Functional heterologous expression of an engineered full length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum
© Currie et al.; licensee BioMed Central Ltd. 2013
Received: 6 November 2012
Accepted: 8 February 2013
Published: 1 March 2013
Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes.
We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to express the cellulosomal component cipA*, an engineered form of the wild-type cipA from C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a ΔcipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome.
This study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA. In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose.
KeywordsThermoanaerobacterium saccharolyticum Clostridium thermocellum Cellulosome Thermophile Anaerobe Ethanol Consolidated bioprocessing
A long sought goal in the cellulosic ethanol field is one-step solubilization and fermentation without added enzymes [1, 2]. Such consolidated bioprocessing, or CBP, is considered to be the ultimate low cost approach for cellulose hydrolysis and fermentation . A successful CBP organism must be capable of solubilizing both cellulose and hemicellulose, and also fermenting the resulting sugars to a useful product (e.g., ethanol) at high yield and titer. Unfortunately, no single organism has yet been found or developed that combines these two essential characteristics .
Two saccharolytic bacteria of interest for development of CBP-enabling microbes are Clostridium thermocellum and Thermoanaerobacterium saccharolyticum, both Gram-positive, thermophilic anaerobes. C. thermocellum exhibits among the highest growth rates on cellulose among described microbes , but lacks the ability to ferment hemicellulose. C. thermocellum’s ability to solubilize crystalline cellulose, as well as other insoluble components of plant biomass, results from its elaborate, multi-protein cellulase complex or cellulosome [4–7]. T. saccharolyticum readily solubilizes hemicellulose and ferments all common sugars in biomass, but does not solubilize cellulose. This bacterium is highly amenable to genetic manipulation, indeed exhibiting natural competence, and has been engineered to make ethanol at high yields and titers [8–11].
The component of the C. thermocellum cellulosome with the highest molecular weight is the scaffoldin protein, CipA, which has been implicated in mediating the enzymatic synergy seen in the cellulosome [7, 12, 13]. The structure of the C. thermocellum CipA includes one cellulose binding domain or CBD, one type II dockerin which is used to associate with cell wall anchoring proteins, and 9 highly conserved type I cohesins interspaced by flexible linker regions . The type 1 cohesin domains bind with high affinity to type 1 dockerin domains present in over 70 catalytically-active enzymes .
Previous studies have largely focused on expressing mini cellulosomes, to date with 4 or fewer cohesin regions, or chimeric “designer” cellulosomes in which cohesin-dockerin pairs from different organisms are used to form complexes with a specified sequence of catalytic proteins [15–31]. Recently a paper reporting in vitro assembly of cellulosomes. To date, there have been no reported attempts to engineer thermophiles to heterologously express a cellulosome, although one attempt has been made to express cellulases .
In C. thermocellum the presence or absence of CipA has little effect on activity on phosphoric acid swollen cellulose (PASC), carboxymethyl cellulose (CMC), or β-Glucan, but when absent, results in over an order of magnitude decrease in activity on microcrystalline cellulose [27, 33]. With this in mind, microcrystalline cellulose was chosen as a test substrate for cellulosome assembly and complementation.
Heterologous expression of a functional cellulosome system in T. saccharolyticum is of interest both for fundamentally-oriented studies of microbial cellulose utilization and as a strategy for developing a CBP-enabling microorganism. The logical point of departure for this endeavor is expression of CipA. Here we endeavor to develop an inducible gene expression system in T. saccharolyticum, synthesize a gene (cipA*) coding for the same amino acid sequence as CipA but with more diversity in the DNA sequences for ease of use, express CipA* in T. saccharolyticum, and demonstrate functionality by complementing ΔcipA mutants of C. thermocellum.
Construction and testing of xylose/xylan inducible promoter system
Design and synthesis of CipA*
Expression and localization of CipA*
CipA, like other cellulosomal components, is secreted via the sec pathway which utilizes an N-terminal signal peptide which is cleaved to liberate the mature protein. In order to assure the His tag’s presence in the mature protein, cipA* was tagged at the C-terminus with a 10X His tag via a linker (GGGTGHHHHHHHHHH) for detection via western blot.
A number of methods to concentrate T. saccharolyticum supernatant were tested including His purification with Ni beads and FPLC. However, the best results were obtained using molecular weight cut off spin columns. Initial attempts included bacterial or mammalian protease inhibitors, but after it became clear that proteolysis was not an issue, their inclusion was discontinued. Xylose was used as an inducer since it was as effective as xylan (Figure 1) but was more practical to use with spin columns. Unlike in E. coli no negative cellular effects were seen as the result of the presence, or induction, of cipA*. The concentrated protein was washed with 20 mM sodium citrate buffer (pH 5.7) to remove residual sugars to prevent heavy warping of the protein bands during migration on a SDS-PAGE gel.
