The limited supply of fossil resources demands the development of renewable alternatives to petroleum-based products. Here, biobased higher alcohols such as isobutanol are versatile platform molecules for the synthesis of chemical commodities and fuels. Currently, their fermentation-based production is limited by the low tolerance of microbial production systems to the end products and also by the low substrate flux into cell metabolism. We developed an innovative cell-free approach, utilizing an artificial minimized glycolytic reaction cascade that only requires one single coenzyme. Using this toolbox the cell-free production of ethanol and isobutanol from glucose was achieved. We also confirmed that these streamlined cascades functioned under conditions at which microbial production would have ceased. Our system can be extended to an array of industrially-relevant molecules. Application of solvent-tolerant biocatalysts potentially allows for high product yields, which significantly simplifies downstream product recovery.

Establishing genetic engineering tools for sustainable production of tailor made fatty acids in the non-conventional, oleaginous yeast Trichosporon oleaginosus.

A economically and ecologically viable, fully integrated yeast oil process.

The quality and regulation of the incident light is crucial in microalgae cultivation processes. Depending on wavelength, spectrum, and intensity, growth characteristics and biochemical composition of these organisms vary. With mainly fluorescent lamps (FL) used previously for illumination, such variabilities could not be studied adequately due to their broad emission spectrum. In contrast, light-emitting diodes (LEDs) emit a very narrow wavelength band and enable flexible photobioreactor designs due to their small size. This review provides a condensed overview on the application of LEDs in microalgal cultivation processes. It summarizes the current availability and applicability of LED technologies as an illumination source for research-focused photobioreactor systems. A particular focus is the use of narrow-wavelength LEDs to address fundamental as well as applied aspects of light color on algae biomass and value-added compound formation. In this respect, the application of internal and external illumination systems is reviewed together with trends in the industrial use of LED systems to intensify algae process efficiency.

Chitin is one of the most abundant biomolecules on earth, occurring in crustacean shells and cell walls of fungi. While the polysaccharide is threatening to pollute coastal ecosystems in the form of accumulating shell-waste, it has the potential to be converted into highly profitable derivatives with applications in medicine, biotechnology, and wastewater treatment, among others. Traditionally this is still mostly done by the employment of aggressive chemicals, yielding low quality while producing toxic by-products. In the last decades, the enzymatic conversion of chitin has been on the rise, albeit still not on the same level of cost-effectiveness compared to the traditional methods due to its multi-step character. Another severe drawback of the biotechnological approach is the highly ordered structure of chitin, which renders it nigh impossible for most glycosidic hydrolases to act upon. So far, only the Auxiliary Activity 10 family (AA10), including lytic polysaccharide monooxygenases (LPMOs), is known to hydrolyse native recalcitrant chitin, which spares the expensive first step of chemical or mechanical pre-treatment to enlarge the substrate surface. The main advantages of enzymatic conversion of chitin over conventional chemical methods are the biocompability and, more strikingly, the higher product specificity, product quality, and yield of the process. Products with a higher Mw due to no unspecific depolymerisation besides an exactly defined degree and pattern of acetylation can be yielded. This provides a new toolset of thousands of new chitin and chitosan derivatives, as the physio-chemical properties can be modified according to the desired application. This review aims to provide an overview of the biotechnological tools currently at hand, as well as challenges and crucial steps to achieve the long-term goal of enzymatic conversion of native chitin into specialty chemical products.

CotB2 catalyzes the first committed step in cyclooctatin biosynthesis of the soil bacterium Streptomyces melanosporofaciens. To date, CotB2 represents the best studied bacterial diterpene synthase. Its reaction mechanism has been addressed by isoptope labeling, targeted mutagenesis and theoretical computations in the gas phase, as well as full enzyme molecular dynamic simulations. By X-ray crystallography different snapshots of CotB2 from the open, inactive, to the closed, active conformation have been obtained in great detail, allowing us to draw detailed conclusions regarding the catalytic mechanism at the molecular level. Moreover, numerous alternative geranylgeranyl diphosphate cyclization products obtained by CotB2 mutagenesis have exciting applications for the sustainable production of high value bioactive substances.

Split inteins link their fused peptide or protein sequences with a peptide bond in an autocatalytic reaction called protein trans-splicing. This reaction is becoming increasingly important for a variety of applications in protein semisynthesis, polypeptide circularisation, construction of biosensors, or segmental isotopic labelling of proteins. However, split inteins exhibit greatly varying solubility, efficiency and tolerance towards the nature of the fused sequences as well as reaction conditions. We envisioned that phage display as an in vitro selection technique would provide a powerful tool for the directed evolution of split inteins with improved properties. As a first step towards this goal, we show that presentation of active split inteins on an M13 bacteriophage is feasible. Two different C-terminal intein fragments of the Ssp DnaB intein, artificially split at amino acid positions 104 and 11, were encoded in a phagemid vector in fusion to a truncated gpIII protein. For efficient production of hybrid phages, the presence of a soluble domain tag at their N-termini was necessary. Immunoblot analysis revealed that the hybrid phages supported protein trans-splicing with a protein or a synthetic peptide, respectively, containing the complementary intein fragment. Incorporation of biotin or desthiobiotin by this reaction provides a straightforward strategy for future enrichment of desired mutants from randomised libraries of the C-terminal intein fragments on streptavidin beads. Protein semisynthesis on a phage could also be exploited for the selection of chemically modified proteins with unique properties.

Isobutanol is deemed to be a next-generation biofuel and a renewable platform chemical.1 Non-natural biosynthetic pathways for isobutanol production have been implemented in cell-based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst.2-6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg(2+) as cofactors. AlsS also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP-dependent enzymes. To unravel catalytically relevant structure-function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg(2+) and in a transition state with a 2-lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.