Two Distinct Enzymes Have Both Phytoene Desaturase and 3,4-Desaturase Activities Involved in Carotenoid Biosynthesis by the Extremely Halophilic Archaeon Haloarcula japonica.

The extremely halophilic archaeon Haloarcula japonica accumulates the C50 carotenoid, bacterioruberin (BR). To reveal the BR biosynthetic pathway, unidentified phytoene desaturase candidates were functionally characterized in the present study. Two genes encoding the potential phytoene desaturases, c0507 and d1086, were found from the Ha. japonica genome sequence by a homology search using the Basic Local Align Search Tool. Disruption mutants of c0507 and d1086 and their complemented strains transformed with expression plasmids for c0507 and d1086 were subsequently constructed. High-performance liquid chromatography (HPLC) ana-lyses of carotenoids produced by these strains revealed that C0507 and D1086 were both bifunctional enzymes with the same activities as both phytoene desaturase (CrtI) and 3,4-desaturase (CrtD). C0507 and D1086 complemented each other during BR biosynthesis in Ha. japonica. This is the first study to identify two distinct enzymes with both CrtI and CrtD activities in an extremely halophilic archaeon.

An omics-based characterization of Wolfiporia cocos reveals three CYP450 members involved in the biosynthetic pathway of pachymic acid.

Wolfiporia cocos is a medicinal mushroom used in China. It biosynthesizes pachymic acid (PA), a main therapeutic triterpene associated with therapies. Nowadays, the unknown PA biosynthesis leads to difficulties in increasing its content in W. cocos. Herein, we report sequencing, assembling, and characterization of the genome and several transcriptomes of W. cocos. Sequence mining determined candidate genes that encode lanosterol synthase, sterol O-acyltransferase, and sterol C-24 methyltransferase likely involved in the steps from lanosterol to PA. Gene cluster analysis identified four CYP450 cDNAs likely involved in the biosynthesis of PA, namely WcCYP64-1, WcCYP64-2, WcCYP52, and WcCYP_FUM15, which were subjected to both overexpression and silencing in mycelia. The overexpression of each of WcCYP64-1, WcCYP52 and WcCYP_FUM15 increased the content of PA, 16α-hydroxytrametenolic acid, eburicoic acid, and tumulosic acid, while the silencing of each gene either significantly or slightly decreased the contents of these four compounds, indicating their involvement in the PA biosynthesis. In addition, different temperatures affected the expression of these genes and the formation of PA. By contrast, the overexpression and silencing of WcCYP64-2 did not alter the formation of these compounds. Taken together, these findings determine more potential steps in the biosynthetic pathway of PA for metabolic engineering.

Activation of secondary metabolite gene clusters in Chaetomium olivaceum via the deletion of a histone deacetylase.

Histone acetylation modifications in filamentous fungi play a crucial role in epigenetic gene regulation and are closely linked to the transcription of secondary metabolite (SM) biosynthetic gene clusters (BGCs). Histone deacetylases (HDACs) play a pivotal role in determining the extent of histone acetylation modifications and act as triggers for the expression activity of target BGCs. The genus Chaetomium is widely recognized as a rich source of novel and bioactive SMs. Deletion of a class I HDAC gene of Chaetomium olivaceum SD-80A, g7489, induces a substantial pleiotropic effect on the expression of SM BGCs. The C. olivaceum SD-80A ∆g7489 strain exhibited significant changes in morphology, sporulation ability, and secondary metabolic profile, resulting in the emergence of new compound peaks. Notably, three polyketides (A1-A3) and one asterriquinone (A4) were isolated from this mutant strain. Furthermore, our study explored the BGCs of A1-A4, confirming the function of two polyketide synthases (PKSs). Collectively, our findings highlight the promising potential of molecular epigenetic approaches for the elucidation of novel active compounds and their biosynthetic elements in Chaetomium species. This finding holds great significance for the exploration and utilization of Chaetomium resources. KEY POINTS: • Deletion of a class I histone deacetylase activated secondary metabolite gene clusters. • Three polyketides and one asterriquinone were isolated from HDAC deleted strain. • Two different PKSs were reported in C. olivaceum SD-80A.

