• Directed evolution was challenged for oversimplifying the claim that “any population can be systematically optimized,” with participants pointing out that survivability valleys and phenotype constraints are often ignored.  

• The Evolugator continuous-culture device was described as an early eukaryotic directed-evolution platform that suppresses fungal wall growth and biofilm formation, enabling a four-month thermotolerance selection in Metarhizium anisopliae for pest-control applications.  

• A yeast-contained, orthogonal DNA replication system using Kluyveromyces lactis components was highlighted; it delivers ~300× higher mutagenesis on a target plasmid while leaving the host genome untouched, providing a clean background for evolution experiments.  

• A “one-pot” DNA assembly concept was proposed in which 10 000 unique 20-mer-overlapping fragments could be joined in a single reaction by evolving a ligase or recombinase that recognizes only perfectly matching ends, eliminating the need for primers and polymerase.  

• Recombinase-polymerase amplification (RPA) was introduced as an isothermal PCR alternative that uses phage-derived recombinases to scan dsDNA for homology and stabilize strand invasion, potentially increasing specificity in Gibson or Golden Gate assemblies.  

• Golden Gate assembly was judged obsolete for large-fragment work; participants favored recombinase-driven or modified Gibson methods to reduce off-target ligation and deletion errors.  

• Brownian-motion limitations for very large DNA molecules finding partners in crowded reactions were discussed, with suggestions to use shorter barcodes or recombinase-mediated active searching to overcome kinetic bottlenecks.