Proteomic and metabolomic analyses reveal metabolic responses to 3-hydroxypropionic acid synthesized internally in cyanobacterium Synechocystis sp. PCC 6803

Abstract

Background: 3-hydroxypropionic acid (3-HP) is an important platform chemical with a wide range of applications. In our previous study, the biosynthetic pathway of 3-HP was constructed and optimized in cyanobacterium Synechocystis sp. PCC 6803, which led to 3-HP production directly from CO₂ at a level of 837.18 mg L^-1 (348.8 mg/g dry cell weight). As the production and accumulation of 3-HP in cells affect cellular metabolism, a better understanding of cellular responses to 3-HP synthesized internally in Synechocystis will be important for further increasing 3-HP productivity in cyanobacterial chassis.

Results: Using a engineered 3-HP-producing SM strain, in this study, the cellular responses to 3-HP internally produced were first determined using a quantitative iTRAQ-LC-MS/MS proteomics approach and a LC-MS-based targeted metabolomics. A total of 2264 unique proteins were identified, which represented about 63 % of all predicted protein in Synechocystis in the proteomic analysis; meanwhile intracellular abundance of 24 key metabolites was determined by a comparative metabolomic 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, along with the abundance of 14 metabolites related to central metabolism. 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. Confirmation of proteomic and metabolomic results with RT-qPCR and gene-overexpression strains of selected genes was also conducted, and the overexpression of three transporter genes putatively involved in cobalt/nickel, manganese and phosphate transporting (i.e., sll0385, sll1598 and sll0679) could lead to an increased 3-HP production in Synechocystis.

Conclusions: The integrative analysis of up-regulated proteome and metabolome data showed that to ensure the high-efficient production of 3-HP and the normal growth of Synechocystis, multiple aspects of cells metabolism including energy, reducing power supply, central carbon metabolism, the stress responses and protein synthesis were enhanced in Synechocystis. The study provides an important basis for further engineering cyanobacteria for high 3-HP production.

Keywords: 3-HP; Biosynthesis; Metabolomics; Proteomics; Response; Synechocystis.

Fig. 1

Cell growth and 3-HP production of the WT and the engineered Synechocystis SM strains. a Growth of the WT and the engineered Synechocystis SM strain; b 3-HP production by the WT and the engineered Synechocystis SM strain

Fig. 2

Distribution, coverage, and functional category of proteins identified in this study. a Distribution of protein identified among different molecular weights; b Coverage of proteins by the identified peptides; c Functional category coverage of the proteins identified

Fig. 3

Targeted LC–MS metabolomic analysis. a PCA plots of the LC–MS metabolomic profiles of the WT and the 3-HP-producing SM strains; b Heatmap analysis of LC–MS metabolomic profiles of the WT and the 3-HP-producing SM strains

Fig. 4

Schematic representation of metabolic responses to 3-HP synthesized internally in Synechocystis. Up-regulated proteins are indicated in the figure

Fig. 5

RT-qPCR analysis of the expression level of selected overexpressed genes in SM strain