Comparative transcriptome analysis reveals major genes, transcription factors and biosynthetic pathways associated with leaf senescence in rice under different nitrogen application.

BACKGROUND: Rice (Oryza sativa L.) is one of the most important food crops in the world and the application of nitrogen fertilizer is an effective means of ensuring stable and high rice yields. However, excessive application of nitrogen fertilizer not only causes a decline in the quality of rice, but also leads to a series of environmental costs. Nitrogen reutilization is closely related to leaf senescence, and nitrogen deficiency will lead to early functional leaf senescence, whereas moderate nitrogen application will help to delay leaf senescence and promote the production of photosynthetic assimilation products in leaves to achieve yield increase. Therefore, it is important to explore the mechanism by which nitrogen affects rice senescence, to search for genes that are tolerant to low nitrogen, and to delay the premature senescence of rice functional leaves. RESULTS: The present study was investigated the transcriptional changes in flag leaves between full heading and mature grain stages of rice (O. sativa) sp. japonica 'NanGeng 5718' under varying nitrogen (N) application: 0 kg/ha (no nitrogen; 0N), 240 kg/ha (moderate nitrogen; MN), and 300 kg/ha (high nitrogen; HN). Compared to MN condition, a total of 10427 and 8177 differentially expressed genes (DEGs) were detected in 0N and HN, respectively. We selected DEGs with opposite expression trends under 0N and HN conditions for GO and KEGG analyses to reveal the molecular mechanisms of nitrogen response involving DEGs. We confirmed that different N applications caused reprogramming of plant hormone signal transduction, glycolysis/gluconeogenesis, ascorbate and aldarate metabolism and photosynthesis pathways in regulating leaf senescence. Most DEGs of the jasmonic acid, ethylene, abscisic acid and salicylic acid metabolic pathways were up-regulated under 0N condition, whereas DEGs related to cytokinin and ascorbate metabolic pathways were induced in HN. Major transcription factors include ERF, WRKY, NAC and bZIP TF families have similar expression patterns which were induced under N starvation condition. CONCLUSION: Our results revealed that different nitrogen levels regulate rice leaf senescence mainly by affecting hormone levels and ascorbic acid biosynthesis. Jasmonic acid, ethylene, abscisic acid and salicylic acid promote early leaf senescence under low nitrogen condition, ethylene and ascorbate delay senescence under high nitrogen condition. In addition, ERF, WRKY, NAC and bZIP TF families promote early leaf senescence. The relevant genes can be used as candidate genes for the regulation of senescence. The results will provide gene reference for further genomic studies and new insights into the gene functions, pathways and transcription factors of N level regulates leaf senescence in rice, thereby improving NUE and reducing the adverse effects of over-application of N.

[Advances in paclitaxel biosynthesis and transcriptional regulation mechanisms].

Paclitaxel, a rare diterpene extracted from the bark of Chinese yew (Taxus chinensis), is renowned for its anti-cancer activity and serves as a primary drug for treating cancers. Due to the exceptionally low content of paclitaxel in the bark, a semi-synthetic method that depletes Chinese yew resources is used in the production of paclitaxel, which, however, fails to meet the escalating clinical demand. In recent years, researchers have achieved significant progress in heterologous biosynthesis and metabolic engineering for the production of paclitaxel. This article comprehensively reviews the advancements in paclitaxel production, encompassing chemical synthesis, heterologous biosynthesis, and cell engineering. It provides an in-depth introduction to the biosynthetic pathway and transcriptional regulation mechanisms of paclitaxel, aiming to provide a valuable reference for further research on paclitaxel biosynthesis.

Discovery, characterization, and engineering of an advantageous Streptomyces host for heterologous expression of natural product biosynthetic gene clusters.

