Quantitative iTRAQ LC–MS/MS Proteomics Reveals Metabolic Responses to Biofuel Ethanol in Cyanobacterial Synechocystis sp. PCC 6803

Abstract

Recent progress in metabolic engineering has led to autotrophic production of ethanol in various cyanobacterial hosts. However, cyanobacteria are known to be sensitive to ethanol, which restricts further efforts to increase ethanol production levels in these renewable host systems. To understand the mechanisms of ethanol tolerance so that engineering more robust cyanobacterial hosts can be possible, in this study, the responses of model cyanobacterial Synechocystis sp. PCC 6803 to ethanol were determined using a quantitative proteomics approach with iTRAQ LC-MS/MS technologies. The resulting high-quality proteomic data set consisted of 24,887 unique peptides corresponding to 1509 identified proteins, a coverage of approximately 42% of the predicted proteins in the Synechocystis genome. Using a cutoff of 1.5-fold change and a p-value less than 0.05, 135 and 293 unique proteins with differential abundance levels were identified between control and ethanol-treated samples at 24 and 48 h, respectively. Functional analysis showed that the Synechocystis cells employed a combination of induced common stress response, modifications of cell membrane and envelope, and induction of multiple transporters and cell mobility-related proteins as protection mechanisms against ethanol toxicity. Interestingly, our proteomic analysis revealed that proteins related to multiple aspects of photosynthesis were up-regulated in the ethanol-treated Synechocystis cells, consistent with increased chlorophyll a concentration in the cells upon ethanol exposure. The study provided the first comprehensive view of the complicated molecular mechanisms against ethanol stress and also provided a list of potential gene targets for further engineering ethanol tolerance in Synechocystis PCC 6803.

A Transcriptional Regulator Sll0794 Regulates Tolerance to Biofuel Ethanol in Photosynthetic Synechocystis sp. PCC 6803

“…The chloramphenicol-resistant transformants were obtained, confirmed for the gene knockout event by PCR and sequencing, and then passed several times on fresh BG11 plates supplemented with 10 g/ml chloramphenicol to achieve complete chromosome segregation (confirmed by PCR). Three transcriptional regulator encoding genes, sll0792, sll0794, and sll1423 that were found differentially regulated by ethanol exposure either at protein or RNA levels according to previous studies (23,24), were selected for construction of gene knockout mutants. The successful knockout mutants were con- 1 The abbreviations used are: CCM, CO2-concentrating mechanism; EMSAs, electrophoretic mobility shift assays; FACS, fluorescence-activated cell sorting; HPLC, high-performance liquid chromatography; IPTG, Isopropyl ␤-D-1-thiogalactopyranoside; iTRAQ, Isobaric tag for relative and absolute quantitation; LC-MS, Liquid chromatography-tandem mass spectrometry; MS, mass spectrometry; PCR, polymerase chain reaction; RT-qPCR, quantitative real-time reverse transcription-PCR; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBE, Tris/Borate/EDTA; TCA, tricarboxylic acid; FSC, forward scatter; SSC, side scatter.…”

“…PCC6803 (hereafter Synechocystis) (23). To identify possible targets related to ethanol tolerance, quantitative iTRAQ-LC-MS/MS based proteomics and RNA-seq based transcriptomics were applied to determine the metabolic response of Synechocystis under ethanol stress, and the results showed that multiple transcriptional regulators were differentially regulated, providing potential gene targets for engineering transcriptional machinery in order to improve ethanol tolerance in Synechocystis (23,24). In this work, by constructing gene knockout mutants and conducting phenotypic analyses, we demonstrated that a transcriptional regulator Sll0794 was involved in ethanol tolerance.…”

“…in an illuminating incubator of 130 rpm at 30°C (HNY-211B Illuminating Shaker, Honor, China) (23,24). Cell density was measured on a UV-1750 spectrophotometer (Shimadzu, Japan) at OD730 or an ELx808 Absorbance Microplate Reader (BioTek, Winooski, VT) at OD630.…”

“…Cells for proteomics analysis were collected by centrifugation at 8000 ϫ g for 10 min at 4°C. To reveal cell phenotype difference, flow cytometric analysis was performed to compare the wild type and the mutant cells under ethanol stress on a FACS Calibur fluorescence-activated cell sorting (FACS) cytometer (Becton Dickinson) as described before (23).…”

Identification of potential microRNA–target pairs associated with osteopetrosis by deep sequencing, iTRAQ proteomics and bioinformatics

“…The process of protein extraction and iTRAQ sample labeling were performed as described by our previous research with minor modifications according to Qiao et al 10,11 Briefly, for the six samples in the osteopetrosis group and the nine samples in the control group, aliquots of PBMCs protein from each subject were pooled respectively. One hundred micrograms of protein from each corresponding group pool protein was digested with trypsin, and labeled according to the iTRAQ protocol (Applied Biosystems).…”