Journal Article

FZJ-2022-03392

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Protein engineering for feedback resistance in 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

Jayaraman, K. ; Trachtmann, N. ; Sprenger, G. A. (Corresponding author) ; Gohlke, H. (Corresponding author)FZJ*

2022
Springer New York

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Please use a persistent id in citations: http://hdl.handle.net/2128/31997  doi:10.1007/s00253-022-12166-9

Abstract: The shikimate pathway delivers aromatic amino acids (AAAs) in prokaryotes, fungi, and plants and is highly utilized in the industrial synthesis of bioactive compounds. Carbon flow into this pathway is controlled by the initial enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS). AAAs produced further downstream, phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), regulate DAHPS by feedback inhibition. Corynebacterium glutamicum, the industrial workhorse for amino acid production, has two isoenzymes of DAHPS, AroF (Tyr sensitive) and AroG (Phe and Tyr sensitive). Here, we introduce feedback resistance against Tyr in the class I DAHPS AroF (AroFcg). We pursued a consensus approach by drawing on structural modeling, sequence and structural comparisons, knowledge of feedback-resistant variants in E. coli homologs, and computed folding free energy changes. Two types of variants were predicted: Those where substitutions putatively either destabilize the inhibitor binding site or directly interfere with inhibitor binding. The recombinant variants were purified and assessed in enzyme activity assays in the presence or absence of Tyr. Of eight AroFcg variants, two yielded > 80% (E154N) and > 50% (P155L) residual activity at 5 mM Tyr and showed > 50% specific activity of the wt AroFcg in the absence of Tyr. Evaluation of two and four further variants at positions 154 and 155 yielded E154S, completely resistant to 5 mM Tyr, and P155I, which behaves similarly to P155L. Hence, feedback-resistant variants were found that are unlikely to evolve by point mutations from the parental gene and, thus, would be missed by classical strain engineering.

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Contributing Institute(s):

1. Bioinformatik (IBG-4)
2. Strukturbiochemie (IBI-7)
3. Jülich Supercomputing Center (JSC)
4. John von Neumann - Institut für Computing (NIC)

Research Program(s):

1. 5111 - Domain-Specific Simulation & Data Life Cycle Labs (SDLs) and Research Groups (POF4-511) (POF4-511)
2. 2171 - Biological and environmental resources for sustainable use (POF4-217) (POF4-217)
3. 2172 - Utilization of renewable carbon and energy sources and engineering of ecosystem functions (POF4-217) (POF4-217)
4. Forschergruppe Gohlke (hkf7_20200501) (hkf7_20200501)
5. 5241 - Molecular Information Processing in Cellular Systems (POF4-524) (POF4-524)

Appears in the scientific report 2022

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Database coverage:
; ; ; BIOSIS Previews ; Biological Abstracts ; Clarivate Analytics Master Journal List ; Current Contents - Agriculture, Biology and Environmental Sciences ; Current Contents - Life Sciences ; DEAL Springer ; Ebsco Academic Search ; Essential Science Indicators ; IF >= 5 ; JCR ; SCOPUS ; Science Citation Index Expanded ; Web of Science Core Collection

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