Improvement of cellulose catabolism in Clostridium cellulolyticum by sporulation abolishment and carbon alleviation
© Li et al.; licensee BioMed Central Ltd. 2014
Received: 6 October 2013
Accepted: 6 February 2014
Published: 20 February 2014
Clostridium cellulolyticum can degrade lignocellulosic biomass, and ferment the soluble sugars to produce valuable chemicals such as lactate, acetate, ethanol and hydrogen. However, the cellulose utilization efficiency of C. cellulolyticum still remains very low, impeding its application in consolidated bioprocessing for biofuels production. In this study, two metabolic engineering strategies were exploited to improve cellulose utilization efficiency, including sporulation abolishment and carbon overload alleviation.
The spo0A gene at locus Ccel_1894, which encodes a master sporulation regulator was inactivated. The spo0A mutant abolished the sporulation ability. In a high concentration of cellulose (50 g/l), the performance of the spo0A mutant increased dramatically in terms of maximum growth, final concentrations of three major metabolic products, and cellulose catabolism. The microarray and gas chromatography–mass spectrometry (GC-MS) analyses showed that the valine, leucine and isoleucine biosynthesis pathways were up-regulated in the spo0A mutant. Based on this information, a partial isobutanol producing pathway modified from valine biosynthesis was introduced into C. cellulolyticum strains to further increase cellulose consumption by alleviating excessive carbon load. The introduction of this synthetic pathway to the wild-type strain improved cellulose consumption from 17.6 g/l to 28.7 g/l with a production of 0.42 g/l isobutanol in the 50 g/l cellulose medium. However, the spo0A mutant strain did not appreciably benefit from introduction of this synthetic pathway and the cellulose utilization efficiency did not further increase. A technical highlight in this study was that an in vivo promoter strength evaluation protocol was developed using anaerobic fluorescent protein and flow cytometry for C. cellulolyticum.
In this study, we inactivated the spo0A gene and introduced a heterologous synthetic pathway to manipulate the stress response to heavy carbon load and accumulation of metabolic products. These findings provide new perspectives to enhance the ability of cellulolytic bacteria to produce biofuels and biocommodities with high efficiency and at low cost directly from lignocellulosic biomass.
KeywordsClostridium cellulolyticum Sporulation spo0A Cellulose catabolism Isobutanol
As the search for affordable and clean energy fuels continues, cellulosic biofuels have become a promising solution because cellulosic biomass is the most abundant renewable feedstock on earth . The key technological barrier to utilize this important renewable resource is the general lack of low-cost technology for overcoming the recalcitrance of cellulosic biomass on a large-scale . Consolidated bioprocessing (CBP), which integrates saccharolytic enzymes production, cellulose fiber degradation, and fermentation of resulting sugars into a single step, is considered a promising technology for significantly reducing the processing cost .
Clostridium cellulolyticum is a mesophilic gram-positive bacterium capable of degrading cellulose via an extracellular enzymatic complex called the cellulosome and fermenting the sugars from cellulose degradation to lactate, acetate, ethanol, hydrogen and CO2. Recently, C. cellulolyticum was engineered to produce isobutanol, a possible alternative to gasoline to fuel combustion engines, directly from cellulose . Therefore, C. cellulolyticum has the potential to be a model system with industrial relevance for the production of biofuels and commodity chemicals directly from plant biomass via CBP. However, its cellulolytic ability and metabolic productivity still need to be improved dramatically in order to meet the requirements of industrial applications. Enzymological properties of the C. cellulolyticum cellulosome have been studied extensively and there is no evidence showing that its cellulolytic system is a limiting factor for cellulose utilization . Therefore, other metabolic engineering strategies need to be exploited to improve the cellulose utilization efficiency.
Hydrolysis of lignocellulose at high concentrations is essential in economical fermentation to ethanol and other valuable products. Also, there are advantages if the metabolic products can be accumulated at high concentrations to reduce the cost of product recovery and lower energy input . However, in nature C. cellulolyticum can rarely find a niche where carbon sources and all other nutrients are plentiful, so it has become well adapted for famine environments after millions of years of evolution . These natural ecosystems are quite different from the fermentation conditions in the laboratory or industrial sites where most nutrient factors have been optimized. Indeed, it has been reported that C. cellulolyticum could not deal with a surfeit of substrates, leading to nicotinamide adenine dinucleotide (NADH) and pyruvate accumulation to toxic levels . This problem was alleviated by heterologous expression of the Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase genes to reduce pyruvate accumulation. As a consequence, cellulose consumption was increased 150% compared to the wild-type (WT) . Meanwhile, it has been reported that high concentration of cellulose and low pH could trigger sporulation and the entry into the stationary phase of C. cellulolyticum, which may be partially responsible for arresting metabolite production . It has been noticed that attachment to cellulose fibers could trigger sporulation in Clostridium thermocellum, indicating a possible connection between sporulation and cellulose degradation. It also has been recognized that the sporulation program could be a hindrance for applying sporulating microbes in fermentations for commodity chemicals production . Since sporulation is not a desirable trait from an industrial point of view in solventogenic Clostridium acetobutylicum, it is of particular importance to investigate how the transcriptional regulation of sporulation impacts solventogenesis. Several key sporulation-related transcriptional regulator genes were inactivated, and the interconnections between differentiation, sporulation and solventogenesis were studied extensively in recent years [13–15].
