Automated Genetic Manipulation for the Construction of Pichia pastoris Cell Factories

As a nonmodel microorganism, Pichia pastoris has unique advantages in recombinant protein expression and natural product biosynthesis. The development of synthetic biology has provided key technical support for the construction of microbial cell factories. However, it faces challenges such as the complexity and diversity of living systems, the necessity for repeated trial and error during research, and the low throughput of manual experiments, which fail to satisfy the demands of various applications. Automated synthetic biotechnology, featured by high throughput, automation, and intelligence, can conduct extensive experiments and expedite the Design-Build-Test-Learn cycle for the construction and optimization of microbial cell factories. This study describes an automated genetic manipulation process for rapid construction of P. pastoris cell factories based on BioFoundry. First, we optimized critical parameters using an automated system, including heat shock temperature, reducing agent, as well as the amounts of donor DNA and sgRNA plasmid, enabling highly efficient heat shock-based transformation and high throughput genome editing in P. pastoris. Specifically, the automated genetic manipulation process enabled the single-site and two-site genome editing efficiency reaching up to 94.6% and 36.3%, respectively. Then, we characterized 96 endogenous promoters in an automated and high throughput manner, including their strengths and time-course features in P. pastoris. Finally, we constructed yeast cell factories using promoters with different strength to produce sesquiterpene α-santalene and α-santalol. Our study provides a reference for the automated genetic manipulation of P. pastoris and other nonmodel yeast species.