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. Author manuscript; available in PMC: 2021 Feb 22.
Published in final edited form as: Nat Chem. 2020 Dec 30;13(2):140–148. doi: 10.1038/s41557-020-00606-w

Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination

James R Marshall 1,#, Peiyuan Yao 1,2,#, Sarah L Montgomery 1, James D Finnigan 3, Thomas W Thorpe 1, Ryan B Palmer 1, Juan Mangas-Sanchez 1, Richard A M Duncan 3, Rachel S Heath 1, Kirsty M Graham 3, Darren J Cook 3, Simon J Charnock 3, Nicholas J Turner 1,✉
PMCID: PMC7116802  EMSID: EMS114756  PMID: 33380742

Abstract

Finding faster and simpler ways to screen protein sequence space to enable the identification of new biocatalysts for asymmetric synthesis remains both a challenge and a rate-limiting step in enzyme discovery. Biocatalytic strategies for the synthesis of chiral amines are increasingly attractive and include enzymatic asymmetric reductive amination, which offers an efficient route to many of these high-value compounds. Here we report the discovery of over 300 new imine reductases and the production of a large (384 enzymes) and sequence-diverse panel of imine reductases available for screening. We also report the development of a facile high-throughput screen to interrogate their activity. Through this approach we identified imine reductase biocatalysts capable of accepting structurally demanding ketones and amines, which include the preparative synthesis of N-substituted β-amino ester derivatives via a dynamic kinetic resolution process, with excellent yields and stereochemical purities.

The discovery and development of novel biocatalysts for applications in organic synthesis has relied on various approaches, which include directed evolution1, in silico computational design2,3, exploration of existing sequence space to find new chemistries4,5, exploiting cofactor versatility to catalyse alternative chemical reactions6 or, indeed, any combination of these methods7. Most of these approaches rely on the availability of high-throughput screening (HTS) methods to interrogate large libraries of variants8. The application of HTS methods is pivotal in other areas of organic chemistry and enabled the development of catalysts9,10, reaction discovery11 and reaction optimization12. However, screening remains a major bottleneck in biocatalysis. In a recent report, although >500 putative haloalkane dehalogenases were identified from the available sequence data13, only 20 different enzymes were characterized for activity in vitro after in silico screening. Previous biocatalytic HTS strategies utilized mass spectrometry14 and microfluidic methods linked to fluorescence15 or absorbance16 to screen the activity of variants17,18. However, these methods often rely on expensive and specialized equipment, which limits their use by the wider community.

The repertoire of biocatalysts for the synthesis of chiral amines is ever increasing and includes lipases19, transaminases19, ammonia lyases20, amine oxidases21,22, P411s23, opine dehydrogenases24, amine dehydrogenases/amino acid dehydrogenases21,24 and N-methylamino acid dehydrogenases25. Unsurprisingly, this expansion was fuelled by the prevalence of the chiral amine moiety across biologically active compounds, active pharmaceutical ingredients (APIs) and agrochemicals24. Asymmetric reductive amination is one of the most important reactions in their synthesis26,27; it accounts for nearly one-quarter of the N-arylation and N-alkylation reactions used in the synthesis of drug candidates28. Chemocatalytic reductive amination can suffer from poor chemoselectivity29, and relies on the use of precious metals to reduce preformed imines30. Similarly, N-alkylation has inherent drawbacks, especially the requirement for genotoxic agents31. Enzymatic approaches can address these issues as they utilize a biodegradable and highly chemo- and stereoselective catalyst under benign conditions, which complies with the metrics of green chemistry19.

Imine reductases (IREDs) have emerged as a synthetically versatile class of biocatalysts for preparing chiral amines32–40. These enzymes are able to synthesize a broad range of primary and secondary, and some tertiary, amine products in comparison with other enzyme families, such as transaminases, and ammonia lyases (which only yield the primary amines) or amine dehydrogenases (which have limited substrate scope)41. This has resulted in the rapid discovery and application of IREDs for the synthesis of various APIs and scaffolds42–44, recently an LSD1 inhibitor (GSK2879552)44. However, certain reductive amination reactions remain a challenge, such as hindered amine partners and conjugated ketones. IRED sequences greatly outnumber other enzyme classes, such as amine oxidases19 and amine dehydrogenases21,45–49, which led to many academic48,49 and industrial groups35,45–47 screening small panels of IREDs (28–95 enzymes). These biocatalysts were either evaluated against only a single substrate or by low-throughput screening methods. Screening IREDs in the area of reductive amination is further hampered by the fact that for each carbonyl substrate there are numerous amine partners, and vice versa32,50. For example, to screen 50 carbonyl substrates with 50 different amines donors across a panel of 400 IRED enzymes requires 106 individual experiments. Therefore, to establish a high-throughput colorimetric screen for IRED activity was identified as a key step in the ability to screen large panels of IRED biocatalysts (Fig. 1).