Motivated by the work mentioned above, we investigated approaches based on manipulating the stress response caused by heavy carbon loading, accumulation of metabolic products, and accompanied physiological changes. Sporulation is a widely used strategy by gram-positive bacteria to increase survival ability in hostile environments by entering a dormant or non-growth state, and forming a robust spore that germinates when conditions are favorable . In C. cellulolyticum, fermentation causes acids accumulation resulting in a drop in pH and accumulation of various metabolic products. Such accumulations can create a less favorable growth condition for C. cellulolyticum, which could trigger the sporulation process and slow down cellulose hydrolysis and metabolism. Therefore, by curbing sporulation in C. cellulolyticum, the fermentation process might be extended to improve cellulose hydrolysis and metabolite production. Furthermore, carbon overload could be alleviated by introducing exogenous pyruvate consumption pathways to further improve cellulose hydrolysis. To test these hypotheses, we disrupted the spo0A gene at locus Ccel_1894 encoding the master-switch transcription factor of sporulation , and introduced a synthetic isobutanol pathway  into WT and spo0A mutant strains to consume excessive pyruvate. Results with strains designed to test these hypotheses are reported in this study.
Group II intron-mediated spo0A inactivation
Characterization of spo0A mutant
Transcriptomic and metabolomic comparison of WT and spo0A mutant
Selected genes with significant expression level changes in spo0A mutant in the category of amino acid transport and metabolism
Acetolactate synthase, large subunit
3-Isopropylmalate dehydratase, small subunit
Tryptophan synthase, beta subunit
Orn/Lys/Arg decarboxylase major region
Metabolites differing in abundance between C. cellulolyticum WT and spo0A mutant cultures
Wild-type growth phase
spo0A growth phase
0.32 ± 0.005
0.46 ± 0.03
0.56 ± 0.003
1.75 ± 0.16
18.7 ± 1.78
17.8 ± 0.89
0.08 ± 0.008
0.20 ± 0.009
0.17 ± 0.03
0.19 ± 0.002
0.95 ± 0.24
1.63 ± 0.22
1.58 ± 0.19
0.04 ± 0.015
0.40 ± 0.05
2.43 ± 0.76
1.25 ± 0.24
0.04 ± 0.015
0.07 ± 0.03
0.06 ± 0.02
0.02 ± 0.01
1.02 ± 0.27
16.51 ± 2.83
26.5 ± 4.12
0.76 ± 0.03
4.20 ± 0.41
3.83 ± 0.27
0.23 ± 0.01
1.1 ± 0.27
0.35 ± 0.16
0.39 ± 0.04
0.15 ± 0.07
0.02 ± 0.006
9.13 ± 1.39
20.2 ± 2.43
21.7 ± 1.63
3.70 ± 0.40
9.38 ± 0.32
7.59 ± 0.46
0.64 ± 0.05
0.74 ± 0.16
3.62 ± 0.03
5.13 ± 0.38
1.43 ± 0.24
12.71 ± 1.72
14.3 ± 2.24
0.05 ± 0.005
3.18 ± 0.05
3.36 ± 0.13
4.65 ± 0.72
3.29 ± 0.22
5.85 ± 0.62
6.72 ± 0.40
1.37 ± 0.07
24.1 ± 3.76
59.3 ± 13.11
87.0 ± 8.80
9.12 ± 1.40
19.48 ± 6.67
35.3 ± 8.65
37.7 ± 8.35
17.3 ± 6.31
12.1 ± 0.18
48.4 ± 7.74
49.8 ± 3.17
38.9 ± 2.94
11.10 min; 246 and 320 m/z
5.1 ± 0.28
13.1 ± 2.9
17.8 ± 2.8
0.61 ± 0.07
2.3 ± 0.50
1.43 ± 0.52
0.07 ± 0.02
7.52 min; 159 and 174 m/z
1.1 ± 0.007
0.98 ± 0.14
1.07 ± 0.09
2.43 ± 0.25
7.56 ± 0.51
7.0 ± 0.31
0.13 ± 0.03
9.58 min; 117, 259, 244, 288 and 303 m/z
0.2 ± 0.03
1.04 ± 0.01
0.52 ± 0.01
0.58 ± 0.11
4.26 ± 1.1
4.66 ± 0.99
0.14 ± 0.03
9.93 min; 331 and 359 m/z
11.7 ± 1.2
55.1 ± 11.6
29.6 ± 10.6
0.66 ± 0.18
2.59 ± 1.20
0.61 ± 0.05
0.07 ± 0.006
10.90 min; 450 m/z
7.94 ± 0.23
5.48 ± 1.19
3.83 ± 0.34
16.0 ± 1.55
21.6 ± 1.37
16.0 ± 1.55
0.08 ± 0.006
Increased cellulose catabolism by the alleviation of carbon overload
It was previously reported that when C. cellulolyticum was grown with high carbon source concentration, high carbon flux was attained, leading to a high level of pyruvate accumulation . As a result, such a catabolic overflow led to an accumulation of intracellular inhibitory compounds that were responsible for the cessation of growth of C. cellulolyticum. To further increase cellulose utilization, the excessively accumulated pyruvate needs to be consumed. A biosynthetic strategy was developed to produce higher alcohols by taking advantage of the amino acid biosynthesis capability to produce 2-keto acids, and then the 2-keto acids were converted to alcohols by 2-keto acid decarboxylase and alcohol dehydrogenase . As the valine, leucine and isoleucine biosynthesis pathways were upregulated based on microarray and GC-MS analyses in the spo0A mutant, we hypothesized that it could be possible to mitigate the pyruvate accumulation and further increase cellulose consumption by driving the pyruvate flow towards amino acid biosynthesis and further diverting the intermediates toward higher alcohols. Thus, two key genes, alsS encoding B. subtilis acetolactate synthase and kivD encoding L. lactis ketoacid decarboxylase, were introduced into both the spo0A mutant and WT to direct the conversion of pyruvate to isobutanol.
