Recombinant protein expression in Escherichia coli is a fundamental technique in molecular biology and biotechnology. This review provides a comprehensive overview of various additivities to enhance the expression levels of soluble recombinant proteins in E. coli. The discussion encompasses five key aspects. Inducer Optimization: strategies for optimizing the inducer concentration to enhance protein expression. Autoinduction system optimization: the examination of glucose, lactose, and glycerol optimization within autoinduction systems to improve protein production. Osmolytes and osmoprotectants: an analysis of the use of osmolytes and osmoprotectants, such as sorbitol and glycine-betaine, to overcome with ease osmotic stress and enhance protein solubility. Ethanol additives: the impact of ethanol on E. coli physiology and its potential to improve recombinant protein expression. Cofactors and metabolic precursors: insights into the addition of cofactors, such as pyridoxal phosphate, riboflavin, thiamine, and pyridoxine, and the utilization of metabolic precursors to enhance the corresponding protein expression. This review highlights both the successful strategies and challenges in recombinant protein expression and provides insights into potential future research directions. Understanding and optimizing these factors is crucial for the efficient production of recombinant proteins for various applications in biotechnology. Furthermore, based on the analyzed data, we propose a straightforward scheme to optimize the additives in the cultivation medium.

Majority of native enzymes are poorly applicable for practical usage: that is why different methods of enzyme modification are used to obtain the biocatalysts with appropriate characteristics....

The analysis of the 3D model structure of the ternary complex of recombinant formate dehydrogenase from soya Glycine max (EC 1.2.1.2., SoyFDH) with bound NAD⁺ and an inhibitor azide ion revealed the presence of hydrophobic Phe290 in the coenzyme-binding domain. This residue should shield the enzyme active site from solvent. On the basis of the alignment of plant FDHs sequences, Asp, Asn and Ser were selected as candidates to substitute Phe290. Computer modeling indicated the formation of two (Ser and Asn) or three (Asp) new hydrogen bonds in such mutants. The mutant SoyFDHs were expressed in Escherichia coli, purified and characterized. All amino acid substitutions increased K_м^HCOO− from 1.5 to 4.1–5.0 mM, whereas the K_м^NAD+ values remained almost unchanged in the range from 9.1 to 14.0 μM, which is close to wt-SoyFDH (13.3 μM). The catalytic constants for F290N, F290D and F290S mutants of SoyFDH equaled 2.8, 5.1 and 4.1 s⁻¹, respectively; while that of the wild-type enzyme was 2.9 s⁻¹. The thermal stability of all mutant SoyFDHs was much higher compared with the wild-type enzyme. The differential scanning calorimetry data were in agreement with the results of thermal inactivation kinetics. The mutations F290S, F290N and F290D introduced into SoyFDH increased the T_m values by 2.9°C, 4.3°C and 7.8°C, respectively. The best mutant F290D exhibited thermal stability similar to that of FDH from the plant Arabidopsis thaliana and exceeded that of the enzymes from the yeast Candida boidinii and the bacterium Moraxella sp. C1.

NAD⁺-dependent formate dehydrogenase from Staphylococcus aureus (SauFDH) is one of the key enzymes responsible for the survival of this pathogen in the form of biofilms. 3D structure of the enzyme might be helpful in the search for highly specific SauFDH inhibitors that can be used as antibacterial agents exactly against S. aureus biofilms. Here, we prepared a recombinant SauFDH in Escherichia coli cells with a yield of 1 g target protein per liter medium. The developed procedure for the enzyme purification allowed to obtain 400 mg of homogenous enzyme with 61% yield. The specific activity of the purified recombinant SauFDH was 20 U per mg protein, which was 2 times higher than the previously reported activities of formate dehydrogenases. We also found crystallization conditions in the course of two rounds of optimization and obtained 200- and 40-µm crystals for the SauFDH apo- and holoenzymes, respectively. X-ray analysis using synchrotron X-ray sources produced diffraction data sufficient for solving the three-dimensional structures of the apo- and holoenzymes with the resolution of 2.2 and 2.7 Å, respectively. Crystals of the apo- and holoforms of SauFDH had different crystal space groups, which suggest coenzyme binding in the SauFDH holoenzyme.

NAD(P)+-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) is actively used in processes of chiral synthesis by oxidoreductases with systems of reduced cof