Biosynthesis Of 3-hydroxypropionic Acid (3-HP) And Its Metabolic Responses In Synechocystis Sp.PCC 6803

3-hydroxypropionic acid(3-HP)is an important platform chemical with a wide range of applications.So far large-scale 3-HP production has been mainly through petroleum-based chemical processes,whose sustainability and environmental issues have attracted widespread attention.With the ability to fix CO2 directly,cyanobacteria have been engineered as an autotrophic microbial cell factory to produce fuels and chemicals.In this study,we constructed the biosynthetic pathway of 3-HP in cyanobacterium Synechocystis sp.PCC 6803,and then optimized the system through the following approaches: i)increasing expression of malonyl-CoA reductase(MCR)gene using different promoters and cultivation conditions;ii)enhancing supply of the precursor malonyl-CoA by overexpressing acetyl-Co A carboxylase and biotinilase;iii)improving NADPH supply by overexpressing the NAD(P)transhydrogenase gene;iv)directing more carbon flux into 3-HP by inactivating the competing pathways of PHA and acetate biosynthesis.Together,the efforts led to a production of 837.18 mg L-1(348.8 mg/g dry cell weight)3-HP directly from CO2 in Synechocystis after 6 days cultivation,demonstrating the feasibility photosynthetic production of 3-HP directly from sunlight and CO2 in cyanobacteria.In addition,the results showed that overexpression of the ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)gene from Anabaena sp.PCC 7120 and Synechococcus sp.PCC 7942 led to no increase of 3-HP production,suggesting CO2 fixation may not be a rate-limiting step for 3-HP biosynthesis in Synechocystis.Current knowledge regarding response mechanisms of cyanobacteria to toxic3-HP is very limited.In this study,the responses of cyanobacteria to 3-HP internally produced were first determined using a quantitative proteomics approach with isobaric tags for relative and absolute quantitation-liquid chromatograph-mass spectrometer(iTRAQ-LC-MS/MS)technologies.In total,2,264 unique proteins were identified,representing about 63% of all predicted protein in the Synechocystis genome.Among all proteins identified,a total of 204 and 123 proteins were found upand down-regulated,respectively.In addition,intracellular abundance of 24 keymetabolites in engineered 3-HP producing strain SM was detected using a LC-MS based technology.The analyses showed that proteins related to oxidative phosphorylation,photosynthesis,ribosome,central carbon metabolism,two-component system and ABC-type transporters were up-regulated,and 14 central metabolites were also up-regulated.The results suggested that supply of ATP,NADPH was increased significantly and precursor malonyl-CoA and acetyl-CoA may also be supplemented to some extent when 3-HP produced a high level in cyanobacteria.Meanwhile,it is expected that the toxicity of 3-HP was also increased.Validation of omic results with RT-qPCR and gene overexpression strains of some related proteins was also conducted.Overall,the study reveals important findings of the metabolic responses to 3-hydroxypropionic acid(3-HP)internally generated in Synechocystis,and provides an important basis for further engineering the cyanobacteria of high 3-HP production.