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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.

Background: alpha-D-Glucosylglycerol (alpha GG) has beneficial functions as a moisturizing agent in cosmetics and potential as a health food material, and therapeutic agent. alpha GG serves as compatible solute in various halotolerant cyanobacteria such as Synechocystis sp. PCC 6803, which synthesizes alpha GG in a two-step reaction: The enzymatic condensation of ADP-glucose and glycerol 3-phosphate by GG-phosphate synthase (GGPS) is followed by the dephosphorylation of the intermediate by the GG-phosphate phosphatase (GGPP). The Gram-positive Corynebacterium glutamicum, an industrial workhorse for amino acid production, does not utilize alpha GG as a substrate and was therefore chosen for the development of a heterologous microbial production platform for alpha GG. Results: Plasmid-bound expression of ggpS and ggpP from Synechocystis sp. PCC 6803 enabled alpha GG synthesis exclusively in osmotically stressed cells of C. glutamicum (pEKEx2-ggpSP), which is probably due to the unique intrinsic control mechanism of GGPS activity in response to intracellular ion concentrations. C. glutamicum was then engineered to optimize precursor supply for alpha GG production: The precursor for alpha GG synthesis ADP-glucose gets metabolized by both the glgA encoded glycogen synthase and the otsA encoded trehalose-6-phosphate synthase. Upon deletion of both genes the alpha GG concentration in culture supernatants was increased from 0.5 mM in C. glutamicum (pEKEx3-ggpSP) to 2.9 mM in C. glutamicum Delta otsA IMglgA (pEKEx3-ggpSP). Upon nitrogen limitation, which inhibits synthesis of amino acids as compatible solutes, C. glutamicum Delta otsA IMglgA (pEKEx3-ggpSP) produced more than 10 mM alpha GG (about 2 g L-1). Conclusions: Corynebacterium glutamicum can be engineered as efficient platform for the production of the compatible solute alpha GG. Redirection of carbon flux towards alpha GG synthesis by elimination of the competing pathways for glycogen and trehalose synthesis as well

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 Delta 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 Delta 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. IMPORTANCE Neu5Ac 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 regul

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.

The ability to use the sialic acid, N-acetylneuraminic acid, Neu5Ac, as a nutrient has been characterized in a number of bacteria, most of which are human pathogens that encounter this molecule because of its presence on mucosal surfaces. The soil bacterium Corynebacterium glutamicum also has a full complement of genes for sialic acid catabolism, and we demonstrate that it can use Neu5Ac as a sole source of carbon and energy and isolate mutants with a much reduced growth lag on Neu5Ac. Disruption of the cg2937 gene, encoding a component of a predicted sialic acid-specific ABC transporter, results in a complete loss of growth of C. glutamicum on Neu5Ac and also a complete loss of [14C]-Neu5Ac uptake into cells. Uptake of [14C]-Neu5Ac is induced by pregrowth on Neu5Ac, but the additional presence of glucose prevents this induction. The demonstration that a member of the Actinobacteria can transport and catabolize Neu5Ac efficiently suggests that sialic acid metabolism has a physiological role in the soil environment.

Recent studies have indicated that nitrogen availability can be an important determinant of primary production in freshwater lakes and that herbivore growth can be limited by low dietary nitrogen availability. Furthermore, a lack of specific essential nitrogenous biochemicals (such as essential amino acids) might be another important constraint on the fitness of consumers. This might be of particular importance for cladoceran zooplankton, which can switch between two alternative reproductive strategies - the production of subitaneously developing and resting eggs. Here, we hypothesize that both the somatic growth and the type of reproduction of the aquatic keystone herbivore Daphnia is limited by the availability of specific essential amino acids in the diet. In laboratory experiments, we investigated this hypothesis by feeding a high quality phytoplankton organism (Cryptomonas) and a green alga of moderate nutritional quality (Chlamydomonas) to a clone of Daphnia pulex with and without the addition of essential amino acids. The somatic growth of D. pulex differed between the algae of different nutritional quality, but not dependent on the addition of dissolved amino acids. However, in reproduction experiments, where moderate crowding conditions at saturating food quantities were applied, addition of the essential amino acids arginine and histidine (but not lysine and threonine) increased the total number and the developmental stage of subitaneous eggs. While D. pulex did not produce resting eggs on Cryptomonas, relatively high numbers of resting eggs were released on Chlamydomonas. When arginine and histidine were added to the green algal diet, the production of resting eggs was effectively suppressed. This demonstrates the high, but previously overlooked importance of single essential amino acids for the reproductive strategy of the aquatic keystone herbivore Daphnia.

Many types of micro-organisms including bacteria are frequently exposed to changes in environmental osmotic conditions. This in particular applies to soil bacteria such as Corynebacterium glutamicum. A change in external osmolarity has direct impact on the bacterial cell volume and hydration. To counteract deleterious effects of sudden hyper- and hypo- osmotic conditions to which they are frequently exposed, these bacteria have developed a range of cellular responses including mechanosensitive channels, which are activated upon sudden hypo- osmotic shifts resulting in the immediate release of small solutes. Based on structural similarity, MscCG (the YggB gene product) of C. glutamicum belongs to the family of MscS-type mechanosensitive channels...

Based on sequence similarity, the mscCG gene product of Corynebacterium glutamicum belongs to the family of MscS-type mechanosensitive channels. In order to investigate the physiological significance of MscCG in response to osmotic shifts in detail, we studied its properties using both patch-clamp techniques and betaine efflux kinetics. After heterologous expression in an Escherichia coli strain devoid of mechanosensitive channels, in patch-clamp analysis of giant E. coli spheroplasts MscCG showed the typical pressure dependent gating behavior of a stretch-activated channel with a current/voltage dependence indicating a strongly rectifying behavior. Apart from that. MscCG is characterized by significant functional differences with respect to conductance, ion selectivity and desensitation behavior as compared to MscS from E. coli. Deletion and complementation studies in C glutamicum showed a significant contribution of MscCG to betaine efflux in response to hypoosmotic conditions. A detailed analysis of concomitant betaine uptake (by the betaine transporter BetP) and efflux (by MscCG) under hyperosmotic conditions indicates that MscCG may act in osmoregulation in C. glutamicum by fine-tuning the steady state concentration of compatible solutes in the cytoplasm which are accumulated in response to hyperosmotic stress. (C) 2010 Elsevier B.V. All rights reserved.