Systematic Large Fragment Deletions in the Genome of Synechococcus elongatus and the Consequent Changes in Transcriptomic Profiles

Abstract

Genome streamlining, as a natural process in the evolution of microbes, has become a common approach for generating ideal chassis cells for synthetic biology studies and industrial applications. However, systematic genome reduction remains a bottleneck in the generation of such chassis cells with cyanobacteria, due to very time-consuming genetic manipulations. Synechococcus elongatus PCC 7942, a unicellular cyanobacterium, is a candidate for systematic genome reduction, as its essential and nonessential genes have been experimentally identified. Here, we report that at least 20 of the 23 over 10 kb nonessential gene regions could be deleted and that stepwise deletions of these regions could be achieved. A septuple-deletion mutant (genome reduced by 3.8%) was generated, and the effects of genome reduction on the growth and genome-wide transcription were investigated. In the ancestral triple to sextuple mutants (b, c, d, e1), an increasingly large number of genes (up to 998) were upregulated relative to the wild type, while slightly fewer genes (831) were upregulated in the septuple mutant (f). In a different sextuple mutant (e2) derived from the quintuple mutant d, much fewer genes (232) were upregulated. Under the standard conditions in this study, the mutant e2 showed a higher growth rate than the wild type, e1 and f. Our results indicate that it is feasible to extensively reduce the genomes of cyanobacteria for generation of chassis cells and for experimental evolutionary studies.

Genome streamlining to improve performance of a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973

“…The other approach is top-down, which involves systemic streamlining of the existing genome based on prior information regarding the core essential genes. Most genome streamlining efforts have relied on traditional and, more recently, the CRISPR-Cas12a/9-mediated genome-editing tools ( 20 , 21 , 24 , 25 ). For strains where the essentiality information is available, the strategy to delete fixed, specified regions of the genome is advantageous as it leaves less room for casualties such as drastic loss of fitness.…”

“…These mutants were further studied to understand the changes in the transcriptomic profile of the cells resulting from the deletions. The CRISPR-Cas12a-editing tool was used to obtain the targeted, specified, and markerless deletions in this study ( 25 ). Although Cas12a is a dynamic and versatile tool, it does not allow the flexibility to explore and identify unknown stretches of dispensable and indispensable regions in the genome of a strain.…”

Genome streamlining to improve performance of a fast-growing cyanobacteriumSynechococcus elongatusUTEX 2973

“…These mutants were further studied to understand the changes in the transcriptomics profile of the cells resulting from the deletions. The CRISPR-Cas12a editing tool was used to obtain the targeted, specified and markerless deletions in this study (25). Though Cas12a is a dynamic and versatile tool, it does not allow the flexibility to explore and identify unknown stretches of dispensable and indispensable regions in the genome of a strain.…”