Prospecting Biochemical Pathways to Implement Microbe-Based Production of the New-to-Nature Platform Chemical Levulinic Acid

Levulinic acid is a versatile platform molecule with potential to be used as an intermediate in the synthesis of many value-added products used across different industries, from cosmetics to fuels. Thus far, microbial biosynthetic pathways having levulinic acid as a product or an intermediate are not known, which restrains the development and optimization of a microbe-based process envisaging the sustainable bioproduction of this chemical. One of the doors opened by synthetic biology in the design of microbial systems is the implementation of new-to-nature pathways, that is, the assembly of combinations of enzymes not observed in vivo, where the enzymes can use not only their native substrates but also non-native ones, creating synthetic steps that enable the production of novel compounds. Resorting to a combined approach involving complementary computational tools and extensive manual curation, in this work, we provide a thorough prospect of candidate biosynthetic pathways that can be assembled for the production of levulinic acid in Escherichia coli or Saccharomyces cerevisiae. Out of the hundreds of combinations screened, five pathways were selected as best candidates on the basis of the availability of substrates and of candidate enzymes to catalyze the synthetic steps (that is, those steps that involve conversions not previously described). Genome-scale metabolic modeling was used to assess the performance of these pathways in the two selected hosts and to anticipate possible bottlenecks. Not only does the herein described approach offer a platform for the future implementation of the microbial production of levulinic acid but also it provides an organized research strategy that can be used as a framework for the implementation of other new-to-nature biosynthetic pathways for the production of value-added chemicals, thus fostering the emerging field of synthetic industrial microbiotechnology.