This study not only included a deeper investigation into the function of PgUGAT252645 but also provides a novel engineering platform for ginsenoside Ro biosynthesis.

Panax japonicus C. A. Meyer is renowned for its significant therapeutic effects and is commonly used worldwide. Its active ingredients, triterpenoid saponins, show variation in content among different tissues. The tissue-specific distribution of saponins is potentially related to the expression of vital genes in the biosynthesis pathway. In this study, the contents of five saponins (ginsenoside Ro, chikusetsusaponin IV, chikusetsusaponin IVa, ginsenoside Rg1, and ginsenoside Rb1) in three different tissues were determined by HPLC. Transcriptome sequencing analysis identified differentially expressed genes (DEGs) involved in triterpenoid saponin biosynthesis, highlighting significant correlations between saponin contents and the expression levels of 10 cytochrome p450 monooxygenase (CYP) and 3 UDP-glycosyltransferase (UGT) genes. Cloning, sequencing, and prokaryotic expression of UGT genes confirmed the molecular weights of UGT proteins. Gene sequence alignment and phylogenetic analysis provided preliminary insights into UGT gene functions. Meanwhile, the function of one UGT gene was characterized in the yeast. These findings advance our understanding of the triterpenoid saponin biosynthesis in P. japonicus and support future research in traditional Chinese medicine (TCM) and synthetic biology.

Background: The excavation and utilization of endophytic fungi from medicinal plants is highly important for the development of new drugs. The endophytic fungus Aspergillus tamarii TPD11, which was isolated and obtained by the authors in the previous stage, can produce a variety of polyphyllins with important potential applications in hemostasis, inflammation and antitumor activities; however, the genomic information of TPD11 is still unknown., Results: In this study, we sequenced and assembled the whole genome of the endophytic fungus A. tamarii TPD11, resolved the genome evolutionary relationships of 24 Aspergillus strains, and phylogenetic analysis of the genomes of 16 strains revealed the evolutionary differences between Aspergillus and Penicillium and the mechanisms of genome expansion and contraction. CAZy annotation analysis revealed that TPD11 obtains nutrients mainly by ingesting starch from the host plant. TPD11 has a biosynthesis-related gene cluster for the synthesis of squalestatin S1, and the silencing of this biosynthesis-related gene cluster might increase the content of polyphyllin. Annotation of 11 UDP-glycosyltransferase genes helps to further reveal the biosynthetic pathway of polyphyllin. In addition, secondary metabolism gene cluster and CAZy analyses confirmed the potential probiotic, insecticidal and antimicrobial activities of TPD11 on host plants., Conclusions: This study reveals the intrinsic mechanism by which endophytic fungi increase the content of polyphyllin, which provides a basis for the synthetic synthesis of the natural product polyphyllin., (© 2024. The Author(s).)

The filamentous Streptomyces are among the most prolific producers of bioactive natural products and are thus attractive chassis for the heterologous expression of native and designed biosynthetic pathways. Although suitable Streptomyces hosts exist, including genetically engineered cluster-free mutants, the approach is currently limited by the relative paucity of synthetic biology tools facilitating the de novo assembly of multicomponent gene clusters. Here, we report a modular system (MoClo) for Streptomyces including a set of adapted vectors and genetic elements, which allow for the construction of complete genetic circuits. Critical functional validation of each of the elements was obtained using the previously reported β-glucuronidase (GusA) reporter system. Furthermore, we provide proof-of-principle for the toolbox in S. albus , demonstrating the efficient assembly of a biosynthetic pathway to flavokermesic acid (FK), an advanced precursor of the commercially valuable carminic acid.

l-Pipecolic acid (L-PA), an essential chiral cyclic nonprotein amino acid, is gaining prominence in the food and pharmaceutical sectors due to its wide-ranging biological and pharmacological properties. Historically, L-PA has been synthesized chemically for commercial purposes. This study introduces a novel and efficient microbial production method for L-PA using engineered strain Saccharomyces cerevisiae BY4743. Initially, an optimized biosynthetic pathway was constructed within S. cerevisiae , converting glucose to L-PA with a yield of 0.60 g/L in a 250 mL shake flask in vivo . Subsequently, a multifaceted engineering strategy was implemented to enhance L-PA production: substrate-enzyme affinity modification, global transcription machinery engineering modification, and Kozak sequence optimization for enhanced L-PA production. Approaches above led to an impressive 8.6-fold increase in L-PA yield, reaching 5.47 g/L in shake flask cultures. Further scaling up in a 5 L fed-batch fermenter achieved a remarkable L-PA concentration of 74.54 g/L. This research offers innovative insights into the industrial-scale production of L-PA.

