Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L−1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.This work was supported by the European Union Project grant H2020-BBI-PPP-2015-2-720297-ENZOX2; the Spanish projects PID2019-106166RB-100-OXYWAVE, PID2020-118968RB-100-LILI, PID2021-123332OB-C21 and PID2019-107098RJ-I00, funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (AEI)/doi: 10.13039/501100011033/; the “Comunidad de Madrid” Synergy CAM project Y2018/BIO-4738-EVOCHIMERA-CM; the Generalitat Valenciana projects CIPROM/2021/079-PROMETEO and SEJI/2020/007; and the PIE-CSIC projects PIE-202040E185 and PIE-201580E042. P.G.d.S. thanks the Ministry of Science, Innovation and Universities (Spain) for her FPI scholarship (BES-2017-080040) and the Ministry of Science and Innovation for her contract as part of the PTQ2020-011037 project funded by MCIN/AEI/10.13039/501100011033 within the NextGenerationEU/PRTR. D.G.-P. thanks Juan de la Cierva Incorporación contract Ref. No.: IJC2020-043725-I, funded by MCIN/AEI/10.13039/501100011033, and the EU NextGenerationEU/PRTR program. K.Ś. thanks to Ministerio de Ciencia e Innovación and Fondo Social Europeo for a Ramón y Cajal contract (Ref. RYC2020-030596-I). We thank the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance with the BL13-XALOC beamline

