Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis
© Wieczorek and Martin; licensee BioMed Central Ltd. 2010
Received: 1 June 2010
Accepted: 14 September 2010
Published: 14 September 2010
The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes.
Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell.
We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals.
Macromolecular enzyme complexes catalyze an array of biochemical and metabolic processes such as the degradation of proteins [1, 2] or recalcitrant polymers  as well as the high-yield synthesis of valuable metabolic products via substrate channeling . From a biotechnological perspective, mimicking such process by incorporating catalytic modules or enzymes of interest within synthetic complexes can significantly enhance the efficiency of such bioprocesses via substrate channeling  and increased enzyme synergy . In a cellulosome, multiple enzymes assemble into a macromolecular complex by their association with a scaffold protein for the efficient degradation of cellulose . In the case of the gram-positive thermophile Clostridium thermocellum, the cellulosome is anchored to the surface of cells, resulting in one of the most efficient systems for bacterial cellulose hydrolysis [3, 7].
Cellulosomal enzymes bear C-terminal type 1 dockerin (dock1) modules, which target them to a central scaffold protein (CipA) via chemically and thermally stable non-covalent interactions with one of nine cohesin (coh) modules . CipA also contains a type 3a cellulose-binding module (CBM3a), allowing the different cellulases to act in synergy on the crystalline substrate, as well as a type 2 dockerin module which binds anchor proteins, ensuring the cellulosome's attachment to the cell [9, 10]. Therefore, the architecture of the cellulosome establishes proximal and synergistic effects of enzymes within the complex when associated with the substrate [8, 11, 12]. These synergistic effects are further augmented by an extra level of synergy resulting from the cellulosome's association with the surface of cells, yielding cellulose-enzyme-microbe (CEM) ternary complexes [6, 7, 13–18]. CEM ternary complexes benefit from the effects of microbe-enzyme synergy, ultimately limiting the escape of hydrolysis products and enzymes, increasing access to substrate hydrolysis products, minimizing the distance products must diffuse before cellular uptake occurs, concentrating enzymes at the substrate surface, protecting hydrolytic enzymes from proteases and thermal degradation, as well as optimizing the chemical environment at the substrate-microbe interface [6, 7, 13–16].
In this work, we describe the cell surface display of small cellulosome scaffold proteins in Lactococcus lactis, a first and necessary step for the eventual engineering of extracellular protein complexes in this, and other bacterial hosts. "Mini" scaffold proteins have been intracellularly expressed and purified from hosts such as Escherichia coli or Bacillus subtilis for the purpose of assembling mini-cellulosomes in vitro [19–23]. The production of mini-cellulosomes in vivo has also been reported in Clostridium acetobutylicum and B. subtilis, however, complex localization was limited to secretion into the culture supernatant [24, 25]. More recently, the surface-display of mini-cellulosomes was described in Saccharomyces cerevisiae, in some cases enabling growth on cellulosic substrates [26–29]. However, there have been no reports on the recombinant assembly of cellulosome-inspired complexes on the surface of bacterial cells. For this purpose, we chose L. lactis, a gram-positive bacterium with established commercial value. L. lactis is of specific interest as it is generally regarded as safe (GRAS), has been used to produce valuable commodity chemicals such as lactic acid  and bioactive compounds , and has been successfully engineered to secrete and/or display on its cell surface, a wide variety of proteins ranging from 9.8 to 165 kDa . The metabolic engineering tools available in conjunction with the successful controlled expression and high-yield production of enzymes and proteins  make it an ideal candidate for the recombinant expression of cellulosomal components. Using L. lactis as a surrogate host, we successfully secreted fragments of CipA (CipAfrags) to the cell surface and the scaffolds retained functionality. All scaffolds containing functional cohesins were capable of associating with an engineered test enzyme, E. coli β-glucuronidase (UidA) fused with a dockerin. We envision expanding on this work to eventually engineer larger scaffolds that will serve as the basis for assembling and immobilizing large extracellular enzyme complexes.
