Pooled CRISPRi screening of the cyanobacterium Synechocystis sp. PCC 6803 for enhanced growth, tolerance, and chemical production

Lun Yao, Kiyan Shabestary, Sara M. Björk, Johannes Asplund-Samuelsson, Haakan N. Joensson, Michael Jahn, Elton P. Hudson

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Nov 08, 2019
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Received Date: 30th October 19

We developed an inducible CRISPRi gene repression library in the cyanobacterium Synechocystis sp. PCC 6803, where all annotated genes are targeted for repression. We used the library to estimate gene fitness in multiple conditions. The library revealed several mutants with increased specific growth rates (up to 17%), and transcriptomics of these mutants revealed common upregulation of genes within photosynthetic electron flow. We challenged the library with L-lactate stress to find more tolerant mutants. Repression of the peroxiredoxin Bcp2 increased growth rate by 49% in the presence of 0.1 M L-lactate. Finally, the library was transformed into a L-lactate-secreting strain, and droplet microfluidics sorting of top producers enriched sgRNAs targeting nutrient assimilation, redox modulation, and cyclic-electron flow. Several clones showed increased productivity in batch cultivations (up to 75%). In some cases, tolerance or productivity was enhanced by partial repression of essential genes, which are difficult to access by transposon insertion.

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This is an abstract of a preprint hosted on an independent third party site. It has not been peer reviewed but is currently under consideration at Nature Communications.

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