Physiological Response of Corynebacterium glutamicum to Indole

Abstract

The aromatic heterocyclic compound indole is widely spread in nature. Due to its floral odor indole finds application in dairy, flavor, and fragrance products. Indole is an inter- and intracellular signaling molecule influencing cell division, sporulation, or virulence in some bacteria that synthesize it from tryptophan by tryptophanase. Corynebacterium glutamicum that is used for the industrial production of amino acids including tryptophan lacks tryptophanase. To test if indole is metabolized by C. glutamicum or has a regulatory role, the physiological response to indole by this bacterium was studied. As shown by RNAseq analysis, indole, which inhibited growth at low concentrations, increased expression of genes involved in the metabolism of iron, copper, and aromatic compounds. In part, this may be due to iron reduction as indole was shown to reduce Fe3+ to Fe2+ in the culture medium. Mutants with improved tolerance to indole were selected by adaptive laboratory evolution. Among the mutations identified by genome sequencing, mutations in three transcriptional regulator genes were demonstrated to be causal for increased indole tolerance. These code for the regulator of iron homeostasis DtxR, the regulator of oxidative stress response RosR, and the hitherto uncharacterized Cg3388. Gel mobility shift analysis revealed that Cg3388 binds to the intergenic region between its own gene and the iolT2-rhcM2D2 operon encoding inositol uptake system IolT2, maleylacetate reductase, and catechol 1,2-dioxygenase. Increased RNA levels of rhcM2 in a cg3388 deletion strain indicated that Cg3388 acts as repressor. Indole, hydroquinone, and 1,2,4-trihydroxybenzene may function as inducers of the iolT2-rhcM2D2 operon in vivo as they interfered with DNA binding of Cg3388 at physiological concentrations in vitro. Cg3388 was named IhtR.

Dynamic Co-Cultivation Process of Corynebacterium glutamicum Strains for the Fermentative Production of Riboflavin

“…Under oxygen-deprivation conditions, C. glutamicum has a better recalcitrance towards furfural and 5-hydroxymethylfurfural than E. coli and S. cerevisiae [74], but aerobic growth, as studied here, is negatively affected. These limitations can be overcome in upcoming projects via adaptive laboratory evolution as shown in C. glutamicum for methanol or indole [49,75] or by genetic engineering as shown for by furfural detoxification by protein FudC [76]. We believe this work is important for application of real SSL, but not within the scope of the study presented here.…”

Genomic and Transcriptomic Investigation of the Physiological Response of the Methylotroph Bacillus methanolicus to 5-Aminovalerate

“…For example, C. glutamicum, which neither synthesizes nor secretes a siderophore, grows faster in the presence of exogenous iron chelators (Liebl et al, 1989), although they are not strictly required (Graf et al, 2019). In C. glutamicum, extracellularly added indole was shown to inhibit growth, to increase expression of iron acquisition genes, and to chelate iron, and a mutant of the iron homeostasis regulator DtxR isolated by ALE improved indole tolerance (Walter et al, 2020). The genome of B. methanolicus does not code for a DtxR homolog according to BLASTx analysis; however, the study presented here also identifies involvement of a regulatory gene, BMMGA3_RS14080, coding for an Rrf2 family transcriptional regulator.…”

“…Taken together, ALE, in particular in combination with transcriptomics, is suitable to select mutants with increased tolerance toward substrates, e.g., methanol (Tuyishime et al, 2018;Hennig et al, 2020;Wang et al, 2020), and products, e.g., indole (Walter et al, 2020). Genome sequencing of ALE strains is straightforward, identifying mutations in genes that may be relevant for the selected phenotype.…”

“…Genome sequencing of ALE strains is straightforward, identifying mutations in genes that may be relevant for the selected phenotype. For microorganisms amenable to gene deletion or gene replacement, reverse engineering of the identified mutations into the parental strain to identify causal mutations among the candidates identified by genome sequencing is the strategy of choice (Hennig et al, 2020;Walter et al, 2020). Due to the lack of genome editing tools for B. methanolicus, we opted to analyze strains overexpressing the mutant genes identified by genome sequencing (Figures 6A-D) in a similar manner as used, e.g., for validating improved cadmium tolerance observed in ALE of a cyanobacterium (Xu et al, 2018).…”

Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being

“…Microbial production normally outcompetes traditional extraction from plant tissues, given drawbacks such as low yield, seasonal variation, and product instability, associated to the latter [87,88]. In comparison with other tested hosts, C. glutamicum shows superior natural robustness against toxic aromatics [89], making it a promising host for the production of aromatic molecules [88,90,91] (Table 2). This trait can be partially attributed to the complex catabolic network for aromatic compounds in C. glutamicum, which needs to be modified prior to establishing polyphenol biosynthesis in this organism [91,92].…”