Synthetic Enzyme-Catalyzed CO2 Fixation Reactions

Abstract

In recent years, (de)carboxylases that catalyze reversible (de)carboxylation have been targeted for application as carboxylation catalysts. This has led to the development of proof-of-concept (bio)synthetic CO₂ fixation routes for chemical production. However, further progress towards industrial application has been hampered by the thermodynamic constraint that accompanies fixing CO₂ to organic molecules. In this Review, biocatalytic carboxylation methods are discussed with emphases on the diverse strategies devised to alleviate the inherent thermodynamic constraints and their application in synthetic CO₂ -fixation cascades.

Keywords: CO2 fixation, enzymes; biocatalysis; carboxylation; cascade reactions.

Scheme 1

A selection of synthetically promising (de)carboxylases acting on distinct but diverse substrate groups and showing site of carboxylation: (a) acting on phenolic compounds; (b) acting on heteroaromatic and nonphenolic aromatic compounds; (c) acting on aliphatic compounds. Organic/divalent metal ions cofactors presented in red. Organic cofactors: ATP=adenosine triphosphate; prFMN=prenylated flavin; TPP=thiamine pyrophosphate; NADPH=nicotinamide adenine dinucleotide phosphate (reduced). Enzymes: PhPC=phenylphosphate carboxylase; pHBD=para‐hydroxybenzoic acid decarboxylase; DBHD=dihydroxybenzoate decarboxylase; PAD=phenolic acid decarboxylase; P2CDC=pyrrole‐2‐carboxylate decarboxylase; I3CDC=indole‐3‐carboxylate decarboxylase; HmfF=2,5‐furan dicarboxylic acid decarboxylase; FDC=(fungal) ferulic acid decarboxylases; PEPC=phosphoenolpyruvate carboxylase; pyruvate decarboxylases; KdcA=branched‐chain α‐ketoacid dehydrogenase; ECR=carboxylating enoyl‐thioester reductases; GCC=glycolyl‐CoA carboxylase.

Scheme 2

Regioselective p‐carboxylation of phenylphosphate, phenol and catechols catalyzed by reversible nonoxidative decarboxylases. (a) Kolbe‐Schmitt carboxylation method (b) Carboxylation of phenyl phosphate catalyzed by phenyl phosphate carboxylase (PhPC) ^[55] (c) Biotransformation performed with phenol decarboxylase from Enterobacter cloacae (Ec_pHBD) ^[57] (d) Biotransformation performed using prFMN dependent 3,4‐dihydroxybenzoic acid decarboxylases (AroY) from E. cloacae (Ec_AroY). ^[40]

Scheme 3

Examples of regioselective ortho‐carboxylation of phenolic compounds catalyzed by recombinant E. coli whole cells containing a 2,3‐dihydroxybenzoic acid decarboxylase (2,3‐DHBD) reported by (a) Wuensch et al., ^[61] (b) Plasch et al., ^[63] and (c) Zhang et al. ^[64] 2,3‐DHBD_Ao=2,3‐dihydroxybenzoic acid decarboxylase from Aspergillus oryzae; 2,6‐DHBD_Rs=2,6‐dihydroxybenzoic acid decarboxylase from Rhizobium sp. and SAD_Tm=salicylic acid decarboxylase from Trichosporon moniliiforme. 2,3‐DHBD_Fo=2,3‐dihydroxybenzoic acid decarboxylase from Fusarium oxysporum. Conversion values (%) obtained with 2,3‐DHBD_Ao, 2,6‐DHBD_Rs, and SAD_Tm are given in black, blue, and pink, respectively.

Scheme 4

Regioselective carboxylation of heteroaromatics: (a) Carboxylation of pyrrole by pyrrole‐2‐carboxylate decarboxylase from Bacillus megaterium (P2CDC_Bm). ^[44] (b) Carboxylation of indole by indole‐3‐carboxylate‐decarboxylase from Arthrobacter nicotianae (I3CDC_At). ^[46] (c) Carboxylation of furoic acid to yield 2,5‐furandicarboxylic acid (FDCA) catalyzed by reversible prFMN‐dependent decarboxylase HmfF ^[42] (d) (de)carboxylation of benzo‐fused O‐, N‐, and S‐containing heteroaromatic carboxylic acids catalyzed by evolved AnFdc variants (I327S or I327N). ^[43] AnFdc=Aspergillus niger ferulic acid decarboxylase.

