Engineering of a chromogenic enzyme screening system based on an auxiliary indole‐3‐carboxylic acid monooxygenase

Abstract Here, we present a proof‐of‐principle for a new high‐throughput functional screening of metagenomic libraries for the selection of enzymes with different activities, predetermined by the substrate being used. By this approach, a total of 21 enzyme‐coding genes were selected, including members of xanthine dehydrogenase, aldehyde dehydrogenase (ALDH), and amidohydrolase families. The screening system is based on a pro‐chromogenic substrate, which is transformed by the target enzyme to indole‐3‐carboxylic acid. The later compound is converted to indoxyl by a newly identified indole‐3‐carboxylate monooxygenase (Icm). Due to the spontaneous oxidation of indoxyl to indigo, the target enzyme‐producing colonies turn blue. Two types of pro‐chromogenic substrates have been tested. Indole‐3‐carboxaldehydes and the amides of indole‐3‐carboxylic acid have been applied as substrates for screening of the ALDHs and amidohydrolases, respectively. Both plate assays described here are rapid, convenient, easy to perform, and adaptable for the screening of a large number of samples both in Escherichia coli and Rhodococcus sp. In addition, the fine‐tuning of the pro‐chromogenic substrate allows screening enzymes with the desired substrate specificity.


| INTRODUC TI ON
In light of the growing importance of biocatalysis, strategies that provide improvements in screening of novel enzymes are of considerable interest. Among other enzymes, aldehyde dehydrogenases (ALDHs), especially exhibiting a broad substrate spectrum, are potential biocatalysts for biotechnology and are applicable in the detoxification of aldehydes, generated during metabolism of different natural and xenobiotic compounds (Kotchoni, Kuhns, Ditzer, Kirch, & Bartels, 2006;Lyu et al., 2017;Singh et al., 2014).
Metagenomics, which helps to circumvent the cultivation of bacteria and select genes directly from the environment, has become a powerful tool in search of new enzymes and metabolic pathways for the industrial biotechnology over the past decades (Allen, Moe, Rodbumrer, Gaarder, & Handelsman, 2009;Maruthamuthu, Jiménez, Stevens, & Elsas, 2016;Suenaga, Ohnuki, & Miyazaki, 2007;Varaljay et al., 2016). Many studies show that the function-based screening or selection approaches permits an effective identification of different biocatalysts, such as lipases/ esterases (Reyes-Duarte, Ferrer, & García-Arellano, 2012), cellulases (Maruthamuthu et al., 2016), and oxygenases (Nagayama et al., 2015), from diverse environmental sources and microbial habitats. However, the common problem in the search for new enzymes is the absence of an appropriate screening system.
The aim of this study was to develop a novel platform for the functional screening of the enzymes, particularly ALDHs. First, we searched for indole-3-carboxylic acid (I3CA)-degrading microorganisms and corresponding genes in metagenomes to determine whether any could transform I3CA to indigo. We have successfully identified Icm encoding gene, which was used for the creation of the screening method. By using the developed approach, we succeeded in a screening of diverse ALDHs with a broad substrate specificity.
Furthermore, the auxiliary Icm enzyme was applied for screening of amidohydrolases using the amide of indole-3-carboxylic acid as a substrate. The Icm was active both in Gram-negative and Gram-positive bacteria, and hence, the enzyme was suitable for a functional screening of enzymes in different hosts.

| Chemicals
Chemicals used in this study are listed in Table A1. Gel resins were purchased from GE Healthcare (Little Chalfont, UK). Restriction endonucleases and DNA polymerases were from Thermo Fisher Scientific (Vilnius, Lithuania). All reagents used in this study were of analytical grade.

| General DNA manipulation
Plasmid preparation, restriction endonuclease digestion, DNA ligation, agarose gel electrophoresis, and other standard recombinant DNA techniques were carried out by standard methods (Sambrook, Fritsch, & Maniatis, 1989). DNA sequencing and primer synthesis were performed commercially at the Macrogen (the Netherlands).
DNA sequences were analyzed with a BLAST program available at the National Center for Biotechnology Information web site (http:// blast.ncbi.nlm.nih.gov/Blast.cgi). Evolutionary analyzes were conducted in MEGA7 (Kumar, Stecher, & Tamura, 2016).

