Investigating ethanol production using the Zymomonas mobilis crude extract

Abstract

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.