Engineering a catabolic pathway in plants for the degradation of 1,2-dichloroethane

Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum ‘Xanthi’) plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater

Lineage-specific gene radiations underlie the evolution of novel betalain pigmentation in Caryophyllales.

Betalain pigments are unique to the Caryophyllales and structurally and biosynthetically distinct from anthocyanins. Two key enzymes within the betalain synthesis pathway have been identified: 4,5-dioxygenase (DODA) that catalyzes the formation of betalamic acid and CYP76AD1, a cytochrome P450 gene that catalyzes the formation of cyclo-DOPA. We performed phylogenetic analyses to reveal the evolutionary history of the DODA and CYP76AD1 lineages and in the context of an ancestral reconstruction of pigment states we explored the evolution of these genes in relation to the complex evolution of pigments in Caryophylalles. Duplications within the CYP76AD1 and DODA lineages arose just before the origin of betalain pigmentation in the core Caryophyllales. The duplications gave rise to DODA-α and CYP76AD1-α isoforms that appear specific to betalain synthesis. Both betalain-specific isoforms were then lost or downregulated in the anthocyanic Molluginaceae and Caryophyllaceae. Our findings suggest a single origin of the betalain synthesis pathway, with neofunctionalization following gene duplications in the CYP76AD1 and DODA lineages. Loss of DODA-α and CYP76AD1-α in anthocyanic taxa suggests that betalain pigmentation has been lost twice in Caryophyllales, and exclusion of betalain pigments from anthocyanic taxa is mediated through gene loss or downregulation. [Correction added after online publication 13 May 2015: in the last two paragraphs of the Summary the gene name CYP761A was changed to CYP76AD1.].S.C. was supported by a grant to IRRI from the Bill and Melinda Gates Foundation and UKAID. This work was supported by a National Science Foundation award (grant numbers DEB 1354048 and DEB 1352907) to S.F.B., M.J.M. and S.A.S., and a NERC Independent Research Fellowship to S.F.B. The 1000 Plants (1KP) initiative, led by G.K.S.W., is funded by the Alberta Ministry of Enterprise and Advanced Education, Alberta Innovates Technology Futures (AITF), Innovates Centre of Research Excellence (iCORE), Musea Ventures and BGI-Shenzhen.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/nph.1344

Are seven amino acid substitutions sufficient to explain the evolution of high l-DOPA 4,5-dioxygenase activity leading to betalain pigmentation? Revisiting the gain-of-function mutants of Bean et al. (2018).

This work revisits a publication by Bean et al. (2018) that reports seven amino acid substitutions are essential for the evolution of l-DOPA 4,5-dioxygenase (DODA) activity in Caryophyllales. In this study, we explore several concerns which led us to replicate the analyses of Bean et al. (2018). Our comparative analyses, with structural modelling, implicate numerous residues additional to those identified by Bean et al. (2018), with many of these additional residues occurring around the active site of BvDODAα1. We therefore replicated the analyses of Bean et al. (2018) to re-observe the effect of their original seven residue substitutions in a BvDODAα2 background, that is the BvDODAα2-mut3 variant. Multiple in vivo assays, in both Saccharomyces cerevisiae and Nicotiana benthamiana, did not result in visible DODA activity in BvDODAα2-mut3, with betalain production always 10-fold below BvDODAα1. In vitro assays also revealed substantial differences in both catalytic activity and pH optima between BvDODAα1, BvDODAα2 and BvDODAα2-mut3 proteins, explaining their differing performance in vivo. In summary, we were unable to replicate the in vivo analyses of Bean et al. (2018), and our quantitative in vivo and in vitro analyses suggest a minimal effect of these seven residues in altering catalytic activity of BvDODAα2. We conclude that the evolutionary pathway to high DODA activity is substantially more complex than implied by Bean et al. (2018)