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Improved riboflavin production using Ashbya gossypii mutant targeting for biorefinery of wasted activated bleaching earth
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[I.26] Improved riboflavin production using Ashbya gossypii mutant targeting for biorefinery of wasted activated bleaching earth Enoch Y. Park 1,2,∗ , Takashi Sgimoto 1 , Yoko Ito 2 , Lies Dwiarti 2 , Masashi Nariyama 2 1
Grad. Sch. Sci. & Technol., Shizuoka University, Japan Dept. of Appl. Biol. Chem., Shizuoka University, Japan Keywords: Riboflavin; Ashbya gossypii; Disparity mutagenesis; Biorefinery 2
At present, riboflavin production is speculated to be more than 5 000 tons/year, which is commercially produced by fermentation using filamentous plant-pathogenic ascomycete Ashbya gossypii. Using A. gossypii, we tried to produce riboflavin from oil waste, such as waste activated bleaching earth (waste ABE) (Park et al., 2004). A large amount of waste ABE is discarded by crude oil refining industries. The ABE has been used for adsorbing the dark color and odor of crude oil, which are caused by chromophoric chloroplast-related materials with different degrees of polymerization. During this oil refinery process, ABE adsorbs 40% of vegetable oil on a weight basis and is disposed as a waste material. Improvement of riboflavin production by Ashbya gossypii was done by disparity mutagenesis technology, producing riboflavin ten-fold the riboflavin that of the wild type strain. Proteomic analysis of improved strain showed the increased expression patterns of riboflavin biosynthetic pathway. DNA micro analysis also showed increased expression levels in purine- and riboflavin-synthetic pathways. On the other hand, enzyme expression levels in beta oxidation and glyoxylation pathway decreased compared to those of wild type strain. When 100 g l−1 rapeseed oil was used as carbon source in the culture of the improved strain, riboflavin concentration increased to 10-fold at seven days of culture. Riboflavin production yield was 0.18 g/g of consumed oil, which was also 10-fold higher than that of the wild type strain. The results show that the improved strain shows potential for improving riboflavin production for practical utilization using vegetable oil as the sole carbon source, and therefore will be contributed to biorefinery technology. Reference Park, Enoch Y., Kato, A., Ming, H., 2004. Riboflavin production using waste activated bleaching earth containing palm oil in the culture of Ashbya gossypii. J. Am. Oil Chem. Soc. 81 (1), 57–62.
doi:10.1016/j.jbiotec.2010.08.204 [I.27] Metabolic engineering of Lactococcus lactis for hyaluronic acid production: effect of co-expression of different gene-sets from has operon and culture conditions Shashi Bala Prasad ∗ , Guhan Jayaraman, K.B. Ramachandran Indian Institute of Technology Madras, India Keywords: Hyaluronic acid; Lactococcus lactis; Metabolic engineering; Has operon Hyaluronic acid (HA) production was metabolically engineered in Lactococcus lactis with three different gene combinations from has operon of pathogenic bacterium S.zooepidemicus. This study showed that the insertion of UDP-glucose pyrophosphorylase (hasC) in addition of hasA and hasB gene significantly improves HA yield. The SJR3 (with hasABC genes) produced more than two fold HA than L.lactisSJR2 (with only hasAB genes) while the
S79
other construct SJR6 with hasABD gene insert (with an additional UDP–N–acetyl glucosamine pyrophosphorylase gene) gave 79% higher HA production than the SJR3 strains in the static flask cultivations. This result indicates that significantly higher HA yield can be obtained by improving the precursor concentrations of the HA biosynthetic pathway. This result also indicates that the concentration of N-acetyl glucosamine in the metabolite pool has greater effect than the glucuronic acid pool on HA production. Since, the production of biomass and HA were limited in static flask cultures due to drastic pH drop because of accumulation of lactic acid, the effect of culture variables such as initial glucose concentration, pH, temperature, pyruvate and N-acetyl glucosamine were studied in batch bioreactor. The effect of initial glucose concentrations (range 20–50 g/l) had a significant effect on enhancing the HA yield. In case of SJR3, a maximum of 3.61 g/l of HA was obtained whereas with SJR6 about 4.5 g/l of HA produced. This is the maximum reported HA yield by any heterologous host till to date. As N-acetyl glucosamine constitute one of the repeating subunit of HA, effect of its addition in culture media was investigated. It was found that HA yield compared to control was enhanced by 58% by the addition of 2 g/l of N-acetyl glucosamine in the media. Addition of pyruvate which is hypothesized to temporarily inhibit the competing glycolytic pathway under feedback control increased the HA yield by 23%. The optimum pH and temperature found to be 7.5 and 3 ◦ C, respectively. doi:10.1016/j.jbiotec.2010.08.205 [I.28] Biosyntheis of bioactive seleno-compunds in Saccharomyces cerevisiae via metabolic and bioprocess engineering V. Mapelli 1,2,∗ , E. Kápolna 2 , P.R. Hillestrøm 2 , L.O. Dragsted 3 , E.H. Larsen 2 , L. Olsson 1 1