BACKGROUND: Streptomyces is renowned for its robust biosynthetic capacity in producing medically relevant natural products. However, the majority of natural products biosynthetic gene clusters (BGCs) either yield low amounts of natural products or remain cryptic under standard laboratory conditions. Various heterologous production hosts have been engineered to address these challenges, and yet the successful activation of BGCs has still been limited. In our search for a valuable addition to the heterologous host panel, we identified the strain Streptomyces sp. A4420, which exhibited rapid initial growth and a high metabolic capacity, prompting further exploration of its potential. RESULTS: We engineered a polyketide-focused chassis strain based on Streptomyces sp. A4420 (CH strain) by deleting 9 native polyketide BGCs. The resulting metabolically simplified organism exhibited consistent sporulation and growth, surpassing the performance of most existing Streptomyces based chassis strains in standard liquid growth media. Four distinct polyketide BGCs were chosen and expressed in various heterologous hosts, including the Streptomyces sp. A4420 wild-type and CH strains, alongside Streptomyces coelicolor M1152, Streptomyces lividans TK24, Streptomyces albus J1074, and Streptomyces venezuelae NRRL B-65442. Remarkably, only the Streptomyces sp. A4420 CH strain demonstrated the capability to produce all metabolites under every condition outperforming its parental strain and other tested organisms. To enhance visualization and comparison of the tested strains, we developed a matrix-like analysis involving 15 parameters. This comprehensive analysis unequivocally illustrated the significant potential of the new strain to become a popular heterologous host. CONCLUSION: Our engineered Streptomyces sp. A4420 CH strain exhibits promising attributes for the heterologous expression of natural products with a focus on polyketides, offering an alternative choice in the arsenal of heterologous production strains. As genomics and cloning strategies progress, establishment of a diverse panel of heterologous production hosts will be crucial for expediting the discovery and production of medically relevant natural products derived from Streptomyces.

Integrated Metabolomics and Transcriptomics Analysis of Flavonoid Biosynthesis Pathway in Polygonatum cyrtonema Hua.

Flavonoids, a class of phenolic compounds, are one of the main functional components and have a wide range of molecular structures and biological activities in Polygonatum. A few of them, including homoisoflavonoids, chalcones, isoflavones, and flavones, were identified in Polygonatum and displayed a wide range of powerful biological activities, such as anti-cancer, anti-viral, and blood sugar regulation. However, few studies have systematically been published on the flavonoid biosynthesis pathway in Polygonatum cyrtonema Hua. Therefore, in the present study, a combined transcriptome and metabolome analysis was performed on the leaf, stem, rhizome, and root tissues of P. cyrtonema to uncover the synthesis pathway of flavonoids and to identify key regulatory genes. Flavonoid-targeted metabolomics detected a total of 65 active substances from four different tissues, among which 49 substances were first study to identify in Polygonatum, and 38 substances were flavonoids. A total of 19 differentially accumulated metabolites (DAMs) (five flavonols, three flavones, two dihydrochalcones, two flavanones, one flavanol, five phenylpropanoids, and one coumarin) were finally screened by KEGG enrichment analysis. Transcriptome analysis indicated that a total of 222 unigenes encoding 28 enzymes were annotated into three flavonoid biosynthesis pathways, which were "phenylpropanoid biosynthesis", "flavonoid biosynthesis", and "flavone and flavonol biosynthesis". The combined analysis of the metabolome and transcriptome revealed that 37 differentially expressed genes (DEGs) encoding 11 enzymes (C4H, PAL, 4CL, CHS, CHI, F3H, DFR, LAR, ANR, FNS, FLS) and 19 DAMs were more likely to be regulated in the flavonoid biosynthesis pathway. The expression of 11 DEGs was validated by qRT-PCR, resulting in good agreement with the RNA-Seq. Our studies provide a theoretical basis for further elucidating the flavonoid biosynthesis pathway in Polygonatum.

Effects of MhMYB1 and MhMYB2 transcription factors on the monoterpenoid biosynthesis pathway in l-menthol chemotype of Mentha haplocalyx Briq.

