ATM-mediated double-strand break repair is required for meiotic genome stability at high temperature.

In eukaryotes, meiotic recombination maintains genome stability and creates genetic diversity. The conserved Ataxia-Telangiectasia Mutated (ATM) kinase regulates multiple processes in meiotic homologous recombination, including DNA double-strand break (DSB) formation and repair, synaptonemal complex organization, and crossover formation and distribution. However, its function in plant meiotic recombination under stressful environmental conditions remains poorly understood. In this study, we demonstrate that ATM is required for the maintenance of meiotic genome stability under heat stress in Arabidopsis thaliana. Using cytogenetic approaches we determined that ATM does not mediate reduced DSB formation but does ensure successful DSB repair, and thus meiotic chromosome integrity, under heat stress. Further genetic analysis suggested that ATM mediates DSB repair at high temperature by acting downstream of the MRE11-RAD50-NBS1 (MRN) complex, and acts in a RAD51-independent but chromosome axis-dependent manner. This study extends our understanding on the role of ATM in DSB repair and the protection of genome stability in plants under high temperature stress.

Two new quinazoline alkaloids produced by Aspergillus versicolor and their antimicrobial activities.

Two new quinazoline alkaloids versicomides G-H (1 and 2), together with seven known compounds, were isolated from Aspergillus versicolor HYQZ-215 obtained from the sediment of Qarhan Salt Lake. Their structures were elucidated by NMR, HRESIMS, and quantum chemical ECD calculations data. The antimicrobial activities of these compounds were evaluated against seven agricultural pathogenic fungi and eight clinically drug-resistant bacteria.

Didepside Formation by the Nonreducing Polyketide Synthase Preu6 of Preussia isomera Requires Interaction of Starter Acyl Transferase and Thioesterase Domains.

Orsellinic acid (OA) derivatives are produced by filamentous fungi using nonreducing polyketide synthases (nrPKSs). The chain-releasing thioesterase (TE) domains of such nrPKSs were proposed to also catalyze dimerization to yield didepsides, such as lecanoric acid. Here, we use combinatorial domain exchanges, domain dissections and reconstitutions to reveal that the TE domain of the lecanoric acid synthase Preu6 of Preussia isomera must collaborate with the starter acyl transferase (SAT) domain from the same nrPKS. We show that artificial SAT-TE fusion proteins are highly effective catalysts and reprogram the ketide homologation chassis to form didepsides. We also demonstrate that dissected SAT and TE domains of Preu6 physically interact, and SAT and TE domains of OA-synthesizing nrPKSs may co-evolve. Our work highlights an unexpected domain-domain interaction in nrPKSs that must be considered for the combinatorial biosynthesis of unnatural didepsides, depsidones, and diphenyl ethers.

Heat stress interferes with formation of double-strand breaks and homolog synapsis.

Meiotic recombination (MR) drives novel combinations of alleles and contributes to genomic diversity in eukaryotes. In this study, we showed that heat stress (36°C-38°C) over the fertile threshold fully abolished crossover formation in Arabidopsis (Arabidopsis thaliana). Cytological and genetic studies in wild-type plants and syn1 and rad51 mutants suggested that heat stress reduces generation of SPO11-dependent double-strand breaks (DSBs). In support, the abundance of recombinase DMC1, which is required for MR-specific DSB repair, was significantly reduced under heat stress. In addition, high temperatures induced disassembly and/or instability of the ASY4- but not the SYN1-mediated chromosome axis. At the same time, the ASY1-associated lateral element of the synaptonemal complex (SC) was partially affected, while the ZYP1-dependent central element of SC was disrupted, indicating that heat stress impairs SC formation. Moreover, expression of genes involved in DSB formation; e.g. SPO11-1, PRD1, 2, and 3 was not impacted; however, recombinase RAD51 and chromosome axis factors ASY3 and ASY4 were significantly downregulated under heat stress. Taken together, these findings revealed that heat stress inhibits MR via compromised DSB formation and homolog synapsis, which are possible downstream effects of the impacted chromosome axis. Our study thus provides evidence shedding light on how increasing environmental temperature influences MR in Arabidopsis.

