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.

Harnessing novel chromosomal integration loci to utilize an organosolv‐derived hemicellulose fraction for isobutanol production with engineered Corynebacterium glutamicum

“…Although they were shown to be non‐relevant for ordinary growth under laboratory conditions, the overall function is to date not clarified in depth and a genetic stability is not guaranteed (Baumgart et al ., 2013). Second, we contemplated non‐essential chromosome sections in the published list of deletable regions (Unthan et al ., 2014). These provide ideal arrays for the integration of genes and exclude lethal effects that arise from disruption of essential genetic structures.…”

“… g. Gene outside (up‐ or downstream) the non‐essential gene cluster (Unthan et al ., 2014); downstream gene is located inside the non‐essential gene cluster.…”

Engineering Corynebacterium glutamicum for fast production of l-lysine and l-pipecolic acid

“…The physiology, biochemistry, and genetics of Llysine biosynthesis are well understood and have been employed in rational development of L-lysine producing strains. For example, the L-lysine production strain GRLys1 (Unthan et al 2015) possesses two gene copies of the key enzyme of L-lysine biosynthesis, aspartokinase (Schrumpf et al 1992;Cremer et al 1991;Kalinowski et al 1991), devoid of feedback inhibition by L-lysine (encoded by lysC T311I ), Electronic supplementary material The online version of this article (doi:10.1007/s00253-016-7682-6) contains supplementary material, which is available to authorized users. two copies of the L-lysine biosynthesis genes asd, dapA, dapB, ddh, and lysA, and two copies of L-lysine export gene lysE (Vrljic et al 1996).…”

Production of 4-Ene-3-ketosteroids in Corynebacterium glutamicum

“…In this sense, a massive deletion of the majority of the non-essential DNA sequences has been recently carried out in order to improve its properties [4].…”