title: Chassis organism from Corynebacterium glutamicum--a top-down approach to identify and delete irrelevant gene clusters.
abstract: 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.
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Systems Biotechnology, Forschungszentrum Jülich, Jülich, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Unthan
authors.forename: Meike
authors.initials: M
authors.lastname: Baumgart
authors.forename: Andreas
authors.initials: A
authors.lastname: Radek
authors.forename: Marius
authors.initials: M
authors.lastname: Herbst
authors.forename: Daniel
authors.initials: D
authors.lastname: Siebert
authors.forename: Natalie
authors.initials: N
authors.lastname: Brühl
authors.forename: Anna
authors.initials: A
authors.lastname: Bartsch
authors.forename: Michael
authors.initials: M
authors.lastname: Bott
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wiechert
authors.forename: Kay
authors.initials: K
authors.lastname: Marin
authors.forename: Stephan
authors.initials: S
authors.lastname: Hans
authors.forename: Reinhard
authors.initials: R
authors.lastname: Krämer
authors.forename: Gerd
authors.initials: G
authors.lastname: Seibold
authors.forename: Julia
authors.initials: J
authors.lastname: Frunzke
authors.forename: Jörn
authors.initials: J
authors.lastname: Kalinowski
authors.forename: Christian
authors.initials: C
authors.lastname: Rückert
authors.forename: Volker F
authors.initials: VF
authors.lastname: Wendisch
authors.forename: Stephan
authors.initials: S
authors.lastname: Noack
mesh_terms: D048230:Corynebacterium glutamicum / Q000235:genetics / Q000254:growth & development*; D020043:Genes, Essential; D005818:Genetic Engineering; D056084:Genetic Fitness; D016680:Genome, Bacterial; D005810:Multigene Family; D010802:Phylogeny; D017384:Sequence Deletion
keywords: Chassis; Corynebacterium glutamicum; Growth studies; Microbial phenotyping; Relevant genes