However, it was shown that strong expression of alsS was toxic to C. cellulolyticum, because no transformants could be obtained when the alsS gene was cloned directly under the ferredoxin promoter from Clostridium pasteurianum. The C. pasteurianum ferredoxin promoter was a strong constitutive promoter for C. cellulolyticum, and was also recognized by Escherichia coli. Although this promoter can drive strong expression of its downstream genes, it is uncontrollable in both C. cellulolyticum and E. coli, causing toxicity in over-expression in C. cellulolyticum and cloning in E. coli. To mitigate the toxicity of foreign gene over-expression in C. cellulolyticum, we decided to utilize inducible promoters recognized by C. cellulolyticum.
Although sporulation abolishment helped C. cellulolyticum improve cellulose utilization and final metabolic product yields in a high concentration of 50 g/l cellulose, the introduction of the partial synthetic isobutanol pathway to consume extra pyruvate did not further increase cellulose utilization in the spo0A mutant. One possible explanation is that a connection between sporulation rate and the availability of amino acids in B. subtilis was reported , and our omics analyses also showed that valine, leucine and isoleucine biosynthesis pathways were altered in the spo0A mutant, indicating sporulation abolishment could probably affect amino acid biosynethsis. The defined media used for the fermentation experiments contained salts, vitamins, minerals, nitrogen and carbon without providing additional amino acids. Therefore, the synthetic pathway might cause a valine or leucine deficient environment during the fermentation. Somehow, such a deficiency had a more severe effect on the spo0A mutant than WT, resulting in unchanged cellulose utilization. However, the introduction of pLyc025 helped WT increase 63% in cellulose utilization. More efforts will be required to clarify what happened to the carbon flux from the additionally consumed cellulose, since the isobutanol production in WT025 was quite low and the concentrations of the other end-products were not significantly altered. To our surprise, in 50 g/l cellulose, the isobutanol production in spo0A025 was even lower than WT025. Although omics analyses showed upregulation of the valine, leucine and isoleucine biosynthesis pathways in the spo0A mutant, the concentration of isobutanol, however, did not show significant increase in the spo0A mutant compared to WT, indicating that additional studies are required to connect omics analyses with synthetic pathway regulations. One possibility for the lower than expected isobutanol production is that several paralogous genes exist in the genome, and upregulation of one gene may not necessarily affect the chemical reactions catalyzed by several proteins. For example, there are two genes encoding acetolactate synthase large subunit, Ccel_0303 and Ccel_3437. Only Ccel_0303 was detected to be up regulated in microarray analysis, but not for Ccel_3437. It is also noticeable that the isobutanol productivity was low in both WT and spo0A transformants in all tested conditions. Several possible reasons could explain this problem. First, although alsS was expressed directly under the cipP promoter, this promoter was relatively less powerful than the ferrodoxin promoter, so that there may not be adequate expression of the two genes, resulting in low productivity. Second, the pathway itself was incomplete and far from optimized. We avoided the inclusion of ilvC, ilvD and adh genes in the construction mainly because the problems of the codon usage of these genes in C. cellulolyticum and multiple gene expression have not yet been solved .
