[1S01m] 10. ASPIRE - Part 1

11:12 AM - 11:24 AM JST(2:12 AM - 2:24 AM UTC)

[1ASPR-03] Accelerating Biomanufacturing through Scalable Biofoundry Workflows: From Multi-Gene Assembly to Enzyme Engineering

○Daehee Lee¹ (1. Korea Research Institute of Bioscience and Biotechnology (Korea))

Keywords:

Biofoundry,Workflow,Engineering Biology,DNA Assembly,Enzyme Engineering

Synthetic biology has transformed traditional biological research into an engineering discipline. At the core of this transformation is the biofoundry, an automated platform essential for accelerating the design-build-test-learn (DBTL) cycles required to engineer advanced biological systems. However, researchers frequently encounter bottlenecks when constructing large-scale combinatorial libraries and optimizing high-performance biocatalysts. To address these limitations, we developed highly scalable, automation-compatible biofoundry workflows that overcome critical challenges in both metabolic pathway prototyping and targeted enzyme engineering.First, we designed EffiModular, a high-throughput multigene assembly platform that integrates the modularity of in vitro Golden Gate Assembly with the high-fidelity in vivo homologous recombination of Saccharomyces cerevisiae. This approach bypasses the need for intermediate PCR amplification and cloning steps, drastically reducing labor and turnaround time. EffiModular enables the reliable, one-step assembly of up to eight transcriptional units with over 80% efficiency. By implementing this system within an automated biofoundry workflow, we successfully constructed a combinatorial expression library of 120 unique β-carotene-producing yeast strains in just three days. Furthermore, we integrated a high-throughput combinatorial strategy that miniaturizes reaction volumes to one microliter, enabling a seamless progression across steps. For rapid screening, we utilize convolutional neural networks (CNNs) and CycleGAN to identify high-activity metabolic pathways directly from colony images.Second, we established semi-automated biofoundry workflows tailored for sequence coevolution-guided enzyme engineering. We utilized SCANEER, a computational sequence coevolution analysis tool, to design smart mutant libraries with high hit rates. Coupled with laboratory automation, we seamlessly processed approximately 100 genetic variants per cycle across three rounds of mutagenesis and screening. We applied this framework to engineer isoprene synthase (IspS), a rate-limiting enzyme in isoprene biosynthesis. The workflow rapidly identified engineered IspS variants exhibiting up to a 4.5-fold increase in catalytic efficiency (kcat/Km) along with enhanced thermostability. Introducing the top-performing IspS variant into the methanotroph Methylococcus capsulatus Bath significantly improved methane-to-isoprene bioconversion, achieving an unprecedented titer of 319.6 mg/L from a single-carbon feedstock.Ultimately, these integrated approaches underscore the power of biofoundries to drive the systematic exploration of genetic design spaces. By combining AI-guided computational design, robust assembly methods, and laboratory automation, we provide a scalable infrastructure that accelerates the development of microbial cell factories and supports the global bioeconomy.