We report the identification of 3,6-dihydroxy-1,2-benzisoxazole (DHB) in a screen of Photorhabdus and Xenorhabdus , whose symbiotic relationship with eukaryotic nematodes favors secondary metabolites that meet several requirements matching those for clinically useful antibiotics. DHB is produced by Photorhabdus laumondii and is selective against the Gram-negative species Escherichia coli, Enterobacter cloacae, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and Acinetobacter baumannii . It is inactive against anaerobic gut bacteria and nontoxic to human cells. Mutants resistant to DHB map to the ubiquinone biosynthesis pathway. DHB binds to 4-hydroxybenzoate octaprenyltransferase (UbiA) and prevents the formation of 4-hydroxy-3-octaprenylbenzoate. Remarkably, DHB itself is prenylated, forming an unusable chimeric product that likely contributes to the toxic effect of this antimicrobial. DHB appears to be both a competitive enzyme inhibitor and a prodrug; this dual mode of action is unusual for an antimicrobial compound., Importance: The spread of resistant pathogens has led to the antimicrobial resistance crisis, and the need for new compounds acting against Gram-negative pathogens is especially acute. From a screen of Photorhabdus symbionts of nematodes, we identified 3,6-dihydroxy-1,2-benzisoxazole (DHB) that acts against a range of Gram-negative bacteria, including Escherichia coli , Enterobacter cloacae , Klebsiella pneumoniae , and Acinetobacter baumannii . DHB had previously been isolated from other bacterial species, but its mechanism of action remained unknown. We show that DHB is unique among antimicrobials, with dual action as an inhibitor of an important enzyme, UbiA, in the biosynthesis pathway of ubiquinone and as a prodrug. DHB is a mimic of the natural substrate, and UbiA modifies it into a toxic product, contributing to the antimicrobial action of this unusual antibiotic. We also uncover the mechanism of DHB selectivity, which depends on a particular fold of the UbiA enzyme., Competing Interests: The authors declare no conflict of interest.

The genus Pseudomonas is a prolific source of specialized metabolites with significant biological activities, including siderophores, antibiotics, and plant hormones. These molecules play pivotal roles in environmental interactions, influencing pathogenicity, inhibiting microorganisms, responding to nutrient limitation and abiotic challenges, and regulating plant growth. These properties mean that pseudomonads are suitable candidates as biological control agents against plant pathogens. Multiple transposon-based screens have identified a Pseudomonas biosynthetic gene cluster (BGC) associated with potent antibacterial and antifungal activities, which produces 7-hydroxytropolone (7-HT). In this study, we show that this BGC also makes 3,7-dihydroxytropolone (3,7-dHT), which has strong antimicrobial activity toward Streptomyces scabies , a potato pathogen. Through metabolomics and reporter assays, we unveil the involvement of cluster-situated genes in generating phenylacetyl-coenzyme A, a key precursor for tropolone biosynthesis via the phenylacetic acid catabolon. The clustering of these phenylacetic acid genes within tropolone BGCs is unusual in other Gram-negative bacteria. Our findings support the interception of phenylacetic acid catabolism via an enoyl-CoA dehydratase encoded in the BGC, as well as highlighting an essential role for a conserved thioesterase in biosynthesis. Biochemical assays were used to show that this thioesterase functions after a dehydrogenation-epoxidation step catalyzed by a flavoprotein. We use this information to identify diverse uncharacterized BGCs that encode proteins with homology to flavoproteins and thioesterases involved in tropolone biosynthesis. This study provides insights into tropolone biosynthesis in Pseudomonas , laying the foundation for further investigations into the ecological role of tropolone production.IMPORTANCE Pseudomonas bacteria produce various potent chemicals that influence interactions in nature, such as metal-binding molecules, antibiotics, or plant hormones. This ability to synthesize bioactive molecules means that Pseudomonas bacteria may be useful as biological control agents to protect plants from agricultural pathogens, as well as a source of antibiotic candidates. We have identified a plant-associated Pseudomonas strain that can produce 3,7-dihydroxytropolone, which has broad biological activity and can inhibit the growth of Streptomyces scabies , a bacterium that causes potato scab. Following the identification of this molecule, we used a combination of genetic, chemical, and biochemical experiments to identify key steps in the production of tropolones in Pseudomonas species. Understanding this biosynthetic process led to the discovery of an array of diverse pathways that we predict will produce new tropolone-like molecules. This work should also help us shed light on the natural function of antibiotics in nature., Competing Interests: The authors declare no conflict of interest.

The Gram-positive model bacterium Bacillus subtilis is used for many biotechnological applications, including the large-scale production of vitamins. For vitamin B5, a precursor for coenzyme A synthesis, there is so far no established fermentation process available, and the metabolic pathways that involve this vitamin are only partially understood. In this study, we have elucidated the complete pathways for the biosynthesis of pantothenate and coenzyme A in B. subtilis . Pantothenate can not only be synthesized but also be taken up from the medium. We have identified the enzymes and the transporter involved in the pantothenate biosynthesis and uptake. High-affinity vitamin B5 uptake in B. subtilis requires an ATP-driven energy coupling factor transporter with PanU (previously YhfU) as the substrate-specific subunit. Moreover, we have identified a salvage pathway for coenzyme A acquisition that acts on complex medium even in the absence of pantothenate synthesis. This pathway requires rewiring of sulfur metabolism resulting in the increased expression of a cysteine transporter. In the salvage pathway, the bacteria import cysteinopantetheine, a novel naturally occurring metabolite, using the cystine transport system TcyJKLMN. This work lays the foundation for the development of effective processes for vitamin B5 and coenzyme A production using B. subtilis ., Importance: Vitamins are essential components of the diet of animals and humans. Vitamins are thus important targets for biotechnological production. While efficient fermentation processes have been developed for several vitamins, this is not the case for vitamin B5 (pantothenate), the precursor of coenzyme A. We have elucidated the complete pathway for coenzyme A biosynthesis in the biotechnological workhorse Bacillus subtilis . Moreover, a salvage pathway for coenzyme A synthesis was found in this study. Normally, this pathway depends on pantetheine; however, we observed activity of the salvage pathway on complex medium in mutants lacking the pantothenate biosynthesis pathway even in the absence of supplemented pantetheine. This required rewiring of metabolism by expressing a cystine transporter due to acquisition of mutations affecting the regulation of cysteine metabolism. This shows how the hidden "underground metabolism" can give rise to the rapid formation of novel metabolic pathways., Competing Interests: The authors declare no conflict of interest.

