Abstract
BackgroundRhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides.ResultsHere we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ΔphbB strains under the same conditions is only slightly affected.ConclusionsIn this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.
Highlights
The purple non-sulphur bacterium Rhodobacter sphaeroides is a microorganism with an extremely adaptable metabolism [1]
We constructed the pBBR_Cas9_NT control vector by cloning the cas9 gene, codon optimized for R. sphaeroides, and a sgRNA expressing module with a non-targeting (NT) spacer in the E. coli-Rhodobacter shuttle vector pBBR1MCS2
The expression of the cas9 gene was under control of the Plac promoter that due to the absence of the lacI repressor gene in the R. sphaeroides genome, has constitutive transcription activity
Summary
The purple non-sulphur bacterium Rhodobacter sphaeroides is a microorganism with an extremely adaptable metabolism [1]. The metabolic versatility of Rhodobacter sphaeroides has raised curiosity for white biotechnological applications. This microorganism has been largely studied for photoheterotrophic hydrogen production and chemoheterotrophic terpene biosynthesis. Reducing formation of by-products like the polymer poly-β-hydroxybutyrate (PHB) holds potential for eliminating competition for carbon and reducing power, as already proven to work for H2 biosynthesis [5, 9]. Rhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. The Cas RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides
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