A key technical achievement of this study was that a quantitative promoter evaluation method was developed using AFP and flow cytometry. It is well known that proper promoters are key elements in metabolic engineering and synthetic biology. Reporter genes, such as those encoding green fluorescent proteins (GFPs) and luciferase, are widely used for the analysis of promoter activities and transcriptional regulation events . However, they all need oxygen for fluorescence and bioluminescence , limiting their applications in anaerobic conditions. The recently engineered oxygen-independent flavin mononucleotide (FMN)-based AFPs (Evoglow series), paved the way for many applications associated with GFPs previously unavailable for anaerobic bacteria, such as in vivo fluorescence imaging, fluorescence-activated flow cytometry and cell sorting. A protocol for promoter screening was developed using the AFP fusion method, and the fluorescence signal intensity was evaluated by flow cytometry and microscopy. To our knowledge, this is the first report of a successful flow cytometry application with AFP, which holds great potential for further development as a high-throughput promoter screening method using flow cytometry and cell sorting. AFP can also be used as an expression reporter assay; the gene of interest is either transcriptionally or translationally fused to AFP so that the expression level of AFP correlating either to the level of transcription or translation can be quantitatively measured by flow cytometry. Using this promoter evaluation protocol, the strength of the ferrodoxin promoter and cipP promoter was compared. Transformants with the pLyc027 plasmid showed differential fluorescent signal response with cellobiose and cellulose, and the transformants with the pLyc017 plasmid had strong signal intensity with both carbon sources.
In this study, we presented two metabolic engineering strategies to improve cellulose utilization in C. cellulolyticum. The spo0A mutant strain abolished sporogenesis and likely became less sensitive to the environmental stress generated by fermentation. In order to alleviate carbon overload, alsS and kivD genes were introduced to divert the excessive carbon to isobutanol production. Both strategies helped WT significantly increase cellulose utilization under high cellulose concentration.
Materials and methods
Media and culture conditions
E. coli TOP10 cells (Invitrogen, Grand Island, NY, USA) were used for cloning and were grown at 37°C in LB medium supplemented with 50 μg/ml kanamycin or 20 μg/ml chloramphenicol, as appropriate. C. cellulolyticum H10 was cultured at 34°C anaerobically in modified VM medium  with 5.0 g/l cellobiose, and 10.0 g/l or 50.0 g/l cellulose as the carbon sources. The complex modified VM medium was supplemented with 2.0 g/l yeast extract and was mainly used for transformation experiments. The defined modified VM medium was supplemented with the vitamin solution and mineral solution as previously described , instead of yeast extract and was used for fermentation and omics experiments. For agar plates, 1.0% (w/v) of Bacto agar was added to the medium. The modified VM medium was prepared anaerobically and was supplemented with 15 μg/ml erythromycin or 15 μg/ml thiamphenicol, as appropriate.
The knock-out plasmid pLyc1217Er was constructed as previously described . The intron integration sites were chosen by calculating all possible sites for insertions into Ccel_1894 using an online intron design tool at http://www.clostron.com. The program predicted multiple intron insertion sites across the gene. Based on the consideration of both optimal gene inactivation and efficient insertion, an anti-sense integration site 60 bp downstream of the start codon was chosen for Ccel_1894. Four PCR primers for this integration design, IBS, EBS1d, EBS2 and EBSu were created by the online intron design tool. The knockout plasmid named pLyc1217Er1894 was constructed as previously described .
The over-expression plasmids were constructed with pJIR750ai as the backbone by removing the promoter, group II intron and ltrA. The ferrodoxin promoter was amplified from pLyc1217Er; the cipP promoter was amplified from C. cellulolyticum genomic DNA; the AFP gene Pp1 was amplified from pGLOW-Pp1-stop (The evoglow basic kit, Evocatal, Monheim am Rhein, Germany); alsS was amplified from pSA69 and kivD was amplified from pSA55 ; the spo0A gene was amplified from C. cellulolyticum genomic DNA. The empty vector was constructed by cutting pJIR750ai with EcoRI and PvuI. The DNA fragments were linked together by standard cloning procedures to generate plasmids, pLyc017, pLyc025, pLyc027, pLyc032, and pSpo0A/over, respectively. A list of primers, plasmids and strains used in this study is presented in Additional file 2.
