Proteomic analyses reveal metabolic responses to 3-hydroxypropionic acid synthesized internally in Synechocystis sp. PCC 6803
Using an engineered 3-HP producing SM strain, in this project, the cellular responses to 3-HP internally produced were first determined using a quantitative iTRAQ-LC-MS/MS proteomics approach. A total of 2,264 unique proteins were identified, which represented about 63% of all predicted protein in Synechocystis in the proteomic analysis of the 3-HP producing strain SM and wild type. Among all identified proteins, 204 proteins were found up- regulated and 123 proteins were found down-regulated, respectively. The proteins related to oxidative phosphorylation, photosynthesis, ribosome, central carbon metabolism, two-component systems and ABC-type transporters were up-regulated. The results suggested that the supply of ATP and NADPH was increased significantly, and the precursor malonyl-CoA and acetyl-CoA may also be supplemented when 3-HP was produced at a high level in Synechocystis.
Sample Processing Protocol
The cells were incubated in a shaking bed (150 rpm) at 30 °C with light intensity of 50 μmol photons m-2 s-1. 0.5 mL 1.0 M NaHCO3 was added to each flask every 24 h, and the culture medium was adjusted to pH 7.5 using 10 N HCl . The cells were grown for up to 6 days. Cell growth (OD730) and the 3-HP biosynthesis were measured through the growth time course. the cells were suspended in the lysis buffer and sonicated in ice. The proteins were reduced with 10 mM DTT then alkylated by 55 mM iodacetamide. Protein mixtures were precipitated at -20 °C overnight. After dissolution in 0.5 M TEAB (Applied Biosystems, Milan, Italy) the pellet was sonicated in ice. Then mixtures were centrifuged at 30 000 x g 4 °C and an aliquot of the supernatant was taken for determination of protein concentration by coomassie blue staining. Total protein (100 μg) taken out of each sample solution was digested with Trypsin Gold (Promega, Madison, WI, USA). The peptides were then dried by vacuum centrifugation and reconstituted in 0.5 M TEAB. Samples were labeled with the iTRAQ. Each fraction was re-suspended and centrifuged, the final peptide concentration was about 0.5 μg/μL on average. 10 μL supernatant was loaded on a LC-20AD nanoHPLC (Shimadzu, Kyoto, Japan) by the autosampler onto a 2 cm C18 trap column. Then, the peptides were eluted onto a 10 cm analytical C18 column (inner diameter 75 μm) packed in-house. Data acquisition was performed as described previously with a TripleTOF 5600 System (AB SCIEX, Concord, ON) fitted with a Nanospray IIIsource (AB SCIEX, Concord, ON) and a pulled quartz tip as the emitter (New Objectives, Woburn, MA). The MS was operated with a RP greater than or equal to 30 000 FWHM for TOF MS scans.
Data Processing Protocol
Original data obtained from the Orbitrap was converted into MGF files with Proteome Discoverer 1.2 (PD 1.2, Thermo Fisher Scientific), and the MGF file was searched. Proteins identification was performed using Mascot search engine (Matrix Science, London, UK; version 2.3.02) with a Synechocystis sequence database. Protein identification was conducted according to our previous publication. For protein quantification, a protein contains at least two unique spectra. The confident ratio change with p-values < 0.05, and only fold changes of >1.2 was considered as significant. Proteins functional annotation was conducted using Blast2GO program against the non-redundant protein database (NR; NCBI). The KEGG database (http://www.genome.jp/kegg/) and the COG database (http://www.ncbi.nlm.nih.gov/COG/) were used to classify and group this identified proteins
Wang Y, Chen L, Zhang W. Proteomic and metabolomic analyses reveal metabolic responses to 3-hydroxypropionic acid synthesized internally in cyanobacterium Synechocystis sp. PCC 6803. Biotechnol Biofuels. 2016 Oct 6;9:209 PubMed: 27757169