School of Pharmaceutical Science and Technology, Tianjin University, Nankai District, 92 Weijin Road, Tianjin, 300072, China.
Biorefining and Advanced Materials Research Center, Scotland's Rural College (SRUC), Kings Buildings, West Mains Road, Edinburgh, EH9 3JG, UK.
Microb Cell Fact. 2021 Jan 21;20(1):20. doi: 10.1186/s12934-021-01510-9.
Many fungi grow as saprobic organisms and obtain nutrients from a wide range of dead organic materials. Among saprobes, fungal species that grow on wood or in polluted environments have evolved prolific mechanisms for the production of degrading compounds, such as ligninolytic enzymes. These enzymes include arrays of intense redox-potential oxidoreductase, such as laccase, catalase, and peroxidases. The ability to produce ligninolytic enzymes makes a variety of fungal species suitable for application in many industries, including the production of biofuels and antibiotics, bioremediation, and biomedical application as biosensors. However, fungal ligninolytic enzymes are produced naturally in small quantities that may not meet the industrial or market demands. Over the last decade, combined synthetic biology and computational designs have yielded significant results in enhancing the synthesis of natural compounds in fungi. In this review, we gave insights into different protein engineering methods, including rational, semi-rational, and directed evolution approaches that have been employed to enhance the production of some important ligninolytic enzymes in fungi. We described the role of metabolic pathway engineering to optimize the synthesis of chemical compounds of interest in various fields. We highlighted synthetic biology novel techniques for biosynthetic gene cluster (BGC) activation in fungo and heterologous reconstruction of BGC in microbial cells. We also discussed in detail some recombinant ligninolytic enzymes that have been successfully enhanced and expressed in different heterologous hosts. Finally, we described recent advance in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) protein systems as the most promising biotechnology for large-scale production of ligninolytic enzymes.
Aggregation, expression, and regulation of ligninolytic enzymes in fungi require very complex procedures with many interfering factors. Synthetic and computational biology strategies, as explained in this review, are powerful tools that can be combined to solve these puzzles. These integrated strategies can lead to the production of enzymes with special abilities, such as wide substrate specifications, thermo-stability, tolerance to long time storage, and stability in different substrate conditions, such as pH and nutrients.
许多真菌作为腐生生物生长，从各种死有机物质中获取营养。在腐生生物中，生长在木材上或受污染环境中的真菌物种已经进化出丰富的产生降解化合物的机制，例如木质素降解酶。这些酶包括一系列强烈的氧化还原电势氧化还原酶，如漆酶、过氧化氢酶和过氧化物酶。产生木质素降解酶的能力使各种真菌物种适合应用于许多行业，包括生物燃料和抗生素的生产、生物修复以及作为生物传感器的生物医学应用。然而，真菌木质素降解酶的产量自然较低，可能无法满足工业或市场需求。在过去的十年中，组合合成生物学和计算设计在提高真菌中天然化合物的合成方面取得了显著成果。在这篇综述中，我们深入探讨了不同的蛋白质工程方法，包括理性、半理性和定向进化方法，这些方法已被用于提高真菌中一些重要木质素降解酶的产量。我们描述了代谢途径工程在优化各个领域感兴趣的化合物合成中的作用。我们强调了合成生物学新技术在真菌生物合成基因簇（BGC）激活和 BGC 在微生物细胞中的异源重建中的作用。我们还详细讨论了一些已成功增强并在不同异源宿主中表达的重组木质素降解酶。最后，我们描述了 CRISPR（Clustered Regularly Interspaced Short Palindromic Repeats）-Cas（CRISPR 相关）蛋白系统作为大规模生产木质素降解酶的最有前途的生物技术的最新进展。
真菌中木质素降解酶的聚集、表达和调控需要非常复杂的程序，其中有许多干扰因素。正如本综述中所解释的，合成和计算生物学策略是解决这些难题的有力工具。这些综合策略可以导致产生具有特殊能力的酶，例如广泛的底物特异性、热稳定性、对长时间储存的耐受性以及在不同底物条件下（如 pH 和营养物质）的稳定性。
