For synthetic biology applications, a robust structural basis is required, which can be constructed either from scratch or in a top‐down approach starting from any existing organism. In this study, we initiated the top‐down construction of a chassis organism from Corynebacterium glutamicum ATCC 13032, aiming for the relevant gene set to maintain its fast growth on defined medium. We evaluated each native gene for its essentiality considering expression levels, phylogenetic conservation, and knockout data. Based on this classification, we determined 41 gene clusters ranging from 3.7 to 49.7 kbp as target sites for deletion. 36 deletions were successful and 10 genome‐reduced strains showed impaired growth rates, indicating that genes were hit, which are relevant to maintain biological fitness at wild‐type level. In contrast, 26 deleted clusters were found to include exclusively irrelevant genes for growth on defined medium. A combinatory deletion of all irrelevant gene clusters would, in a prophage‐free strain, decrease the size of the native genome by about 722 kbp (22%) to 2561 kbp. Finally, five combinatory deletions of irrelevant gene clusters were investigated. The study introduces the novel concept of relevant genes and demonstrates general strategies to construct a chassis suitable for biotechnological application.

Phosphoenolpyruvate-dependent glucose phosphorylation via the phosphotransferase system (PTS) is the major path of glucose uptake in Corynebacterium glutamicum , but some growth from glucose is retained in the absence of the PTS. The growth defect of a deletion mutant lacking the general PTS component HPr in glucose medium could be overcome by suppressor mutations leading to the high expression of inositol utilization genes or by the addition of inositol to the growth medium if a glucokinase is overproduced simultaneously. PTS-independent glucose uptake was shown to require at least one of the inositol transporters IolT1 and IolT2 as a mutant lacking IolT1, IolT2, and the PTS component HPr could not grow with glucose as the sole carbon source. Efficient glucose utilization in the absence of the PTS necessitated the overexpression of a glucokinase gene in addition to either iolT1 or iolT2 . IolT1 and IolT2 are low-affinity glucose permeases with K _s values of 2.8 and 1.9 mM, respectively. As glucose uptake and phosphorylation via the PTS differs from glucose uptake via IolT1 or IolT2 and phosphorylation via glucokinase by the requirement for phosphoenolpyruvate, the roles of the two pathways for l -lysine production were tested. The l -lysine yield by C. glutamicum DM1729, a rationally engineered l -lysine-producing strain, was lower than that by its PTS-deficient derivate DM1729Δ hpr , which, however, showed low production rates. The combined overexpression of iolT1 or iolT2 with ppgK , the gene for PolyP/ATP-dependent glucokinase, in DM1729Δ hpr enabled l -lysine production as fast as that by the parent strain DM1729 but with 10 to 20% higher l -lysine yield.