Many microbial biosynthetic gene clusters (BGCs) are inactive under standard laboratory conditions, making characterization of their products difficult. Silent BGCs are likely activated by specific cues in their natural environment, such as the presence of competitors. Growth conditions such as coculture with other microbes, which more closely mimic natural environments, are practical strategies for inducing silent BGCs. Here, we utilize coculture to activate BGCs in nine actinobacteria strains. We observed increased production of the ferrous siderophores siderochelin A and B during coculture of Amycolatopsis strain WAC04611 and Tsukamurella strain WAC06889b. Furthermore, we identified the siderochelin BGC in WAC04611 and discovered that the GntR-family transcription factor sidR3 represses siderochelin production. Deletion of the predicted aminotransferase sidA abolished production of the carboxamides siderochelin A/B and led to the accumulation of the carboxylate siderochelin D. Finally, we deleted the predicted hydroxylase sidB and established that it is essential for siderochelin production. Our findings show that microbial coculture can successfully activate silent BGCs and lead to the discovery and characterization of unknown BGCs for molecules like siderochelin.IMPORTANCESiderophores are vital iron-acquisition elements required by microbes for survival in a variety of environments. Furthermore, many siderophores are essential for the virulence of various human pathogens, making them a possible target for antibacterials. The significance of our work is in the identification and characterization of the previously unknown BGC for the siderophore siderochelin. Our work adds to the growing knowledge of siderophore biosynthesis, which may aid in the future development of siderophore-targeting pharmaceuticals and inform on the ecological roles of these compounds. Furthermore, our work demonstrates that combining microbial coculture with metabolomics is a valuable strategy for identifying upregulated compounds and their BGCs., Competing Interests: The authors declare no conflict of interest.

Gray mold caused by Botrytis elliptica is one of the most determinative factors of lily growth and has become a major threat to lily productivity. However, the nature of the lily B. elliptica interaction remains largely unknown. Here, comparative transcriptomic and metabolomic were used to investigate the defense responses of resistant ('Sorbonne') and susceptible ('Tresor') lily cultivars to B. elliptica infection at 24 hpi. In total, 1326 metabolites were identified in 'Sorbonne' and 'Tresor' after infection, including a large number of phenylpropanoids. Specifically, the accumulation of four phenylpropanes, including eriodictyol, hesperetin, ferulic acid, and sinapyl alcohol, was significantly upregulated in the B. elliptica -infected 'Sorbonne' compared with the infected 'Tresor', and these phenylpropanes could significantly inhibit B. elliptica growth. At the transcript level, higher expression levels of F3'M , COMT , and CAD led to a higher content of resistance-related phenylpropanes (eriodictyol, ferulic acid, and sinapyl alcohol) in 'Sorbonne' following B. elliptica infection. It can be assumed that these phenylpropanes cause the resistance difference between 'Sorbonne' and 'Tresor', and could be the potential marker metabolites for gray mold resistance in the lily. Further transcriptional regulatory network analysis suggested that members of the AP2/ERF, WRKY, Trihelix, and MADS-M-type families positively regulated the biosynthesis of resistance-related phenylpropanes. Additionally, the expression patterns of genes involved in phenylpropanoid biosynthesis were confirmed using qRT-PCR. Therefore, we speculate that the degree of gray mold resistance in the lily is closely related to the contents of phenylpropanes and the transcript levels of the genes in the phenylpropanoid biosynthesis pathway. Our results not only improve our understanding of the lily's resistance mechanisms against B. elliptica , but also facilitate the genetic improvement of lily cultivars with gray mold resistance.

Tetrapyrroles such as heme, chlorophyll, and vitamin B 12 are essential for various metabolic pathways. They derive from 5-aminolevulinic acid (5-ALA), which can be synthesized by a single enzyme (5-ALA synthase or AlaS, Shemin pathway) or by a two-enzyme pathway. The genomes of some bacteriophages from aquatic environments carry various tetrapyrrole biosynthesis genes. Here, we analyze available metagenomic datasets and identify alaS homologs (viral alaS, or valaS) in sequences corresponding to marine and freshwater phages. The genes are found individually or as part of complete or truncated three-gene loci encoding heme-catabolizing enzymes. Amino-acid sequence alignments and three-dimensional structure prediction support that the valaS sequences likely encode functional enzymes. Indeed, we demonstrate that is the case for a freshwater phage valaS sequence, as it can complement an Escherichia coli 5-ALA auxotroph, and an E. coli strain overexpressing the gene converts the typical AlaS substrates glycine and succinyl-CoA into 5-ALA. Thus, our work identifies valaS as an auxiliary metabolic gene in phage sequences from aquatic environments, further supporting the importance of tetrapyrrole metabolism in bacteriophage biology., (© 2024. The Author(s).)

