High-yield production of coenzyme F 420 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.
Competing Interests: Declaration of competing interest The authors declare that there are no competing interests.
(Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)