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/.

Microginins are a large family of cyanobacterial lipopeptide protease inhibitors. A hybrid polyketide synthase/non-ribosomal peptide synthetase biosynthetic gene cluster (BGC) found in several microginin-producing strainsmicwas proposed to encode the production of microginins, based on bioinformatic analysis. Here, we explored a cyanobacterium, Microcystis aeruginosa LEGE 91341, which contains a mic BGC, to discover 12 new microginin variants. The new compounds contain uncommon amino acids, namely, homophenylalanine (Hphe), homotyrosine (Htyr), or methylproline, as well as a 3-aminodecanoic acid (Ada) residue, which in some variants was chlorinated at its terminal methyl group. We have used direct pathway cloning (DiPaC) to heterologously express the mic BGC from M. aeruginosa LEGE 91341 in Escherichia coli, which led to the production of several microginins. This proved that the mic BGC is, in fact, responsible for the biosynthesis of microginins and paves the way to accessing new variants from (meta)­genome data or through pathway engineering.

Certain cyanobacteria of the secondary metabolite-rich order Nostocales can establish permanent symbioses with a large number of cycads, by accumulating in their coralloid roots and shifting their metabolism to dinitrogen fixation. Here, we report the discovery of two new lipoglycopeptides, desmamides A ( 1 ) and B ( 2 ), together with their aglycone desmamide C ( 3 ), from the nostocalean cyanobacterium Desmonostoc muscorum LEGE 12446 isolated from a cycad ( Cycas revoluta ) coralloid root. The chemical structures of the compounds were elucidated using a combination of 1D and 2D NMR spectroscopy and mass spectrometry. The desmamides are decapeptides featuring O -glycosylation of tyrosine (in 1 and 2 ) and an unusual 3,5-dihydroxy-2-methyldecanoic acid residue. The biosynthesis of the desmamides was studied by substrate incubation experiments and bioinformatics. We describe herein the dsm biosynthetic gene cluster and propose it to be associated with desmamide production. The discovery of this class of very abundant (>1.5% d.w.) bacterial lipoglycopeptides paves the way for exploration of their potential role in root endosymbiosis.

Lactylates are an important group of molecules in the food and cosmetic industries. A series of natural halogenated 1-lactylates – chlorosphaerolactyaltes (<b>1</b>-<b>4</b>) – were recently reported from <i>Sphaerospermopsis</i> sp. LEGE 00249. Here, we identify the <i>cly</i> biosynthetic gene cluster, containing all the necessary functionalities to generate and release the natural lactylates. Using a combination of stable isotope-labeled precursor feeding and bioinformatic analysis, we propose that dodecanoic acid and pyruvate are the key building blocks in the biosynthesis of <b>1</b>-<b>4</b>. We additionally report minor analogues of these molecules<b> </b>with varying alkyl chains. The discovery of the <i>cly</i> gene cluster paves the way to accessing industrially-relevant lactylates through pathway engineering.

Lactylates are an important group of molecules in the food and cosmetic industries. A series of natural halogenated 1-lactylates – chlorosphaerolactyaltes (<b>1</b>-<b>4</b>) – were recently reported from <i>Sphaerospermopsis</i> sp. LEGE 00249. Here, we identify the <i>cly</i> biosynthetic gene cluster, containing all the necessary functionalities to generate and release the natural lactylates. Using a combination of stable isotope-labeled precursor feeding and bioinformatic analysis, we propose that dodecanoic acid and pyruvate are the key building blocks in the biosynthesis of <b>1</b>-<b>4</b>. We additionally report minor analogues of these molecules<b> </b>with varying alkyl chains. The discovery of the <i>cly</i> gene cluster paves the way to accessing industrially-relevant lactylates through pathway engineering.