Erythritol production on wheat straw using Trichoderma reesei
© Jovanović et al.; licensee Springer 2014
Received: 26 January 2014
Accepted: 28 January 2014
Published: 29 May 2014
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© Jovanović et al.; licensee Springer 2014
Received: 26 January 2014
Accepted: 28 January 2014
Published: 29 May 2014
We overexpressed the err1 gene in the Trichoderma reesei wild-type and in the cellulase hyperproducing, carbon catabolite derepressed strain Rut-C30 in order to investigate the possibility of producing erythritol with T. reesei. Two different promoters were used for err1 overexpression in both strains, a constitutive (the native pyruvat kinase (pki) promoter) and an inducible one (the native β-xylosidase (bxl1) promoter). The derived recombinant strains were precharacterized by analysis of err1 transcript formation on D-xylose and xylan. Based on this, one strain of each type was chosen for further investigation for erythritol production in shake flasks and in bioreactor experiments. For the latter, we used wheat straw pretreated by an alkaline organosolve process as lignocellulosic substrate. Shake flask experiments on D-xylose showed increased erythritol formation for both, the wild-type and the Rut-C30 overexpression strain compared to their respective parental strain. Bioreactor cultivations on wheat straw did not increase erythritol formation in the wild-type overexpression strain. However, err1 overexpression in Rut-C30 led to a clearly higher erythritol formation on wheat straw.
Erythritol is a four-carbon sugar alcohol, which is applied as flavor enhancer, formulation aid, humectant, stabilizer, thickener, and as low-calorie sweetener, of which the latter is the main utilization. Compared with other polyols yielding about 2 cal/g, erythritol yields only up to 0.2 cal/g, which is due to the fact that erythritol does not undergo systemic metabolism in the human body but is excreted unchanged in the urine (Moon et al. 2010). Additionally, as a small molecule, it is easily absorbed already in the upper intestine and therefore, causes less digestive distress than other sweeteners (Livesey 2001). Since erythritol is not assimilated by Streptococcus mutans it is non-cariogenic. Furthermore it has some favorable physical and chemical properties: it is thermally stable (no decomposition or colorization at 200℃ for 1 h), better crystallizeable than sucrose, and less hygroscopic (Kasumi 1995). The negative enthalpy of solution leads to a cooling effect when dissolved. The sweetness of erythritol is plain with very weak after-taste. In a 10% (w/v) solution it has 60-80% the sweetness of sucrose. It has a natural occurrence in several foods including beer, sake, wine, soy sauce, water melon, pear, and grape. The tolerance of erythritol by animals and humans was intensively studied (Munro et al. 1998). No adverse toxicological effects were observed. Also no carcinogenic, mutagenic or teratogenic potential or effects on fertility could be detected. Therefore, erythritol is a sugar substitute with a growing market and optimization of its production remains an issue.
Current biotechnological production of erythritol use osmophilic yeasts like Aureobasidium sp., Trichosporonoides sp., Torula sp., and Candida magnoliae. As substrate a highly concentrated glucose (typically 40% (w/v)) solution is applied, which is gained from chemically and enzymatically hydrolyzed wheat- and cornstarch. The hydrolyzed starch serves as carbon source and causes a high osmotic pressure that pushes the yeast to produce the osmolyte erythritol (Moon et al. 2010). Although these processes reach 40% (w/w) yields of D-glucose to erythritol conversion, they depend on D-glucose as starting material. With regard to (socio)economical issues, starch-derived D-glucose is not a preferable substrate. Therefore, it would be an interesting alternative to use organisms that can utilize non-food, lignocellulosic biomass for the production of erythritol.
