Enabling pathway design by multiplex experimentation and machine learning.

The remarkable metabolic diversity observed in nature has provided a foundation for sustainable production of a wide array of valuable molecules. However, transferring the biosynthetic pathway to the desired host often runs into inherent failures that arise from intermediate accumulation and reduced flux resulting from competing pathways within the host cell. Moreover, the conventional trial and error methods utilized in pathway optimization struggle to fully grasp the intricacies of installed pathways, leading to time-consuming and labor-intensive experiments, ultimately resulting in suboptimal yields. Considering these obstacles, there is a pressing need to explore the enzyme expression landscape and identify the optimal pathway configuration for enhanced production of molecules. This review delves into recent advancements in pathway engineering, with a focus on multiplex experimentation and machine learning techniques. These approaches play a pivotal role in overcoming the limitations of traditional methods, enabling exploration of a broader design space and increasing the likelihood of discovering optimal pathway configurations for enhanced production of molecules. We discuss several tools and strategies for pathway design, construction, and optimization for sustainable and cost-effective microbial production of molecules ranging from bulk to fine chemicals. We also highlight major successes in academia and industry through compelling case studies. • Provided a detailed meta-analysis of pathway engineering with emphasis on multiplex experimentation and machine learning. • Summarized tools and techniques for pathway design, construction, and optimization in prokaryotic and eukaryotic hosts. • Highlighted the major successes in the field both on the academic and industrial side through case studies. • Discussed challenges and future perspectives for pathway design. [ABSTRACT FROM AUTHOR]