The plasmids were transformed into C. cellulolyticum by electroporation. The electroporation-competent cells and methylated plasmids were prepared as previously described . For each transformation, a 100-μl cell suspension was mixed with 2.0 μg of methylated plasmid DNA. The cells were electroporated in 2-mm gap electroporation cuvettes (BTX, Hawthorne, NY, USA) with a Gene Pulser Xcell electroporator (Bio-Rad, Hercules, CA, USA) inside an anaerobic chamber. The competent cells were transformed by a square wave protocol: the voltage was 1.25 kV, and the time constant was 5 ms. After electroporation, the cells were recovered and plated as previously described , and supplemented with 15 μg/ml erythromycin or 15 μg/ml thiamphenicol as appropriate. The plates were incubated at 34°C anaerobically in BD GasPak plastic bags until single colonies appeared.
All fermentation experiments were run in the defined media with cellobiose or Avicel PH101 crystalline cellulose (FMC BioPolymer, Philadelphia, PA, USA) as the carbon source. Cultures were initially grown in 10 ml of cellobiose medium to an optical density (OD)600 of 0.7 to 0.9. These cultures were then used to inoculate either 10 ml cellobiose medium (5.0 g/l) or 100 ml cellulose medium (10 g/l or 50 g/l) at 0.5% (v/v) with three biological replicates for each strain. In 50 g/l cellulose fermentation, pH was adjusted to 7.4 by injecting 5 M NaOH every 48 hours. The pH was monitored by a Cardy Twin micro pH meter (Spectrum Technologies, Aurora, IL, USA). The NaOH injection volumes ranged from 50 μl to 600 μl, depending on the pH values measured before and after injections. The cellular growth was estimated by total protein measurement. The cells were lysed by 0.2 N NaOH/1% w/v SDS for 60 minutes at room temperature, and followed by neutralization with 0.8 N HCl. The total protein was measured with the BCA Protein Assay Kit (Pierce, Rockford, IL, USA), using bovine serum albumin as a standard. The cellulose consumption was determined with glucose as the standard as previously described  with minor modifications. The residual cellulose was washed and suspended in distilled water. For attached cell lysis, the suspension was heated at 100°C for 30 minutes. The residual cellulose was further washed with distilled water and hydrolyzed into soluble sugars with 65% H2SO4. An aliquot of a 200-μl sample was mixed with 200 μl 5% phenol and 1,000 μl 98% H2SO4 and incubated for 30 minutes at room temperature. Absorbance at 490 nm was determined by a FLUOstar OPTIMA microplate reader (BMG Labtech, Cary, NC, USA), as described previously . For fermentation product analyses, the samples were filtered through 0.2-μm filters, acidified by 0.025% sulfuric acid and analyzed for lactate, acetate, ethanol, and isobutanol concentrations using HPLC with an Agilent 1200 system (Agilent Technologies, Santa Clara, CA, USA) equipped with a variable-wavelength (190 to 600 nm) detector (with UV absorption measured at 245 nm) and an ion-exclusion column (Aminex HPX-87H; 300 mm × 7.8 mm; Bio-Rad, Hercules, CA, USA) operating at 55°C . The mobile phase consisted of 0.025% sulfuric acid at a flow rate of 0.6 ml/minute.
C. cellulolyticum genomic DNA was extracted using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA): 10 μg of genomic DNA was digested with EcoRI, which does not cut the inserted intron fragment, and was separated by agarose gel electrophoresis. The DNA transfer, cross-link, hybridization and detection experiments were performed as previously described .
Heat-survival assay for sporulation
C. cellulolyticum strains were grown in defined VM cellulose medium (50 g/l) for 18 days to allow spores to form. Cells were harvested by centrifugation, and suspended and diluted appropriately in anaerobic PBS buffer. Cultures were heated at 80°C for 10 minutes to kill vegetative cells, followed by plating on cellobiose (5.0 g/l) agar plates. Sporulation frequency was calculated as colony-forming unit(cfu)/ml enumerated before and after heat-treatment in triplicates.
Transcriptional profile changes were analyzed from triplicate cultures by a whole genome microarray designed and synthesized by NimbleGen (Roche, Madison, WI, USA). DNA microarrays used in this study covered 3078 of the 3390 annotated protein-coding sequences of the C. cellulolyticum genome; the probe length was 70-mer and synthesized in triplicate for each gene. Total RNA was collected and isolated for all samples taken at log phase with Trizol and the Qiagen RNA miniprep method as previously described . The fluorescent dye Cyanine 3 was used for labeling of cDNA from total RNA by reverse transcription PCR with random primers. Genomic DNA (gDNA) was labeled with Cyanine 5 and co-hybridized with Cyanine 3-labeled cDNA onto the microarray slide at 42°C on a Hybridization Station (MAUI, BioMicro Systems, Salt Lake City, UT, USA) for 16 h with mixing. Microarray slide washing and image scanning, raw data processing, and fold-change calculation of the gene expression levels were performed as described previously [34, 35].
