Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes

Abstract

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-lactatesecreting 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.

Engineering of NADPH Supply Boosts Photosynthesis-Driven Biotransformations

“…This approach could even be partnered with existing screening technologies, such as CRISPRi-based screenings. 54 …”

Over Expression of the Cyanobacterial Pgr5-Homologue Leads to Pseudoreversion in a Gene Coding for a Putative Esterase in Synechocystis 6803

“…It was annotated as an esterase, identified in a transposon library screen [ 60 ]. Disruption of slr1916 resulted in an altered chlorophyll fluorescence kinetic profile, high chlorophyll and PSI content under high light and glucose sensitivity [ 60 ], similar to that observed in pmgA [ 61 , 62 , 63 ]. It was shown that slr1916 is essential for growth under photomixotrophic conditions [ 64 ].…”

Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species

“…One major aim of cyanobacterial synthetic biology is the development of new tools and strategies to facilitate stringent and precise control of gene expression. A wide variety of new molecular tools and genetic parts to tune gene expression are now available for use by the research community ( Englund et al, 2016 ; Kim et al, 2017 ; Ferreira et al, 2018 ; Kelly et al, 2018 ; Vasudevan et al, 2019 ; Yao et al, 2020 ). The increase in availability of well-characterized genetic parts has allowed rational design, a core process to the synthetic biology paradigm, to be more routinely employed in the engineering of new cyanobacterial strains.…”

“…The increase in availability of well-characterized genetic parts has allowed rational design, a core process to the synthetic biology paradigm, to be more routinely employed in the engineering of new cyanobacterial strains. Nevertheless, the majority of synthetic biology work in cyanobacteria has thus far concentrated on characterizing genetic elements that control gene transcription (e.g., promoters, CRISPRi) or translation modulation (e.g., ribosomal binding sites (RBS), riboswitches, small RNAs) ( Huang and Lindblad, 2013 ; Camsund et al, 2014 ; Ma et al, 2014 ; Immethun et al, 2017 ; Kelly et al, 2018 ; Sun et al, 2018 ; Behle et al, 2020 ; Yao et al, 2020 ). Transcription terminators are also key transcriptional control elements, but far fewer studies have examined their roles in regulating gene expression in cyanobacteria.…”