In a previous work (Jovanovic et al. 2013) we characterized the erythrose reductases (Err1) from the filamentous ascomycota Trichoderma reesei (telemorph Hypocrea jecorina (Kuhls et al. 1996)), Aspergillus niger, and Fusarium graminearium (telemorph Gibberella zeae), which are all very potent degraders of biomass. It turned out that the Err1 of T. reesei and A. niger showed comparable activities, whereas the Err1 from F. graminearium had a considerably lower activity (Jovanovic et al. 2013). In the present study we focused on the potential of producing erythritol in T. reesei from lignocellulosic biomass. The native lignocellulose-degrading enzymes of the fungus have already broad application in industry, i.e. in pulp and paper (Buchert et al. 1998; Noé et al. 1986; Welt and Dinus 1995), food and feed (Galante et al. 1993; Lanzarini and Pifferi 1989; Walsh et al. 1993), and textile industries (Koo et al. 1994, Kumar et al. 1994, Pedersen et al. 1992) as well as in biofuel production (Hahn-Hägerdal et al. 2006; Himmel et al. 2007; Ragauskas et al. 2006). As such a strong producer of cellulases and hemicellulases (a genome-wide search using the JGI Genome Portal (http://genome.jgipsf.org/Trire2/Trire2.home.html) revealed for T. reesei 10 celluloytic and 16 xylanolytic enzyme-encoding genes (Martinez et al. 2008)) it is likely that T. reesei is able to grow on cheap biowaste material like wheat straw as the sole carbon source. This is supported by former reports on T. reesei capable of growing on lignocellulosic material (Acebal et al. 1986; Dashtban et al. 2013).
In this study we used wheat straw that was pretreated by an alkaline organosolve process (Fackler et al. 2012) to remove the lignin up to a residual concentration of about 1% (w/w), which makes the cellulose and hemicellulose more easily accessible for the fungus. We investigated a T. reesei wild-type strain and the strain Rut-C30. Rut-30 is a cellulase hyperproducing, carbon catabolite derepressed mutant (Montenecourt and Eveleigh 1979), which is the parental strain of most industrially used T. reesei strains (Peterson and Nevalainen 2012; Derntl et al. 2013). In both strains the err1 gene was overexpressed using either the native, constitutive promoter from the pyruvate kinase encoding gene (pki) or the native, inducible promoter from the β-xylosidase 1 encoding gene (bxl1). The overexpression strains were screened for enhanced err1 transcript formation and the best ones where then cultivated on D-xylose and wheat straw for investigating their erythritol production capacity.
The T. reesei strains QM6a Δ tmus53 (Steiger et al. 2011) and Rut-C30 (ATCC 56765), which was derived from the wild-type strain QM6a by one UV-light and two N-methyl-N’-nitro-N-nitrosoguanidine mutation steps (Montenecourt and Eveleigh 1979), were maintained on 3% malt extract (MEX) agar. The recombinant T. reesei strains QPEC1, QBEC2, RPEC1, and RBEC2 generated during this study, were maintained on MEX agar containing 250 μl/l hygromycin B (Merck, Darmstadt, Germany).
Purification of transformed strains by streak out of spores was done on MEX agar containing 250 μl/l hygromycin B and 500 μl/l IGEPAL®; CA-630 (Sigma-Aldrich, St. Louis, MO, USA).
Cultivation in shake flasks was performed in 250-ml-Erlenmeyer flasks containing 50 ml Mandels-Andreotti (MA) medium (Mandels 1985) supplemented with 1% (w/v) D-xylose or 1% (w/v) birch-wood xylan. For inoculation 109 conidia per liter were used. Growth conditions were pH 5, 30℃, and 160 rpm shaking rate. Mycelia and supernatant were seperated by filtration. For short-term storage, harvested mycelia were shock-frozen and kept in liquid nitrogen, supernatants were kept at -20℃.
Oligonucleotides used during the study
Sequence (5’ – 3’)
For the construction of pBJ-PEC1 the vector pRLM e x30 (Mach et al. 1994), which contains the hph gene flanked by the pki promoter (ppki) and the cbh2 terminator, was used. The hph gene was removed by NsiI/XbaI digestion and subsequently, err1 that was excised from pJET-1.2 also by NsiI/XbaI digestion, was inserted.
For the construction of pBJ-BEC2 ppki was excised from pBJ-PEC1 with XhoI/XbaI digestion and replaced by pbxl1, excised from pJET-1.2 with SalI/XbaI digestion.
For QM6a Δ tmus53 protoplast transformation was performed as described in (Gruber et al. 1990). 5 g of either pBJ-PEC1 or pBJ-BEC2 and 1 μg pAN7, which confers hygromycin B resistance (Punt et al. 1987), were co-transformed into the fungal genome.
Rut-C30 was transformed with the Biolistic®; PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, Hercules, CA, USA) according to a modified protocol originally described in (Te’o et al. 2002). 5 μg of either pBJ-PEC1 or pBJ-BEC2 and 1 μg pAN7, which confers hygromycin B resistance (Punt et al. (Punt et al. 1987)), were co-transformed into the fungal genome.
