- Email: [email protected]
Astaxanthin-producing strain breeding by genome shuffling
S310
Abstracts / Journal of Biotechnology 136S (2008) S290–S344
treatment, inhibitory by-products produced that complicate downstream biological process. Therefore, it is pivotal to establish a cost-effective inulinase (EC 3.2.1.7) production. In this study, we isolated the DNA sequence encoding the mature peptide sequence of the exo-inulinase gene from Kluyveromyces marxianus CBS 6556 and expressed in Pichia pastoris X-33. The purified recombinant enzyme (reINU) gave a specific activity of 13,100 U mg−1 , which was about 100-fold higher that reported for the wild type protein isolated from K. marxianus CBS6556 (Rouwenhorst et al., 1988, 1990). Ferguson plot analysis showed that secretion expressed reINU was a 169 kDa dimer. Detailed analytical assays showed that the optimal temperature and pH for reINU were 60 ◦ C and pH 4.0, respectively. It was found that hydrolysis activity was about 20% higher in the presence of Mn2+ . reINU was stable for over 3 days at room temperature. When employed for hydrolysis of the juice of the fresh tuber (water/tuber, 1:1) of Helianthus tuberosus at 50 ◦ C for 1 h, total reduced sugar reached 65 g l−1 , and 85% of the total sugar of the raw material was released based on ion chromatography analysis. In conclusion, we have developed an overproduction system for inulinase that can now be used as biocatalysts for fructose syrups industry and biomass conversion research. Keywords: Exo-inulinase; Secretory expression; Enzyme References Peters, D., 2006. Carbohydrates for fermentation. Biotechnol. J. 1, 806–814. Rouwenhorst, R.J., Visser, L.E., Van der Baan, A.A., et al., 1988. Production, distribution, and kinetic properties of inulinase in continuous cultures of Kluyveromyces marxianus CBS 6556. Appl. Environ. Microbiol. 54, 1131–1137. Rouwenhorst, R.J., Hensing, M., Verbakel, J., et al., 1990. Structure and properties of the extracellular inulinase of Kluyveromyces marxianus CBS 6556. Appl. Environ. Microbiol. 56, 3337–3345.
doi:10.1016/j.jbiotec.2008.07.1905
shoulder at 330 nm, which is typical of fungal laccases. The highest enzyme affinity was obtained with syringaldazine, and it could not oxidize tyrosine. The ESI-Tandem MS of the laccase were obtained from three peptides, and no relevant homologies with other laccases produced by different microorganisms were found. So the internal peptides of the protein were unique, supporting its consideration as a novel laccase produced by the newly isolated strain (Fernaud et al., 2006). The fungus exhibited visible decolorization towards malachite green, crystal violet and remazol brilliant blue R. The laccase treatment also improved bleaching efficiency of different pulps and depressed heat-induced color reversion of bleached pulps. Acknowledgements Financial support from National Natural Science Foundation of China (No. 30570044) is gratefully acknowledged. References Fernaud, J.R., Marina, A., González, K., Vázquez, J., Falcón, M.A., 2006. Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum. Appl. Microbiol. Biotechnol. 70, 212–221. Jimenez-Juarez, N., Roman-Miranda, R., Baeza, A., Sánchez-Amat, A., VazquezDuhalt, R., Valderrama, B., 2005. Alkali and halide-resistant catalysis by the multipotent oxidase from Marinomonas mediterranea. J. Biotechnol. 117, 73–82.
doi:10.1016/j.jbiotec.2008.07.1906 V1-P-032 Astaxanthin-producing strain breeding by genome shuffling Zongbao Zheng, Xueming Zhao ∗
V1-P-031
School of Chemical Engineering and Technology, Edinburgh-Tianjin Joint Centre for Systems Biology and Synthetic Biology, Tianjin University, Tianjin 300072, China
A novel alkaline laccase produced by Monilia sp. M5-3 and its potential in dye decolorization and pulp bleaching
E-mail address: [email protected] (X. Zhao).