Fig. 1. A workflow and approximate time frame for generating a metagenomic library with examples of how this platform was used to expand the biocatalytic toolbox as applied to iReDs.

Fig. 1

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Various environmental niches were sampled (1) to obtain DNA from the environment without the need for pre-culturing of the organism. DNA was sequenced through a Mi-Seq Illumina platform. Sequence reads (contigs) were assembled, processed and deposited to the relevant database (2). Query sequences of known enzymes were used to identify putative sequences, which were then cloned and expressed heterologously in E. coli (3). Enzymes were arrayed into mictotitre plates for screening (4), which then enabled expansion of the biocatalytic scope across various avenues, which included synthetic scopes and operational properties (bottom). bp, base pairs.

We set out to harness the wealth and diversity of data offered by metagenomics51–54 to explore considerable, unchartered sequence space to discover new IREDs with an expanded substrate range and enhanced thermostability via an arrayment of wild-type enzymes into microtitre plates and the development of a HTS.

Results and discussion

Identification and cloning of metagenomic IREDs

The metagenomic libraries investigated in this study were accessed from terrestrial and coastal geographical sites across the United Kingdom (Supplementary Section 3.2.1) in compliance with the Nagoya Protocol55. The libraries contain over 2.5 billion base pairs of DNA, which highlights the wealth of data available for interrogation. The diversity of the taxa between each metagenomic library is visualized in Supplementary Fig. 3.1.3, which shows a clear distinction between the hierarchies of each metagenome. Manipulation and subsequent mining of this sequence data was performed using the bioinformatics platform ProzOMIGO (developed at Prozomix). The platform enabled an efficient mining of libraries for putative IREDs and ensured that a concise but diverse dataset was obtained. In total, more than 677 full-length metagenomic sequences were returned (Supplementary Section 3.2.4). After the removal of redundant sequences, the putative panel size was trimmed to 302 putative sequences such that no sequence relationship had a >95% sequence identity. The diverse sequence space explored is highlighted by enzymes such as pIR-110, which shares one of lowest primary sequence identities in public databases, for which at 63% the closest related sequence is from Chryseobacterium piperi. pIR-110 was mined in the preliminary stages of the development of this panel, and has been applied to the synthesis of enantiomerically pure substituted azepanes56. A sequence similarity network (Supplementary Fig. 3.2.4) that compared this selection of sequences to previously described IREDs47 and other families of NAD(P)H-dependent oxidoreductases was constructed. This network demonstrates that a similar but diverse sequence space was interrogated and expanded (when compared with those previously described)47, and also identified a direct relationship between 4-hydroxy-tetrahydrodipicolinate reductases (EC 1.17.1.8) and IREDs, which suggests either activity promiscuity or an evolutionary relationship. To generate further sequence diversity, the selected metagenomic sequences were augmented with 90 genomic IREDs to create a panel of putative IREDs from prokaryotic and eukaryotic sources that covered fungi, plantae, animals, proteobacteria and gram-positive bacteria (Supplementary Section 3.2.6)57. A total of 392 putative IRED genes were cloned into pET28a and heterologously expressed in Escherichia coli BL21(DE3) (Supplementary Section 3.3.2).