Regulated expression of CipAfrags yields the surface-display of scaffold proteins
NucA-CipAfrag proteins are localized to the cell wall of L. lactis
Cell surface displayed CipAfrag scaffolds bind UidA-dock1
The effect of the N-terminal nuclease reporter on secretion efficiency was also analyzed by comparing the binding capacity of L. lactis harboring the pAW300 series (nuclease fusions) with cells harboring the pAW500 (nuclease deficient) series of vectors. Initially included as a reporter to facilitate detection of exported scaffolds, we hypothesized that the nuclease fusion might also increase secretion efficiency, as has been previously observed [35, 38]. Removal of NucA had no detrimental effects on scaffold display for all constructs (Fig. 4B), as similar amounts of anchor-containing scaffolds were located to the cell surface. Furthermore, removal of NucA resulted in a fourfold increase in the amount of coh1-coh2-cwa successfully displayed when compared to its NucA-containing counterpart. The presence of NucA appeared to interfere with the secretion of supernatant-targeted scaffolds from the cell, given that the cwa-deficient variants of coh1, coh9, and CBM3a-coh3 remained associated with the cell to a much larger extent than their NucA-deficient counterparts (Fig. 4).
Several recent studies have reported on the recombinant expression of mini cellulosome scaffold proteins in Saccharomyces cerevisiae [26–29]. In these examples, the potential application of the engineered strains for the direct conversion of cellulosic biomass to ethanol was the driving factor for choosing S. cerevisiae as a host. However, many more platform strains have been or are now being developed that will produce ethanol, biofuels other than ethanol, and non-biofuel chemicals [5, 14, 40–47]. The economics of these processes would be greatly improved if these engineered microbes could use cellulosic substrates. With this goal in mind, the first logical step in establishing this system was the successful secretion and display of cohesin-bearing scaffold proteins. Previous studies have demonstrated that controlled gene expression in L. lactis can reduce toxicity and increase net protein yields [33, 48, 49]. In our study, the constitutive expression of the scaffold proteins consistently led to cellular toxicity, a problem that was solved by delaying the onset of gene expression until the cells had reached mid log-phase. In cell division, higher concentrations of recombinant cell wall-targeted proteins are localized to the septum, the site of cell wall biosynthesis . It is thus likely that over-expression of our scaffold proteins targeted to the extracytoplasmic space early in the growth phase impaired cell wall biosynthesis and ultimately resulted in cell death. Removal of NucA from the scaffolds decreased or eliminated cellular toxicity for all cohesin-containing constructs (Fig. 2), and we thus suspect that accumulation of NucA in the cytoplasm may also contribute to this observed lag in the onset of growth when induced at t = 0 hrs. In addition, as a larger proportion of scaffolds lacking a cell wall anchor remained trapped in the cell wall when fused with NucA, it is also likely that part of this observed reduction in toxicity is due to a decrease in the amounts of recombinant proteins being trapped in the cell wall and ultimately disrupting its integrity.
Quantification of cell surface displayed proteins in lactic acid bacteria was previously reported using fluorescence-activated cell sorting, flow cytometry, or whole-cell ELISA . In our assay, functionality of the displayed CipAfrag scaffold proteins could be tested directly through binding with a dockerin-containing reporter enzyme, attesting that the number of cohesins detected was a direct quantification of those that retained biochemical function. Of the four expressed CipA fragments containing at least one cohesin (coh1, coh9, coh1-coh2, CBM3a-coh3), coh1 was displayed with the highest efficiency (~9 × 103 scaffolds per cell). Due to its small size and decreased number of modules compared with coh1-coh2 and CBM3a-coh3, we attribute part of this increase in display to the decrease in size of the scaffold itself. However, coh1 was also displayed more efficiently than coh9, which is approximately the same size and similar in primary amino acid sequence. One possible explanation may relate to the position of coh1 relative to coh9 on native CipA scaffold. Coh1 is located at the N-terminus of the 200 kDa scaffold CipA, adjacent to the processing site of the signal peptide by the sec-pathway machinery of C. thermocellum . It is possible that the increase in secretion efficiency of coh1 when compared with coh9 may be in some part due to differences in amino acid content adjacent to the signal peptide, possibly increasing its accessibility to the chaperones involved in its transport to the extracytoplasmic space . This, however, does not account for the differences in display efficiency between NucA-coh1 and NucA-coh9, as in both cases, NucA is adjacent to the signal sequence. The amount of sequence identity among cohesins perhaps provides a better explanation for these observed differences. Of the nine cohesin modules on CipA, cohesins 3 through 8 show between 96 to 100% sequence identity, whereas among the remaining cohesins, coh1 and coh9 show the least amount of sequence identity (69 and 75%, respectively) . These differences in amino acid content may translate into differences in folding and solubility of the recombinantly expressed modules.
