Abstract
Computational pathway design and retro-biosynthetic approaches can facilitate the development of innovative biochemical production routes, biodegradation strategies, and the funneling of multiple precursors into a single bioproduct. However, effective pathway design necessitates a comprehensive understanding of biochemistries, enzyme activities, and thermodynamic feasibility. Herein, we introduce novoStoic2.0, an integrated platform that combines tools for estimating overall stoichiometry, designing de novo synthesis pathways, assessing thermodynamic feasibility, and selecting enzymes. novoStoic2.0 offers a unified web-based interface as a part of the AlphaSynthesis platform (http://novostoic.platform.moleculemaker.org/) tailored for the synthesis of thermodynamically viable pathways as well as the selection of enzymes for re-engineering required for novel reaction steps. We exemplify the utility of the platform to identify novel pathways for hydroxytyrosol synthesis, which are shorter than the known pathways and require reduced cofactor usage. In summary, novoStoic2.0 aims to streamline the process of pathway design contributing to the development of sustainable biotechnological solutions.