Development of a 384-well plate high-throughput IRED screen

To interrogate the activity of all putative IREDs towards the combinatorial reductive amination, the aim was to develop a quick and simple HTS. By utilizing the reversibility of IRED-catalysed redox chemistry, we were able to determine the enzyme activity though a colorimetric screen (termed IREDy-to-go). The nicotinamide cofactor generated on the IRED-mediated oxidation of a target amine with NAD(P)+ could be detected by the known diaphorase-mediated reduction of the tetrazolium salt 5, which led to the formation of the corresponding red/pink formazan dye 6 (Fig. 2a)58 generating a tetrazolium-based IRED screen. Screening for the oxidation of a target amine is advantageous because two different carbonyl and amine substrates can be tested simultaneously as oxidation can take place on either C–N bond (Supplementary Scheme 5.5.1). Optimization and initial colorimetric screen validation was undertaken using salsolidine 1 (Fig. 1) and N-methylcyclohexylamine 2a, and a small selection of IREDs (Supplementary Section 4.1). (Note: a full list of all ketone, amine and imine substrates described here is given in Supplementary Section 2; the convention used for reductive amination reactions is that amine 2a results from the reductive amination of carbonyl 2 with amine a). Based on these initial results and with the number of enzymes identified, liquid-handling robots were utilized to generate >1,000 copies of this 384 IRED plate, which allowed for the rapid characterization of their activity. These plates are freely available through Prozomix (Supplementary Section 4.3).

Fig. 2. 384 iReDy-to-go screening of amine 2b combined with biotransformation data for the reductive amination of 2 with b mapped phylogenetically to show the overall distribution of activity.

Fig. 2

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a, A heatmap representative of IREDy-to-go screen of 2b in which conversion (%, determined by a GC–flame ionization detector) for the reductive amination of 2 and b are overlaid to each well for each IRED. IREDy-to-go data generated by measuring the end point read at λ = 490 nm is also shown behind the data generated by a GC–flame ionization detector as a heat map. A deeper red colour represents a higher absorbance. b, The distribution of activity and stereoselectivity mapped phylogenetically across all 384 IREDs for the reductive amination of 2 with b shown as bars with a ■red–white gradient in which darker red represents a higher conversion up to >99%. The multi-value bar chart represents the % conversion (as the total height), with the percentage of each enantiomer for the reduction of 4 to 3 with grey representing % (S)-3 ▶ and light purple denoting % (R)-3 ▶. The expression is represented by the outer blue gradient, in which low expression is shown in light blue ● and good expression in dark blue ●. The parental organism of each IRED (if known) is highlighted through tip labels, clades and branches (centre): plantae IREDs ■, fungal IREDs ■, human IREDs ■, cyanobacteria IREDs ■ and bacterial IREDs ■ and ■.

Further validation of screen was undertaken with N-cyclopropylcyclohexylamine 2b and 2-phenylpiperidine 3, which were tested against the entire panel of 384 enzymes using the colorimetric screen (Fig. 2a). In parallel, analytical scale biotransformations were performed across all 384 enzymes for the corresponding reductive amination of ketone 2 and amine b and the reduction of imine 4 (Fig. 1) to assess the correlation between activity in the oxidative (IREDy-to-go) and reductive (synthetic) reaction directions. The panel exhibited excellent activity for each synthetic transformation, and over one-quarter of the enzymes gave >99% conversion for both types of reactions. As expected, both enantiomers of 3 could be accessed via the reduction of imine 4, 70% (S)-3 and 30% (R)-3 (Supplementary Table 5.4.1). The heat map of the 384-well plate (Fig. 2a) shows the correlation between the spectrophotometric colorimetric data for the oxidation of amine 2b (colour intensity) and conversion for the reductive amination of 2 and b (numerical data). Interestingly, some enzymes were not active towards the oxidation of 2b, but were active towards the reductive amination of 2b, which is further explored in Supplementary Section 4.4.3. In addition, it was observed that nine enzymes consistently changed colour independent of the presence of amine or diaphorase during the colorimetric screening; these enzymes are explored in Supplementary 4.4.5 and were not selected for further reductive amination reactions. However, enzymes identified via the oxidative screen were able to efficiently catalyse the corresponding imine reduction (4 to 3) or reductive amination (for the synthesis of 2a) when using glucose dehydrogenase (GDH) to recycle the nicotinamide cofactor (Supplementary Section 4.1).

To assess the overall sequence–characteristic relationship within the IRED panel, conversions and selectivities for both the cyclic imine reduction of 4 to 3 and the reductive amination of 2 and b were mapped phylogenetically alongside the relative enzyme expression level (Fig. 2b). In all cases, enzymes that possess reductive amination activity also catalysed imine reduction; for example, pIR-122 gave a >99% conversion for the conversion of 4 to 3 (>99% e.e. (S)-3) and also for 2 and b to 2b (Supplementary 5.4). Interestingly, pIR-014 (Arabidopsis thaliana) and pIR-202 (Aspergillus fumigatus Z5) had a preference for reductive amination, which gave high conversion to 2b (both >99%), but a low conversion to 3 (5% and 16% respectively) (Supplementary 5.4). Only 12 IREDs showed activity towards imine reduction but not towards reductive amination (Supplementary Table 5.4.1). These observations agree with reports of the natural cyclic imine-reducing role of these enzymes under investigation59,60.