Wenzhu Tang, Jian Zhao ∗ , Yinbo Qu ∗ State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China E-mail addresses: [email protected] (J. Zhao), [email protected] (Y. Qu). Laccase can catalyze the oxidation of a variety of organic and inorganic substrates and it has attracted great scientific interest for the biotechnological application. Many processes involve high pH condition, but there are few reports on laccases with high activity and stability under alkaline conditions. In this study, we screened for strains which produce alkaline laccases from environmental samples by plate tests using the indicator compound guaiacol. Monilia sp. M5-3 showed extracellular laccase activity as high as 24.46 U/ml with 2,6-dimethoxyphenol as substrate in pH 7.0. The purified laccase gave a single band on SDS-PAGE. The pH optima of the laccase were 7.0 for 2,6-dimethoxyphenol, and 8.0 for syringaldazine respectively, and it remained quite stable within the pH range of 5.0–10.0 after 300 h incubation at 4 ◦ C. These properties are unusual because most fungal laccases have pH optima around 4–6 when measured with phenolic substrates and no activity above pH 8.0 (Jimenez-Juarez et al., 2005). The stability of the laccase over a broad pH range is also worthy of note. This combination of attractive characteristics makes it an ideal candidate for industrial application. The spectrum of the laccase showed a peak at 600 nm, and a
Astaxanthin is a high-value additive carotenoid with different physiological functions. Phaffia rhodozyma has the natural ability of producing astaxanthin and has some advantages just like shorter period of fermentation, easy to culture in a high density. Genome shuffling has been applied to strain breeding widely for its high efficiency (Visser et al., 2003; Johnson and Schroeder, 1995; Zhang et al., 2002). Wildtype strain P. rhodozyma As 2.1557 was treated with ultraviolet radiation and NTG, three mutants were selected with the selective pressure of 75 mol/L DPA. Among the three, the highest concentration of astaxanthin was 1.188 mg/L, which was 48% higher than that of As 2.1557. Then we studied the conditions for protoplast preparation, regeneration and fusion. At the optimal lysozyme treating conditions obtained through response surface methodology, the product of protoplast preparation and regeneration was above 60%. The protoplasts were inactivated by UV and heat respectively and then fusion between biparental inactivated protoplasts was carried out. The three strains obtained after complex mutation and another mutant preserved were selected as parent strains for genome shuffling. After 2 cycles of recursive protoplast fusion, a shuffled strain named P. rhodozyma F2-11 was selected, the concentration of astaxanthin was 1.950 mg/L, which was 1.43 higher than As 2.1557. Comparing the flux distribution, we found that the pentose phosphate flux of shuffled strain was lower than the mutant, while the EMP and TCA cycle flux were higher. Pyruvate dehydrogenase was still inefficient and about 32% pyruvate was excreted. The enzyme assays proved that the activities of pyruvate dehydrogenase in shuffled strains were only a little
Abstracts / Journal of Biotechnology 136S (2008) S290–S344
higher, but there is much improve on the activities of ATP: citrate lyase which is affinitive with the acetyl coenzyme A (a precursor of astaxanthin). That somehow illuminated the mechanism that the shuffled strains could over-produce astaxanthin.
doi:10.1016/j.jbiotec.2008.07.1908
Acknowledgement
V1-P-034
Supported by NSFC-20536040, 973-2007CB707802. References Johnson, E., Schroeder, W., 1995. Astaxanthin from the yeast Phaffia rhodozyma. Stud. Mycol. 38, 81–89. Visser, H., van Ooyen, J., Verdoes, J.C., 2003. Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous. FEMS Yeast Res. 4, 221–231. Zhang, Y., Perry, K., Vinci, V., Powell, K., Stemmer, W.P., del Cardayré, S.B., 2002. Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415, 644–646.
doi:10.1016/j.jbiotec.2008.07.1907 V1-P-033 Isolation of demulsifying strains and characterization of a biodemulsifier produced by Alcaligenes sp. Liu Jia ∗ , Huang Xiang-feng, Lu Li-jun, Yang Dian-hai, Zhou Qi College of Environmental Science & Engineering, State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shanghai 200092, China
S311
Mukherjee, S., Das, P., Sen, R., 2006. Towards commercial production of microbial surfactants. Trends Biotechnol. 24, 509–515.
