Comparative genomics reveals the molecular determinants of rapid growth of the cyanobacterium <i>Synechococcus elongatus</i> UTEX 2973.

Abstract

Cyanobacteria are emerging as attractive organisms for sustainable bioproduction. We previously described <i>Synechococcus elongatus</i> UTEX 2973 as the fastest growing cyanobacterium known. <i>Synechococcus</i> 2973 exhibits high light tolerance and an increased photosynthetic rate and produces biomass at three times the rate of its close relative, the model strain <i>Synechococcus elongatus</i> 7942. The two strains differ at 55 genetic loci, and <i>s</i>ome of these loci must contain the genetic determinants of rapid photoautotrophic growth and improved photosynthetic rate. Using CRISPR/Cpf1, we performed a comprehensive mutational analysis of <i>Synechococcus</i> 2973 and identified three specific genes, <i>atpA</i>, <i>ppnK</i>, and <i>rpaA</i>, with SNPs that confer rapid growth. The fast-growth-associated allele of each gene was then used to replace the wild-type alleles in <i>Synechococcus</i> 7942. Upon incorporation, each allele successively increased the growth rate of <i>Synechococcus</i> 7942; remarkably, inclusion of all three alleles drastically reduced the doubling time from 6.8 to 2.3 hours. Further analysis revealed that our engineering effort doubled the photosynthetic productivity of <i>Synechococcus</i> 7942. We also determined that the fast-growth-associated allele of <i>atpA</i> yielded an ATP synthase with higher specific activity, while that of <i>ppnK</i> encoded a NAD⁺ kinase with significantly improved kinetics. The <i>rpaA</i> SNPs cause broad changes in the transcriptional profile, as this gene is the master output regulator of the circadian clock. This pioneering study has revealed the molecular basis for rapid growth, demonstrating that limited genetic changes can dramatically improve the growth rate of a microbe by as much as threefold.