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.

Targeted top-down strategies for genome reduction are considered to have a high potential for providing robust basic strains for synthetic biology and industrial biotechnology. Recently, we created a library of 26 genome-reduced strains of Corynebacterium glutamicum carrying broad deletions in single gene clusters and showing wild-type-like biological fitness. Here, we proceeded with combinatorial deletions of these irrelevant gene clusters in two parallel orders, and the resulting library of 28 strains was characterized under various environmental conditions. The final chassis strain C1* carries a genome reduction of 13.4% (412 deleted genes) and shows wild-type-like growth behavior in defined medium with d-glucose as carbon and energy source. Moreover, C1* proves to be robust against several stresses (including oxygen limitation) and shows long-term growth stability under defined and complex medium conditions. In addition to providing a novel prokaryotic chassis strain, our results comprise a large strain library and a revised genome annotation list, which will be valuable sources for future systemic studies of C. glutamicum.

Corynebacterium glutamicum metabolizes sialic acid (Neu5Ac) to fructose-6-phosphate (fructose-6P) via the consecutive activity of the sialic acid importer SiaEFGI, N-acetylneuraminic acid lyase (NanA), N-acetylmannosamine kinase (NanK), N-acetylmannosamine-6P epimerase (NanE), N-acetylglucosamine-6P deacetylase (NagA), and glucosamine-6P deaminase (NagB). Within the cluster of the three operons nagAB, nanAKE, and siaEFGI for Neu5Ac utilization a fourth operon is present, which comprises cg2936, encoding a GntR-type transcriptional regulator, here named NanR. Microarray studies and reporter gene assays showed that nagAB, nanAKE, siaEFGI, and nanR are repressed in wild-type (WT) C. glutamicum but highly induced in a ⌬nanR C. glutamicum mutant. Purified NanR was found to specifically bind to the nucleotide motifs A [AC]G[CT][AC]TGATGT C[AT][TG]ATGT[AC]TA located within the nagA-nanA and nanR-sialA intergenic regions. Binding of NanR to promoter regions was abolished in the presence of the Neu5Ac metabolism intermediates GlcNAc-6P and N-acetylmannosamine-6-phosphate (ManNAc-6P). We observed consecutive utilization of glucose and Neu5Ac as well as fructose and Neu5Ac by WT C. glutamicum, whereas the deletion mutant C. glutamicum ⌬nanR simultaneously consumed these sugars. Increased reporter gene activities for nagAB, nanAKE, and nanR were observed in cultivations of WT C. glutamicum with Neu5Ac as the sole substrate compared to cultivations when fructose was present. Taken together, our findings show that Neu5Ac metabolism in C. glutamicum is subject to catabolite repression, which involves control by the repressor NanR. IMPORTANCENeu5Ac utilization is currently regarded as a common trait of both pathogenic and commensal bacteria. Interestingly, the nonpathogenic soil bacterium C. glutamicum efficiently utilizes Neu5Ac as a substrate for growth. Expression of genes for Neu5Ac utilization in C. glutamicum is here shown to depend on the transcriptional regulator NanR, which is the first GntR-type regulator of Neu5Ac metabolism not to use Neu5Ac as effector but relies instead on the inducers GlcNAc-6P and ManNAc-6P. The identification of conserved NanR-binding sites in intergenic regions within the operons for Neu5Ac utilization in pathogenic Corynebacterium species indicates that the mechanism for the control of Neu5Ac catabolism in C. glutamicum by NanR as described in this work is probably conserved within this genus. The Gram-positive Corynebacterium glutamicum is mostly known for its application in the industrial production of amino acids, mainly L-glutamate and L-lysine (1, 2), and has become a versatile cell factory for the production of various commodity products (3-5). C. glutamicum utilizes a large variety of sugars and organic acids as sources of carbon and energy (6, 7) and additionally has been genetically engineered for the utilization of alternative feedstocks such as starch, glycerol, xylose, glucuronic acid, and N-acetylglucosamine (8-12). In contrast to many other bacterial species, C. glutamic...

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