High-throughput characterization across the 384 enzyme panel

We next examined a structurally diverse panel of substrates, which included primary, secondary and tertiary amines, as well as APIs to characterize the enzyme panel. Fig. 3 depicts the number of hits for each compound detected by the colorimetric screen (determined by the difference of absorbance between the assay and blank plate (Supplementary Section 4.5)). In general, six- and seven-membered cyclic amines, which included bioactive alkaloids 1 (73 hits) and 9 (46 hits), proved to be well accepted across the enzyme panel, and phenylazepane rac-7 gave the most hits (103). The screening of linear amines also generated large numbers of hits (for example, 2c with 67 hits) including several natural products and APIs such as rasagiline 14d which had 48 hits.

Fig. 3. High-throughput characterization employing the colorimetric screen, a chart showing the substrates presented to the colorimetric screen alongside the number of definitive enzyme hits; the method for the number of hits calculated is given in Supplementary Section 4.5.

Fig. 3

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The IRED-mediated oxidation of the target amine is shown (top) in which the hydride is delivered to the cofactor for regeneration, which leads to subsequent reduction of 5 to 6 mediated by the diaphorase to generate a red colorimetric change. The number of enzyme hits is shown above the structure of the corresponding compound. The red colour scale in each tile is representative of the number of hits generated through the screen with the given substrate. A scale for the number of hits is also shown below the chart. A variety of amines was screened, which included five-, six- and seven-membered heterocycles (1, 3, 7, 9, 19, 20 and 21), linear, allylic and benzylic amines (2a, 2b, 2c, 2f, 10d, 11e, 12f, 13a, 13b, 13f, 13j, 13k, 15a, 22a, 23d, 24k and 25b), N-substituted β-amino ester derivatives (26b, 27b, 28b and 29b) and several APIs (14d, 16g and 18i).

For the most active substrate/enzyme combinations (for example, 2a with pIR-09 and pIR-16), colour formation began rapidly at the point of substrate addition, which correlates well with the fact that both pIR-9 and pIR-16 performed well in the analytical screening (Fig. 4).

Fig. 4. Analytical scale reductive aminations.

Fig. 4

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Reductive aminations of 2, 12, 13, 15, 27, 30, 31, 32, 33 and 34 with a, b, d, e, f, i, k and l. IRED catalyst conversions and e.e. (brackets) were determined by GC and are given below the product. APIs are i 72, ii 63, iii 73, iV 73, V 68, Vi 68 and Viii 74. See Supplementary Table 6.1 for the assignment of (A) and (B). * indicates a chiral centre. ND, not determined.

Identification of IREDs for expanded reductive amination reactions

IREDs that accept acetophenone. The 2-arylethylamine moiety is frequently found in APIs and therefore a useful target for biocatalytic reductive amination (Fig. 4); however, the precursor acetophenone 13 and derivatives for this reaction are typically poor substrates for current wild-type IREDs36,61,62. Six acetophenone-derived racemic amines (13a–13k and 15a) were assessed using the colorimetric screen (Supplementary Table 5.5.4). 13b afforded the most hits (45) (Fig. 3, row 4). Interestingly, these amine substrates underwent enzyme-dependant regiocomplementary modes of C–N oxidation, both of which resulted in colour formation during the screen but could be detected by GC–mass spectroscopy after extraction. For example, during the pIR-117-catalysed oxidation of N-(1-phenylethyl)cyclohexanamine 13j (Supplementary Scheme 5.5.1), both acetophenone 13 and cyclohexanone 2 could be detected. This enzyme could potentially allow access to two different synthetic routes to 13j catalysed by IREDs.

Reductive amination of acetophenone 13 with methylamine a had an excellent conversion (>99%) with the top three IREDs identified via the colorimetric screen (Fig. 4). Both enantiomers of 13a could be accessed with different IREDs (pIR-9 or pIR-16 generated (S)-13a and pIR-361 generated (R)-13a). Some conversion (7%), but a high e.e. (>99%) was also observed for the precursor to orvepitant63 (15a) using pIR-361 identified in the screen, which presents a future engineering target.

