Ceren Alkim

Engineering of a Synthetic Metabolic Pathway for the Assimilation of (d)-Xylose into Value-Added Chemicals

A synthetic pathway for (d)-xylose assimilation was stoichiometrically evaluated and implemented in Escherichia coli strains. The pathway proceeds via isomerization of (d)-xylose to (d)-xylulose, phosphorylation of (d)-xylulose to obtain (d)-xylulose-1-phosphate (X1P), and aldolytic cleavage of the latter to yield glycolaldehyde and DHAP. Stoichiometric analyses showed that this pathway provides access to ethylene glycol with a theoretical molar yield of 1. Alternatively, both glycolaldehyde and DHAP can be converted to glycolic acid with a theoretical yield that is 20% higher than for the exclusive production of this acid via the glyoxylate shunt. Simultaneous expression of xylulose-1 kinase and X1P aldolase activities, provided by human ketohexokinase-C and human aldolase-B, respectively, restored growth of a (d)-xylulose-5-kinase mutant on xylose. This strain produced ethylene glycol as the major metabolic endproduct. Metabolic engineering provided strains that assimilated the entire C2 fraction into the central metabolism or that produced 4.3 g/L glycolic acid at a molar yield of 0.9 in shake flasks.

Evolutionary engineering and transcriptomic analysis of nickel-resistant Saccharomyces cerevisiae.

Increased exposure to nickel compounds and alloys due to industrial development has resulted in nickel pollution and many pathological effects on human health. However, there is very limited information about nickel response, transport, and tolerance in eukaryotes. To investigate nickel resistance in the model eukaryote Saccharomyces cerevisiae, evolutionary engineering by batch selection under gradually increasing nickel stress levels was performed. Nickel hyper-resistant mutants that could resist up to 5.3 mM NiCl2 , a lethal level for the reference strain, were selected. The mutants were also cross-resistant against iron, cobalt, zinc, and manganese stresses and accumulated more than twofold higher nickel than the reference strain. Global transcriptomic analysis revealed that 640 upregulated genes were related to iron homeostasis, stress response, and oxidative damage, implying that nickel resistance may share common mechanisms with iron and cobalt resistance, general stress response, and oxidative damage.

Protein Engineering Methods and Applications

Protein engineering is the design of new enzymes or proteins with new or desirable functions. It is based on the use of recombinant DNA technology to change amino acid sequences. The first papers on protein engineering date back to early 1980ies: in a review by Ulmer (1983), the prospects for protein engineering, such as X-ray crystallography, chemical DNA synthesis, computer modelling of protein structure and folding were discussed and the combination of crystal structure and protein chemistry information with artificial gene synthesis was emphasized as a powerful approach to obtain proteins with desirable properties (Ulmer, 1983). In a later review in 1992, protein engineering was mentioned as a highly promising technique within the frame of biocatalyst engineering to improve enzyme stability and efficiency in low water systems (Gupta, 1992). Today, owing to the development in recombinant DNA technology and high-throughput screening techniques, protein engineering methods and applications are becoming increasingly important and widespread. In this Chapter, a chronological review of protein engineering methods and applications is provided.

Evolutionary engineering of yeast.

Evolutionary engineering is an inverse metabolic engineering strategy which is based on increasing genetic diversity and screening large populations for desired phenotypes. This strategy is highly advantageous in certain situations over rational metabolic engineering approaches, since there is little or no requirement of detailed genetic background information for the trait of interest. Here, we describe the experimental methodology for selecting stress-resistant yeast strains via evolutionary engineering approach by either serial batch or chemostat cultivations.

Physiological and transcriptomic analysis of a salt-resistant Saccharomyces cerevisiae mutant obtained by evolutionary engineering

Salt-resistant yeast strains are highly demanded by industry due to the exposure of yeast cells to high concentrations of salt, in various industrial bioprocesses. The aim of this study was to perform a physiological and transcriptomic analysis of a salt-resistant Saccharomyces cerevisiae (S. cerevisiae) mutant generated by evolutionary engineering. NaCl-resistant S. cerevisiae strains were obtained by ethyl methanesulfonate (EMS) mutagenesis followed by successive batch cultivations in the presence of gradually increasing NaCl concentrations, up to 8.5% w/v of NaCl (1.45 M). The most probable number (MPN) method, high-performance liquid chromatography (HPLC), and glucose oxidase/peroxidase method were used for physiological analysis, while Agilent yeast DNA microarray systems were used for transcriptome analysis. NaCl-resistant mutant strain T8 was highly cross-resistant to LiCl and highly sensitive to AlCl3. In the absence of NaCl stress, T8 strain had significantly higher trehalose and glycogen levels compared to the reference strain. Global transcriptome analysis by means of DNA microarrays showed that the genes related to stress response, carbohydrate transport, glycogen and trehalose biosynthesis, as well as biofilm formation, were upregulated. According to gene set enrichment analysis, 548 genes were upregulated and 22 downregulated in T8 strain, compared to the reference strain. Among the 548 upregulated genes, the highest upregulation was observed for the FLO11 (MUC1) gene (92-fold that of the reference strain). Overall, evolutionary engineering by chemical mutagenesis and increasing NaCl concentrations is a promising approach in developing industrial strains for biotechnological applications.