The efficient refactoring of natural product biosynthetic gene clusters (BGCs) for activating silent BGCs is a central challenge for the discovery of new bioactive natural products. Herein, we have developed a simple and robust CRISETR (CRISPR/Cas9 and RecET-mediated Refactoring) technique, combining clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 and RecET, for the multiplexed refactoring of natural product BGCs. By this approach, natural product BGCs can be refactored through the synergistic interaction between RecET-mediated efficient homologous recombination and the CRISPR/Cas9 system. We first performed a proof-of-concept validation of the ability of CRISETR, and CRISETR can achieve simultaneous replacement of four promoter sites and marker-free replacement of single promoter site in natural product BGCs. Subsequently, we applied CRISETR to the promoter engineering of the 74-kb daptomycin BGC containing a large number of direct repeat sequences for enhancing the heterologous production of daptomycin. We used combinatorial design to build multiple refactored daptomycin BGCs with diverse combinations of promoters different in transcriptional strengths, and the yield of daptomycin was improved 20.4-fold in heterologous host Streptomyces coelicolor A3(2). In general, CRISETR exhibits enhanced tolerance to repetitive sequences within gene clusters, enabling efficient refactoring of diverse and complex BGCs, which would greatly accelerate discovery of novel bioactive metabolites present in microorganism., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Houttuynia cordata, also known as Yuxingcao in Chinese, is a perennial herb in the Saururaceae family. It is highly regarded for its medicinal properties, particularly in treating respiratory infections and inflammatory conditions, as well as boosting the human immune system. However, a lack of genomic information has hindered research on the functional genomics and potential improvements of H. cordata. In this study, we present a near-complete assembly of H. cordata genome and investigate the biosynthetic pathway of flavonoids, specifically quercetin, using genomics, transcriptomics, and metabolomics analyses. The genome of H. cordata diverged from that of Saururus chinensis around 33.4 million years ago; it consists of 2.24 Gb with 76 chromosomes (4n = 76) and has undergone three whole-genome duplication (WGD) events. These WGDs played a crucial role in shaping the H. cordata genome and influencing the gene families associated with its medicinal properties. Through metabolomics and transcriptomics analyses, we identified key genes involved in the β-oxidation process for biosynthesis of houttuynin, one of the volatile oils responsible for the plant's fishy smell. In addition, using the reference genome, we identified genes involved in flavonoid biosynthesis, particularly quercetin metabolism, in H. cordata. This discovery has important implications for understanding the regulatory mechanisms that underlie production of active pharmaceutical ingredients in traditional Chinese medicine. Overall, the high-quality genome assembly of H. cordata serves as a valuable resource for future functional genomics research and provides a solid foundation for genetic improvement of H. cordata for the benefit of human health., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Terpenoids are known for their diverse structures and broad bioactivities with significant potential in pharmaceutical applications. However, natural products with low yields are usually ignored in traditional chemical analysis. Feature-based molecular networking (FBMN) was developed recently to cluster compounds with similar skeletons, which can highlight trace amounts of unknown compounds. Fusoxypene A is a sesterterpene synthesized by Fusarium oxysporum fusoxypene synthase (FoFS) with a unique 5/6/7/3/5 ring system. In this study, the FoFS-containing biosynthetic gene cluster was identified from F. oxysporum FO14005, and an efficient FBMN-based strategy was established to characterize four new sesterterpenoids, fusoxyordienoid A-D (1-4), based on a small-scale fermentation strategy. A cytochrome P450 monooxygenase, FusB, was found to be involved in the functionalization of fusoxypene A at C-17 and C-24 and responsible for the hydroxylation of fusoxyordienoid A at C-1 and C-8. This study highlights the potential of FBMN as a powerful tool for the discovery and characterization of natural compounds with low abundance. KEY POINTS: Combined small-scale fermentation and FBMN for rapid discovery of fusoxyordienoids Characterization of four new fusoxyordienoids with 5/6/7/3/5 ring system Biosynthetic pathway elucidation via tandem expression and substrate feeding., (© 2024. The Author(s).)