Detailed metabolomic profiles were analyzed from triplicate cultures by GC-MS, using an Agilent Technologies Inc. (Santa Clara, CA, USA) 5975C inert XL gas chromatograph-mass spectrometer, fitted with an Rtx-5MS with Integra-guard (5% diphenyl/95% dimethyl polysiloxane) 30 m × 250 μm × 0.25-μm film-thickness capillary column. Supernatants of C. cellulolyticum cultures grown with 5.0 g/l cellobiose were collected at log phase, stationary phase and late stationary phase and centrifuged at 10,000 rpm for 10 minutes at 4°C to remove precipitates. Aliquots containing 250 μl of supernatant and 10 μl of sorbitol (1.0 g/L aqueous) were transferred by pipette to a vial and stored at -20°C overnight. The samples were thawed and concentrated to dryness under a stream of N2. The internal sorbitol standard was added to correct for subsequent differences in trimethylsilyl derivatization efficiency and changes in sample volume during heating. Three replicate samples at each phase were analyzed per microbial strain as previously described . Briefly, the standard quadrupole GC-MS was operated with splitless injection and analyses were conducted in the electron impact (70 eV) ionization mode, with 6 full-spectrum (50 to 650 Da) scans per second.
Real-time PCR quantification
In order to validate microarray hybridization results, five genes were selected for further analysis with real-time PCR. Reverse transcription was conducted by using SuperScript® III Reverse Transcriptase (Invitrogen, Grand Island, NY, USA). cDNA products were diluted as appropriate and used as the templates. Quantitative real-time PCR was performed using iTaq SYBR Green Supermix with ROX (Bio-Rad, Hercules, CA, USA) on Bio-Rad iQ5. Gene-specific primers used for transcript quantification are listed in Additional file 2. The thermal cycling conditions were as follows: 95°C for 3 minutes, 40 cycles of 95°C for 15 s, 55°C for 15 s and 72°C for 45 s. The recA gene was used as an internal calibrator . Relative expressional level was calculated with the Pfaffl Method .
Microscopy and flow cytometry
Promoter strength was evaluated by fluorescent microscopy and flow cytometry. Transformants were grown to middle log phase. Samples were washed twice with anaerobic PBS buffer and suspended in the same buffer as well. Slides were imaged using Olympus BX51 fluorescence microscope equipped with an Olympus DP71 digital camera; the optical filter was set with excitation at 490 nm and emission at 525 nm for the green fluorescence. Flow cytometry analysis was performed on a BD Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA, USA). All samples were diluted with anaerobic PBS buffer approximately 106 to 108 times from the original cultures to similar concentrations. The run limit was set up as 10,000 events with slow flow rate. The threshold was set up as 40,000 on FSC-H. The samples were run through the flow cytometer automatically following the manufacturer’s instructions. The fluorescence was detected with an FL1 detector with a 530/30 filter. The data were collected and analyzed with the CFlow software.
anaerobic fluorescent protein
gas chromatography–mass spectrometry
green fluorescent protein
high performance liquid chromatography
open reading frame
This work was supported mainly by the NSF EPSCoR Program through the award EPS 0814361 and partially by the BioEnergy Science Center, a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. This manuscript has been co-authored by a contractor of the US Government under contract DE-AC05-00OR22725. We thank Dr Joy D Van Nostrand for discussions and proofreading of this manuscript.
- Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS: Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 2002, 66: 506-577. 10.1128/MMBR.66.3.506-577.2002View ArticleGoogle Scholar
- Li Y, Irwin DC, Wilson DB: Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A. Appl Environ Microbiol 2007, 73: 3165-3172. 10.1128/AEM.02960-06View ArticleGoogle Scholar
- Lynd LR, van Zyl WH, McBride JE, Laser M: Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol 2005, 16: 577-583. 10.1016/j.copbio.2005.08.009View ArticleGoogle Scholar
- Desvaux M: Clostridium cellulolyticum : model organism of mesophilic cellulolytic clostridia. FEMS Microbiol Rev 2005, 29: 741-764. 10.1016/j.femsre.2004.11.003View ArticleGoogle Scholar
- Higashide W, Li Y, Yang Y, Liao JC: Metabolic engineering of Clostridium cellulolyticum for production of isobutanol from cellulose. Appl Environ Microbiol 2011, 77: 2727-2733. 10.1128/AEM.02454-10View ArticleGoogle Scholar
- Kristensen JB, Felby C, Jorgensen H: Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnol Biofuels 2009, 2: 11. 10.1186/1754-6834-2-11View ArticleGoogle Scholar
- Petitdemange ECF, Giallo J, Gaudin C: Clostridium cellulolyticum sp. nov., a cellulolytic, mesophilic: species from decayed grass. Int J Syst Bacteriol 1984, 34: 155-159. 10.1099/00207713-34-2-155View ArticleGoogle Scholar
- Guedon E, Desvaux M, Payot S, Petitdemange H: Growth inhibition of Clostridium cellulolyticum by an inefficiently regulated carbon flow. Microbiology 1999, 145: 1831-1838. 10.1099/13500872-145-8-1831View ArticleGoogle Scholar
- Guedon E, Desvaux M, Petitdemange H: Improvement of cellulolytic properties of Clostridium cellulolyticum by metabolic engineering. Appl Environ Microbiol 2002, 68: 53-58. 10.1128/AEM.68.1.53-58.2002View ArticleGoogle Scholar
- Desvaux M, Petitdemange H: Sporulation of Clostridium cellulolyticum while grown in cellulose-batch and cellulose-fed continuous cultures on a mineral-salt based medium. Microb Ecol 2002, 43: 271-279. 10.1007/s00248-001-0043-7View ArticleGoogle Scholar
- Wiegel J, Dykstra M: Clostridium thermocellum : adhesion and sporulation while adhered to cellulose and hemicellulose. Appl Microbiol Biotechnol 1984, 20: 59-65.View ArticleGoogle Scholar
- Papoutsakis ET: Engineering solventogenic clostridia. Curr Opin Biotechnol 2008, 19: 420-429. 10.1016/j.copbio.2008.08.003View ArticleGoogle Scholar
- Tracy BP, Jones SW, Papoutsakis ET: Inactivation of sigma(E) and sigma(G) in Clostridium acetobutylicum illuminates their roles in Clostridial-cell-form biogenesis, granulose synthesis, solventogenesis, and spore morphogenesis. J Bacteriol 2011, 193: 1414-1426. 10.1128/JB.01380-10View ArticleGoogle Scholar
- Jones SW, Tracy BP, Gaida SM, Papoutsakis ET: Inactivation of sigmaF in Clostridium acetobutylicum ATCC 824 blocks sporulation prior to asymmetric division and abolishes sigmaE and sigmaG protein expression but does not block solvent formation. J Bacteriol 2011, 193: 2429-2440. 10.1128/JB.00088-11View ArticleGoogle Scholar
- Bi CH, Jones SW, Hess DR, Tracy BP, Papoutsakis ET: SpoIIE is necessary for asymmetric division, sporulation, and expression of sigma(F), sigma(E), and sigma(G) but does not control solvent production in Clostridium acetobutylicum ATCC 824. J Bacteriol 2011, 193: 5130-5137. 10.1128/JB.05474-11View ArticleGoogle Scholar
- Paredes CJ, Alsaker KV, Papoutsakis ET: A comparative genomic view of clostridial sporulation and physiology. Nat Rev Microbiol 2005, 3: 969-978. 10.1038/nrmicro1288View ArticleGoogle Scholar
- Molle V, Fujita M, Jensen ST, Eichenberger P, Gonzalez-Pastor JE, Liu JS, Losick R: The Spo0A regulon of Bacillus subtilis . Mol Microbiol 2003, 50: 1683-1701. 10.1046/j.1365-2958.2003.03818.xView ArticleGoogle Scholar
- Atsumi S, Hanai T, Liao JC: Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 2008, 451: 86-89. 10.1038/nature06450View ArticleGoogle Scholar
- Hoch JA: Genetic analysis of pleiotropic negative sporulation mutants in Bacillus Subtilis . J Bacteriol 1971, 105: 896-901.Google Scholar
- Huang IH, Waters M, Grau RR, Sarker MR: Disruption of the gene (spo0A) encoding sporulation transcription factor blocks endospore formation and enterotoxin production in enterotoxigenic Clostridium perfringens type A. FEMS Microbiol Lett 2004, 233: 233-240. 10.1111/j.1574-6968.2004.tb09487.xView ArticleGoogle Scholar
- Ravagnani A, Jennert KC, Steiner E, Grunberg R, Jefferies JR, Wilkinson SR, Young DI, Tidswell EC, Brown DP, Youngman P, Morris JG, Young M: Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia. Mol Microbiol 2000, 37: 1172-1185. 10.1046/j.1365-2958.2000.02071.xView ArticleGoogle Scholar