L. lactis was engineered to display a scaffold containing 2 cohesin modules (coh1-coh2). Based on a 1:1 binding ratio of the enzyme-cohesin and assuming equivalent expression and secretion, we expected this strain to bind twice the amount of UidA when compared to scaffolds of similar size but containing a single cohesin module (i.e. CBM3a-coh3). However, coh1-coh2 bound similar amounts of UidA as CBM3a-coh3 (Fig 4B). This reduction in UidA binding was not attributed to CipAfrag size differences, since both mature scaffolds have a theoretical molecular weight of 68 kDa, suggesting that other factors affected secretion and display efficiency. In fact, protein size is not regarded as a major bottleneck for protein secretion in L. lactis, as the size of successfully secreted heterologous proteins ranges from 6.9 kDa to a staggering 165 kDa . We hypothesize that the substitution of a cohesin module by CBM3a may have enhanced secretion by increasing the rate of folding of the scaffold into its soluble form. A similar effect was recently reported with the fusion of the highly insoluble Clostridium cellulovorans cellulase CelL with the CBM of cellulase CelD, which resulted in dramatic increases in its solubility .
Comparisons between amounts of UidA binding to cells expressing CipAfrags with or without the cwaM6 domain revealed that the cell wall anchor motif significantly increased the amounts of functional scaffolds displayed on the cell (Fig. 4). With NucA present, CipAfrags lacking cwaM6 remained cell-associated to a larger extent (Fig. 3) and bound UidA (Fig 4), suggesting that NucA fusion proteins remained trapped in the cell wall for reasons other than covalent cross-linking by the sortase, but yet the cohesin modules were accessible to UidA. This phenomenon is well-documented in other studies of protein secretion in L. lactis, as in some cases the fusion of two generally well-secreted proteins results in changes in the folding of the hybrid protein, and deficiencies in their release from cells [37, 54]. While the exact mechanism of this phenomenon is not clear, hydrophobic domains resulting from fusing two recombinant proteins may promote cell wall association .
Until now, all attempts to anchor enzymes on the surface of a bacterium have been limited to a single enzyme per anchor [33, 35, 36, 38, 50, 55–61]. In our system, multiple enzymes could theoretically associate with scaffolds containing a corresponding number of cohesins. We used purified β-glucuronidase fused to a dockerin module as a probe to establish proper display and function of the cohesins, but envision co-expression of enzymes and scaffold in a subsequent development of the strain. We thus envision that further development of this cellulosome-inspired system may contribute to the efficient bioconversion of substrates into industrially relevant fuels and commodity chemicals, and that tailor-designed synthetic macromolecular complexes could be engineered to contain large permutations and combinations of desired enzymes of interest.
Bacterial strains and plasmids
Strains and plasmids used in this study.