Scheme 5

Regioselective β‐carboxylation of (p‐hydroxy)styrenes (a) Regioselective β‐carboxylation of para‐hydroxystyrenes by applying E. coli whole cells expressing co‐factor free phenolic acid decarboxylases (PADs)/ferulic acid decarboxylase (FDC) from bacterial sources. ^[52] Enzymes: Phenolic acid decarboxylase (PAD) from Bacillus amyloliquefaciens (PAD_Ba); from Mycobacterium colombiense (PAD_Mc), and ferulic acid decarboxylase (FDC) from Enterobacter sp. (FDC_Es). For clarity, conversion values (%) obtained with PAD_Ba, PAD_Mc and FDC_Es have been given in black, blue, and pink, respectively. (b) Regioselective β‐carboxylation of styrene catalyzed by prFMN‐bound ferulic acid decarboxylase from Aspergillus niger (AnFdc). ^[43]

Scheme 6

Mechanism of FDC‐catalyzed (de)carboxylation of phenylacrylic acids (e. g., cinnamic acid) vs PAD‐mediated (de)carboxylation of phenolic analogues (e. g., p‐coumaric acid). (a) Mechanism for decarboxylation of cinnamic acid by covalent catalysis using the prFMN iminium involving an initial 1,3‐dipolar cycloaddition between the dipolarophile of the substrate and the azomethine ylide‐like species of prFMN^iminium resulting in a cycloadduct species Int1. The Glu282 side chain mediates the protonation step to form Int3.[ ⁷⁰ , ⁷¹ ] The decarboxylation product styrene is released through a cycloelimination process. (i–v)=1,3‐dipolar cycloaddition, decarboxylation, CO₂ to Glu 282 exchange, protonation and cycloelimination, respectively. (b) A general acid‐base mechanism employed by phenolic acid decarboxylases (PADs) and enabled by an essential phenolic moiety.[ ⁵² , ⁷² ]

Scheme 7

One‐step CO₂‐fixation transformations for oxy‐functionalized aliphatic compounds. (a) Carboxylation of acetaldehyde to yield pyruvic acid with CO₂, catalyzed by pyruvate decarboxylase. ^[33] (b) One‐step CO₂‐fixation route to the corresponding α‐keto acid, although, owing to severe thermodynamic limitation of this reaction, significant carboxylation was only achieved when the carboxylate was removed from the equilibrium by further enzymatic derivatization. ^[34] (c) Conversion of phosphoenolpyruvate+CO₂ into oxaloacetate catalyzed by phosphoenolpyruvate carboxylase (PEPC).[ ⁸⁰ , ⁸¹ ]

Scheme 8

Enzymatic carboxylation of coenzyme A (CoA) thioesters. (a) Conversion of acetyl CoA into ethylmalonyl CoA through a multistep reaction involving a carboxylation step, catalyzed by a Rhodobacter sphaeroides lysate. ^[82] (b) Carboxylation of propionyl CoA catalyzed by crotonyl CoA carboxylase/reductase (CCR) ^[82] (c) Substrate scope of carboxylating enoyl thioester reductases (ECRs). Three ECRs (RevT from Streptomyces sp., CinF from Streptomyces sp., and EcrSh from Streptomyces hygroscopicus) showed broad substrate scope for α,β‐unsaturated CoA‐thioesters. RevT, CinF, EcrSh afforded moderate to excellent conversion of the presented substrates (40–100 % conversion). ^[36]

Scheme 9

In situ CO₂ capture and utilization for carboxylation. (a) Linking amine‐mediated CO₂ capture to carboxylation reaction catalyzed by 2,3‐dihydroxybenzoic acid decarboxylase from Aspergillus oryzae (2,3_DHBD_Ao). ^[97] (b) Linking in situ capture of CO₂ mediated by carbonic anhydrase and its utilization for the carboxylation of phosphoenoylpyruvate (PEP) catalyzed by PEP carboxylase.[ ⁸⁰ , ⁸¹ ]

Scheme 10

Enzyme‐catalyzed CO₂ fixation linked to chemical derivatization of the carboxylate product. (a) A strategy applying quaternary ammonium salt to shift the reaction equilibrium towards 1,6‐DHBD_Rs‐catalyzed carboxylation of resorcinol. The carboxylation step is linked to the precipitation of the carboxylate in one pot. The product was recovered by acid hydrolysis. 1,6‐DHBD_Rs=1,6‐dihydroxybenzoic acid decarboxylase. ^[100] (b) Pyruvate:ferredoxin oxidoreductase (PFOR)‐catalyzed CO₂ fixation linked with derivatization using semicarbazide. ^[107]

Scheme 11

Enzymatic CO₂‐fixation cascade for the conversion of ethanol and CO₂ into lactic acid through pyruvate decarboxylase (PyDC)‐catalyzed carboxylation. ^[108]

Scheme 12

One‐pot enzymatic cascade for the conversion of aldehyde into amino acid involving CO₂‐fixation step catalyzed by TPP‐dependent decarboxylase (KdCA) and a reductive amination step catalyzed either by a transaminase (YbdL) or an amine dehydrogenase (LeuDH). ^[34] TPP=thiamine pyrophosphate, LeuDH=leucine dehydrogenase.