IREDs that accept dimethylamine

The dimethylamine moiety is also often found in APIs, which include (S)-dapoxetine64 and rivastigmine65, but existing IREDs have shown a preference for unhindered amines and pyrrolidines. We initially screened for activity towards amine 2f, which resulted in 26 hits. The three enzymes that exhibited the greatest colour change during the colorimetric assay were selected for the biocatalytic reductive amination of various carbonyls with dimethylamine f (Fig. 4). Hydrocinnamaldehyde 12 and dimethylamine (10 equiv.) underwent pIR-23-catalysed reductive coupling to give the tertiary amine product 12f (86% conversion). Dimethylamine could also be coupled with ketones 2, 30 and 31, which allowed access to five- and six-membered sterically crowded carbocyclic amines and gave moderate to good conversions (43–70%; Fig. 4).

IREDs for the synthesis of N-substituted β-amino esters

IREDs have been shown to be effective catalysts for the preparation of N-substituted-δ-amino esters and γ-lactams32,38. However, the synthesis of N-substituted β-amino esters, which are important building blocks in the pharmaceutical industry (Fig. 4), has not been reported66,67. To determine whether the IRED panel could be used to synthesize this class of amines, 27b was presented to the colorimetric screen, which resulted in eight hits (Supplementary Section 4.5). Subsequent analytical biotransformations with these IREDs demonstrated that both enantiomers of 27b could be accessed from β-keto ester 27 and cyclopropylamine b with a high conversion (>99%) and excellent enantioselectivity (>99% e.e.) (Fig. 4). Using the same set of eight IREDs, cyclopropylamine b underwent an efficient reductive amination with β-keto esters 32 and 33, as well as β-keto esters 34 and 42 (Supplementary Table 5.5.5). Interestingly, activity decreased with increasing size of the substituent at the β-position (Fig. 4)

α-Substituted β-amino esters derivatives are important chiral building blocks for the synthesis of antifungal agents and alkaloids68. Owing to the keto–enol tautomerism, the α-position of β-keto esters is prone to racemization in an aqueous medium69. Thus, we decided to examine the potential for the IRED-catalysed dynamic kinetic resolution (DKR) of these species during the reductive amination processes. The desired amine products of reductive amination (28b and 29b) were tested against the panel of IREDs using the colorimetric screen (Supplementary 4.6). All 15 hits (Table 1) selected were able to perform reductive amination of the corresponding β-keto esters with cyclopropylamine b. By selecting complementary metagenomic IREDs, three of the four possible isomers of 28b and 29b could be accessed by enzyme-mediated DKR, with a good-to-excellent conversion (56–99%), moderate-to-excellent diastereoselectivity (up to a diastereomeric ratio (d.r.) > 99:1) and good-to-excellent enantioselectivity (up to 99% e.e.). Employing a combination of pIR-361 and pIR-241, this generated complementary enantiomers of both products with excellent enantioselectivities (99% e.e. and 98% e.e., respectively, for the preparation of 28b). In some cases, an alcohol by-product was observed through the likely reduction of 28 and 29 when employing crude cell-free extracts (CFEs) in the transformations. The use of purified pIR-117 and pIR-361 eliminated these side products, which suggests that alcohol formation could be attributed to endogenous E. coli enzyme activity70.

Table 1. Synthesis of diastereomeric carbocyclic |S-amino esters through a DKR with IREDs.

