Summary The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR-interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a Protospacer Adjacent Motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R-loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg2+-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization.

Hyphal tip cells of Aspergillus nidulans are > 100 µm-long, which challenges intracellular traffic. In spite of the basic and applied interest of the secretory pathway of filamentous fungi, only recently has it been investigated in detail. We used InuA, an inducible and highly glycosylated inulinase, and mutations affecting different intracellular membranous compartments, to investigate the route by which the enzyme traffics to the extracellular medium. InuA is core-N-glycosylated in the ER and hyperglycosylated during transit across the Golgi. Hyperglycosylation was prevented by ts mutations in sarA impeding ER exit, and in sedV and rabO dissipating the early Golgi, but not by mutations in the TGN regulators hypA and hypB , implicating the early Golgi in cargo glycosylation. podB1 (cog2 ) affecting the COG complex also prevents glycosylation, without disassembling early Golgi cisternae. That InuA exocytosis is prevented by inactivation of any of the above genes shows that it follows a conventional secretory pathway. However, ablation of RabB regulating early endosomes (EEs), but not of RabS , its equivalent in late endosomes, also prevents InuA accumulation in the medium, indicating that EEs are specifically required for InuA exocytosis. This work provides a framework to understand the secretion of enzyme cargoes by industrial filamentous fungi.

Deoxypodophyllotoxin is present in the roots of . This compound is cytotoxic on its own, but it can also be converted into podophyllotoxin, which is in high demand as a precursor for the important anticancer drugs etoposide and teniposide. In this study, deoxypodophyllotoxin is extracted from roots by supercritical carbon dioxide extraction. The process is simple and scalable. The supercritical carbon dioxide method extracts 75 - 80% of the total deoxypodophyllotoxin content, which is comparable to a single extraction by traditional Soxhlet. However, less polar components are extracted. The activity of the supercritical carbon dioxide extract containing deoxypodophyllotoxin was assessed by demonstrating that the extract arrests A549 and HeLa cells in the G/M phase of the cell cycle. We conclude that biologically active deoxypodophyllotoxin can be extracted from by supercritical carbon dioxide extraction. The method is solvent free and more sustainable compared to traditional methods.

The pharmaceutically important anticancer drugs etoposide and teniposide are derived from podophyllotoxin, a natural product isolated from roots of Podophyllum hexandrum growing in the wild. The overexploitation of this endangered plant has led to the search for alternative sources. Metabolic engineering aimed at constructing the pathway in another host cell is very appealing, but for that approach, an in-depth knowledge of the pathway toward podophyllotoxin is necessary. In this chapter, we give an overview of the lignan pathway leading to podophyllotoxin. Subsequently, we will discuss the engineering possibilities to produce podophyllotoxin in a heterologous host. This will require detailed knowledge on the cellular localization of the enzymes of the lignan biosynthesis pathway. Due to the high number of enzymes involved and the scarce information on compartmentalization, the heterologous production of podophyllotoxin still remains a tremendous challenge. At the moment, research is focusing on the last step(s) in the conversion of deoxypodophyllotoxin to (epi)podophyllotoxin and 4′-demethyldesoxypodophyllotoxin by plant cytochromes.

Background and aim The endangered Podophyllum hexandrum is an important industrial source of podophyllotoxin, which is a precursor for the anticancer drugs etoposide and teniposide. Attempts to obtain podophyllotoxin through cell cultures or chemical synthesis have still a long way to go before being economical feasible. The objective of this study was to increase the root formation and podophyllotoxin production of P. hexandrum cultivated in a glasshouse. Methods Root formation and podophyllotoxin production of P. hexandrum in sand or peat-perlite soil at 15°C or 25°C was determined. Furthermore, the influence of methyl jasmonate on the podophyllotoxin production was determined. Results More root formation was observed in peat-perlite soil than in sand soil. Furthermore, root formation was higher at 15°C than at 25°C. This resulted in the highest podophyllotoxin production per plant in peat-perlite at 15°C (160 ± 22 mg/plant d.w.). Furthermore, methyl jasmonate treatment of the leaves increased the podophyllotoxin production in the roots by 21%. Conclusion We were able to cultivate P. hexandrum in a glasshouse in the Netherlands and improve the root formation and podophyllotoxin production. This paves the way for large-scale cultivation of P. hexandrum in the temperate latitudes for the production of the pharmaceutical interesting podophyllotoxin.