Machine Learning-Guided Optimization of p-Coumaric Acid Production in Yeast.

Industrial biotechnology uses Design-Build-Test-Learn (DBTL) cycles to accelerate the development of microbial cell factories, required for the transition to a biobased economy. To use them effectively, appropriate connections between the phases of the cycle are crucial. Using p-coumaric acid (pCA) production in Saccharomyces cerevisiae as a case study, we propose the use of one-pot library generation, random screening, targeted sequencing, and machine learning (ML) as links during DBTL cycles. We showed that the robustness and flexibility of the ML models strongly enable pathway optimization and propose feature importance and Shapley additive explanation values as a guide to expand the design space of original libraries. This approach allowed a 68% increased production of pCA within two DBTL cycles, leading to a 0.52 g/L titer and a 0.03 g/g yield on glucose.

Combinatorial optimization of pathway, process and media for the production of p-coumaric acid by Saccharomyces cerevisiae.

Microbial cell factories are instrumental in transitioning towards a sustainable bio-based economy, offering alternatives to conventional chemical processes. However, fulfilling their potential requires simultaneous screening for optimal media composition, process and genetic factors, acknowledging the complex interplay between the organism's genotype and its environment. This study employs statistical design of experiments to systematically explore these relationships and optimize the production of p-coumaric acid (pCA) in Saccharomyces cerevisiae. Two rounds of fractional factorial designs were used to identify factors with a significant effect on pCA production, which resulted in a 168-fold variation in pCA titre. Moreover, a significant interaction between the culture temperature and expression of ARO4 highlighted the importance of simultaneous process and strain optimization. The presented approach leverages the strengths of experimental design and statistical analysis and could be systematically applied during strain and bioprocess design efforts to unlock the full potential of microbial cell factories.