Advances in Enzymatic Oxyfunctionalization of Aliphatic Compounds

Biotechnology Advances xxx (xxxx) xxx Contents lists available at ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv Advances in enzymatic oxyfunctionalization of aliphatic compounds Carmen Aranda a,1,2, Juan Carro b,1, Alejandro Gonzalez-Benjumea´ a, Esteban D. Babot a, Andres´ Olmedo a, Dolores Linde b, Angel T. Martínez b,*, Ana Guti´errez a,* a Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC, Reina Mercedes 10, 41012 Seville, Spain b Centro de Investigaciones Biologicas´ "Margarita Salas" (CIB), CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain ARTICLE INFO ABSTRACT Keywords: Selective oxyfunctionalizations of aliphatic compounds are difficultchemical reactions, where enzymes can play Peroxygenases an important role due to their stereo- and regio-selectivity and operation under mild reaction conditions. P450 UPO monooxygenases are well-known biocatalysts that mediate oxyfunctionalization reactions in different living P450 organisms (from bacteria to humans). Unspecific peroxygenases (UPOs), discovered in fungi, have arisen as Biocatalysis “dream biocatalysts” of great biotechnological interest because they catalyze the oxyfunctionalization of Oxyfunctionalization Hydroxylation aliphatic and aromatic compounds, avoiding the necessity of expensive cofactors and regeneration systems, and Epoxidation only depending on H2O2 for their catalysis. Here, we summarize recent advances in aliphatic oxy Alkanes functionalization reactions by UPOs, as well as the molecular determinants of the enzyme structures responsible Fatty acids for their activities, emphasizing the differences found between well-known P450s and the novel fungal Terpenoids peroxygenases. 1. Introduction compounds is of interest for the production of new more active drugs and for the safety testing of their metabolites in the organism to assess The activation of C–H bonds in aliphatic and other compounds for human risks and ensure clinical safety of new therapeutic agents their oxyfunctionalization is of great interest for the chemical industry. (Atrakchi, 2009; Baillie et al., 2002). On the other hand, epoxidation and Those bonds are thermodynamically strong and rather inert and, other oxygenation reactions are under the spotlight due to the current therefore, their activation is an intricate task (Balcells et al., 2010; Wang and potential commercial applications of the obtained products. Epox et al., 2017; Xue et al., 2017). Not only is the activation of the said bonds ides from vegetable oils and their hydrolysis or transesterification difficult,but also that of molecular oxygen is hindered due to its ground products are of interest for several industrial applications such as sta triplet state. Transition metals are currently employed for this purpose, bilizers and plasticizers (Jia et al., 2016; Kandula et al., 2014), and are but the outcome of the reactions is not desirable due to the poor selec also promising intermediates for the production of polyols (Zhang et al., tivity they display (Roduner et al., 2013), so biocatalysis can present 2014a), polyurethanes (Zhang et al., 2014b), biolubricants (Borugadda different advantages. and Goud, 2014) and epoxy resins (Xia and Larock, 2010), among other The addition of oxygen to aliphatic hydrocarbons and biobased uses. lipids, which are cheap and widespread feedstocks, may convert them The main advantage of using biocatalysis in the synthesis of these into very valuable compounds, such as building blocks or pharmaceu compounds is the enzymatic selectivity, which is in contrast with the use ticals. In this review, we will focus in two types of oxygenation reactions, of small-molecule chemical catalysts. Normally, enzymes engulf the namely hydroxylation and epoxidation. The reaction of hydroxylation is substrates in such a way that their positioning inside the catalyst re one of the most common ways of drug metabolism leading to: i) stricts the regions that can be altered. Thus, regio- and even stereo- detoxification processes by increasing hydrophilicity of more hydro selectivity arises. Moreover, the economic and environmental benefits phobic compounds for subsequent excretion; or ii) bioactivation pro that make biocatalytic methods attractive include the biodegradable and cesses producing reactive metabolites. The “in vitro” synthesis of these nontoxic nature of biocatalysts and their ability to perform their * Corresponding authors. E-mail addresses: [email protected] (A.T. Martínez), [email protected] (A. Gutierrez).