ReviewRecent advances in artificial enzyme cascades for the production of value-added chemicals
Introduction
In recent decades, numerous achievements were made in the green and sustainable production of high-value chemicals through the development of selective and efficient catalysts (Chapman et al., 2018), simplification of reaction routes (Rogers and Jensen, 2019), and the use of greener reaction media (Erythropel et al., 2018, Potdar et al., 2015). One of the most significant achievement is employing enzymes as catalysts, which often gives high efficiency, high chemo-, regio-, and stereo-selectivity, under mild reaction conditions. Thus far, enzyme catalysed synthesis has been widely applied for the manufacturing of chemicals in pharmaceutical industry (Sheldon and Brady, 2019), fine chemical industry (Pereira et al., 2018, Seo et al., 2018, Yuan et al., 2018), energy industry (Wong et al., 2019), food and agriculture industries (Garzon-Posse et al., 2018, Wu et al., 2020). Recently, enzyme-catalysed cascade reactions have received increasing attention (Schrittwieser et al., 2018, Wu and Li, 2018).
Organic synthesis of high-value chemicals are generally multi-step reactions performed via step-by-step process which requires the isolation and purification of the compounds produced in each step. Cascade reaction allows two or more chemical reactions simultaneously or sequentially performed in a single reaction vessel without the isolation of intermediates, thus is simpler and more attractive (Schrittwieser et al., 2018). The development of cascade reactions with chemical catalyst is difficult due to the incompatibility of reaction conditions. Inspired by the metabolic pathways in living cells which comprised multiple enzymes, researchers began to mimic this natural system and combine enzymes in one pot to perform cascade reactions for chemical synthesis (Muschiol et al., 2015, Santacoloma et al., 2011). This could avoid the isolation of intermediates and minimize the generation of wastes. In addition, enzyme cascades could overcome the possible thermodynamic hurdles and avoid inhibition by intermediates (Li et al., 2016), thus improving the product yield. Furthermore, the process time and units could be significantly reduced which in turn decreased the cost of operation and energy utilization. As a result, many artificial cascade reactions were recently developed for the production of value-added chemicals by using and combining enzymes from different organisms in one-pot. These artificial enzyme cascades are clearly distinguished from the natural metabolic pathways (Schrittwieser et al., 2018), providing efficient synthesis of non-natural chemicals or improved synthesis of natural chemicals. Enzyme cascade reactions could be implemented in vitro or in vivo, and have significant advantages over chemical synthesis methods. For instance, in vitro enzyme cascade allows to perform one-pot reactions with high flexibility for adjusting enzyme amounts for each reaction step to enable the full conversion, and also provides cleaner reactions. On the other hand, in vivo enzyme cascade using whole-cells allows to perform one-pot reactions in simpler and cheaper way, and also provides cofactor regeneration system using native cellular metabolic pathways which is highly wanted for many oxidations and reductions. Many production methods for both natural and non-natural high-value chemicals from simple starting materials were established using artificial enzyme cascade which shows high potential for industrialization. For example, 80.4 g/L of β-alanine was successfully produced from fumaric acid via E. coli cells co-expressing ʟ-aspartase (AspA) and ʟ-aspartate-α-decarboxylase (PanD) with a conversion of 95.3% in 12 h (Qian et al., 2018). The production of 13.3 g/L vanillin from ferulic acid (100 mM) was achieved with E. coli cells co-expressing newly identified thermophilic phenolic acid decarboxylase (Pad) and aromatic dioxygenase (Ado) in 18 h (Ni et al., 2018). Recently, the production of GSK2879552, a lysine-specific demethylase-1 inhibitor, was demonstrated by GlaxoSmithKline using a two-enzyme cascade comprising ketoreductase and engineered imine reductase with 99.5% ee (Schober et al., 2019).
New enzyme cascades consisting of different enzymatic reactions, such as oxidation, reduction, hydrolysis, amination, isomerization, and condensation, were developed for the synthesis of high-value chemicals such as chiral alcohols (Wu et al., 2014), amines (Rinaldi et al., 2020), aldehydes (Ni et al., 2018), ketones (Hohagen et al., 2017), hydroxy acids (Hou et al., 2019), amino acids (Liu et al., 2018a, Xu et al., 2020), dicarboxylic acids (Wang et al., 2020), lactones and esters, etc (Wu and Li, 2018). Majority of the reported cascades consists of two or three enzymes (Table 1). In addition, a clear trend can be seen in the increase of multi-step cascades to expand the substrates and products scopes, enriching the manufacturing methods for high-value chemicals from simple starting materials (Table 2). To account for sustainability, the use of bio-based substrates and renewable feedstocks as starting materials for cascade reactions are receiving increasing attention.
The rapid development of multi-step artificial enzyme cascades in recent years is partially due to the recent advances in synthetic biology, protein engineering, metabolic engineering, and DNA sequencing techniques. The design of a multi-enzyme cascade mainly utilizes two strategies namely ‘forward’ and ‘reverse’ (retrosynthesis) design (Wu and Li, 2018). In the ‘forward design’, the substrate is fixed as the starting point, and enzyme reactions are added to the cascade until a desired product with high-value was obtained. This method can be considered as a value-added process and applied to starting materials that are cheap and easily available in large quantities, such as styrene, amino acids, and sugars. The ‘reverse design’ strategy refers to the retrosynthetic analysis commonly used in total synthesis, by fixing the target product and establishing an enzyme cascade that produces the target product from a desired starting material.