Synthesis of compatible solute ectoine from halophilic bacterium and structural analysis of synthease gene Yajun Lang 1,∗ , Linghua Zhang 1 , Shinichi Nagata 2 , Yunji Wang 1 1
College of Environmental Science and Engineering, Dalian Maritime University, 1 Lingshui Road, Dalian 116026, PR China 2 Environmental Biochemistry Division, Research Center for Inland Seas, Kobe University, 5-1-1 Fukae, Higashinada-ku, Kobe 658-0022, Japan E-mail address: [email protected] (Y. Lang). Halophilic bacterium DL08 was screened from seawater in China. The strain 16S rDNA sequence had similarity (100%) with Cobetia marina DSM 4741 (AJ306890). The strain can grow in MM63 medium (Larsen et al., 1987) with 3 M NaCl. NMR and HPLC–MS results indicated that the strain accumulated ectoine in medium (Onraedt et al., 2005) with 1 M NaCl. During batch fermentation (10 L fermentor), ectoine synthesis amount were 1454.91 mg/L. The 5231 bp nucleotide sequence including structural gene of ectoine synthease (EctA, EctB, EctC) was obtained through TAIL-PCR. The results from 5231 bp nucleotide sequence analysis showed that three open reading frames (ORF) consisted of 558 bp, 1323 bp and 393 bp.
E-mail address: [email protected] (J. Liu). Four screening methods, colorimetric assay, blood-plate hemolysis method, surface tension activity and oil spreading technique were introduced to isolate demulsifying strains from 6 different bacteria source samples. Four demulsifying strains with high demulsification ratio were obtained, which are qualified in demulsification test of kerosene model emulsion (Hamme et al., 2003). It is recommended to use surface tension and oil spreading methods in pre-screening and validate the results in demulsification test with kerosene model emulsions. A strain, was successfully isolated from petroleum-contaminated soil and identified as Alcaligenes sp. Its physicochemical properties, demulsification capability and utilization of waste oil were further investigated. The valid part of the bio-demulsifier in demulsifying the model emulsion was confirmed as the cell wall coherent material. TLC and FIR were introduced to identify the valid part as one kind of lipopeptide. It achieved 96% and 50% of emulsion breaking ratio in W/O (water in oil) and O/W (oil in water) model emulsion, respectively, which implied that the strain was more suitable to break W/O emulsion (Kosaric, 1992). From the dynamic change of kerosene breaking ratio, emulsion breaking ratio and water breaking ratio during demulsification process, it was found that this bio-demulsifier reacted with the continuous phase of the emulsion prior to its reaction with the dispersed phase. With oil waste as carbon source, the demulsifier produced by Alcaligenes sp. showed high demulsifying capability. Moreover, the production of demulsifier was 4.6 times of that generated with kerosene as the carbon source. This will greatly reduce the production cost of bio-demulsifier and make the industrial application possible (Mukherjee et al., 2006). References Hamme, J.D., Singh, A., Ward, O.P., 2003. Recent advances in petroleum microbiology. Microbiol. Mol. Biol. Rev. 67, 503–549. Kosaric, N., 1992. Biosurfactants in industry. Pure Appl Chem. 64, 1731–1737.
References Larsen, P.I., Sydnes, L.K., Landfald, B., Strom, A.R., 1987. Osmoregulation in Escherichia coli by accumulation of organic osmolytes: betaines, glutamic acid, and trehalose. Arch. Microbiol. 147, 1–7. Onraedt, A.E., Walcarius, B.A., Soetaert, W.K., Vandamme, E.J., 2005. Optimization of ectoine synthesis through fed-Batch fermentation of Brevibacterium epidermis. Biotechnol. Prog. 21, 1206–1212.
