ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2012, том 48, № 2, с. 243-248
UDC 663.18
CONSTRUCTION OF THE INDUSTRIAL ETHANOL-PRODUCING STRAIN OF Saccharomyces cerevisiae ABLE TO FERMENT CELLOBIOSE
AND MELIBIOSE © 2012 L. Zhang, Z.-P. Guo, Z.-Y. Ding, Z.-X. Wang, G.-Y. Shi
The Key Laboratory of Industrial Biotechnology, Ministry of Education; Center for Bioresources & Bioenergy, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China e-mail: biomass_jnu@126.com Received December 29, 2010
The gene mel1, encoding a-galactosidase in Schizosaccharomyces pombe, and the gene bgl2, encoding and P-glucosidase in Trichoderma reesei, were isolated and co-expressed in the industrial ethanol-producing strain of Saccharomyces cerevisiae. The resulting strains were able to grow on cellobiose and melibiose through simultaneous production of sufficient extracellular a-galactosidase and P-glucosidase activity. Under aerobic conditions, the growth rate of the recombinant strain GC1 co-expressing 2 genes could achieve 0.29 OD600 h-1 and a biomass yield up to 7.8 g l 1 dry cell weight on medium containing 10.0 g l 1 cellobiose and 10.0 g l-1 melibiose as sole carbohydrate source. Meanwhile, the new strain of S. cerevisiae CG1 demonstrated the ability to directly produce ethanol from microcrystalline cellulose during simultaneous sacchari-fication and fermentation process. Approximately 36.5 g l-1 ethanol was produced from 100 g of cellulose supplied with 5 g l-1 melibose within 60 h. The yield (g of ethanol produced/g of carbohydrate consumed) was 0.44 g/g, which corresponds to 88.0% of the theoretical yield.
Tremendous researches have been devoted to producing fuel ethanol from cellulosic raw materials, and cellulases are key factors in solving this problem. The two-step conversion of biomass to ethanol involves the enzymatic hydrolysis of cellulosic biomass to produce reducing sugars, and the conversion of the resulting sugars to ethanol. However, this is a very costly process due to the recalcitrance of cellulose, and therefore the low yield and high cost of the enzymatic hydrolysis process [1]. P-Glucosidases working synergistically with endoglucanases (EC 3.2.1.4) and exoglucanases (EC 3.2.1.91) on the degradation of cellulose [2] not only catalyze the final step in the degradation of cellulose, but also stimulate the extent of cellulose hydrolysis by relieving the cellobiose-mediated inhibition of exoglucanase and endoglucanase [3, 4].
Development of a yeast strain capable of producing ethanol by fermenting cellulosic substrates has received a great deal of interest over recent years. The advantages using this microorganism include: (i) high ethanol productivity and tolerance, (ii) large cells size, which simplify their separation from the culture broth and (iii) resistance to viral infection [5]. Although Saccharomyces cerevisiae is one of the most suitable microorganisms for practical purposes, it cannot degrade polysaccharides such as cellobiose. Since cellobiose (and longer chain cello-oligosaccharides) is the major soluble by-product of cellulose hydrolysis, its efficient utilization is of primary importance to cellulose bio-degradation process development. Enzymatic hydrolysis of cellobiose requires the action of P-glu-
cosidases. This heterogeneous group of enzymes displays broad substrate specificity towards cellobiose, cello-oligosaccharides and different aryl- and alkyl-P-D-glucosides. P-Glucosidases were found widely in animals, plants, fungi and bacteria [6]. Though many efforts have been done to express heterogenous gene of P-glucosides in yeast and bacteria to improve the ethanol productivity, the strains they used mostly are hap-loid auxotrophic strains and primarily for laboratory research [7-11]. In addition, melibiose, a disaccha-ride containing glucose and galactose linked through a-1,4 glycosidic bond, is one of the main non-reducing saccharides not effectively utilized during S. cere-visiae-mediated very high gravity ethanol fermentation from starchy materials such as wheat, corn, and cassava. Thus, it is necessary to enhance the ability of the yeast to ferment melibiose to improve the utilization rate of these materials and ethanol yield.
In this study, the gene mel1, encoding a-galactosidase in Schizosaccharomyces pombe, and the gene bgl2, encoding P-glucosidase in Trichoderma reesei, were isolated and separately expressed or co-expressed in the industrial ethanol-producing strain of Saccharomyces cerevisiae. The resulting strains were studied under anaerobic conditions and ethanol production from cellobiose and melibiose was achieved by expressing these genes.
MATERIALS AND METHODS
Yeast strains and media. E. coli JM109 {recAl supE44 endA1 HsdRH (rK, mK) gyrA96 relAl thi-1 (lac-proAB) [F', traD36proAB+ lacIq lacZM15]} (Stratagene, USA) was used for plasmid transformation and propagation. T. reesei was grown in a medium containing (g/l): bean cake powder — 45.0, wheat bran — 10.0, corn meal — 20.0, KH2PO4 - 5.0, CaCl2 - 3.0, NH4Cl - 5.0.
The industrial yeast, S. cerevisiae CICIMY0086 (http://cicim-cu.sytu.edu.cn/, ethanol producing yeast used in industrial plants) was used for genetic manipulation. The yeasts including S. pombe were routinely grown in a medium composed of 1% yeast extract, 2% bactopeptone, and 2% glucose (YEPD), solid media contained 2% agar. For selection of yeast transformants, geneticin (G418) was added with the final concentration 300 ^g/ml. Incubation conditions were standardized on the rotary shaker with 150 rpm at 30°C.
