Enzymatic C-H activation of aromatic compounds

through CO2 fixationGodwin A. Aleku1, Annica Saaret1, Ruth T. Bradshaw-Allen1, Sasha R. Derrington1, Gabriel R.

Titchiner1, Irina Gostimskaya1, Deepankar Gahloth1, David A. Parker2, Sam Hay1, David

Leys1*

1Manchester Institute of Biotechnology, School of Chemistry, University of Manchester,

Manchester, UK.2 Innovation/Biodomain, Shell International Exploration and Production Inc., Westhollow

Technology Center, Houston, USA.

*corresponding author: david.leys@manchester.ac.uk

The direct C-H carboxylation of aromatic compounds is an attractive route

to the corresponding carboxylic acids, but remains challenging under mild

conditions. It has been proposed that the first step in the anaerobic

microbial degradation of recalcitrant aromatic compounds is a UbiD-

mediated carboxylation. In this study, we use the UbiD-enzyme, ferulic acid

decarboxylase in combination with a carboxylic acid reductase to create

aromatic degradation inspired cascade reactions. These lead to efficient

functionalization of styrene through CO2 fixation. We reveal that rational

structure-guided laboratory evolution can expand the substrate scope,

resulting in activity on a range of mono- and bi-cyclic aromatic compounds

through a single mutation. Selected variants demonstrate a 150-fold

improvement in the coumarillic acid to benzofuran + CO2 conversion and

unlock reactivity towards naphthoic acid. Our data demonstrates that

UbiD-mediated C-H activation is a versatile tool for the transformation of

aryl/alkene compounds and CO2 into commodity chemicals.

Introduction

Carboxylic acids can be prepared through direct C-H carboxylation of aromatic

compounds, a synthetically challenging reaction under mild conditions [1-4].

Enzymatic carboxylation has recently emerged as an attractive alternative and

green route to produce carboxylic acids, frequently making use of

(de)carboxylases involved in secondary metabolism [5]. Unfortunately, many

(de)carboxylases are restricted in substrate scope, and efficient carboxylation

requires high concentrations of [CO2] (Fig 1a). In contrast, the widespread UbiD

family of enzymes offers a particularly diverse range of natural substrates,

ranging from cinnamic acid and related compounds to furan or indole-containing

heteroaromatic carboxylic acids [6]. The general reaction mediated by UbiD

enzymes, the interconversion of alkene/aryl + CO2 with the corresponding

unsaturated/aromatic carboxylic acid, is mediated by the recently described

prenylated FMN cofactor (prFMN, [7]; Fig 1b).

The UbiD cofactor is synthesised by the associated UbiX, a flavin

prenyltransferase that extends the isoalloxazine FMN ring system through

prenylation with a 4th non-aromatic ring [8,9]. Oxidative maturation of the UbiX

product leads to formation of the prFMNiminium form of the cofactor. This species

has azomethine ylide characteristics, and has been proposed to form

cycloadducts with dipolarophile species such as , -α β unsaturated carboxylic acids

[10]. Indeed, a reversible 1,3 dipolar cycloaddition process has been

demonstrated to underpin the decarboxylation of cinnamic acid [11] (Fig 1b). It

is unclear to what extent the unusual 1,3 dipolar cycloaddition-based mechanism

is used in case of aromatic substrates, given the inherent barrier presented for

cycloaddition in such compounds [12]. Indeed, alternative proposals for prFMN-

based covalent catalysis have been put forward in case of activated aromatic

substrates [13,14].

While the majority of UbiDs act as decarboxylases, a subset is proposed to

function in the carboxylative direction at ambient [CO2], such as the Thauera

aromatica phenolphosphate carboxylase [15]. This enzyme converts the

phosphorylated phenol substrate to 4-hydroxybenzoic acid, thus coupling

carboxylation to dephosphorylation through an unknown mechanism. Arguably,

the most challenging reaction proposed to be catalyzed by UbiD enzymes is the

activation of highly stable aromatic compounds such as naphthalene and

benzene, a reversible process that is coupled to a second, irreversible step by

conversion of the aromatic carboxylic acid product into the corresponding acyl-

CoA thioester ([16,17]; Fig 1c). This process is believed to be essential to

anaerobic degradation of these compounds by some microbial cultures, but

corresponding enzyme activity has yet to be conclusively demonstrated.

The chemistry that underpins microbial benzene/naphthalene degradation has

inspired us to design and develop cascade reactions enabling efficient

functionalization of terminal alkenes to the corresponding aldehyde, alcohol,

amide or amine derivatives through ambient CO2 fixation. We sought to mimic

and validate the proposed pathway for microbial anaerobic degradation of

benzene/naphtalene by coupling the model UbiD enzyme Aspergillus niger

ferulic acid decarboxylase (AnFdc) (which catalyses cinnamic acid to styrene +

CO2 interconversion [10,11]) with carboxylic acid reductase (CAR) [18]. The

latter is capable of catalysing the irreversible conversion of aromatic carboxylic

acids to the corresponding aldehydes in an ATP- and NADPH-dependent manner

(Fig 1c). We demonstrate that efficient functionalization of styrene can be

achieved through CO2 fixation at ambient conditions through the use of Fdc/CAR

cascade reactions. We also establish that structure-guided laboratory evolution

can expand the substrate scope of ferulic acid decarboxylases, resulting in

activity on a range of mono- and bi-cyclic aromatic compounds. This extends the

product profile of the Fdc/CAR system enabling the C-H activation of

(hetero)aromatic compounds.

