Thermotargetron for modifying thermophilic microorganisms

The Thermotargetron is a genetic tool for modifying thermophilic microorganisms using a type-II intron element, TeI3c‑4c. It allows efficient gene inactivation across a broad host range without needing selection markers and can target short gene sequences.

Background

Renewable fuels like bioethanol are increasingly vital due to rising global energy demands, limited fossil fuel reserves, and climate change concerns. Thermophilic microorganisms, especially Clostridium thermocellum, are promising candidates for bioethanol production due to their ability to degrade plant biomass and ferment various sugars at high temperatures, reducing contamination risks and facilitating ethanol removal. However, their industrial application is hindered by their natural deficiencies such as the inability to utilize pentose sugars and low tolerance to ethanol and other hydrolysates.

Although genome sequencing has enabled metabolic engineering of C. thermocellum, existing gene disruption methods, primarily based on homologous recombination, are inefficient due to high transformation frequency requirements and low gene disruption efficiency. Therefore, there is a critical need for novel gene targeting methods to enhance the metabolic engineering of C. thermocellum and other industrially significant thermophiles.

Technology description

The Thermotargetron system is a genetic modification tool designed for thermophilic microorganisms, utilizing a thermophilic type-II intron element known as TeI3c-4c. This element is characterized by specific nucleotide sequences and is integrated into a targeting vector, facilitating precise gene targeting inactivation in thermophiles.

The system is highly efficient in gene disruption and allows for easy modification of the targeting site. It supports a broad host range, including both Gram-positive and Gram-negative bacteria, and operates without the need for nutritional deficiency selection markers. Additionally, the system is effective even with low transformation efficiencies and can inactivate short gene sequences ranging from 100 to 200 base pairs.

What differentiates the Thermotargetron system is its high efficiency and flexibility in genetic inactivation. Unlike traditional methods, it does not require nutritional deficiency markers, making the process simpler and more versatile. The ability to target both Gram-positive and Gram-negative bacteria expands its applicability across various thermophilic microorganisms. Moreover, the system's effectiveness with low transformation efficiencies and its capability to inactivate short gene sequences make it a powerful tool for genetic engineering. This broad host range and ease of modifying targeting sites significantly reduce the time and complexity associated with constructing targeting vectors, positioning the Thermotargetron as an advanced and efficient solution for genetic modifications in thermophiles.