Lichen-forming fungi (LFF) are prolific producers of functionally and structurally diverse secondary metabolites, most of which are taxonomically exclusive and play lineage-specific roles. To date, widely distributed, evolutionarily conserved biosynthetic pathways in LFF are not known. However, this idea stems from polyketide derivatives, since most biochemical research on lichens has concentrated on polyketide synthases (PKSs). Here, we present the first systematic identification and comparison of terpene biosynthetic genes of LFF using all the available Lecanoromycete reference genomes and 22 de novo sequenced ones (111 in total, representing 60 genera and 23 families). We implemented genome mining and gene networking approaches to identify and group the biosynthetic gene clusters (BGCs) into networks of similar BGCs. Our large-scale analysis led to the identification of 724 terpene BGCs with varying degrees of pairwise similarity. Most BGCs in the dataset were unique with no similarity to a previously known fungal or bacterial BGC or among each other. Remarkably, we found two BGCs that were widely distributed in LFF. Interestingly, both conserved BGCs contain the same core gene, i.e., putatively a squalene/phytoene synthase (SQS), involved in sterol biosynthesis. This indicates that early gene duplications, followed by gene losses/gains and gene rearrangement are the major evolutionary factors shaping the composition of these widely distributed SQS BGCs across LFF. We provide an in-depth overview of these BGCs, including the transmembrane, conserved, variable and LFF-specific regions. Our study revealed that lichenized fungi do have a highly conserved BGC, providing the first evidence that a biosynthetic gene may constitute essential genes in lichens., (© 2024. The Author(s).)

The use of compounds produced by hosts or symbionts for defence against antagonists has been identified in many organisms, including in fungus-farming termites (Macrotermitinae). The obligate mutualistic fungus Termitomyces plays a pivotal role in plant biomass decomposition and as the primary food source for these termites. Despite the isolation of various specialized metabolites from different Termitomyces species, our grasp of their natural product repertoire remains incomplete. To address this knowledge gap, we conducted a comprehensive analysis of 39 Termitomyces genomes, representing 21 species associated with members of five termite host genera. We identified 754 biosynthetic gene clusters (BGCs) coding for specialized metabolites and categorized 660 BGCs into 61 biosynthetic gene cluster families (GCFs) spanning five compound classes. Seven GCFs were shared by all 21 Termitomyces species and 21 GCFs were present in all genomes of subsets of species. Evolutionary constraint analyses on the 25 most abundant GCFs revealed distinctive evolutionary histories, signifying that millions of years of termite-fungus symbiosis have influenced diverse biosynthetic pathways. This study unveils a wealth of non-random and largely undiscovered chemical potential within Termitomyces and contributes to our understanding of the intricate evolutionary trajectories of biosynthetic gene clusters in the context of long-standing symbiosis., (© 2024. The Author(s).)

Mammals host a remarkable diversity and abundance of gut microbes. Biosynthetic gene clusters (BGCs) in microbial genomes encode biologically active chemical products and play an important role in microbe-host interactions. Traditionally, the exploration of gut microbial metabolic functions has relied on the pure culture method. However, given the limited amounts of microbes being cultivated, insights into the metabolism of gut microbes in mammals continued to be very limited. In this study, we adopted a computational pipeline for mining the metagenomic data (named taxonomy-guided identification of biosynthetic gene clusters, TaxiBGC) to identify experimentally verified BGCs in 373 metagenomes across 53 mammalian species in an unbiased manner. We demonstrated that polyketides (PKs) and nonribosomal peptides (NRPs) are representative of mammals, and the products derived from them were associated with cell-cell communication and resistance to inflammation. Large carnivores had the highest number of BGCs, followed by large herbivores and small mammals. We also observed that the large mammals had more common BGCs that aid in the biosynthesis of a variety of natural products. However, small mammals not only had fewer BGCs but were also unique to each species. Our results provide novel insights into the mining of metagenomic data sets to identify active BGCs and their products across mammals.IMPORTANCEThe gut microbes host numerous biosynthetic gene clusters (BGCs) that biosynthesize natural products and impact the host's physiology. Historically, our understanding of BGCs in mammalian gut microbes was largely based on studies on cultured isolates; however, only a small fraction of mammal-associated microbes have been investigated. The biochemical diversity of the mammalian gut microbiota is poorly understood. Metagenomic sequencing contains data from a vast number of organisms and provides information on the total gene content of communities. Unfortunately, the existing BGC prediction tools are designed for individual microbial genomes. Recently, a BGC prediction tool called the taxonomy-guided identification of biosynthetic gene clusters (TaxiBGC) that directly mine the metagenome was developed. To gain new insights into the microbial metabolism, we used TaxiBGC to predict BGCs from 373 metagenomes across 53 mammalian species representing seven orders. Our findings elucidate the functional activities of complex microbial communities in the gut., Competing Interests: The authors declare no conflict of interest.