Results

Coupling AnFdc to CAR results in efficient CO2 fixation - To test whether

combination of a UbiD-enzyme with CAR could yield effective carboxylation

under ambient conditions, we used wild-type AnFdc [10,11] with styrene as the

model substrate. In absence of CAR, incubation of purified AnFdc or lyophilised

E. coli whole cells containing AnFdc with 5 mM styrene 1 in the presence 0.5-1 M

KHCO3 yielded cinnamic acid 2, albeit in low conversion (<2%). In contrast, a

one-pot reaction containing purified Tsukamurella paurometabola carboxylic

acid reductase (TpCAR; [19]) and AnFdc, converted 5 mM styrene 1 to

cinnamaldehyde 3 with up to 25% conversion via the cinnamic acid intermediate

(in presence of 0.05-0.5 M KHC03 or (NH4)HCO3 and with supply of

stoichiometric amounts of NADPH and ATP). Given that the biotransformation of

the intermediate cinnamic acid 2 using E. coli whole cells expressing TpCAR

afforded 97% conversion to cinnamyl alcohol 4, (due to endogenous alcohol

dehydrogenase (EcADH) activity, Extended Data Figs 1, 2), we sought to

implement the three step AnFdc-TpCAR-EcADH cascade (Fig 2). In order to

obtain high conversion of styrene 1 to cinnamyl alcohol 4, these catalysts were

assembled in one-pot, and conversion in the presence of KHCO3 was monitored.

In this case, AnFdc was utilised as purified enzyme or lyophilised cell free extract

(CFE), whereas TpCAR/EcADH were employed using E. coli whole-cell

preparation. This revealed the formation of cinnamyl alcohol as the major

product, with cinnamaldehyde and cinnamic acid only detected in trace amounts

(Fig 2, Extended Data Fig 2a). Furthermore, optimum conversion levels were

obtained at 40-60 mM KHCO3, revealing full conversion occurs near

stoichiometric molar ratios of styrene and KHCO3 (as the CO2 source; Extended

Data Fig 1b). Biotransformation conversions were similar when using AnFdc as

lyophilised CFE preparation or when using related homologues such as

Saccharomyces cerevisiae (ScFdc; Extended Data Fig 1e).

Expanding the AnFdc - CAR product scope - To further demonstrate the

potential for formation of a wide range of compounds via AnFdc-catalysed CO2

fixation, we took advantage of a recently established protocol for CAR-mediated

NADPH-free amidation [20]. We optimized the NADPH-independent TpCAR-

catalysed amidation process for cinnamic acid 2 in 500 mM (NH4)HCO3, leading

to the formation of cinnamamide 5 from 2 in up to 75 % conversion (Extended

Data Fig 3a). By coupling purified preparations AnFdc with TpCAR in an in vitro

one-pot biotransformation reaction with stoichiometric amount of ATP in 500

mM (NH4)HCO3 buffer, styrene 1 was converted to cinnamamide 5 in up to 14 %

conversion (Fig 2). The inherent reactivity of the CAR aldehyde product offers

further opportunities to extend the product profile. In order to demonstrate

access to N-alkylated secondary amines, we linked the Fdc-CAR system to a

biocatalytic reductive amination step, employing Cystobacter ferrugineus imine

reductase (CfIRED; [21]) as the reductive aminase, while Segniliparus rugosus

CAR (SrCAR) ([22]) was utilised for the carboxylate reduction. Using 2.5

equivalents of cyclopropylamine as a primary amine source, we found the three

step AnFdc-SrCAR-CfIRED cascade afforded the secondary amine 6 from styrene

1 in up to 93% conversion (Fig 2, Extended Data Figs 2c, 3).

AnFdc is a weak heteroaromatic acid decarboxylase - Encouraged by the fact

the AnFdc-CAR system enables conversion of terminal alkenes such as styrene to

respectively aldehydes, amides, secondary amines or allylic alcohols via

stoichiometric carboxylation, we next aimed to extend our approach towards the

functionalisation of (hetero)-aromatic compounds which are prevalent medicinal

and synthetic scaffolds. Unfortunately many UbiD-enzymes that act on such

compounds are oxygen-sensitive and/or require cofactor reconstitution when

produced through heterologous expression [6,7,13,14]. We thus took advantage

of the robust nature of the AnFdc enzyme and set out to evaluate and expand the

scope for activity with (hetero)aromatic acids. We identified putative alternative

substrates of AnFdc by screening a compound library comprising structurally

diverse (hetero)aromatic acids (Extended Data Fig 4) for inhibitory effect on

the decarboxylation of cinnamic acid 2. Compounds that acted as competitive

inhibitors were rescreened for AnFdc decarboxylation activity under

biotransformation conditions, enabling the detection of very weak

decarboxylation activity of the wild-type AnFdc towards the heteroaromatic

acids 7 and 8 with conversion of up to 5% achieved (Fig. 3a). In contrast, no

such activity could be observed with compounds 9 and 10, even though these

compounds exhibited competitive inhibition towards decarboxylation of 2. This

data suggests that AnFdc can potentially mimic other UbiD-enzymes such as

those involved in benzene/napthalene degradation, if the activity with

(hetero)aromatic compounds could be improved. We employed semi-rational

laboratory evolution of AnFdc to explore whether the enzyme could be evolved

to readily accept (hetero)aromatic compounds, and thus demonstrate the

potential for C-H activation of recalcitrant molecules by the UbiD-enzyme family.

