Tackling the Catch-22 Situation of Optimizing a Sensor and a Transporter System in a Whole-Cell Microbial Biosensor Design for an Anthropogenic Small Molecule

Abstract

Whole-cell biosensors provide a convenient detection tool for the high-throughput screening of genetically engineered biocatalytic activity. But establishing a biosensor for an anthropogenic molecule requires both a custom transporter and a transcription factor. This results in an unavoidable “Catch-22” situation in which transporter activity cannot be easily confirmed without a biosensor and a biosensor cannot be established without a functional transporter in a host organism. We overcame this type of circular problem while developing an adipic acid (ADA) sensor. First, leveraging an established cis,cis-muconic acid (ccMA) sensor, an annotated ccMA transporter MucK, which is expected to be broadly responsive to dicarboxylates, was stably expressed in the genome of Pseudomonas putida to function as a transporter for ADA, and then a PcaR transcription factor (endogenous to the strain and naturally induced by β-ketoadipic acid, BKA) was diversified and selected to detect the ADA molecule. While MucK expression is otherwise very unstable in P. putida under strong promoter expression, our optimized mucK expression was functional for over 70 generations without loss of function, and we selected an ADA sensor that showed a specificity switch of over 35-fold from BKA at low concentrations (typically <0.1 mM of inducers). Our ADA and BKA biosensors show high sensitivity (low detection concentration <10 μM) and dynamic range (∼50-fold) in an industrially relevant organism and will open new avenues for high throughput discovery and optimization of enzymes and metabolic pathways for the biomanufacture of these molecules. In particular, the novel ADA sensor will aid the discovery and evolution of efficient biocatalysts for the biological recycling of ADA from the degradation of nylon-6,6 waste.

Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations

“…The methodology described here eliminates the bottleneck of testing individual variants of genes and promoters by presenting a protocol for assaying the combinatorial effect of several mutations at once, drastically reducing the time and money required to obtain genetic variants with desirable qualities. The efficacy of the method described here is illustrated by several of our original research papers (Jha et al, 2014;Jha et al, 2016;Jha et al, 2018;Shin et al, 2022), which have successfully employed this approach. The versatility and usefulness of this high-throughput screening method applied to large genetic libraries is therefore evidenced in published literature.…”

“…In such cases, randomization of specific regions in the promoter such as −35/−10 sites, can tune down the constitutive promoter, resulting in stable expression of the downstream gene. The approach was successfully applied in tuning down the expression of mucK transporter gene for stable expression from a constitutive promoter in Pseudomonas putida (Shin et al, 2022).…”

“…Targeting only very relevant residues with computationally informed mutations, or conservative mutations such as neutral drift mutations (Lynch and Hill, 1986), can simplify library generation, reduce library size, and decrease noise and workload. In silico ligand docking in a comparative model to guide the design of a focused library was successfully used to engineer several biosensors for small molecules (Jha et al, 2015;Jha et al, 2016;Shin et al, 2022).…”

“…The ability to diversify multiple positions in a given sequence at once provides a significantly faster route to arriving at a combination of mutations providing appreciable alterations in phenotype. In addition, even imperfect predicted structures can be used to great effect by indicating promising residues to target for mutagenesis (Jha et al, 2016;Shin et al, 2022).…”

“…Frontiers in Bioengineering and Biotechnology frontiersin.org approach using large libraries of diverse genetic variants, can be successfully employed in any project requiring random, or semirational design of pathways, proteins, and promoter variants. We have successfully applied this method to optimize promoter activity (Bentley et al, 2020;Pardo et al, 2020;Shin et al 2022), alter ligand binding (Jha et al, 2016;Shin et al 2022), reduce enzyme inhibition (Jha et al, 2019), construct biosensors (Jha et al, 2014;Jha et al, 2015) increase enzyme efficiency (Jha and Strauss, 2020), and improve thermostability and expression of an enzyme (Harrington et al, 2017), while taking advantage of the high throughput efficiency of flow cytometry.…”