Engineering RNA life cycle to optimize economy of microbial energy: application to the bioconversion of biomass-derived carbon sources – RIB-ECO

Our aim is to reduce the energy-dissipating RNA life-cycle and to reallocate the energy saved towards cell growth and end-product formation. This approach provides a novel integrated vision of microbial physiology, coupling regulation of RNA life-cycle and metabolism.

To analyze the impact of the RNA life cycle on the E. coli physiology we have constructed several modified mutants in the mRNA lifecycle by targeting 11 enzymes of the RNA degradation process. Growth of these different strains and their control as well as their ability to adapt to changes in carbon sources were characterized for a wide range of carbon sources (glycolytic substrates: glucose, D-gluconate, L-arabinose, D- xylose, fructose, D-glucuronate; glucogenic substrates: pyruvate, succinate and acetate). This screening step made it possible to select the most relevant targets for examining the links between the mRNA life cycle and energy metabolism and in particular the role of PAPI, an ATP consuming enzyme involved in the degradation of mRNAs.
We then studied the effect of PAPI deletion or overexpression on E. coli physiology by characterizing energy allocation, growth and product formation in controlled bioreactor environments. We also analyzed the effect of the PAPI deletion on the mRNA life cycle by measuring the half-lives of all the mRNAs in growing cells and the residual (not degraded) mRNA concentrations in the absence of growth. We now seek to explain and relate the large changes observed in energy metabolism and the RNA life cycle through modeling approaches. In order to identify the metabolic reactions most impacted by the overexpression or deletion of PAPI, we integrated external metabolite and biomass quantifications in stoichiometric models of the E. coli central metabolism. In parallel, we have developed a mathematical model which describes the mechanism of degradation of cellular mRNAs. This model allows the regulation of the degradation mechanisms by endoribonucleases to be analyzed, in particular the competition of mRNAs for their binding to RNase E. Our approach is modular and could be extended to take into account the energy aspects of mRNA degradation and the role of PAPI. This will be a first step towards coupling energy metabolism with mRNA metabolism.

The expected results of the project are engineered microbes with accelerated growth and improved productivity. In addition to PAPI which consumes ATP, we have identified other interesting enzyme targets in particular related to the metabolism of xylose which is one of the constituents of plant biomass. In the next months, we will combine these various disruptions in the RNA life cycle with the aim of significantly reducing the energy-consuming cycle and reallocating the saved energy towards the synthesis of a heterologous protein.

Publications
-Roux C, Etienne TA, Hajnsdorf E, Ropers D, Carpousis AJ, Cocaign-Bousquet M, Girbal L. The essential role of mRNA degradation in understanding and engineering E. coli metabolism. Biotechnol Adv. 2021 Jul 21:107805. doi: 10.1016/j.biotechadv.2021.107805. Epub ahead of print. PMID: 34302931.
-TA. Etienne, M Cocaign-Bousquet, D Ropers, 2020, Competitive effects in bacterial mRNA decay. Journal of Theoretical Biology 504(7)110333. doi.org/10.1016/j.jtbi.2020.110333
-M. Lejars & E. Hajnsdorf «The world of asRNAs in Gram-negative and Gram-positive bacteria« BBA - Gene Regulatory Mechanisms 1863 (2020) 194489
-M. Lejars, A. Kobayashi & E. Hajnsdorf «Physiological roles of antisense RNAs in prokaryotes« Biochimie 164 (2019) 3e16

Posters
-Roux C, Hajnsdorf E, Girbal L, Cocaign-Bousquet M (2021) Modulation of RNA polyadenylation induces strong metabolic rearrangements in Escherichia coli. World Microbe Forum 20-24 June 2021. Online Worldwide.

Conferences
-Roux C, Hajnsdorf E, Girbal L, Cocaign-Bousquet M (2021) Modulation of RNA polyadenylation induces strong metabolic rearrangements in Escherichia coli. RNAocc2021, 3-4 June 2021, Montpellier, France.

: Improving the performance of microbial platforms to transform biomass into bioproducts is crucial for the development of sustainable and economically relevant biotechnological processes. Metabolic engineering to optimize expression of native or heterologous pathways often leads to significant energy-limitation, which reduces cell growth and product productivity. The challenge of the RIB-ECO project is to improve microbial energy efficiency through RIBonucleotide ECOnomy for applications in biotechnology. Our aim is to reduce the energy-dissipating RNA life cycle and to reallocate this energy saved towards cell growth and end-product formation using biomass-derived carbon sources. This approach provides a novel integrated vision of microbial physiology, coupling regulation of RNA life-cycle and metabolism. Knowledge gained will allow us to understand and to predict the adaptation of microbes to environmental changes and propose innovative strategies for strain optimization. The expected results of the project are engineered microbes with accelerated growth and productivity, and reduced lag phase when growing on non-preferential carbon sources. In these optimized microbes, the energetic limitation of growth will be reduced by modulating the futile RNA life cycle. The project will be exemplified in Escherichia coli bacterial cells and applied to the valorization of vegetal biomass-derived carbon sources. Optimization of microbial energetics is a generic strategy, relevant to any microorganism of industrial interest (natural and engineered strains) and to any other carbon sources. Increase the economical sustainability of the biotechnology sector will contribute to the bioeconomy development.