Structure of AnFdc heteroaromatic acid complexes – To provide detailed

understanding of what governs AnFdc substrate specificity with respect to

(hetero)aromatic compounds, and thus guide downstream semi-rational

laboraoty evolution experiments, we determined high-resolution structures of

the wild-type enzyme in complex with respectively the weak substrate 8 and the

inhibitors 9 and 10 (Fig 3b, Extended data Fig 5). Complexes were obtained by

soaking AnFdc crystals with 100 mM - 500 mM ligand stock. Comparison of the

various ligand complex structures with previously determined cinnamic acid

substrate complexes [11] reveals aromatic ligands bind at the active site in two

distinct binding modes, one of which resembles a catalytically relevant

conformation. In this case the ligand carboxylic acid moiety is located in the

active site CO2 / Glu282 binding pocket. This places the substrate Calpha in close

proximity of the prFMNiminium C1’ atom (at ~3.1 Å). Given that wild-type AnFdc

depends on reversible cycloaddition to catalyse interconversion of cinnamic acid

2 with styrene 1 and CO2 (Fig 1b), the relative position of both the substrate

Calpha and Cbeta carbon atoms with respect to the prFMN iminium cofactor C1’ and

C4a is crucial. In the case of compounds 8 and 9, the Cbeta is located further

away from the prFMN C4a compared to cinnamic acid substrates (3.8 Å

compared to 3.3 Å; no catalytic binding mode was observed for 10). Hence, the

weak activity observed with 7 and 8 could be accounted for through an

alternative mechanism only requiring formation of a single substrate-

prFMNiminium bond (i.e. Calpha-C1’), or because these compounds are able to

explore conformations that minimize the Cbeta-C4a distance. Such

conformations are likely to require concomitant motion of residues M283 and

I327 that restrict the access and mobility of the more bulky aromatic compounds

in the active site.

Evolution of AnFdc towards aromatic substrates – Using the insights gained

from the wt AnFdc structural studies, we evaluated the effect of M283 and I327

on ligand position/conformational freedom on the desired reactivity. Using a

semi-rational laboratory evolution approach, we constructed NDT libraries of

M283 and I327 and assessed in vivo activity for the decarboxylation of 7 or 8 by

reversed-phase HPLC. Five variants (bearing bulky hydrophobic residues at

M283 or small non-hydrophobic residues at I327) displayed improved activity

towards compound 7 with I327S and I327N variants affording 95% and 61%

conversion respectively when used as whole-cell preparation, and both variants

afforded >99% conversion when employed as purified enzyme preparation

(Extended data Fig 6). In order to validate and understand the effect of the

I327S mutation, we determined high resolution structures of I327S variants in

complex with 8 or 9. These structures confirm subtle changes have occurred in

the active site and relative position of ligand/prFMNiminium cofactor as a result of

the mutation (Fig 3c, Extended data Fig 5 ). The reduction in side chain size at

position 327 is accompanied by a minor rotation of the ligand in the active site,

reducing the Cbeta-C4a distance by ~0.1 Å to 3.7 Å. While this distance remains

larger than the 3.3 Å observed for the cinnamic acid (WT enzyme complex),

conformational heterogeneity in the position of the M283 side chain induced by

the I327S mutation is likely to offer opportunity for further transient rotational

movement of the substrate along the Calpha-C1’ axis, and hence additional

reduction of the Cbeta-C4a distance. Crucially, the I327S structure with 2-

naphthoic acid 10 reveals this compound is now able to occupy a catalytically

relevant conformation, with both Calpha-C1’ and Cbeta-C4a distances closely

resembling the cinnamic acid distances observed previously (respectively 3.0 Å

and 3.4 Å).

AnFdC I327S/N-catalysed aromatic decarboxylation - An assessment of the

substrate scope of AnFdc I327S and I327N variants against a wide range of

(hetero)aromatic carboxylic acids was performed in the decarboxylation

direction, revealing an extended substrate repertoire for both variants.

Conversion of >99% was achieved with 7, using the purified AnFdc I327S, and

this variant displayed a total turnover number of >5000 constituting a 150-fold

improvement in specific activity when compared to the wild-type (Fig 4a,

Extended data Fig 6). Furthermore, related compounds such as 8 and 9 were

decarboxylated by the I327S variant, affording conversion of 42% and 93%

respectively. In addition, a range of bicyclic and monocyclic heteroaromatic

(di)carboxylic acids were also decarboxylated (Fig 4a). The AnFdc variants

displayed excellent regioselectivity for oxygen and sulphur-containing

heterocycles (7, 9, vs 12, 13, Fig 4a, Extended data Fig 6) displaying clear

preference towards heterocyclic 2-carboxylic acids (e.g. 7, 9) and rejecting the

heterocyclic 3-carboxylate 12 and 13. Remarkably, 2-naphthoic acid 10 was

decarboxylated by AnFdc I32S/N albeit in low conversion (7 %), representing

the first example of an isolated enzyme capable of decarboxylating non-activated

aromatic carboxylic acids (Fig 4a, Extended Data Figs 6, Fig 7). This is in

agreement with the observed catalytically active binding mode as revealed from

structural studies, Fig. 3c.

DFT computational studies of aromatic decarboxylation - In order to gain

further mechanistic insight into decarboxylation of aromatic acids by AnFdc

I327S, we used density functional theory (DFT) calculations to study the

formation of the covalent adduct and subsequent decarboxylation reaction.

