关键词: 合成生物学, 启动子工程, 调控机制, 改造策略, 诱导信号, 动态代谢调控, 人工智能从头设计

Abstract:

Synthetic biology is of vital importance to the green biomanufacturing industry and sustainable development strategies of our country. Promoter is the core-component of synthetic biology, playing a significant role in highly efficient and fine-tuning expression and regulation of target genes at the transcriptional level. Herein we summarized and discussed the key progress and future frontiers of microbial promoter engineering, particularly for prokaryotic microorganisms. Firstly, we introduced the basic DNA sequence characteristics of promoters and the regular mechanism for promoter recognition and transcription-initiation by RNA polymerase sigma factors. Inducible mechanisms for both negative and positive regulation were particularly highlighted with the typical lac operator of Escherichia coli as an example. Then, effective strategies for obtaining improved-promoters were summarized, which were roughly divided into two categories: endogenous promoter mutation and heterologous promoter replacement. For the endogenous promoter mutation, the following strategies, e.g. point mutation toward sigma factor consensus sequence, coupling optimization of -35 and -10 regions with RBS sequence, random mutation or saturation mutagenesis of UP element or spacer sequences accompanying with promoter library construction and high-throughput screening were emphasized. For the heterologous promoter replacement, strategies such as substituting the native promoter into stronger ones from other microorganisms, introducing phage-source chimeric promoters, tuning the constitutive promoter into inducible pattern and integrating positive regulator(s), were mainly discussed. We further sorted out the representative inducers for inducible promoters reported so far, including both chemical molecules and physical signals. Progress in constitutive promoters of non-model and model microbial organisms were simply summarized as well. Next, arising from the breakthrough development of dynamic metabolic regulation and artificial intelligence (AI), we proposed that the innovative research on identification and evolution of new and unique promoters with dynamic-response features and AI de novo design for promoters with novel/superior functions will be the new frontiers of promoter engineering. Finally, we analyzed the challenging scientific issues in the microbial promoter engineering, from the viewpoint of both basic research and large-scale applications; and further discussed the research priority coupling with the vigorous development of synthetic biology.

Key words: synthetic biology, promoter engineering, regulation mechanism, evolution strategies, induction signals, dynamic metabolic regulation, AI de novo design