Molecular Evolution of Peptide Ligands with Custom-Tailored Characteristics for Targeting of Glycostructures

As an advanced approach to identify suitable targeting molecules required for various diagnostic and therapeutic interventions, we developed a procedure to devise peptides with customizable features by an iterative computer-assisted optimization strategy. An evolutionary algorithm was utilized to breed peptides in silico and the “fitness” of peptides was determined in an appropriate laboratory in vitro assay. The influence of different evolutional parameters and mechanisms such as mutation rate, crossover probability, gaussian variation and fitness value scaling on the course of this artificial evolutional process was investigated. As a proof of concept peptidic ligands for a model target molecule, the cell surface glycolipid ganglioside G_M1, were identified. Consensus sequences describing local fitness optima were reached from diverse sets of L- and proteolytically stable D lead peptides. Ten rounds of evolutional optimization encompassing a total of just 4400 peptides lead to an increase in affinity of the peptides towards fluorescently labeled ganglioside G_M1 by a factor of 100 for L- and 400 for D-peptides.     

Tracing hotspots of soil erosion in high mountain environments: how forensic science based on plant eDNA can lead the way. An opinion

Plant and Soil - High mountain environments are among the most fragile on Earth. Due to anthropogenic disturbances and the exposure to extreme weather events, the rates of soil erosion have...     

Mutants for photosystem I subunit D of Arabidopsis thaliana: effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions

In Arabidopsis thaliana, the D-subunit of photosystem I (PSI-D) is encoded by two functional genes, PsaD1 and PsaD2, which are highly homologous. Knock-out alleles for each of the loci have been identified by a combination of forward and reverse genetics. The double mutant psad1-1 psad2-1 is seedling-lethal, high-chlorophyll-fluorescent and deficient for all tested PSI subunits, indicating that PSI-D is essential for photosynthesis. In addition, psad1-1 psad2-1 plants show a defect in the accumulation of thylakoid multiprotein complexes other than PSI. Of the single-gene mutations, psad2 plants behave like wild-type (WT) plants, whereas psad1-1 markedly affects the accumulation of PsaD mRNA and protein, and photosynthetic electron flow. Additional effects of the psad1-1 mutation include a decrease in growth rate under greenhouse conditions and downregulation of the mRNA expression of most genes involved in the light phase of photosynthesis. In the same mutant, a marked decrease in the levels of PSI and PSII polypeptides is evident, as well as a light-green leaf coloration and increased photosensitivity. Increased dosage of PsaD2 in the psad1-1 background restores the WT phenotype, indicating that PSI-D1 and PSI-D2 have redundant functions.     

Discovery of a novel styrene monooxygenase originating from the metagenome

Oxygenases form an interesting class of biocatalysts, as they typically perform oxygenations with exquisite chemo-, regio-, and/or enantioselectivity. It has been observed that, once heterologously expressed in Escherichia coli, some oxygenases are able to form the blue pigment indigo. We have exploited this characteristic to screen a metagenomic library derived from loam soil and identified a novel oxygenase. This oxygenase shows 50% sequence identity with styrene monooxygenases from pseudomonads (StyA). Only a limited number of homologs can be found in the genome sequence database, indicating that this biocatalyst is a member of a relatively small family of bacterial monooxygenases. The newly identified monooxygenase catalyzes the epoxidation of styrene and styrene derivatives and forms the corresponding (S)-epoxides with excellent enantiomeric excess [e.g., (S)-styrene oxide is formed with >99% enantiomeric excess, ee] and therefore is named styrene monooxgenase subunit A (SmoA). SmoA shows high enantioselectivity towards aromatic sulfides [e.g., (R)-ethyl phenyl sulfoxide is formed with 92% ee]. This excellent enantioselectivity in combination with the moderate sequence identity forms a clear indication that SmoA from a metagenomic origin represents a new enzyme within the small family of styrene monooxygenases.