| Screening of soil samples and gene cloning
About 1 g of soil samples were suspended in 1 ml 0.9% w/v NaCl solution, and 50 μl aliquots were spread on the agar plates supplemented with 1 mM I3CA. The plates were incubated at 30°C for 48 hr and were subsequently visually inspected for colonies producing the blue indigo pigment. Chromosomal DNA was isolated from the blue pigment producing bacteria, digested with the PstI restriction endonuclease and ligated in the pUC19 vector. Escherichia coli DH5α was used for screening of blue colonies on the plates supplemented with 1 mM I3CA.
For the screening assay, the pKVIABam8 encoding the icm gene was digested with BamHI and PscI and subcloned to the BamHI and PagI restriction sites of pACYC184 vector and resulted plasmid was designated pACYC-KVIA. For construction of expression vectors, icm gene was PCR-amplified with primers KviaEcoR and KviaNde2F (Table 1)  was amplified with primers Kvia-IBA3-F and Kvia-IBA3-R, digested with Eco31I and ligated into Eco31I-digested pASK-IBA3, resulting in pASK-IBA3-KVIA. For cloning of aldehyde dehydrogenase Vmix gene, it was PCR-amplified with primers VmixHindR and VmixNdeF (Table 1) and DNA from the metagenome clone Vmix as template. PCR product was digested with NdeI/HindIII restriction endonucleases and ligated into pNitRT1 previously digested with the corresponding enzymes to obtain pNitRT-Vmix. For construction of C-terminal His 6 -tagged amidohydrolase, MO13 gene was PCR-amplified with primers am13F and am13R2 (Table 1) and pMO13 as DNA matrix. PCR product was digested with NdeI/XhoI restriction endonucleases and ligated into pET-21a(+) previously digested with the corresponding enzymes to obtain pET21-MO13. Electrocompetent cells were prepared as described previously (Nakashima & Tamura, 2004b;Stanislauskiene et al., 2012) and used for transformation.

| Construction of the metagenomic library and screening for enzymes
For the construction of environmental DNA libraries, surface soils (0-15 cm) from a different fields in district Vilnius (Lithuania) were collected. The environmental DNA was isolated from samples using

| Bioconversion of aldehydes or carboxylic acids by whole cells
The E. coli or R. erythropolis SQ1 cells transformed with the appropriate plasmids were grown aerobically in LB containing appropriated antibiotic at 30°C until optical density reached 0.8 (A 600 ), then 0.5 mM of IPTG was added and cells were grown aerobically at 30°C for 12 hr. Cells were harvested by centrifugation, washed with 50 mM potassium phosphate buffer (pH 7.2), suspended in the same buffer and used as the whole cells. Then, 1 mM solutions of substrates were added, and bioconversion reactions were carried out at 30°C with shaking at 180 rpm for 1-24 hr. The conversion was followed by changes in UV absorption spectrum in 200-400 nm range or by HPLC/MS analysis, as described previously (Stankevičiūtė et al., 2016).

| Monooxygenase activity assay
The monooxygenase activity was evaluated from the decrease of the absorbance at 340 nm due to oxidation of NADH or NADPH (ε 340 = 6,220 M/cm), using spectrophotometer and was performed at room temperature. Simultaneously, reaction mixtures were incubated overnight at 30°C and inspected for the formation of blue precipitate. A total reaction volume of 1 ml contained 50 mM Tris-HCl, pH 7.5, 1 mM I3CA, different amounts (1-20 mM) of NADH or NADPH and 50 μM of flavin (FAD, FMN or riboflavin). Reactions were initiated by adding 2.5 μg of the purified enzyme or 20 μl of the soluble fraction (approx. 10 μg of total protein).

