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  1. Abstract

    Microalgae are promising biological factories for diverse natural products. Microalgae tout high productivity, and their biomass has value in industrial products ranging from biofuels, feedstocks, food additives, cosmetics, pharmaceuticals, and as alternatives to synthetic or animal‐derived products. However, harvesting microalgae to extract bioproducts is challenging given their small size and suspension in liquid growth media. In response, technologic developments have relied upon mechanical, chemical, thermal, and biological means to dewater microalgal suspensions and further extract bioproducts. In this review, the effectiveness and considerations were evaluated for the implementation of microalgae harvesting techniques. Nonbiological methods—filtration, chemical, electrical, and magnetic nanoparticle flocculation, centrifugation, hydrothermal liquefaction, and solvent‐based extraction, as well as biological coculture‐based methods are included. Recent advances in coculture algae‐flocculation technologies that involve bacteria and fungi are summarized. These produce a variety of natural bioproducts, which show promise in fuel and food additive applications. Furthermore, this review addresses the developments of genetic tools and resources to optimize the productivity and harvesting of microalgae or to provide new bioproducts via heterologous expression. Finally, a glimpse of future biotechnologies that will converge to produce, harvest, and process microalgae using sustainable and cost‐effective methods is offered.

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  2. Summary

    The mint family (Lamiaceae) is well documented as a rich source of terpene natural products. More than 200 diterpene skeletons have been reported from mints, but biosynthetic pathways are known for just a few of these.

    We crossreferenced chemotaxonomic data with publicly available transcriptomes to select common selfheal (Prunella vulgaris) and its highly unusual vulgarisin diterpenoids as a case study for exploring the origins of diterpene skeletal diversity in Lamiaceae. Four terpene synthases (TPS) from the TPS‐a subfamily, including two localised to the plastid, were cloned and functionally characterised. Previous examples of TPS‐a enzymes from Lamiaceae were cytosolic and reported to act on the 15‐carbon farnesyl diphosphate. Plastidial TPS‐a enzymes using the 20‐carbon geranylgeranyl diphosphate are known from other plant families, having apparently arisen independently in each family.

    All four new enzymes were found to be active on multiple prenyl‐diphosphate substrates with different chain lengths and stereochemistries. One of the new enzymes catalysed the cyclisation of geranylgeranyl diphosphate into 11‐hydroxy vulgarisane, the likely biosynthetic precursor of the vulgarisins.

    We uncovered the pathway to a rare diterpene skeleton. Our results support an emerging paradigm of substrate and compartment switching as important aspects of TPS evolution and diversification.

