Microbial production of plant derived, biologically active compounds has the potential to provide economic and ecologic alternatives to existing low productive, plant-based processes. Current production of the pharmacologically active cyclic triterpenoid betulinic acid is realized by extraction from the bark of plane tree or birch. Here, we reengineered the reported betulinic acid pathway into Saccharomyces cerevisiae and used this novel strain to develop efficient fermentation and product purification methods. Fed-batch cultivations with ethanol excess, using either an ethanol-pulse feed or controlling a constant ethanol concentration in the fermentation medium, significantly enhanced production of betulinic acid and its triterpenoid precursors. The beneficial effect of excess ethanol was further exploited in nitrogen-limited resting cell fermentations, yielding betulinic acid concentrations of 182 mg/L, and total triterpenoid concentrations of 854 mg/L, the highest concentrations reported so far. Purification of lupane-type triterpenoids with high selectivity and yield was achieved by solid-liquid extraction without prior cell disruption using polar aprotic solvents such as acetone or ethyl acetate and subsequent precipitation with strong acids. This study highlights the potential of microbial production of plant derived triterpenoids in S. cerevisiae by combining metabolic and process engineering.

BackgroundChimeric virus-like particles (VLP) allow the display of foreign antigens on their surface and have proved valuable in the development of safe subunit vaccines or drug delivery. However, finding an inexpensive production system and a VLP scaffold that allows stable incorporation of diverse, large foreign antigens are major challenges in this field.ResultsIn this study, a versatile and cost-effective platform for chimeric VLP development was established. The membrane integral small surface protein (dS) of the duck hepatitis B virus was chosen as VLP scaffold and the industrially applied and safe yeast Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) as the heterologous expression host. Eight different, large molecular weight antigens of up to 412 amino acids derived from four animal-infecting viruses were genetically fused to the dS and recombinant production strains were isolated. In all cases, the fusion protein was well expressed and upon co-production with dS, chimeric VLP containing both proteins could be generated. Purification was accomplished by a downstream process adapted from the production of a recombinant hepatitis B VLP vaccine. Chimeric VLP were up to 95% pure on protein level and contained up to 33% fusion protein. Immunological data supported surface exposure of the foreign antigens on the native VLP. Approximately 40 mg of chimeric VLP per 100 g dry cell weight could be isolated. This is highly comparable to values reported for the optimized production of human hepatitis B VLP. Purified chimeric VLP were shown to be essentially stable for 6 months at 4 °C.ConclusionsThe dS-based VLP scaffold tolerates the incorporation of a variety of large molecular weight foreign protein sequences. It is applicable for the display of highly immunogenic antigens originating from a variety of pathogens. The yeast-based production system allows cost-effective production that is not limited to small-scale fundamental research. Thus, the dS-based VLP platform is highly efficient for antigen presentation and should be considered in the development of future vaccines.

The presence of both inversion (P ) and time-reversal (T ) symmetries in solids leads to well-known double degeneracy of electronic bands (Kramers degeneracy). When the degeneracy is lifted, spin textures can be directly observed in momentum space, as in topological insulators or in strong Rashba materials. The existence of spin textures with Kramers degeneracy, however, is very difficult to observe directly. Here, we use quantum interference measurements combined with first-principle band structure calculations to provide evidence for the existence of hidden entanglement between spin and momentum in antiperovskite-type 3D Dirac material Sr3SnO. We find robust weak antilocalization (WAL) independent of the position of EF, whereas clear signature of weak localization (WL) develops only when EF shifts away from the Dirac node by doping. The observed WAL signal at low doping is fitted using a single interference channel which implies that the different Dirac valleys are mixed by disorder. Notably, this mixing does not suppress WAL, suggesting contrasting interference physics compared to graphene. We identify scattering among axially spin-momentum locked states as a key process that leads to a spin orbital entanglement, giving rise to robust WAL. Our work sheds light on the subtle role of spin and pseudospin when both could contribute to the same quantum effect. arXiv:1806.08712v1 [cond-mat.mes-hall]

A variety of different applications render terpenes and terpenoids attractive research targets. A promising but so far insufficiently explored family of terpenoids are the fusicoccanes that comprise a characteristic 5-8-5 fused tricyclic ring system. Besides herbicidal effects, these compounds also show apoptotic and anti-tumour effects on mammalian cells. The access to fusicoccanes from natural sources is scarce. Recently, we introduced a metabolically engineered Saccharomyces cerevisiae strain to enable the heterologous fermentation of the shared fusicoccane–diterpenoid precursor, fusicocca-2,10(14)-diene. Here, we show experiments towards the identification of bottlenecks in this process. The suppression of biosynthetic by-products via medium optimisation was found to be an important aspect. In addition, the fermentation process seems to be improved under oxygen limitation conditions. Under fed-batch conditions, the fermentation yield was reproducibly increased to approximately 20 mg/L. Furthermore, the impact of the properties of the terpene synthase on the fermentation yield is discussed, and the preliminary studies on the engineering of this key enzyme are presented.

Downstream processing of biochemical products strongly depends on the interaction between biomolecules and material surfaces that can be influenced greatly by the pH level. In this study, the influence of the pH value on the adsorption of the amino acids glycine, alanine, and lysine in MFI-type zeolite was investigated using density functional theory (DFT) and microcalorimetric experiments. Different pH values were modeled by varying the amino acids protonation states. The investigated protonation states exhibit two different adsorption motifs in the pores, with the neutral α-C-amino group motifs being significantly less stable than the protonated ones. In the case of neutral glycine and alanine, the adsorption energy is insufficient to overcome the adsorption of the four water molecules usually present in the pores. This result explains the pH-dependent adsorption behavior that has also been observed experimentally and provides an avenue for designing efficient adsorption processes.