MAIN CONCLUSION: Transcription factors MhMYB1 and MhMYB2 correlate with monoterpenoid biosynthesis pathway in l-menthol chemotype of Mentha haplocalyx Briq, which could affect the contents of ( -)-menthol and ( -)-menthone. Mentha haplocalyx Briq., a plant with traditional medicinal and edible uses, is renowned for its rich essential oil content. The distinct functional activities and aromatic flavors of mint essential oils arise from various chemotypes. While the biosynthetic pathways of the main monoterpenes in mint are well understood, the regulatory mechanisms governing different chemotypes remain inadequately explored. In this investigation, we identified and cloned two transcription factor genes from the M. haplocalyx MYB family, namely MhMYB1 (PP236792) and MhMYB2 (PP236793), previously identified by our research group. Bioinformatics analysis revealed that MhMYB1 possesses two conserved MYB domains, while MhMYB2 contains a conserved SANT domain. Yeast one-hybrid (Y1H) analysis results demonstrated that both MhMYB1 and MhMYB2 interacted with the promoter regions of MhMD and MhPR, critical enzymes in the monoterpenoid biosynthesis pathway of M. haplocalyx. Subsequent virus-induced gene silencing (VIGS) of MhMYB1 and MhMYB2 led to a significant reduction (P < 0.01) in the relative expression levels of MhMD and MhPR genes in the VIGS groups of M. haplocalyx. In addition, there was a noteworthy decrease (P < 0.05) in the contents of ( -)-menthol and ( -)-menthone in the essential oil of M. haplocalyx. These findings suggest that MhMYB1 and MhMYB2 transcription factors play a positive regulatory role in ( -)-menthol biosynthesis, consequently influencing the essential oil composition in the l-menthol chemotype of M. haplocalyx. This study serves as a pivotal foundation for unraveling the regulatory mechanisms governing monoterpenoid biosynthesis in different chemotypes of M. haplocalyx.

Metabolomics and transcriptomics reveal the mechanism of alkaloid synthesis in Corydalis yanhusuo bulbs.

Corydalis yanhusuo W.T. Wang is a traditional herb. Benzylisoquinoline alkaloids (BIAs) are the main pharmacological active ingredients that play an important role in sedation, relieving pain, promoting blood circulation, and inhibiting cancer cells. However, there are few studies on the biosynthetic pathway of benzylisoquinoline alkaloids in Corydalis yanhusuo, especially on some specific components, such as tetrahydropalmatine. We carried out widely targeted metabolome and transcriptomic analyses to construct the biosynthetic pathway of benzylisoquinoline alkaloids and identified candidate genes. In this study, 702 metabolites were detected, including 216 alkaloids. Protoberberine-type and aporphine-type alkaloids are the main chemical components in C. yanhusuo bulbs. Key genes for benzylisoquinoline alkaloids biosynthesis, including 6-OMT, CNMT, NMCH, BBE, SOMT1, CFS, SPS, STOX, MSH, TNMT and P6H, were successfully identified. There was no significant difference in the content of benzylisoquinoline alkaloids and the expression level of genes between the two suborgans (mother-bulb and son-bulb). The expression levels of BIA genes in the expansion stage (MB-A and SB-A) were significantly higher than those in the maturity stage (MB-C and SB-C), and the content of benzylisoquinoline alkaloids was consistent with the pattern of gene regulation. Five complete single genes were likely to encode the functional enzyme of CoOMT, which participated in tetrahydropalmatine biosynthesis in C. yanhusuo bulbs. These studies provide a strong theoretical basis for the subsequent development of metabolic engineering of benzylisoquinoline alkaloids (especially tetrahydropalmatine) of C. yanhusuo.

Metabolic Blockade-Based Genome Mining of Sea Anemone-Associated Streptomyces sp. S1502 Identifies Atypical Angucyclines WS-5995 A-E: Isolation, Identification, Biosynthetic Investigation, and Bioactivities.