Nitrogen containing biobased chemicals produced using enzymes

Today, nitriles are produced by introducing the nitrogen functionality in hydrocarbons originated from fossil resources under high pressure and temperature. Environmental concerns associated with the use of fossil resources, as shown in chapter 1, stimulate the need to produce chemicals in a more sustainable way. Renewable resources such as amino acids from biomass, that already contain nitrogen in their molecule, are investigated as alternative starting materials to produce biobased nitriles. In this thesis the enzyme vanadium chloroperoxidase (VCPO) was used as catalyst to produce biobased nitriles from amino acids via the oxidative decarboxylation reaction. Industrially relevant nitriles such as acrylonitrile and succinonitrile, can be prepared starting from amino acids. For example, glutamic acid (Glu) – the most abundant non-essential amino acid in biomass – can be converted to acrylonitrile via the intermediate 3-cyanopropanoic acid (GluCN). The oxidative decarboxylation reaction of other biomass derived amino acids was investigated as well. Glu can be fully converted into GluCN with high selectivity using the enzyme VCPO, H2O2 and catalytic amounts of NaBr. In contrast, under the same reaction conditions the oxidative decarboxylation of aspartic acid (Asp) resulted in low conversion and selectivity towards the nitrile. In chapter 2, it was investigated how two chemically similar amino acids, Glu and Asp, react differently towards the oxidative decarboxylation. For this, the conversion of Glu and Asp was investigated as a function of bromide concentration. In presence of catalytic amount of bromide (0.1 equiv.), Glu resulted in full conversion and high selectivity. It was shown that by increasing the amount of bromide present in the reaction mixture to 2 equiv., the conversion of Asp was increased from 15% to 100% and its selectivity towards 2-cyanoacetic acid (AspCN) from 45% to 80%. It was concluded that the difference in reactivity must be due to the difference of one carbon atom in the side chain between Asp and Glu and the proximity of the side chain to the reactive alpha functionalities of the amino acids. It was hypothesised that the alpha functionalities in Asp are stabilised in intra- or intermolecular interactions with the side chain carboxyl functionality which prevents Asp to react in a similar manner as Glu. The influence of the side chain functionality and the side chain length of amino acids towards the reactivity of alpha functionalities with respect to oxidative decarboxylation was further investigated for different amino acids (chapter 3). It was shown that the conversion can be modified as a function of the concentration of NaBr for all amino acids tested. Only two amino acids, Glu and aminoadipic acid, can be fully converted into nitriles with catalytic amounts of NaBr (0.04 equiv.). For all other amino acids with aliphatic, hydroxy, carboxyl and methyl ester functionalities tested, a minimum amount of NaBr present in the solution (≥ 0.4 equiv.) is required to reach full conversion. It was concluded that the length of the side chain does not make a significant difference for the selectivity, as previously proposed. However, the position of the functionality on the side chain (β-carbon) in relation to the bromination centre could hinder the production of nitriles by oxidative decarboxylation by reducing the reaction rate of the bromination. It was shown that while functional groups like aliphatic, hydroxyl or methyl ester show no significant influence on the reactivity of amino acids, the carboxyl functionality has a positive effect during the oxidative decarboxylation reaction. An addition to the known reaction mechanism was proposed for the amino acids with carboxyl functionality at the side chain. It is proposed that the side chain carboxyl functionality is involved in a self-catalysis mechanism. The elucidation of the exact reaction mechanism could enable reactions of mixtures of amino acids at lower concentration of NaBr for the production of biobased nitriles. To further enhance the sustainability of the oxidative decarboxylation of amino acids, the in situ production of H2O2 was explored in chapter 4. The direct use of oxygen by alcohol oxidase (AOX) was investigated as alternative to the hydrogen peroxide originated from the energy-intensive anthraquinone process. The conversion of ethanol to the volatile acetaldehyde was selected for the half redox reaction of AOX due to the easiness of the downstream processing, e.g. by pervaporation of acetaldehyde. The cascade AOX-VCPO was used for in situ production of hydrogen peroxide for fast halogenation reactions and oxidation reactions via halogenation. For the first time, the oxidative decarboxylation of glutamic acid - an oxidation reaction via halogenation - was shown to be possible using the cascade AOX-VCPO. For this reaction, the two enzymes had to be separated in two reactors due to inhibition of AOX caused by HOBr – the product of VCPO. However, the fast halogenation reactions such as the bromination of monochlorodimedone, using the cascade AOX-VCPO was possible in one reactor. Oxygen availability in aqueous solutions, scaling up as well as the reaction kinetics need to be further addressed. The feasibility of the conversion of Glu into GluCN – an intermediate in the production of biobased acrylonitrile, was evaluated in chapter 5. The production of GluCN by VCPO and H2O2 (Scenario 3) was compared in a techno-economic assessment with other alternative biobased routes i.e. the production of GluCN by NaOCl (Scenario 1), by the cascade AOX-VCPO (Scenario 4) and by oxygen with a Ru catalyst (Scenario 5). It was found that by replacing NaOCl with VCPO-H2O2 the energy requirements of the process is reduced by a factor of 1.5 for the production of 1 t GluCN. This is mainly as a result of performing the reaction at 25°C, eliminating the need of cooling below room temperature (4°C) as in the case of NaOCl. The mass balance is slightly improved as selectivity close to 100% can be achieved by VCPO-H2O2 system and a significant reduction in waste was achieved. By further replacing NaOCl with oxygen in Scenario 4 and 5 the cost-benefit margin was increased significantly. Based on the cost-benefit analysis the only scenario with a positive cost-benefit margin of 194 €/t GluCN is Scenario 4, owed to the co-production of acetaldehyde which is a valuable product. The sensitivity analysis of Scenario 4 and 5 where the price of different compounds was changed, shows that the price of Glu and GluCN are the parameters that influence the economics of the process the most. At the moment, the price of the substrate, Glu, and the price of the product, GluCN – the intermediate in the production of acrylonitrile or succinonitrile – are too high to be competitive with the fossil based nitriles. As the price of Glu is already a best case scenario the use of cheaper sources of amino acids, e.g. crude mixtures of amino acids, should be tested. To produce the biobased nitriles, constraints should be applied to polluting industries to increase the price of fossil-based nitriles and as a result make the biobased nitriles more competitive. In chapter 6, the results presented in chapters 2-5 and their implications are discussed. Suggestions for future research and concluding remarks are also provided.</p

Synthesis and Conformational Analysis of the First 3-Oxa-7-thia-1- r-azabicyclo[3.3.0]-c-5-octane Single Functionalised at the C-5 Position

International audienc

Enzymatic halogenation and oxidation using an alcohol oxidase-vanadium chloroperoxidase cascade

The chemo-enzymatic cascade which combines alcohol oxidase from Hansenula polymorpha (AOXHp) with vanadium chloroperoxidase (VCPO), for the production of biobased nitriles from amino acids was investigated. In the first reaction H2O2 (and acetaldehyde) are generated from ethanol and oxygen by AOXHp. H2O2 is subsequently used in the second reaction by VCPO to produce HOBr in situ. HOBr is required for the non-enzymatic oxidative decarboxylation of glutamic acid (Glu) to 3-cyanopropanoic acid (CPA), an intermediate in the production of biobased acrylonitrile. It was found that during the one pot conversion of Glu to CPA by AOXHp-VCPO cascade, AOXHp was deactivated by HOBr. To avoid deactivation, the two enzymes were separated in two fed-batch reactors. The deactivation of AOXHp by HOBr appeared to depend on the substrate: an easily halogenated compound like monochlorodimedone (MCD) was significantly converted in one pot by the cascade reaction of AOXHp and VCPO, while conversion of Glu did not occur under those conditions. Apparently, MCD scavenges HOBr before it can inactivate AOXHp, while Glu reacts slower, leading to detrimental concentrations of HOBr. Enzymatically generated H2O2 was used in a cascade reaction involving halogenation steps to enable the co-production of biobased nitriles and acetaldehyde

