Genome‐scale metabolic modeling reveals key features of a minimal gene set

Mesoplasma florum , a fast‐growing near‐minimal organism, is a compelling model to explore rational genome designs. Using sequence and structural homology, the set of metabolic functions its genome encodes was identified, allowing the reconstruction of a metabolic network representing ˜ 30% of its protein‐coding genes. Growth medium simplification enabled substrate uptake and product secretion rate quantification which, along with experimental biomass composition, were integrated as species‐specific constraints to produce the functional i JL208 genome‐scale model (GEM) of metabolism. Genome‐wide expression and essentiality datasets as well as growth data on various carbohydrates were used to validate and refine i JL208. Discrepancies between model predictions and observations were mechanistically explained using protein structures and network analysis. i JL208 was also used to propose an in silico reduced genome. Comparing this prediction to the minimal cell JCVI‐syn3.0 and its parent JCVI‐syn1.0 revealed key features of a minimal gene set. i JL208 is a stepping‐stone toward model‐driven whole‐genome engineering. SYNOPSIS The first genome‐scale metabolic model for the near‐minimal bacterium Mesoplasma florum is reported. Comparing the model‐driven prediction of a M . florum genome reduction scenario to a closely related minimal cell reveals key features of a minimal gene set. iJL208, the first genome‐scale metabolic model for the near‐minimal organism Mesoplasma florum , comprises 370 reactions and accounts for ˜ 30% of the total gene count in the genome. Model‐driven predictions are validated through the integration of extensive experimental data, including gene expression datasets and growth phenotypes on various sugars. A robust M . florum genome reduction scenario is predicted using gene essentiality data and transcription units, resulting in a minimal genome containing 535 protein‐coding genes. A detailed comparison of this prediction to the phylogenetically related minimal cell JCVI‐syn3.0 reveals key features of a minimal gene set. Graphical Abstract The first genome‐scale metabolic model for the near‐minimal bacterium Mesoplasma florum is reported. Comparing the model‐driven prediction of a M . florum genome reduction scenario to a closely related minimal cell reveals key features of a minimal gene set.