Intrinsic and Extrinsic Programming of Product Chain Length and Release Mode in Fungal Collaborating Iterative Polyketide Synthases.

Combinatorial biosynthesis with fungal polyketide synthases (PKSs) promises to produce unprecedented bioactive "unnatural" natural products (uNPs) for drug discovery. Genome mining of the dothideomycete Rhytidhysteron rufulum uncovered a collaborating highly reducing PKS (hrPKS)-nonreducing PKS (nrPKS) pair. These enzymes produce trace amounts of rare S-type benzenediol macrolactone congeners with a phenylacetate core in a heterologous host. However, subunit shuffling and domain swaps with voucher enzymes demonstrated that all PKS domains are highly productive. This contradiction led us to reveal novel programming layers exerted by the starter unit acyltransferase (SAT) and the thioesterase (TE) domains on the PKS system. First, macrocyclic vs linear product formation is dictated by the intrinsic biosynthetic program of the TE domain. Next, the chain length of the hrPKS product is strongly influenced in trans by the off-loading preferences of the nrPKS SAT domain. Last, TE domains are size-selective filters that facilitate or obstruct product formation from certain priming units. Thus, the intrinsic programs of the SAT and TE domains are both part of the extrinsic program of the hrPKS subunit and modulate the observable metaprogram of the whole PKS system. Reconstruction of SAT and TE phylogenies suggests that these domains travel different evolutionary trajectories, with the resulting divergence creating potential conflicts in the PKS metaprogram. Such conflicts often emerge in chimeric PKSs created by combinatorial biosynthesis, reducing biosynthetic efficiency or even incapacitating the system. Understanding the points of failure for such engineered biocatalysts is pivotal to advance the biosynthetic production of uNPs.

Rational Reprogramming of O-Methylation Regioselectivity for Combinatorial Biosynthetic Tailoring of Benzenediol Lactone Scaffolds.

O-Methylation modulates the pharmacokinetic and pharmacodynamic (PK/PD) properties of small-molecule natural products, affecting their bioavailability, stability, and binding to targets. Diversity-oriented combinatorial biosynthesis of new chemical entities for drug discovery and optimization of known bioactive scaffolds during drug development both demand efficient O-methyltransferase (OMT) biocatalysts with considerable substrate promiscuity and tunable regioselectivity that can be deployed in a scalable and sustainable manner. Here we demonstrate efficient total biosynthetic and biocatalytic platforms that use a pair of fungal OMTs with orthogonal regiospecificity to produce unnatural O-methylated benzenediol lactone polyketides. We show that rational, structure-guided active-site cavity engineering can reprogram the regioselectivity of these enzymes. We also characterize the interplay of engineered regioselectivity with substrate plasticity. These findings will guide combinatorial biosynthetic tailoring of unnatural products toward the generation of diverse chemical matter for drug discovery and the PK/PD optimization of bioactive scaffolds for drug development.

Effect of A Polyphenol-Rich Canarium album Extract on the Composition of the Gut Microbiota of Mice Fed a High-Fat Diet.

This study investigated the influence of Canarium album extract (CAext) on intestinal microbiota composition of mice fed a high-fat diet (HFD). Kun Ming (KM) mice were fed either a normal chow diet or a HFD for six weeks. At the seventh week, HFD-fed mice were gavaged daily with saline, or a different dose of CAext for four weeks, respectively. Then, the composition of the gut microbiota was analyzed by high-throughput sequencing technology. Analysis of fecal microbial populations, grouped by phyla, showed significant increases of Firmicutes and Verrucomicrobia, but a decrease of Bacteroidetes in all CAext-fed mice. Particularly, CAext gavage in a low dose or a medium dose caused a significant increase in the proportion of Akkermansia. These findings suggested that CAext can alter the gut microbiota composition of HFD-fed mice, and had a potential prebiotic effects on Akkermansia.