Fungal genomic DNA was isolated by phenol-chloroform extraction, using a FastPrep®;-24 (MP Biomedicals, Santa Ana, CA, USA) for cell disruption. About 100 mg of mycelia was transferred to 400 μl DNA extraction buffer (0.1 M Tris-HCl pH 8.0, 1.2 M NaCl, 5 mM EDTA) and grounded with glass beads (0.37 g 0.01 0.1 mm, 0.25 g 1 mm, 1 piece 3 mm) using the FastPrep. Afterwards, the mixture was immediately put on 65℃, supplemented with 9 μM RNase A, and incubated for 30 min. Then 200 μl of phenol (pH 7.9) and 200 μl of a chloroform-isoamyl alcohol-mixture (25:1) were added, with vigorous mixing following each addition. Phases were separated by centrifugation (12000 g, 10 min, 4℃) and the aqueous phase was transferred into a new vial. DNA was precipitated by addition of the 0.7-fold volume of isopropanol to the aqueous phase. After 20 min incubation at room temperature (RT) the DNA was separated by centrifugation (20000 g, 20 min, 4℃) and washed with 500 μl ethanol (70%). The air-dried DNA pellet was solubilised in 50 μl Tris-HCl (10 mM, pH 7.5) at 60℃.
RNA extraction from fungal mycelia was performed with peqGOLD TriFast™ (peqlab, Erlangen, Germany) according to the manufacturer’s procedure, using a FastPrep®;-24 (MP Biomedicals) for cell disruption. RNA quantity and quality were determined with a NanoDrop 1000 (Thermo Scientific). A 260 nm/280 nm ratio of at least 1.8 was stipulated for further sample processing. cDNA synthesis was performed with RevertAid™H Minus First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s procedure using 0.5 μg of RNA.
where E is cycling efficiency, C is the threshold cycling number, r is the reference gene, t the target gene and a 0 marks the sample which is used as the reference (Pfaffl 2001).
For the probe preparation 500 ng of err1 cDNA, 5 μl 10x Klenow buffer (Thermo Scientific), and 6.5 μl 100 μM random hexamer primer (Thermo Scientific) were filled up with double distilled water (ddH2O) to a final volume of 39 μl and incubated at 95℃ for 5-10 min. The reaction mixture was put on ice and 5 μl Biotin PCR Labeling Mix (Jena Bioscience, Jena, Germany) and 1 μl Klenow fragment exo- (Thermo Scientific) were added. The mixture was filled up with ddH2O to a final volume of 50 μl and incubated at 37℃ for 24 h. For DNA precipitation 10 μl LiCl (4 M) and 200 μl ethanol (96%) were added. After incubation at RT for 15 min, at ice for 15 min, and at -20℃ for 1 h, the DNA was separated by centrifugation with 20000 g at 4℃ for 30 min. The pellet was washed with 500 μl ethanol (70%), followed by centrifugation with 20000 g at 4℃ for 10 min. After drying the pellet at 50℃ for about 10 min, it was dissolved in 100 μl ddH2O. The quality of the probe was tested by agarose gel electrophoresis, and the concentration was determined with the NanoDrop 1000 (Thermo Scientific).
