Abstract
Cyanobacteria, the largest phylum of prokaryotes, perform oxygenic photosynthesis and are regarded as the ancestors of the plant chloroplast and the purveyors of the oxygen and biomass that shaped the biosphere. Nowadays, cyanobacteria are attracting a growing interest in being able to use solar energy, H2O, CO2 and minerals to produce biotechnologically interesting chemicals. This often requires the introduction and expression of heterologous genes encoding the enzymes that are not present in natural cyanobacteria. However, only a handful of model strains with a well-established genetic system are being studied so far, leaving the vast biodiversity of cyanobacteria poorly understood and exploited. In this study, we focused on the robust unicellular cyanobacterium Cyanothece PCC 7425 that has many interesting attributes, such as large cell size; capacity to fix atmospheric nitrogen (under anaerobiosis) and to grow not only on nitrate but also on urea (a frequent pollutant) as the sole nitrogen source; capacity to form CO2-sequestrating intracellular calcium carbonate granules and to produce various biotechnologically interesting products. We demonstrate for the first time that RSF1010-derived plasmid vectors can be used for promoter analysis, as well as constitutive or temperature-controlled overproduction of proteins and analysis of their sub-cellular localization in Cyanothece PCC 7425. These findings are important because no gene manipulation system had been developed for Cyanothece PCC 7425, yet, handicapping its potential to serve as a model host. Furthermore, using this toolbox, we engineered Cyanothece PCC 7425 to produce the high-value terpene, limonene which has applications in biofuels, bioplastics, cosmetics, food and pharmaceutical industries. This is the first report of the engineering of a Cyanothece strain for the production of a chemical and the first demonstration that terpene can be produced by an engineered cyanobacterium growing on urea as the sole nitrogen source.
Highlights
Cyanobacteria, the oldest and largest phylum of prokaryotes that perform the plant-like photosynthesis (Schirrmeister et al, 2015), are regarded as the ancestors of the plant chloroplast (Ponce-Toledo et al, 2017) and the purveyors of the oxygen that shaped our biosphere (Hamilton et al, 2016)
We developed a simple and efficient protocol for the conjugative transfer to Cyanothece PCC 7425 of the plasmid vectors derived from the broad-host-range plasmid RSF1010 that we previously constructed for gene manipulation in Synechocystis PCC 6803 and Synechococcus PCC 7942 (Marraccini et al, 1993; Mermet-Bouvier and Chauvat, 1994; Mazouni et al, 2004). We showed that these vectors replicate autonomously in Cyanothece PCC 7425 where they can be used for facile (i) promoter analysis, (ii) high-level, constitutive or temperature-controlled, protein productions, and (iii) analysis of sub-cellular localization of proteins
As Cyanothece PCC 7425 possesses the full panoply of genes encoding the uptake and catabolism of urea that frequently occurs in natural waters (Veaudor et al, 2019), we have tested its capability to grow on urea as the sole nitrogen source
Summary
Cyanobacteria, the oldest and largest phylum of prokaryotes that perform the plant-like photosynthesis (Schirrmeister et al, 2015), are regarded as the ancestors of the plant chloroplast (Ponce-Toledo et al, 2017) and the purveyors of the oxygen that shaped our biosphere (Hamilton et al, 2016). In colonizing most waters (fresh, brackish and marine) and soils (even deserts) biotopes, cyanobacteria have developed as widely diverse organisms. Their genomes vary in size (1.44–12.07 Mb) and organization (presence/absence of plasmids and linear chromosomes in addition to their circular chromosome) (Cassier-Chauvat et al, 2016). Cyanobacteria are interesting models to study how cells divide and pass their morphology on to their progeny (Cassier-Chauvat and Chauvat, 2014). The disparities of the genome size, cell morphology and metabolism of cyanobacteria should prompt us to analyze a larger number of evolutionary-distant models to better understand and distinguish the common and species-specific aspects of cyanobacteria (Cassier-Chauvat and Chauvat, 2018)
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