Similarities in the structure of the transcriptional repressor AmtR in two different space groups suggest a model for the interaction with GlnK

Sevvana, Madhumati; Hasselt, Kristin; Grau, F.C.; Burkovski, Andreas; Muller, Yves A.

doi:10.1107/s2053230x17002485

Cited by 2 publications

(3 citation statements)

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“…RNAseq analysis revealed higher expression of genes belonging to the AmtR regulon of nitrogen starvation in GluA T5 and the parental strain GluA T0 than in GluA T7, which carries GltB E686Q (Additional file 2 : Tables S1 and S2). Binding to operator DNA by homodimeric AmtR is not released by a small-molecule effector, but by GlnK when adenylylated at tyrosine residue 51 [ 61 ], likely in a 6:6 stoichiometric (AmtR 2 ) 3 –(GlnK 3 ) 2 complex [ 62 ]. The adenylation status of PII-type signal transduction protein GlnK is controlled by adenyltransferase GlnD under nitrogen starvation, which is most likely perceived as ammonium limitation [ 63 ].…”

Section: Discussionmentioning

confidence: 99%

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum

et al. 2021

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Background The demand for biobased polymers is increasing steadily worldwide. Microbial hosts for production of their monomeric precursors such as glutarate are developed. To meet the market demand, production hosts have to be improved constantly with respect to product titers and yields, but also shortening bioprocess duration is important. Results In this study, adaptive laboratory evolution was used to improve a C. glutamicum strain engineered for production of the C5-dicarboxylic acid glutarate by flux enforcement. Deletion of the l-glutamic acid dehydrogenase gene gdh coupled growth to glutarate production since two transaminases in the glutarate pathway are crucial for nitrogen assimilation. The hypothesis that strains selected for faster glutarate-coupled growth by adaptive laboratory evolution show improved glutarate production was tested. A serial dilution growth experiment allowed isolating faster growing mutants with growth rates increasing from 0.10 h−1 by the parental strain to 0.17 h−1 by the fastest mutant. Indeed, the fastest growing mutant produced glutarate with a twofold higher volumetric productivity of 0.18 g L−1 h−1 than the parental strain. Genome sequencing of the evolved strain revealed candidate mutations for improved production. Reverse genetic engineering revealed that an amino acid exchange in the large subunit of l-glutamic acid-2-oxoglutarate aminotransferase was causal for accelerated glutarate production and its beneficial effect was dependent on flux enforcement due to deletion of gdh. Performance of the evolved mutant was stable at the 2 L bioreactor-scale operated in batch and fed-batch mode in a mineral salts medium and reached a titer of 22.7 g L−1, a yield of 0.23 g g−1 and a volumetric productivity of 0.35 g L−1 h−1. Reactive extraction of glutarate directly from the fermentation broth was optimized leading to yields of 58% and 99% in the reactive extraction and reactive re-extraction step, respectively. The fermentation medium was adapted according to the downstream processing results. Conclusion Flux enforcement to couple growth to operation of a product biosynthesis pathway provides a basis to select strains growing and producing faster by adaptive laboratory evolution. After identifying candidate mutations by genome sequencing causal mutations can be identified by reverse genetics. As exemplified here for glutarate production by C. glutamicum, this approach allowed deducing rational metabolic engineering strategies.

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Section: Discussionmentioning

confidence: 99%

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum

et al. 2021

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“…Based on the crystal structure of C. glutamicum AmtR, a model for interaction of AmtR with adGlnK has been proposed. In this model, two adGlnK trimers bind to an AmtR hexamer and block the binding of the AmtR operator (Sevvana et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…Deletion of the glnK gene abolishes the upregulation of AmtR-controlled genes, and the derepression of genes under AmtR only occurs if GlnK is adenylylated at position 51, since exchanging Tyr51 for phenylalanine resulted in an identical phenotype to those observed for glnK deletion mutants (Nolden et al, 2001;Beckers et al, 2005). Whereas the interaction between AmtR and its target DNA has been well characterized, the details and structural implications of the adenylylation of GlnK as well as the mechanism by which adGlnK derepresses AmtR-controlled gene transcription currently remain largely elusive (Palanca & Rubio, 2016;Sevvana et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Crystal structures of adenylylated and unadenylylated P_IIprotein GlnK fromCorynebacterium glutamicum

Grau¹,

Burkovski²,

Muller³

2021

Acta Crystallogr D Struct Biol

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PII proteins are ubiquitous signaling proteins that are involved in the regulation of the nitrogen/carbon balance in bacteria, archaea, and some plants and algae. Signal transduction via PII proteins is modulated by effector molecules and post-translational modifications in the PII T-loop. Whereas the binding of ADP, ATP and the concomitant binding of ATP and 2-oxoglutarate (2OG) engender two distinct conformations of the T-loop that either favor or disfavor the interaction with partner proteins, the structural consequences of post-translational modifications such as phosphorylation, uridylylation and adenylylation are far less well understood. In the present study, crystal structures of the PII protein GlnK from Corynebacterium glutamicum have been determined, namely of adenylylated GlnK (adGlnK) and unmodified unadenylylated GlnK (unGlnK). AdGlnK has been proposed to act as an inducer of the transcription repressor AmtR, and the adenylylation of Tyr51 in GlnK has been proposed to be a prerequisite for this function. The structures of unGlnK and adGlnK allow the first atomic insights into the structural implications of the covalent attachment of an AMP moiety to the T-loop. The overall GlnK fold remains unaltered upon adenylylation, and T-loop adenylylation does not appear to interfere with the formation of the two major functionally important T-loop conformations, namely the extended T-loop in the canonical ADP-bound state and the compacted T-loop that is adopted upon the simultaneous binding of Mg-ATP and 2OG. Thus, the PII-typical conformational switching mechanism appears to be preserved in GlnK from C. glutamicum, while at the same time the functional repertoire becomes expanded through the accommodation of a peculiar post-translational modification.

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Similarities in the structure of the transcriptional repressor AmtR in two different space groups suggest a model for the interaction with GlnK

Cited by 2 publications

References 29 publications

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum

Crystal structures of adenylylated and unadenylylated P_IIprotein GlnK fromCorynebacterium glutamicum

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Similarities in the structure of the transcriptional repressor AmtR in two different space groups suggest a model for the interaction with GlnK

Cited by 2 publications

References 29 publications

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum

Crystal structures of adenylylated and unadenylylated PIIprotein GlnK fromCorynebacterium glutamicum

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Crystal structures of adenylylated and unadenylylated P_IIprotein GlnK fromCorynebacterium glutamicum