- Email: [email protected]
Study on antibiotic substances metabolized by Monascus purpureus
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
S35
I1-P-031
I1-P-032
Expression of Vitreoscilla hemoglobin enhances growth and production of riboflavin in recombinant Bacillus subtilis
Study on antibiotic substances metabolized by Monascus purpureus
Yunxia Duan 1 , Shuobo Shi 1 , Tao Chen 1 , Xun Chen 1 , Yi Gong 2 , Shengli Yang 2 , Xueming Zhao 1,∗
Zhao Shuxin ∗ , Zeng Luyan, Li Yi
1
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Edinburgh-Tianjin Joint Research Centre for Systems Biology and Synthetic Biology, Tianjin University, Tianjin 300072, China 2 Shanghai Research Center of Biotechnology, Academic Sinica, Shanghai 200233, China E-mail address: [email protected] (X. Zhao).
Riboflavin is a commercially important vitamin used for pharmaceuticals, feed additives, cosmetics, and food industry. The whole riboflavin biosynthesis is an energy consumption step from the sum riboflavin biosynthesis reaction (Zamboni, 2003). Vitreoscilla hemoglobin (VHb), which is produced by Vitreoscilla in hypoxic environments can enhance the ATP synthesis rate and microaerobic respiration efficiency (Chen and Bailey, 1994; Kallio et al., 1994). In order to further enhance riboflavin biosynthesis, vgb gene which encodes VHb was integrated into the chromosome of genetically engineered strain B. subtilis PY and its influence on physiological characters has been investigated. The apparent phenotype with more rapid specific growth rate, higher oxygen uptake rate and higher biomass was achieved under oxygen-limited conditions in batch cultivation. A metabolic flux distribution analysis discloses that VHb can also facilitate oxygen delivering, generate sufficient oxygen flux and direct interaction with the respiratory apparatus of the cell and thus relieve the overflow metabolism. Therefore, VHb-expression strains have a lower adenosine triphosphate (ATP) synthesis rate from substrate-level phosphorylation but a higher overall ATP production rate under microaerobic conditions. The increased higher oxygen uptake rate, as well as decreased by-products could explain the increased biomass and riboflavin production in VHb-expression strain. The expression of VHb also improved the biomass and yield of riboflavin compared with the control strain under fed-batch fermentation, and 28% higher biomass and about 20% improvement of the yield of riboflavin were obtained. Acknowledgement
Tianjin Key Laboratory of Industrial Microbiology, Tianjin University of Science & Technology, Tianjin 300457, China E-mail address: [email protected] (Z. Shuxin). Monascus has been used for thousands of years in China, after fermentation whose products (Red qu) contains many high value substances. Nowadays, the public has recognized the importance of Monascus products. The focus of this study is to explore the antibiotic substances except citrinin and pigments. First, we crushed the mycelium by ultrasonic crusher, then filtrated by vacuum pump and decolored by active carbon. Second, HPLC analyzes (Wang et al., 2005) the decolored sample. The result demonstrated that citrinin has reduced to 88–98% of its base level after decolored. The content of citrinin was reduced from 0.058 g/ml to 0.002 g/ml. Pigment analysis showed that after decolored, yellow and red pigments were reduced from 12.8 U/g and 17.1 U/g to 0.036 U/g and 0.016 U/g. From the research, we can confirm that citrinin and pigments have no impact on bacteriostatic effect of the decolored sample. Third, disc papers was used to ascertain bacteriostatic effect of decolored sample that has ability to inhibited Bacillus cereus and Staphylococcus aureus and has no effect on E. coli and Saccharmoyces cerevisiae. From this experiment, we can conclude that there was some other antibiotic substance in Red qu except citrinin and pigments. Forth, using dialysis bag technique dialyses the sample, then test different parts respectively. From the result, we can confirm that the substance with bacteriostatic effect was below 1 kDa. Qualitative analysis (Zhang and Guo, 2003; Boubekeur et al., 2007) showed that antibiotic substance contains the carbohydrate and amino acid, and the substance was identified as carbohydrate-peptide linkage by the thin-layer chromatography. References Boubekeur, Badji, Abdellah, Mostefaoui, Nasserdine, Sabaou, Ahmed, Lebrihi, 2007. Isolation and partial characterization of antimicrobial compounds from a new strain Nonomuraea sp. NM94. J. Ind. Microbiol. Biotechnol. 34, 403–412. Wang, Y.Z., Ju, X.L., Zhou, Y.G., 2005. The variability of citrinin production in Monascus type cultures. Food Microbiol. 22, 145–148. Zhang, X.M., Guo, S.T., 2003. Isolation and identification of the glycopeptide from -conglycincin soy protein hydrolysate. Food Sci. 10, 26–29.