Identification and role analysis of an intermediate produced by a polygenic mutant of Monascus pigments cluster in Monascus ruber M7.

Monascus pigments (Mps) are a group of azaphilonic secondary metabolites produced by Monascus spp. via a polyketide pathway. A mutant deleted an about 30 kb region of Mps gene cluster from Monascus ruber M7 was isolated previously, which produces a high amount of a light yellow pigment. The current study revealed that the mutant named ΔMpigJ-R lost proximate eight genes of the Mps gene cluster in M. ruber M7 through genetic analysis at DNA and RNA levels. The produced light yellow material was identified as a benzaldehyde derivative named as 6-(4-hydroxy-2-oxopentyl)-3-methyl-2, 4-dioxocyclohexane carb-aldehyde (M7PKS-1) by FT-IR, NMR, and MS. The sodium acetate-1-(13)C feeding experiment indicated that M7PKS-1 was a product produced from polyketide pathway. Finally, the feeding of M7PKS-1 helped to induce and regain Mps production of the mutants (ΔMpigA and ΔMpigE) which were previously unable to biosynthesize Mps and proved that M7PKS-1 was an initial intermediate of Mps. The results in this study provide a line of action to unveil Monascus pigments biosynthesis pathway.

MpigE, a gene involved in pigment biosynthesis in Monascus ruber M7.

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries. However, MP biosynthesis pathway is still a controversy, and only few related genes have been reported. In this study, the function of MpigE, a gene involved in MP biosynthesis in Monascus ruber M7, was analyzed. The results revealed that the disruption, complementation, and overexpression of MpigE in M. ruber M7 had very little effects on the growth and phenotypes except MPs. The MpigE deletion strain (∆MpigE) just yielded four kinds of yellow MPs and very little red pigments, while the wild-type strain M. ruber M7 produced a MP complex mixture including three (orange, red, and yellow) categories of MP compounds. Two of the four yellow MPs produced by ∆MpigE were the same as those yielded by M. ruber M7. The MpigE complementation strain (∆MpigE::MpigE) recovered the ability to generate orange and red MPs as M. ruber M7. The MP types produced by the MpigE overexpression strain (M7::PtrpC-MpigE) were consistent with those of M. ruber M7, while the color value was about 1.3-fold as that of M. ruber M7 (3,129 U/g red kojic). For the production of citrinin, the disruption of MpigE almost had no influence on the strain, whereas the overexpression of MpigE made citrinin decrease drastically in YES fermentation. This work will make a contribution to the study on the biosynthesis pathway of MPs in M. ruber.

Indole alkaloids from endophytic fungus Robillarda sessilis and their antibacterial activity.

Three undescribed indole alkaloids, fusarindoles F and G (1 and 2), and chlamydosporin B (3), together with five known compounds (4-8) were isolated from Robillarda sessilis. Their structures were elucidated based on NMR, UV, HRESIMS, and ECD calculation. Fusarindole F (1) own unusual asymmetric bis-indole structure. Compounds 5, 6, 7 exhibited moderate antibacterial activity against methicillin-resistant Staphylococcus aureus with a MIC value of 12.5 µg/mL. According to molecular docking experiment, the target proteins of compound 7 against methicillin-resistant S. aureus may be ELANE, MAOB and STAT3.

Emerindanols A and B: Two Bipolyhydroindenol Sesterterpenes with 5/6-6/5 Coupled Ring System Discovered by Genome Mining.

Genome mining of Emericella sp. XL-029 achieved a new type E sesterterpene synthase, EmES, which affored a novel bipolyhydroindenol sesterterpene, emerindanol A. Heterologous coexpression with the upstream P450 oxidase revealed C-4 hydroxylated product, emerindanol B. Notably, emerindanols A and B represented the first sesterterpenes featuring a unique 5/6-6/5 coupled ring system. EmES was postulated to initiate through C1-IV-V pathway and convert the fused ring intermediate into the final coupled ring product through a spiro skeleton.