Our initial attempts using the standard 100°C denaturation in preparation for running an SDS-PAGE resulted in protein cleavage as others have also observed for cipA[36, 37]. However, when the denaturation temperature was dropped, or time was decreased, we obtained a single intact band.
The wild type C. thermocellum performed well with respect to cellulose hydrolysis as measured by dry weight. The observed low product formation and unfermented sugars seen with wild type C. thermocellum are most likely the result of the low starting pH and the lack of pH control resulting in a discontinuation of metabolic, but not enzymatic hydrolysis activity later in growth. No strain of T. saccharolyticum alone appeared to have any effect on cellulose, but grew entirely on the supplied xylose, nor did the wild type strain of T. saccharolyticum rescue the cellulose hydrolysis defect in C. thermocellum strain DS11. Only T. saccharolyticum expressing cipA* (strain DHC15) was able to restore cellulose hydrolysis functionality to the C. thermocellum ΔcipA (strain DS11).
Finally, we wished to confirm that populated cellulosomes were being formed. Cellulosomes were purified via affinity digestion from co-cultures between DHC15 and DS11 and compared via native PAGE to those from wild type C. thermocellum and concentrated supernatants from DHC15 and DS11 grown indivigually (Additional file 2: Figure S3). As expected neither DHC15 nor DS11 were able to form cellulosomes when grown independently. When DHC15 and DS11 were grown together cellulosomes were produced with an identical native PAGE migration as those from wild type C. thermocellum.
These data demonstrate that the T. saccharolyticum-produced CipA* is capable of gathering and displaying functional cellulosomal enzymes. In addition, the appearance of the pellet further supported the removal of cellulose from the wild type C. thermocellum and the co-cultures of cipA* and ΔcipA as these dry pellets were nearly translucent, suggesting only cell debris, rather than the white cellulose observed in the other samples.
Here we report the expression of an engineered CipA* under the control of a novel inducible promoter in T. saccharolyticum which allowed for the assembly of active cellulosomes when co-cultured with a cipA deletion strain of C. thermocellum.
The wild-type cipA gene has multiple sections with essentially identical DNA sequences, corresponding to the 9 type-I dockerin regions . These repeated sequences can be problematic by complicating sequence verification via routine sequencing technology and could also lead to unwanted partial gene deletion via homologous recombination. Many strains of E. coli used to heterologously express cellulosomal proteins for biochemical studies are recA- [24, 27, 38]. However, studies such as this one which seek to integrate cellulase genes into the chromosome via native host machinery must use recA+ strains thereby exacerbating the challenge of homologous recombination. By using cipA*, designed to avoid repeated sequences, routine sequencing proceeded without difficulty and homologous recombination was not observed.
Toxicity has been observed by other groups working with heterologous expression of cipA, and were solved, at least in part, by using inducible promoters in E. coli and Lactococcus lactis[23, 27]. For this and other reasons we wished to express cipA* under the control of an inducible promoter. As there have been no inducible promoter systems described for T. saccharolyticum we designed and tested one based on xynA’s promoter. We chose this promoter due to the fact that XynA has been shown to be non-essential  and thus could be replaced with a gene of interest. We found that the xynA promoter avoided toxicity effects in both E. coli and T. saccharolyticum, although apparent toxicity was encountered using the pta/ack promoter.
In past in vitro heterologous cellulosome expression reports significant hydrolysis of microcrystalline cellulose was either not achieved or not tested, with one exception reaching 45% hydrolysis [15, 17, 18, 23, 25, 28–31]. In the data reported here if we remove the contribution of cell mass to the dry weights and compare that to the uninoculated bottles we see that total cellulose solubilization is between 98 and 100 percent for the co-culture of the CipA* expressing T. saccharolyiticum and the wild-type C. thermocellum, and between 71 and 93 percent for the CipA* expressing T. saccharolyiticum and C. thermocellum ΔcipA. While the trans-complementation co-culture can achieve close to wild type co-culture solubilization in some cases, it is rather variable. The lower hydrolysis from the CipA* expressing T. saccharolyticum and C. thermocellum ΔcipA co-culture, as compared to that of wild-type C. thermocellum, may be the result of one or more effects. First, while the cohesin dockerin interaction is quite robust, it is possible that when produced by two different strains the assembly of mature cellulosomes occurs less efficiently than if the components are being secreted simultaneously from the same cell [39, 40]. Second, the assembled cellulosomes are incapable of adhering to the surface of the more metabolically active T. saccharolyticum, and thus are either present free in the media or bound to the surface of C. thermocellum’s via the native anchor proteins [41–43]. This could result in local product inhibition, and may contribute to the lower than expected hydrolysis [3, 44–46].
