Pierre Schoenmakers (Aachen / DE), Axel Ihl (Aachen / DE), Claudia Tahiraj (Aachen / DE), Ramineh Rad (Bochum / DE), Isabelle Weickardt (Toulouse / FR), Stéphane Guillouet (Toulouse / FR), Ulf-Peter Apfel (Bochum / DE), Lars Lauterbach (Aachen / DE)
Introduction: Cupriavidus necator efficiently sequesters CO2, utilizing H2 to recycle cofactors through its native hydrogenases1. Advancing synthetic biology tools is crucial to redirect carbon to valuable products like N-heterocycles. Since C. necator is an obligate aerobe, autotrophic cultivation usually entails hazardous pressurized knallgas.
Goals: We developed synthetic biology tools and safe electro-fermentation methods for C. necator, enabling electricity-driven biotransformations and pioneering the development of novel pathways for N-heterocycle synthesis2,3.
Methods: Our engineered C. necator features two genomic integrations: 1) rhamnose-inducible dCas9 for gene repression; 2) arabinose-inducible T7 RNA polymerase-cytidine deaminase fusion (MT7) for gene expression and pathway hypermutation. A newly developed reactor incorporating a biocompatible electrolyzer regenerates hydrogen in-situ4, with integrated sensors for precise monitoring of dissolved- and headspace gasses.
Results: Genomic integration of inducible MT7 and dCas9 systems regulated gene expression and enabled pathway evolution. These tools were applied to accelerate the development of new-to-nature pathways for N-heterocycle biosynthesis. Furthermore, an electro-fermentation reactor ensured safe and efficient synthesis of isopropanol, using a previously engineered strain5, as a proof-of-concept.
Conclusions: Genomic integration of molecular tools significantly enhanced future C. necator strain development. Additionally, the developed electro-fermentation reactor was successfully implemented for isopropanol production. The utility of this reactor extends to newly developed strains for biosynthesis of N-heterocycles.
References:
Al-Shameri A., Siebert D. L., Sutiono S., Lauterbach L. & Sieber V. Nat. Commun. 14, (2023).2. Al-Shameri A., Petrich M-C M., Puring K.j., Apfel U.-P., Nestl B.M., Lauterbach L., Chemie - Int. Ed. 59, 10929–10933 (2020).3. Al-Shameri A., Borlinghaus N., Schneller P., Nestl B.M., Lauterbach L., Green Chem. 21, 1396–1400 (2019).4. Rad, R. et al. Cell Reports Phys. Sci. 4, 101526 (2023)Grousseau E. et al. Appl. Microbiol. Biotechnol. 98, 4277–4290 (2014).