doi:10.1016/j.jbiotec.2008.07.1909 V1-P-035 Genome shuffling to improve the ethanol production of Saccharomyces cerevisiae Guo-li Gong ∗ , Chang-lu Wang, Mian-hua Chen, Zhi-qiang Chen, Yu-rong Wang College of Food Engineering & Biotechnology, Tian Jin University of Science &Technology, Tianjin 300457, PR China E-mail address: [email protected] (G.-l. Gong). Ethanol has become an alternative fuel to alleviate the high demand for gasoline. Genome shuffling by recursive protoplast fusion of the strains with different genotypes (Zhang et al., 2002; Stephanopoulos, 2002), is an efficient method for improving the production of metabolites by microbes (Patnaik et al., 2002; Dai and Copley, 2004; Gong et al., 2007). This study aimed improving the ethanol yield of S. cerevisiae by genome shuffling technique.More than 100 natural strains of S. cerevisiae were isolated and characterized to screen for ethanol production strains from various soil samples. We obtained 5 yeasts, ES-1, ES-2, ES-3, ES-4 and ES5, which respectively yield 40 g/l, 51 g/l, 63 g/l, 68 g/l and 70 g/l. These strains were separately incubated and prepared protoplasts. The prepared protoplasts were mixed to be mutagenized using
Abstracts / Journal of Biotechnology 136S (2008) S290–S344
treatment, inhibitory by-products produced that complicate downstream biological process. Therefore, it is pivotal to establish a cost-effective inulinase (EC 3.2.1.7) production. In this study, we isolated the DNA sequence encoding the mature peptide sequence of the exo-inulinase gene from Kluyveromyces marxianus CBS 6556 and expressed in Pichia pastoris X-33. The purified recombinant enzyme (reINU) gave a specific activity of 13,100 U mg−1 , which was about 100-fold higher that reported for the wild type protein isolated from K. marxianus CBS6556 (Rouwenhorst et al., 1988, 1990). Ferguson plot analysis showed that secretion expressed reINU was a 169 kDa dimer. Detailed analytical assays showed that the optimal temperature and pH for reINU were 60 ◦ C and pH 4.0, respectively. It was found that hydrolysis activity was about 20% higher in the presence of Mn2+ . reINU was stable for over 3 days at room temperature. When employed for hydrolysis of the juice of the fresh tuber (water/tuber, 1:1) of Helianthus tuberosus at 50 ◦ C for 1 h, total reduced sugar reached 65 g l−1 , and 85% of the total sugar of the raw material was released based on ion chromatography analysis. In conclusion, we have developed an overproduction system for inulinase that can now be used as biocatalysts for fructose syrups industry and biomass conversion research. Keywords: Exo-inulinase; Secretory expression; Enzyme References Peters, D., 2006. Carbohydrates for fermentation. Biotechnol. J. 1, 806–814. Rouwenhorst, R.J., Visser, L.E., Van der Baan, A.A., et al., 1988. Production, distribution, and kinetic properties of inulinase in continuous cultures of Kluyveromyces marxianus CBS 6556. Appl. Environ. Microbiol. 54, 1131–1137. Rouwenhorst, R.J., Hensing, M., Verbakel, J., et al., 1990. Structure and properties of the extracellular inulinase of Kluyveromyces marxianus CBS 6556. Appl. Environ. Microbiol. 56, 3337–3345.
doi:10.1016/j.jbiotec.2008.07.1905
shoulder at 330 nm, which is typical of fungal laccases. The highest enzyme affinity was obtained with syringaldazine, and it could not oxidize tyrosine. The ESI-Tandem MS of the laccase were obtained from three peptides, and no relevant homologies with other laccases produced by different microorganisms were found. So the internal peptides of the protein were unique, supporting its consideration as a novel laccase produced by the newly isolated strain (Fernaud et al., 2006). The fungus exhibited visible decolorization towards malachite green, crystal violet and remazol brilliant blue R. The laccase treatment also improved bleaching efficiency of different pulps and depressed heat-induced color reversion of bleached pulps. Acknowledgements Financial support from National Natural Science Foundation of China (No. 30570044) is gratefully acknowledged. References Fernaud, J.R., Marina, A., González, K., Vázquez, J., Falcón, M.A., 2006. Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum. Appl. Microbiol. Biotechnol. 70, 212–221. Jimenez-Juarez, N., Roman-Miranda, R., Baeza, A., Sánchez-Amat, A., VazquezDuhalt, R., Valderrama, B., 2005. Alkali and halide-resistant catalysis by the multipotent oxidase from Marinomonas mediterranea. J. Biotechnol. 117, 73–82.
doi:10.1016/j.jbiotec.2008.07.1906 V1-P-032 Astaxanthin-producing strain breeding by genome shuffling Zongbao Zheng, Xueming Zhao ∗
V1-P-031
School of Chemical Engineering and Technology, Edinburgh-Tianjin Joint Centre for Systems Biology and Synthetic Biology, Tianjin University, Tianjin 300072, China
A novel alkaline laccase produced by Monilia sp. M5-3 and its potential in dye decolorization and pulp bleaching
E-mail address: [email protected] (X. Zhao).
