- Email: [email protected]
Enhanced production of itaconic acid by transformed fungal cells of Aspergillus terreus harboring Vitreoscilla Hemoglobin Gene
Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S114–S134 FM-P14 Enhanced production of itaconic acid by transformed fungal cells of Aspergillus terreus harboring Vitreoscilla Hemoglobin Gene Woo-Shik Shin,1 Yoon-Hee Kim,1 Yong-Seob Jeong,2 Chulhwan Park,3 Sangyong Kim,4 Dohoon Lee,4 Jongdae Lee,4 and Gie-Taek Chun1 Kangwon National University, Chunchon, Kangwon-do, Republic of Korea 1 Chonbuk National University, Jeonju, Jeonbuk, Republic of Korea 2 Kwangwoon University, Seoul, Republic of Korea 3 and Korea Institute of Industrial Technology, Cheonan, Chungcheongnam-do, Republic of Korea 4 Itaconic acid utilized in the manufacture of various synthetic resins is a dicarboxylic acid biosynthesized by fungal cells of Aspergillus terreus in a branch of the TCA cycle via decarboxylation of cis-aconitate. It is well known that highly branched filamentous morphology results in high viscosities of culture broth in fungal cell fermentations, leading to significant reduction in mass and oxygen transfer capacity (1). In this study, for more facilitated utilization of dissolved oxygen by the high-yielding mutants, an efficient expression vector with Vitreoscilla hemoglobin(VHb) gene plus gpdA promoter was constructed and then introduced into the high-yielding producers (2). It was found through Southern blot analysis that 8 copies of VHb gene were integrated into the transformants' chromosomes. Notably, a resulting transformant harboring the wild VHb gene showed approximately 40% higher productivity of itaconic acid than the parallel nontransformed strains in the shake flask fermentations performed under the identical fermentation conditions. Furthermore, production stability of most of the strains further screened from the transformants was greatly enhanced, exhibiting sharp contrast to the results obtained from the corresponding nontransformants. Based on these consequences, it was concluded that optimal supply of oxygen was prerequisite for the enhanced biosynthesis of itaconic acid as well as production stability of the high-yielding producers.
S123
lene succinic acid) is a dicarboxylic acid utilized in the manufacturing of various synthetic resins. In this study, miniaturized fermentation methods were developed for rapid and large screening of overproducing mutants of itaconic acid, using both 12-well and 24-well microrplates in both growth and production cultures (1). It was found that for enhanced production of itaconic acid, morphology of the producers should be induced into the highly filamentous forms in the growth cultures. Under these culture conditions, loose-pelleted morphology of optimum sizes (less than 0.5 mm in diameter) were successfully induced in the microplate production-cultures, which turned out to be prerequisite for the active biosynthesis of itaconic acid. In addition, in the production fermentation stage, maintenance of high-level of dissolved oxygen was observed to be very important for higher production of itaconic acid. Notably, it was observed that pH of the fermentation broth should be maintained at very low level of around 2.0 during the later stage of production cultures, demonstrating that the productivity was considerably dependent upon the initial pH of the production medium (2). The effects of various surfactants on itaconic acid production were also investigated, since the higher productivity of itaconic acid by the high-yielding mutants was considered to be possibly due to the special roles of membrane permeability of the producers. References 1. Rowlands, R. T.: Industrial strain improvement: mutagenesis and random screening procedures, Enzyme Microbiol., 6, 99-104 (1984). 2. Riscaldati, E., Moresi, M., Federici, F., and Petruccioli, M.: Effect of pH and stirring rate on itaconate production by Aspergillus terreus, J. Biotechnol., 83, 219-230 (2000).
doi:10.1016/j.jbiosc.2009.08.360
References
FM-P16
1. Lin, Y. H., Li, Y. F., Huang, M. C., and Tsai Y. C.: Intracellular expression of Vitreoscilla hemoglobin in Aspergillus terreus to alleviate the effect of a short break in aeration during culture, Biotechnol. Lett., 26, 1067-1072 (2004).
A Novel Lipid Detection for the Fungus, Acremonium chrysogenum M35
2. Zhang, L., Li, Y., Wang, Z., Xia, Y., Chen, W., and Tang, K.: Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering, Biotechnol. Adv., 25, 123-136 (2007).
Hyun Yong Shin, and Seung Wook Kim
doi:10.1016/j.jbiosc.2009.08.359
Various plant oils and fatty acids have been used to reduce the catabolic repression of cell metabolism during the production of antibiotics and secondary metabolites (1, 2). Oils and fatty acids are rapidly absorbed through the membrane of fungi. However, no rapid analytical methods to evaluate accumulated oils and fatty acids in fungi have been developed to date. In this study, a novel method in which oil red O is used for the identification of intracellular lipid deposition in Acremonium chrysogenum M35 was developed. A. chrysogenum M35 were immersed in working solution of prewarmed (70 °C) oil red O solution and then heat treated at 70 °C for 60 min. When the sample was heat treated at 70 °C using 3–4% oil red O the lipid contents was well observed in the cell. Lipid droplets in A. chrysogenum were then detected without using cell section steps. The storage of lipids that had been converted from rice oil or fatty acid was then investigated in A. chrysogenum M35. Lipid droplets in A. chrysogenum M35 were successfully stained in the rice oil and fatty acid supplemented media. Through the use of modified oil red O analysis, a better understanding of the effect of carbon source supplementation on cephalosporin C (CPC) production and lipid accumulation in A. chrysogenum M35 is provided.