Marine symbiotic and epiphyte microorganisms are sources of bioactive or structurally novel natural products. Metabolic blockade-based genome mining has been proven to be an effective strategy to accelerate the discovery of natural products from both terrestrial and marine microorganisms. Here, the metabolic blockade-based genome mining strategy was applied to the discovery of other metabolites in a sea anemone-associated Streptomyces sp. S1502. We constructed a mutant Streptomyces sp. S1502/Δstp1 that switched to producing the atypical angucyclines WS-5995 A-E, among which WS-5995 E is a new compound. A biosynthetic gene cluster (wsm) of the angucyclines was identified through gene knock-out and heterologous expression studies. The biosynthetic pathways of WS-5995 A-E were proposed, the roles of some tailoring and regulatory genes were investigated, and the biological activities of WS-5995 A-E were evaluated. WS-5995 A has significant anti-Eimeria tenell activity with an IC50 value of 2.21 µM. The production of antibacterial streptopyrroles and anticoccidial WS-5995 A-E may play a protective role in the mutual relationship between Streptomyces sp. S1502 and its host.

Cell-free biosynthesis and engineering of ribosomally synthesized lanthipeptides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with diverse chemical structures and potent biological activities. A vast majority of RiPP gene clusters remain unexplored in microbial genomes, which is partially due to the lack of rapid and efficient heterologous expression systems for RiPP characterization and biosynthesis. Here, we report a unified biocatalysis (UniBioCat) system based on cell-free gene expression for rapid biosynthesis and engineering of RiPPs. We demonstrate UniBioCat by reconstituting a full biosynthetic pathway for de novo biosynthesis of salivaricin B, a lanthipeptide RiPP. Next, we delete several protease/peptidase genes from the source strain to enhance the performance of UniBioCat, which then can synthesize and screen salivaricin B variants with enhanced antimicrobial activity. Finally, we show that UniBioCat is generalizable by synthesizing and evaluating the bioactivity of ten uncharacterized lanthipeptides. We expect UniBioCat to accelerate the discovery, characterization, and synthesis of RiPPs.

Discovery of fungal onoceroid triterpenoids through domainless enzyme-targeted global genome mining.

Genomics-guided methodologies have revolutionized the discovery of natural products. However, a major challenge in the field of genome mining is determining how to selectively extract biosynthetic gene clusters (BGCs) for untapped natural products from numerous available genome sequences. In this study, we developed a fungal genome mining tool that extracts BGCs encoding enzymes that lack a detectable protein domain (i.e., domainless enzymes) and are not recognized as biosynthetic proteins by existing bioinformatic tools. We searched for BGCs encoding a homologue of Pyr4-family terpene cyclases, which are representative examples of apparently domainless enzymes, in approximately 2000 fungal genomes and discovered several BGCs with unique features. The subsequent characterization of selected BGCs led to the discovery of fungal onoceroid triterpenoids and unprecedented onoceroid synthases. Furthermore, in addition to the onoceroids, a previously unreported sesquiterpene hydroquinone, of which the biosynthesis involves a Pyr4-family terpene cyclase, was obtained. Our genome mining tool has broad applicability in fungal genome mining and can serve as a beneficial platform for accessing diverse, unexploited natural products.

Enhancing astaxanthin accumulation through the expression of the plant-derived astaxanthin biosynthetic pathway in Dunaliella salina.

Dunaliella salina, a microalga that thrives under high-saline conditions, is notable for its high ß-carotene content and the absence of a polysaccharide cell wall. These unique characteristics render it a prime candidate as a cellular platform for astaxanthin production. In this study, our initial tests in an E. coli revealed that ß-ring-4-dehydrogenase (CBFD) and 4-hydroxy-ß-ring-4-dehydrogenase (HBFD) genes from Adonis aestivalis outperformed ß-carotene hydroxylase (BCH) and ß-carotene ketolase (BKT) from Haematococcus pluvialis counterparts by two-fold in terms of astaxanthin biosynthesis efficiency. Subsequently, we utilized electroporation to integrate either the BKT gene or the CBFD and HBFD genes into the genome of D. salina. In comparison to wild-type D. salina, strains transformed with BKT or CBFD and HBFD exhibited inhibited growth, underwent color changes to shades of red and yellow, and saw a nearly 50% decline in cell density. HPLC analysis confirmed astaxanthin synthesis in engineered D. salina strains, with CBFD + HBFD-D. salina yielding 134.88 ± 9.12 µg/g of dry cell weight (DCW), significantly higher than BKT-D. salina (83.58 ± 2.40 µg/g). This represents the largest amount of astaxanthin extracted from transgenic D. salina, as reported to date. These findings have significant implications, opening up new avenues for the development of specialized D. salina-based microcell factories for efficient astaxanthin production.