Similar to previous work [11], an active site ‘cluster’ model was generated based

on the crystal structure of AnFdc I327S with non-covalently bound 2-naphthoic

acid 10 (Extended Data Fig 9). Stable covalent substrate-prFMN adducts were

found for both coumarillic 7 and naphthoic acid 10 substrates, corresponding to

two distinct species prior to decarboxylation (Int1cyclo and Int1open) and the

decarboxylation product Int2CO2. In agreement with solution studies, the

potential energy profile for coumarillic acid decarboxylation is substantially

lower than for naphthoic acid (Extended Data Fig 9e). In both cases, the

cycloadduct Int1cyclo is substantially higher in energy than the Int1open species,

and decarboxylation proceeds from Int1open. This suggests that, instead of a

concerted cycloaddition between the prFMN azomethine ylide dipole and the

aromatic substrate leading to the formation of the pyrrolidine adduct Int1cyclo, a

stepwise process is followed, whereby only the Ca-C1’ bond is fully formed in the

Int1open adduct. While Int1open resembles a putative arenium ion or Wheland

intermediate following aromatic electrophilic addition [23], analysis of the

charge distribution suggests through-space interactions between the closely

stacked prFMN and substrate aromatic planes (Cβ-C4a distance for the Int1open is

2.73 Å for 7 and 2.77 Å for 10) appear to avoid significant charge separation

between the aromatic substrate and prFMN (Extended Data Fig 9e). Similar

through-space electronic interactions have previously been used to control

abiotic electrophilic aromatic substitutions [24].

I327S/N plus CAR affords aromatic C-H activation - The AnFdc I327S/N

variants thus offer the possibility of using Fdc-CAR cascade reactions to

functionalise aromatic compounds through CO2 fixation. We employed AnFdc

I327S for the functionalisation of benzofuran 18 via transient formation of 7 as a

model reaction (Fig 4b). We demonstrated reduction of benzofuran-2-carboxylic

(coumarillic) acid 7 using E. coli whole cells expressing TpCAR afforded 1-

benzofuran-2-ylmethanol 20. To form the three step AnFdc I327S-TpCAR-EcADH

cascade reaction, purified AnFdc I327S or CFE was combined with fresh resting

E. coli whole-cells expressing TpCAR. This system converted benzofuran 18 to 1-

benzofuran-2-ylmethanol 20 with supply of 60-100 mM KHCO3 (Fig 4b,

Extended Data Fig 6c). Similarly, the use of purified AnFdc I327S and TpCAR or

SrCAR in (NH4)HCO3 reaction buffer leads to conversion of 18 to the

corresponding amide 21 in up to 18 % conversion (~1% of the carboxylic acid

intermediate 7 was detected). Finally, the use of a three step AnFdc, I327S-SrCAR

and reductive aminase from Aspergillus oryzae (AspRedAm; [25]) leads to

approximately 22% of the substrate converted, affording the corresponding

secondary amine 22 (13 %) (Fig 4b, Extended Data Fig 8).

Discussion - Taken together, our data confirms UbiD-mediated

(de)carboxylation can readily occur for a wide range of substrates, including

heteroaromatic and non-activated aromatic compounds. While the reaction

equilibrium is poised towards decarboxylation at ambient conditions, carboxylic

acids formed can be irreversibly converted to a range of C+1 compounds using

cascade reaction systems, thus achieving efficient CO2 fixation and substrate

activation. Hence, this confirms a similar system can operate in those microbial

cultures degrading benzene/naphthalene under anaerobic conditions that

contain suitable UbiD carboxylases [16,17]. While the aromatic stability of these

substrates renders them recalcitrant to transformation, prFMNiminium mediated

carboxylation offers a route to activation of these compounds. The energetic

barriers imposed by such transformation might offer insights into the

remarkably slow growth rates of such cultures. The one-pot biocatalytic

cascades developed enable the functionalisation of unactivated terminal alkenes

such as styrene derivatives or (hetero)aromatic compounds via carboxylation at

near stoichiometric supply of CO2. When using CAR mediated conversion as the

irreversible second step, the inherent reactivity of the aldehyde generated offers

opportunities to access an extensive product range. Given the broad substrate

tolerance of CAR enzymes towards , -α β unsaturated carboxylic acids as well as

(hetero)aromatic acids [26] is complementary to the substrate scope of

(evolved) Fdc, the Fdc-CAR system can be exploited to provide access to a broad

range of industrially important products. Additionally, it is likely that coupling

Fdc-mediated CO2-fixation to other irreversible and synthetically useful

enzymatic reactions such as enoate reduction [27] or CoA-ligation [28] can

further extend the scope for UbiD-mediated CO2 transformation into commodity

chemicals. This approach expands the repertoire of existing biocatalytic

functionalisation of alkenes [29], and contributes a new route to using CO2 as a

chemical building block [30,31].

Acknowledgements

This work was supported by the grants BBSRC BB/K017802 (DL) and ERC pre-

FAB 695013 (DL). We acknowledge assistance via use of the Manchester Protein

Structure Facility and Diamond Light Source for access (proposal numbers

MX12788 and MX17773) which contributed to the results presented here. We

also acknowledge the assistance given by IT Services and the use of the

Computational Shared Facility at The University of Manchester. We thank James

Marshall and Nicholas J. Turner (Manchester) for kindly providing the CfIRED

plasmid. D.L. is a Royal Society Wolfson Merit Award holder.

