Paper title: Bridging continuous and discrete evolution through a controllable, hypermutagenic phage-bacteria system

Publishing journal: Nature Microbiology

Impact factor: 19.4

Client: National University of Singapore (NUS)

GentleGen provides services: Gene Synthesis

Gene editing and directed evolution have always been challenges in accelerating bioengineering. Recently, Julius Fredens' team at the National University of Singapore (NUS) published a study in the journal Nature Microbiology introducing a brand-new solution: a controllable phage-bacterial directed evolution platform called LySE (Lysis Selection and Evolution). This platform balances high-speed flux for continuous evolution with precise controllability for discrete evolution, supports the evolution of 40kb ultra-large gene clusters, and completely solves the three major pain points of traditional technologies: off-target, cheating mutation, and fragment limitation.

Long-term bottleneck: Directional evolution can never be a perfect solution

Directed evolution is a core technology for synthetic biology, enzyme modification, microbial breeding, and optimization of plastic degradation pathways, but mainstream systems still face irreconcilable shortcomings. Traditional discrete evolution models are precisely controllable, traceable, and free from off-target host interference, but they are slow to iterate, have low throughput, and long R&D cycles;

In contrast, continuous evolution represented by PACE is fast and highly automated, but it suffers from uncontrollable evolutionary trajectories, easy accumulation of cheating mutations, and genomic off-target mutations, making precise attribution difficult. More importantly, traditional continuous evolution only supports small gene fragments within 8kb, making it completely incompatible with complex metabolic pathways, multiprotein complexes, and other large gene cluster modifications, which has long limited the advancement of complex microbial engineering.

NUS Major Innovation: LySE Hybrid Evolution System

To address industry bottlenecks, the Julius Fredens team at the National University of Singapore developed the LySE cleavage screening and evolution system, establishing a new evolutionary paradigm of "discrete quality control + continuous high-speed iteration." It is currently one of the few universal platforms capable of enabling precise and rapid evolution of ultra-large gene clusters. The system's core consists of two major innovation modules:

First, the team engineered an ultra-high mutation T7 DNA polymerase, fused with diadenine cytosine deaminase, achieving uniform and comprehensive mutation. The mutation efficiency reached 160,000 times that of the E. coli background (3.82×10⁻⁵ per base), and the mutations targeted only the target phage plasmid, without damaging the host genome, eliminating off-target and cheating mutations at the source.

Second, relying on T7 phages, a high and low MOI cleavage-transduction cycle system was constructed: rapid hypermutation and phage packaging release were completed during the high-infection multiple stages, while the low-infection complex stage transferred the mutant gene cluster into a new host. Each evolutionary cycle refreshed host cells, retaining the quality control nodes of discrete evolution and traceability and controllability, while achieving rapid iterative cycles of continuous evolution.

Core Advantage: Completely Crushes Traditional Evolution Technology

Compared to mainstream solutions like PACE and ALE, LySE's advantages are extremely prominent:

1. Breaks the 8kb fragment limit, supports up to 40kb ultra-large gene cluster evolution, adapting to complete metabolic pathway modification;
2. Mutations precisely target sequences, with phenotypic changes precisely matching genotype mutations, greatly enhancing experimental credibility;
3. Simple operation, no need for high-end equipment, supports high-throughput automated operation, and is highly versatile.

Dual-scenario validation: The evolution effect is significantly superior to traditional solutions

• Antibiotic resistance evolves rapidly

Against the tetA resistance gene, after only five evolutionary cycles, the strain's tigecycline resistance increased 25-fold, mutations remained stable in target plasmids, and traits were completely preserved after transplantation into a new host. In contrast, the resistance evolved by traditional ALEs depends on host genomic mutations, and resistance is directly lost after transfer, making it impractical.

• Optimization of PET plastic degradation pathways

The core ethylene glycol degradation gene gox0313 used for evolutionary modification was synthesized by GentleGen. The research team established a complete PET monomer degradation pathway based on this gene, completing targeted evolution through the LySE system. After five rounds of iterative optimization, the biomass using ethylene glycol as the sole carbon source increased by 50.9%, successfully screening key synergistic mutations such as gox0313 and glcD. In contrast, the ALE system did not produce any effective abrupt changes in the target pathway, making precise channel optimization impossible.

Value Summary: Reshaping the Directed Evolution Paradigm of Microorganisms

LySE successfully solves the industry challenges of directed evolution: "fast but not accurate, accurate but not fast, and large fragments ineffective." It is suitable for basic research such as protein engineering and gene pathway mechanism analysis, as well as supporting industrial scenarios such as plastic biodegradation, carbon-neutral engineering strains, industrial enzyme modification, and screening of novel antibacterial targets.

This technology has already applied for an international patent and possesses complete independent intellectual property rights. It is expected to become the standardization mainstream tool for next-generation directional evolution in synthetic biology, promoting complex microbial engineering from trial-and-error iteration to a new stage of precise and efficient evolution.

Gene synthesis: automated large-scale preparation

Over 80% of GentleGen's gene synthesis team members have more than five years of frontline industry experience and have participated in several major domestic synthetic biology projects, enabling them to provide clients with fast and stable gene synthesis services. At the same time, based on customers' downstream experimental needs, we can provide one-stop solutions including personalized design communication, codon optimization, and carrier construction proposals.

GentleGen can provide related services. For detailed information about related services, please send an email to marketing@gentlegen.com.