Reconstruction and metabolic profiling of the genome-scale metabolic network model of Pseudomonas stutzeri A1501.

Pseudomonas stutzeri A1501 is a non-fluorescent denitrifying bacteria that belongs to the gram-negative bacterial group. As a prominent strain in the fields of agriculture and bioengineering, there is still a lack of comprehensive understanding regarding its metabolic capabilities, specifically in terms of central metabolism and substrate utilization. Therefore, further exploration and extensive studies are required to gain a detailed insight into these aspects. This study reconstructed a genome-scale metabolic network model for P. stutzeri A1501 and conducted extensive curations, including correcting energy generation cycles, respiratory chains, and biomass composition. The final model, iQY1018, was successfully developed, covering more genes and reactions and having higher prediction accuracy compared with the previously published model iPB890. The substrate utilization ability of 71 carbon sources was investigated by BIOLOG experiment and was utilized to validate the model quality. The model prediction accuracy of substrate utilization for P. stutzeri A1501 reached 90 %. The model analysis revealed its new ability in central metabolism and predicted that the strain is a suitable chassis for the production of Acetyl CoA-derived products. This work provides an updated, high-quality model of P. stutzeri A1501for further research and will further enhance our understanding of the metabolic capabilities.

Engineering Escherichia coli for Poly-ß-hydroxybutyrate Production from Methanol.

The naturally occurring one-carbon assimilation pathways for the production of acetyl-CoA and its derivatives often have low product yields because of carbon loss as CO2. We constructed a methanol assimilation pathway to produce poly-3-hydroxybutyrate (P3HB) using the MCC pathway, which included the ribulose monophosphate (RuMP) pathway for methanol assimilation and non-oxidative glycolysis (NOG) for acetyl-CoA (precursor for PHB synthesis) production. The theoretical product carbon yield of the new pathway is 100%, hence no carbon loss. We constructed this pathway in E. coli JM109 by introducing methanol dehydrogenase (Mdh), a fused Hps-phi (hexulose-6-phosphate synthase and 3-phospho-6-hexuloisomerase), phosphoketolase, and the genes for PHB synthesis. We also knocked out the frmA gene (encoding formaldehyde dehydrogenase) to prevent the dehydrogenation of formaldehyde to formate. Mdh is the primary rate-limiting enzyme in methanol uptake; thus, we compared the activities of three Mdhs in vitro and in vivo and then selected the one from Bacillus methanolicus MGA3 for further study. Experimental results indicate that, in agreement with the computational analysis results, the introduction of the NOG pathway is essential for improving PHB production (65% increase in PHB concentration, up to 6.19% of dry cell weight). We demonstrated that PHB can be produced from methanol via metabolic engineering, which provides the foundation for the future large-scale use of one-carbon compounds for biopolymer production.