Comparative genomics reveals the molecular determinants of rapid growth of the cyanobacteriumSynechococcus elongatusUTEX 2973

Abstract

Cyanobacteria are emerging as attractive organisms for sustainable bioproduction. We previously describedSynechococcus elongatusUTEX 2973 as the fastest growing cyanobacterium known.Synechococcus2973 exhibits high light tolerance and an increased photosynthetic rate and produces biomass at three times the rate of its close relative, the model strainSynechococcus elongatus7942. The two strains differ at 55 genetic loci, andsome 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 ofSynechococcus2973 and identified three specific genes,atpA,ppnK, andrpaA, with SNPs that confer rapid growth. The fast-growth–associated allele of each gene was then used to replace the wild-type alleles inSynechococcus7942. Upon incorporation, each allele successively increased the growth rate ofSynechococcus7942; 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 ofSynechococcus7942. We also determined that the fast-growth–associated allele ofatpAyielded an ATP synthase with higher specific activity, while that ofppnKencoded a NAD+kinase with significantly improved kinetics. TherpaASNPs 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.