Stevioside (5-10%) and rebaudioside-A (2-4%) are well-characterized diterpene glycosides found in leaves of Stevia rebaudiana known to have natural sweetening properties with zero glycaemic index. Stevioside has after-taste bitterness, whereas rebaudioside-A is sweet in taste. The ratio of rebaudioside-A to stevioside needs to be changed in order to increase the effectiveness and palatability of this natural sweetener. Plant-specific miRNAs play a significant role in the regulation of metabolic pathways for the biosynthesis of economically important secondary metabolites. In this study inhibition of miRNA through antisense technology was employed to antagonize the repressive action of miRstv_7 on its target mRNAs involved in the steviol glycosides (SGs) biosynthesis pathway. In transgenic plants expressing anti-miRstv_7, reduced expression level of endogenous miRstv_7 was observed than the non-transformed plants. As a result, enhanced expression of target genes, viz. KO (Kaurene oxidase), KAH (Kaurenoic acid-13-hydroxylase), and UGT76G1 (UDP-glycosyltransferase 76G1) led to a significant increase in the rebaudioside-A to stevioside ratio. Furthermore, metabolome analysis revealed a significant increase in total steviol glycosides content as well as total flavonoids content. Thus, our study can be utilized to generate more palatable varieties of Stevia with improved nutraceutical values including better organoleptic and antioxidant properties., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background. Bacteria significantly influence human health and disease, with bacterial biosynthetic gene clusters (BGCs) being crucial in the microbiome-host and microbe-microbe interactions. Gap statement. Despite extensive research into BGCs within the human gut microbiome, their roles in the oral microbiome are less understood. Aim. This pilot study utilizes high-throughput shotgun metagenomic sequencing to examine the oral microbiota in different niches, particularly focusing on the association of BGCs with periodontitis. Methodology. We analysed saliva, subgingival plaque and supragingival plaque samples from periodontitis patients ( n =23) and controls ( n =16). DNA was extracted from these samples using standardized protocols. The high-throughput shotgun metagenomic sequencing was then performed to obtain comprehensive genetic information from the microbial communities present in the samples. Results. Our study identified 10 742 BGCs, with certain clusters being niche-specific. Notably, aryl polyenes and bacteriocins were the most prevalent BGCs identified. We discovered several 'novel' BGCs that are widely represented across various bacterial phyla and identified BGCs that had different distributions between periodontitis and control subjects. Our systematic approach unveiled the previously unexplored biosynthetic pathways that may be key players in periodontitis. Conclusions. Our research expands the current metagenomic knowledge of the oral microbiota in both healthy and periodontally diseased states. These findings highlight the presence of novel biosynthetic pathways in the oral cavity and suggest a complex network of host-microbe and microbe-microbe interactions, potentially influencing periodontal disease. The BGCs identified in this study pave the way for future investigations into the role of small-molecule-mediated interactions within the human oral microbiota and their impact on periodontitis.

Background: Diviner's sage (Salvia divinorum; Lamiaceae) is the source of the powerful hallucinogen salvinorin A (SalA). This neoclerodane diterpenoid is an agonist of the human Κ-opioid receptor with potential medical applications in the treatment of chronic pain, addiction, and post-traumatic stress disorder. Only two steps of the approximately twelve step biosynthetic sequence leading to SalA have been resolved to date., Results: To facilitate pathway elucidation in this ethnomedicinal plant species, here we report a chromosome level genome assembly. A high-quality genome sequence was assembled with an N50 value of 41.4 Mb and a BUSCO completeness score of 98.4%. The diploid (2n = 22) genome of ~ 541 Mb is comparable in size and ploidy to most other members of this genus. Two diterpene biosynthetic gene clusters were identified and are highly enriched in previously unidentified cytochrome P450s as well as crotonolide G synthase, which forms the dihydrofuran ring early in the SalA pathway. Coding sequences for other enzyme classes with likely involvement in downstream steps of the SalA pathway (BAHD acyl transferases, alcohol dehydrogenases, and O-methyl transferases) were scattered throughout the genome with no clear indication of clustering. Differential gene expression analysis suggests that most of these genes are not inducible by methyl jasmonate treatment., Conclusions: This genome sequence and associated gene annotation are among the highest resolution in Salvia, a genus well known for the medicinal properties of its members. Here we have identified the cohort of genes responsible for the remaining steps in the SalA pathway. This genome sequence and associated candidate genes will facilitate the elucidation of SalA biosynthesis and enable an exploration of its full clinical potential., (© 2024. The Author(s).)

Objective: Evaluation of Nepeta cataria as a host with specific endogenous metabolite background for transient expression and metabolic engineering of secondary biosynthetic sequences., Results: The reporter gene gfp::licBM3 as well as three biosynthetic genes leading to the formation of the cannabinoid precursor olivetolic acid were adopted to the modular cloning standard GoldenBraid, transiently expressed in two chemotypes of N. cataria and compared to Nicotiana benthamiana. To estimate the expression efficiency in both hosts, quantification of the reporter activity was carried out with a sensitive and specific lichenase assay. While N. benthamiana exhibited lichenase activity of 676 ± 94 μmol g -1  s -1 , N. cataria cultivar '1000', and the cultivar 'Citriodora' showed an activity of 37 ± 8 μmol g -1  s -1 and 18 ± 4 μmol g -1  s -1 , respectively. Further, combinatorial expression of genes involved in cannabinoid biosynthetic pathway acyl-activating enzyme 1 (aae1), olivetol synthase (ols) and olivetolic acid cyclase (oac) in N. cataria cv. resulted presumably in the in vivo production of olivetolic acid glycosides., Conclusion: Nepeta cataria is amenable to Agrobacterium-mediated transient expression and could serve as a novel chassis for the engineering of secondary metabolic pathways and transient evaluation of heterologous genes., (© 2024. The Author(s).)

