Hercules Incorporated, Corporate Research, 500 Hercules Road, Wilmington, DE 19808 and 2 Kairos Scientific, 10225 Barnes Canyon Road, #A110, San Diego, CA 92121, USA
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Abstract |
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Keywords: alcohol oxidation/digital imaging spectroscopy/directed evolution/galactose oxidase/high-throughput screening
Abbreviations: ABTS, 2,2'-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid 4CN, 4-chloronaphthol EPP, error-prone PCR GO, galactose oxidase
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Introduction |
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![]() | (1) |
Although standard chemical methods exist for oxidizing alcohols, these traditional methods rely on heavy metal-containing compounds such as chromium(VI) reagents and are performed in organic solvents (ten Brink et al., 2000). In addition, industrial chemical oxidation reactions give low yields of the desired aldehyde owing to their tendency to proceed to the carboxylic acid. In contrast, the use of enzymes provides a clear benefit, in terms of both environmental impact and yield of desired product.
Guar, a natural polymer isolated from seeds of the guar plant and other sources, can be oxidized enzymatically to yield an aldehyde-bearing polymer called oxidized guar (Figure 1). This compound can be used as an additive in paper manufacturing to confer mechanical strength to paper products (Chiu et al., 1996
; Dasgupta, 1996
; Brady and Leibfried, 2000
). For our purposes, the activity of GO towards guar is of particular interest. The enzyme oxidizes the galactose side chains of guar, under ambient conditions of temperature and pressure, in aqueous solution. However, when isolated from its native host, GO is insufficiently active to catalyze economically the desired conversion on an industrial scale. We therefore undertook to improve its specific activity by in vitro evolution.
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A frequently observed property of polymers is that they form viscous solutions or are difficult to dissolve. Guar is no exception. When dissolved at a concentration of 1% in water, it has a viscosity of 1000 cP. Because viscous solutions or insoluble materials are difficult to handle using typical high-throughput screening systems, polymers can be problematic substrates for directed evolution. To realize fully the promise of enzymes in the chemical and pharmaceutical industries (Marrs et al., 1999; Chartrain et al., 2000
), a general solution to this situation is desirable.
It has previously been demonstrated that digital imaging can be interfaced with mutagenesis to isolate mutants having a desired phenotype with high efficiency (Arkin et al., 1990; Delagrave et al., 1995
; Youvan et al., 1995
). Bylina et al. (Bylina et al., 1999
, 2000
) described an instrument and methods for screening enzyme variants that is more efficient than robotics-based high-throughput systems. This system, known commercially as `Kcat', combines single-pixel imaging spectroscopy with a solid-phase screening format; it can be applied to a broad range of problems because it relies on simple, widely known colorimetric activity assays. As we will show, this technology enables screens on solid substrates or highly viscous substrate solutions that are refractory to traditional high-throughput screening methods.
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Materials and methods |
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Plasmid pGAO11 comprising the entire gaoA open-reading-frame (orf) (GenBank No. M86819) (McPherson et al., 1992), plasmid pPICZ
gaoA encoding GO fused to the yeast alpha factor under control of the AOX1 promoter and a rat anti-GO polyclonal antibody were kindly provided by Professor Michael McPherson of Leeds University, UK. Enzymes were obtained from Roche Molecular Biochemicals (Indianapolis, IN), New England Biolabs (Beverly, MA), Epicenter (Madison, WI) and Sigma (St. Louis, MO). Oligonucleotides were synthesized by Operon (Alameda, CA). Kits from Qiagen (Valencia, CA) were used for plasmid preparation and extraction. DH10B competent cells were obtained from Life Technologies (Grand Island, NY).
DNA manipulations
Molecular biology techniques described by Sambrook et al. (Sambrook et al., 1989) were generally followed. The pBADmyc/his vectors (Invitrogen, Carlsbad, CA) were used for recombinant expression of GO in Escherichia coli. The entire GO orf was subcloned into pBADmyc/his by digesting an overlap PCR product with SphI and HindIII and ligating this fragment to similarly digested vector. A silent XhoI restriction site at the 5' end of the GO orf and a 3' HindIII site immediately after two engineered stop codons were introduced by the oligos used for overlap PCR. The resulting construct, in which the GO orf was not in frame with the C-terminal myc/his tag provided by the vector, was designated pBADGO6. A silent KpnI site was engineered into pBADGO6 to yield clone pBADGOK3. This latter construct was used as a wild-type (WT) control in subsequent experiments. The GO orfs of both plasmids were sequenced completely.
