# novoStoic2.0: An integrated framework for pathway synthesis, thermodynamic evaluation, and enzyme selection

## TL;DR

novoStoic2.0 is a new platform that helps design efficient biosynthetic pathways by combining pathway synthesis, thermodynamic analysis, and enzyme selection in one tool.

## Contribution

novoStoic2.0 integrates pathway synthesis, thermodynamic evaluation, and enzyme selection into a unified platform for streamlined biosynthetic design.

## Key findings

- novoStoic2.0 enables the design of thermodynamically viable biosynthetic pathways with reduced cofactor usage.
- The platform identified shorter hydroxytyrosol synthesis pathways compared to existing routes.
- Integration of multiple biosynthesis tasks improves consistency and accelerates experimental implementation.

## 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.

Designing biosynthetic pathways for novel chemical targets often involves a series of non-trivial tasks: estimating stoichiometries, assessing thermodynamic feasibility, and selecting appropriate enzymes, typically addressed using separate tools, which can lead to inconsistencies and hinder the transition from computational design to experimental implementation. To address this, we developed novoStoic2.0, an integrated platform that unifies these tasks into a single workflow. It supports the construction of biosynthetic routes that are thermodynamically viable, meaning they are energetically favorable and chemically feasible, and helps identify which steps may require enzyme re-engineering. This streamlines the transition from computational design to experimental implementation. We demonstrated the platform’s capabilities by designing biosynthetic pathways for hydroxytyrosol, a compound with both industrial and biomedical relevance as an antioxidant. The resulting routes are shorter than known alternatives and reduce cofactor requirements, offering more efficient options for microbial production. By combining pathway construction, thermodynamic analysis, and enzyme selection in a coherent system, novoStoic2.0 simplifies and strengthens early-stage biosynthesis planning. We see this as a step toward faster, more reliable development of sustainable production routes in synthetic biology and metabolic engineering.