| Aldehyde dehydrogenase activity assay
For colorimetric assay, the cells were disrupted by sonication and the cell-free extracts were used to analyze the ALDH activity as described in (Bianchi et al., 2017). In brief, the obtained supernatants

| Amidohydrolase activity assay
A total reaction volume of 0.5 ml contained 50 mM Tris-HCl, pH 8.5, and 1 mM of appropriate substrate. Reactions were initiated by adding 2.5 μg of the purified enzyme. The progress of the reaction was followed by changes in UV absorption spectrum in 200-600 nm range or by HPLC/MS analysis, as described previously (Stankevičiūtė et al., 2016).

| Cloning and identification of indole-3carboxylate monooxygenase
To screen enzymes displaying an indigo-forming activity in the presence of I3CA, two approaches were used. Initially, several blue colonies-forming bacteria were screened using soil samples and the agar plates supplemented with I3CA. One of these isolates, KVIA, was chosen for further studies. The analysis of the 16S rRNA F I G U R E 1 Characterization of Icm-KVIA. (a) Evolutionary relationship of decarboxylating flavin-dependent oxidoreductases. The evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei, 1987), the evolutionary distances were computed using the number of differences method (Nei & Kumar, 2000) and are in the units of the number of amino acid differences per sequence. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. (b) Hydroxylation reactions performed by Icm-related enzymes. SalH, salicylate-1-hydroxylase (EC 1.14.13.1); Hbh, 4-hydroxybenzoate hydroxylase (EC 1.14.13.64); Abh, 4-aminobenzoate 1-monooxygenase (EC 1.14.13.27); PhzS, 5-methylphenazine- Moreover, those enzymes are also active toward indole (Eaton & Chapman, 1995). In contrast, the enzymes encoded by the KVIA, MILC, and NVS clones were unrelated to any known dioxygenase and showed the highest sequence similarity to the experimentally characterized monooxygenases such as 5-methylphenazine-1-car- from Pseudomonas putida (Uemura et al., 2016), 6-hydroxynicotinic acid 3-monooxygenases NicC from P. putida and Bordetella bronchiseptica (Hicks et al., 2016), 5-methyl phenazine-1-carboxylate-1monooxygenase PhzS from P. aeruginosa (Mavrodi et al., 2001),  (Tsuji, Ogawa, Bando, & Sasaoka, 1986). The relationship between similar enzymes is shown in the phylogenetic tree (Figure 1a).