Ku70 and ku80 null mutants improve the gene targeting frequency in Monascus ruber M7.

Normally, gene targeting by homologous recombination occurs rarely during a transformation process since non-homologous recombination is predominant in filamentous fungi. In our previous researches, the average gene replacement frequency (GRF) in Monascus ruber M7 was as low as 15 %. To develop a highly efficient gene targeting system for M. ruber M7, two M. ruber M7 null mutants of ku70 (MrΔku70) and ku80 (MrΔku80) were constructed which had no apparent defects in the development including vegetative growth, colony phenotype, microscopic morphology and spore yield compared with M. ruber M7. In addition, the production of some significant secondary metabolites such as pigments and citrinin had no differences between the two disruptants and the wild-type strain. Further results revealed that the GRFs of triA (encoding a putative acetyltransferase) were 42.2 % and 61.5 % in the MrΔku70 and MrΔku80 strains, respectively, while it was only about 20 % in M. ruber M7. Furthermore, GRFs of these two disruptants at other loci (the pigE, fmdS genes in MrΔku70 and the ku70 gene in MrΔku80) were investigated, and the results indicated that GRFs in the MrΔku70 strain and the MrΔku80 strain were doubled and tripled compared with that in M. ruber M7, respectively. Therefore, the ku70 and ku80 null mutants of M. ruber M7, especially the ku80-deleted strain, will be excellent hosts for efficient gene targeting.

Deletion of pigR gene in Monascus ruber leads to loss of pigment production.

Pigments produced by Monascus are traditional food colorants and are widely used as dietary supplements. Since genes involving in pigment biosynthesis have not been reported, we describe the identification of a putative pigment-regulatory gene (pigR) obtained by molecular analysis of an albino strain of Monascus ruber M7. In the pigR-deleted strain (ΔpigR), neither the pigments nor pigR expression were detected by HPLC or reverse-transcription PCR, respectively, whereas the introduction of the pigR, together with a constitutive trpC promoter into ΔpigR, caused it to produce 5.4 U of red pigments/g dry mycelia, about 12-fold higher than Monascus ruber M7 (0.46 U/g dry mycelia). Thus pigR up-regulates pigment production in Monascus ruber M7.

Cloning and Functional Characterization of the Polyketide Synthases Based on Genome Mining of Preussia isomera XL-1326.

Fungal polyketides (PKs) are one of the largest families of structurally diverse bioactive natural products biosynthesized by multidomain megasynthases, in which thioesterase (TE) domains act as nonequivalent decision gates determining both the shape and the yield of the polyketide intermediate. The endophytic fungus Preussia isomera XL-1326 was discovered to have an excellent capacity for secreting diverse bioactive PKs, i.e., the hot enantiomers (±)-preuisolactone A with antibacterial activity, the single-spiro minimoidione B with α-glucosidase inhibition activity, and the uncommon heptaketide setosol with antifungal activity, which drive us to illustrate how the unique PKs are biosynthesized. In this study, we first reported the genome sequence information of P. isomera. Based on genome mining, we discovered nine transcriptionally active genes encoding polyketide synthases (PKSs), Preu1-Preu9, of which those of Preu3, Preu4, and Preu6 were cloned and functionally characterized due to possessing complete sets of synthetic and release domains. Through heterologous expression in Saccharomyces cerevisiae, Preu3 and Preu6 could release high yields of orsellinic acid (OA) derivatives [3-methylorsellinic acid (3-MOA) and lecanoric acid, respectively]. Correspondingly, we found that Preu3 and Preu6 were clustered into OA derivative synthase groups by phylogenetic analysis. Next, with TE domain swapping, we constructed a novel "non-native" PKS, Preu6-TEPreu3, which shared a very low identity with OA synthase, OrsA, from Aspergillus nidulans but could produce a large amount of OA. In addition, with the use of Preu6-TEPreu3, we synthesized methyl 3-methylorsellinate (synthetic oak moss of great economic value) from 3-MOA as the substrate, and interestingly, 3-MOA exhibited remarkable antibacterial activities, while methyl 3-methylorsellinate displayed broad-spectrum antifungal activity. Taken together, we identified two novel PKSs to biosynthesize 3-MOA and lecanoric acid, respectively, with information on such kinds of PKSs rarely reported, and constructed one novel "non-native" PKS to largely biosynthesize OA. This work is our first step to explore the biosynthesis of the PKs in P. isomera, and it also provides a new platform for high-level environment-friendly production of OA derivatives and the development of new antimicrobial agents.