For the Southern blot 15 μg chromosomal DNA of each strain used in this study was digested in a triple digestion with 5 μl of each NdeI, SalI, and BglII (each 10 U/ μl, Thermo Scientific), using 10x Buffer O (Thermo Scientific). The reaction mixtures were filled up with ddH2O to a final volume of 100 μl and then split into 20 μl aliquots for digestion at 37℃ over night (o/n). After digestion, samples were incubated at 70℃ for 20 min and the completion of digestion controlled by agarose gel electrophoresis. Digestion aliquots of each sample were pooled and concentrated to a final volume of 10 - 20 μl and applied to a 1% agarose gel. As length standard 5 μl Gene Ruler 1 kb DNA Ladder (Thermo Scientific) was used. Using the Mini-Sub®; Cell system (Bio-Rad Laboratories) the gel was run at 80 V for 1 h in TAE buffer, and afterwards incubated in 0.4 M NaOH and 0.6 M NaCl, and then in 0.5 M Tris (pH 7.5) and 1.5 M NaCl for 30 min each. The DNA was transferred to a Biodyne B membrane (Pall Corporation, Port Washington, NY, USA) by a capillary blot with 10x saline-sodium citrate (SSC) buffer (1.5 M NaCl, 0.15 M sodium citrate, pH 7.2) o/n. After blotting, the membrane was incubated in 0.4 M NaOH, and then in 0.2 M Tris (pH 7.5) for 1 min each. Cross-linking was performed with a GS Gene Linker UV chamber (Bio-Rad Laboratories) using program C3 and 150 mJoule on the wet membrane. For pre-hybridization, the membrane was incubated at 65℃ for 3 h in 20 ml Southern blot hybridization buffer (25% (v/v) 20x SSC, 10% (v/v) 50x Denhardt’s solution, 0.2% (v/v) EDTA (0.5 M, pH 8.0), 0.05 M NaH2PO4, 0.1% (w/v) SDS, 0.5% (w/v) BSA), supplemented with 100 μg/ml single stranded salmon sperm DNA and freshly denaturated (10 min at 95℃) probe. For hybridization, the membrane was incubated at 65℃ o/n in 10 ml Southern blot hybridization buffer. The membrane was washed twice at RT for 5 min in 50 ml 2x SSC supplemented with 0.1% (w/v) SDS, followed by washing twice at 65℃ for 15 min with 50 ml 0.1x SSC, supplemented with 0.1% (w/v) SDS. After incubation at RT for 10 min in Southern blot blocking solution (125 mM NaCl, 17 mM Na2HPO4, 8 mM NaH2PO4, 0.5% (w/v) SDS, pH 7.2), the membrane was incubated light-protected at RT for 30 min in Southern blot blocking solution supplemented with 1 μg/ml Dylight 650-labeled Streptavidin (Thermo Scientific). The membrane was washed 4 times light-protected at RT for 10 min in 50 ml 1:10 diluted Southern blocking solution and then scanned with Typhoon FLA 9500 (GE Healthcare Life Sciences, Buckinghamshire, England) set for Alexa Fluor 647 at 1000 V.
Cultivation was performed in 2-l-bench top bioreactors (Bioengineering AG, Wald, Swiss), containing 1.3 l fermentation medium ((NH4)2SO4 3.50 g/l, KH2PO4 5.00 g/l, MgSO4.7H2O 1.25 g/l, NaCl 0.625 g/l, peptone from Casein 1.25 g/l, Tween®; 80 0.625 g/l), supplemented with 1.5 ml/l trace element solution (FeSO4.7H2O 0.90 mM, MnSO4.H2O 0.50 mM, ZnSO4.7H2O 0.24 mM, CaCl2.7H2O 0.68 mM), 1.7% (w/v) wheat straw (pretreated by an alkaline organosolv process for lignin removal (Fackler et al. 2012) (Annikki, Graz, Austria)), and Antifoam Y-30 Emulsion (1 ml/bioreactor). For inoculation 109 conidia per liter were used. Agitation rate was 500 rpm, temperature was 28℃, and aeration rate was 0.5 vvm.
Mycelia from shake flask cultures were ground under liquid nitrogen. The powder was suspended in 3 ml distilled water and sonicated using a Sonifier®; 250 Cell Disruptor (Branson, Danbury, CT, USA) (power 70%, duty cycle 40%, power for 3 min, on ice). Insoluble compounds were separated by centrifugation (20000 g, 10 min, 4℃), the clear supernatant was used for further processing.
Supernatants from shake flask cultures were used directly for further processing.
For samples from cultivation in bioreactors 30 ml of the whole cultivation broth were first mechanically disrupted with a potter for 1 min, then sonicated, and afterwards centrifuged as described above for mycelia from shake flask cultures.
Sample preparation for GC was done in triplicates as follows: 300 μl of the clear supernatant (prepared as described above), supplemented with 10 ng myo-inositol as internal standard, was gently mixed with 1.2 ml ethanol (96%) and incubated for 30 min at RT for protein precipitation. The precipitate was separated by centrifugation (20000 g, 10 min, 4℃). Samples were dried under vacuum and thereafter silylated (50 μl pyridine, 250 μl hexamethyldisilazane, 120 μl trimethylsilyl chloride). For quantitative erythritol determination a GC equipment (Agilent Technologies, Santa Clara, CA, USA) with a HP-5-column (30 m, inner diameter 0.32 mm, film 0.26 μm) (Agilent) was used. The mobile phase consisted of helium with a flow of 1.4 l/min, the column temperature was as follows: 150℃ for 1 min, ramping 150 220℃ (ΔT 4℃/min), ramping 220 - 320℃ (ΔT 20℃/min), 320℃ for 6.5 min. Detection was performed with FID at 300℃. The retention times were determined using pure standard substances.
