Expression and purification of Mycobacterium tuberculosis F420-dependent glucose-6-phosphate dehydrogenase enzyme using Escherichia coli

Since their discovery in Mycobacterium tuberculosis (Mtb), F420-dependent enzymes have been identified as both important drug targets and potential industrial biocatalysts, including for bioremediation of otherwise recalcitrant substrates. Mtb-FGD1, utilizes glucose 6-phosphate (G6P) as an electron donor for the reduction of F420. Current expression systems for Mtb-FGD1 use Mycobacterium smegmatis as host, because of the tendency for it to form inclusion bodies in E. coli. However, large scale recombinant protein production using M. smegmatis is slow and costly and the organism is not generally recognized as safe. Here, we report a faster, cheaper and safer approach for the expression of fully functional Mtb-FGD1 in E. coli using cold-adapted GroEL/ES as chaperones. Our approach yielded ∼70 mg of protein per litre (L) of culture. The purified enzyme catalysed the reduction of F420 to F420.H2 in the presence of G6P, and the re-oxidation of the F420.H2 to F420 when coupled to Tfu-FNO, which is a thermostable oxidoreductase that utilizes F420 for the reversible oxidation of NADPH. This latter finding provides opportunity for the utilization of Mtb-FGD1 as an industrial biocatalyst or in the detoxification of environmental contaminants such as malachite green, picrate and aflatoxin.