An engineered Calvin-Benson-Bassham cycle for carbon dioxide fixation in Methylobacterium extorquens AM1

Abstract

Organisms are either heterotrophic or autotrophic, meaning that they cover their carbon requirements by assimilating organic compounds or by fixing inorganic carbon dioxide (CO). The conversion of a heterotrophic organism into an autotrophic one by metabolic engineering is a long-standing goal in synthetic biology and biotechnology, because it ultimately allows for the production of value-added compounds from CO. The heterotrophic Alphaproteobacterium Methylobacterium extorquens AM1 is a platform organism for a future C1-based bioeconomy. Here we show that M. extorquens AM1 provides unique advantages for establishing synthetic autotrophy, because energy metabolism and biomass formation can be effectively separated from each other in the organism. We designed and realized an engineered strain of M. extorquens AM1 that can use the C1 compound methanol for energy acquisition and forms biomass from CO by implementation of a heterologous Calvin-Benson-Bassham (CBB) cycle. We demonstrate that the heterologous CBB cycle is active, confers a distinct phenotype, and strongly increases viability of the engineered strain. Metabolic C-tracer analysis demonstrates the functional operation of the heterologous CBB cycle in M. extorquens AM1 and comparative proteomics of the engineered strain show that the host cell reacts to the implementation of the CBB cycle in a plastic way. While the heterologous CBB cycle is not able to support full autotrophic growth of M. extorquens AM1, our study represents a further advancement in the design and realization of synthetic autotrophic organisms.

Improved recombinant expression and purification of functional plant Rubisco

“…As the pET28 and pET11a plasmids (for expression of Rubisco and chaperonins, respectively) share the pBR322 origin of replication, the long‐term coexistence of these plasmids may result in variation of copy number and thus fluctuations in expression levels . Thus, applications sensitive to fluctuations of Rubisco expression, such as directed evolution screening or metabolic engineering , may require the use of compatible plasmids.…”

A synthetic Calvin cycle enables autotrophic growth in yeast

“…This enables the cell to utilize CO 2 as a carbon source and to grow as an autotrophic organism. By splitting the metabolism into an energy generation module and a carbon fixation module, it is possible to decouple energy generation from carbon fixation 6,12 . This modular design is crucial to demonstrate CO 2 fixation by the engineered P. pastoris strains.…”

“…In a recent study, the assimilatory pathway of Methylobacterium extorquens AM1 was blocked by deleting three native genes ( ccrΔ , glyAΔ and ftfLΔ ). RuBisCO and Prk were overexpressed, resulting in a strain that can incorporate CO 2 into biomass using methanol as energy source, but no continuous growth on CO 2 as sole carbon source was observed 12 . So far, it was not possible to construct a synthetic autotrophic organism which can readily grow on CO 2 as a sole carbon source to form its entire biomass.…”

Expanding Metabolic Capabilities Using Novel Pathway Designs: Computational Tools and Case Studies

“…Directed evolution experiments enabled evolving CO 2 fixation for biomass synthesis and pyruvate oxidation for electrons source . Similarly, the methanol‐consuming bacteria Methylobacterium extorquens AM1 was recently engineered to (partially) assimilate CO 2 by introducing the required CBB enzymes and deleting essential genes for methanol assimilation . Imbalances between the CBB operation added to the constant drain of its metabolites for growth were suggested as possible causes for the incomplete hemiautotrophy.…”

“…Each color corresponds to a different application where a particular product formation and/or substrate uptake was sought to be improved. References are color‐coded green; purple; and yellow . B) Simplified representation of the first synthetic in vitro pathway for CO 2 fixation (CETCH cycle).…”