An Integrated Approach to Determine the Boundaries of the Azaphilone Pigment Biosynthetic Gene Cluster of Monascus ruber M7 Grown on Potato Dextrose Agar.

Monascus-type azaphilone pigments (MonAzPs) are produced in multi-thousand ton quantities each year and used as food colorants and nutraceuticals in East Asia. Several groups, including ours, described MonAzPs biosynthesis as a highly complex pathway with many branch points, affording more than 110 MonAzP congeners in a small group of fungi in the Eurotiales order. MonAzPs biosynthetic gene clusters (BGCs) are also very complex and mosaic-like, with some genes involved in more than one pathway, while other genes playing no apparent role in MonAzPs production. Due to this complexity, MonAzPs BGCs have been delimited differently in various fungi. Since most of these predictions rely primarily on bioinformatic analyses, it is possible that genes immediately outside the currently predicted BGC borders are also involved, especially those whose function cannot be predicted from sequence similarities alone. Conversely, some peripheral genes presumed to be part of the BGC may in fact lay outside the boundaries. This study uses a combination of computational and transcriptional analyses to predict the extent of the MonAzPs BGC in Monascus ruber M7. Gene knockouts and analysis of MonAzPs production of the mutants are then used to validate the prediction, revealing that the BGC consists of 16 genes, extending from mrpigA to mrpigP. We further predict that two strains of Talaromyces marneffei, ATCC 18224 and PM1, encode an orthologous but non-syntenic MonAzPs BGC with 14 genes. This work highlights the need to use comprehensive, integrated approaches for the more precise determination of secondary metabolite BGC boundaries.

Two novel eremophylane acetophenone conjugates from Colletotrichum gloeosporioides, an endophytic fungus in Salvia miltiorrhiza.

Two novel eremophylane acetophenone conjugates, colletotricholides A (1) and B (2), were isolated from the solid fermentation cultures of an endophytic fungus Colletotrichum gloeosporioides XL1200 isolated from the aerial parts of Salvia miltiorrhiza. The chemical structures of 1-2 were characterized by extensive spectroscopic methods and single-crystal X-ray crystallography. Structurally, compounds 1-2 are two unusual eremophylane acetophenone conjugates originating from the hybrid pathways of polyketide synthase and sesquiterpene synthase. In addition, compounds 1-2 were inactive against tested pathogens.

(±)-Preisomide: A new alkaloid featuring a rare naturally occurring tetrahydro-2H-1,2-oxazin skeleton from an endophytic fungus Preussia isomera by using OSMAC strategy.

A new alkaloid, named (±)-preisomide (1), together with five known polyketides (2-6), were isolated from an endophytic fungus Preussia isomera in Panax notoginseng by using one strain-many compounds (OSMAC) strategy. Their structures were identified by extensive spectroscopic experiments and comparison with literature data. Structurally, compound 1 possessed a rare naturally occurring tetrahydro-2H-1,2-oxazin ring. Compound 6 displayed significant antibacterial activity against multidrug-resistant Enterococcus faecium, methicinllin-resistant Staphylococcus aureus and multidrug-resistant Enterococcus faecalis with an MIC value of 25 µg/mL, as well as moderate antifungal activity against Gibberella saubinetii with an MIC value of 50 µg/mL.