´ 1 These two authors equally contributed to the work. 2 Current address: Johnson Matthey, Cambridge Science Park U260, Milton Road, Cambridge CB4 0FP, UK. https://doi.org/10.1016/j.biotechadv.2021.107703 Received 7 November 2020; Received in revised form 17 January 2021; Accepted 25 January 2021 Available online 3 February 2021 0734-9750/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article as: Carmen Aranda, Biotechnology Advances, https://doi.org/10.1016/j.biotechadv.2021.107703 C. Aranda et al. Biotechnology Advances xxx (xxxx) xxx selective reactions under mild conditions (Sheldon and Woodley, 2018). or two oxygen atoms (thus known as dioxygenases, not included in the Thanks to advances in genetic and protein engineering, allowing for the present review) from O2 (e.g. the well-known P450s) or H2O2 (e.g. the easier production of better biocatalysts, and the new methods of reaction more recently discovered UPOs, also acting as monooxygenases) to engineering (Wohlgemuth, 2017; Woodley, 2019), biocatalytic methods different substrates (Fig. 1) introducing new functionalities into them. are emerging as an alternative to traditional synthetic chemistry. As a These enzymes are of great interest for synthetic chemistry since they consequence, the amount of processes able to be carried out at prepar can act even on the most chemically inert positions of aliphatic and ative scales (even in some cases at industrial scale) is continuously other compounds (Dong et al., 2018). growing (Chakrabarty et al., 2020; Wiltschi et al., 2020). However, the use of biocatalysts also presents some disadvantages. In general, en zymes introducing oxygen atoms display rather low turnover numbers 2.1. Classical monooxygenases due to their intrinsic complex mechanisms, often require multiprotein interactions and cofactors and, in many cases, there exist uncoupling of Monooxygenases are the main natural biocatalysts to mediate oxy electron transfer and product formation, giving rise to reactive oxygen functionalizations, being P450s the largest and most diverse group species (Girhard et al., 2015; Holtmann and Hollmann, 2016). (Munro et al., 2018). P450s are heme-containing enzymes that catalyze In this review, we highlight the advances in enzymatic oxy the insertion of one atom of oxygen (Fig. 2) through the electrophilic functionalization of a number of aliphatic compounds and the current species Compound I. Most P450s require nicotinamide cosubstrates and availability of new and promising oxygenation biocatalysts. Special NAD(P)H-driven redox partners for function, to transform the hydride emphasis is made on the reactions catalyzed by the self-sufficient un donation from NAD(P)H into two consecutively single electron transfer specific peroxygenases (UPOs), which have been termed “dream bio enabling O2 activation and formation of compound I. catalysts” owing to their unique features (Wang et al., 2017) that make There are some P450s, grouped into classes VII and VIII, that possess them an appealing starting point for biotechnological applications. both the P450 (heme) and the reductase domains fused, which makes Moreover, comparison with other oxygenases, such as cytochrome P450 them of high relevance for synthetic chemistry (Ciaramella et al., 2017). monooxygenases (P450s), is included in some cases. In addition to the Typical examples of these enzymes are the P450RhF from Rhodococcus sp. reactions that these enzymes catalyze, a smattering of their structure- (Roberts et al., 2003) and the naturally-fused and more studied P450 function relationships, as well as the biotechnological efforts to render from Bacillus megaterium (P450BM3), the firstof this kind to be described “ ” them better biocatalysts, are provided. (Whitehouse et al., 2012). Other naturally fused ( self-sufficient ) en zymes mentioned in this review are the P450s from Labrenzia aggregata 2. Oxyfunctionalization biocatalysts (P450LaMO) (Yin et al., 2014) and those found in thermophilic Amyco latopsis thermoflava, Jahorihella thermophila and Thermobispora bispora Oxygenases are biocatalysts able to transfer one oxygen atom (in the (Tavanti et al., 2018). Besides, artificial chimeric proteins have been case of monooxygenases, where the second oxygen is reduced to water) constructed in which the P450 catalyst of interest and a reductase domain of choice are fused. This simplifies the catalysis to the only Fig. 1. Aliphatic oxyfunctionalizations by classical two-domain P450s and fungal UPOs differing in their intra- or extra-cellular location, the former being associated with lower operational stability and dependence on a source of reducing power. 2 C. Aranda et al. Biotechnology Advances xxx (xxxx) xxx Fig. 2. Comparison of reactive compound I formation by reductive activation of O2 in classical P450s or by H2O2 in peroxygenases. addition of

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