Unusual Sesquiterpenes from Streptomyces olindensis DAUFPE 5622.

In nature, the vast majority of sesquiterpenes are produced by type I mechanisms, and glycosylated sesquiterpenes are rare in actinobacteria. Streptomyces olindensis DAUFPE 5622 produces the sesquiterpenes olindenones A-G, a new class of rearranged drimane sesquiterpenes. Olindenones B-D are oxygenated derivatives of olindenone A, while olindenones E-G are analogs glycosylated with dideoxysugars. 13C-isotope labeling studies demonstrated olindenone A biosynthesis occurs via the methylerythritol phosphate (MEP) pathway and suggested the rearrangement is only partially concerted. Based on the structures, one potential mechanism of olindenone A formation proceeds by cyclization of the linear terpenoid precursor, likely occurring via a terpene cyclase-mediated type II mechanism whereby the terminal alkene of the precursor is protonated, triggering carbocation-driven cyclization followed by rearrangement. Diphosphate hydrolysis may occur either before or after cyclization. Although a biosynthetic route is proposed, the terpene cyclase gene responsible for producing olindenones currently remains unidentified.

Validating a Promoter Library for Application in Plasmid-Based Diatom Genetic Engineering.

While diatoms are promising synthetic biology platforms, there currently exists a limited number of validated genetic regulatory parts available for genetic engineering. The standard method for diatom transformation, nonspecific introduction of DNA into chromosomes via biolistic particle bombardment, is low throughput and suffers from clonal variability and epigenetic effects. Recent developments in diatom engineering have demonstrated that autonomously replicating episomal plasmids serve as stable expression platforms for diverse gene expression technologies. These plasmids are delivered via bacterial conjugation and, when combined with modular DNA assembly technologies, provide a flexibility and speed not possible with biolistic-mediated strain generation. In order to expand the current toolbox for plasmid-based engineering in the diatom Phaeodactylum tricornutum, a conjugation-based forward genetics screen for promoter discovery was developed, and application to a diatom genomic DNA library defined 252 P. tricornutum promoter elements. From this library, 40 promoter/terminator pairs were delivered via conjugation on episomal plasmids, characterized in vivo, and ranked across 4 orders of magnitude difference in reporter gene expression levels.

MIBiG 3.0: a community-driven effort to annotate experimentally validated biosynthetic gene clusters.

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/.

Vibrio fluminensis sp. nov. isolated from the skin of Southern Atlantic sharpnose-pufferfish (Canthigaster figueiredoi).

This manuscript provides the description of the bacterial strain A621T characterized by Gram negative motile rods, presenting green circular colonies on TCBS. It was obtained from the skin of the sharpnose pufferfish Canthigaster figueredoi (Tetraodontidae Family), collected in Arraial do Cabo, located in the Rio de Janeiro region, Brazil. Optimum growth occurs at 20-28 °C in the presence of 3% NaCl. The Genome sequence of the novel isolate consisted of 4.224 Mb, 4431 coding genes and G + C content of 44.5%. Genomic taxonomy analysis based on average amino acid (AAI), genome-to-genome-distance (GGDH) and phylogenetic reconstruction placed (A621T= CBAS 741T = CAIM 1945T = CCMR 150T) into a new species of the genus Vibrio (Vibrio fluminensis sp. nov). The genome of the novel species contains four gene clusters (~ 56.17 Kbp in total) coding for different types of bioactive compounds that hint to several possible ecological roles in the sharpnose pufferfish host.

Proximity proteomics in a marine diatom reveals a putative cell surface-to-chloroplast iron trafficking pathway.

Iron is a biochemically critical metal cofactor in enzymes involved in photosynthesis, cellular respiration, nitrate assimilation, nitrogen fixation, and reactive oxygen species defense. Marine microeukaryotes have evolved a phytotransferrin-based iron uptake system to cope with iron scarcity, a major factor limiting primary productivity in the global ocean. Diatom phytotransferrin is endocytosed; however, proteins downstream of this environmentally ubiquitous iron receptor are unknown. We applied engineered ascorbate peroxidase APEX2-based subcellular proteomics to catalog proximal proteins of phytotransferrin in the model marine diatom Phaeodactylum tricornutum. Proteins encoded by poorly characterized iron-sensitive genes were identified including three that are expressed from a chromosomal gene cluster. Two of them showed unambiguous colocalization with phytotransferrin adjacent to the chloroplast. Further phylogenetic, domain, and biochemical analyses suggest their involvement in intracellular iron processing. Proximity proteomics holds enormous potential to glean new insights into iron acquisition pathways and beyond in these evolutionarily, ecologically, and biotechnologically important microalgae.

Multiplexed Knockouts in the Model Diatom Phaeodactylum by Episomal Delivery of a Selectable Cas9.

