DNA amplification and reverse transcription enzymes have proven to be invaluable in fast and reliable diagnostics and research applications because of their processivity, specificity, and robustness. Our study focused on the production of mutant Taq DNA polymerase and mutant M-MLV reverse transcriptase in the expression hosts Vibrio natriegens and Escherichia coli under various expression conditions. We also examined nonspecific extracellular production in V. natriegens. Intracellularly, M-MLV was produced in V. natriegens at the level of 11% of the total cell proteins (TCPs) compared with 16% of TCPs in E. coli. We obtained a soluble protein that accounted for 11% of the enzyme produced in V. natriegens and 22% of the enzyme produced in E. coli. Taq pol was produced intracellularly in V. natriegens at the level of 30% of TCPs compared with 26% of TCPs in E. coli. However, Taq pol was almost non-soluble in E. coli, whereas in V. natriegens, we obtained a soluble protein that accounted for 23% of the produced enzyme. We detected substantial extracellular production of Taq pol. Thus, V. natriegens is a suitable alternative host with the potential for production of recombinant proteins.

N-glycosylation of recombinant proteins using bacterial glycosylation system has proven to be a valuable although developing tool ultimately applicable to various industries. When used for enzyme engineering, it offers the possibility of increased stability or immobilization route and thus increasing effectiveness of e.g. biotransformation or other biocatalysis procedures. One such promising enzyme is alcohol dehydrogenase (ADH) for use in redox biotransformation reactions. Given the current possibilities of recombinant enzyme production, including major advances in glycoengineering and glycoprotein production in bacterial organisms, the aim of this work was the production of thermotolerant ADH from Rhodococcus ruber (RrADH) in glycosylated form in Escherichia coli. We have successfully developed a dual plasmid expression system enabling glycosylation of target proteins utilizing a glyco-tag approach. We were able to produce RrADH in soluble form and at the same time we detected a bacterial glycan conjugated to RrADH as well as the activity of the enzyme. The glycan bound to recombinant enzyme can be used for oriented covalent immobilization of the enzyme, which would increase the potential for its practical application in biotransformation of various compounds.