Biology subjects

Dronsella, B.

Publications and source records attributed to Dronsella, B..

4 recordsLinked to original sources

bioRxiv2024

System-level characterization of engineered and evolved formatotrophic E. coli strains

One-carbon compounds, such as formate, are promising and sustainable feedstocks for microbial bioproduction of fuels and chemicals. Growth of Escherichia coli on formate was recently achieved by introducing the reductive glycine pathway (rGlyP) into its genome, which is theoretically the most energy-efficient aerobic formate assimilation pathway. While adaptive laboratory evolution was used to enhance the growth rate and biomass yield significantly, still the best performing formatotrophic E. coli strain did not approach the theoretical optimal biomass yield of the rGlyP. In this study, we investigated these previously engineered formatotrophic E. coli strains to find out why the biomass yield was sub-optimal and how it may be improved. Through a combination of metabolic modelling, genomic and proteomic analysis, we identified several potential metabolic bottlenecks and future targets for optimization. This study also reveals further insights in the evolutionary mutations and related changes in proteome allocation that supported the already substantially improved growth of formatotrophic E. coli strain. This systems-level analysis provides key insights to realize high-yield, fast growing formatotrophic strains for future bioproduction.

synthetic biology↗

bioRxiv2022

Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains empowers synthetic formate assimilation

AO_SCPLOWBSTRACTC_SCPLOWFormate is a promising, water-soluble C1 feedstock for biotechnology since it can be efficiently produced from CO2--but very few industrially-relevant hosts have been engineered towards formatotrophy. Here, the non-pathogenic soil bacterium Pseudomonas putida was adopted as a platform for synthetic formate assimilation. The metabolism of genome-reduced variants of P. putida was rewired to establish synthetic auxotrophies that could be functionally complemented by expressing components of the reductive glycine (rGly) pathway. The rGly pathway mediates the formate [->] glycine [->] serine transformations that yield pyruvate, ultimately assimilated into biomass. We adopted a modular engineering approach, dividing C1 assimilation in segments composed of both heterologous activities (sourced from Methylorubrum extorquens) and native reactions. Promoter engineering of chromosomally-encoded functions coupled to modular expression of rGly pathway elements enabled growth on formate as carbon source and acetate for energy supply. Adaptive laboratory evolution of two lineages of engineered P. putida formatotrophs significantly reduced doubling times to ca. 15 h. During evolution, two catabolic regimes became predominant in independently evolved clones, either via glycine hydroxymethylation (GlyA) or oxidation (ThiO). Taken together, our results expand the landscape of microbial platforms for C1-based biotechnological production towards supporting a formate bioeconomy. GO_SCPLOWRAPHICALC_SCPLOW AO_SCPLOWBSTRACTC_SCPLOW O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/499488v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@10b12edorg.highwire.dtl.DTLVardef@7a4eaorg.highwire.dtl.DTLVardef@1dc101aorg.highwire.dtl.DTLVardef@a52d5a_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Activating silent glycolysis bypasses in Escherichia coli

All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocks and reducing power. To identify whether alternative metabolic routes of glycolysis can operate in E. coli, we complementarily employed in silico design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model and identified two potential pathways within the metabolic network of this organism replacing canonical Embden-Meyerhof-Parnas (EMP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal, the other via serine biosynthesis and degradation. Then, we implemented both pathways in E. coli strains harboring defective EMP glycolysis. Surprisingly, the pathway via methylglyoxal immediately operated in a triosephosphate isomerase deletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression of methylglyoxal synthase was necessary for implementing a functional methylglyoxal pathway. Furthermore, we engineered the serine shunt which converts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, to explore which of these alternatives would emerge by natural selection we performed an adaptive laboratory evolution study using an enolase deletion strain. The evolved mutants were shown to use the serine shunt. Our study reveals the flexible repurposing of metabolic pathways to create new metabolite links and rewire central metabolism.

synthetic biology↗

bioRxiv2020

Awakening a latent carbon fixation cycle in Escherichia coli

Carbon fixation is one of the most important biochemical processes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of Escherichia coli native enzymes. We identify the GED (Gnd-Entner-Doudoroff) cycle as the simplest pathway that can operate with high thermodynamic driving force. This autocatalytic route is based on reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (Gnd), followed by reactions of the Entner-Doudoroff pathway, gluconeogenesis, and the pentose phosphate pathway. We demonstrate the in vivo feasibility of this new-to-nature pathway by constructing E. coli gene deletion strains whose growth on pentose sugars depends on the GED shunt, a linear variant of the GED cycle which does not require the regeneration of Ru5P. Several metabolic adaptations, most importantly the increased production of NADPH, assist in establishing sufficiently high flux to sustain this growth. Our study exemplifies a trajectory for the emergence of carbon fixation in a heterotrophic organism and demonstrates a synthetic pathway of biotechnological interest.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