This work was supported by NSFC-20536040, 973-2007CB 707802 and TJ-05YFGZGX04500.
doi:10.1016/j.jbiotec.2008.07.069
References
I1-P-033
Chen, R., Bailey, J.E., 1994. Energetic effect of Vitreoscilla hemoglobin expression in Escherichia coli: an on-line 31P NMR and saturation transfer study. Biotechnol. Prog. 10, 360–364. Kallio, P.T., Kim, D.J., Tsai, P.S., Bailey, J.E., 1994. Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions. Eur. J. Biochem. 219, 201–208. Zamboni, N., 2003. Metabolic engineering of respiration for improved riboflavin production and elucidation of NADPH metabolism in Bacillus subtilis. Ph.D. Thesis. Switzerland.
Enhancing riboflavin production by genetic modification of purine pathway in Bacillus subtilis
doi:10.1016/j.jbiotec.2008.07.068
E-mail address: [email protected] (X. Zhao).
Shuobo Shi ∗ , Tao Chen, Xun Chen, Qiuli Wu, Yu Gan, Xueming Zhao Department of Biochemical Engineering, School of Chemical Engineering and Technology, Edinburgh-Tianjin Joint Centre for Systems Biology and Synthetic Biology, Tianjin University, Tianjin 300072, China
Riboflavin is an essential nutrient for humans and animals and it is an important food additive. By means of classical mutagenesis and gene manipulations, Bacillus subtilis has turned into an excellent riboflavin producer strain (Perkins et al., 1999; Van Loon et al., 1996). By further increasing riboflavin operon dosages or over-expression of the key riboflavin biosynthesis gene ribA in an
S35
I1-P-031
I1-P-032
Expression of Vitreoscilla hemoglobin enhances growth and production of riboflavin in recombinant Bacillus subtilis
Study on antibiotic substances metabolized by Monascus purpureus
Yunxia Duan 1 , Shuobo Shi 1 , Tao Chen 1 , Xun Chen 1 , Yi Gong 2 , Shengli Yang 2 , Xueming Zhao 1,∗
Zhao Shuxin ∗ , Zeng Luyan, Li Yi
1
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Edinburgh-Tianjin Joint Research Centre for Systems Biology and Synthetic Biology, Tianjin University, Tianjin 300072, China 2 Shanghai Research Center of Biotechnology, Academic Sinica, Shanghai 200233, China E-mail address: [email protected] (X. Zhao).
Riboflavin is a commercially important vitamin used for pharmaceuticals, feed additives, cosmetics, and food industry. The whole riboflavin biosynthesis is an energy consumption step from the sum riboflavin biosynthesis reaction (Zamboni, 2003). Vitreoscilla hemoglobin (VHb), which is produced by Vitreoscilla in hypoxic environments can enhance the ATP synthesis rate and microaerobic respiration efficiency (Chen and Bailey, 1994; Kallio et al., 1994). In order to further enhance riboflavin biosynthesis, vgb gene which encodes VHb was integrated into the chromosome of genetically engineered strain B. subtilis PY and its influence on physiological characters has been investigated. The apparent phenotype with more rapid specific growth rate, higher oxygen uptake rate and higher biomass was achieved under oxygen-limited conditions in batch cultivation. A metabolic flux distribution analysis discloses that VHb can also facilitate oxygen delivering, generate sufficient oxygen flux and direct interaction with the respiratory apparatus of the cell and thus relieve the overflow metabolism. Therefore, VHb-expression strains have a lower adenosine triphosphate (ATP) synthesis rate from substrate-level phosphorylation but a higher overall ATP production rate under microaerobic conditions. The increased higher oxygen uptake rate, as well as decreased by-products could explain the increased biomass and riboflavin production in VHb-expression strain. The expression of VHb also improved the biomass and yield of riboflavin compared with the control strain under fed-batch fermentation, and 28% higher biomass and about 20% improvement of the yield of riboflavin were obtained. Acknowledgement
