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Microbiology and Biotechnology Letters

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Microbial Biotechnology (MB)  |  Protein Structure, Function, and Engineering

Microbiol. Biotechnol. Lett. 2022; 50(2): 240-244

https://doi.org/10.48022/mbl.2203.03008

Received: March 28, 2022; Revised: April 15, 2022; Accepted: April 18, 2022

Directed Evolution of a β-Glucosidase for Improved Functions as a Reporter in Protein Expression

Ho-Dong Lim, So-Young Han, Gi-Hye Park, Dae-Eun Cheong, and Geun-Joong Kim*

Department of Biological Sciences and Research Center of Ecomimetics, College of Natural Sciences, Chonnam National University, Gwangju 61186, Republic of Korea

Correspondence to :
Geun-Joong Kim,       gjkim@chonnam.ac.kr

Abstract

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Precisely reliable and quantitative reporters can provide phenotypes that are consistent with research goals in protein expression. Here, we developed an improved reporter mATglu III 5 by directed evolution using a versatile β-glucosidase ATglu derived from Agrobacterium tumefaciens. When expressed in hosts, a vector containing this mutant distinctly showed a colored or fluorescent phenotype, according to the supplemented substrate, without any inducer. Analysis of mATglu III 5 showed it to be fully functional in fusion state with oligomeric proteins, especially under non-induction conditions, thereby offering an alternative to conventional reporters.

Keywords: Versatile reporter, β-glucosidase, directed evolution, activity staining

Graphical Abstract


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As an indispensable tool of biological research, reporter system can be used as a fundamental technique to detect or prove whether a gene of interest is expressed when it is introduced into the corresponding system. This is a crucial step if the expressed protein from the target gene could not be detected obviously by a typical phenotypic change such a difference in cell growth or morphology [1, 2]. Typically, successfully implemented reporters are mainly derived from green fluorescent protein GFP because it requires no auxiliary cofactors or substrates [3]. However, it has the time-consuming maturation step and also needs an exposure to toxic UV for fluorescence emission. Thus, superfolder GFP [4] and mCherry [5] with improved functions have been developed and widely used.

As an alternative, β-galactosidase is a widely used reporter that can be easily detected by fluorogenic and chromogenic assay using specific substrates [6]. However, an intrinsic drawback of lacZ is that its transcript is unstable, thus highly affecting the efficiency of translation [7]. Therefore, more diverse reporters still need to be developed as alternatives for basic and applied research [8, 9].

A β-glucosidase (ATglu) from Agrobacterium tumefaciens was originally screened from related enzyme library based on its ability to convert indican to indigo blue [10]. During characterization, the purified enzyme also generated a fluorescent blue color from 4-methylumbeliferyl- β-D-glucopyranoside (MUG) and a dark blue color from 5-bromo-4-chloro-3-indoxyl-β-D-galactoside (X-gal) as shown in the related enzymes [11]. These versatile properties of ATglu make it possible to be used as a reporter for protein expression [12]. However, this enzyme showed a relatively low expression level in soluble fraction and an unstable fusion ability. Thus, further applications have not been attempted [10].

We here used directed evolution to obtain ATglu mutants (mATglu) with improved function as a reporter in-vivo. The gene (GenBank, EHH05948.1) encoding ATglu was amplified from the genomic DNA of A. tumefaciens by PCR using a pair of primers (forward, 5'-ATAGAGCTCATGACCGATCCCCAAACG-3'; reverese, 5'-CCCAAGCTTTCACCCCTTCACCACACCA-3'). The resulting DNA fragment was subcloned into pTrc99A vector using restriction enzyme sites SacI and HindIII, transformed into E. coli XL1-Blue, and then purified from the corresponding transformant. Subsequently, error prone PCR was performed and the resulting genes were used to generate a library according to the typical procedure reported previously [13]. Positive clones with high activity on LB-agar plate containing 50 μg/ml ampicillin were screened using visual inspection with Xgal (1 mM) as a substrate by eye. Resulting genes from primarily screened six clones were amplified by PCR using the same primers shown above and in-vitro recombination was performed by DNA shuffling using identical procedures as reported previously [13]. After generation of shuffled gene library, probable candidates with higher activity toward X-gal were also screened from approximately 150,000 clones using the same procedure as described above. Consequently, five clones were finally screened from the mutant library after three rounds of DNA shuffling. As shown in Fig. 1A, all mutants revealed better activities on the solid plate than the wild type ATglu. Among them, two mutants, III 4 and III 5, showed relatively higher activities. Deduced amino acid sequences by DNA sequencing of all mutants revealed one or two point mutations in each case (Fig. 1B). As expected, mutated residues were distinctly biased due to combinatorial shuffling of beneficial mutation-bearing DNA fragments.

