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@ARTICLE{Jayaraman:909762,
author = {Jayaraman, Kumaresan and Trachtmann, Natalia and Sprenger,
Georg A. and Gohlke, Holger},
title = {{P}rotein engineering for feedback resistance in
3-deoxy-{D}-arabino-heptulosonate 7-phosphate synthase},
journal = {Applied microbiology and biotechnology},
volume = {106},
issn = {0171-1741},
address = {New York},
publisher = {Springer},
reportid = {FZJ-2022-03392},
pages = {6505–6517},
year = {2022},
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.},
cin = {IBG-4 / IBI-7 / JSC / NIC},
ddc = {570},
cid = {I:(DE-Juel1)IBG-4-20200403 / I:(DE-Juel1)IBI-7-20200312 /
I:(DE-Juel1)JSC-20090406 / I:(DE-Juel1)NIC-20090406},
pnm = {5111 - Domain-Specific Simulation $\&$ Data Life Cycle Labs
(SDLs) and Research Groups (POF4-511) / 2171 - Biological
and environmental resources for sustainable use (POF4-217) /
2172 - Utilization of renewable carbon and energy sources
and engineering of ecosystem functions (POF4-217) /
Forschergruppe Gohlke $(hkf7_20200501)$ / 5241 - Molecular
Information Processing in Cellular Systems (POF4-524)},
pid = {G:(DE-HGF)POF4-5111 / G:(DE-HGF)POF4-2171 /
G:(DE-HGF)POF4-2172 / $G:(DE-Juel1)hkf7_20200501$ /
G:(DE-HGF)POF4-5241},
typ = {PUB:(DE-HGF)16},
pubmed = {36109385},
UT = {WOS:000854705600002},
doi = {10.1007/s00253-022-12166-9},
url = {https://juser.fz-juelich.de/record/909762},
}