Escherichia coli alcohol dehydrogenase YahK is a protein that binds both iron and zinc.

Background: Previous studies have highlighted the catalytic activity of Escherichia coli alcohol dehydrogenase YahK in the presence of coenzyme nicotinamide adenine dinucleotide (NAD) and metal zinc. Notably, competitive interaction between iron and zinc ligands has been shown to influence the catalytic efficiency of several key proteases. This study aims to unravel the intricate mechanisms underlying YahK's catalytic action, with a particular focus on the pivotal roles played by metal ions zinc and iron. Methods: The purified YahK protein from E. coli cells cultivated in LB medium was utilized to investigate its metal-binding properties through UV-visible absorption measurements and determination of metal content. Subsequently, the effects of excess zinc and iron on the metal-binding ability and alcohol dehydrogenase activity of the YahK protein were explored using M9 minimal medium. Furthermore, site-directed mutagenesis technology was employed to determine the iron-binding site location within the YahK protein. Polyacrylamide gel electrophoresis was conducted to examine the relationship between iron and zinc with respect to the YahK protein. Results: The study confirmed the presence of iron and zinc in the YahK protein, with the zinc-bound form exhibiting enhanced catalytic activity in alcohol dehydrogenation reactions. Conversely, the presence of iron appears to play a pivotal role in maintaining overall stability of the YahK protein. Furthermore, experimental findings indicate that excessive zinc within M9 minimal medium can competitively bind to iron-binding sites on YahK, thereby augmenting its alcohol dehydrogenase activity. Conclusion: The dynamic binding of YahK to iron and zinc unveils its intricate regulatory mechanism as an alcohol dehydrogenase, thereby highlighting the possible physiological role of YahK in E. coli and its significance in governing cellular metabolic processes. This discovery provides a novel perspective for further investigating the specific impact of metal ion binding on YahK and E. coli cell metabolism.

A low-cost and eco-friendly recombinant protein expression system using copper-containing industrial wastewater.

The development of innovative methods for highly efficient production of recombinant proteins remains a prominent focus of research in the biotechnology field, primarily due to the fact that current commercial protein expression systems rely on expensive chemical inducers, such as isopropyl ß-D-thiogalactoside (IPTG). In our study, we designed a novel approach for protein expression by creating a plasmid that responds to copper. This specialized plasmid was engineered through the fusion of a copper-sensing element with an optimized multiple cloning site (MCS) sequence. This MCS sequence can be easily customized by inserting the coding sequences of target recombinant proteins. Once the plasmid was generated, it was introduced into an engineered Escherichia coli strain lacking copA and cueO. With this modified E. coli strain, we demonstrated that the presence of copper ions can efficiently trigger the induction of recombinant protein expression, resulting in the production of active proteins. Most importantly, this expression system can directly utilize copper-containing industrial wastewater as an inducer for protein expression while simultaneously removing copper from the wastewater. Thus, this study provides a low-cost and eco-friendly strategy for the large-scale recombinant protein production. To the best of our knowledge, this is the first report on the induction of recombinant proteins using industrial wastewater.