CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
ACS Synth Biol. 2024 Jun 21;13(6):1893-1905. doi: 10.1021/acssynbio.4c00175. Epub 2024 Jun 2.
Gas-fermenting species hold tremendous promise for one-carbon biomanufacturing. To unlock their full potential, it is crucial to unravel and optimize the intricate regulatory networks that govern these organisms; however, this aspect is currently underexplored. In this study, we employed pooled CRISPR interference (CRISPRi) screening to uncover a wide range of functional transcription factors (TFs) in , a representative species of gas-fermenting , with a special focus on TFs associated with the utilization of carbon resources. Among the 425 TF candidates, we identified 75 and 68 TF genes affecting the heterotrophic and autotrophic growth of , respectively. We focused our attention on two of the screened TFs, NrdR and DeoR, and revealed their pivotal roles in the regulation of deoxyribonucleoside triphosphates (dNTPs) supply, carbon fixation, and product synthesis in , thereby influencing the strain performance in gas fermentation. Based on this, we proceeded to optimize the expression of in by adjusting its promoter strength, leading to an improved growth rate and ethanol synthesis of when utilizing syngas. This study highlights the effectiveness of pooled CRISPRi screening in gas-fermenting species, expanding the horizons for functional genomic research in these industrially important bacteria.
产甲烷菌在一碳生物制造方面具有巨大的潜力。为了充分发挥它们的潜力，揭示和优化调控这些生物体的复杂调控网络至关重要；然而，这一方面目前还未得到充分探索。在这项研究中，我们采用 pooled CRISPR 干扰（CRISPRi）筛选技术，揭示了产甲烷菌中广泛的功能转录因子（TFs），特别关注与碳资源利用相关的 TFs。在 425 个 TF 候选物中，我们分别鉴定出了 75 个和 68 个影响 异养和自养生长的 TF 基因。我们重点关注了筛选出的两个 TF，NrdR 和 DeoR，并揭示了它们在调控脱氧核糖核苷三磷酸（dNTPs）供应、碳固定和产物合成中的关键作用，从而影响菌株在气体发酵中的性能。基于这一点，我们通过调整其启动子强度来优化 在 中的表达，当利用合成气时，这导致了 的生长速率和乙醇合成的提高。本研究强调了 pooled CRISPRi 筛选在产甲烷菌中的有效性，为这些在工业上重要的细菌的功能基因组研究拓展了新的视野。