- Harris LM, Welker NE, Papoutsakis ET: Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824. J Bacteriol 2002, 184: 3586-3597. 10.1128/JB.184.13.3586-3597.2002View ArticleGoogle Scholar
- Guedon E, Desvaux M, Petitdemange H: Kinetic analysis of Clostridium cellulolyticum carbohydrate metabolism: importance of glucose 1-phosphate and glucose 6-phosphate branch points for distribution of carbon fluxes inside and outside cells as revealed by steady-state continuous culture. J Bacteriol 2000, 182: 2010-2017. 10.1128/JB.182.7.2010-2017.2000View ArticleGoogle Scholar
- Graves MC, Rabinowitz JC: In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for “extended” promoter elements in gram-positive organisms. J Biol Chem 1986, 261: 11409-11415.Google Scholar
- Gal L, Pages S, Gaudin C, Belaich A, Reverbel-Leroy C, Tardif C, Belaich JP: Characterization of the cellulolytic complex (cellulosome) produced by Clostridium cellulolyticum . Appl Environ Microbiol 1997, 63: 903-909.Google Scholar
- Abdou L, Boileau C, de Philip P, Pages S, Fierobe HP, Tardif C: Transcriptional regulation of the Clostridium cellulolyticum cip-cel operon: a complex mechanism involving a catabolite-responsive element. J Bacteriol 2008, 190: 1499-1506. 10.1128/JB.01160-07View ArticleGoogle Scholar
- Drepper T, Eggert T, Circolone F, Heck A, Krauss U, Guterl JK, Wendorff M, Losi A, Gartner W, Jaeger KE: Reporter proteins for in vivo fluorescence without oxygen. Nat Biotechnol 2007, 25: 443-445. 10.1038/nbt1293View ArticleGoogle Scholar
- Mader U, Homuth G, Scharf C, Buttner K, Bode R, Hecker M: Transcriptome and proteome analysis of Bacillus subtilis gene expression modulated by amino acid availability. J Bacteriol 2002, 184: 4288-4295. 10.1128/JB.184.15.4288-4295.2002View ArticleGoogle Scholar
- Knoppova M, Phensaijai M, Vesely M, Zemanova M, Nesvera J, Patek M: Plasmid vectors for testing in vivo promoter activities in Corynebacterium glutamicum and Rhodococcus erythropolis . Curr Microbiol 2007, 55: 234-239. 10.1007/s00284-007-0106-1View ArticleGoogle Scholar
- Li Y, Tschaplinski TJ, Engle NL, Hamilton CY, Rodriguez M Jr, Liao JC, Schadt CW, Guss AM, Yang Y, Graham DE: Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations. Biotechnol Biofuels 2012, 5: 2. 10.1186/1754-6834-5-2View ArticleGoogle Scholar
- Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC, Minton NP: The ClosTron: mutagenesis in Clostridium refined and streamlined. J Microbiol Methods 2009, 80: 49-55.View ArticleGoogle Scholar
- Atsumi S, Wu TY, Eckl EM, Hawkins SD, Buelter T, Liao JC: Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes. Appl Microbiol Biotechnol 2010, 85: 651-657. 10.1007/s00253-009-2085-6View ArticleGoogle Scholar
- Hemme CL, Fields MW, He Q, Deng Y, Lin L, Tu QC, Mouttaki H, Zhou AF, Feng XY, Zuo Z, Ramsay BD, He Z, Wu L, Van Nostrand J, Xu J, Tang YJ, Wiegel J, Phelps TJ, Zhou J: Correlation of genomic and physiological traits of thermoanaerobacter species with biofuel yields. Appl Environ Microbiol 2011, 77: 7998-8008. 10.1128/AEM.05677-11View ArticleGoogle Scholar
- He Q, Huang KH, He ZL, Alm EJ, Fields MW, Hazen TC, Arkin AP, Wall JD, Zhou JZ: Energetic consequences of nitrite stress in Desulfovibrio vulgaris Hildenborough, inferred from global transcriptional analysis. Appl Environ Microbiol 2006, 72: 4370-4381. 10.1128/AEM.02609-05View ArticleGoogle Scholar
- Liang YT, He ZL, Wu LY, Deng Y, Li GH, Zhou JZ: Development of a common Oligonucleotide reference standard for microarray data normalization and comparison across different microbial communities. Appl Environ Microbiol 2010, 76: 1088-1094. 10.1128/AEM.02749-09View ArticleGoogle Scholar
- Yang S, Tschaplinski TJ, Engle NL, Carroll SL, Martin SL, Davison BH, Palumbo AV, Rodriguez M Jr, Brown SD: Transcriptomic and metabolomic profiling of Zymomonas mobilis during aerobic and anaerobic fermentations. BMC Genomics 2009, 10: 34. 10.1186/1471-2164-10-34View ArticleGoogle Scholar
- Stevenson DM, Weimer PJ: Expression of 17 genes in Clostridium thermocellum ATCC 27405 during fermentation of cellulose or cellobiose in continuous culture. Appl Environ Microbiol 2005, 71: 4672-4678. 10.1128/AEM.71.8.4672-4678.2005View ArticleGoogle Scholar
- Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001, 29: e45. 10.1093/nar/29.9.e45View ArticleGoogle Scholar
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