Author contributions

G.  A. A & R. T. B.  performed enzyme inhibition studies, mutagenesis and protein

engineering experiments. G. A. A. & I. G.  performed crystallisation and D.L.

determined associated crystal structures.  G. A. A., S. R. D. & D. G.  identified

suitable carboxylic acid reductases. G. A. A., S. R. D. & G. R. T. identified suitable

reductive aminases. G. A. A. & R. T. B. performed decarboxylation

biotransformations. G. A. A. constructed CO2-fixation cascades, and performed

and analysed cascade biotransformations. A.S. & S.H. performed computational

studies. G. A. A. & D. L. designed experiments and wrote initial draft of the

manuscript. D.P. advised on all aspects. All authors discussed the results and

commented on the manuscript. D. L. initiated and coordinated the project.

Competing interests

The authors declare no competing interests.

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Figure legends

Fig 1. UbiD (de)carboxylases offer new routes to unsaturated carboxylic

acid derivates. a) Previous biocatalytic carboxylation work using large molar

excess of KHCO3 and applying enzymes with narrow substrate scope ([5]; i.e.

phenolic acid decarboxylase, PAD and 2,3-dihydroxybenzoic acid decarboxylase,

DHBD). b) A chemical scheme of a model UbiD reaction, interconversion of

styrene + CO2 with cinnamic acid, using covalent catalysis by the prFMNiminium

cofactor mediated by a reversible 1,3 dipolar cycloaddition process [10,11]. c)

The proposed pathway for microbial anaerobic benzene/naphthalene

degradation mediated through initial UbiD mediated carboxylation coupled to

irreversible CoA-ester production by benzoyl-CoA ligase (BCL) [16,17].

Fig 2. CO2-fixation synthetic cascades linking AnFdc and CAR-catalysed

reactions for the functionalisation of terminal alkenes. One-pot reaction

containing AnFdc and resting E. coli whole-cells expressing TpCAR converts

styrene 1 to cinnamyl alcohol 4 (95% conversion) via AnFDc catalysed

carboxylation, CAR-catalysed carboxylate reduction and a carbonyl reduction

step catalysed by E. coli endogenous ADH (EcADH). A one-pot two-enzyme in

vitro cascade containing AnFDC and CAR, and excluding NADPH afforded the

conversion of styrene 1 to cinnamide 5 (14% conversion) via cinnamic acid 2.

An alternative one-pot three-step cascade applying the CfIRED imine reductase

to catalyse the reductive coupling of the resulting aldehyde to a primary amine

provides access to the secondary amine 6 (93% conversion). Conversion values

were determined from HPLC/GC-MS analyses of biotransformation reactions.

Biotransformation products (2, 3, 4, 5) were confirmed by HPLC analyses

against commercial authentic reference standards; product 6 was confirmed by

analysis of the MS spectra.

Fig 3. Structure guided laboratory evolution of AnFdc. a) Wild-type AnFdc

demonstrates weak decarboxylation activity with hetero-aromatic bicyclic

compounds 7 and 8, but not 9 and 10. b) Overlay of the crystal structures of

AnFdc WT with compounds 8 and 10 and cinnamic acid 2 (individual structures

and omit electron densities are shown in Extended Data Fig 6). Key amino acids

are shown in atom coloured sticks (green carbons), while compound 8 (cyan

carbons) is shown in the catalytically active conformation (grey carbons),

compound 10 (magenta carbons) binds in a non-catalytic mode. Grey dotted

lines indicate hydrogen bonds established with the carboxylic acid moiety of

compound 8, while red dotted lines indicate the distance between the prFMN C1’

and C4a atoms and the compound 8 Calpha and Cbeta. Residues M283 and I327

restrict compound 8 from binding in a conformation whereby the Calpha-beta

double bond overlays with the corresponding cinnamic acid Calpha-Cbeta atoms.

c) Overlay of the crystal structures of AnFdc I327S with compounds 9 and 10

(individual structures and electron densities are shown in Extended Data Fig 6).

Key amino acids are shown in atom coloured sticks (green carbons), while

compound 9 (blue carbons) is shown in the catalytically active I327S

conformation as well as the conformation observed in the WT structure (in grey

carbons), compound 10 (magenta carbons) now binds in a catalytic mode as

compared to the corresponding WT complex (see panel b). Grey dotted lines

indicate hydrogen bonds established with the carboxylic acid moiety of

compound 10, while red dotted lines indicate the distance between the prFMN

C1’ and C4a atoms and the ligand 10 Calpha and Cbeta. The latter are near

identical to the previously determined relative position of cinnamic acid-prFMN

[11].

Fig 4. Enzymatic C-H activation of (hetero)aromatic compounds by the I327S

AnFdc – CAR system a) Expanded substrate panel for AnFdc variant I327S. b)

The use of I327S AnFdc in cascade reactions similar to those shown in Fig 2

allows enzymatic activation of benzofuran 18 through CO2 fixation at ambient

conditions. Conversion values were determined from HPLC/GC-MS analyses of

biotransformation reactions. Biotransformation products were confirmed by

HPLC/GC-MS analyses against commercial authentic reference standards, except

for product 22 which was confirmed by analysis of the MS spectra.

Fig 1

Fig 2.

Fig 3.

Fig 4.

Online Methods

No statistical methods were used to predetermine sample size. The experiments were

not randomized, and investigations were not blinded to allocation during experiments

and outcome assessment.

