Photoautotrophic production of fuels and chemicals by cyanobacteria typically gives lower volumetric productivities and titers than heterotrophic production. Cyanobacteria cultures become light limited above an optimal cell density, so that this substrate is not supplied to all cells sufficiently. Here, we investigate genetic strategies for a two-phase cultivation, where biofuel-producing Synechocystis cultures are limited to an optimal cell density through inducible CRISPR interference (CRISPRi) repression of cell growth. Fixed CO₂ is diverted to ethanol or n-butanol. Among the most successful strategies was partial repression of citrate synthase gltA. Strong repression (>90%) of gltA at low culture densities increased carbon partitioning to n-butanol 5-fold relative to a nonrepression strain, but sacrificed volumetric productivity due to severe growth restriction. CO₂ fixation continued for at least 3 days after growth was arrested. By targeting sgRNAs to different regions of the gltA gene, we could modulate GltA expression and carbon partitioning between growth and product to increase both specific and volumetric productivity. These growth arrest strategies can be useful for improving performance of other photoautotrophic processes.

中文翻译：

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有针对性的抑制必需基因，以抑制蓝藻中的生长并增加碳分配和生物燃料滴度。

与异养生产相比，通过蓝细菌进行光养养生产的燃料和化学品通常具有较低的容积生产率和效价。蓝细菌培养物的光被限制在最佳细胞密度以上，因此该底物未充分提供给所有细胞。在这里，我们研究了两阶段培养的遗传策略，其中通过产生可诱导的CRISPR干扰（CRISPRi）抑制细胞生长，将产生生物燃料的集胞藻培养物限制在最佳细胞密度。固定的CO ₂被转移到乙醇或正丁醇中。最成功的策略之一是部分抑制柠檬酸合酶gltA。强烈抑制（> 90％）gltA在低培养密度下，相对于非抑制菌株，碳分配到正丁醇的碳分配增加了5倍，但由于严格的生长限制而牺牲了体积生产率。生长停止后，CO ₂固定持续至少3天。通过将sgRNA靶向gltA基因的不同区域，我们可以调节GltA表达和生长与产物之间的碳分配，从而提高比生产率和容积生产率。这些生长停滞策略可用于改善其他光合自养过程的性能。