Error-prone PCR was performed as described (Leung et al., 1989). PCR products were cloned using XhoI and HindIII sites to make mutant libraries, except for the libraries generated using clones GO.1-3 or 8-1 as templates, which were cloned using a PstI site internal to the GO orf and HindIII.
The expression `manual recombination' simply refers to the construction of double and triple mutants from the single mutants obtained by error-prone PCR using standard molecular cloning techniques. Double mutant (C383S/Y436H) was constructed by subcloning a SmaIKpnI fragment from the C383S-containing clone into the Y436H-containing vector. The triple mutant was constructed by subcloning a SmaIBstXI C383S/Y436H-containing fragment into the V494A-containing vector.
pPICZ8-1 was constructed by PCR amplification of the GO orf of clone 8-1 using a 3' primer that introduces an XbaI restriction site downstream of the stop codon. The resulting PCR product was digested with PstI and XbaI and cloned into pPICZ
K3, a Pichia expression vector encoding the wild-type GO protein fused to mating factor alpha. The construction of this vector and its use in Pichia pastoris will be described elsewhere (M.McPherson et al., in preparation.)
All mutants were sequenced completely in their orf. The 310 Genetic Analyzer (PE Biosystems, Foster City, CA) and sequencing reagents were used according to the manufacturer's instructions. Sequencing data were analyzed using Sequencher (Gene Codes, Ann Arbor, MI).
Solid-phase assay
Solid phase assays and their use with the Kcat system have been described previously (Bylina et al., 2000) but additional details specific to this work are provided below. Freshly transformed cells were applied to a polyester 0.2 µm pore size membrane (Osmonics, Minnetonka, MN) that was placed on the surface of a growth plate (LB-agar containing 25 µg/ml carbenicillin, 100 µg/ml ampicillin). After overnight growth, the membrane was lifted from the growth plate and transferred to an induction plate (LB-agar containing 25 µg/ml carbenicillin, 100 µg/ml ampicillin, 0.2% L-arabinose and 0.5 mM CuSO4). This induction step was performed for at least 4 h at 26°C. To lyse the microcolonies on the membrane prior to assay, the membrane was placed in a chloroform vapor chamber for 45 s. Note that in each of these steps, microcolonies were always `facing up' so that no colony lifts were performed. Also, microcolonies were generally grown and induced such that their final radii were uniform and <0.4 mm.
Methylgalactose (5 mM) assay plates were made by pouring the following into polystyrene petri dishes: 1% agarose (Biorad, Hercules, CA) in 50 mM potassium phosphate buffer (pH 7), 5 mM methyl--D-galactopyranoside (methylgalactose) (Sigma), 0.5 mM CuSO4, 1.5 mM 4-chloronaphthol (4-CN) (Pierce Chemical, Rockford, IL), 0.1 ml soybean or horseradish peroxidase dilution. Membranes bearing lysed microcolonies were transferred to assay plates and immediately introduced into a Kcat instrument (Kairos Scientific, Santa Clara, CA). This instrument was described in detail by Bylina and colleagues (Bylina et al., 1999
, 2000
).
Guar assay plates had a similar composition to methylgalactose assay plates except that the following modifications were necessary. Hot (>50°C) solutions of 2% guar (cationic guar, 80H1F; Hercules, Wilmington, DE) and 2% agarose, both prepared in 50 mM potassium phosphate buffer (pH 7), were first mixed in hot centrifuge tubes. CuSO4 and 4-CN were added and mixed thoroughly using a `vortex'. The resulting solution was centrifuged briefly to remove bubbles and poured into hot petri dishes. After solidification, a peroxidase solution was spread homogeneously on to the surface of the guar/agarose gel and allowed to diffuse into the gel at 4°C overnight.
