The metabolic redox regime ofPseudomonas putidatunes its evolvability towards novel xenobiotic substrates

Abstract

During evolution of biodegradation pathways for xenobiotic compounds, the transition towards novel substrates of Rieske non-heme iron oxygenases borne by environmental bacteria is frequently associated with faulty reactions. Such reactions release reactive oxygen species (ROS), endowed with high mutagenic potential. The present work studies how the operation of a given metabolic network by a bacterial host may either foster or curtail the still-evolving biochemical pathway for catabolism of 2,4-dinitrotoluene (2,4-DNT). To this end, the genetically tractable strain Pseudomonas putida EM173 was chromosomally implanted with a Tn7 construct carrying the whole genetic complement (recruited from the environmental isolate Burkholderia sp. R34) necessary for complete biodegradation of 2,4-DNT. By using reporter technology and direct measurements of ROS formation, we observed that the engineered P. putida strain experienced oxidative stress when catabolizing the nitroaromatic substrate. However, ROS was neither translated into significant activation of the SOS response to DNA damage nor resulted in a mutagenic regime (unlike Burkholderia sp. R34, the original host of the pathway). To inspect whether the tolerance of P. putida to oxidative insults could be traced to its characteristic reductive redox regime, we artificially lowered the pool of NAD(P)H by conditional expression of a water forming, NADH-specific oxidase. Under the resulting low-NAD(P)H status, 2,4-DNT triggered a conspicuous mutagenic and genomic diversification scenario. These results indicate that the background biochemical network of environmental bacteria ultimately determines the evolvability of metabolic pathways. Moreover, the data explains the efficacy of some bacteria such as Pseudomonads to host and evolve new catabolic routes.IMPORTANCESome environmental bacteria evolve new capacities for aerobic biodegradation of chemical pollutants by adapting pre-existing redox reactions to recently faced compounds. The process typically starts by co-option of enzymes of an available route to act on the chemical structure of the substrates-to-be. The critical bottleneck is generally the first biochemical step and most of the selective pressure operates on reshaping the initial reaction. In Rieske non-heme iron oxygenases, the interim uncoupling of the novel substrate to the old enzymes results in production of highly mutagenic ROS. In this work, we demonstrate that the background metabolic regime of the bacterium that hosts an evolving catabolic pathway (e.g. biodegradation of the xenobiotic 2,4-DNT) determines whether the cells would either adopt a genetic diversification regime or a robust ROS-tolerant state. These results expose new perspectives to contemporary attempts for rational assembly of whole-cell biocatalysts, as pursued by present-day metabolic engineering.

Biotransformation of 2,4‐dinitrotoluene in a phototrophic co‐culture of engineered Synechococcus elongatus and Pseudomonas putida

“…These results are indicative of the biotransformation of 2,4‐DNT and, as reported previously, P . putida strains carrying the dnt gene cluster were not able to grow on 2,4‐DNT as the only carbon source (Akkaya et al , ). A separate quantitative analysis of the absorbance spectra of P. putida EM·DNT·S supernatants focused upon the first 8 h following 2,4‐DNT addition, revealing a characteristic absorbance rise at 420 nm (between 1‐4 h post‐inoculation).…”

“…We observed that the soluble fraction of 2,4‐DNT declined significantly within the first 4 h, and dropped to undetectable levels by 22 h. This kinetic pattern of 2,4‐DNT transformation is similar to what has been reported for Burkholderia sp. R34 (Pérez‐Pantoja et al , ) and other engineered P. putida strains (Akkaya et al , , ). Soluble levels of 2,4‐DNT declined in cultures of P. putida EM·S as well (Fig.…”

“…Selection for the genomically integrated cscB module was achieved by addition of 12.5 μg ml −1 chloramphenicol to the culture medium. Engineered P. putida strains were constructed essentially as reported previously (Martínez‐García et al , ; Löwe et al , ,b, ; Akkaya et al , ). During routine cultivation, P. putida strains carrying an integrated dnt gene cluster were maintained in the presence of 25 μg ml −1 gentamicin, while strains transformed with plasmid pSEVA221· cscRABY were maintained with 50 μg ml −1 kanamycin.…”

Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

“…arising from the native genomic and metabolic properties of the cells) or exogenous (i.e. due to the burden that producing a chemical compound at high yields usually imposes on the engineered cells) (Akkaya et al, 2018). Tackling this problem is therefore one of the major challenges in bringing bacterial chassis to actual industrial-scale bioproduction.…”

Evolving metabolism of 2,4‐dinitrotoluene triggers SOS‐independent diversification of host cells

“…While the phenomenon that starts with ROS production brought about by deficient metabolization of DNT and ends with a high‐mutagenesis regime in B. cepacia R34 is well‐documented (Perez‐Pantoja et al, ; Akkaya et al, ), the molecular mechanisms behind the process are not clear. ROS can react directly with free or DNA‐bound nucleotides and generate variants (e.g., 8‐oxoguanine) that pair with the wrong partners in the helix and thus generate mutations.…”