Present in the genomes of bacteria and eukaryotic organelles, group II introns are an ancient class of ribozymes and retroelements that are believed to have been the ancestors of nuclear pre-mRNA introns. Despite long-standing speculation, there is limited understanding about the actual pathway by which group II introns evolved into eukaryotic introns. In this review, we focus on the evolution of group II introns themselves. We describe the different forms of group II introns known to exist in nature and then address how these forms may have evolved to give rise to spliceosomal introns and other genetic elements. Finally, we summarize the structural and biochemical parallels between group II introns and the spliceosome, including recent data that strongly support their hypothesized evolutionary relationship.

Accurate and complete identification of mobile elements is a challenging task in the current era of sequencing, given their large numbers and frequent truncations. Group II intron retroelements, which consist of a ribozyme and an intron-encoded protein (IEP), are usually identified in bacterial genomes through their IEP; however, the RNA component that defines the intron boundaries is often difficult to identify because of a lack of strong sequence conservation corresponding to the RNA structure. Compounding the problem of boundary definition is the fact that a majority of group II intron copies in bacteria are truncated.

Previously, DNA microarrays analysis showed that, in co-culture with Bacillus subtilis, a biosynthetic gene cluster anchored with a nonribosomal peptides synthetase of Aspergillus niger is downregulated. Based on phylogenetic and synteny analyses, we show here that this gene cluster, NRRL3_00036-NRRL3_00042, comprises genes predicted to encode a nonribosomal peptides synthetase, a FAD-binding domain-containing protein, an uncharacterized protein, a transporter, a cytochrome P450 protein, a NAD(P)-binding domain-containing protein and a transcription factor. We overexpressed the in-cluster transcription factor gene NRRL3_00042. The overexpression strain, NRRL3_00042^OE, displays reduced growth rate and production of a yellow pigment, which by mass spectrometric analysis corresponds to two compounds with masses of 409.1384 and 425.1331. We deleted the gene encoding the NRRL3_00036 nonribosomal peptides synthetase in the NRRL3_00042^OE strain. The resulting strain reverted to the wild-type phenotype. These results suggest that the biosynthetic gene cluster anchored by the NRRL3_00036 nonribosomal peptides synthetase gene is regulated by the in-cluster transcriptional regulator gene NRRL3_00042, and that it is involved in the production of two previously uncharacterized compounds.

Mitogenic growth factors are major cost drivers in serum-free media, contributing up to 95% of the total cost. Here, we present a streamlined workflow detailing cloning, expression testing, protein purification, and bioactivity screening that allows for low-cost production of bioactive growth factors including basic fibroblast growth factor and transforming growth factor β1. This generalized procedure can be used for multiple families of growth factors with minor modification, and the outputs are bioactive and suitable for cell culture applications. For complete details on the use and execution of this protocol, please refer to Venkatesan, et al.¹.

Genomics analysis confirmed the status of filamentous fungi as a rich source of novel secondary metabolites; however, the discovery of these compounds is hampered by the cryptic nature of their biosynthetic pathways under laboratory conditions. Consequently, despite substantial research effort over the past decades, much of the secondary metabolome remains uncharacterized in fungal organisms. Our manual curation of biosynthetic gene clusters (BGCs) in the Aspergillus niger NRRL3 genome revealed that only 13 of 86 BGCs have had their cognate secondary metabolite products confirmed or reliably inferred. We also identified 60 transcription factors (TFs) associated with cryptic BGCs. To further characterize A. niger secondary metabolism, we created a collection of strains each overexpressing a single BGC-associated TF. We analyzed the strain collection using a standardized pipeline where we monitored phenotypic changes and compound production using mass spectrometry. Strains showing evidence of secondary metabolism activation were selected for gene expression analysis. Our approach resulted in the production of multiple potentially novel secondary metabolites and linked a specific BGC to tensidol production in A. niger. More broadly, this study found evidence counter to the existing paradigm of BGC expression controlled by colocalized TFs, lending credence to the emerging picture of a complex regulatory network governing fungal secondary metabolism.