Cupriavidus necator H16 is a “Knallgas” bacterium with the ability to utilize various carbon sources and has been employed as a versatile microbial cell factory to produce a wide range of value-added compounds. However, limited genome engineering, especially gene regulation methods, has constrained its full potential as a microbial production platform. The advent of CRISPR/Cas9 technology has shown promise in addressing this limitation. Here, we developed an optimized CRISPR interference (CRISPRi) system for gene repression in C. necator by expressing a codon-optimized deactivated Cas9 (dCas9) and appropriate single guide RNAs (sgRNAs). CRISPRi was proven to be a programmable and controllable tool and could successfully repress both exogenous and endogenous genes. As a case study, we decreased the accumulation of polyhydroxyalkanoate (PHB) via CRISPRi and rewired the carbon fluxes to the synthesis of lycopene. Additionally, by disturbing the expression of DNA mismatch repair gene mutS with CRISPRi, we established CRISPRi-Mutator for genome evolution, rapidly generating mutant strains with enhanced hydrogen peroxide tolerance and robustness in microbial electrosynthesis (MES) system. Our work provides an efficient CRISPRi toolkit for advanced genetic manipulation and optimization of C. necator cell factories for diverse biotechnology applications.

中文翻译：

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建立 CRISPRi 以实现 Cupriavidus necator 的可编程基因抑制和基因组进化


Cupriavidus necator H16 是一种“Knallgas”细菌，能够利用各种碳源，并已被用作多功能微生物细胞工厂来生产各种增值化合物。然而，有限的基因组工程，特别是基因调控方法，限制了其作为微生物生产平台的全部潜力。 CRISPR/Cas9 技术的出现有望解决这一限制。在这里，我们开发了一种优化的 CRISPR 干扰 (CRISPRi) 系统，通过表达密码子优化的失活 Cas9 (dCas9) 和适当的单引导 RNA (sgRNA)，用于C. necator中的基因抑制。 CRISPRi被证明是一种可编程和可控的工具，可以成功地抑制外源和内源基因。作为一个案例研究，我们通过 CRISPRi 减少了聚羟基脂肪酸酯 (PHB) 的积累，并将碳通量重新连接到番茄红素的合成。此外，通过用CRISPRi干扰DNA错配修复基因mutS的表达，我们建立了用于基因组进化的CRISPRi-Mutator，快速生成具有增强的过氧化氢耐受性和微生物电合成（MES）系统稳健性的突变菌株。我们的工作为各种生物技术应用的C. necator细胞工厂的高级遗传操作和优化提供了高效的 CRISPRi 工具包。