Figure 1.Screening and sequence analyses of directed-evolved β-glucosidase with improved function. (A) Activity staining of screened clones on LB agar plate containing X-gal as a substrate. Isolated recombinant plasmids from screened clones were re-transformed into E. coli XL1-Blue cells, followed by spreading onto LB-ampicillin plates containing X-gal (1 mM) and incubated overnight. (B) Deduced amino acid sequences of mutation residues in screened genes by DNA sequencing. Faint vertical black lines indicated silent mutation residues that caused no change in amino acid.

To further analyze the expression patterns and relative activities of mutated enzymes, screened clones were inoculated into the same LB liquid medium and cultured at 37℃ and 200 rpm for 8 h. Resulting cells was reseeded (1%, v/v) and further grown in the same medium for 24 h under the conditions without any inducer. These cells were harvested by centrifugation at 10,000 g for 2 min and then washed with phosphate-buffered saline (PBS). After cell lysis by untrasonication, the supernatant was subjected to native-PAGE and zymogram analysis using three substrates (fluorescent MUG, 0.1 mM; chromogenic X-gal and indican, 1 mM) according to the previous study [12]. As shown in Fig. 2A, mutated enzymes had higher activities on solid gel than the wild-type enzyme. Especially, zymogram activities of two mutants, III 4 and III 5, against X-gal and indican were the highest among clones. Active protein bands of the two mutants toward MUG were also higher than those of the wild type. We intentionally selected two clones (III 4 and III 5) for further analyses of expression level and solubility by SDS-PAGE using the same supernatant.

Figure 2.Zymogram and SDS-PAGE analyses of mutant β-glucosidases with improved function. (A) Zymogram assays on native gels using MUG, X-gal, and indican as substrates. An aliquot (10 μg) of supernatant was mixed with native sample buffer and loaded onto 8% (w/v) native-PAGE. These separated gels were washed in PBS buffer and then overlaid with 1% (w/v) agarose gel containing each substrate. These gels were incubated at 37℃ for 5-10 min (MUG) or 1 h (X-gal and indican). PC, pTrc99A-ATglu (wild type); NC, pTrc99A; 1-5, pTrc99A-mATglu III 1-5. (B) SDS-PAGE was also conducted on a 12 % acrylamide gel using the same crude extract of mutants III 4 and III 5 cells. Lane 1, control cell with empty vector; Lane 2, ATglu; Lane 3, mATglu III 4; and Lane 4, mATglu III 5.

As shown in Fig. 2B, both mutants showed 2.8 to 3.3 folds increase in the expression level, resulting in proportional increases in soluble fractions. These differences in expression level and solubility were not observed under the induction condition with IPTG (data not shown). Additionally, only marginal increases (1.1- 1.3 folds) in specific activities of two mutant enzymes were detected by using indican and MUG as substrates. Therefore, activity differences in the screening step were mainly linked to expression level and solubility under non-induction conditions, not enhanced activity. This assumption is partly supported by the located mutation residing far away from the activity site (residues 355 to 365) of the related glycosyl hydrolase family I. These characters of the mutated enzymes were unexpected. However, they are suitable for monitoring protein expression as reporters under non-invasive (without any interference by a stressful inducer IPTG) conditions. Considering these points, we finally selected mutant III 5 as a plausible candidate for further analyses in terms of fusion ability with the target protein.

