Integrated OMICS guided engineering of biofuel butanol-tolerance in photosynthetic Synechocystis sp. PCC 6803

Abstract

Background: Photosynthetic cyanobacteria have been recently proposed as a 'microbial factory' to produce butanol due to their capability to utilize solar energy and CO2 as the sole energy and carbon sources, respectively. However, to improve the productivity, one key issue needed to be addressed is the low tolerance of the photosynthetic hosts to butanol.

Results: In this study, we first applied a quantitative transcriptomics approach with a next-generation RNA sequencing technology to identify gene targets relevant to butanol tolerance in a model cyanobacterium Synechocystis sp. PCC 6803. The results showed that 278 genes were induced by the butanol exposure at all three sampling points through the growth time course. Genes encoding heat-shock proteins, oxidative stress related proteins, transporters and proteins involved in common stress responses, were induced by butanol exposure. We then applied GC-MS based metabolomics analysis to determine the metabolic changes associated with the butanol exposure. The results showed that 46 out of 73 chemically classified metabolites were differentially regulated by butanol treatment. Notably, 3-phosphoglycerate, glycine, serine and urea related to general stress responses were elevated in butanol-treated cells. To validate the potential targets, we constructed gene knockout mutants for three selected gene targets. The comparative phenotypic analysis confirmed that these genes were involved in the butanol tolerance.

Conclusion: The integrated OMICS analysis provided a comprehensive view of the complicated molecular mechanisms employed by Synechocystis sp. PCC 6803 against butanol stress, and allowed identification of a series of potential gene candidates for tolerance engineering in cyanobacterium Synechocystis sp. PCC 6803.

Figure 1

Reproducibility of RNA-seq transcriptomic analysis. Two biological replicates of butanol-treated samples were plotted. Normalized expression RPKM values were used. Correlation coefficient is indicated inside.

Figure 2

PCA score plot of metabolomic profiles of Synechocystis along the treatment time course. Samples with or without butanol treatments were indicated by different colors. The biological replicates were circled for the same conditions.

Figure 3

Comparative analysis of butanol tolerance of wild-type strain and mutants. A) Growth time courses of wild type, Δsll0690, Δslr0947 and Δslr1295 mutants in normal BG11 medium; B) Growth time courses of wild type, Δsll0690 mutant, Δslr0947 mutant and Δslr1295 mutant in BG11 media supplemented with 0.25% (v/v) butanol.