Liquid-phase assay
Km and Vmax were measured to compare the variant enzymes to the wild-type. The velocity of each reaction (A405/min) was determined by a linear fit to the increase in absorbance for the first 2 min of reaction. The velocity versus concentration of methyl-
-D-galactose was then fitted to the MichaelisMenten equation to determine Km and Vmax.
A single colony was used to inoculate 3.0 ml of LB containing 60 µg/ml of carbenicillin, 0.002% of L-arabinose and 0.32 mM CuSO4. The culture was grown for 24 h at 26°C with shaking to yield a saturated culture. Cleared cell lysate was used in the assays without further purification and could be stored at 4°C for several days. All components except the lysate were dissolved in buffer containing 50 mM potassium phosphate, 1 mM CuSO4, pH 7.0. The 250 µl assay mixture contained 1 mM ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid] (Sigma), 1 U/ml horseradish peroxidase (HRP) (Sigma), a variable amount of methyl--D-galactose (Sigma) and 1.0 µl of the culture lysate. The concentrations of methyl-
-D-galactose (methylgalactose) varied from 0.72 to 200 mM.
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Results and discussion |
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To detect GO activity, we employ a coupled assay that generates a colored product. In this assay, hydrogen peroxide, a byproduct of GO activity (see Equation 11), is used by a peroxidase to transform 4-chloronaphthol (4CN) or ABTS into colored compounds. When the assay is performed in the liquid phase, ABTS is used to generate a soluble green compound. In the solid phase, 4CN is used, yielding an insoluble compound absorbing visible light maximally at 550 nm. The solid-phase assay, a general description of which was provided by Bylina and colleagues (Bylina et al., 2000), can be used to visualize the activity of GO in bacterial colonies, as described below.
About 40005000 bacterial microcolonies (each <0.4 mm in size) can be grown and induced on a single microporous membrane and subsequently exposed to chloroform vapor to lyse the bacterial cells. Colony lifts are conveniently avoided by growing the microcolonies directly on the surface of a membrane that lies on the surface of a growth medium such as LB-agar. After chloroform lysis, the membrane is transferred to the surface of an agarose gel (solid phase) containing a GO substrate such as methylgalactose or guar, as well as CuSO4, 4CN and horseradish peroxidase. The resulting assay plate is immediately inserted into a Kcat instrument, as described in detail by Bylina and colleagues (Bylina et al., 1999).
The instrument periodically captures a digital image of the membrane illuminated at a specified wavelength (in this case, 550 nm). Color develops in the microcolonies as a result of GO activity and the stored digital images are used to compute an absorbance versus time plot for each pixel in the image. The kinetic data associated with each pixel can be displayed and compared to determine rapidly which mutant colony on the assay plate is most active. By computing and comparing the activities of individual pixels, we avoid the complex problem of resolving contiguous microcolonies on the membrane without losing any useful information (Yang et al, 2000).
Figure 2 illustrates a typical output of the imaging and analysis software. Pixels in the image were color-coded according to their relative activity: red pixels corresponded to the most active colony on the entire assay plate. The red colony corresponded to a mutant that was subsequently shown to have increased activity, as discussed below.
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In vitro evolution of GO using methylgalactose
The structure of guar (Figure 1) suggested that methylgalactose could be used as a proxy for this compound. We investigated the usefulness of this proxy substrate in directed evolution by comparing mutants isolated using methylgalactose with mutants isolated using guar.
The pedigree shown in Figure 3 summarizes how several mutants of the present study were obtained. The figure also clearly illustrates how the different mutations identified in the first round of mutagenesis and screening were used to generate a highly active GO variant (clone 8-1) and other mutants described below.