Construction of the strains. The plasmid for expressing P-glucosidase was constructed. The total RNA of T. reesei was extracted using with a guanidine thiocyanate-phenyl-chloroform method [12]. Poly A+ mRNA was isolated from the obtained total RNA of T. reesei using Oligoex Kit (Qiagen, Germany) according to the manufacturer's instructions. The gene bgl2, encoding P-glucosidase in T. reesei, was obtained by PCR amplification using Qiagen One Step RT-PCR kit from the poly A+ mRNA with primers P1(5'-CCG-GAAZ2EATGTTGCCCAAGGACTTTCAGTGGG-3') and P2(5'-CCC^^2AAATTTCCCCTTTGA AGA-AGCATCAGG-3') [13, 14] containing EcoRI and HindIII sit, respectively. A 1538-bp PCR fragment including the entire coding region for P-glucosidase was obtained. This EcoR I/HindIII digested fragment was inserted into the vector pYX212 (Ingenenius MBV-028-10) at the same sit, resulting plasmid pYX-BGL. Kanamycin resistance gene which confers resistance to geneticin in S. cerevisiae was isolated from the vector pPIC9K and was inserted into the downstream of the target gene in pYX-BGL resulting in the plasmid pYX-BGL-Km. The P-glucosidase expressing cassette including TPI (triosephosphate isomerase) promoter, and geneticin resistance gene was isolated from the plasmid pYX-BGL-Km with primers P3, 5'-AAC-TTAACTTCCGGCCACTTGAATGCTGGTAGAA-AGAGAAGTTCCTCTTCTGTTAACGGGAGCG-TAATGGTGATGGAA-3' and P4, 5'-TAATTCTTCA-ATCATGTCCGGCAGGTTCTTCATTGGGTAGT TGTTGTAAACGATGAGATATCATGCGTAGTCA-GGCAC-3'. A 54-bp gene fragment (underlined) of the S. cerevisiae glycerol phosphate dehydrogenase gene (GDP1) was added to each primer used as homologous integration site. After purification, this gpdT-PTPI-BGLII-Km-gpdT fragment was introduced into the industrial alcoholic yeast by the lithium acetate method [15]. The recombinants were screened on the
YEPD plate containing 300 ^g/ml G418. Correct insertion of the gene into the target locus was verified by PCR.
The gene mell was amplified by PCR form the ge-nomic DNA of S. pombe using primers P5(5'-CCGG-GATCCTTGCCACATTCGCCTCCGTA- 3'), and P6 (5'-CCCGGAZCCATCATGTGCTAGGTCGATTCTG-GT-3') containing BamH I sit on both ends. This BamH I digested fragment was inserted into the same sit of vector pYX212, resulting in the plasmid pYX-MEL. After that, G418 resistance gene was inserted into the downstream of the gene mell and the resulted plasmid was designated pYX-MEL-Km. The a-galactosidase expressing cassette including TPI promoter, and G418 resistance gene was isolated from the plasmid pYX-MEL-Km with primers P7 (5'-ATGTAATAAGCAAA-CAAGCACGAATGGGGAAAGCCTATGTGCAA-TCACCAAGGTAACGGGAGCGTAATGGTGAT-GGAA-3') and P8 (5'-TCGTGAACTTCTCTGCA-TGTGATTATCCCTTGGGCGGATTGACCGTTAA-GCAATGAGATATCATGCGTAGTCAGGCAC-3'). A 54-bp gene fragment (underlined) of the S. cerevisiae glycerol phosphate dehydrogenase (gdp2) gene was added to each primer used as homologous integration site. After purification, this gpd2'-PTPI-Mel-Km-gpd2' fragment was introduced into the industrial alcoholic yeast. The recombinants were screened on the YEPD plate containing 300 ^g/ml G418. Correct insertion of the gene into the target locus was verified by PCR.
For co-expressing 2 genes, the recombinant strain expressing P-glucosidase was re-transformed by the a-galactosidase expressing cassette and higher concentration of G418 and nearly 800 ^g/ml was used which was determined by the resistance experiment of the initial recombinant. Correct insertion of the gene into the target locus was verified by PCR.
Measurement of enzyme activity. The recombinant strains were cultivated at 30°C for 48 h in YEPD medium and the resulting fermentation fluid was used as enzyme solution. Activities of P-glucosidase and a-galactosidase were determined by measuring pNP (p-nitro-phenol) concentration derived from pNPG. To assay the P-glucosidase activity, the reaction mixture (final volume, 4.0 ml) containing 0.2 ml of enzyme solution, 1.8 ml of 0.2 M Na2HPO4 and 2.0 ml of5.0 mM 4-nitro-phenyl-P-D-glucopyranoside (Sigma, USA) in 0.1 M citric acid buffer (pH 4.5), was mixed and incubated at 30°C for 10 min. Reaction was stopped by adding 2.0 ml of 1.0 M Na2CO3. The enzyme reaction was monitored by spectrophotometry (400 nm) at room temperature for 5 min [16]. For measuring the activity of a-galactosidase, 4-nitrophenyl-P-D-glucopyrano-side was substituted by 4-nitrophenyl-a-D-galactopy-ranoside used as chromogenic substrate [16]. One unit
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