graphic file with name EMS114756-i001.jpg
n = 2 n = 1
IRED Conversion (%) Alcohol (%) d.r. (cis:trans) e.e. (%) IRED Conversion (%) Alcohol (%) d.r. (cis:trans) e.e. (%)
pIR-85 >99 22 >99:1 96 pIR-85 66 1 >99:1 89
pIR-90 95 42 97:3a –55a pIR-104 67 0.4 >99:1 94
pIR-104 >99 9 >99:1 96 pIR-107 62 0.4 >99:1 93
pIR-107 98 14 >99:1 95 pIR-114 71 0.6 >99:1 93
pIR-114 >99 11 >99:1 95 pIR-117 78 0.2 40:60a –99a
pIR-117 >99 15 48:52b –87b pIR-128 62 0.6 >99:1 89
pIR-128 98 12 >99:1 93 pIR-234 61 0.6 >99:1 49
pIR-219 79 60 >99:1 90 pIR-238 56 0.4 >99:1 86
pIR-238 92 14 >99:1 94 pIR-241 74 0.6 >99:1 97
pIR-241 >99 13 >99:1 98 pIR-258 74 0.9 88:12a –92a
pIR-258 >99 18 90:10a –51a pIR-351 80 0.4 >99:1 91
pIR-351 >99 12 >99:1 92 pIR-356 72 0.6 >99:1 95
pIR-356 >99 14 >99:1 96 pIR-358 94 0.8 >99:1 97
pIR-358 >99 12 >99:1 82 pIR-360 69 0 >99:1 93
pIR-361 >99 13 81:19a –99a pIR-361 82 0 48:52a –99a
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The e.e. and d.r. were determined by chiral GC. The results shown represent access to three-quarters of the isomers of 28b and 29b and representative of cis-1B,2A.

a

Representative of cis-1A,2B.

b

Representative of trans-1B,2B.

Preparative-scale asymmetric synthesis of N-substituted β-amino esters

The synthetic utility of the metagenomic IREDs was exemplified by a series of preparative-scale reactions performed under optimized conditions at 50 mM ketone loading (Fig. 5). Excellent yields were obtained for the reductive amination of β-keto ester 27 with 2 equiv. amine b catalysed by the CFEs of pIR-117 or pIR-356. Further β-amino esters (S)- or (R)-27i, (A)- or (B)- 27d and (A)- or (B)-27e were also obtained by employing enantiocomplementary IREDs pIR-117 or pIR-355 in good-to-very good yields (31–78%) and excellent in e.e. (>99%) (definition of the assignment of (A) and (B) is given in Supplementary Table 6.1). Preparative-scale transformations were also achieved for a range of diastereomerically enriched β-amino esters (Fig. 5). DKR-mediated reductive amination of α-substituted β-keto esters yielded 28b and 29b with good-to-excellent yields (57–92%), and excellent d.r. values (99:1) for pIR-241- and pIR-358- catalysed reactions. Overall, less alcohol from the competing enzyme-catalysed ketone reduction (&3%) was observed on this scale, as confirmed by gas chromatography (GC)– flame ionization detector and NMR spectroscopy.

Fig. 5. Preparative-scale asymmetric reductive aminations of β-keto esters.

Fig. 5

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Products of the preparative-scale synthesis shown with corresponding IRED with isolated yield. For the complete figure, which includes % conversion and % e.e., see Supplementary Figure 5.6.1. The e.e. and d.r. were determined by chiral GC analysis. NaPi, sodium phosphate. (A)- and (B)- represent the absolute configuration of the product.

Investigation into the thermostability of metagenomic wild-type IREDs

To show the versatility of the platform, we employed the colorimetric screen to identify thermotolerant IREDs. A 384-well plate that contained only IRED CFEs and buffer was incubated at 70 °C for one hour, prior to the addition of the screen components. Of these, 22 gave a colour change using 2b as a substrate, which suggests that these were thermotolerant IREDs. Simple residual activity studies after incubation of all 22 IREDs at increasing temperatures were used to assess which enzymes retained activity at the highest temperature. Both fungal-derived pIR-9 (A. lentulus) and pIR-202 (A. fumigatus Z5)71 retained activity up to 60 °C. Circular dichroism was utilized to calculate the melting temperature (T m) of pIR-09 (53.5 ± 0.5 °C) and further thorough residual activity study was also performed for validation (thermal inactivation (T 50) = 49.5 ± 0.2 °C) (Supplementary Fig. 3.4.1). These thermotolerant IREDs are useful templates for directed evolution.

Conclusion

To summarize, we describe the identification, cloning, production, HTS and synthetic application of the largest panel (384) of IREDs reported to date. Exploration of metagenomic libraries and genomic databases resulted in the identification of >300 novel enzymes that covered previously undescribed sequence space. The wild-type biocatalysts were arrayed into lyophilized 384-well microtitre plates (in two different formats, colorimetric screen and CFE), which allowed a simple and rapid characterization of these enzymes to enable the identification of active biocatalysts.