Krauss et al. found that CipA purified from E. coli was populated with cellulosomal enzymes present in C. thermocellum supernatants and had near wild-type activity on microcrystalline cellulose . These results led the authors to conclude that the cohesin dockerin interactions are the primary means of cellulosome assembly, an interpretation which our work also supports. Whereas Krauss et al. reported cellulosome assembly in vitro, we demonstrate here assembly from components produced by growing cultures with heterologous production using a host that has a temperature optimum compatible with that of the C. thermocellum cellulosome.
In both Krauss et al. and this study, cellulosomes derived from components produced in separate organisms but otherwise unmodified are found to exhibit similar, although somewhat lower, activity on crystalline cellulose as compared to controls with components produced by a single organism. By contrast, studies involving “designer cellulosomes”, in which specific catalytic components bind in a specific order to chimeric scaffoldins, either report several-fold lower activity on crystalline cellulose compared to controls or do not report activity on crystalline cellulose at all. This difference could be because of the importance of a diverse population of cellulosomes with randomly-combined catalytic components, the smaller size of chimeric scaffoldins used in designer cellulosome work, or a combination. The model system reported here, featuring full-sized engineered scaffoldins, is a promising platform for understanding these effects.
An unexpected result of the expression and purification of CipA* in T. saccharolyticum was the observed instability upon high temperature treatment with SDS-PAGE loading buffer present. While a similar effect has been reported by Morag and Lamed in C. thermocellum, the conditions used to achieve this result are markedly different [36, 37]. In the previous reports, purified protein was subjected to low pH (3.5) or low ionic strength (dialyzed against double distilled water overnight) which resulted in the cleavage of an Asp-Pro peptide bond present in the cohesin domain of CipA. In contrast, no harsh conditions were applied to CipA* from T. saccharolyticum with supernatant pHs staying above 5.8 and with no dialysis treatment applied. This may indicate a considerable difference between the extracellular environment developed by cultures of C. thermocellum compared to that of T. saccharolyticum, and could be important in future attempts at heterologous cellulosome expression.
Combined with the native ability of T. saccharolyticum to utilize hemicellulose and the availability of engineered strains that produce ethanol at high yield and titer [8, 11], a strain of T. saccharolyticum with the ability to solubilize cellulose would be a strong candidate organism for CBP. Our results, including expression and secretion of a functional, engineered, full-length CipA, represent a step toward developing such an organism. In addition, we demonstrate a model system in which understanding cellulolytic organisms and their enzyme systems can be tested by systematically reconstructing them.
Microorganisms and growth media
The parent strain for all T. saccharolyticum strains is M1442 , engineered with deletions of the genes for phosphotransacetylase, acetate kinase, and lactate dehydrogenase and expressing genes for urea utilization from C. thermocellum which serve to buffer acid production . C. thermocellum DSM1313 was obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany). C. thermocellum strain DS11, a cipA deletion mutant, was generated in our laboratory and is derived from C. thermocellum DSM1313 Δhpt and was supplied by D. G. Olson . All T. saccharolyticum strains were grown in modified DSMZ M122 medium  with 10 g/l xylose or xylan where noted, 0.5 g/l urea at pH 6.3 and 55°C unless otherwise stated. C. thermocellum strains were grown in M122 with 10 g/l cellobiose, pH 7.0 at 55°C. All cultures were grown in a Coy anaerobic chamber under a nitrogen, carbon dioxide, and hydrogen gas mix unless otherwise noted.
