College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen 518060, China.
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China; Plant Immunity Center, Haixia Institute of Science and Technology, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Microbiol Res. 2022 Jun;259:127011. doi: 10.1016/j.micres.2022.127011. Epub 2022 Mar 21.
Trichoderma reesei has extraordinary potential for high-level protein production at large scales, and it need to be further explored through genetic engineering tools to obtain a thorough understanding of its cellular physiology. Understanding the genetic factors involved in the intrinsic regulatory network is crucial; without this information, there would be restrictions in expressing genes of interest. Past and present studies are concentrated on the application and expansion of novel expression systems using synthetic biology concepts. These approaches involve either using previously established promoters that are strong or genetically engineered promoters. Genomic and transcriptomic methods have also been employed to isolate strong promoters and expression systems such as light-inducible expression systems, copper-inducible expression systems, L-methionine inducible promoters, and Tet-On expression system etc. AIMS OF REVIEW: In this review, we will highlight various research endeavors related to tunable and constitutive promoters; the role of different promoters in homologous and heterologous protein expression; the identification of innovative promoters, and strategies that may be beneficial for future research aimed at improving and enhancing protein expression in T. reesei.
The characterization of new promoters and implementation of novel expression systems that will result in a significant extension of the molecular toolbox that is accessible for the genetic engineering of innovative strains of T. reesei. Genetically engineered strong inducible promoters such as Pcbh1 through replacement of transcriptional repressors (cre1, ace1) with transcriptional activators (xyr1, ace2, ace3, hap2/3/5) and synthetic expression systems can result in elevated production of endoglucanases (EGLs), β-glucosidases (BGLs), and cellobiohydrolases (CBHs). Strong constitutive promoters such as Pcdna1 can be converted into genetically engineered synthetic hybrid promoters by integrating the activation region of strong inducible promoters, which can allow the induction and expression of cellulases even on repressing media. More efforts are necessary to identify innovative promoters and novel expression strategies for the enhanced expression of desirable proteins at industrial scales.
里氏木霉具有在大规模生产中高水平表达蛋白质的巨大潜力，需要通过遗传工程工具进一步探索，以深入了解其细胞生理学。理解参与内在调节网络的遗传因素至关重要；没有这些信息，表达感兴趣的基因就会受到限制。过去和现在的研究都集中在应用和扩展使用合成生物学概念的新型表达系统上。这些方法要么使用先前建立的强启动子，要么使用遗传工程化的启动子。基因组学和转录组学方法也被用于分离强启动子和表达系统，如光诱导表达系统、铜诱导表达系统、L-甲硫氨酸诱导启动子和 Tet-On 表达系统等。
在这篇综述中，我们将重点介绍各种与可调谐和组成型启动子相关的研究努力；不同启动子在同源和异源蛋白表达中的作用；新型启动子的鉴定，以及可能对未来旨在改善和增强里氏木霉中蛋白质表达的研究有益的策略。
新启动子的特征和新型表达系统的实施，将极大地扩展可用于里氏木霉遗传工程的创新菌株的分子工具包。通过用转录激活子（xyr1、ace2、ace3、hap2/3/5）替换转录阻遏物（cre1、ace1）来替换新型组成型强诱导启动子 Pcbh1，以及合成表达系统，可以导致内切葡聚糖酶（EGLs）、β-葡萄糖苷酶（BGLs）和纤维二糖水解酶（CBHs）的产量增加。通过整合强诱导启动子的激活区，将强组成型启动子 Pcdna1 转化为遗传工程合成杂交启动子，可以允许在抑制性培养基上诱导和表达纤维素酶。需要更多的努力来识别创新的启动子和新的表达策略，以在工业规模上增强理想蛋白的表达。