In total, 36 amine substrates were screened against the panel and identified enantiocomplementary biocatalysts for the preparation of three important molecular scaffolds. The enzymatic synthesis of N-substituted β-amino esters, which includes α-substituted derivatives via a DKR process, was demonstrated and afforded products in high yield and excellent enantio- and diastereoselectivity. We also identified IREDs capable of accepting structurally demanding ketones and amines for the synthesis of high-value chiral amines. Finally, the introduction of a heat-treatment step resulted in the identification of thermotolerant IREDs, which represent excellent candidates for use as industrial biocatalysts or for further protein engineering.

The overall process from the initial enzyme discovery to generation and screening of the 384-well IRED plates took approximately two-and-a-half to three months (Fig. 1). For other families of enzymes in which there exists a similar diversity of sequence space in metagenomic and genomic libraries, together with a HTS method, we imagine similar timescales could be achieved. The panel of IREDs reported here is available and can be accessed by any researcher. We anticipate that further screening will reveal new enzyme activities within the sphere of imine reduction and reductive amination, and thereby expand the synthetic application of this family of enzymes.

Methods

General

A full description of the materials and methods, characterization of the compounds and the software used can be found in the Supplementary Information.

Access to metagenomic sequence space

Metagenomic IREDs were identified within a variety of sample sites across the United Kingdom (Supplementary Section 3.2.1). Metagenomic data were processed using ProzOMIGO (Prozomix). All the metagenome sequencing results are uploaded into ProzOMIGO and protein sequence BLAST databases created using the BlastGO function (completing a six-frame translation and open reading frame (ORF) prediction from all the assembled contigs). Known IREDs from the literature were used as query sequences for the mining approach. The returned targets were then filtered using the FilterGO tool, which eliminated any targets that shared more than a 95% sequence identity to a characterized IRED or any other IRED in the returned targets. All the selected IREDs were extracted from ProzOMIGO in a FASTA format and sent to TWIST Biosciences for codon optimization and synthesis as DNA fragments. Synthetic gene fragments were then cloned into pET28a.

Preparation and performing the colorimetric screen

For the low-throughput IREDy-to-go screens, all the low-throughput development was performed using 96-well microtitre plates (Sarstedt). Lyophilized powder of the supernatant of the lysate of IRED interest was equilibrated to room temperature and an appropriate stock solution was prepared with Tris-HCl buffer (0.1 M, pH 9.0). A mastermix was made up of the following components: amine substrate, INT, NADP+ and PRO-DIA-001, all in Tris-HCl buffer (pH 9.0). Selected IREDs: pIR-023 (CfIRED), pIR-49 (IR-22), pIR-63 (PcIRED), pIR-64, pIR-65, pIR-068, pIR-102 (mIRED 02), pIR-110 (mIRED 10), AdRedAm, AspRedAm and (+/−)-pET28a47,56. The concentrations of the components were subject to change depending on the optimization being performed as well as the buffer component and pH. Reactions were monitored spectrophotometrically at λ = 490 nm and by manual observation. For the full protocol and accompanying notes of reaction, as well as the specific concentrations in each reaction, see Supplementary Section 4.

The 384-well IREDy-to-go plates were generated using the robotics instrument Thermo Matrix PlateMate Plus liquid handler (Thermofisher Scientific), which was driven using the software Thermo Scientific Matrix ControlMate (Thermofisher Scientific) (see Supplementary Section 4.3). A mastermix solution (50 μl) was aspirated into each well with a 384-pipette handler. The mastermix was made up of PRO-DIA-001, NADP+ and IRED (lyophilized powder of supernatant of lysate) suspended in Tris-HCl buffer (0.1 M, pH 9.0). In conjunction with the generation screening plates, 384 plates that contained 0.5 mg of lyophilized powder of the supernatant of the lysate was also generated for the user’s own assay requirements. Post-aspiration, both plates were subjected to lyophilization.

To perform the 384 IREDy-to-go screen, a mastermix reagent was made up to 25 ml of 0.125 mg ml–1 INT and 10 mM amine substrate in Tris-HCl buffer (0.1 M) adjusted to pH 9.0. the mastermix (50 μl) was aliquoted to each well of the plate. The plate was then spun down at 1,500 r.p.m. for 1 min and the red membrane was removed. The plate was incubated (in the dark) at 30 °C for 24 h. An absorbance reading at λ = 490 nm was taken at 0, 1, 4 and 24 h. Expected false positives in wells B02, D08, F12, H21, I02, I07, K16, O01, O08 and P17 were unaccounted for. The same procedure was followed for the 384 IREDy-to-go blank plate except that no amine was added to the mastermix. For an extended method on performing colorimetric screen, see Supplementary Section 4.2.

