Over the past decade, researchers have tried to decipher the borderline between serine proteases and carboxylic ester hydrolases; although they share the same catalytic triad, the factor that distinguishes their substrate preference still remains unknown. So far, the total inversion of the catalytic character has not yet been fulfilled; only minor improvements of the promiscuous amidase activity have been achieved for the carboxylic ester hydrolases compared to their natural activities, even though different sophisticated approaches of rational design have been applied. In the present study we used a combination of directed evolution and rational design to improve the promiscuous amidase activity of a Bacillus subtilis esterase. A double mutant was identified with 7.5 fold higher relative reaction specificity for the promiscuous amidase reaction with both aromatic and aliphatic substrates, whereas the esterase activity was not significantly affected. The mutation that resulted from the directed evolution experiments was not in the direct vicinity of the active site but affected the substrate binding through a π–π stacking network.

Cutaneotrichosporon curvatus DSM 101032 is an oleaginous yeast that can be isolated from various habitats and is capable of producing substantial amounts of polyunsaturated fatty acids. Here, we present the first draft genome sequence of any C. curvatus species.

Despite the increasing relevance, ranging from academic research to industrial applications, only a limited number of non-conventional, oleaginous Yarrowia lipolytica strains are characterized in detail. Therefore, we analyzed three strains in regard to their metabolic and physiological properties, especially with respect to important characteristics of a production strain. By investigating different cultivation conditions and media compositions, similarities and differences between the distinct strain backgrounds could be derived. Especially sugar alcohol production, as well as an agglomeration of cells were found to be connected with growth at high temperatures. In addition, sugar alcohol production was independent of high substrate concentrations under these conditions. To investigate the genotypic basis of particular traits, including growth characteristics and metabolite concentrations, genomic analysis were performed. We found sequence variations for one third of the annotated proteins but no obvious link to all phenotypic features.

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The oleaginous yeast Yarrowia lipolytica is capable of accumulating large amounts of fatty acids in the form of lipids which can serve as a platform polymer for a variety of applications. Additionally, through heterologous gene expression, Y. lipolytica is capable of producing carotenoids. Due to the observation that Y. lipolytica exhibits a high phenotypic inter-strain variability, robotics and microwell scale cultivations are applied to examine 12 wild type strains of a Y. lipolytica collection. The strains are characterized with respect to their metabolic capabilities for fatty acid production as well as genetically manipulated to produce beta-carotene. Furthermore, growth and production behavior of the strain collection at different temperatures as well as initial pH are assessed. A molecular discrimination between the strains is achieved by internal transcribed spacer (ITS)-sequencing and polymerase chain reaction (PCR)-based methods. The best performing strain with respect to lipid production produces ≈2% lipids per dry cell weight (DCW) and 8 mg g −1 beta-carotenoids in yeast nitrogen base (YNB) media. All strains show robust growth from 28 to 34°C. Practical Applications: Unlocking nature's potential by screening natural isolates shows that even on inter-strain level sufficient phenotypic diversity may arise. Automated growth-based characterization of beta-caroteneproducing strains in terms of media composition, effect of initial pH, and temperature tolerance shows that with modern cultivation techniques, rapid characterization of strain collections is feasible. Combining results of beta-carotene and lipid formation could help to balance both pathways to improve the storage of hydrophobic compounds in the lipid droplets. The generalized findings could be further transferred to improve the production of any valuable compound, derived from the mevalonate pathway.

Despite the increasing relevance, ranging from academic research to industrial applications, only a limited number of nonconventional, oleaginous Yarrowia lipolytica strains are characterized in detail.Therefore, we analyzed three strains in regard to their metabolic and physiological properties and in respect to important characteristics of a production strains. A flow cytometry method was set up to evaluate their fitness in a rapid manner. By investigating different cultivation conditions and media compositions, similarities and differences between the distinct strain backgrounds could be derived.Especially sugar alcohol production, as well as a agglomeration of cells were found to be connected with growth at high temperatures. In addition, sugar alcohol production was independent of high substrate concentrations under these conditions. To investigate particular traits, including growth characteristics and metabolite concentrations, genomic analysis were performed. We found sequence variations for one third of the annotated proteins but no obvious link to all phenotypic features.

No abstract