Su-Lim Choi

A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments

BackgroundAlkaline phosphatase (AP) catalyzes the hydrolytic cleavage of phosphate monoesters under alkaline conditions and plays important roles in microbial ecology and molecular biology applications. Here, we report on the first isolation and biochemical characterization of a thermolabile AP from a metagenome.ResultsThe gene encoding a novel AP was isolated from a metagenomic library constructed with ocean-tidal flat sediments from the west coast of Korea. The metagenome-derived AP (mAP) gene composed of 1,824 nucleotides encodes a polypeptide with a calculated molecular mass of 64 kDa. The deduced amino acid sequence of mAP showed a high degree of similarity to other members of the AP family. Phylogenetic analysis revealed that the mAP is shown to be a member of a recently identified family of PhoX that is distinct from the well-studied classical PhoA family. When the open reading frame encoding mAP was cloned and expressed in recombinant Escherichia coli, the mature mAP was secreted to the periplasm and lacks an 81-amino-acid N-terminal Tat signal peptide. Mature mAP was purified to homogeneity as a monomeric enzyme with a molecular mass of 56 kDa. The purified mAP displayed typical features of a psychrophilic enzyme: high catalytic activity at low temperature and a remarkable thermal instability. The optimal temperature for the enzymatic activity of mAP was 37°C and complete thermal inactivation of the enzyme was observed at 65°C within 15 min. mAP was activated by Ca2+ and exhibited maximal activity at pH 9.0. Except for phytic acid and glucose 1-phosphate, mAP showed phosphatase activity against various phosphorylated substrates indicating that it had low substrate specificity. In addition, the mAP was able to remove terminal phosphates from cohesive and blunt ends of linearized plasmid DNA, exhibiting comparable efficiency to commercially available APs that have been used in molecular biology.ConclusionsThe presented mAP enzyme is the first thermolabile AP found in cold-adapted marine metagenomes and may be useful for efficient dephosphorylation of linearized DNA.

Toward a Generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits

Large-scale screening of enzyme libraries is essential for the development of cost-effective biological processes, which will be indispensable for the production of sustainable biobased chemicals. Here, we introduce a genetic circuit termed the Genetic Enzyme Screening System that is highly useful for high-throughput enzyme screening from diverse microbial metagenomes. The circuit consists of two AND logics. The first AND logic, the two inputs of which are the target enzyme and its substrate, is responsible for the accumulation of a phenol compound in cell. Then, the phenol compound and its inducible transcription factor, whose activation turns on the expression of a reporter gene, interact in the other logic gate. We confirmed that an individual cell harboring this genetic circuit can present approximately a 100-fold higher cellular fluorescence than the negative control and can be easily quantified by flow cytometry depending on the amounts of phenolic derivatives. The high sensitivity of the genetic circuit enables the rapid discovery of novel enzymes from metagenomic libraries, even for genes that show marginal activities in a host system. The crucial feature of this approach is that this single system can be used to screen a variety of enzymes that produce a phenol compound from respective synthetic phenyl-substrates, including cellulase, lipase, alkaline phosphatase, tyrosine phenol-lyase, and methyl parathion hydrolase. Consequently, the highly sensitive and quantitative nature of this genetic circuit along with flow cytometry techniques could provide a widely applicable toolkit for discovering and engineering novel enzymes at a single cell level.

Mesh-integrated microdroplet array for simultaneous merging and storage of single-cell droplets

We constructed a mesh-grid integrated microwell array which enables easy trapping and consistent addition of droplets. The grid acts as a microchannel structure to guide droplets into the microwells underneath, and also provides open access for additional manipulation in a high-throughput manner. Each droplet in the array forms a stable environment of pico-litre volume to implement a single-cell-based assay.

High-throughput screening system based on phenolics-responsive transcription activator for directed evolution of organophosphate-degrading enzymes

Synthetic organophosphates (OPs) have been used as nerve agents and pesticides due to their extreme toxicity and have caused serious environmental and human health problems. Hence, effective methods for detoxification and decontamination of OPs are of great significance. Here we constructed and used a high-throughput screening (HTS) system that was based on phenolics-responsive transcription activator for directed evolution of OP-degrading enzymes. In the screening system, phenolic compounds produced from substrates by OP-degrading enzymes bind a constitutively expressed transcription factor DmpR, initiating the expression of enhanced green fluorescent protein located at the downstream of the DmpR promoter. Fluorescence intensities of host cells are proportional to the levels of phenolic compounds, enabling the screening of OP-degrading enzymes with high catalytic activities by fluorescence-activated cell sorting. Methyl parathion hydrolase from Pseudomonas sp. WBC-3 and p-nitrophenyl diphenylphosphate were used as a model enzyme and an analogue of G-type nerve agents, respectively. The utility of the screening system was demonstrated by generating a triple mutant with a 100-fold higher k(cat)/K(m) than the wild-type enzyme after three rounds of directed evolution. The contributions of individual mutations to the catalytic efficiency were elucidated by mutational and structural analyses. The DmpR-based screening system is expected to be widely used for developing OP-degrading enzymes with greater potential.