Wenzhu Tang, Jian Zhao ∗ , Yinbo Qu ∗ State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China E-mail addresses: [email protected] (J. Zhao), [email protected] (Y. Qu). Laccase can catalyze the oxidation of a variety of organic and inorganic substrates and it has attracted great scientific interest for the biotechnological application. Many processes involve high pH condition, but there are few reports on laccases with high activity and stability under alkaline conditions. In this study, we screened for strains which produce alkaline laccases from environmental samples by plate tests using the indicator compound guaiacol. Monilia sp. M5-3 showed extracellular laccase activity as high as 24.46 U/ml with 2,6-dimethoxyphenol as substrate in pH 7.0. The purified laccase gave a single band on SDS-PAGE. The pH optima of the laccase were 7.0 for 2,6-dimethoxyphenol, and 8.0 for syringaldazine respectively, and it remained quite stable within the pH range of 5.0–10.0 after 300 h incubation at 4 ◦ C. These properties are unusual because most fungal laccases have pH optima around 4–6 when measured with phenolic substrates and no activity above pH 8.0 (Jimenez-Juarez et al., 2005). The stability of the laccase over a broad pH range is also worthy of note. This combination of attractive characteristics makes it an ideal candidate for industrial application. The spectrum of the laccase showed a peak at 600 nm, and a
Astaxanthin is a high-value additive carotenoid with different physiological functions. Phaffia rhodozyma has the natural ability of producing astaxanthin and has some advantages just like shorter period of fermentation, easy to culture in a high density. Genome shuffling has been applied to strain breeding widely for its high efficiency (Visser et al., 2003; Johnson and Schroeder, 1995; Zhang et al., 2002). Wildtype strain P. rhodozyma As 2.1557 was treated with ultraviolet radiation and NTG, three mutants were selected with the selective pressure of 75 mol/L DPA. Among the three, the highest concentration of astaxanthin was 1.188 mg/L, which was 48% higher than that of As 2.1557. Then we studied the conditions for protoplast preparation, regeneration and fusion. At the optimal lysozyme treating conditions obtained through response surface methodology, the product of protoplast preparation and regeneration was above 60%. The protoplasts were inactivated by UV and heat respectively and then fusion between biparental inactivated protoplasts was carried out. The three strains obtained after complex mutation and another mutant preserved were selected as parent strains for genome shuffling. After 2 cycles of recursive protoplast fusion, a shuffled strain named P. rhodozyma F2-11 was selected, the concentration of astaxanthin was 1.950 mg/L, which was 1.43 higher than As 2.1557. Comparing the flux distribution, we found that the pentose phosphate flux of shuffled strain was lower than the mutant, while the EMP and TCA cycle flux were higher. Pyruvate dehydrogenase was still inefficient and about 32% pyruvate was excreted. The enzyme assays proved that the activities of pyruvate dehydrogenase in shuffled strains were only a little
Abstracts / Journal of Biotechnology 136S (2008) S290–S344
higher, but there is much improve on the activities of ATP: citrate lyase which is affinitive with the acetyl coenzyme A (a precursor of astaxanthin). That somehow illuminated the mechanism that the shuffled strains could over-produce astaxanthin.
doi:10.1016/j.jbiotec.2008.07.1908
Acknowledgement
V1-P-034
Supported by NSFC-20536040, 973-2007CB707802. References Johnson, E., Schroeder, W., 1995. Astaxanthin from the yeast Phaffia rhodozyma. Stud. Mycol. 38, 81–89. Visser, H., van Ooyen, J., Verdoes, J.C., 2003. Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous. FEMS Yeast Res. 4, 221–231. Zhang, Y., Perry, K., Vinci, V., Powell, K., Stemmer, W.P., del Cardayré, S.B., 2002. Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415, 644–646.
doi:10.1016/j.jbiotec.2008.07.1907 V1-P-033 Isolation of demulsifying strains and characterization of a biodemulsifier produced by Alcaligenes sp. Liu Jia ∗ , Huang Xiang-feng, Lu Li-jun, Yang Dian-hai, Zhou Qi College of Environmental Science & Engineering, State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shanghai 200092, China
S311
Mukherjee, S., Das, P., Sen, R., 2006. Towards commercial production of microbial surfactants. Trends Biotechnol. 24, 509–515.
