1-Propanol Production of S. Cerevisiae Engineering 2-Ketobutyrate Biosynthetic Pathway

As a means of handling global issues like warming impact and depletion of fossil resources, biofuels and products from sustainable biomass resource have received a lot of attention in recent years. Especially, since alcohols are available as next-generation fuels and building blocks of chemicals, they have been extensively studied. Therefore, we focused on 1-propanol. 1-propanol is generically used as solvent, food additive, paint, cosmetics and chemical intermediate like n-propylamine. However, a natural microbial producer of 1-propanol is yet to be identified.  

  The budding yeast S. cerevisiae is expected to become a producer strain of 1-propanol. As S. cerevisiae has been used to produce ethanol, it possesses tolerances to alcohols and other stresses during fermentation. The tolerance to high concentrations of alcohol should be useful for the industrial production of 1-propanol. Furthermore, S. cerevisiae utilizing not only glucose but also xylose as sugar have been developed to expand application range of biomass. For these reasons, S. cerevisiae would be more suitable for the production of 1-propanol than other microbial hosts.

  Recently, it has been reported that the production of various alcohols from a-keto acids is enabled by using 2-Keto acid decarboxylase (KDC) and alcohol/aldehyde dehydrogenase (ADH) in metabolic engineering (Atsumi et al., 2008). Therefore, 1-propanol can be produced from 2-ketobutyrate (2KB). By using this method, 2.1 mM of 1-propanol was produced from 8 g/L of 2KB in E. coli (Atsumi et al., 2008). Therefore, it would be expected to convert 2KB produced from threonine into 1-propanol by reaction with KDC and ADH in S. cerevisiae.

  Additionally, the pathway producing 2KB without passing through threonine was constructed in E. coli. Although E. coli primarily have no citramalate synthase (cimA), E. coli learn to produce 2KB from pyruvate through citramalate by introducing this heterogenous gene. With this method, 4.5 g/L of 1-propanol was produced in E. coli (Shen and Liao, 2013). Therefore, it would be expected to produce 2KB from pyruvate through citramalate by introducing cimA and methylmalate dehydratase (leuC and leuD) into yeast.

  Furthermore, the yeast deleting acetolactate synthase (ILV2) in competing pathway produced 60 mg/L of 1-propanol (Eden et al., 2001). Against such a background, we tried to develop metabolically engineered S. cerevisiae suitable for the production of 1-propanol by the combination of overexpression of genes for 1-propanol biosynthesis pathway and deletion of competing pathway.