Chemicals and reagents

Chemicals and reagents including all substrates and reference product standards

(except compounds 6 and 22) of highest purity were purchased from Sigma-Aldrich

(Poole, Dorset, UK), or Fluorochem (Hadfield, Derbyshire, UK). HPLC solvents were

obtained from Sigma-Aldrich (Poole, Dorset, UK) or ROMIL (Waterbeach, Cambridge,

UK) and GC gases from BOC gases (Guildford, UK). Enzyme nicotinamide cofactors

NADP+ and NADPH were purchased from Bio Basic (Markham, Ontario, Canada), and

Adenosine triphosphate (ATP) was sourced from Sigma-Aldrich (Poole, Dorset, UK).

Production and preparation of AnFdcUbiX biocatalysts

Cloning, expression and purification of AnFdcUbiX were performed according to

previously described methods [10,32] and AnFdcUbiX whole-cell biocatalyst was prepared

as described previously [32].

Preparation of CARSfp whole-cell Biocatalyst

Cloning of carboxylic acid reductases (TpCAR and SrCAR) and phosphopantetheine

transferase (Sfp) was performed as previously reported [22]. A single colony of

recombinant E. coli BL21 (DE3) containing pET28-b-FL-TpCAR and Sfp from Bacillus

subtilis (pCDF1b-Sfp) was inoculated into 10 mL lysogeny broth (LB) (1% tryptone,

0.5% yeast extract, 1% NaCl) and incubated overnight at 37 °C in an orbital shaker at

200 rpm. This starter culture was used as the inoculum. A 2 L flask with 500 mL LB was

supplemented with kanamycin (30 g mLμ -1) and streptomycin (50 g mLμ -1) and

inoculated with 5 mL of starter culture. Cultivation was performed at 37 °C in an orbital

shaker with 200 rpm shaking. At an optical density (OD600 nm) of between 0.6 and 0.8,

isopropyl -D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2β

mM to induce protein expression. Incubation was continued at 20 °C and 200 rpm for 18

h. Cells from a 500 mL culture were harvested by centrifugation at 6000 rpm for 15 min,

and washed in sodium phosphate buffer (NaPi, 50 mM, pH 7.5). Harvested cells

containing CARSfp biocatalyst system were used as fresh resting whole-cell biocatalyst or

purified according to previously reported method [22].

Biotransformation procedures for Fdc-catalysed decarboxylation

(i) For Fdc-catalysed decarboxylation of acrylic acid derivatives, e.g. cinnamic acid 2

using purified holo-Fdc, a 500 µL reaction mixture contained cinnamic acid 5 mM (or up

to 20 mM), 2% (v/v) dimethylsulfoxide (DMSO) and 0.2-1mg mL -1 of purified Fdcubix in

NaPi (50 mM or 100mM, pH 6).

(ii) For Fdc-catalysed decarboxylation of (hetero)aromatic carboxylic acid, e.g.

benzuran-2-carboxylic acid 7, a 500 µL reaction contained 0.5-1mg AnFdc I327S/N,

(hetero)aromatic carboxylic acid 2 mM (or up to 20 mM for benzofuran-2-carboxylic

acid), 2% (v/v) dimethylsulfoxide (DMSO) in NaPi (50 mM, pH 6).

(iii) For Fdc-catalysed decarboxylation reaction using the whole-cell biocatalyst, a

typical 500 µL reaction mixture contained 5 mM carboxylic acid substrate, 2% (v/v)

DMSO and fresh resting E. coli whole cells containing the overexpressed FdcUbiX (to a

final optical density, OD 600 nm of 30), or 30 mg of the lyophilized whole-cell biocatalyst

in NaPi (50 mM, pH 6).

Reaction mixture in a 2 mL tightly closed glass vials were incubated at 30 °C with

shaking at 250 rpm for 18 h. The reaction was stopped by addition of 3 volumes of

acetonitrile and vigorous mixing. The reaction mixtures were centrifuged (15°C, 13 000

rpm, 20 min); the clear supernatant was further centrifuged and analysed by RP-HPLC

(Agilent system,Santa Clara, CA, USA, equipped with a G1379A degasser, G1312A binary

pump, a G1367A well plate autosampler unit, a G1316A temperature-controlled column

compartment and a G1315C diode array detector). ARP-HPLC column, syncronis; C18;

250 mm length, 4.6 mm diameter, 5 m particle size (Thermo Scientific; Waltham, MAμ

USA) was used.

Where analysis of biotransformations were performed on the GC-MS, an equal volume of

EtOAc (containing a known concentration of an internal standard where necessary) was

added to biotransformation mixture, vigorously mixed, centrifuged and the organic

layer was extracted. The aqueous layer was then acidified to a pH of ~2 and further

extracted with EtOAc and centrifugation (15 °C, 13 000 rpm, 10 min) to improve the

separation of phases. The organic layers were combined and dried over anhydrous

MgSO4 and samples were analysed by GC-MS (Agilent 5977A Series GC/MSD System

with an Agilent 7890B Series GC coupled to Mass Selective Detector. Column: 30 m DB‐WAX column with 0.25 mm inner diameter and 0.25 m film thickness (Agilent, Santaμ

Clara, CA, USA) was used. Analysis method: Inlet temperature: 240 °C, detector

temperature: 250 °C, MS source 230 °C, helium flow: 1.2 mL min−1; oven temperature

40–240 °C, 15 °C min−1).

Construction and screening of AnFdc I327NDT and M283NDT libraries

Site-saturation mutant libraries were generated using the following overlapping primer

sets: AnFdc I327NDT, forward: CCCATACGATGNDTGGCAGCCTGGCC (5'>3'); reverse:

GGCCAGGCTGCCAHNCATCGTATGGG (5'>3'). AnFdc M283NDT, forward:

GGCCCGTTTGGTGAANDTCATGGCTATATTTTC (5'>3'); reverse:

GAAAATATAGCCATGAHNTTCACCAAACGGGCC (5'>3').