Unusual differences in the reactivity of glutamic and aspartic acid in oxidative decarboxylation reactions

Amino acids are potential substrates to replace fossil feedstocks for the synthesis of nitriles via oxidative decarboxylation using vanadium chloroperoxidase (VCPO), H2O2 and bromide. Here the conversion of glutamic acid (Glu) and aspartic acid (Asp) was investigated. It was observed that these two chemically similar amino acids have strikingly different reactivity. In the presence of catalytic amounts of NaBr (0.1 equiv.), Glu was converted with high selectivity to 3-cyanopropanoic acid. In contrast, under the same reaction conditions Asp showed low conversion and selectivity towards the nitrile, 2-cyanoacetic acid (AspCN). It was shown that only by increasing the amount of NaBr present in the reaction mixture (from 0.1 to 2 equiv.), could the conversion of Asp be increased from 15% to 100% and its selectivity towards AspCN from 45% to 80%. This contradicts the theoretical hypothesis that bromide is recycled during the reaction. NaBr concentration was found to have a major influence on reactivity, independent of ionic strength of the solution. NaBr is involved not only in the formation of the reactive Br+ species by VCPO, but also results in the formation of potential intermediates which influences reactivity. It was concluded that the difference in reactivity between Asp and Glu must be due to subtle differences in inter- and intramolecular interactions between the functionalities of the amino acids.</p

Unusual differences in the reactivity of glutamic and aspartic acid in oxidative decarboxylation reactions

Amino acids are potential substrates to replace fossil feedstocks for the synthesis of nitriles via oxidative decarboxylation using vanadium chloroperoxidase (VCPO), H2O2 and bromide. Here the conversion of glutamic acid (Glu) and aspartic acid (Asp) was investigated. It was observed that these two chemically similar amino acids have strikingly different reactivity. In the presence of catalytic amounts of NaBr (0.1 equiv.), Glu was converted with high selectivity to 3-cyanopropanoic acid. In contrast, under the same reaction conditions Asp showed low conversion and selectivity towards the nitrile, 2-cyanoacetic acid (AspCN). It was shown that only by increasing the amount of NaBr present in the reaction mixture (from 0.1 to 2 equiv.), could the conversion of Asp be increased from 15% to 100% and its selectivity towards AspCN from 45% to 80%. This contradicts the theoretical hypothesis that bromide is recycled during the reaction. NaBr concentration was found to have a major influence on reactivity, independent of ionic strength of the solution. NaBr is involved not only in the formation of the reactive Br+ species by VCPO, but also results in the formation of potential intermediates which influences reactivity. It was concluded that the difference in reactivity between Asp and Glu must be due to subtle differences in inter- and intramolecular interactions between the functionalities of the amino acids.</p

Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids

10 páginas.- 4 figuras.- 2 tablas.- 30 referencias.- Supporting information for this article is given via a link at the end of the document https://onlinelibrary.wiley.com/doi/10.1002/anie.202217372he hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. Here, we have engineered a highly regioselective fungal peroxygenase for the w-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward sub-terminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g/L in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (w-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83% ee through the (S)-enantiomer.The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. Here, we have engineered a highly regioselective fungal peroxygenase for the w-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward sub-terminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g/L in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (w-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83% ee through the (S)-enantiomer.This work was supported by the European Union Project grant H2020-BBI-PPP-2015-2-720297-ENZOX2, the I+D+I PID2019-106166RB-I00-OXYWAVE Spanish project funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (AEI)/doi: 10.13039/501100011033/, the PID2020-118968RB-I00 LILI project from the Spanish MCIN/AEI/10.13039/501100011033, the ‘Comunidad de Madrid’ Synergy CAM project Y2018/BIO-4738-EVOCHIMERA-CMand the PIE-CSIC projects PIE-202040E185 and PIE-201580E042. PGS thanks the Ministry of Science, Innovation and Universities (Spain) for her FPI scholarship (BES-2017-080040) and to the Ministry of Science and Innovation for her contract as part of PTQ2020-011037 project funded by MCIN/AEI/10.13039/501100011033 within the NextGenerationEU/PRTR.DGP thanks Juan de la Cierva Incorporación contract Ref nº: IJC2020-043725-I, funded by MCIN / AEI / 10.13039/501100011033, and the EUNextGenerationEU/PRTR program. The authors thank the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance with the BL13-XALOC beamline.Peer reviewe