Marine diatoms are eukaryotic microalgae that play significant ecological and biogeochemical roles in oceans. They also have significant potential as organismal platforms for exploitation to address biotechnological and industrial goals. In order to address both modes of research, sophisticated molecular and genetic tools are required. We presented here new and improved methodologies for introducing CRISPR-Cas9 to the model diatom Phaeodactylum tricornutum cells and a streamlined protocol for genotyping mutant cell lines with previously unknown phenotypes. First, bacterial-conjugation was optimized for the delivery of Cas9 by transcriptionally fusing Cas9 to a selectable marker by the 2A peptide. An episome cloning strategy using both negative and positive selection was developed to streamline CRISPR-episome assembly. Next, cell line picking and genotyping strategies, that utilize manual sequencing curation, TIDE sequencing analysis, and a T7 endonuclease assay, were developed to shorten the time required to generate mutants. Following this new experimental pipeline, both single-gene and two-gene knockout cell lines were generated at mutagenesis efficiencies of 48% and 25%, respectively. Lastly, a protocol for precise gene insertions via CRISPR-Cas9 targeting was developed using particle-bombardment transformation methods. Overall, the novel Cas9 episome design and improved genotyping methods presented here allow for quick and easy genotyping and isolation of Phaeodactylum mutant cell lines (less than 3 weeks) without relying on a known phenotype to screen for mutants.

Biosynthesis of the neurotoxin domoic acid in a bloom-forming diatom.

Oceanic harmful algal blooms of Pseudo-nitzschia diatoms produce the potent mammalian neurotoxin domoic acid (DA). Despite decades of research, the molecular basis for its biosynthesis is not known. By using growth conditions known to induce DA production in Pseudo-nitzschia multiseries, we implemented transcriptome sequencing in order to identify DA biosynthesis genes that colocalize in a genomic four-gene cluster. We biochemically investigated the recombinant DA biosynthetic enzymes and linked their mechanisms to the construction of DA's diagnostic pyrrolidine skeleton, establishing a model for DA biosynthesis. Knowledge of the genetic basis for toxin production provides an orthogonal approach to bloom monitoring and enables study of environmental factors that drive oceanic DA production.

Diatom centromeres suggest a mechanism for nuclear DNA acquisition.

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequence features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.

Refinement of the Diatom Episome Maintenance Sequence and Improvement of Conjugation-Based DNA Delivery Methods.

Conjugation of episomal plasmids from bacteria to diatoms advances diatom genetic manipulation by simplifying transgene delivery and providing a stable and consistent gene expression platform. To reach its full potential, this nascent technology requires new optimized expression vectors and a deeper understanding of episome maintenance. Here, we present the development of an additional diatom vector (pPtPBR1), based on the parent plasmid pBR322, to add a plasmid maintained at medium copy number in Escherichia coli to the diatom genetic toolkit. Using this new vector, we evaluated the contribution of individual yeast DNA elements comprising the 1.4-kb tripartite CEN6-ARSH4-HIS3 sequence that enables episome maintenance in Phaeodactylum tricornutum. While various combinations of these individual elements enable efficient conjugation and high exconjugant yield in P. tricornutum, individual elements alone do not. Conjugation of episomes containing CEN6-ARSH4 and a small sequence from the low GC content 3' end of HIS3 produced the highest number of diatom exconjugant colonies, resulting in a smaller and more efficient vector design. Our findings suggest that the CEN6 and ARSH4 sequences function differently in yeast and diatoms, and that low GC content regions of greater than ~500 bp are a potential indicator of a functional diatom episome maintenance sequence. Additionally, we have developed improvements to the conjugation protocol including a high-throughput option utilizing 12-well plates and plating methods that improve exconjugant yield and reduce time and materials required for the conjugation protocol. The data presented offer additional information regarding the mechanism by which the yeast-derived sequence enables diatom episome maintenance and demonstrate options for flexible vector design.

Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase.

Phosphorylation and activation of ribosomal S6 protein kinase is an important link in the regulation of cell size by the target of rapamycin (TOR) protein kinase. A combination of selective inhibition and RNA interference were used to test the roles of members of the PP2A subfamily of protein phosphatases in dephosphorylation of Drosophila S6 kinase (dS6K). Treatment of Drosophila Schneider 2 cells with calyculin A, a selective inhibitor of PP2A-like phosphatases, resulted in a 7-fold increase in the basal level of dS6K phosphorylation at the TOR phosphorylation site (Thr398) and blocked dephosphorylation following inactivation of TOR by amino acid starvation or rapamycin treatment. Knockdown of the PP2A catalytic subunit increased basal dS6K phosphorylation and inhibited dephosphorylation induced by amino acid withdrawal. In contrast, depletion of the catalytic subunits of the other two members of the subfamily did not enhance dS6K phosphorylation. Knockdown of PP4 caused a 20% decrease in dS6K phosphorylation and knockdown of PP6 had no effect. Knockdown of the Drosophila B56-2 subunit resulted in enhanced dephosphorylation of dS6K following removal of amino acids. In contrast, knockdown of the homologs of the other PP2A regulatory subunits had no effects. Knockdown of the Drosophila homolog of the PP2A/PP4/PP6 interaction protein alpha4/Tap42 did not affect S6K phosphorylation, but did induce apoptosis. These results indicate that PP2A, but not other members of this subfamily, is likely to be a major S6K phosphatase in intact cells and is consistent with an important role for this phosphatase in the TOR pathway.