Chalmers University of Technology, Sweden Danish Technical University, Denmark 3 Copenhagen University, Denmark Keywords: Saccharomyces cerevisiae; Se-metabolites; LC–ICP–MS; LC–ESI–MS/MS 2
Selenium (Se) is an essential element for many organisms as it is present under the form of Se-cysteine in Se-proteins. The main sources of Se for animals are edible plants able to accumulate Se from the soil and store it under organic forms. Some of Se organic forms bioavailable for animals, such as Se-methyl-selenocysteine (SeMCys), have been proven to have cancer-preventing effects if regularly introduced into the diet. Since Se content in plants is highly susceptible to environmental factors, the intake of Se is often insufficient to result in beneficial effects. Therefore, the use of Seenriched yeast as food supplement is made available to avoid Se shortage. The yeast Saccharomyces cerevisiae does not require Se as essential element, but is able to metabolise and accumulate Se. This work shows a study of Se-metabolism in yeast that lead to the definition of a metabolic engineering strategy and to an optimized bioprocess to increase the levels of beneficial Se-compounds in yeast. After a preliminary study on Se uptake dynamics and Se-metabolite profile in yeast, a recombinant yeast strain was generated, by introducing heterologous genes from plants belonging to Brassicaceae and Fabaceae families. Due to the delicate balance between toxic and beneficial effects of Se, the cultivation process was carefully optimized in terms of medium composition and Se feeding. The established bioprocess allowed minimizing the toxicity of Se and redirecting Se fluxes towards the biosynthesis of SeMCys at the same time.
[I.26] Improved riboflavin production using Ashbya gossypii mutant targeting for biorefinery of wasted activated bleaching earth Enoch Y. Park 1,2,∗ , Takashi Sgimoto 1 , Yoko Ito 2 , Lies Dwiarti 2 , Masashi Nariyama 2 1
Grad. Sch. Sci. & Technol., Shizuoka University, Japan Dept. of Appl. Biol. Chem., Shizuoka University, Japan Keywords: Riboflavin; Ashbya gossypii; Disparity mutagenesis; Biorefinery 2
At present, riboflavin production is speculated to be more than 5 000 tons/year, which is commercially produced by fermentation using filamentous plant-pathogenic ascomycete Ashbya gossypii. Using A. gossypii, we tried to produce riboflavin from oil waste, such as waste activated bleaching earth (waste ABE) (Park et al., 2004). A large amount of waste ABE is discarded by crude oil refining industries. The ABE has been used for adsorbing the dark color and odor of crude oil, which are caused by chromophoric chloroplast-related materials with different degrees of polymerization. During this oil refinery process, ABE adsorbs 40% of vegetable oil on a weight basis and is disposed as a waste material. Improvement of riboflavin production by Ashbya gossypii was done by disparity mutagenesis technology, producing riboflavin ten-fold the riboflavin that of the wild type strain. Proteomic analysis of improved strain showed the increased expression patterns of riboflavin biosynthetic pathway. DNA micro analysis also showed increased expression levels in purine- and riboflavin-synthetic pathways. On the other hand, enzyme expression levels in beta oxidation and glyoxylation pathway decreased compared to those of wild type strain. When 100 g l−1 rapeseed oil was used as carbon source in the culture of the improved strain, riboflavin concentration increased to 10-fold at seven days of culture. Riboflavin production yield was 0.18 g/g of consumed oil, which was also 10-fold higher than that of the wild type strain. The results show that the improved strain shows potential for improving riboflavin production for practical utilization using vegetable oil as the sole carbon source, and therefore will be contributed to biorefinery technology. Reference Park, Enoch Y., Kato, A., Ming, H., 2004. Riboflavin production using waste activated bleaching earth containing palm oil in the culture of Ashbya gossypii. J. Am. Oil Chem. Soc. 81 (1), 57–62.
