Genetic, Genomics, and Responses to Stresses in Cyanobacteria: Biotechnological Implications

Abstract

Cyanobacteria are widely-diverse, environmentally crucial photosynthetic prokaryotes of great interests for basic and applied science. Work to date has focused mostly on the three non-nitrogen fixing unicellular species Synechocystis PCC 6803, Synechococcus PCC 7942, and Synechococcus PCC 7002, which have been selected for their genetic and physiological interests summarized in this review. Extensive “omics” data sets have been generated, and genome-scale models (GSM) have been developed for the rational engineering of these cyanobacteria for biotechnological purposes. We presently discuss what should be done to improve our understanding of the genotype-phenotype relationships of these models and generate robust and predictive models of their metabolism. Furthermore, we also emphasize that because Synechocystis PCC 6803, Synechococcus PCC 7942, and Synechococcus PCC 7002 represent only a limited part of the wide biodiversity of cyanobacteria, other species distantly related to these three models, should be studied. Finally, we highlight the need to strengthen the communication between academic researchers, who know well cyanobacteria and can engineer them for biotechnological purposes, but have a limited access to large photobioreactors, and industrial partners who attempt to use natural or engineered cyanobacteria to produce interesting chemicals at reasonable costs, but may lack knowledge on cyanobacterial physiology and metabolism.

First in vivo analysis of the regulatory protein CP12 of the model cyanobacterium Synechocystis PCC 6803: Biotechnological implications

“…We are interested in the engineering of cyanobacteria for the photosynthetic production of high-value compounds, such as the terpenes bisabolene and limonene which can be used to produce drugs, flavors, fragrances and biofuels ( Lin and Pakrasi, 2019 ; Chenebault et al, 2020 ). Synechocystis PCC 6803 is well-suited for this purpose because it possesses the methylerythritol 4-phosphate (MEP) pathway that produces GPP (geranyl-diphosphate) and FPP (farnesyl-diphosphate), the metabolites that can be transformed into terpenes by heterologous (plant) terpene synthases produced in appropriate recombinant strains ( Lin and Pakrasi, 2019 ; Cassier-Chauvat et al, 2021 ). As three MEP-pathway enzymes require NADPH ( Lin and Pakrasi, 2019 ) whose level is dependent on CP12 ( Figures 4A , B ), we tested the possible influence of CP12 on terpene production.…”

“…Every cyanobacteria possess at least one CP12 protein ( Stanley et al, 2013 ), which had not been studied in vivo . Consequently, we have analyzed the single CP12 protein (canonical, four-Cys, Ssl3364 in CyanoBase) of Synechocystis PCC 6803, the unicellular cyanobacterium that is intensively studied for basic and applied science thanks to its powerful genetics ( Cassier-Chauvat et al, 2021 ) and its capability to grow photoautotrophically (light + CO 2 ), photomixotrophically (low light + CO 2 + glucose) or heterotrophically (catabolism of exogenous glucose in absence of photosynthesis; Rippka et al, 1979 ; Veaudor et al, 2020 ). Using targeted gene deletion, site-directed mutagenesis and phenotypic analysis of the resulting mutants we showed for the first time that the unique CP12 protein of Synechocystis PCC 6803 is dispensable to cell growth under continuous light or light/dark regimes ( Figure 1 ).…”

“…Finally, it is known that Synechocystis PCC 6803 is widely-used for the photosynthetic production of high-value chemicals, such as terpenes that can be used for the production of drugs, flavors, fragrances and biofuels ( Lin and Pakrasi, 2019 ; Chenebault et al, 2020 ). Indeed, Synechocystis PCC 6803 possesses the methylerythritol 4-phosphate (MEP) pathway that produces the GPP and FPP metabolites, which can be transformed into terpenes, after introduction and expression of heterologous genes encoding (plant) terpene synthase ( Lin and Pakrasi, 2019 ; Cassier-Chauvat et al, 2021 ). As three MEP-pathway enzymes require NADPH ( Lin and Pakrasi, 2019 ), the level of which is controlled by CP12 ( Figures 4A , B ), we have tested the influence of CP12 on terpene production.…”

Generation of Synthetic Shuttle Vectors Enabling Modular Genetic Engineering of Cyanobacteria

“…Accordingly, this has been accompanied by the development of recombination and selection strategies as well as the search for suitable integration sites. 16,40,63 However, genetic segregation in a polyploid organism, such as Synechocystis, is a laborious procedure and all of the more time consuming when carried out sequentially, even if neutral sites are targeted. Moreover, targeting endogenous plasmids via homologous recombination directly is accompanied by the same problems and appears to be even more difficult compared to chromosomal sites.…”

“…Some cyanobacteria, including Synechocystis , are naturally competent, enabling the uptake of exogenous DNA and to integrate it into their genome via homologous recombination . Although neutral chromosomal sites for the integration of heterologous genes devoid of pleiotropic effects are available, the method is impaired by chromosome polyploidy, which requires time-consuming genetic segregation. Moreover, the number of neutral sites is inevitably limited, which requires additional alternatives to introduce genes.…”