A synthetic microbial biosensor for high-throughput screening of lactam biocatalysts

Abstract

Biocatalytic cyclization is highly desirable for efficient synthesis of biologically derived chemical substances, such as the commodity chemicals ε-caprolactam and δ-valerolactam. To identify biocatalysts in lactam biosynthesis, we develop a caprolactam-detecting genetic enzyme screening system (CL-GESS). The Alcaligenes faecalis regulatory protein NitR is adopted for the highly specific detection of lactam compounds against lactam biosynthetic intermediates. We further systematically optimize the genetic components of the CL-GESS to enhance sensitivity, achieving 10-fold improvement. Using this highly sensitive GESS, we screen marine metagenomes and find an enzyme that cyclizes ω-amino fatty acids to lactam. Moreover, we determine the X-ray crystal structure and catalytic residues based on mutational analysis of the cyclase. The cyclase is also used as a helper enzyme to sense intracellular ω-amino fatty acids. We expect this simple and accurate biosensor to have wide-ranging applications in rapid screening of new lactam-synthesizing enzymes and metabolic engineering for lactam bio-production.

Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

“…A similar observation was reported in the directed evolution of NitR to detect ε-caprolactam[65]. Homology modelling and docking of the NitR-L117F mutant showed a shorter distance between the effector and the mutated amino acid inside the binding-pocket compared to the WT, which suggested an association with the improved sensing[65]. Therefore the docking results for Van2 could indicate an association between the altered specificity pattern in Van2 with the availability of the position of the new effector in the binding pocket.…”

“…Interestingly, homology modelling and docking of the active in vivo mutants, Van2 and PcaV M113S N114A, with vanillin, indicated an interaction between the aldehyde functional group of vanillin and the serine at position 113. A similar observation was reported in the directed evolution of NitR to detect ε-caprolactam[65]. Homology modelling and docking of the NitR-L117F mutant showed a shorter distance between the effector and the mutated amino acid inside the binding-pocket compared to the WT, which suggested an association with the improved sensing[65].…”

Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid

“…This TAL sensor displays elevated background expression as compared to wild-type AraC, as well as relaxed substrate specificity and relatively low sensitivity to TAL (half-maximum dose response occurs at 4 mM TAL concentration). More interesting applications of a TAL biosensor, or a biosensor that responds to similar, minimal polyketides such as orsellinic acid (OA), lie in the identification of novel PKS variants whose poor functional expression and/or low catalytic activity demand significantly greater sensitivity and/or specificity than AraC-TAL1 (Yeom et al 2018). A similar constraint was recently described by Thompson et al, for the case of engineering biosynthesis of the nylon precursor caprolactam (Thompson et al 2020).…”

Recent Advances of L-ornithine Biosynthesis in Metabolically Engineered Corynebacterium glutamicum

“…However, random mutations and labor-intensive screening methods remain as bottleneck issues. Recently, high-throughput screening of mutant strains was developed using a metabolite biosensor, which can measure the metabolite concentrations based on the fluorescence signals or antibiotic selective pressure (Hammer and Avalos, 2016;Lin et al, 2017;Yeom et al, 2018). Due to an improvement in the screening efficiency using a metabolite biosensor, the traditional irrational breeding strategy is considered a promising method for the construction of engineered strains (Zheng et al, 2018).…”