Published in ACS Catalysis
Loop-mediated isothermal amplification (LAMP) is a powerful DNA amplification technique celebrated for its simplicity, efficiency, and precision. With its exceptional specificity, sensitivity, speed, and ease of use, LAMP has become a cornerstone in molecular point-of-care testing (POCT), driving innovations in disease diagnostics, genetically modified food detection, and beyond. At the heart of this technology lies Bst DNA polymerase, the core enzyme whose performance—spanning polymerase activity, strand displacement capability, and thermostability—directly determines the speed and accuracy of LAMP reactions.
Leveraging cutting-edge fluorescence-activated droplet sorting (FADS) technology, Yeasen has successfully evolved a thermostable Bst DNA polymerase facilitating high-temperature LAMP assays at 70 °C, thereby eliminating the common issue of false positives in LAMP assays. It has been published in ACS Catalysis, a leading journal in biocatalysis (Impact factor 11.3)
For the full ducument, visit: https://doi.org/10.1021/acscatal.4c07614.
Pioneering Technology Meets Expertise
The enzyme is developed through ultra-high-throughput FADS platform. This sophisticated system integrates optical, electrical, signal acquisition, and microfluidic components, featuring custom-designed chips for droplet generation, microinjection, and droplet detection and sorting. Built on droplet microfluidic technology, the FADS platform enables single-cell-level screening of vast mutant libraries, processing over 10 million mutants daily with unparalleled efficiency.
Figure 1. FADS Ultra-High-Throughput Enzyme Screening Platform
a. The system comprises key components including a light source, signal generator, voltage amplifier, digital power supply, photomultiplier tube (PMT), data acquisition card, and high-speed camera.
b. Droplet generation chip.
c. Electric field-triggered picoliter injection chip.
d. Fluorescence-activated droplet sorting chip.
Meanwhile, Yeasen Biotech brought its deep expertise in enzyme application and modification to the table. The team designed an innovative ultra-high-throughput screening strategy for Bst DNA polymerase, utilizing strand displacement activity probes tailored for the FADS platform. Through a combination of semi-rational design, random mutation, and DNA recombination, they constructed mutant libraries encapsulated in microdroplets at the single-cell level. After three rigorous rounds of screening, two standout mutants—M2 and M3—emerged as top performers.
Figure 2. Schematic of Bst DNA Polymerase Molecular Evolution via FADS
a. A multi-site saturation mutagenesis library was established through semi-rational design (different colors represent distinct amino acid mutations).
b. Based on positive mutants from the first round of screening, a random mutagenesis library was created to enhance thermostability (red dots indicate superior mutation sites from the previous round, while other colored dots represent additional amino acid mutations).
c. DNA recombination was performed on mutants from the second round of screening (red dots denote high-performing mutation sites identified in prior rounds).
d. After three rounds of screening, mutants with improved thermostability and strand displacement activity were successfully isolated.
e. The library was transformed into Escherichia coli BL21 (DE3) cells. Following IPTG induction, each cell expressed a unique Bst DNA polymerase variant (different colors represent different mutants).
f. When individual cells were encapsulated in the reaction system, fluorescence was absent in the droplets due to a quenching group on the primer.
g. Lysis reagents disrupted the cell structure, releasing Bst DNA polymerase for strand displacement reactions. Subsequently, the primer was displaced, triggering fluorescence emission in the droplet.
h. Positive droplets were enriched in the FADS sorting system.
Redefining LAMP Performance
Building on these mutants, we developed a specialized LAMP reaction system that showcased remarkable improvements. Experimental results revealed that the engineered mutants slashed the time to characteristic peak detection from 35 minutes to under 10 minutes at a stable 55°C, far surpassing the reaction rates of the widely used imported Bst 2.0 system across standard temperature ranges. Even more impressively, these mutants demonstrated resilience at extreme temperatures up to 70°C, enabling the team to pioneer High-Temperature LAMP (HT-LAMP) detection technology. This innovation tackles a long-standing challenge in traditional LAMP—false positives—offering a game-changing solution for diagnostic accuracy.
Figure 3. Establishment and Screening Process of the FADS System
a. Bright-field and fluorescence images of cells cultured at different time points in microreactors.
b. Bright-field and fluorescence images of microreactors expressing wild-type (WT) and mutant libraries (incubated for 30 minutes).
c. Enrichment of positive strains from the first round of evolution.
d. Enrichment of positive strains from the second round of evolution.
e. Enrichment of positive strains from the third round of evolution.
The breakthroughs didn’t stop there. The team also developed lyophilized LAMP reagents based on these enhanced Bst DNA polymerase mutants. Compared to the wild-type enzyme, the mutants boosted reagent storage stability by two orders of magnitude, retaining activity for over six months even under accelerated conditions at 50°C. These advancements position the evolved Bst DNA polymerase as a commercially viable core enzyme with vast potential in LAMP-based applications.
Figure 4. Performance of Bst DNA Polymerase Mutants in LAMP Detection
a. Performance of Bst DNA polymerase mutants in LAMP detection across different temperatures.
b. Compared to NEB’s Bst DNA polymerase (green), mutants M2 (orange) and M3 (red) exhibited significantly higher activity against various targets at different temperatures.
c. Performance of mutant M2 in false-positive experiments.
d. Performance of mutant M3 in false-positive experiments.
e. Performance of Bst DNA polymerase mutants in thermal acceleration tests at 50°C.
Unlocking New Insights
Beyond practical applications, the researchers delved into the molecular mechanisms behind the mutants’ superior performance. Their analysis revealed a novel mechanism enhancing strand displacement activity, leading to the proposal of the “hydrophobic blade” hypothesis. This discovery provides a theoretical foundation for further optimizing Bst DNA polymerase, paving the way for future innovations.
Figure 5. Structural Analysis of Bst DNA Polymerase
a. Illustration of the substructure of Bst DNA polymerase, depicting its structural positioning with the template and primer (PDB: 3TAN).
b. Structural changes before and after the N204R mutation.
c. Structural changes before and after the I359L mutation.
d. Structural changes before and after the F445I mutation.
e. Structural changes before and after the E461K and N462K mutations.
f. Structural changes before and after the D428K and Y429W mutations.
A Leap Forward in Enzyme Evolution
This study marks the first successful application of ultra-high-throughput droplet screening to Bst DNA polymerase evolution. The resulting high-activity mutants have dramatically accelerated LAMP reaction times, enhanced enzyme thermostability, and ensured long-term reagent stability, transforming a wild-type enzyme into a commercially valuable asset. These achievements underscore the transformative potential of FADS technology in molecular enzyme evolution.
From Research to Reality
Building on this research, Yeasen Biotech has brought the thermostable DNA polymerase to market as Hieff Super Bst DNA Polymerase (2,000 U/μL, Cat#14410). This product is now available for industrial and research applications. Interested in exploring its potential? Contact us today for product trials!