Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterised neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria, and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31-81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology.

中文翻译：

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用于改造高产蓝藻聚球藻的工具箱。 PCC 11901

聚球藻属。 PCC 11901 (PCC 11901) 是一种快速生长的海洋蓝藻菌株，具有持续生物量积累到非常高的细胞密度的能力，与商业相关的异养生物所达到的密度相当。然而，用于生物技术应用的 PCC 11901 基因设计工具是有限的。在这里，我们描述了一套基于 CyanoGate MoClo 系统的工具，以释放 PCC 11901 的工程潜力。首先，我们描述了适合稳定基因组整合的中性位点，即使在高细胞密度下也不会影响生长。其次，我们测试了一套组成型启动子、终止子和诱导型启动子，包括 2,4-二乙酰基间苯三酚 (DAPG) 诱导型 PhlF 阻遏系统，该系统之前尚未在蓝细菌中得到证实，并显示出严格的调控和 228 倍的动态感应范围。最后，我们开发了基于 DAPG 诱导的 dCas9 的 CRISPR 干扰 (CRISPRi) 系统和使用 CRISPR-Cas12a 生成无标记突变体的模块化方法。根据我们的研究结果，PCC 11901 对基于 CRISPRi 的抑制高度敏感，并在使用 Cas12a 进行单插入 (31-81%) 和多重双插入 (25%) 基因组编辑时表现出高效率。我们预计这些工具将为采用 PCC 11901 作为工程生物学和绿色生物技术的强大模型菌株奠定基础。