A comprehensive review of the molecular and genetic mechanisms underlying gum and resin synthesis in Ferula species.

Ferula spp. are plants that produce oleo-gum-resins (OGRs), which are plant exudates with various colors. These OGRs have various industrial applications in pharmacology, perfumery, and food. The main constituents of these OGRs are terpenoids, a diverse group of organic compounds with different structures and functions. The biosynthesis of OGRs in Ferula spp., particularly galbanum, holds considerable economic and ecological importance. However, the molecular and genetic underpinnings of this biosynthetic pathway remain largely enigmatic. This review provides an overview of the current state of knowledge on the biosynthesis of OGRs in Ferula spp., highlighting the major enzymes, genes, and pathways involved in the synthesis of different terpenoid classes, such as monoterpenes, sesquiterpenes, and triterpenes. It also examines the potential of using omics techniques, such as transcriptomics and metabolomics, and genome editing tools, such as CRISPR/Cas, to increase the yield and quality of Ferula OGRs, as well as to create novel bioactive compounds with enhanced properties. Moreover, this review addresses the current challenges and opportunities of applying gene editing in Ferula spp., and suggests some directions for future research and development.

The GPI-anchor biosynthesis pathway is critical for syncytiotrophoblast differentiation and placental development.

The glycosylphosphatidylinositol (GPI) biosynthetic pathway in the endoplasmic reticulum (ER) is crucial for generating GPI-anchored proteins (GPI-APs), which are translocated to the cell surface and play a vital role in cell signaling and adhesion. This study focuses on two integral components of the GPI pathway, the PIGL and PIGF proteins, and their significance in trophoblast biology. We show that GPI pathway mutations impact on placental development impairing the differentiation of the syncytiotrophoblast (SynT), and especially the SynT-II layer, which is essential for the establishment of the definitive nutrient exchange area within the placental labyrinth. CRISPR/Cas9 knockout of Pigl and Pigf in mouse trophoblast stem cells (mTSCs) confirms the role of these GPI enzymes in syncytiotrophoblast differentiation. Mechanistically, impaired GPI-AP generation induces an excessive unfolded protein response (UPR) in the ER in mTSCs growing in stem cell conditions, akin to what is observed in human preeclampsia. Upon differentiation, the impairment of the GPI pathway hinders the induction of WNT signaling for early SynT-II development. Remarkably, the transcriptomic profile of Pigl- and Pigf-deficient cells separates human patient placental samples into preeclampsia and control groups, suggesting an involvement of Pigl and Pigf in establishing a preeclamptic gene signature. Our study unveils the pivotal role of GPI biosynthesis in early placentation and uncovers a new preeclampsia gene expression profile associated with mutations in the GPI biosynthesis pathway, providing novel molecular insights into placental development with implications for enhanced patient stratification and timely interventions.

Biosynthetic gene clusters with biotechnological applications in novel Antarctic isolates from Actinomycetota.

