Szabolcs Lányi

Cultivating conditions optimization of the anaerobic digestion of corn ethanol distillery residuals using response surface methodology

This study investigated the individual and interactive effects of three factors — temperature, inoculum/substrate ratio (ISR) and inoculum typology — on the anaerobic digestion of corn ethanol distillery wastewater. Biochemical methane potential assays planned with factorial design with two independent quantitative variables on three levels (ISR: 1:1, 2:1 and 3:1; temperature: 30°C, 33.5°C, 37°C) and one independent qualitative variable (inoculum type: suspended, granular, mixed) have been performed. Response Surface Methodology has been used to study the effect of the factors with the aim of maximizing the specific methane yields (YCH4) obtainable with this substrate. The results show that all three investigated factors influence in a significant matter the YCH4, the ISR having the strongest effect on it. The temperature has significant influence on the YCH4 only in combination with high ISR values. The optimal conditions for the maximum YCH4 (551 mL CH4 g−1 VSadded) have been found at 37°C operating temperature, ISR=3:1 and using granular inoculum. These conditions gave rise to a 4-fold increase of YCH4 with respect to the worst combination of factors (YCH4=129 mL g−1 VSadded for the suspended inoculum type, at 30°C and ISR=1:1). The results improve the knowledge on the digestion of this substrate, providing information for successful process up-scaling.

Heterologous Expression and Purification of Recombinant Proapoptotic Human Protein Smac/Diablo with EGFP as Fusion Partner

New proteins as molecular targets in development of therapies are discovered every day. However, study of their interactions with other proteins or binding partners in complex cellular environments has its limits. Therefore, high-yield production of these proteins in heterologous systems is a valid necessity, while obtaining these proteins linked to suitable fluorescent markers represents a step ahead in protein-protein interaction studies and cellular or subcellular localization. In this study, we present production of human SMAC/Diablo recombinant protein with EGFP as a fusion partner. High-yield expression of the fusion protein was carried out in E. coli RosettaTM(DE3)pLysS strain, and an acceptable purity of the protein was obtained after affinity chromatography purification and gel filtration. The obtained protein can be further used in protein-protein interaction studies, whereas our method represents a cost-effective and efficient production method for EGFP-fused proteins, applicable for a number of therapeutically important polypeptides.

In silico and in vivo stability analysis of a heterologous biosynthetic pathway for 1,4-butanediol production in metabolically engineered E. coli

Recently, several approaches have been published in order to develop a functional biosynthesis route for the non-natural compound 1,4-butanediol (BDO) in E. coli using glucose as a sole carbon source or starting from xylose. Among these studies, there was reported as high as 18 g/L product concentration achieved by industrial strains, however BDO production varies greatly in case of the reviewed studies. Our motivation was to build a simple heterologous pathway for this compound in E. coli and to design an appropriate cellular chassis based on a systemic biology approach, using constraint-based flux balance analysis and bi-level optimization for gene knock-out prediction. Thus, the present study reports, at the “proof-of concept” level, our findings related to model-driven development of a metabolically engineered E. coli strain lacking key genes for ethanol, lactate and formate production (ΔpflB, ΔldhA and ΔadhE), with a three-step biosynthetic pathway. We found this strain to produce a limited quantity of 1,4-BDO (.89 mg/L BDO under microaerobic conditions and .82 mg/L under anaerobic conditions). Using glycerol as carbon source, an approach, which to our knowledge has not been tackled before, our results suggest that further metabolic optimization is needed (gene-introductions or knock-outs, promoter fine-tuning) to address the redox potential imbalance problem and to achieve development of an industrially sustainable strain. Our experimental data on culture conditions, growth dynamics and fermentation parameters can consist a base for ongoing research on gene expression profiles and genetic stability of such metabolically engineered E. coli strains.