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The PROACTIVE project aims at harnessing natural mechanism of generation of diversity and selection in order to accelerate the evolution in vivo of biodegradative pathways for aromatic xenobiotics, one of the most prominent groups of persistent organic pollutants. Three specific objectives were initially pursued:

Objective 1. Reconstruction and expression of ancestral catabolic enzymes.
Objective 2. Setup and validation of an in vivo method for one-step random assembly of multiple genes.
Objective 3. Setup and validation of conditional in vivo methods for intensive mutagenesis on evolvable catabolic operons.

The initial stage of PROACTIVE consisted of bioinformatic approaches for the phylogenetic reconstruction of enzymes families involved in biodegradation. Six different subfamilies of monooxygenases representing diverse substrate specificities were selected for maximum likelihood and bayesian phylogenetic analysis. The phylogenies obtained were used to predict protein ancestral sequences of each oxygenase subfamily and a consensus ancestral sequence was generated. These predicted sequences encode putative oxygenases owing a distinct substrate profile and represent for the first time the application of ancestral reconstruction to biodegradative enzymes. The purpose of these predicted sequences was to be incorporated in assembled catabolic operons as indicated by the second objective. In addition, the prediction of ancestral aromatic oxygenases has an added value since shed light on the evolutionary origin of monooxygenases with singular substrates as pentachlorophenol or octylphenol.

As a platform for straightforward assessment of in vivo methods for intensive mutagenesis on evolvable operons we performed the functional reconstitution of multipart biodegradative pathways conferring challenging catabolic phenotypes. Three different catabolic routes were chosen to use as model pathways: The TOL pathway for xylene degradation (xyl genes), the 2,4-dichlorophenoxyacetate pathway (tfd genes) and the 2,4-dinitrotoluene pathway (dnt genes, F1). These pathways were chosen because are encoded by a large number of genes that can be separated in genetic modules suitable for reconstitution in heterologous hosts and subsequently to be assembled in vivo as a single operon. Each pathway was divided in different cloned fragments to be expressed independently under control of heterologous expression systems and its functionality was evaluated in P. putida and E. coli. The reconstitution of TOL pathway was especially successful since a functional TOL pathway was expressed in E. coli, a goal previously not achieved in the field of biodegradation. It should be noted that several reports have shown that the functional expression of xyl genes conferring a catabolic phenotype is restricted only to bacteria closely related to the original host of the TOL plasmid, severely impairing its utility in bioaugmentation. By setting out the genetic reconstitution of the TOL degradation pathway in a heterologous host we established the basis to create minimal variants of the natural TOL plasmid with improved properties for bioaugmentation as well as a strategy to expand the functional host range, by means of recruiting reliable regulatory circuits de-bugged of undesired metabolic signals.

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