Cell-free systems have become valuable investigating tools for metabolic engineering research due to their easy access to metabolism without the interference of the membrane. Therefore, we applied Zymomonas mobilis cell-free system to investigate whether ethanol production is controlled by the genes of the metabolic pathway or is limited by cofactors. Initially, different glucose concentrations were added to the extract to determine the crude extract's capability to produce ethanol. Then, we investigated the genes of the metabolic pathway to find the limiting step in the ethanol production pathway. Next, to identify the bottleneck gene, a systemic approach was applied based on the integration of gene expression data on a cell-free metabolic model. ZMO1696 was determined as the bottleneck gene and an activator for its enzyme was added to the extract to experimentally assess its effect on ethanol production. Then the effect of NAD+ addition at the high concentration of glucose (1 M) was evaluated, which indicates no improvement in efficiency. Finally, the imbalance ratio of ADP/ATP was found as the controlling factor by measuring ATP levels in the extract. Furthermore, sodium gluconate as a carbon source was utilized to investigate the expansion of substrate consumption by the extract. 100% of the maximum theoretical yield was obtained at 0.01 M of sodium gluconate while it cannot be consumed by Z. mobilis. This research demonstrated the challenges and advantages of using Z. mobilis crude extract for overproduction.

The cell-free system has emerged as a strong platform for producing chemical molecules and pathway debugging. However, cofactor regeneration in this system is also a challenge. To solve cofactor challenges and pathway debugging in the bioethanol pathway, we use the Zymomonas cell-free system to produce bioethanol and investigate cofactor challenges. First, to determine the ability of this system to produce bioethanol, different concentrations of glucose were added to the extract. At low glucose concentrations (0.01 and 0.1 M), the maximum theoretical yield of bioethanol produced was close to 100%, but with increasing glucose concentrations, this amount decreased. Next, sodium gluconate, as an intermediate component, in the ED pathway, was experimentally assessed. At 0.01 M of sodium gluconate, the maximum theoretical yield of 100%; was obtained. Then, to identify the ED pathway bottleneck, a systemic approach based on metabolic modeling was applied. A regulatory compound that controlled the bioethanol reaction was obtained from the BRENDA database. The regulator was added to the Zymomonas crude extract and its effect on bioethabol production was experimentally assessed. Then the effect of NAD+, at the concentration of 1 M glucose, was investigated, which does not affect increasing efficiency. Finally, by measuring ATP and glucose remaining in the culture medium, the imbalance ratio of ADP/ATP was found as the limiting step in producing bioethanol. This research demonstrated the highlighted role of cofactors in bioethanol production and the power of Zymomonas crude extract in reaching a high molar yield of bioethanol production.