E. coli NEB 5-alpha competent cells (New England Biolabs, Hitchin, UK) were

transformed with Dpn1-treated PCR products. Six colonies were sequenced from each

library to ascertain even distribution of mutations. Plates containing >60 colonies were

washed with 5 mL LB into a falcon tube and the plasmids were isolated using Qiagen

miniprep kits. The extracted plasmid mixture was used to transform E. coli BL21 (DE3)

cells. Protein expression was performed in deep-well plates; each well contained a

single E. coli BL21 (DE3) colony (bearing AnFdc variant in pET-30a (+) vector) in 800 Lμ

LB supplemented with kanamycin (30 g mLμ -1). The culture was then incubated at 37 °C

in an orbital shaker at 300 rpm, induced at OD600 nm between 0.6 and 0.8 with IPTG (final

concentration of 0.2 mM). Further cultivation was continued at 20 °C, 300 rpm shaking

for 18 h. Cell cultures were harvested at 4000 rpm for 20 min, washed with NaPi (50

mM, pH 7.5) and were used for biotransformations as fresh resting cells.

Biotransformation reactions was performed in deep-well plates; 250 L NaPμ i buffer (50

mM, pH 6) containing 2 mM substrate and 2 % (v/v) DMSO was added to pellets in each

well, sealed and incubated at 30 °C with 250 rpm shaking for 18 h, after which the

reaction was stopped by addition of 3 volumes of MeCN. Product formation was

monitored using RP-HPLC. Five mutants (displaying best activity) identified from the 36

transformants initially screened were further rescreened using cell-free extract.

Variants of interests were purified and further characterised.

Identification of competitive inhibitors and alternative substrates

A compound library containing aromatic and heteroaromatic carboxylic acids was

constructed. The inhibitory effect of these compounds on the AnFdc-catalysed

decarboxylation of cinnamic acid 2 was investigated; AnFdcUbiX (0.1 mg mL-1) was

incubated with 5 mM 2 in the presence of 0-20 mM inhibitor (in 100-200 mM NaPi

buffer, pH 7.5), and the conversion of 2 was determined after 30 min incubation.

(Hetero)aromatic carboxylic acids that inhibited the decarboxylation of 2 were

rescreened under 24 h biotransformation conditions to assess for decarboxylation

product. In all cases, control reactions lacking AnFdcUbiX were performed.

Biotransformation procedures for the conversion of cinnamic acid to cinnamyl alcohol

Conversion of cinnamic acid 2 to cinnamyl alcohol 4 via the intermediate

cinnamaldehyde 3 was achieved using recombinant resting E. coli cells expressing CARsfp

and containing an endogenous alcohol dehydrogenase (EcADH); glucose was added to

enable in situ generation of cofactors. A typical 500 uL reaction mixture contained 5 mM

cinnamic acid 2, 5% v/v DMSO, E. coli resting whole-cells containing over-expressed

CARsfp to the final OD600 nm of 30 in NaPi or Tris-HCl (50 mM, pH 7.5). Reaction mixtures in

2 mL Eppendorf tubes were incubated at 30 °C with 250 rpm shaking for 18 h, after

which samples were prepared for RP-HPLC.

One-pot biotransformation for the conversion of styrene to cinnamaldehyde via CO2-

fixation

For Fdc-CAR mediated conversion of styrene 1 to cinnamaldehyde 3 in one pot, a typical

1 mL reaction mixture contained 5 mM styrene 1, 2% v/v DMSO, purified FdcUbiX (1-2mg

mL-1), purified CARsfp (0.6-1 mg mL-1), 10-200 mM KHCO3, 10 mM NADPH and 10 mM

ATP, 15 mM MgCl2 in Tris-HCl buffer (50 mM, pH 7.5). Reaction mixtures in 2 mL tightly-

closed glass vials were incubated at 30 °C with 250 rpm shaking for 18 h, after which the

enzyme was inactivated by the addition of 3 volumes of MeCN, vigorously mixed and

samples were prepared for RP-HPLC.

One-pot biotransformation for the conversion of alkene or heteroaromatic to the

corresponding alcohol via CO2-fixation

For Fdc-CAR-ADH mediated conversion of styrene 1 to cinnamyl alcohol 4 in one pot, a

typical 1 mL reaction mixture contained 5 mM 1, 2% v/v DMSO, AnFdcUbiX either as

purified form (1-2 mg mL-1) or lyophilized cell free extract (10mg), CARsfp as fresh

resting whole-cells preparation (to the final OD 600 nm of 50), 20 mM D-glucose and 10-

200 mM KHCO3 in NaPi (50 mM, pH 7.5). Reaction mixtures in 2 mL tightly-closed glass

vials were incubated at 30 °C with 250 rpm shaking for 18 h, after which the enzyme

was inactivated by the addition of two volumes of MeCN and vigorously mixed. The

reaction mixtures were centrifuged (15 °C, 13 000 rpm, 10 min); the clear supernatant

was filtered and analysed by RP-HPLC.

