Periodic Reporting for period 4 - NOVCARBFIX (Analysis, Design and Experimental Evolution of Novel Carbon Fixation Pathways) Reporting period: 2020-07-01 to 2020-12-31 Summary of the context and overall objectives of the project With the need to feed our growing human population and increasing evidence for the threat of global warming, a means of channeling greenhouse gasses such as CO2 into food, feed or fuel is a good avenue to simultaneously deal with two of humanity’s major challenges. In nature, carbon fixation by autotrophic organisms is the gateway of inorganic carbon into the living world and is the process which all living organisms depend on for energy and food. Several pathways have evolved to use CO2 as a carbon source but the Calvin-Benson-Bassham (CBB) cycle is by far the most dominant and relies on the carboxylation activity of Ribulose Bisphosphate Carboxylase/Oxygenase (Rubisco), a relatively inefficient enzyme because of both its relatively slow catalytic rate and its inability to distinguish efficiently between O2 and CO2. This issue has been addressed by evolution in many autotrophic organisms that developed a solution in the form of carbon concentrating mechanisms (CCMs), encapsulating Rubisco in an environment rich with CO2 and poor with O2. In agriculture, improvement of the carbon fixation process might increase crop yield and would lead to a more efficient use of resources such as water or land. Additionally, in the biotechnology industry a versatile biological platform able to create compounds of interest from CO2 could be of great significance in halting CO2 atmospheric accumulation. With the ERC support, we were able to introduce a non-native CBB cycle into E. coli, a model heterotrophic organism, gaining the ability to grow on CO2 as a sole carbon source in an environment of 10% partial pressure CO2, rendering the bacteria a synthetic autotroph. Beyond this breakthrough we also characterized essential mutations leading to this phenotype and made progress in shedding light on their molecular mechanism. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We demonstrated how a combination of rational metabolic rewiring, recombinant expression, and laboratory evolution has led to the biosynthesis of various significant fractions of the cellular biomass (30%, 80% and 100%) directly from CO2 by a fully functional Calvin-Benson-Bassham (CBB) cycle in E. coli. In the evolved bacterial strains, CO2 fixation is performed via a non-native CBB cycle, while reducing power and energy are obtained by oxidizing a supplied organic compound (pyruvate, acetate or formate). For our lab-evolution strategy, we applied continuous-culturing of engineered E. coli strains in chemostats under starvation conditions for organic carbon sources. Abundant external supply of reducing power source and CO2 with simultaneous expression of heterologous CO2 fixation machinery by the evolving bacteria has led to strong selective pressure to utilize CO2 as an alternative carbon source for generation of major biomass constituents. Periodic sampling of the evolving population in the course of a few months (2-12) enabled isolation of mutants with desired growth phenotypes. Subsequent characterization of the evolved strain using 13C-labeling experiments and analysis by liquid chromatography coupled to mass-spectrometry validates the desired novel metabolic setups. Whole genome sequencing of the strains by next-generation-sequencing tools allows highlighting the genetic and biochemical basis for the adaptation to the novel trophic mode of the bacteria. The successful evolution of a non-native carbon fixation pathway in E. coli and generation of a fully autotrophic E. coli strikingly demonstrates the capacity for rapid trophic-mode evolution of metabolism applicable to biotechnology and sustainability. The work was presented to many audiences around the world, published in the journal Cell and reviewed in many publications studying the work. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In spite of widespread interest for renewable energy storage and more sustainable food production, so far industrially-relevant heterophic model organisms could not be engineered to use CO2 as the sole carbon source. In this project we achieved this transformation on laboratory timescales. We constructed and evolved Escherichia coli to produce all biomass carbon directly from CO2. Reducing power and energy, but not carbon, is supplied via the one-carbon molecule formate, which can be produced electrochemically. Rubisco and phosphoribulokinase were co-expressed with formate dehydrogenase to enable CO2 fixation and reduction via the Calvin-Benson-Bassham cycle. Autotrophic growth was achieved following several months of continuous laboratory evolution in a chemostat under intensifying organic carbon limitation and confirmed via isotopic labeling. This is well beyond the state of the art and takes the community to new fronts and challenges. 28601-illustration-33140-ron-milo-shmuel-and-roee-v2-fig1.jpg
