Xie Hao, Bourgade Barbara, Stensjö Karin, Lindblad Peter
Microbial Chemistry, Department of Chemistry-Ångström Laboratory, Uppsala University, Uppsala, Sweden.
College of Bioengineering, Sichuan University of Science and Engineering, Yibin, Sichuan, China.
Microb Cell Fact. 2025 May 13;24(1):104. doi: 10.1186/s12934-025-02727-8.
Cyanobacteria of the genera Synechocystis and Synechococcus have emerged as promising platforms for metabolic engineering endeavors aimed at converting carbon dioxide into valuable fuels and chemicals, thus addressing the pressing energy demand and mitigating global climate change. Notably, Synechocystis sp. strain PCC 6803 (Synechocystis) has been engineered to produce isobutanol (IB) and 3-methyl-1-butanol (3M1B) via heterologous expression of α-ketoisovalerate decarboxylase (Kivd). Despite these advances, the achieved IB/3M1B titers remain low. CRISPR interference (CRISPRi), an emerging tool for targeted gene repression, has demonstrated success in various cellular systems to enhance biochemical productivity.
In this study, we developed a dCas12a-mediated CRISPRi system (CRISPRi-dCas12a) that effectively blocked the transcriptional initiation/elongation of essential gene(s), resulting in up to 60% gene repression in Synechocystis. Subsequently, the CRISPRi-dCas12a system was successfully integrated into an IB/3M1B producer strain, where it exhibited target gene repression under optimal cultivation conditions. To identify gene targets involved in metabolic pathways potentially limiting IB/3M1B biosynthesis, we initially designed a CRISPR RNA (crRNA) library targeting fifteen individual gene(s), where repression of ten genes significantly increased IB/3M1B production per cell. Moreover, a synergetic effect was observed on IB/3M1B production by designing a single crRNA targeting multiple genes for simultaneous repression. A final strain HX106, featuring dual repression of ppc and gltA, both involved in the TCA cycle, resulted in 2.6-fold and 14.8-fold improvement in IB and 3M1B production per cell, respectively.
Our findings underscore the effectiveness of the CRISPRi-dCas12a system in Synechocystis for identifying competing pathways and redirecting carbon flux to enhance IB/3M1B production. Furthermore, this study established a solid groundwork for utilizing an expanded CRISPRi-crRNA library to undertake genome-wide exploration of potential competing pathways not only for IB/3M1B biosynthesis but also for other diverse biofuels and biochemical production processes.
集胞藻属（Synechocystis）和聚球藻属（Synechococcus）的蓝细菌已成为代谢工程领域颇具前景的平台，旨在将二氧化碳转化为有价值的燃料和化学品，从而满足紧迫的能源需求并缓解全球气候变化。值得注意的是，聚球藻PCC 6803株（集胞藻）已通过异源表达α-酮异戊酸脱羧酶（Kivd）进行工程改造以生产异丁醇（IB）和3-甲基-1-丁醇（3M1B）。尽管取得了这些进展，但所实现的IB/3M1B滴度仍然很低。CRISPR干扰（CRISPRi）是一种新兴的靶向基因抑制工具，已在各种细胞系统中成功提高了生化产物的产量。
在本研究中，我们开发了一种dCas12a介导的CRISPRi系统（CRISPRi-dCas12a），该系统有效地阻断了必需基因的转录起始/延伸，在集胞藻中导致高达60%的基因抑制。随后，CRISPRi-dCas12a系统成功整合到IB/3M1B生产菌株中，在最佳培养条件下表现出对靶基因的抑制作用。为了确定参与可能限制IB/3M1B生物合成的代谢途径的基因靶点，我们最初设计了一个靶向15个单个基因的CRISPR RNA（crRNA）文库，其中对10个基因的抑制显著提高了每个细胞的IB/3M1B产量。此外，通过设计单个crRNA靶向多个基因进行同时抑制，在IB/3M1B生产中观察到了协同效应。最终菌株HX106对参与三羧酸循环的ppc和gltA进行双重抑制，导致每个细胞的IB和3M1B产量分别提高了2.6倍和14.8倍。
我们的研究结果强调了CRISPRi-dCas12a系统在集胞藻中用于识别竞争途径和重新引导碳通量以提高IB/3M1B产量的有效性。此外，本研究为利用扩展的CRISPRi-crRNA文库进行全基因组探索潜在竞争途径奠定了坚实基础，这些途径不仅涉及IB/3M1B生物合成，还涉及其他各种生物燃料和生化生产过程。