It is well-known that a reporter protein should have a suitable fusion ability to monitor the expression of the target protein in-vivo. To test this property, various fusion partners, kanamycin acetyltransferase (KAT, monomer), GFPuv (dimer), and DsRed (tetramer) were arbitrary chosen as model proteins and fused with Cterminal region of the mutant III 5 using a GS(3) liker. The resulting clone was cultured to an OD600 of 2.0 and then harvested by centrifugation. After washing with PBS, appropriate amounts of cells (105 cells/ml) were mixed with a PBS buffer containing 0−50 μg/ml of kanamycin. Then 10 μl of the mixture was dropped onto LB solid medium with 1 mM X-gal and incubated at 37℃ for 24 h. As shown in Fig. 3A, cells harboring the empty and the recombinant vector carrying a III 5 gene did not grow when the concentration of kanamycin was higher than 5 μg/ml. In contrast, cells expressing III 5-KAT fusion protein grew well when the concentration of kanamycin was 50 μg/ml. The activity of β-glucosidase in fusion state was also detected in grown cells based on a blue color using X-gal as a substrate.

Figure 3.Analyses of fusion ability of the mutant III 5 with monomeric and oligomeric fusion partners. (A) Inhibitory concentration (IC) assay for KAT. IC assay showed differences in growth of recombinant cells in the presence of kanamycin at a concentration of 0-50 ppm. (B) For zymogram assay, an aliguot (5-20 μg) of the supernatant was analyzed on a 8% native-PAGE under the same conditions of Fig. 1. Lane 1, positive control (mATglu); Lane 2, negative control (empty vector); Lane 3, III 5-GFPuv. (C) Fluorescence microscopy images of III 5-DsRed fusion proteins. The clone expressing III 5-DsRed was cultured at 37℃ for 24 h with shaking at 200 rpm. After harvesting by centrifugation, cell images were captured after further incubation with 0.1 mM MUG for 10 min. (D) Comparison of relative fluorescence intensities of III 5 using MUG as a substrate and DsRed in the fusion state. The data represent the mean values observed in three separate experiments.

Two representative fluorescence reporters, GFPuv and DsRed, were also fused with III 5 to monitor whether III 5 could function in a fusion state with oligomeric proteins. The supernatant from cells expressing III 5- GFPuv fusion protein was prepared according to the same procedure above and loaded on a native-PAGE (8%), and further attempted to confirm activity toward 0.1 mM MUG. The resulting zymogram revealed clearly detectable active bands corresponding to the fusion protein on acrylamide gels. As a control, non-fused III 5 was also detected clearly in the corresponding size (Fig. 3B). We also examined fluorescence images of tetrameric DsRed-fused proteins in cells with a fluorescence microscope (Olympus, Japan) using cultivated cells [14]. Expectedly, the same spots of two fluorescence, red from DsRed and blue from catalytic activity of III 5 toward MUG, overlapped (Fig. 3C), although a spot within a cell has more blue fluorescence and thus hardly detected red fluorescence due to slow maturation time [15] and lower molecular ratio of DsRed than MUG. These phenomena were further supported by fluorometric assay (Infinite M200, TECAN) using supernatant from the same culture. As shown in Fig. 3D, the emitting fluorescence from MUG was more proportionally increased than that of DsRed in the supernatant used.

Here we developed a reporter system using a directed evolved β-glucosidase, which is more reliable and less restrictive than the wild type enzyme. Although further validation in a practical study is needed, a versatile substrate spectrum and potential function of this reporter under non-induction condition could be used for various gene expression systems.

Acknowledgments

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This work was supported by a grant (grant number: 20170305) from the Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries, Korea. This work was also supported by a grant (grant number: 2021R1A2C1006734) of the Basic Science Research program of the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology of Korea (MEST), Republic of Korea.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

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