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@ARTICLE{Weber:1006586,
author = {Weber, Douglas and de Souza Bastos, Lucas and Winkler,
Margit and Ni, Yeke and Aliev, Abil E. and Hailes, Helen C.
and Rother, Doerte},
title = {{M}ulti-enzyme catalysed processes using purified and
whole-cell biocatalysts towards a 1,3,4-substituted
tetrahydroisoquinoline},
journal = {RSC Advances},
volume = {13},
number = {15},
issn = {2046-2069},
address = {London},
publisher = {RSC Publishing},
reportid = {FZJ-2023-01727},
pages = {10097 - 10109},
year = {2023},
abstract = {In this work, two multi-enzyme catalysed processes to
access a 1,3,4-substituted tetrahydroisoquinoline (THIQ),
using either purified enzymes or lyophilised whole-cell
catalysts, are presented. A key focus was the first step in
which the reduction of 3-hydroxybenzoic acid (3-OH-BZ) into
3-hydroxybenzaldehyde (3-OH-BA) was catalysed by a
carboxylate reductase (CAR) enzyme. Incorporation of the
CAR-catalysed step enables substituted benzoic acids as the
aromatic components, which can potentially be obtained from
renewable resources by microbial cell factories. In this
reduction, the implementation of an efficient cofactor
regeneration system of both ATP and NADPH was crucial. Two
different recycling approaches, either using purified
enzymes or lyophilised whole-cells, were established and
compared. Both of them showed high conversions of the acid
into 3-OH-BA $(>80\%).$ However, the whole-cell system
showed superior performance because it allowed the
combination of the first and second steps into a one-pot
cascade with excellent HPLC yields $(>99\%,$ enantiomeric
excess (ee) ≥ $95\%)$ producing the intermediate
3-hydroxyphenylacetylcarbinol. Moreover, enhanced substrate
loads could be achieved compared to the system employing
only purified enzymes. The third and fourth steps were
performed in a sequential mode to avoid cross-reactivities
and the formation of several side products. Thus,
(1R,2S)-metaraminol could be formed with high HPLC yields
$(>90\%,$ isomeric content (ic) ≥ $95\%)$ applying either
purified or whole-cell transaminases from Bacillus
megaterium (BmTA) or Chromobacterium violaceum (Cv2025).
Finally, the cyclisation step was performed using either a
purified or lyophilised whole-cell norcoclaurine synthase
variant from Thalictrum flavum (ΔTfNCS-A79I), leading to
the formation of the target THIQ product with high HPLC
yields $(>90\%,$ ic > $90\%).$ As many of the educts applied
are from renewable resources and a complex product with
three chiral centers can be gained by only four highly
selective steps, a very step- and atom efficient approach to
stereoisomerically pure THIQ is shown.},
cin = {IBG-1},
ddc = {540},
cid = {I:(DE-Juel1)IBG-1-20101118},
pnm = {2172 - Utilization of renewable carbon and energy sources
and engineering of ecosystem functions (POF4-217)},
pid = {G:(DE-HGF)POF4-2172},
typ = {PUB:(DE-HGF)16},
pubmed = {37006360},
UT = {WOS:000962147800001},
doi = {10.1039/D3RA01210G},
url = {https://juser.fz-juelich.de/record/1006586},
}