Simultaneous improvement of catalytic activity and thermal stability of tyrosine phenol-lyase by directed evolution

The tyrosine phenol‐lyase from Symbiobacterium toebii was engineered to improve both its stability and catalytic activity by the application of random mutagenesis and subsequent reassembly of the acquired mutations. Activity screening of the random library produced four mutants with a two‐fold improved activity, whereas parallel screening after heat treatment at 65 °C identified three mutants with half‐inactivation temperatures improved by up to 5.6 °C. The selected mutants were then reassembled using the staggered extension PCR method, and subsequent screening of the library produced seven mutants with up to three‐fold improved activity and half‐inactivation temperatures improved by up to 11.2 °C. Sequence analyses revealed that the stability‐improved hits included A13V, E83K and T407A mutations, whereas the activity‐improved hits included the additional T129I or T451A mutation. In particular, the A13V mutation was propagated in the hits with improved stability during the reassembly–screening process, indicating the critical nature of the N‐terminal moiety for enzyme stability. Furthermore, homology modeling of the enzyme structure revealed that most of the stability mutations were located around the dimer–dimer interface, including the N‐terminus, whereas the activity‐improving mutations were located further away, thereby minimizing any interference that would be detrimental to the co‐improvement of the stability and catalytic activity of the enzyme.

Quantitative analyses of individual sugars in mixture using FRET‐based biosensors

Molecular biosensors were developed and applied to measure individual sugars in biological mixtures such as bacterial culture broths. As the sensing units, four sugar‐binding proteins (SBPs for allose, arabinose, ribose, and glucose) were selected from the Escherichia coli genome and connected to a cyan fluorescent protein and yellow fluorescent protein via dipeptide linkers (CFP‐L‐SBP‐YFP). The putative sensors were randomized in the linker region (L) and then investigated with regard to the intensity of fluorescence resonance energy transfer on the binding of the respective sugars. As a result, four representatives were selected from each library and examined for their specificity using 16 available sugars. The apparent dissociation constants of the allose, arabinose, ribose, and glucose sensors were estimated to be 0.35, 0.36, 0.17, and 0.18 μM. Finally, the sugar sensors were applied to monitor the consumption rate of individual sugars in an E. coli culture broth. The individual sugar profiles exhibited a good correlation with those obtained using an HPLC method, confirming that the biosensors offer a rapid and easy‐to‐use method for monitoring individual sugars in mixed compositions.

Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate

Cellulose-binding domain (CBD) enriches cellulolytic enzymes on cellulosic surfaces and contributes to the catalytic efficiency by increasing enzyme-substrate complex formations. Thus, high affinity CBDs are essential for the development of efficient cellulose-degrading enzymes. Here, we present a microtiter plate-based assay system to measure the binding affinity of CBDs to cellulose. The assay uses a periplasmic alkaline phosphatase (AP) as a fusion reporter and its activity is detected using a fluorogenic substrate, 4-methylumbelliferyl phosphate. Lignocellulose discs of 6 mm in diameter were used as substrates in 96-well plate. As a result, the enzyme-linked assay detected the binding of CBDs on the cellulosic discs in a highly sensitive manner, detecting from 0.05 to 1.0 μg/mL of APCBD proteins, which is several hundred times more sensitive than conventional protein measurements. The proposed method was applied to compare the binding affinity of different CBDs from Cellulomonas fimi to lignocellulose discs.

Evolution of enzymes with new specificity by high-throughput screening using DmpR-based genetic circuits and multiple flow cytometry rounds

Genetic circuit-based biosensors are useful in detecting target metabolites or in vivo enzymes using transcription factors (Tx) as a molecular switch to express reporter signals, such as cellular fluorescence and antibiotic resistance. Herein, a phenol-detecting Tx (DmpR) was employed as a critical tool for enzyme engineering, specifically for the rapid analysis of numerous mutants with multiple mutations at the active site of tryptophan-indole lyase (TIL, EC 4.1.99.1). Cellular fluorescence was monitored cell-by-cell using flow cytometry to detect the creation of phenolic compounds by a new tyrosine-phenol-lyase (TPL, EC 4.1.99.2). In the TIL scaffold, target amino acids near the indole ring (Asp137, Phe304, Val394, Ile396 and His463) were mutated randomly to construct a large diversity of specificity variations. Collection of candidate positives by cell sorting using flow cytometry and subsequent shuffling of beneficial mutations identified a critical hit with four mutations (D137P, F304D, V394L, and I396R) in the TIL sequence. The variant displayed one-thirteenth the level of TPL activity, compared with native TPLs, and completely lost the original TIL activity. The findings demonstrate that hypersensitive, Tx-based biosensors could be useful critically to generate new activity from a related template, which would alleviate the current burden to high-throughput screening.