FM-P15 Large and rapid screening of high yielding Mutants of Itaconic Acid Through Optimized Miniaturized Culture system, and Investigation of Culture Conditions Woo-Shik Shin,1 Yoon-Hee Kim,1 Yong-Seob Jeong,2 Chulhwan Park,3 Sangyong Kim,4 Dohoon Lee,4 Jongdae Lee,4 and Gie-Taek Chun1 Kangwon National University, Chunchon, Kangwon-do, Republic of Korea 1 Chonbuk National University, Jeonju, Jeonbuk, Republic of Korea 2 Kwangwoon University, Seoul, Republic of Korea 3 and Korea Institute of Industrial Technology, Cheonan, Chungcheongnam-do, Republic of Korea 4 Produced by fungal cells of Aspergillus terreus in a branch of the TCA cycle via decarboxylation of cis-aconitate, itaconic acid (methy-
Korea University, Seoul, Republic of Korea
S123
lene succinic acid) is a dicarboxylic acid utilized in the manufacturing of various synthetic resins. In this study, miniaturized fermentation methods were developed for rapid and large screening of overproducing mutants of itaconic acid, using both 12-well and 24-well microrplates in both growth and production cultures (1). It was found that for enhanced production of itaconic acid, morphology of the producers should be induced into the highly filamentous forms in the growth cultures. Under these culture conditions, loose-pelleted morphology of optimum sizes (less than 0.5 mm in diameter) were successfully induced in the microplate production-cultures, which turned out to be prerequisite for the active biosynthesis of itaconic acid. In addition, in the production fermentation stage, maintenance of high-level of dissolved oxygen was observed to be very important for higher production of itaconic acid. Notably, it was observed that pH of the fermentation broth should be maintained at very low level of around 2.0 during the later stage of production cultures, demonstrating that the productivity was considerably dependent upon the initial pH of the production medium (2). The effects of various surfactants on itaconic acid production were also investigated, since the higher productivity of itaconic acid by the high-yielding mutants was considered to be possibly due to the special roles of membrane permeability of the producers. References 1. Rowlands, R. T.: Industrial strain improvement: mutagenesis and random screening procedures, Enzyme Microbiol., 6, 99-104 (1984). 2. Riscaldati, E., Moresi, M., Federici, F., and Petruccioli, M.: Effect of pH and stirring rate on itaconate production by Aspergillus terreus, J. Biotechnol., 83, 219-230 (2000).
doi:10.1016/j.jbiosc.2009.08.360
References
FM-P16
1. Lin, Y. H., Li, Y. F., Huang, M. C., and Tsai Y. C.: Intracellular expression of Vitreoscilla hemoglobin in Aspergillus terreus to alleviate the effect of a short break in aeration during culture, Biotechnol. Lett., 26, 1067-1072 (2004).
A Novel Lipid Detection for the Fungus, Acremonium chrysogenum M35
2. Zhang, L., Li, Y., Wang, Z., Xia, Y., Chen, W., and Tang, K.: Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering, Biotechnol. Adv., 25, 123-136 (2007).
Hyun Yong Shin, and Seung Wook Kim
doi:10.1016/j.jbiosc.2009.08.359
Various plant oils and fatty acids have been used to reduce the catabolic repression of cell metabolism during the production of antibiotics and secondary metabolites (1, 2). Oils and fatty acids are rapidly absorbed through the membrane of fungi. However, no rapid analytical methods to evaluate accumulated oils and fatty acids in fungi have been developed to date. In this study, a novel method in which oil red O is used for the identification of intracellular lipid deposition in Acremonium chrysogenum M35 was developed. A. chrysogenum M35 were immersed in working solution of prewarmed (70 °C) oil red O solution and then heat treated at 70 °C for 60 min. When the sample was heat treated at 70 °C using 3–4% oil red O the lipid contents was well observed in the cell. Lipid droplets in A. chrysogenum were then detected without using cell section steps. The storage of lipids that had been converted from rice oil or fatty acid was then investigated in A. chrysogenum M35. Lipid droplets in A. chrysogenum M35 were successfully stained in the rice oil and fatty acid supplemented media. Through the use of modified oil red O analysis, a better understanding of the effect of carbon source supplementation on cephalosporin C (CPC) production and lipid accumulation in A. chrysogenum M35 is provided.
FM-P15 Large and rapid screening of high yielding Mutants of Itaconic Acid Through Optimized Miniaturized Culture system, and Investigation of Culture Conditions Woo-Shik Shin,1 Yoon-Hee Kim,1 Yong-Seob Jeong,2 Chulhwan Park,3 Sangyong Kim,4 Dohoon Lee,4 Jongdae Lee,4 and Gie-Taek Chun1 Kangwon National University, Chunchon, Kangwon-do, Republic of Korea 1 Chonbuk National University, Jeonju, Jeonbuk, Republic of Korea 2 Kwangwoon University, Seoul, Republic of Korea 3 and Korea Institute of Industrial Technology, Cheonan, Chungcheongnam-do, Republic of Korea 4 Produced by fungal cells of Aspergillus terreus in a branch of the TCA cycle via decarboxylation of cis-aconitate, itaconic acid (methy-
Korea University, Seoul, Republic of Korea