Plasmid and strain construction
Strains, plasmids, and primers used in this study
Description and characteristics
T. sacch. M1442
High titer ethanol producing strain
Lee et al., 2011 
T. sacch. DHC6
T. sacch ΔxynA::Clo1313_2747
T. sacch. DHC15
T. sacch ΔxynA::cipA* (wild type sec tag)
T. sacch. DHC16
T. sacch ΔxynA::cipA* (with a deletion in the sec tag)
C. therm. DSM 1313
C. therm wild type obtained from DSMZ culture collection
C. therm. DS11
C. therm 1313 ΔcipA
Olson et al. 2013 
E. coli TOP 10
S. cerevisiae FY2
Uracil Auxotroph used for homologous recombination
Winston et. al, 1995 
Description and characteristics
Deletes xynA and replaces it with a kanamycin resistance gene and a removable marker
Replaces xynA with Clo1313_2747, a kanamycin resistance gene, and a removable marker
Replaces xynA with cipA*, a kanamycin resistance gene, and a removable marker
Replaces xynA with Δsec tag cipA*, a kanamycin resistance gene, and a removable marker
Reverse transcription PCR
RNA was isolated from cultures of T. saccharolyticum incubated overnight at 55°C in modified M122  with 10 g/l glucose, xylose, or xylan. RNA was purified with the QIAGEN RNeasy Mini Kit and stored at −80°C. cDNA was generated with the QIAGEN QuantiTect Reverse Transcription Kit. The resulting cDNA was examined for the presence of the transcripts of interest using the primers listed in Table 1.
Insertion of CipA* into the chromosome
Insertion of cipA* into the chromosome under the control of the xynA promoter was achieved via double homologous recombination, selected for by the presence of a kanamycin resistance marker on a nonreplicative plasmid pMC213. The sites of recombination were directly upstream of the start codon and downstream of the stop codon in the xynA open reading frame. The size of these regions of homology was 1000 base pairs each, and left the ribosome binding site from xynA intact.
Western blot analysis of his-tagged proteins
T. saccharolyticum strains were grown to 2/3 maximum OD600. Cells were pelleted, supernatants were filter sterilized and concentrated with Vivaspin 20, PES 10,000 molecular weight cut off centrifugal concentrators (Sartorius Stedim Biotech) as per the manufacturer’s instructions. Samples were washed with two volumes of sodium citrate buffer (20 mM, pH 5.7) at 15°C. Pellets were treated with 20 mg/ml lysozyme in SET buffer (40 mM EDTA, 50 mM Tris–HCl, pH 8.0, 0.75 M sucrose) for 10 minutes to remove the cell wall, pelleted and resuspended in lysis buffer (10 mM Tris pH 7, 0.2% SDS, 1 mM DTT) and incubated at 55°C for 15 minutes. Proteins were denatured at 55°C in loading buffer (5X loading buffer: 6.25 ml 1 M Tris pH 6.8, 2 ml glycerol, 7.3 g SDS, bromophenol blue 0.1%, final pH 6.8). Total protein from either supernatants or lysed cell pellets were analyzed via Western blot with mouse Penta-His (Cat. No. 34660, QIAGEN Inc.) primary, and goat anti-mouse peroxidase conjugate (Cat. No. 31439, Thermo Sci.) secondary antibody. Detection was performed with Western Lightning ECL substrate (PerkinElmer, Waltham, MA) and detected on Kodak X-ray film.
50 ml co-cultures were grown in 115 ml nitrogen flushed anaerobic serum bottles agitated in an incubator at 55°C. The medium for the co-cultures was modified DSMZ M122  with 10 g/l xylose and 10 g/l Sigmacell 101 (a microcrystalline cellulose similar to avicel) and 0.5 g/l cellobiose to assist in the initial growth of C. thermocellum. The initial pH was pH 6.3, previously demonstrated to be suitable for co-cultures between these two organisms . Co-cultures were allowed to grow for 5 days. Cellulosomes were purified via affinity digestion of PASC .
Product formation and dry weight
Dry weight was determined by pelleting the remaining cellulose via centrifugation for 10 minutes at 8000 g, washing twice with deionized water, and drying at 45°C under vacuum for 2 days. Dried pellets were then weighed to determine residual solids. The contribution of cell mass was taken into account with controls containing 10 g/l cellobiose in place of Sigmacell. Residual sugars and ethanol in the supernatants were quantified by HPLC using an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA). Nucleotide sequence accession number.The sequences reported in this paper have been deposited in the GenBank database (accession no. KC675188 [pMC200], KC675189 [pMC212], KC675190 [pMC213], and KC675191 [pMC223]).
Cellulose binding domain
Phosphoric acid swollen cellulose
We would like to thank Dr. Joe Shaw and Dr. Erin Wiswall of Mascoma Corporation for providing strains, plasmids, methodological training, and advice. We would like to thank Alicia Eve Ballok for critical reading of the manuscript.
This research was supported by Mascoma Corporation, Lebanon NH, the Department of Energy under Award Number DE-FC36-07G017057, and by the BioEnergy Science Center (BESC), Oak Ridge National Laboratory. The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.
“This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.”
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