Tianjin Key Laboratory of Industrial Microbiology, Tianjin University of Science & Technology, Tianjin 300457, China E-mail address: [email protected] (Z. Shuxin). Monascus has been used for thousands of years in China, after fermentation whose products (Red qu) contains many high value substances. Nowadays, the public has recognized the importance of Monascus products. The focus of this study is to explore the antibiotic substances except citrinin and pigments. First, we crushed the mycelium by ultrasonic crusher, then filtrated by vacuum pump and decolored by active carbon. Second, HPLC analyzes (Wang et al., 2005) the decolored sample. The result demonstrated that citrinin has reduced to 88–98% of its base level after decolored. The content of citrinin was reduced from 0.058 g/ml to 0.002 g/ml. Pigment analysis showed that after decolored, yellow and red pigments were reduced from 12.8 U/g and 17.1 U/g to 0.036 U/g and 0.016 U/g. From the research, we can confirm that citrinin and pigments have no impact on bacteriostatic effect of the decolored sample. Third, disc papers was used to ascertain bacteriostatic effect of decolored sample that has ability to inhibited Bacillus cereus and Staphylococcus aureus and has no effect on E. coli and Saccharmoyces cerevisiae. From this experiment, we can conclude that there was some other antibiotic substance in Red qu except citrinin and pigments. Forth, using dialysis bag technique dialyses the sample, then test different parts respectively. From the result, we can confirm that the substance with bacteriostatic effect was below 1 kDa. Qualitative analysis (Zhang and Guo, 2003; Boubekeur et al., 2007) showed that antibiotic substance contains the carbohydrate and amino acid, and the substance was identified as carbohydrate-peptide linkage by the thin-layer chromatography. References Boubekeur, Badji, Abdellah, Mostefaoui, Nasserdine, Sabaou, Ahmed, Lebrihi, 2007. Isolation and partial characterization of antimicrobial compounds from a new strain Nonomuraea sp. NM94. J. Ind. Microbiol. Biotechnol. 34, 403–412. Wang, Y.Z., Ju, X.L., Zhou, Y.G., 2005. The variability of citrinin production in Monascus type cultures. Food Microbiol. 22, 145–148. Zhang, X.M., Guo, S.T., 2003. Isolation and identification of the glycopeptide from -conglycincin soy protein hydrolysate. Food Sci. 10, 26–29.
This work was supported by NSFC-20536040, 973-2007CB 707802 and TJ-05YFGZGX04500.
doi:10.1016/j.jbiotec.2008.07.069
References
I1-P-033
Chen, R., Bailey, J.E., 1994. Energetic effect of Vitreoscilla hemoglobin expression in Escherichia coli: an on-line 31P NMR and saturation transfer study. Biotechnol. Prog. 10, 360–364. Kallio, P.T., Kim, D.J., Tsai, P.S., Bailey, J.E., 1994. Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions. Eur. J. Biochem. 219, 201–208. Zamboni, N., 2003. Metabolic engineering of respiration for improved riboflavin production and elucidation of NADPH metabolism in Bacillus subtilis. Ph.D. Thesis. Switzerland.
Enhancing riboflavin production by genetic modification of purine pathway in Bacillus subtilis
doi:10.1016/j.jbiotec.2008.07.068
E-mail address: [email protected] (X. Zhao).
Shuobo Shi ∗ , Tao Chen, Xun Chen, Qiuli Wu, Yu Gan, Xueming Zhao Department of Biochemical Engineering, School of Chemical Engineering and Technology, Edinburgh-Tianjin Joint Centre for Systems Biology and Synthetic Biology, Tianjin University, Tianjin 300072, China
Riboflavin is an essential nutrient for humans and animals and it is an important food additive. By means of classical mutagenesis and gene manipulations, Bacillus subtilis has turned into an excellent riboflavin producer strain (Perkins et al., 1999; Van Loon et al., 1996). By further increasing riboflavin operon dosages or over-expression of the key riboflavin biosynthesis gene ribA in an