Science for Life Laboratory, KTH - Royal Institute of Technology, SE-171 21, Stockholm, Sweden.
Department of Protein Science, KTH - Royal Institute of Technology, SE-106 91, Stockholm, Sweden.
Nat Commun. 2020 Apr 3;11(1):1666. doi: 10.1038/s41467-020-15491-7.
Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803, where we aim to target all genes for repression. We track the growth of all library members in multiple conditions and estimate gene fitness. The library reveals several clones with increased growth rates, and these have a common upregulation of genes related to cyclic electron flow. We challenge the library with 0.1 M L-lactate and find that repression of peroxiredoxin bcp2 increases growth rate by 49%. Transforming the library into an L-lactate-secreting Synechocystis strain and sorting top lactate producers enriches clones with sgRNAs targeting nutrient assimilation, central carbon metabolism, and cyclic electron flow. In many examples, productivity can be enhanced by repression of essential genes, which are difficult to access by transposon insertion.
蓝藻是光合作用的模式生物，在生物技术应用中具有吸引力。为了帮助研究蓝藻中的基因型-表型关系，我们在集胞藻 PCC 6803 中开发了一种诱导型 CRISPRi 基因抑制文库，我们旨在针对所有基因进行抑制。我们在多种条件下跟踪所有文库成员的生长情况，并估计基因适应性。该文库揭示了几个生长速度提高的克隆，这些克隆中与循环电子流相关的基因上调。我们用 0.1 M L-乳酸挑战文库，发现过氧化物酶 bcp2 的抑制可使生长速度提高 49%。将文库转化为分泌 L-乳酸的集胞藻菌株，并对产乳酸量最高的细胞进行分选，可富集针对营养物质同化、中心碳代谢和循环电子流的 sgRNA 靶向克隆。在许多情况下，通过抑制必需基因可以提高生产力，而这些基因通过转座子插入是难以达到的。
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