Preparative-scale biotransformations

Optimized conditions were implemented for biotransformations on the preparative scale (50 ml) and performed starting from β-keto esters (25–50 mM), amine (50–250 mM), CFEs of the IREDs (from 2.5 g wet cells), d-glucose (62.5–125 mM), 8 mg of NADP+, 3 mg of GDH (150 U) and 50 ml of sodium phosphate buffer (100 mM, pH 7.0) with 4% v/v dimethysulfoxide, and the pH was adjusted to 7.6 with 1 M HCl. The reaction mixture was shaken at 30 °C and 180 r.p.m. The reaction was quenched by the addition of sodium carbonate (4.24 g, 40 mmol) and extracted with ethyl acetate/ cyclohexane (1/1 v/v) three times (50 ml) with centrifugation (15 °C, 4,000 g, 10 min) to improve the separation of the phases. The organic layers were combined and dried over anhydrous MgSO4. After removal of the solvent under reduced pressure, the residue was dissolved with 5 ml of diethyl ether. A solution of HCl in diethyl ether (2 M, 2 ml) was added, the ether layer decanted and the remaining solid or oil washed with diethyl ether (3 × 5 ml) and dried under vacuum to afford the desired product.

Supplementary Material

Supplementary material

Acknowledgements

We thank the Industrial Biotechnology Innovation Centre (IBioIC) and Biotechnology and Biological Sciences Research Council (BBSRC) for awarding the CASE studentship to J.R.M. from Prozomix Ltd. P.Y. was supported by a CSC scholarship and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (Grant no. 2016166). T.W.T. was supported by a BBSRC CASE studentship awarded by Pfizer. S.L.M. was supported by a BBSRC CASE studentship from Johnson Matthey. R.B.P. and R.S.H. were supported by the European Research Council (ERC Grant no. 742987). J.M.-S. was funded by grant BB/M006832/1 from the UK Biotechnology and Biological Sciences Research Council. S.J.C., D.J.C., J.D.F., R.A.M.D. and K.M.G. acknowledge the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 685474 for supporting MetaFluidics. N.J.T. is grateful to the ERC for the award of an Advanced Grant (Grant no. 742987). We thank D. Heyes of the Manchester Institute of Biotechnology (MIB) for assistance in gathering the circular dichroism data. We thank Y. Qi of Prozomix Ltd for screening of the Prozomix diaphorases. Prozomix and J.R.M., P.Y., S.L.M., T.W.T., R.B.P., J.M.-S., R.S.H. and N.J.T. also thank other staff of Prozomix for their support.

Footnotes

Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author contributions

N.J.T. and S.J.C. devised and supervised the project. J.M.-S. and R.S.H. managed the project. J.R.M., J.D.F, K.M.G., S.L.M. and D.J.C. performed the identification, cloning and expression of the enzymes. J.R.M., J.D.F., R.A.M.D. and S.J.C. were involved in the design, development and implementation of the colorimetric screen and 384-well plates. J.R.M. and P.Y. carried out the high-throughput characterization of the enzymes. J.R.M. and P.Y. performed the analytical scale biotransformations. P.Y. conducted the preparative-scale biotransformations. P.Y. and R.B.P. synthesized the chemical standards. J.R.M. and T.W.T. undertook the thermostability studies. N.J.T., S.J.C., J.R.M., P.Y., R.S.H., J.M.-S., J.D.F. and T.W.T. wrote the manuscript and generated the figures.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information is available for this paper at https://doi.org/10.1038/s41557-020-00606-w.

Correspondence and requests for materials should be addressed to N.J.T.

Data availability

Data supporting the results and conclusions are available within this paper and the Supplementary Information. Both the IREDy-to-go screen and a duplication of the screening plate without the colorimetric screening components containing 0.5 mg of crude lysate of each IRED in each well are freely available through Prozomix Ltd.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material
EMS114756-supplement-Supplementary_material.pdf (39.6MB, pdf)

Data Availability Statement

Data supporting the results and conclusions are available within this paper and the Supplementary Information. Both the IREDy-to-go screen and a duplication of the screening plate without the colorimetric screening components containing 0.5 mg of crude lysate of each IRED in each well are freely available through Prozomix Ltd.

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