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Based on previously reported observations, (Wells, 1990; Chen and Arnold, 1991
) it is apparent that the underlying mechanism for the stepwise improvements of phenotype seen in many directed evolution experiments involves mostly the accumulation of mutations whose effects on the phenotype (e.g. free energy of catalysis or thermostability) are roughly additive. It is therefore expected that many mutations can be combined in a single clone to yield a variant that is several-fold better than WT. We manually recombined (MR) the three mutations discussed above into a double mutant (6-1) and a triple mutant (8-1). The latter showed an ~16-fold higher activity than WT towards methylgalactose. A further round of EPP and screening was carried out using clone 8-1 as template or parent molecule. Among seven mutants isolated (Table I
), clone 7.5.1 showed a slight improvement (20%) in activity towards methylgalactose compared with its parent. Compared with WT, clone 7.5.1 was 19-fold more active on methylgalactose. The data presented so far suggest that a proxy substrate is adequate to identify variants, such as C383S, that have improved activity towards a problematic substrate such as guar.
Screening GO mutants using guar
We wished to determine whether Kcat technology could also be used to identify mutants with improved activity directly on highly viscous polysaccharides. Guar assay plates (see Materials and methods) were used to screen the WT-derived EPP library described above. A key observation was that the substitution C383S, as well as a new substitution of the same residue, C383G, were identified in two of five mutants isolated (mutant GO.05A, not shown in Table I because it is essentially identical with GO.110, encoding C383S as its only substitution and mutant GG51R, Table I
). Another variant that we identified, GG41R encoding substitution N535D (Table I
), showed that even slight improvements in GO activity could be detected using this assay format, as verified by liquid-phase measurements.
Mutants have improved intrinsic activity
We measured and compared the activities in the liquid phase of several mutants, both on guar and on methylgalactose (Figure 4). This assay was similar to the solid-phase assay used to screen mutants, but required ABTS as a chromogenic substrate instead of 4CN. We measured the activity of cell lysates on 1% solutions of low molecular weight guar produced by acid hydrolysis and ultrafiltration. This guar had significant but reduced viscosity, permitting reproducible liquid-phase measurements on a limited number of samples. Size-exclusion chromatography and HPLC showed that the resulting hydrolyzed guar had a narrow distribution of molecular weights and that cleavage did not preferentially release galactose side-chains (Lei Qiao, personal communication.) Vmax and Km were measured for the same mutants using methylgalactose as a substrate in the liquid-phase assay (Table I
). Figure 4
shows the correlation between the activity of mutants on guar (
OD 405/min) versus methylgalactose (Vmax/Km).
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Although E.coli is a convenient organism for carrying out directed evolution, it is not always ideal for the expression of large amounts of protein. We are therefore interested in improving the intrinsic characteristics of an enzyme, such as kinetic parameters, rather than potentially host-specific characteristics relating, for example, to expression. The Michaelis constant, Km, can be measured via liquid-phase assay of cell lysates without determining total enzyme concentration. This permits a rapid identification of GO variants that are more active due to an improvement in their catalytic properties rather than improved expression or solubility in E.coli. As shown in Table I, a decrease in Km for methylgalactose of approximately 3-fold is observed for GO mutants carrying a C383S substitution. An additional 1.7-fold increase in Vmax is also observed for this mutant.
As a further verification that mutants such as 8-1 are intrinsically more active, the genes for wild-type GO and clone 8-1 were introduced in expression vector pPICZ and expressed in two host strains of the yeast Pichia pastoris. Table II
shows that the Km of wild-type and GO variant 8-1 were very similar to the E.coli data. The Km of 8-1 was again found to be decreased approximately by a factor of three relative to wild-type. Total activity (Vmax) is higher in the mutant strains than wild-type.
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Conclusion
We have shown that a very high-throughput digital imaging screen can be applied to the isolation of enzyme variants with increased activity towards polymeric substrates. The screen can be performed on highly viscous substrates such as guar or on a small molecule proxy such as methylgalactose to isolate useful mutants. Our data also indicate that guar-specific phenotypes exist, supporting the notion that a guar-based screen would be preferable. This latter conclusion reinforces the significance of the screening method described here because it enables the evolution of enzymes to functionalize valuable substrates such as guar, cellulose, carboxymethylcellulose and other polymers that have problematic physical properties such as high viscosity.
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Notes |
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Acknowledgments |
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References |
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Received August 23, 2000; revised November 29, 2000; accepted December 29, 2000.