Lytic polysaccharide monooxygenases:a crystallographer's view on a new class of biomass-degrading enzymes

Lytic polysaccharide monooxygenases (LPMOs) are a new class of microbial copper enzymes involved in the degradation of recalcitrant polysaccharides. They have only been discovered and characterized in the last 5–10 years and have stimulated strong interest both in biotechnology and in bioinorganic chemistry. In biotechnology, the hope is that these enzymes will finally help to make enzymatic biomass conversion, especially of lignocellulosic plant waste, economically attractive. Here, the role of LPMOs is likely to be in attacking bonds that are not accessible to other enzymes. LPMOs have attracted enormous interest since their discovery. The emphasis in this review is on the past and present contribution of crystallographic studies as a guide to functional understanding, with a final look towards the future

Renal haemodynamics are not related to genotypes in offspring of parents with essential hypertension.

Introduction. The pathogenesis of essential hypertension (EH) has a major genetic component and is associated with renal abnormalities. Normotensive offspring of hypertensive parents are likely to develop EH and are a suitable population for identifying possible relations between genetic and renal abnormalities.Methods. We investigated if renin-angiotensin-aldosterone system associated genotypes (angiotensinogen [M235T] and ACE [I/D]) are related to blood pressure (BP), renal haemodynamics and sodium excretion in sex and age-matched (18—35 years) healthy Caucasian offspring of either two parents with EH (n=101, EH-offspring) or two normotensive parents (n=50, controls). The alpha-adducin polymorphism (G460W) was also investigated.Results. Compared to controls, BP, heart rate, renal vascular resistance (RVR) and urinary sodium excretion were, respectively, 5%, 7%, 15% and 20% higher in EH-offspring. In controls, the TT-genotype of the M235T angiotensinogen polymorphism was associated with higher BP and higher plasma angiotensinogen. By contrast, in EHoffspring the TT-genotype was associated with lower BP and unchanged plasma angiotensinogen. Plasma angiotensinogen correlated positively with BP in EH-offspring, with a similar tendency (p=0.08) in controls. The distributions of the three candidate polymorphisms were similar in EH-offspring and controls. There were no associations between any of the polymorphisms and any of the renal parameters measured.Conclusion. The markedly greater RVR, proportionally larger than the greater BP, supports a role for RVR in the pathogenesis of EH. The lack of association between the candidate polymorphisms and the investigated parameters, even in this homogenous and for hypertension strongly predisposed group, suggests that the polymorphisms investigated do not play important roles in the pathogenesis of hypertension

Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

BACKGROUND: The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed. RESULTS: The new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ΔFaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 10(6) macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A. CONCLUSION: The new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this

Physiological characterization of secondary metabolite producing Penicillium cell factories

Abstract Background Penicillium species are important producers of bioactive secondary metabolites. However, the immense diversity of the fungal kingdom is only scarcely represented in industrial bioprocesses and the upscaling of compound production remains a costly and labor intensive challenge. In order to facilitate the development of novel secondary metabolite producing processes, two routes are typically explored: optimization of the native producer or transferring the enzymatic pathway into a heterologous host. Recent genome sequencing of ten Penicillium species showed the vast amount of secondary metabolite gene clusters present in their genomes, and makes them accessible for rational strain improvement. In this study, we aimed to characterize the potential of these ten Penicillium species as native producing cell factories by testing their growth performance and secondary metabolite production in submerged cultivations. Results Cultivation of the fungal species in controlled submerged bioreactors showed that the ten wild type Penicillium species had promising, highly reproducible growth characteristics in two different media. Analysis of the secondary metabolite production using liquid chromatography coupled with high resolution mass spectrometry proved that the species produced a broad range of secondary metabolites, at different stages of the fermentations. Metabolite profiling for identification of the known compounds resulted in identification of 34 metabolites; which included several with bioactive properties such as antibacterial, antifungal and anti-cancer activities. Additionally, several novel species–metabolite relationships were found. Conclusions This study demonstrates that the fermentation characteristics and the highly reproducible performance in bioreactors of ten recently genome sequenced Penicillium species should be considered as very encouraging for the application of native hosts for production via submerged fermentation. The results are particularly promising for the potential development of the ten analysed Penicillium species for production of novel bioactive compounds via submerged fermentations