10.1002/anie.202108037
Angewandte Chemie International Edition
RESEARCH ARTICLE
The interest in optically active ionones, including both
we optimized the enzyme by directed evolution and could improve
enantiomers of g-dihydroionone (5), has stimulated the
the conversion of nerylacetone ((Z)-2) to (R)-g-dihydroionone 5 by
development of numerous methods for their synthesis.[5] However, two orders of magnitude to 79% in 48h. It should be noted, that
the most obvious synthesis route toward these compounds is
currently missing, namely the asymmetric cation-olefin cyclization
of pseudoionone (1) or a suitable derivative thereof. It appears
that carbocation formation at the unpolar isoprene end of the
linear chain in combination with enantiospecific folding of the
linear C13 precursor to form a monocycle is difficult to achieve with
classical asymmetric catalysis.
during the preparation of this manuscript, a study by the Hauer
group was published, which similarly reports the biocatalytic
production of (R)- g-dihydroionone (5) by an engineered SHC from
Alicyclobacillus acidocaldarius. After five rounds of directed
evolution, the authors identified an AacSHC variant with four
mutations, which exhibited excellent selectivity (99.5% ee) and
conversion (89%) in seven days.[23]
In contrast, squalene-hopene cyclases (SHCs), which belong
to the class II terpene cyclases, are capable of locking linear
terpenoid substrates in defined chiral conformations, which allows
to achieve polyene cyclizations with perfect stereocontrol.
Consequently, SHCs have great potential as industrial
biocatalysts for the production of enantiopure cyclic terpenoids. A
widely spread model reaction is the cyclization of the linear C30
triterpene squalene (7) into the pentacyclic products hopene (8)
and hopanol (9), through the generation of five new C-C bonds
and nine new stereocenters (Scheme 3).[6] The reaction is initiated
by the protonation of the unactivated terminal isoprene unit with
the unusually acidic middle aspartate of the DXDD active site
motif. The excellent chemo-, regio-, and stereocontrol over the
polycyclization cascade is achieved through pre-folding of the
In our report, we thus confirm the exciting observation that it
is possible to obtain (R)-selective monocyclizations via SHC
biocatalysis (>99% ee) yet using the distinct AciSHC enzyme
(51.6% sequence identity to AacSHC). In addition, we observed
that all of our AciSHC variants exhibited exquisite selectivity in the
transformation of the geometric geranylacetone (2) isomers:
While the Z-isomer yielded the desired monocyclic (R)-5 product,
the E-isomer led to the formation of the bicyclic enolether (S,S)-4.
Biochemical and docking studies helped us to understand the
mechanistic basis of the observed sterodivergent and
enantioselective cyclization reactions. Harnessing this knowledge,
we ultimately succeeded to additionally obtain the enantio-
complementary (S)-5 (>99.9% ee) through the application of an
appropriately chosen SHC-substrate pair.
substrate in
a product-like conformation, stabilization and
shielding of the highly reactive carbocation intermediates from
side reactions, and a selective termination through base assisted
proton elimination or addition of water.[7,8] Terpene cyclases from
the SHC family are promiscuous enzymes and accept molecules
Results and Discussion
In our quest to create an efficient biocatalyst for the
enantioselective production of (dihydro-)ionones, we aimed to
identify an SHC enzyme with the capability to generate
monocyclic products from either (E/Z)-geranylacetone (2) or
(E/Z)-pseudoionone (1). AacSHC,[24] ZmoSHC1,[24] and
engineered variants of these enzymes[25] were previously
reported to be inactive towards 1 and were found to convert 2
exclusively into the bicyclic product 4. Consequently, we chose to
explore the SHC diversity beyond these heavily studied variants
by setting up a comprehensive screening panel of 31 wild-type
enzymes, selected to span all major clades of the phylogenetic
tree (Figure S1). The screening library consisted of 13 previously
characterized class II terpene cyclases from the SHCs family and
18 novel SHC homologs, which were identified through the
presence of two defining PFAM domains for type II triterpene
cyclases (PF13249, PF13243) and the SHC-family specific DXDD
active site motif (Table S2). As thermostable enzyme scaffolds
can be superior starting points for protein engineering and
directed evolution approaches,[26] ten of the novel sequences
were explicitly chosen to originate from thermophilic bacteria.
To characterize our SHC library and evaluate the biocatalysts’
potential for (dihydro)ionone production, we overexpressed the
enzymes in E. coli BL21(DE3) and carried out whole-cell
biotransformations with 10 mM squalene (7), 10 mM (E/Z)-
geranylacetone (2), and 10 mM (E/Z)-pseudoionone (1). Product
formation was analyzed using gas chromatography coupled to
mass spectrometry equipped with a flame ionization detector
(GC-MS-FID) (Figure 1). Nineteen of the investigated SHCs
showed activity towards at least one substrate. Notably, nine of
the active enzymes correspond to novel SHC homologs, with
sequence identities to experimentally characterized variants
between 52.6% and 82.9%. These results validate our
ranging from C10 monoterpenoids[9] to C35 squalene analogues[10]
,
and the cyclization reaction can be initiated through protonation
of unactivated olefins, carbonyls, and epoxides.[11] This is in
contrast to other main families of class II terpene cyclases:
oxidosqualene cyclases are limited to substrates containing an
epoxide functional group for initial protonation,[12] while class II
diterpene cyclases such as ent-copalyl diphosphate synthases
are generally only active towards the diphosphate containing
substrate geranylgeranylpyrophosphate.[13]
Importantly, SHCs have proven to be highly evolvable:
Engineered SHC variants with not more than three mutations
enabled
a
viable industrial-scale process to obtain
Ambrofix®,[14,15] as well as dramatically increased activity and
altered chemo- and stereoselectivity of cyclization reactions with
mono- and sesquiterpenoids, such as geraniol,[11] farnesol,[16] or
citronellal.[17] A limitation of SHCs, however, is their strict (S)-
enantioselectivity at the stereocenter formed after the first
cyclization of all polyisoprenoids tested so far (an overview of
products is given in reviews [18,19]).
Here, we report our efforts to gain enantio-complementary
access to valuable monocyclic terpenoids such as (R)- and (S)- g-
dihydroionone (5) via SHC catalysis. Even though the natural
diversity of SHC sequences is vast,[20,21] most of the work on non-
native substrates has thus far focused on two enzyme variants
from Alicyclobacillus acidocaldarius (AacSHC) and Zymomonas
mobilis (ZmoSHC1)[19] and only one study reported a screening
panel consisting of 12 wild-type enzymes.[22] Thus, to identify
enzymes capable of synthesizing (R)- and (S)- g-dihydroionone
(5), we opted for a screening approach based on an SHC wild-
type library which included 18 novel SHC homologs. Building on
the ability of
a newly identified SHC from Acidothermus
cellulolyticus to generate the monocyclic (R)- g-dihydroionone (5),
2
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