Synthesis of compatible solute ectoine from halophilic bacterium and structural analysis of synthease gene Yajun Lang 1,∗ , Linghua Zhang 1 , Shinichi Nagata 2 , Yunji Wang 1 1
College of Environmental Science and Engineering, Dalian Maritime University, 1 Lingshui Road, Dalian 116026, PR China 2 Environmental Biochemistry Division, Research Center for Inland Seas, Kobe University, 5-1-1 Fukae, Higashinada-ku, Kobe 658-0022, Japan E-mail address: [email protected] (Y. Lang). Halophilic bacterium DL08 was screened from seawater in China. The strain 16S rDNA sequence had similarity (100%) with Cobetia marina DSM 4741 (AJ306890). The strain can grow in MM63 medium (Larsen et al., 1987) with 3 M NaCl. NMR and HPLC–MS results indicated that the strain accumulated ectoine in medium (Onraedt et al., 2005) with 1 M NaCl. During batch fermentation (10 L fermentor), ectoine synthesis amount were 1454.91 mg/L. The 5231 bp nucleotide sequence including structural gene of ectoine synthease (EctA, EctB, EctC) was obtained through TAIL-PCR. The results from 5231 bp nucleotide sequence analysis showed that three open reading frames (ORF) consisted of 558 bp, 1323 bp and 393 bp.
E-mail address: [email protected] (J. Liu). Four screening methods, colorimetric assay, blood-plate hemolysis method, surface tension activity and oil spreading technique were introduced to isolate demulsifying strains from 6 different bacteria source samples. Four demulsifying strains with high demulsification ratio were obtained, which are qualified in demulsification test of kerosene model emulsion (Hamme et al., 2003). It is recommended to use surface tension and oil spreading methods in pre-screening and validate the results in demulsification test with kerosene model emulsions. A strain, was successfully isolated from petroleum-contaminated soil and identified as Alcaligenes sp. Its physicochemical properties, demulsification capability and utilization of waste oil were further investigated. The valid part of the bio-demulsifier in demulsifying the model emulsion was confirmed as the cell wall coherent material. TLC and FIR were introduced to identify the valid part as one kind of lipopeptide. It achieved 96% and 50% of emulsion breaking ratio in W/O (water in oil) and O/W (oil in water) model emulsion, respectively, which implied that the strain was more suitable to break W/O emulsion (Kosaric, 1992). From the dynamic change of kerosene breaking ratio, emulsion breaking ratio and water breaking ratio during demulsification process, it was found that this bio-demulsifier reacted with the continuous phase of the emulsion prior to its reaction with the dispersed phase. With oil waste as carbon source, the demulsifier produced by Alcaligenes sp. showed high demulsifying capability. Moreover, the production of demulsifier was 4.6 times of that generated with kerosene as the carbon source. This will greatly reduce the production cost of bio-demulsifier and make the industrial application possible (Mukherjee et al., 2006). References Hamme, J.D., Singh, A., Ward, O.P., 2003. Recent advances in petroleum microbiology. Microbiol. Mol. Biol. Rev. 67, 503–549. Kosaric, N., 1992. Biosurfactants in industry. Pure Appl Chem. 64, 1731–1737.
References Larsen, P.I., Sydnes, L.K., Landfald, B., Strom, A.R., 1987. Osmoregulation in Escherichia coli by accumulation of organic osmolytes: betaines, glutamic acid, and trehalose. Arch. Microbiol. 147, 1–7. Onraedt, A.E., Walcarius, B.A., Soetaert, W.K., Vandamme, E.J., 2005. Optimization of ectoine synthesis through fed-Batch fermentation of Brevibacterium epidermis. Biotechnol. Prog. 21, 1206–1212.
doi:10.1016/j.jbiotec.2008.07.1909 V1-P-035 Genome shuffling to improve the ethanol production of Saccharomyces cerevisiae Guo-li Gong ∗ , Chang-lu Wang, Mian-hua Chen, Zhi-qiang Chen, Yu-rong Wang College of Food Engineering & Biotechnology, Tian Jin University of Science &Technology, Tianjin 300457, PR China E-mail address: [email protected] (G.-l. Gong). Ethanol has become an alternative fuel to alleviate the high demand for gasoline. Genome shuffling by recursive protoplast fusion of the strains with different genotypes (Zhang et al., 2002; Stephanopoulos, 2002), is an efficient method for improving the production of metabolites by microbes (Patnaik et al., 2002; Dai and Copley, 2004; Gong et al., 2007). This study aimed improving the ethanol yield of S. cerevisiae by genome shuffling technique.More than 100 natural strains of S. cerevisiae were isolated and characterized to screen for ethanol production strains from various soil samples. We obtained 5 yeasts, ES-1, ES-2, ES-3, ES-4 and ES5, which respectively yield 40 g/l, 51 g/l, 63 g/l, 68 g/l and 70 g/l. These strains were separately incubated and prepared protoplasts. The prepared protoplasts were mixed to be mutagenized using