Decoupling growth from product synthesis is a promising strategy to increase carbon partitioning and maximize productivity in cell factories. However, reduction in both substrate uptake rate and metabolic activity in the production phase are an underlying problem for upscaling. Here, we used CRISPR interference to repress growth in lactate-producing Synechocystis sp. PCC 6803. Carbon partitioning to lactate in the production phase exceeded 90%, but CO₂ uptake was severely reduced compared to uptake during the growth phase. We characterized strains during the onset of growth arrest using transcriptomics and proteomics. Multiple genes involved in ATP homeostasis were regulated once growth was inhibited, which suggests an alteration of energy charge that may lead to reduced substrate uptake. In order to overcome the reduced metabolic activity and take advantage of increased carbon partitioning, we tested a novel production strategy that involved alternating growth arrest and recovery by periodic addition of an inducer molecule to activate CRISPRi. Using this strategy, we maintained lactate biosynthesis in Synechocystis for 30 days in a constant light turbidostat cultivation. Cumulative lactate titers were also increased by 100% compared to a constant growth-arrest regime, and reached 1 g/L. Further, the cultivation produced lactate for 30 days, compared to 20 days for the non-growth arrest cultivation. Periodic growth arrest could be applicable for other products, and in cyanobacteria, could be linked to internal circadian rhythms that persist in constant light.

将增长与产品合成脱钩是一种很有前景的策略，可以增加细胞工厂的碳分配并最大限度地提高生产力。然而，生产阶段底物吸收率和代谢活性的降低是放大的潜在问题。在这里，我们使用 CRISPR 干扰来抑制产乳酸的集胞藻的生长。PCC 6803. 生产阶段碳分配为乳酸超过 90%，但 CO ₂与生长阶段的吸收相比，吸收严重减少。我们使用转录组学和蛋白质组学对生长停滞开始期间的菌株进行了表征。一旦生长受到抑制，参与 ATP 稳态的多个基因就会受到调节，这表明能量电荷的改变可能导致底物吸收减少。为了克服降低的代谢活性并利用增加的碳分配，我们测试了一种新的生产策略，该策略涉及通过定期添加诱导剂分子来激活 CRISPRi 来交替生长停滞和恢复。使用这种策略，我们维持了集胞藻中乳酸的生物合成在恒光恒浊培养中培养 30 天。与恒定生长停滞方案相比，累积乳酸滴度也增加了 100%，达到 1 g/L。此外，与非生长停滞培养的 20 天相比，培养产生了 30 天的乳酸。周期性生长停滞可能适用于其他产品，在蓝藻中，可能与在恒定光照下持续存在的内部昼夜节律有关。