Bioscience Evidence 2024, Vol.14, No.3, 131-142 http://bioscipublisher.com/index.php/be 131 Review and Progress Open Access Application of Synthetic Biology in Directed Evolution to Enhance Enzyme Catalytic Efficiency Wenzhong Huang Biomass Research Center, Hainan Institute of Tropical Agricultural Resouces, Sanya, 572025, Hainan, China Corresponding author email: wenzhong.huang@hitar.org Bioscience Evidence, 2024, Vol.14, No.3 doi: 10.5376/be.2024.14.0015 Received: 03 May, 2024 Accepted: 06 Jun., 2024 Published: 21 Jun., 2024 Copyright © 2024 Huang, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Huang W.Z., 2024, Application of synthetic biology in directed evolution to enhance enzyme catalytic efficiency, Bioscience Evidence, 14(3): 131-142 (doi: 10.5376/be.2024.14.0015) Abstract Synthetic biology and directed evolution are at the forefront of modern biotechnology, offering unprecedented opportunities to enhance enzyme catalytic efficiency for industrial applications. This study provides a comprehensive overview of these fields, starting with an introduction to the principles of synthetic biology and the fundamentals of directed evolution, emphasizing their significance in improving enzyme performance. We explore various methods in directed evolution, including random and site-directed mutagenesis techniques and high-throughput screening methods, which are crucial for identifying variants with superior catalytic properties. The study also delves into the synthetic biology tools that have revolutionized directed evolution, such as CRISPR/Cas systems, recombinant DNA technology, and computational tools for enzyme design. Through detailed case studies, we highlight the successful application of these approaches in enhancing enzymes for biofuel production, pharmaceutical synthesis, food industry applications, and environmental bioremediation. The discussion extends to recent advances in enzyme engineering, showcasing significant achievements in catalytic efficiency improvements and the integration of synthetic biology with directed evolution. We also address the challenges and limitations in the field, including technical hurdles, scalability issues, and ethical considerations. Finally, we outline future perspectives, focusing on emerging technologies like genome editing and artificial intelligence, which hold the potential to further advance enzyme engineering. This study concludes with a reflection on the long-term goals and implications for the future of synthetic biology and directed evolution in industrial biotechnology. Keywords Synthetic biology; Directed evolution; Enzyme catalytic efficiency; Protein engineering; Industrial biotechnology 1 Introduction Synthetic biology is an interdisciplinary field that combines principles from biology, engineering, and computer science to design and construct new biological parts, devices, and systems, or to redesign existing biological systems for useful purposes. One of the most powerful tools in synthetic biology is directed evolution, a method that mimics the process of natural selection to evolve proteins or nucleic acids towards a user-defined goal. Directed evolution involves iterative cycles of mutagenesis and selection to generate and identify variants with enhanced or novel properties (Cobb et al., 2013; Zeymer and Hilvert, 2018). This approach has been instrumental in overcoming the limitations of rational design, which often requires detailed structural and mechanistic knowledge that may not be available (Markel et al., 2019). Enzymes are nature's catalysts, facilitating biochemical reactions with remarkable specificity and efficiency. Their catalytic prowess has been harnessed in various industrial applications, including the production of pharmaceuticals, biofuels, food and beverages, and environmental protection (Chen and Arnold, 2020; Planas-Iglesia et al., 2021). However, natural enzymes are not always optimized for industrial conditions, which can differ significantly from their native environments. Enhancing enzyme catalytic efficiency through directed evolution can lead to significant improvements in process efficiency, cost-effectiveness, and sustainability (Otten et al., 2020). For instance, enzymes with improved stability and activity under harsh industrial conditions can reduce the need for extreme temperatures or pH levels, thereby saving energy and reducing environmental impact (Currin et al., 2021). This study aims to explore the application of synthetic biology, particularly directed evolution, in enhancing enzyme catalytic efficiency. We will discuss the latest advances in directed evolution techniques, including
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