Fig. 2.

Bottom-up validation of the pentanol biosynthetic pathway using a modular approach. (A) Validation of module 3 was achieved by feeding trans-2-pentenoate and monitoring production of valerate and pentanol. Effects of formate supplementation, the replacement of Bcd with Ter_Td (trapping carbon flow toward the forward direction of pentanol synthesis), and the overexpression of Fdh1_Sc and PDH_m (boosting intracellular NADH availability) on pentanol synthesis were examined with respect to product titers and selectivities (pentanol/valerate molar ratios). (B) Two versions of module 2, module 2S with an hbd-crt gene pair and module 2R with a phaB-phaJ1 gene pair, were then stacked onto module 3. The combined modules (module 2S+3 or module 2R+3) were examined across three pathway variants. (C) The propionyl-CoA synthesis module (module 1) was then introduced into the best-performing variant from the previous test with module 2S+3, and the combined pathway (module 1+2S+3) was examined for direct pentanol synthesis from glucose or glycerol. The product profiles, including carboxylic acids and n-alcohols, and product selectivities (alcohol/acid molar ratios) are shown. (D) Effect of gene knockouts, including the mdh gene in the production host genome and the adhE_opt gene in the pentanol biosynthetic pathway, on synthesis of carboxylic acids and n-alcohols. NT, not tested.