6-Phosphogluconolactonase is critical for the efficient functioning of the pentose phosphate pathway.

The metabolic networks of microorganisms are remarkably robust to genetic and environmental perturbations. This robustness stems from redundancies such as gene duplications, isoenzymes, alternative metabolic pathways, and also from non-enzymatic reactions. In the oxidative branch of the pentose phosphate pathway (oxPPP), 6-phosphogluconolactone hydrolysis into 6-phosphogluconate is catalysed by 6-phosphogluconolactonase (Pgl) but in the absence of the latter, the oxPPP flux is thought to be maintained by spontaneous hydrolysis. However, in Δpgl Escherichia coli, an extracellular pathway can also contribute to pentose phosphate synthesis. This raises question as to whether the intracellular non-enzymatic reaction can compensate for the absence of 6-phosphogluconolactonase and, ultimately, on the role of 6-phosphogluconolactonase in central metabolism. Our results validate that the bypass pathway is active in the absence of Pgl, specifically involving the extracellular spontaneous hydrolysis of gluconolactones to gluconate. Under these conditions, metabolic flux analysis reveals that this bypass pathway accounts for the entire flux into the oxPPP. This alternative metabolic route-partially extracellular-sustains the flux through the oxPPP necessary for cell growth, albeit at a reduced rate in the absence of Pgl. Importantly, these findings imply that intracellular non-enzymatic hydrolysis of 6-phosphogluconolactone does not compensate for the absence of Pgl. This underscores the crucial role of Pgl in ensuring the efficient functioning of the oxPPP.

Dissecting carbon metabolism of Yarrowia lipolytica type strain W29 using genome-scale metabolic modelling.

Yarrowia lipolytica is a widely-used chassis cell in biotechnological applications. It has recently gained extensive research interest owing to its extraordinary ability of producing industrially valuable biochemicals from a variety of carbon sources. Genome-scale metabolic models (GSMMs) enable analyses of cellular metabolism for engineering various industrial hosts. In the present study, we developed a high-quality GSMM iYli21 for Y. lipolytica type strain W29 by extensive manual curation with Biolog experimental data. The model showed a high accuracy of 85.7% in predicting nutrient utilization. Transcriptomics data were integrated to delineate cellular metabolism of utilizing six individual metabolites as sole carbon sources. Comparisons showed that 302 reactions were commonly used, including those from TCA cycle, oxidative phosphorylation, and purine metabolism for energy and material supply. Whereas glycolytic reactions were employed only when glucose and glycerol used as sole carbon sources, gluconeogenesis and fatty acid oxidation reactions were specifically employed when fatty acid, alkane and glycerolipid were the sole carbon sources. Further test of 46 substrates for generating 5 products showed that hexanoate outcompeted other compounds in terms of maximum theoretical yield owing to the lowest carbon loss for energy supply. This newly generated model iYli21 will be a valuable tool in dissecting metabolic mechanism and guiding metabolic engineering of this important industrial cell factory.