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  3. Sarrocco, Sabrina (Ed.)
    Harnessing the plant microbiome has the potential to improve agricultural yields and protect plants against pathogens and/or abiotic stresses, while also relieving economic and environmental costs of crop production. While previous studies have gained valuable insights into the underlying genetics facilitating plant-fungal interactions, these have largely been skewed towards certain fungal clades (e.g. arbuscular mycorrhizal fungi). Several different phyla of fungi have been shown to positively impact plant growth rates, including Mortierellaceae fungi. However, the extent of the plant growth promotion (PGP) phenotype(s), their underlying mechanism(s), and the impact of bacterial endosymbionts on fungal-plant interactions remain poorly understood for Mortierellaceae. In this study, we focused on the symbiosis between soil fungus Linnemannia elongata (Mortierellaceae) and Arabidopsis thaliana (Brassicaceae), as both organisms have high-quality reference genomes and transcriptomes available, and their lifestyles and growth requirements are conducive to research conditions. Further, L . elongata can host bacterial endosymbionts related to Mollicutes and Burkholderia . The role of these endobacteria on facilitating fungal-plant associations, including potentially further promoting plant growth, remains completely unexplored. We measured Arabidopsis aerial growth at early and late life stages, seed production, and used mRNA sequencing to characterize differentially expressed plant genes in response to fungal inoculation with and without bacterial endosymbionts. We found that L . elongata improved aerial plant growth, seed mass and altered the plant transcriptome, including the upregulation of genes involved in plant hormones and “response to oxidative stress”, “defense response to bacterium”, and “defense response to fungus”. Furthermore, the expression of genes in certain phytohormone biosynthetic pathways were found to be modified in plants treated with L . elongata . Notably, the presence of Mollicutes- or Burkholderia- related endosymbionts in Linnemannia did not impact the expression of genes in Arabidopsis or overall growth rates. Together, these results indicate that beneficial plant growth promotion and seed mass impacts of L . elongata on Arabidopsis are likely driven by plant hormone and defense transcription responses after plant-fungal contact, and that plant phenotypic and transcriptional responses are independent of whether the fungal symbiont is colonized by Mollicutes or Burkholderia -related endohyphal bacteria. 
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  4. The process of fermenting tofu extends back thousands of years and is an indispensable part of Chinese culture. Despite a cultural resurgence in fermented foods and interest in microbiomes, there is little knowledge on the microbial diversity represented in fermented ‘hairy’ tofu, known locally in China as Mao tofu. High-throughput metagenomic sequencing of the ITS, LSU and 16S rDNA was used to determine Mao tofu’s fungal and bacterial community diversity across four wet markets in Yunnan, China. The results show that hairy tofu in this region consists of around 170 fungal and 365 bacterial taxa, and that microbial taxa differ between markets. Diversity also differed based on the specific niche of the tofu block, comparing the outside rind-like niche to that of the inside of the tofu block. Machine learning random forest models were able to accurately classify both the market and niche of sample origin. An over-abundance of yeast and Geotrichum was found, and Mucor (Mucoromycota) was abundant in the outside rind-like niche, which consists of the visible ‘hairy’ mycelium. The majority of the bacterial OTUs belonged to Proteobacteria, Firmicutes, and Bacteroidetes, with Acinetobacter, Lactobacillus, Sphingobacterium and Flavobacterium the most abundant genera. Putative fungal pathogens of plants (Cercospora, Diaporthe, Fusarium) and animals (Metarhizium, Entomomortierella, Pyxidiophora, Candida, Clavispora) were also detected, as were putative bacterial pathogens identified as Legionella. Non-fungal eukaryotic taxa detected by LSU amplicon sequencing included soybean (Glycine max), Protozoa, Metazoa (e.g., Nematoda and Platyhelminthes), Rhizaria and Chromista, indicating that additional biodiversity exists in the hairy tofu microbiome. 
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  5. null (Ed.)
    High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest. 
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  6. null (Ed.)
    Interactions between plant-associated fungi and their hosts are characterized by a continuous crosstalk of chemical molecules. Specialized metabolites are often produced during these associations and play important roles in the symbiosis between the plant and the fungus, as well as in the establishment of additional interactions between the symbionts and other organisms present in the niche. Serendipita indica, a root endophytic fungus from the phylum Basidiomycota, is able to colonize a wide range of plant species, conferring many benefits to its hosts. The genome of S. indica possesses only few genes predicted to be involved in specialized metabolite biosynthesis, including a putative terpenoid synthase gene (SiTPS). In our experimental setup, SiTPS expression was upregulated when the fungus colonized tomato roots compared to its expression in fungal biomass growing on synthetic medium. Heterologous expression of SiTPS in Escherichia coli showed that the produced protein catalyzes the synthesis of a few sesquiterpenoids, with the alcohol viridiflorol being the main product. To investigate the role of SiTPS in the plant-endophyte interaction, an SiTPS-over-expressing mutant line was created and assessed for its ability to colonize tomato roots. Although overexpression of SiTPS did not lead to improved fungal colonization ability, an in vitro growth-inhibition assay showed that viridiflorol has antifungal properties. Addition of viridiflorol to the culture medium inhibited the germination of spores from a phytopathogenic fungus, indicating that SiTPS and its products could provide S. indica with a competitive advantage over other plant-associated fungi during root colonization. 
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