Sorbicillinoids are a diverse group of yellow secondary metabolites that are produced by a range of not closely related ascomycetes, including Penicillium chrysogenum, Acremonium chrysogenum, and Trichoderma reesei. They share a similarity to the name-giving compound sorbicillin, a hexaketide. Previously, a conserved gene cluster containing two polyketide synthases has been identified as the source of sorbicillin, and a model for the biosynthesis of sorbicillin in P. chrysogenum has been proposed. In this study, we deleted the major genes of interest of the cluster in T. reesei, namely sor1, sor3, and sor4. Sor1 is the homolog of P. chrysogenum SorA, which is the first polyketide synthase of the proposed biosynthesis pathway. Sor3 is a flavin adenine dinucleotide (FAD)-dependent monooxygenase, and its homolog in P. chrysogenum, SorC, was shown to oxidize sorbicillin and 2′,3′-dihydrosorbicillin to sorbicillinol and 2′,3′-dihydrosorbicillinol, respectively, in vitro. Sor4 is an FAD/flavin mononucleotide-containing dehydrogenase with an unknown function. We measured the amounts of synthesized sorbicillinoids throughout growth and could verify the roles of Sor1 and Sor3 in vivo in T. reesei. In the absence of Sor4, two compounds annotated to dihydrosorbicillinol accumulate in the supernatant and only small amounts of sorbicillinol are synthesized. Therefore, we suggest extending the current biosynthesis model about Sor4 reducing 2′,3′-dihydrosorbicillin and 2′,3′-dihydrosorbicillinol to sorbicillinol and sorbicillinol, respectively. Sorbicillinol turned out to be the main chemical building block for most sorbicillinoids, including oxosorbicillinol, bisorbicillinol, and bisvertinolon. Further, we detected the sorbicillinol-dependent synthesis of 5-hydroxyvertinolide at early time points, which contradicts previous models for biosynthesis of 5-hydroxyvertinolide. Finally, we investigated whether sorbicillinoids from T. reesei have a growth limiting effect on the fungus itself or on plant pathogenic fungi or on pathogenic bacteria.

Penicillium chrysogenum (renamed P. rubens) is the most studied member of a family of more than 350 Penicillium species that constitute the genus. Since the discovery of penicillin by Alexander Fleming, this filamentous fungus is used as a commercial β-lactam antibiotic producer. For several decades, P. chrysogenum was subjected to a classical strain improvement (CSI) program to increase penicillin titers. This resulted in a massive increase in the penicillin production capacity, paralleled by the silencing of several other biosynthetic gene clusters (BGCs), causing a reduction in the production of a broad range of BGC encoded natural products (NPs). Several approaches have been used to restore the ability of the penicillin production strains to synthetize the NPs lost during the CSI. Here, we summarize various re-activation mechanisms of BGCs, and how interference with regulation can be used as a strategy to activate or silence BGCs in filamentous fungi. To further emphasize the versatility of P. chrysogenum as a fungal production platform for NPs with potential commercial value, protein engineering of biosynthetic enzymes is discussed as a tool to develop de novo BGC pathways for new NPs.

Secondary metabolism in Penicillium chrysogenum was intensively subjected to classical strain improvement (CSI), the resulting industrial strains producing high levels of β-lactams. During this process, the production of yellow pigments, including sorbicillinoids, was eliminated as part of a strategy to enable the rapid purification of β-lactams. Here we report the identification of the polyketide synthase (PKS) gene essential for sorbicillinoid biosynthesis in P. chrysogenum . We demonstrate that the production of polyketide precursors like sorbicillinol and dihydrosorbicillinol as well as their derivatives bisorbicillinoids requires the function of a highly reducing PKS encoded by the gene Pc21g05080 ( pks13 ). This gene belongs to the cluster that was mutated and transcriptionally silenced during the strain improvement program. Using an improved β-lactam-producing strain, repair of the mutation in pks13 led to the restoration of sorbicillinoid production. This now enables genetic studies on the mechanism of sorbicillinoid biosynthesis in P. chrysogenum and opens new perspectives for pathway engineering. IMPORTANCE Sorbicillinoids are secondary metabolites with antiviral, anti-inflammatory, and antimicrobial activities produced by filamentous fungi. This study identified the gene cluster responsible for sorbicillinoid formation in Penicillium chrysogenum , which now allows engineering of this diverse group of compounds.

Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203–424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.

Summary: Penicillium chrysogenum is a filamentous fungus that is used to produce β‐lactams at an industrial scale. At an early stage of classical strain improvement, the ability to produce the yellow‐coloured sorbicillinoids was lost through mutation. Sorbicillinoids are highly bioactive of great pharmaceutical interest. By repair of a critical mutation in one of the two polyketide synthases in an industrial P. chrysogenum strain, sorbicillinoid production was restored at high levels. Using this strain, the sorbicillin biosynthesis pathway was elucidated through gene deletion, overexpression and metabolite profiling. The polyketide synthase enzymes SorA and SorB are required to generate the key intermediates sorbicillin and dihydrosorbicillin, which are subsequently converted to (dihydro)sorbillinol by the FAD ‐dependent monooxygenase SorC and into the final product oxosorbicillinol by the oxidoreductase SorD. Deletion of either of the two pks genes not only impacted the overall production but also strongly reduce the expression of the pathway genes. Expression is regulated through the interplay of two transcriptional regulators: SorR1 and SorR2. SorR1 acts as a transcriptional activator, while SorR2 controls the expression of sorR1 . Furthermore, the sorbicillinoid pathway is regulated through a novel autoinduction mechanism where sorbicillinoids activate transcription.

Considerable amounts of information is available on the complex carbohydrates that are mobilized and utilized by the seed to support early seedling development. These events occur after radicle has protruded from the seed. However, scarce information is available on the role of the endogenous soluble carbohydrates from the embryo in the first hours of germination. The present work analysed how the soluble carbohydrate reserves in isolated maize embryos are mobilized during 6–24 h of water imbibition, an interval that exclusively embraces the first two phases of the germination process. It was found that sucrose constitutes a very significant reserve in the scutellum and that it is efficiently consumed during the time in which the adjacent embryo axis is engaged in an active metabolism. Sucrose transporter was immunolocalized in the scutellum and in vascular elements. In parallel, a cell-wall invertase activity, which hydrolyses sucrose, developed in the embryo axis, which favoured higher glucose uptake. Sucrose and hexose transporters were active in the embryo tissues, together with the plasma membrane H⁺-ATPase, which was localized in all embryo regions involved in both nutrient transport and active cell elongation to support radicle extension. It is proposed that, during the initial maize germination phases, a net flow of sucrose takes place from the scutellum towards the embryo axis and regions that undergo elongation. During radicle extension, sucrose and hexose transporters, as well as H⁺-ATPase, become the fundamental proteins that orchestrate the transport of nutrients required for successful germination.