Actinomycetota have been widely described as valuable sources for the acquisition of secondary metabolites. Most microbial metabolites are produced via metabolic pathways encoded by biosynthetic gene clusters (BGCs). Although many secondary metabolites are not essential for the survival of bacteria, they play an important role in their adaptation and interactions within microbial communities. This is how bacteria isolated from extreme environments such as Antarctica could facilitate the discovery of new BGCs with biotechnological potential. This study aimed to isolate rare Actinomycetota strains from Antarctic soil and sediment samples and identify their metabolic potential based on genome mining and exploration of biosynthetic gene clusters. To this end, the strains were sequenced using Illumina and Oxford Nanopore Technologies platforms. The assemblies were annotated and subjected to phylogenetic analysis. Finally, the BGCs present in each genome were identified using the antiSMASH tool, and the biosynthetic diversity of the Micrococcaceae family was evaluated. Taxonomic annotation revealed that seven strains were new and two were previously reported in the NCBI database. Additionally, BGCs encoding type III polyketide synthases (T3PKS), beta-lactones, siderophores, and non-ribosomal peptide synthetases (NRPS) have been identified, among others. In addition, the sequence similarity network showed a predominant type of BGCs in the family Micrococcaceae, and some genera were distinctly grouped. The BGCs identified in the isolated strains could be associated with applications such as antimicrobials, anticancer agents, and plant growth promoters, among others, positioning them as excellent candidates for future biotechnological applications and innovations. KEY POINTS: • Novel Antarctic rare Actinomycetota strains were isolated from soil and sediments • Genome-based taxonomic affiliation revealed seven potentially novel species • Genome mining showed metabolic potential for novel natural products.

Tomato root specialized metabolites evolved through gene duplication and regulatory divergence within a biosynthetic gene cluster.

Tremendous plant metabolic diversity arises from phylogenetically restricted specialized metabolic pathways. Specialized metabolites are synthesized in dedicated cells or tissues, with pathway genes sometimes colocalizing in biosynthetic gene clusters (BGCs). However, the mechanisms by which spatial expression patterns arise and the role of BGCs in pathway evolution remain underappreciated. In this study, we investigated the mechanisms driving acylsugar evolution in the Solanaceae. Previously thought to be restricted to glandular trichomes, acylsugars were recently found in cultivated tomato roots. We demonstrated that acylsugars in cultivated tomato roots and trichomes have different sugar cores, identified root-enriched paralogs of trichome acylsugar pathway genes, and characterized a key paralog required for root acylsugar biosynthesis, SlASAT1-LIKE (SlASAT1-L), which is nested within a previously reported trichome acylsugar BGC. Last, we provided evidence that ASAT1-L arose through duplication of its paralog, ASAT1, and was trichome-expressed before acquiring root-specific expression in the Solanum genus. Our results illuminate the genomic context and molecular mechanisms underpinning metabolic diversity in plants.

Engineering Streptomyces sp. CPCC 204095 for the targeted high-level production of isatropolone A by elucidating its pathway-specific regulatory mechanism.

BACKGROUND: Isatropolone A and C, produced by Streptomyces sp. CPCC 204095, belong to an unusual class of non-benzenoid aromatic compounds and contain a rare seven-membered ring structure. Isatropolone A exhibits potent activity against Leishmania donovani, comparable to the only oral drug miltefosine. However, its variably low productivity represents a limitation for this lead compound in the future development of new anti-leishmaniasis drugs to meet unmet clinical needs. RESULTS: Here we first elucidated the regulatory cascade of biosynthesis of isatropolones, which consists of two SARP family regulators, IsaF and IsaJ. Through a series of in vivo and in vitro experiments, IsaF was identified as a pathway-specific activator that orchestrates the transcription of the gene cluster essential for isatropolone biosynthesis. Interestingly, IsaJ was found to only upregulate the expression of the cytochrome P450 monooxygenase IsaS, which is crucial for the yield and proportion of isatropolone A and C. Through targeted gene deletions of isaJ or isaS, we effectively impeded the conversion of isatropolone A to C. Concurrently, the facilitation of isaF overexpression governed by selected promoters, prompted the comprehensive activation of the production of isatropolone A. Furthermore, meticulous optimization of the fermentation parameters was conducted. These strategies culminated in the attainment of an unprecedented maximum yield-980.8 mg/L of isatropolone A-achieved in small-scale solid-state fermentation utilizing the genetically modified strains, thereby establishing the highest reported titer to date. CONCLUSION: In Streptomyces sp. CPCC 204095, the production of isatropolone A and C is modulated by the SARP regulators IsaF and IsaJ. IsaF serves as a master pathway-specific regulator for the production of isatropolones. IsaJ, on the other hand, only dictates the transcription of IsaS, the enzyme responsible for the conversion of isatropolone A and C. By engineering the expression of these pivotal genes, we have devised a strategy for genetic modification aimed at the selective and high-yield biosynthesis of isatropolone A. This study not only unveils the unique regulatory mechanisms governing isatropolone biosynthesis for the first time, but also establishes an essential engineering framework for the targeted high-level production of isatropolone A.

