Making industry-ready enzymes that operate at high temperatures

Scientists at Los Alamos National Laboratory engineered a “fluorogenic reporter” protein that works at high temperatures such as 70 degrees Celsius (158 degrees Fahrenheit) and signals its function by emitting a green glow. Researchers can evaluate the stability or function of enzymes by tagging them with this green-glowing reporter.

The reporter, Thermostable GlowTag, makes it easy to screen large libraries of proteins quickly for specific, industry-relevant characteristics such as a long shelf life or retained activity at high temperatures. The team took advantage of thermophilic microbes — microorganisms that naturally thrive in hot environments — as hosts to produce these proteins.

Proteins have a wide range of biotechnological applications. They can be used as therapeutic agents, antibodies, reagents for research and diagnostics, and as functional catalysts for biomanufacturing.

• The challenge is that many proteins need cold storage and can also rapidly lose function at the high temperatures needed for certain industrial processes.
• Some proteins naturally function at high temperatures, but finding and studying them requires working with a host organism that can also survive at high temperatures conditions.

The advantages of Thermostable GlowTag (YFAST) include: 

• Enables rapid, high-throughput screening at high temperatures.
• Introduces a thermostable fluorogenic reporter that provides an optical response that correlates to the quantity of the protein it is fused to.
• Improves screening accuracy by using direct selection rather than elimination-based methods, reducing false positives.
• Expands access to thermostable enzymes by allowing common mesophilic enzymes to be engineered for heat resistance.
• Allows for screening and optimization of proteins from thermophilic sources.
  Reduces storage and operational costs by producing enzymes that remain stable without refrigeration.

The Los Alamos team computationally designed libraries of variants of a low temperature fluorogenic reporter, YFAST, with thermostability in mind and directly screened the variants in a thermophilic microbial host that thrives in extreme heat. 

• Through this process, the team developed a fluorogenic protein called hsFAST or Thermostable GlowTag. 
• hsFAST can now be used as a “fluorescent tag” to screen thermostable variants of other industrial proteins for biological applications.
• hsFAST is also useful a tag to report other conditions inside a microbial host cell.

Contrary to some other thermostable fluorescent reporters such as TGP (thermal green protein, also developed at the Lab), the new reporter hsFAST can function in an oxygen-free environment, a characteristic highly relevant for applications in thermophiles, which mostly exist in anaerobic conditions.

• Biofuels
• Waste Management
• Chemical and Pharmaceutical Manufacturing
• Food and Beverage Processing
• Textiles and Detergents

By allowing mesophilic enzymes to be re-engineered for high-heat environments, reducing reliance on cold storage, and streamlining the screening process with direct fluorescence-based selection, this technology overcomes key bottlenecks in enzyme development and opens the door to more robust, cost-effective and versatile enzymes for industrial and environmental applications.