2 ml cultivation broth were centrifuged at 20000 g for 10 min at 4℃. The supernatant was discarded and the pellet resuspended in 3 ml 0.1 M NaOH before sonication with a Sonifier®; 250 Cell Disruptor (Branson) (power 70%, duty cycle 40%, power 20 s, pause 40 s, 10 cycles, on ice). The sonicated samples were incubated for 3 h at RT. After centrifugation (20000 g, 10 min, 4℃) the supernatant was used to determine protein concentration with a Bradford assay. Therefore, 20 μl diluted sample (1:10 - 1:100) were added to 1 ml 1:5 diluted Bradford Reagens (Bio-Rad Laboratories) and incubated for exactly 10 min at RT before measuring the absorption on a V-630 UV-Vis spectrophotometer (Jasco, Tokio, Japan) at 595 nm. As standard bovine serum albumin in concentrations from 10 - 100 μg/ml was used.
The by an alkaline organosolve process pretreated wheat straw (Fackler et al. 2012) used in our experiments, turned out to be a very well utilizeable substrate for T. reesei cultivation. In contrary to other pretreatment processes, this method does not require any chemicals or catalysts that subsequently inhibit fungal growth. The alcohol, which is used in the process as organic solvent, can be sufficiently removed by washing. The achieved removal of lignin (up to a residual share of 1%) (Fackler et al. 2012) makes the utilizeable cellulose and hemicellulose enough accessible for the fungus so that even direct inoculation with conidia was possible with this substrate as sole carbon source.
The comparison of the recombinant strains with their respective parentals showed that the overexpression of err1 was successful an led to an increase in erythritol formation. In case of the wild-type and its recombinant strains this effect was more pronounced in shake flask cultivations on D-xylose, whereas Rut-C30 and its recombinant strains yielded better results in the bioreactor cultivation on pretreated wheat straw. Not only the relative increase of erythritol concentration in the recombinant strains compared to the parental strain was higher, but also the total amount of erythritol produced was about 10-fold increased compared to the wild-type and its recombinant strains. This observation can be explained by the fact that Rut-C30 is a cellulase hyperproducing, carbon catabolite derepressed strain (Montenecourt and Eveleigh 1979), which makes it very likely that it better utilizes a complex substrate like wheat straw. This assumption is supported by the observed increased biomass formation and enhanced xylanase activity produced. Concerning the promoters used, the constitutive pki promoter seems to be favorable, since the erythritol production peak was slightly higher and this high level remained for a longer period (54 h). It should be mentioned that an even higher maximum might occur between the samples taken. However, cultivation time turned out to be an important factor for the erythritol formation, since after the peak of production, the erythritol concentration drops about as fast as it rises in the beginning. Accordingly, the elimination of the back reaction can be considered as one of the main targets of further strain improvement. Since we found that in T. reesei erythritol is not exported to the media, but accumulated in the cell, presumably, the most efficient way to prevent the back reaction would be to force the fungus to secrete the erythritol. This strategy would also be favorable in consideration of the osmotic balance of the cell. Taking into account that in case of erythritol production methods using yeasts, erythritol can be found in the supernatant (see e.g. Ryu et al. 2000; Rymowicz et al. 2009; Sawada et al. 2009), in yeasts must exist a transport system for erythritol that probably can be introduced into T. reesei. Another strategy to improve erythritol formation could be to reduce the accumulation of other polyols. This would on the one hand provide additional starting material for the erythritol production, and at the same time it would prevent an additional rise of the intracellular osmotic pressure by these substances.
Concluding this, in the present study we demonstrated that the production of erythritol on the renewable, non-food substrate wheat straw, using T. reesei is possible. The alkaline organosolve pretreatment process used for the wheat straw is compatible for subsequent fungal growth and provides an easily utilizeable substrate. Moreover, strain modification by overexpression of err1 led to increased erythritol formation on this substrate.
This study was supported by Annikki GmbH, by two grants from the Austrian Science Fund (FWF): [P20192, P24851] given to RLM and ARMA, respectively, by an Innovative Project of Vienna University of Technology (Demo-Tech), and by a doctoral program of Vienna University of Technology (AB-Tec).
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