L. lactis HtrA NZ9000
MG1363 (nisRK genes on the chromosome)
E. coli TG1
supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5 (rK- mK-) [F' traD36 proAB lacI qZ ΔM15]
E. coli DH5α
fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17
E. coli BL21 (DE3)
F - ompT gal dcm lon hsdS B (r B - m B - ) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])
Eryr, Ampr; pBS::pIL252::t trpA ::P59::rbs usp45 ::sp Usp45 -nucA-cwaM6-t1t2
Eryr, Ampr; pBS::pIL252::t trpA ::P59::rbs usp45 ::sp Usp45 -nucA-t1t2
Eryr; P nisA ::rbs nisA ::uidA
J. Seegers a
Eryr; P nisA ::rbsnisA::spUsp45-nucA
Ampr; t trpA ::P nisA ::rbs usp45 ::spUsp45-nucA
Ampr; t trpA ::P59::rbs usp45 ::spUsp45-nucA
Ampr; t trpA ::P nisA ::rbs nisA ::spUsp45-nucA
Ampr; t trpA ::P nisA ::rbs nisA ::spUsp45
Eryr, Ampr; pBS::pIL252::t trpA ::P59::rbs usp45 ::spUsp45-nucA-MCS-cwaM6-t1t2
Eryr, Ampr; pBS::pIL252::t trpA ::P59::rbs usp45 ::spUsp45-nucA-MCS-t1t2
Eryr, Ampr; pBS::pIL252::t trpA ::P59::rbs usp45 ::spUsp45-nucA-lacZα-cwaM6-tlt2
Eryr, Ampr; pBS::pIL252::t trpA ::P59::rbs usp45 ::spUsp45-nucA- lacZα-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P59::rbs usp45 ::spUsp45-nucA-lacZα-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P59::rbs usp45 ::spUsp45-nucA- lacZα-tlt2
Ampr; pGEMT::with cloned coh9 from cipA
Ampr; pGEMT::with cloned coh1 from cipA
Ampr; pGEMT::with cloned coh1-coh2 from cipA
Ampr; pGEMT::with cloned cbm3a-coh3 from cipA
Ampr; pGEMT::with cloned cbm3a from cipA
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs usp45 ::spUsp45-nucA-LacZα-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs usp45 ::spUsp45-nucA-LacZα-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-lacZα-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-lacZα-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-coh9-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-coh9-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-coh1-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-coh1-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-coh1-coh2-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-coh1-coh2-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-cbm3a-coh3-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-cbm3a-coh3-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-cbm3a-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-nucA-cbm3a-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-lacZα-cwa M6 -tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-lacZα-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-coh9-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-coh9-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-coh1-cwa M6 -tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-coh1-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-coh1-coh2-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-coh1-coh2-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-cbm3a-coh3-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-cbm3a-coh3-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-cbm3a-cwaM6-tlt2
Cmr, Ampr; pBS::pIL252::t trpA ::P nisA ::rbs nisA ::spUsp45-cbm3a-tlt2
Knr; pET28(b)::with cloned dock1 from celS
Assembly of cassettes for scaffold protein expression and targeting
Primers used in this study.
Cloning of cipA fragments from C. thermocellum
Five unique cipA fragments were PCR-amplified from C. thermocellum genomic DNA using primer pairs g-h, i-j, g-k, l-m and n-m (Table 2), ligated into pGEM-T (Promega) and sequenced to verify the integrity of the gene sequence. The resulting pGEM plasmids were digested with Asc I-Not I to release the cipA gene fragments and these were ligated into pAW004ZC and pAW005ZC. The cipA fragments were chosen on the basis of containing a single cohesin (coh1 or coh9), two cohesins of identical specificity (coh1-coh2), one cohesin and a cellulose-binding module (coh3-CBM3a) and only a cellulose-binding module (CBM3a) (Fig. 1). The resulting spUsp45-nucA-cipAfrag-cwaM6 cassettes were under control of the P 59 promoter and contained rbs usp45 . The same cipA fragments were cloned into pAW104 and pAW105 for inducible expression of the scaffold proteins.
