Abstract
1-Butanol is the most popular one of biomass energy because its energy content is similar with petroleum. On the other hand, 1-butanol is hydrophobic and the vapor pressure is lower than ethanol, so 1-butanol has the higher convenience of transport and storage. We think 1-butanol has the potential to become a substitute for petroleum. The previous study has successfully developed a synthetic amino acid metabolic pathways to transform itself to the production of 1-butanol from ketoacids. This study uses glucose as carbon source and citramalate pathway which is one of synthetic amino acid metabolic pathway as 1-butanol production pathway. In this pathway, 1-propanol is an inevitable byproduct competing for the precursor (2-ketobutyrate) needed for 1-butanol. In previous study, we found the ratio of 1-butanol and 1-propanol is low (Butanol：Propanol = 1:6). We hope to regulate the gene at pathway branch point expression (ex. change the replication origin, operon structure and ribosomal binding site), reduce the toxicity of norvaline which derived from precursor of 1-butanol, promote the specificity of reactive enzyme to the precursor of 1-butanol and add acetate to medium when cell induced to promote the ratio of 1-butanol to 1-propanol. The experimental results show the extent of our regulation of gene at pathway branch-point expression can enhance the ratio of 1-butanol to 1-propanol about 15 times higher than the previous study. The ratio of 1-butanol to 1-propanol promoted from 15 times to 30 times by promoting the specificity of reactive enzyme to 1-butanol precursor and we found the titer of 1-butanol is low to 0.49 g/L. We observed when utilizing Kivd mutant (V461A) produce 1-pentanol compared to wild-type Kivd improved by 11%, so we think Kivd (V461A) has the higher specificity of the precursor of 1-pentanol than 1-butanol. The experiment result of adding acetate into medium show the titer of 1-butanol significantly enhance whatever wild-type Kivd or Kivd(V461A), so we can confirm adding sodium acetate has a positive effect to 1-butanol production. The experiment results of reducing the toxicity of norvaline are not good because the titers of 1-butanol and 1-propanol are low to 0.08 g/L and 0.02 g/L and the ratio is not promotion. We don’t have a good reason to explain these results. From the results, we think how can the same time enhance the titer of 1-butanol production and reduce the amounts of by-products become an important issue on the future.