Comparative genomics reveals the molecular determinants of rapid growth of the cyanobacterium Synechococcus elongatus UTEX 2973

Abstract

Cyanobacteria are emerging as attractive organisms for sustainable bioproduction. We previously described Synechococcus elongatus UTEX 2973 as the fastest growing cyanobacterium known. Synechococcus 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 Synechococcus elongatus 7942. The two strains differ at 55 genetic loci, and some 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 Synechococcus 2973 and identified three specific genes, atpA, ppnK, and rpaA, with SNPs that confer rapid growth. The fast-growth-associated allele of each gene was then used to replace the wild-type alleles in Synechococcus 7942. Upon incorporation, each allele successively increased the growth rate of Synechococcus 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 Synechococcus 7942. We also determined that the fast-growth-associated allele of atpA yielded an ATP synthase with higher specific activity, while that of ppnK encoded a NAD⁺ kinase with significantly improved kinetics. The rpaA 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.

Keywords: Synechococcus; cyanobacteria; growth; photosynthesis.

Fig. 1.

Growth of Synechococcus 2973, Synechococcus 7942, and mutant strains. (A) Representative growth curves of Synechococcus 7942 with different SNPs from Synechococcus 2973 incorporated individually. (B) Representative growth curves of Synechococcus 7942 with SNPs from Synechococcus 2973 incorporated in combinations. (C) Doubling times of both Synechococcus strains and mutants thereof (n = 3). (D) Whole-chain oxygen evolution of both wild-type strains and Synechococcus 7942 with various allele combinations from Synechococcus 2973 (n = 3). Allele 1, ATP synthase; allele 2, NAD⁺ kinase; allele 3, both coding SNPs and promoter deletion in rpaA. Growth was performed in a multicultivator bioreactor with 5% CO₂, 900 µmol⋅m⁻²⋅s⁻¹ light, 38 °C.

Fig. 2.

NAD⁺ kinase activity in the presence of various concentrations of competitive inhibitors. (A) Activity with added NADPH. (B) Activity with added NADH (n = 3).

Fig. 3.

ATP synthase activity of the Synechococcus 2973 allele vs. the Synechococcus 7942 allele (n = 3).

Fig. 4.

Local alignments of the SNPs that are involved in rapid growth in various cyanobacteria. (A) ATP synthase. (B) NAD⁺ kinase. (C) RpaA. The SNPs in Synechococcus 7942 and Synechococcus 2973 are indicated by boxes.