For the inducible expression of the fusion proteins under the control of P nisA with an intact ribosome-binding site from the nisA gene (rbs nisA ), spUsp45-nucA was PCR-amplified from pAW004ZC using primers q and r, creating a Bsp HI cut site at the 5' end of the PCR product. The PCR product was digested with Bsp HI and Xho I and ligated to pSIP502 digested with Nco I-Xho I, effectively replacing the gusA gene with spUsp45-nucA, retaining the first lysine of the signal peptide, and yielding pSIPSPNUC. For the insertion of an upstream transcriptional terminator and removal of nucA, a 1500-bp Sap I-Xba I fragment was temporarily removed from pAW104, and was ligated to similarly cut pUC19, yielding vector pUC104. To introduce the E. coli transcriptional terminator from the tryptophan synthase operon (t trpA ) upstream of P nisA and to introduce a Bgl II cut site, a 200-bp fragment containing t trpA was PCR-amplified from pVE5524 using primers s and t, digested with AflI II-Nru I and ligated to similarly-cut pUC104, yielding pUC104mod. Plasmid pSIPSPNUC was digested with Bgl II-Xho I and ligated to similarly-digested pUC104mod, yielding vector pUC304. This was the base vector harboring the t trpA -P nisA -rbs nisA -spUsp45-nucA cassette, which was digested with Apa I-Asc I and ligated into the pAW100 series of vectors. Inserting this cassette into Apa I-Eco RV digested pAW110 and pAW111, yielding pAW301 and pAW302, respectively, created controls lacking cipA fragments for expression of nucA alone. For deletion of the nucA reporter and construction of the pAW500 series, pUC304 was digested with Sal I-Xho I and self-ligated, yielding vector pUC504. The t trpA -P nisA -rbs nisA -spUsp45 cassette was released via digestion with Apa I-Asc I, gel-purified, and ligated to similarly-cut pAW100 series vectors, yielding the pAW500 series of vectors. This cassette was also ligated into similarly cut pAW104 and pAW105 yielding base vectors containing the lacZ-α stuffer fragment. The final expression vectors for this study included the pAW300 series of vectors for inducible expression and targeting of NucA-fused scaffolds, and the pAW500 series of vectors for inducible expression and targeting of scaffolds lacking the N-terminal NucA reporter (Fig. 1).
Expression and localization of CipAfrags in L. lactis
L. lactis HtrANZ9000 was transformed with the pAW300 and pAW500 series of vectors for the controlled expression of scaffolds. It contains chromosomal copies of the nisR and nisK genes necessary for nisin-inducible expression of cassettes under control of the nisA promoter, and is deficient in a major extracellular housekeeping protease, which has been shown previously to be responsible for the proteolysis of exported recombinant proteins . Growth curves were used to evaluate the potential of growth inhibition caused by the over-expressed CipAfrag proteins. Growth curves were performed in 96 well plates and cells were induced with 10 ng nisin/mL at inoculation (t = 0 hrs), 4 hrs post-inoculation (t = 4 hrs) or were not induced. For the expression of CipAfrag proteins in L. lactis HtrA NZ9000, overnight cultures were diluted 1/50 into fresh GM17 medium and were induced with 10 ng nisin/mL when an OD600 ≈ 0.3 was reached (4 hrs). After 20 hrs growth, successful CipAfrag secretion was evaluated using a nuclease assay consisting of spotting cells on TBD-agar and observing pink color formation . For analysis of NucA-CipAfrag proteins in various cellular locations, cell fractionation was performed as described previously , with the addition of lysostaphin (0.6 mg/mL) . Aliquots of proteins were blotted on TBD-agar plates and formation of a pink color was analyzed after a 1-hr incubation at 37°C.