Biosynthetic potential of the sediment microbial subcommunities of an unexplored karst ecosystem and its ecological implications.

Microbial communities from various environments have been studied in the quest for new natural products with a broad range of applications in medicine and biotechnology. We employed an enrichment method and genome mining tools to examine the biosynthetic potential of microbial communities in the sediments of a coastal sinkhole within the karst ecosystem of the Yucatán Peninsula, Mexico. Our investigation led to the detection of 203 biosynthetic gene clusters (BGCs) and 55 secondary metabolites (SMs) within 35 high-quality metagenome-assembled genomes (MAGs) derived from these subcommunities. The most abundant types of BGCs were Terpene, Nonribosomal peptide-synthetase, and Type III polyketide synthase. Some of the in silico identified BGCs and SMs have been previously reported to exhibit biological activities against pathogenic bacteria and fungi. Others could play significant roles in the sinkhole ecosystem, such as iron solubilization and osmotic stress protection. Interestingly, 75% of the BGCs showed no sequence homology with bacterial BGCs previously reported in the MiBIG database. This suggests that the microbial communities in this environment could be an untapped source of genes encoding novel specialized compounds. The majority of the BGCs were identified in pathways found in the genus Virgibacillus, followed by Sporosarcina, Siminovitchia, Rhodococcus, and Halomonas. The latter, along with Paraclostridium and Lysinibacillus, had the highest number of identified BGC types. This study offers fresh insights into the potential ecological role of SMs from sediment microbial communities in an unexplored environment, underscoring their value as a source of novel natural products.

Integrated Untargeted Metabolome, Full-Length Sequencing and Transcriptome Analyses Reveal the Mechanism of Flavonoid Biosynthesis in Blueberry (Vaccinium spp.) Fruit.

As a highly economic berry fruit crop, blueberry is enjoyed by most people and has various potential health benefits, many of which are attributed to the relatively high concentrations of flavonoids. To obtain more accurate and comprehensive transcripts, the full-length transcriptome of half-highbush blueberry (Vaccinium corymbosum/angustifolium cultivar Northland) obtained using single molecule real-time and next-generation sequencing technologies was reported for the first time. Overall, 147,569 consensus transcripts (average length, 2738 bp; N50, 3176 bp) were obtained. After quality control steps, 63,425 high-quality isoforms were obtained and 5030 novel genes, 3002 long non-coding RNAs, 3946 transcription factor genes (TFs), 30,540 alternative splicing events, and 2285 fusion gene pairs were identified. To better explore the molecular mechanism of flavonoid biosynthesis in mature blueberry fruit, an integrative analysis of the metabolome and transcriptome was performed on the exocarp, sarcocarp, and seed. A relatively complete biosynthesis pathway map of phenylpropanoids, flavonoids, and proanthocyanins in blueberry was constructed. The results of the joint analysis showed that the 228 functional genes and 42 TFs regulated 78 differentially expressed metabolites within the biosynthesis pathway of phenylpropanoids/flavonoids. O2PLS analysis results showed that the key metabolites differentially accumulated in blueberry fruit tissues were albireodelphin, delphinidin 3,5-diglucoside, delphinidin 3-O-rutinoside, and delphinidin 3-O-sophoroside, and 10 structural genes (4 Vc4CLs, 3 VcBZ1s, 1 VcUGT75C1, 1 VcAT, and 1 VcUGAT), 4 transporter genes (1 VcGSTF and 3 VcMATEs), and 10 TFs (1 VcMYB, 2 VcbHLHs, 4 VcWD40s, and 3 VcNACs) exhibited strong correlations with 4 delphinidin glycosides. These findings provide insights into the molecular mechanisms of flavonoid biosynthesis and accumulation in blueberry fruit.