Scheme 13

Enzymatic CO₂‐fixation cascades for the conversion of aromatic compounds into alcohols and amines. ^[43] (a) CO₂‐fixation cascades enabling the conversion of styrene into the corresponding allylic alcohol and amine. ^[43] (b) One‐pot three‐step enzymatic CO₂‐fixation cascade for the conversion of styrene into cinnamyl alcohol, involving a sequence of Fdc‐catalyzed carboxylation, CAR‐catalyzed carboxylate reduction, and carbonyl reduction catalyzed by endogenous alcohol dehydrogenase. ^[43] (c) One‐pot three‐step artificial CO₂‐fixation cascade involving a sequence of Fdc‐catalyzed carboxylation, CAR‐catalyzed carboxylate reduction and reductive aminase(RedAm)‐catalyzed reductive amination, enabling the conversion of styrene into the corresponding secondary amines; reaction contained (NH₄)HCO₃ as CO₂ source and cyclopropylamine as the primary amine source, and incorporated GDH‐based recycling of NADPH. ^[43] (d) One‐pot artificial CO₂‐fixation cascade enabling the conversion of styrene into the corresponding cinnamide involving AnFDC‐catalyzed carboxylation linked to CAR‐catalyzed amidation using NH₄HCO₃ as source of both CO₂ and NH₃. (e) CO₂‐fixation cascades enabling the conversion of benzofuran into the corresponding alcohol, amine, and amide featuring similar conditions to those for (b), (c), and (d) respectively but employing the engineered heteroaromatic (de)carboxylase AnFDC I327S as the carboxylation catalyst. ^[43] TpCAR=Tsukamurella paurometabola carboxylic acid reductase; SrCAR=Segniliparus rugosus carboxylic acid reductase; AnFdc=ferulic acid decarboxylase from Aspergillus niger; CfIRED=Cystobacter ferrugineus imine reductase; GDH=glucose dehydrogenase.

Scheme 14

Artificially developed CETCH cycle applied to the synthesis of malate. The multienzyme cascade employs 17 enzymes catalyzing 13 linear enzymatic steps including two reductive carboxylation steps catalyzed by CCR, and additional auxiliary steps for cofactor recycling. A 520 μL reaction was capable of fixing 1080 μM CO₂ and afforded 540 μM malate in 90 min. ^[23] PCO=propionyl‐CoA oxidase; CCR=crotonyl‐CoA carboxylase/reductase; MCL=β‐methylmalyl‐CoA lyase; MAS=malate synthase. Propionyl‐CoA: 60; acrylyl‐CoA: 70; methylmalonyl‐CoA: 70 a; crotonyl‐CoA: 46; ethylmalonyl‐CoA: 46 a; methylmalyl‐CoA: 71; glyoxylate:72; malate: 73.

Scheme 15

Artificially developed TaCo CO₂‐fixation cascade and its application in de novo synthesis. (a) TaCo‐based enzymatic cascade enabling conversion of glycolyl‐CoA to glycerate. ^[37] (b) Conversion of glycolate to glycerate using a TaCo based enzymatic cascade. (c). Conversion of ethylene glycol to glycerate in a 4‐step TaCo‐based enzymatic cascade. Enzymes: GCS=glycolyl‐CoA synthetase; GCC=glycolyl‐CoA carboxylase; TCR=tartronyl‐CoA reductase; GOx0313=alcohol dehydrogenase from Gluconobacter oxydans; PduP=aldehyde dehydrogenase from Rhodopseudomonas palustris BisB18.

Scheme 16

Engineered ferulic acid decarboxylase for (de)carboxylation of (heteroaromatic) carboxylates. Substrate profiling study of AnFdc variants I327S/N using purified enzyme preparation of I327S (denoted as A) or I327N (denoted as B), as well as the cell‐free extract of I327S (denoted as C). ^[43] AnFdc is a prenylated flavin (prFMN)‐dependent reversible decarboxylase from Aspergillus niger.

Scheme 17

Synthetic scope of prFMN dependent ferulic acid decarboxylases (FDCs) for (de)carboxylation of aliphatic carboxylic acids.[ ⁴¹ , ⁷³ , ¹³⁶ ]