Expression and purification of CipAfrag-binding β-glucuronidase
The E. coli β-glucuronidase (UidA) was engineered to have a C-terminal dock1 module for binding onto CipAfrag scaffolds, as well as an N-terminal 6 × His-tag for protein purification. The dock1 module of the C. thermocellum celS gene was amplified from C. thermocellum genomic DNA using primers u and v (Table 2). PCR products were digested with Eco RI-Not I and ligated to similarly-digested pET28(b), yielding pETdock1. The uidA gene lacking a stop codon was amplified using primers w and x and pSIP502 as template. The PCR product was digested with Nhe I-Eco RI and ligated to similarly-cut pET28(b) and pETdock1, yielding His-tagged UidA proteins with and without a dock1 module (pETUdock1 and pETU). His-tagged proteins were expressed in E. coli BL21(DE3). Cultures were induced at an OD600 of 0.5 with 1 mM IPTG and incubated for an additional 5 hrs at 37°C. Cells were harvested (1000 × g, 10 min, 4°C) and cell pellets were kept overnight at -80°C. Thawed cell pellets were suspended in 50 mM phosphate buffer, pH 7.5, containing 300 mM NaCl. Samples were subjected to sonication (15 sec pulse, 5 sec between pulses, 2 min total process time) and lysates were loaded on approximately 10 mL of Ni-NTA sepharose resin. The resin was washed with phosphate buffer (50 mM, pH 6.0) containing 300 mM NaCl and 20 mM imidazole and eluted using the same buffer containing 250 mM imidazole. Fifty μL of each elution fraction were added to 450 μL GUS buffer containing 50 mM sodium phosphate buffer (pH 7), 10 mM β-mercaptoethanol, 1 mM ethylenediaminetetraacetic acid and 0.1% (v/v) Triton X-100. Samples were heated for 1 min, after which p-nitrophenyl-β-D-glucuronide was added to a final concentration of 4 mg/mL . The UidA-containing fractions were identified by the appearance of a yellow color. Proteins from the elution fractions showing UidA activity were visualized by SDS-PAGE on a 12% (w/v) gel to identify fractions containing the highest purity of enzyme. The specific activities of UidA-dock1 and UidA were determined by colorimetric assays in a thermostated UV-Vis spectrophotometer (Cary 50 WinUv) at 405 nm, using a 1 cm (L) cuvette, and the known molar extinction coefficient of p-nitrophenol being 18 000 M-1 cm-1. Quantification of the proteins was done using a Bradford protein assay kit (Pierce) and BSA as a standard. Specific activities were used to evaluate the amount of enzyme bound to cells in the in vivo binding assay described below.
Binding of β-glucuronidase to L. lactis
L. lactis HtrA NZ9000 cells harboring the pAW300 or pAW500 series of vectors, as well as the plasmid-free strain were grown overnight in GM17 medium. Cultures were diluted 1/50 in 5 mL of fresh media and grown for an additional 4 hrs (OD600 ≈ 0.3) after which cells were induced with 10 ng nisin/mL for scaffold expression. After 20 hrs of growth, cells from 1-mL of culture were harvested (4,300 × g, 5 min, 4°C) washed once in phosphate buffer (50 mM, pH 6.0) containing 300 mM NaCl and suspended in 100 μL of purified UidA-dock1 or UidA at a concentration 100 μg/mL. To ensure that saturation of all cohesin sites was achieved, binding assay with 200 μg UidA-dock1/mL was tested for L. lactis harboring pAW328. Binding was carried out at 4°C for 10 hrs. Cells were then washed 6 times to eliminate residual enzyme activity and suspended in 100 μL of phosphate buffer (50 mM, pH 6.0) containing 300 mM NaCl for detection of β-glucuronidase activity. For quantification of bound UdiA-dock1, 50 μL of washed cells were analyzed for β-glucuronidase activity. Reactions were stopped with 250 μL of 1 M sodium carbonate once a yellow color appeared, and the duration of each assay was recorded. The specific activities of the purified UidA-dock1 and UidA were used to determine the amount of enzyme bound onto the L lactis cells. Using the calculated molecular weight of UidA-dock1 and the known amount of cells present in each sample, the average number of enzyme units bound per cell was estimated. Assuming a 1:1 cohesin to dockerin ratio, the number of enzymes present per cell also is a representation of the number of cohesins present on the cell surface. The calculated molecular weight of the scaffolds was used to estimate the net amount of recombinant protein anchored to cells in respective cultures. Experiments were repeated twice and true biological replicates (independent colonies and cultures) were performed in triplicate for all samples.
We are grateful to Dr. Alexandra Gruss and Dr. Isabelle Poquet for providing base expression vectors for LAB as well as strains of L. lactis. The authors would like to acknowledge Dr. Andy Ekins for his help in reviewing the manuscript. This work was supported by research grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) (grant numbers 312357-06 and 330781-06) the Canada Foundation for Innovation (grant number 202359) and a Canada Research Chair to V.J.J.M. A.S.W. is the recipient of graduate scholarships from NSERC and the Fonds Québécois de la Recherche sur la Nature et les Technologies.
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