One-pot biotransformation for the conversion of terminal alkene or heteroaromatic to the

corresponding amine via CO2-fixation

For the one-pot, three-step Fdc-CAR-RedAm-mediated conversion of styrene 1 to the

corresponding N-alkylated cinnamylamine 6, a typical 1 mL reaction mixture contained

5 mM 1, 5% v/v DMSO, purified AnFdcUbiX (1.5 mg mL-1), purified SrCAR (0.6 mg mL-1 ),

0.8 mg mL-1 purified imine reductase/reductive aminase, 12.5 mM cyclopropylamine,

10 mM ATP, 15 mM MgCl2, 20 mM D-glucose , 1 mM NADP+, 0.2 mg mL-1 purified GDH, in

100 mM or 500mM (NH4)HCO3 buffer, pH8. Reaction mixtures in 2 mL tightly closed

glass vials were incubated at 30 °C with 250 rpm shaking for 18 h. The reaction was then

basified with NaOH to pH >12; 500 uL of EtoAc added, vigorously mixed, centrifuged (15

°C, 13 000 rpm, 5 min); and organic layer collected. The remaining aqueous layer was

then acidified to pH ~2 and further extracted with EtOAc with centrifugation (15 °C, 13

000 rpm, 10 min). The organic layers were combined and dried over anhydrous MgSO4

and samples were analysed by GC-MS. The same reaction conditions were used for the

conversion of benzofuran 18 to the corresponding N-alkylated amine derivative 22.

Products 6 and 22 obtained from analytic biotransformation reactions were confirmed

from analysis of the MS spectra. GCMS analyses were performed on Agilent 5977A

Series GC/MSD System with an Agilent 7890B Series GC coupled to Mass Selective

Detector. Data analysis was performed using GC/MSD MassHunter Data Acquisition and

ChemStation Data Analysis. A 30 m x 0.25 mm x 0.1 m VF-5HT column (Agilent, Santaμ

Clara, CA, USA) was used. The parameters of the method include: Inlet temperature =

240C, detector temperature = 250C, MS source= 230 C, helium flow = 1.2 mL min-1;

oven temperature between 50 - 360C, 30C min-1.

One-pot biotransformation for the conversion of terminal alkene or heteroaromatic to the

corresponding amide via CO2-fixation

For the one-pot, two step carboxylation-amidation cascade mediated by Fdc-CAR system

for the conversion of styrene 1 to the corresponding cinnamide 5, a typical 1 mL

reaction mixture contained 5 mM 1, 2% v/v DMSO, purified AnFdcUbiX (1-2 mg mL-1),

purified SrCAR (0.6 mg mL-1 ), 10 mM ATP, 15 mM MgCl2, in 500 mM (NH4)HCO3 buffer,

pH 8. Reaction mixtures in 2 mL tightly closed glass vials were incubated at 30 °C with

250 rpm shaking for 18 h. The reaction was then basified with NaOH to pH >12; 500 uL

of EtoAc added, vigorously mixed, centrifuged (15 °C, 13 000 rpm, 5 min); and organic

layer collected. The remaining aqueous layer was then acidified to pH ~2 and further

extracted with EtOAc with centrifugation (15 °C, 13 000 rpm, 10 min). The organic

layers were combined and dried over anhydrous MgSO4 and samples were analysed by

GC-MS.

Protein crystallization and structure determination

Crystallisation was performed by sitting drop vapor diffusion using a seed stock based

on previously established crystallization conditions for AnFdc ([10]; 0.2 M potassium

thiocyanate, Bis-Tris propane pH 6.5, 20% w/v PEG 3350 by mixing 0.05 L seed stock,μ

0.25 L protein solution and 0.3 L reservoir solution at 4 °C). Where FMN containingμ μ

AnFdc was used, production, purification and crystallisation were performed as

previously described [11]. Crystals were cryoprotected in reservoir solution

supplemented with 10% PEG 200 and flash cooled in liquid nitrogen. To obtained co-

crystals, the protein was incubated with 1-5 mM ligand prior to the crystallisation while

ligand soaks was performed in cryoprotectant solution supplemented with 100 mM-500

mM ligand stock solution in MeOH. Diffraction data were collected at Diamond

beamlines and data were processed using XDS [33]. Iterative refinement and manual

model building were performed with REFMAC5 and COOT [34]. Final data collection and

refinement statistics are given in Supplementary Table 1.

Computational studies

Active site DFT ‘cluster’ models comprising ~350 atoms (figure B) were built from the

crystal structure of AnFdc I327S with non-covalently bound naphthoic acid bound and

modelled at the B3LYP/6-31G(d,p) level of theory with the D3 version of Grimme’s

dispersion with Becke–Johnson damping [35] and a generic polarizable continuum with

= 5.7 using the polarizable continuum model. Calculations were performed usingε

Gaussian 09 revision D.01, the models and additional details are given in Extended

Data Fig. 9 and are similar to our previous work [11], but make use of a larger model

comprising more of the active site.

Data availabilityThe atomic coordinates and experimental data have been deposited in the

Protein Data Bank with accessions codes 6TIH, 6TIJ, 6TIO, 6TIN, 6TIL, 6TIB, 6TIC

and 6TIE. All other data are available from the corresponding author on

reasonable request.

Methods only References

32. Aleku, G.A. et al. Terminal alkenes from acrylic acid derivatives via non-oxidative enzymatic

decarboxylation by ferulic acid decarboxylases. ChemCatChem 10, 3736-3745 (2018).

33. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

34. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D

Biol. Crystallogr. 67, 235–242 (2011).

35. Grimme, S., Ehrlich, S. & Goerigk, L. Effect of the damping function in dispersion corrected

density functional theory. J. Comp. Chem. 32, 1456–1465 (2011).