Age is a prominent risk factor for cardiometabolic disease, often leading to heart structural and functional changes. However, precise molecular mechanisms underlying cardiac remodeling and dysfunction exclusively resulting from physiological aging remain elusive. Previous research demonstrated age-related functional alterations in baboons, analogous to humans. The goal of this study is to identify early cardiac molecular alterations preceding functional adaptations, shedding light on the regulation of age-associated changes. Unbiased transcriptomics of left ventricle samples are performed from female baboons aged 7.5-22.1 years (human equivalent ≈30-88 years). Weighted-gene correlation network and pathway enrichment analyses are performed, with histological validation. Modules of transcripts negatively correlated with age implicated declined metabolism-oxidative phosphorylation, tricarboxylic acid cycle, glycolysis, and fatty-acid β-oxidation. Transcripts positively correlated with age suggested a metabolic shift toward glucose-dependent anabolic pathways, including hexosamine biosynthetic pathway (HBP). This shift is associated with increased glycosaminoglycan synthesis, modification, precursor synthesis via HBP, and extracellular matrix accumulation, verified histologically. Upregulated extracellular matrix-induced signaling coincided with glycosaminoglycan accumulation, followed by cardiac hypertrophy-related pathways. Overall, these findings revealed a transcriptional shift in metabolism favoring glycosaminoglycan accumulation through HBP before cardiac hypertrophy. Unveiling this metabolic shift provides potential targets for age-related cardiac diseases, offering novel insights into early age-related mechanisms., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Docynia delavayi is an economically significant fruit species with a high market potential due to the special aroma of its fruit. Here, a 653.34 Mb high-quality genome of D. delavayi was first reported, of which 93.8 % of the sequences (612.98 Mb) could be anchored to 17 chromosomes, containing 48,325 protein-coding genes. Ks analysis proved that two whole genome duplication (WGD) events occurred in D. delavayi, resulting in the expansion of genes associated with terpene biosynthesis, which promoted its fruit-specific aroma production. Combined multi-omics analysis, α-farnesene was detected as the most abundant aroma substance emitted by D. delavayi fruit during storage, meanwhile one α-farnesene synthase gene (AFS) and 15 transcription factors (TFs) were identified as the candidate genes potentially involved in α-farnesene biosynthesis. Further studies for the regulation network of α-farnesene biosynthesis revealed that DdebHLH, DdeERF1 and DdeMYB could activate the transcription of DdeAFS. To our knowledge, it is the first report that MYB TF plays a regulatory role in α-farnesene biosynthesis, which will greatly facilitate future breeding programs for D. delavayi., Competing Interests: Declaration of competing interest The authors declare no conflict of interest in this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pyrimidine nucleosides, as intermediate materials of significant commercial value, find extensive applications in the pharmaceutical industry. However, the current production of pyrimidine nucleosides largely relies on chemical synthesis, creating environmental problems that do not align with sustainable development goals. Recent progress in systemic metabolic engineering and synthetic biology has enabled the synthesis of natural products like pyrimidine nucleosides through microbial fermentation, offering a more sustainable alternative. Nevertheless, the intricate and tightly regulated biosynthetic pathways involved in the microbial production of pyrimidine nucleosides pose a formidable challenge. This study focuses on metabolic engineering and synthetic biology strategies aimed at enhancing pyrimidine nucleoside production. These strategies include gene modification, transcriptional regulation, metabolic flux analysis, cofactor balance optimization, and transporter engineering. Finally, this research highlights the challenges involved in the further development of pyrimidine nucleoside-producing strains and offers potential solutions in order to provide theoretical guidance for future research endeavors in this field., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Benzylisoquinoline alkaloids (BIAs) are a class of secondary metabolites that possess diverse pharmaceutical properties and are exclusively accumulated in specific plant genera. The Pictet-Spengler condensation, catalyzed by norcoclaurine synthase (NCS), represents a key enzymatic reaction in the biosynthetic pathway of BIAs. While NCS genes have been identified in several plant families such as Papaveraceae, Berberidaceae, and Ranunculaceae, no NCS genes have been reported in Menispermaceae, which is another genus known to accumulate BIAs. Here, NCSs were isolated and functionally characterized from the Menispermaceae family plant Stephania tetrandra. In vitro enzyme assay identified two functional StNCSs which could catalyze the formation of (S)-norcoclaurine. These functionally characterized genes were then integrated into engineered yeast to enable the production of norcoclaurine. Phylogenetic analysis of the NCS enzymes revealed that the StNCSs predominantly clustered into two clades. The functional StNCSs clustered with known NCSs, highlighting the presence of a specific NCS catalytic domain. This study not only provides additional genetic components for the synthetic biology-based production of BIAs in yeast but also contributes to the understanding of the phylogenetic relationships and structure-function relationship of NCS genes involved in the origin and production of BIAs., (© 2023 Wiley Periodicals LLC.)

Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis-independent recombinase-assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum. We then integrated a non-ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum, which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

The formation of rice aroma is a complex process that is influenced by genetic and environmental factors. More than 500 fragrance compounds have been documented in fragrant rice, among which 2-AP dominates the aroma of rice. This paper introduced the identification of OsBadh2 in the biosynthesis of 2-AP in rice. Then, non-enzymatic and enzymatic pathways of the 2-AP biosynthesis have been comprehensively investigated. In detail, 2-AP biosynthesis-associated enzyme, such as OsBADH2, OsP5CS, OsGAD, OsGAPDH, OsProDH, OsOAT, OsODC and OsDAO, have been summarized, while MG and fatty acids are also implicated in modulating the biosynthesis of 2-AP by providing the acetyl groups. Moreover, extensive collections of traditional fragrant rice varieties have been collated, together with the OsBadh2 haplotypes of 312 fragrant rice germplasm in China. And finally, genetic engineering of OsBadh2 and other genes in the 2-AP biosynthesis to develop fragrant rice are discussed., 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 Masson SAS. All rights reserved.)

