Title : Robot-aided high-throughput engineering of FLS enzyme to establish in vivo synthetic C1 metabolism
Abstract:
Climate change poses the greatest environmental threat humanity has ever faced, primarily driven by the combustion of fossil fuels, which accounts for over 85 % of global CO2 emissions and contributes to the anthropogenic greenhouse effect. To mitigate these processes, it is essential to not only reduce the use of fossil fuels and lower emissions but also actively capture and convert CO2 in the atmosphere. In the context of a sustainable circular economy, the capture and conversion of atmospheric and industrial CO2 into chemical products via biotechnological processes represents a promising route. One way to achieve this is the construction and engineering of synthetic methylotrophic microbes, which use CO2-based methanol as their sole carbon source. However, a major challenge is the toxicity of formaldehyde, an intermediate product of the metabolic conversion of methanol, even in small quantities. To address this challenge, a synthetic enzyme called formolase is used to convert formaldehyde directly into dihydroxyacetone. This pathway requires the implementation of only two enzymes in synthetic methylotrophic host organisms for efficient methanol assimilation. So far, the parental formolase enzyme exhibits low activity, restricting its practical applications. To address this limitation, the enzyme was subjected to iterative rounds of directed evolution. The key to the success of the directed evolution process was a high-throughput screening approach utilizing an automated procedure. By subjecting the enzyme to this screening, a novel FLS variant with higher expression levels and improved tolerance to formaldehyde in
E. coli was identified. Moreover, this variant displayed a 30 % decrease in KM for the formose reaction and exhibited increased thermostability by 5 °C. The implementation of the FLS pathway offers several advantages. Firstly, it enables the direct conversion of formaldehyde into dihydroxyacetone, eliminating the need for multiple intermediate steps. This simplification of the pathway not only reduces energy and resource requirements but also enhances overall efficiency. Secondly, the streamlined nature of the pathway, requiring only two enzymes, facilitates its integration into synthetic methylotrophic host organisms. The semi-rational approach employed in this study combined rational design and directed evolution, leveraging existing knowledge of the enzyme's structure and function to guide the design of mutations while allowing for random mutations to explore new possibilities. This balanced approach proved successful in generating a highly improved FLS variant.
Audience Take Away
- The audience will understand the benefits of using synthetic enzymes and directed evolution techniques in enhancing enzyme performance for tailored applications.
- This research provides a practical solution for improving the efficiency of methanol assimilation, which can benefit researchers working in the field of sustainable and resource- efficient biotechnological processes.
- The presented work provides valuable insights and knowledge with respect to application of automated high-throughput processes, their optimization and their limitations.
- Specifically, the identification of an improved FLS variant opens up possibilities for the production of CO2-based products and fuels, contributing to the circular economy and reducing carbon emissions.
