As researchers engineer cyanobacteria for biotechnological applications, we must consider potential environmental release of these organisms. Previous theoretical work has considered cyanobacterial containment through elimination of the CO₂-concentrating mechanism (CCM) to impose a high-CO₂ requirement (HCR), which could be provided in the cultivation environment but not in the surroundings. In this work, we experimentally implemented an HCR containment mechanism in Synechococcussp. strain PCC7002 (PCC7002) through deletion of carboxysome shell proteins and showed that this mechanism contained cyanobacteria in a 5% CO₂ environment. We considered escape through horizontal gene transfer (HGT) and reduced the risk of HGT escape by deleting competence genes. We showed that the HCR containment mechanism did not negatively impact the performance of a strain of PCC7002 engineered for L-lactate production. We showed through coculture experiments of HCR strains with ccm-containing strains that this HCR mechanism reduced the frequency of escape below the NIH recommended limit for recombinant organisms of one escape event in 10⁸ CFU.

中文翻译：

---

高CO ₂需求作为遏制转基因蓝细菌的机制

当研究人员为生物技术应用设计蓝细菌时，我们必须考虑这些生物的潜在环境释放。先前的理论工作已经考虑了通过消除CO ₂浓缩机制（CCM）来施加高CO ₂需求（HCR）来遏制蓝细菌，这可以在栽培环境中提供，但不能在周围环境中提供。在这项工作中，我们实验性地在Synechococcus sp中实现了HCR遏制机制。菌株PCC7002（PCC7002）通过缺失羧基核壳蛋白而显示出这种机制在5％CO _2中包含蓝细菌环境。我们考虑通过水平基因转移（HGT）逃逸，并通过删除能力基因来降低HGT逃逸的风险。我们表明，HCR遏制机制不会对专为L-乳酸生产而设计的PCC7002菌株的性能产生负面影响。我们通过HCR菌株与含ccm菌株的共培养实验表明，这种HCR机制将逃逸频率降低到10 ⁸ CFU中一次逃逸事件的重组生物的NIH推荐极限以下。