Anthocyanins are high-value natural compounds, but to date, their production still mainly relies on extraction from plants. A five-step metabolic pathway was constructed in probiotic Lactococcus lactis NZ9000 for rapid, stable, and glycosylated anthocyanin biosynthesis using chalcone as a substrate. The genes were cloned from anthocyanin-rich blueberry: chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS), and UDPG-flavonoid 3-O-glycosyltransferase (3GT). Using HR, the polysaccharide pellicle (PSP) segment of the cell wall polysaccharide synthesis (cwps) gene cluster from L. lactis NZ9000 was cloned into vector p15A-Cm-repDE. Then, CHI and F3H were placed sequentially under the control of NZProm 3 of this gene cluster in the vector, which was transformed into L. lactis NZ9000 to obtain Strain A. Furthermore, Strain B was constructed by placing F3H-DFR-ANS and 3GT under NZProm 2 and 3, respectively. Using LC-MS/MS analysis, several types of anthocyanins, including callistephin chloride, oenin chloride, malvidin O-hexoside, malvidin 3,5-diglucoside, and pelargonidin 3-O-malonyl-malonylhexoside, increased in the supernatant of the co-culture of Strains A and B compared to that of L. lactis NZ9000. This is the first time that a five-step metabolic pathway has been developed for anthocyanin biosynthesis in probiotic L. lactis NZ9000. This work lays the groundwork for novel anthocyanin production by a process involving the placement of several biosynthesis genes under the control of a gene cluster., (© 2024 Wiley‐VCH GmbH.)

Understanding human, animal, and environmental microbiota is essential for advancing global health and combating antimicrobial resistance (AMR). We investigate the oral and gut microbiota of 48 animal species in captivity, comparing them to those of wildlife animals. Specifically, we characterize the microbiota composition, metabolic pathways, AMR genes, and biosynthetic gene clusters (BGCs) encoding the production of specialized metabolites. Our results reveal a high diversity of microbiota, with 585 novel species-level genome bins (SGBs) and 484 complete BGCs identified. Functional gene analysis of microbiomes shows diet-dependent variations. Furthermore, by comparing our findings to wildlife-derived microbiomes, we observe the impact of captivity on the animal microbiome, including examples of converging microbiome compositions. Importantly, our study identifies AMR genes against commonly used veterinary antibiotics, as well as resistance to vancomycin, a critical antibiotic in human medicine. These findings underscore the importance of the 'One Health' approach and the potential for zoonotic transmission of pathogenic bacteria and AMR. Overall, our study contributes to a better understanding of the complexity of the animal microbiome and highlights its BGC diversity relevant to the discovery of novel antimicrobial compounds., (© 2024. The Author(s).)

Neohesperidin is a flavonoid glycoside widely used in the food and pharmaceutical industries. The current production of neohesperidin mainly relies on extraction from plants. Microbial fermentation demonstrates a promising prospect as an environmentally friendly, efficient, and economical method. In this study, we designed and constructed the biosynthetic pathway of neohesperidin in an Escherichia coli strain by introducing the glycosyltransferase UGT73B2 from Arabidopsis thaliana , rhamnose synthase Vv RHM-NRS from Vitis vinifera , and rhamnose transferase Cm 1,2RhaT from Citrus maxima . After optimization of the module and the uridine diphosphate (UDP)-glucose synthetic pathway, the engineered strain produced 4.64 g/L neohesperidin in a 5 L bioreactor, and the molar conversion rate of hesperetin was 45.8%. This has been the highest titer reported to date for the biosynthesis of neohesperidin in microorganisms. This study lays a foundation for the construction and application of strains with high yields of neohesperidin and provides a potential choice for the microbial production of other flavonoid glycosides.

Capsicum (pepper) is among the most economically important species worldwide, and its fruits accumulate specialized metabolites with essential roles in plant environmental interaction and human health benefits as well as in conferring their unique taste. However, the genetics underlying differences in metabolite presence/absence and/or accumulation remain largely unknown. In this study, we carried out a genome-wide association study as well as generating and characterizing a novel backcross inbred line mapping population to determine the genetic architecture of the pepper metabolome. This genetic analysis provided over 1,000 metabolic quantitative trait loci (mQTL) for over 250 annotated metabolites. We identified 92 candidate genes involved in various mQTLs. Among the identified loci, we described and validated a gene cluster of eleven UDP-glycosyltransferases (UGTs) involved in monomeric capsianoside biosynthesis. We additionally constructed the gene-by-gene-based biosynthetic pathway of pepper capsianoside biosynthesis, including both core and decorative reactions. Given that one of these decorative pathways, namely the glycosylation of acyclic diterpenoid glycosides, contributes to plant resistance, these data provide new insights and breeding resources for pepper. They additionally provide a blueprint for the better understanding of the biosynthesis of species-specific natural compounds in general., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)