Detailed profiling of carbon fixation of in silico synthetic autotrophy with reductive tricarboxylic acid cycle and Calvin-Benson-Bassham cycle in Esherichia coli using hydrogen as an energy source

Carbon fixation is the main route of inorganic carbon in the form of CO2 into the biosphere.In nature,RuBisCO is the most abundant protein that photosynthetic organisms use to fix CO2 from the atmosphere through the Calvin-Benson-Bassham(CBB)cycle.However,the CBB cycle is limited by its low catalytic rate and low energy efficiency.In this work,we attempt to integrate the reductive tricarboxylic acid and CBB cycles in silico to further improve carbon fixation capacity.Key heterologous enzymes,mostly carboxylating enzymes,are inserted into the Esherichia coli core metabolic network to assimilate CO2 into biomass using hydrogen as energy source.Overall,such a strain shows enhanced growth yield with simultaneous running of dual carbon fixation cycles.Our key results include the following.(i)We identified two main growth states:carbon-limited and hydrogenlimited;(ii)we identified a hierarchy of carbon fixation usage when hydrogen supply is limited;and(iii)we identified the alternative sub-optimal growth mode while performing genetic perturbation.The results and modeling approach can guide bioengineering projects toward optimal production using such a strain as a microbial cell factory.