High-yield production of coenzyme F₄₂₀ in Escherichia coli by fluorescence-based screening of multi-dimensional gene expression space

Coenzyme F 420 is involved in bioprocesses such as biosynthesis of antibiotics by streptomycetes, prodrug activation in Mycobacterium tuberculosis, and methanogenesis in archaea. F 420 -dependent enzymes also attract interest as biocatalysts in organic chemistry. However, as only low F 420 levels are produced in microorganisms, F 420 availability is a serious bottleneck for research and application. Recent advances in our understanding of the F 420 biosynthesis enabled heterologous overproduction of F 420 in Escherichia coli, but the yields remained moderate. To address this issue, we rationally designed a synthetic operon for F 420 biosynthesis in E. coli. However, it still led to the production of low amounts of F 420 and undesired side-products. In order to strongly improve yield and purity, a screening approach was chosen to interrogate the gene expression-space of a combinatorial library based on diversified promotors and ribosome binding sites. The whole pathway was encoded by a two-operon construct. The first module ("core") addressed parts of the riboflavin biosynthesis pathway and F O synthase for the conversion of GTP to the stable F 420 intermediate F O . The enzymes of the second module ("decoration") were chosen to turn F O into F 420 . The final construct included variations of T7 promoter strengths and ribosome binding site activity to vary the expression ratio for the eight genes involved in the pathway. Fluorescence-activated cell sorting was used to isolate clones of this library displaying strong F 420 -derived fluorescence. This approach yielded the highest titer of coenzyme F 420 produced in the widely used organism E. coli so far. Production in standard LB medium offers a highly effective and simple production process that will facilitate basic research into unexplored F 420 -dependent bioprocesses as well as applications of F 420 -dependent enzymes in biocatalysis.