Monasone Naphthoquinone Biosynthesis and Resistance in Monascus Fungi.

Despite the important biological activities of natural product naphthoquinones, the biosynthetic pathways of and resistance mechanisms against such compounds remain poorly understood in fungi. Here, we report that the genes responsible for the biosynthesis of Monascus naphthoquinones (monasones) reside within the gene cluster for Monascus azaphilone pigments (MonAzPs). We elucidate the biosynthetic pathway of monasones by a combination of comparative genome analysis, gene knockouts, heterologous coexpression, and in vivo and in vitro enzymatic reactions to show that this pathway branches from the first polyketide intermediate of MonAzPs. Furthermore, we propose that the monasone subset of biosynthetic genes also encodes a two-tiered resistance strategy in which an inducible monasone-specific exporter expels monasones from the mycelia, while residual intracellular monasones may be rendered nontoxic through a multistep reduction cascade.IMPORTANCE The genes for Monascus naphthoquinone (monasone) biosynthesis are embedded in and form a composite supercluster with the Monascus azaphilone pigment biosynthetic gene cluster. Early biosynthetic intermediates are shared by the two pathways. Some enzymes encoded by the supercluster play double duty in contributing to both pathways, while others are specific for one or the other pathway. The monasone subcluster is independently regulated and inducible by elicitation with competing microorganisms. This study illustrates genomic and biosynthetic parsimony in fungi and proposes a potential path for the evolution of the mosaic-like azaphilone-naphthoquinone supercluster. The monasone subcluster also encodes a two-tiered self-resistance mechanism that models resistance determinants that may transfer to target microorganisms or emerge in cancer cells in case of naphthoquinone-type cytotoxic agents.

Orange, red, yellow: biosynthesis of azaphilone pigments in Monascus fungi.

Monascus azaphilone pigments (MonAzPs) are very widely used as food colorants, but their biosynthetic pathway has remained poorly characterized for more than half a century. In this study, the individual steps of MonAzPs biosynthesis in Monascus ruber M7 were elucidated by a combination of targeted gene knockouts, heterologous gene expression, and in vitro chemical and enzymatic reactions. This study describes the first rational engineering of MonAzPs biosynthesis and provides a roadmap for future pathway engineering efforts directed towards the selective production of the most valuable pigments and serves as a model for the biosynthesis of fungal azaphilones in general.

Inactivation of the global regulator LaeA in Monascus ruber results in a species-dependent response in sporulation and secondary metabolism.

The nuclear regulator LaeA has been proven to globally govern fungal development and secondary metabolism, but its function may be species-dependent, even though its amino acid sequences are well conserved in numerous fungi. Herein we identified the LaeA in Monascus ruber M7 (MrLaeA), and verified its role to mediate growth, sporulation and secondary metabolism. Results showed that the radial growth rate of the selected MrlaeA knock-out mutant (MrΔlaeA-22) was significantly faster than that of the parental strain M. ruber M7, and growth was accompanied by the formation of an abnormal colony phenotype with more abundant aerial hyphae. Interestingly, conidia production of the MrΔlaeA-22 strain was about thrice that of M. ruber M7, but ascospores were not observed in the MrΔlaeA-22 strain. Additionally, compared to M. ruber M7, MrΔlaeA-22 exhibited drastically reduced production of multiple secondary metabolites, especially those of the six well-known Monascus pigments and citrinin. Simultaneously, the selected MrlaeA complementation strain (MrΔlaeA::laeA-45) nearly recovered the capacity for sporulation and secondary metabolism observed in the parental strain. These results demonstrate that MrLaeA regulates not only secondary metabolism, but also asexual and sexual differentiation in M. ruber, but some of its regulation appears to differ from other fungi.