In this review, recent progresses (mostly from the year 2015 to date) on the enzyme cascade reactions for the production of high-value chemicals were discussed (Fig. 1). Specifically, we review the important 2-step cascades for the conversion of alcohols to amines, a highly wanted green chemistry reaction in pharmaceutical manufacturing; 2-step cascades combining diverse reactions for the functionalisation of simple chemicals; multi-step cascade conversion of styrenes, epoxides, cyclic alkanes, and aromatic compounds to high-value chemicals; multi-step cascade conversion of bio-based substrates such as ʟ-phenylalanine (ʟ-phe) to value-added chemicals; and the combination of enzyme cascades with natural biosynthesis pathways to produce fine chemicals from renewable feedstocks such as glucose and glycerol. The challenges in the development of enzyme cascades are addressed, and the possible future research directions on enzyme cascades are also briefly discussed. The detailed history, classification of cascades (France et al., 2016, Köhler and Turner, 2015, Schrittwieser et al., 2018), as well as process technology (Xue and Woodley, 2012) were reviewed recently, thus not being covered in this review.
Section snippets
Bioproduction of amines from alcohols via oxidation-amination enzyme cascades
Amines, especially chiral amines, are very important and valuable chemicals widely used in the pharmaceutical and agrochemical industries: ʟ-3,4-dihydroxyphenylalanine (ʟ-DOPA) is used for the treatment of Parkinson's disease (Wei et al., 2008); sitagliptin is used as anti-hyperglycaemic drug (Savile et al., 2010); (S)-ketamine is used as anaesthesia (Chen and Lu, 2019); ʟ-phosphinothricin and (S)-metolachlor are used as herbicides (Blaser and Spindler, 1997, Cao et al., 2020); (R)-metalaxyl is
Enzyme cascades involving epoxide hydrolase
In recent years, many new 2-step artificial enzyme cascades were developed for versatile enantioselective synthesis. In an early proof-of-concept study, an epoxidation-hydrolysis enzyme cascade was demonstrated for the dihydroxylation of styrene by using styrene monooxygenase (SMO) and epoxide hydrolase (SpEH) (Xu et al., 2009). Wu et al. further refined this system by engineering E. coli (SSP1) co-expressing SMO and SpEH for S-enantioselective dihydroxylation of styrene and eight derivatives
Enzyme cascades involving styrene oxide isomerase
Styrene is a cheap and readily available aromatic petrochemical, being an attractive industrial feedstock for producing aromatic compounds. In the past few years, several enzyme cascades were developed to produce high value aromatic chemicals from styrene and its derivatives (Wu et al., 2017a, Wu et al., 2017b, Wu et al., 2016). Wu et al. reported an epoxidation-isomerization-reduction enzyme cascade comprising SMO, styrene oxide isomerase (SOI) from Pseudomonas sp. VLB120, and a
Cascade biotransformation of styrene oxide
The asymmetric synthesis of (R)-phenylglycinol from racemic styrene oxide was achieved by using a hydrolysis-oxidation-amination cascade (Fig. 5a) (Sun et al., 2019) containing a mutant of epoxide hydrolase (VrEH2M263V), a glycerol dehydrogenase (EaGDH), and a mutant of ω-transaminase (VfTAY150F/V153A). An AlaDH was used to regenerate the cofactor NAD+ and remove by-products pyruvate. To further drive the thermodynamic equilibrium of the cascade reaction towards product generation, in situ
Cascade biotransformation of ʟ-phenylalanine and ʟ-tyrosine
ʟ-Phenylalanine (ʟ-phe) is a bio-based substrate that could be obtained by microbial fermentation. For instance, an industrial production strain Corynebacterium glutamicum has achieved the fermentative production of ʟ-phe at 57 g/L (Sanchez et al., 2018). Recently, E. coli strains were successfully engineered to produce ʟ-phe from glucose at 62.47 g/L (Ding et al., 2016) and 72.9 g/L (Liu et al., 2018b), respectively. A 2-step enzyme cascade for deamination-decarboxylation reaction could
Bioproduction of high-value chemicals from renewable feedstocks via multi-step artificial enzyme cascades coupled with natural biosynthesis pathway
Glucose is a renewable feedstock that could be obtained from nature through hydrolysis of non-edible lignocellulosic biomass such as sugarcane bagasse. Similarly, glycerol could be obtained as a by-product from biodiesel synthesis, therefore considered as sustainable starting material. As the society is moving towards green and sustainable processes, production of high-value chemicals from renewable feedstocks such as glucose and glycerol is of great interest. E. coli could efficiently grow on
Challenges and future outlook
The demonstration of the efficiency of artificial enzyme cascades for organic synthesis using simple and easily available substrates attracted increasing attention from industries. However, to fulfil the requirements of industrial applications, further increase of enzyme activity and development of new enzymes are essential. With the help of bioinformatics and gene sequencing technologies, finding new enzymes with desired properties from the nature is a promising solution. In order to speed up
Conclusions
One-pot artificial enzyme cascades have become a useful tool for green and sustainable synthesis, proving high efficiency and selectivity, avoiding intermediate isolation, and minimizing waste generation. 2-step cascades were developed for functionalising simple substrates to produce versatile chemicals. Multi-step cascades were engineered to convert petrochemicals and bio-based substrates to high-value chemicals. Bioproduction of value-added chemicals from renewable feedstocks were also
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank the financial support from the National Research Foundation (NRF) Singapore under its Advanced Manufacturing and Engineering Individual Research Grant (Project No. A1783c0014) administered by the Agency for Science, Technology and Research.
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