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-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.
Highlights
Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications
We have primarily used the CRISPRi repression library to identify potential mutants with improved industrial phenotypes, which we validated with screening of individual clones
An increase in PSI activity at high light can dissipate pressure in the electron-transport chain; a similar phenomenon was reported to contribute to high-light tolerance and faster growth in Synechococcus elongatus UTEX 2973 compared to its close relative Synechococcus PCC 794246
Summary
Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. Cyanobacteria are model organisms for photosynthetic electron flow, photorespiration, and the circadian clock[1,2,3] In addition to their massive ecological importance, biotechnological applications of cyanobacteria have been proposed, such as microbial cell factories, where metabolism is engineered to synthesize chemicals from CO2 using energy derived from light[4,5,6]. We provide support for previous computational predictions that alteration of the ATP/ NADPH balance can improve bioproduction in cyanobacteria By screening both growth and productivity, this platform can yield mutants that trade biomass formation for increased productivity of a target compound. While we have focused on biotechnological traits of interest here, the CRISPRi library can be used to explore the connection between growth and robustness in many conditions, all while allowing access to modulation of essential genes. The data for all competition experiments performed with the sgRNA library can be accessed through an interactive web application [https://m-jahn.shinyapps.io/ShinyLib/]
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