A transcriptional regulator Sll0794 regulates tolerance to biofuel ethanol in photosynthetic Synechocystis sp. PCC 6803

Abstract

To improve ethanol production directly from CO2 in photosynthetic cyanobacterial systems, one key issue that needs to be addressed is the low ethanol tolerance of cyanobacterial cells. Our previous proteomic and transcriptomic analyses found that several regulatory proteins were up-regulated by exogenous ethanol in Synechocystis sp. PCC6803. In this study, through tolerance analysis of the gene disruption mutants of the up-regulated regulatory genes, we uncovered that one transcriptional regulator, Sll0794, was related directly to ethanol tolerance in Synechocystis. Using a quantitative iTRAQ-LC-MS/MS proteomics approach coupled with quantitative real-time reverse transcription-PCR (RT-qPCR), we further determined the possible regulatory network of Sll0794. The proteomic analysis showed that in the Δsll0794 mutant grown under ethanol stress a total of 54 and 87 unique proteins were down- and up-regulated, respectively. In addition, electrophoretic mobility shift assays demonstrated that the Sll0794 transcriptional regulator was able to bind directly to the upstream regions of sll1514, slr1512, and slr1838, which encode a 16.6 kDa small heat shock protein, a putative sodium-dependent bicarbonate transporter and a carbon dioxide concentrating mechanism protein CcmK, respectively. The study provided a proteomic description of the putative ethanol-tolerance network regulated by the sll0794 gene, and revealed new insights on the ethanol-tolerance regulatory mechanism in Synechocystis. As the first regulatory protein discovered related to ethanol tolerance, the gene may serve as a valuable target for transcription machinery engineering to further improve ethanol tolerance in Synechocystis. All MS data have been deposited in the ProteomeXchange with identifier PXD001266 (http://proteomecentral.proteomexchange.org/dataset/PXD001266).

Fig. 1.

Growth of the wild-type strain and the Δsll0794 mutant in BG11 with or without 1.5% ethanol (v/v).

Fig. 2.

Flow cytometric analysis of the wild-type strain and the Δsll0794 mutant grown in the BG11 medium supplemented with 1. 5% ethanol (v/v) at 24, 48, and 72 h, respectively.

Fig. 3.

Reproducibility between biological replicates. Wild-type A, and Δsll0794 mutant B, respectively; Distribution of iTRAQ log ratios of the detected proteins between Δsll0794 biological replicate 1 and the wild-type C, and between Δsll0794 biological replicate 2 and the wild-type D.

Fig. 4.

EMSAs to assess the interaction of the transcriptional regulator Sll0794 with putative promoter regions of selective target genes. A, SDS-PAGE analysis of expression and purification of His₆-Sll0794 in E. coli. Lane1: the crude extract of E. coli BL21 (DE3) harboring pET46–0794 without being induced by IPTG; Lanes 2 and 3: the supernatant and the precipitate fractions of E. coli BL21(DE3) harboring pET46–0794 induced by IPTG at a final concentration of 0.2 mm at 18 °C overnight; Lane 4: the purified His₆-Sll0794 protein; Lane M: protein markers. B, EMSAs of the promoter regions of ssr2061, slr1512, sll1514, and slr1838 with purified His₆-Sll0794. The amounts of His₆-Sll0794 (μm) used were as indicated and 10 ng each of 5′-cy5-labeled probes was added in each of the EMSAs reactions. The results for other eight probes investigated in this study were with the same patterns as ssr2061 and thus not presented.

Fig. 5.

Visualization of putative Sll0794 binding motifs. The motif is represented by a sequence logo generated by the WebLogos software (37).