Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway in E. coli.

One-carbon (C1) compounds, such as methanol, have recently gained attention as alternative low-cost and non-food feedstocks for microbial bioprocesses. Considerable research efforts are thus currently focused on the generation of synthetic methylotrophs by transferring methanol assimilation pathways into established bacterial production hosts. In this study, we used an iterative combination of dry and wet approaches to design, implement and optimize this metabolic trait in the most common chassis, E. coli. Through in silico modelling, we designed a new route that "mixed and matched" two methylotrophic enzymes: a bacterial methanol dehydrogenase (Mdh) and a dihydroxyacetone synthase (Das) from yeast. To identify the best combination of enzymes to introduce into E. coli, we built a library of 266 pathway variants containing different combinations of Mdh and Das homologues and screened it using high-throughput 13C-labeling experiments. The highest level of incorporation of methanol into central metabolism intermediates (e.g. 22% into the PEP), was obtained using a variant composed of a Mdh from A. gerneri and a codon-optimized version of P. angusta Das. Finally, the activity of this new synthetic pathway was further improved by engineering strategic metabolic targets identified using omics and modelling approaches. The final synthetic strain had 1.5 to 5.9 times higher methanol assimilation in intracellular metabolites and proteinogenic amino acids than the starting strain did. Broadening the repertoire of methanol assimilation pathways is one step further toward synthetic methylotrophy in E. coli.

Benzo(a)pyrene inhibits the role of the bioturbator Tubifex tubifex in river sediment biogeochemistry.

The interactions between invertebrates and micro-organisms living in streambed sediments often play key roles in the regulation of nutrient and organic matter fluxes in aquatic ecosystems. However, benthic sediments also constitute a privileged compartment for the accumulation of persistent organic pollutants such as PAHs or PCBs that may affect the diversity, abundance and activity of benthic organisms. The objective of this study was to quantify the impact of sediment contamination with the PAH benzo(a)pyrene on the interaction between micro-organisms and the tubificid worm, Tubifex tubifex, which has been recognized as a major bioturbator in freshwater sediments. Sedimentary microcosms (slow filtration columns) contaminated or not with benzo(a)pyrene (3 tested concentrations: 0, 1 and 5 mg kg(-1)) at the sediment surface were incubated under laboratory conditions in the presence (100 individuals) or absence of T. tubifex. Although the surface sediment contaminations with 1 mg kg(-1) and 5 mg kg(-1) of benzo(a)pyrene did not affect tubificid worm survival, these contaminations significantly influenced the role played by T. tubifex in biogeochemical processes. Indeed, tubificid worms stimulated aerobic respiration, denitrification, dehydrogenase and hydrolytic activities of micro-organisms in uncontaminated sediments whereas such effects were inhibited in sediments polluted with benzo(a)pyrene. This inhibition was due to contaminant-induced changes in bioturbation (and especially bio-irrigation) activities of worms and their resulting effects on microbial processes. This study reveals the importance of sublethal concentrations of a contaminant on ecological processes in river sediments through affecting bioturbator-microbe interactions. Since they affect microbial processes involved in water purification processes, such impacts of sublethal concentrations of pollutants should be more often considered in ecosystem health assessment.