| Application of Icm as an auxiliary enzyme for functional screening of aldehyde dehydrogenases
Despite the fact that Icm activity was not detected in vitro, E. coli cells harboring the icm gene readily produced a blue indigo dye on the agar plates supplemented with I3CA. This property was further exploited to create a system for a functional screening of metagenomic libraries. The idea was to use the appropriate substrate, for example indole-3-carboxaldehyde, which would be converted to I3CA by the target enzyme, in this case ALDH. Then, Icm as an auxiliary enzyme would oxidize I3CA into indigo; hence, the colored E. coli colonies would indicate the presence of the active ALDH (Figure 2).
To test such screening platform, the icm gene was subcloned into the pACYC184 vector, compatible with the pUC19, which was used for creation of metagenomic DNA libraries. The E. coli DH5α cells transformed with pACYC-KVIA produced blue colonies on the agar plates supplemented with I3CA (0.01 mM of I3CA in the medium was sufficient for the formation of blue pigment (Figure 1d), but only white colonies were observed when indole-3-carboxaldehyde was used as a substrate. Therefore, this strain was further used for screening of metagenomic libraries.
Twenty-one metagenomic libraries were created using the pUC19 plasmid and DNA isolated from soil. Each library contained clones with inserts of ~3-15 kb average size, yielding approximately 0.5 Gb of total cloned genomic DNA per library. In order to screen F I G U R E 3 Sequence diversity of metagenomic aldehyde dehydrogenases. The evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei, 1987), the evolutionary distances were computed using the number of differences method (Nei & Kumar, 2000) and are in the units of the number of amino acid differences per sequence. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The optimal tree with the sum of branch length = 2,362.75195313 is shown. All positions containing gaps and missing data were eliminated. A total of 351 positions in the final dataset were used for data analysis  for ALDH activity, about 30,000 clones per library were spread on LB agar supplemented with indole-3-carboxaldehyde. In this way, 52 indigo-forming clones were identified. The clones producing indigo without the presence of Icm (the false positives, i.e., most of such clones encoded Baeyer-Villiger monooxygenases, data not shown) as well as redundant clones were omitted resulting in 20 unique hits harboring the distinct genomic fragments. The sequence analysis of the screened ALDH-positive clones revealed the presence of genes encoding the proteins that were 73%-99% identical to the known sequences in the NCBI databank and homologous to ALDHs (19 clones), and molybdopterin xanthine dehydrogenase (one clone; see Table A2). Thus, the proposed functional screening approach was suitable for identification of hits expressing ALDHs (Table 2). To gain insight into the phylogenetic relationship of all selected enzymes, the phylogenetic tree was constructed ( Figure 3). As revealed by comparison between UniProtKB/SwissProt sequences, nine ALDHs, that is, pDON4, pALDGA1, JU61, pALD442, pER2AH2, Vmix, pALDJU6, pALDBS21, and pALD458 were closest to vanillin dehydrogenase, pALDMO9 was related to B. subtilis vanillin dehydrogenase. pEMMO, pALDMO11, and UraGR were related to NAD(+)-dependent benzaldehyde dehydrogenase and pALDBSal to NAD(P)-dependent benzaldehyde dehydrogenase. The sequences of clones pRG1, pEGA1, and pALDSV3 were closest to betaine aldehyde dehydrogenase.
To analyze a substrate specificity of the screened enzymes, the bioconversion of substrates by whole cells was monitored by UV-Vis spectrophotometer and products of the reaction were confirmed by HPLC-MS analysis (Table 3). For some substrates, the colorimetric assay based on the formation of formazan by the cell-free extracts was applied (Table 4). Thirteen derivatives of indole-3-carboxyaldehyde were tested. The most preferred substrates among the tested ones were 5-bromindole-3-carboxaldehyde, 6-benzyloxyindole-3carboxaldehyde, and 1H-benzo[g]indole-3-carboxaldehyde, which were oxidized by 18 ALDHs (Table 3). Only one strain (pALDR177) could oxidize 2-phenylindole-3-carboxaldehyde. The whole cells with an empty vector (E. coli DH5α/pUC19) did not show any activity on the tested substrates, confirming that the ALDHs were encoded by the metagenomic inserts. Even though among aldehydes without indole ring, the favorable substrate was 3-hydroxybenzaldehyde, which was oxidized by 19 clones, the hits showed very different substrate specificity (Table 4), and hence, the offered screening platform allowed the identification of ALDHs both of different structures and catalytic properties.

| Screening of amidohydrolases
Moreover, this amidase was able to regioselectively deprotect lysine in N ε position when N α ,N ε -di-Z-L-lysine or N α -Boc-N ε -Z-L-lysine was used as substrates.

| CON CLUS IONS
In this study, we have successfully identified a monooxygenase (Icm) active toward indole-3-carboxylic acid. The indigo formation due to activity of Icm allowed the development of a simple system for functional screening of enzymes from the metagenomic libraries. We showed that different enzymes, for example, ALDHs or amidohydrolases could be identified depending on the used substrate. Moreover, the system might be easily extended for screening other activities as shown in Figure 2. The only requirement is that the product of enzymatic reaction would be indole-3-carboxylic acid (with or without substituents in the indole ring), which could

ACK N OWLED G EM ENT
We are grateful to Kristė Šalkauskienė for technical assistance.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.

E TH I C S S TATEM ENT
None required.

DATA ACCE SS I B I LIT Y
All DNA sequences are submitted to GenBank. The sequences ob-