Systems and Methods for Continuous Evolution of Enzymes.

Directed evolution generates novel biomolecules with desired functions by iteratively diversifying the genetic sequence of wildtype biomolecules, relaying the genetic information to the molecule with function, and selecting the variants that progresses towards the properties of interest. While traditional directed evolution consumes significant labor and time for each step, continuous evolution seeks to automate all steps so directed evolution can proceed with minimum human intervention and dramatically shortened time. A major application of continuous evolution is the generation of novel enzymes, which catalyze reactions under conditions that are not favorable to their wildtype counterparts, or on altered substrates. The challenge to continuously evolve enzymes lies in automating sufficient, unbiased gene diversification, providing selection for a wide array of reaction types, and linking the genetic information to the phenotypic function. Over years of development, continuous evolution has accumulated versatile strategies to address these challenges, enabling its use as a general tool for enzyme engineering. As the capability of continuous evolution continues to expand, its impact will increase across various industries. In this review, we summarize the working mechanisms of recently developed continuous evolution strategies, discuss examples of their applications focusing on enzyme evolution, and point out their limitations and future directions.

High-sensitivity whole-genome recovery of single viral species in environmental samples.

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. This work presents a method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A novel gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. Validation experiments using a sewage sample spiked with two known viruses demonstrate the method's efficacy. We achieve 100% recovery of the spiked-in SV40 (Simian virus 40, 5243bp) genome sequence with uniform coverage distribution, and approximately 99.4% for the larger HAd5 genome (Human Adenovirus 5, 35938bp). Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables targeted characterizations of rare viral species and whole-genome amplification of single genomes for accessing the mutational profile in single virus genomes, contributing to an improved understanding of viral ecology.