doi:10.1016/j.jbiotec.2010.08.204 [I.27] Metabolic engineering of Lactococcus lactis for hyaluronic acid production: effect of co-expression of different gene-sets from has operon and culture conditions Shashi Bala Prasad ∗ , Guhan Jayaraman, K.B. Ramachandran Indian Institute of Technology Madras, India Keywords: Hyaluronic acid; Lactococcus lactis; Metabolic engineering; Has operon Hyaluronic acid (HA) production was metabolically engineered in Lactococcus lactis with three different gene combinations from has operon of pathogenic bacterium S.zooepidemicus. This study showed that the insertion of UDP-glucose pyrophosphorylase (hasC) in addition of hasA and hasB gene significantly improves HA yield. The SJR3 (with hasABC genes) produced more than two fold HA than L.lactisSJR2 (with only hasAB genes) while the
S79
other construct SJR6 with hasABD gene insert (with an additional UDP–N–acetyl glucosamine pyrophosphorylase gene) gave 79% higher HA production than the SJR3 strains in the static flask cultivations. This result indicates that significantly higher HA yield can be obtained by improving the precursor concentrations of the HA biosynthetic pathway. This result also indicates that the concentration of N-acetyl glucosamine in the metabolite pool has greater effect than the glucuronic acid pool on HA production. Since, the production of biomass and HA were limited in static flask cultures due to drastic pH drop because of accumulation of lactic acid, the effect of culture variables such as initial glucose concentration, pH, temperature, pyruvate and N-acetyl glucosamine were studied in batch bioreactor. The effect of initial glucose concentrations (range 20–50 g/l) had a significant effect on enhancing the HA yield. In case of SJR3, a maximum of 3.61 g/l of HA was obtained whereas with SJR6 about 4.5 g/l of HA produced. This is the maximum reported HA yield by any heterologous host till to date. As N-acetyl glucosamine constitute one of the repeating subunit of HA, effect of its addition in culture media was investigated. It was found that HA yield compared to control was enhanced by 58% by the addition of 2 g/l of N-acetyl glucosamine in the media. Addition of pyruvate which is hypothesized to temporarily inhibit the competing glycolytic pathway under feedback control increased the HA yield by 23%. The optimum pH and temperature found to be 7.5 and 3 ◦ C, respectively. doi:10.1016/j.jbiotec.2010.08.205 [I.28] Biosyntheis of bioactive seleno-compunds in Saccharomyces cerevisiae via metabolic and bioprocess engineering V. Mapelli 1,2,∗ , E. Kápolna 2 , P.R. Hillestrøm 2 , L.O. Dragsted 3 , E.H. Larsen 2 , L. Olsson 1 1
Chalmers University of Technology, Sweden Danish Technical University, Denmark 3 Copenhagen University, Denmark Keywords: Saccharomyces cerevisiae; Se-metabolites; LC–ICP–MS; LC–ESI–MS/MS 2
Selenium (Se) is an essential element for many organisms as it is present under the form of Se-cysteine in Se-proteins. The main sources of Se for animals are edible plants able to accumulate Se from the soil and store it under organic forms. Some of Se organic forms bioavailable for animals, such as Se-methyl-selenocysteine (SeMCys), have been proven to have cancer-preventing effects if regularly introduced into the diet. Since Se content in plants is highly susceptible to environmental factors, the intake of Se is often insufficient to result in beneficial effects. Therefore, the use of Seenriched yeast as food supplement is made available to avoid Se shortage. The yeast Saccharomyces cerevisiae does not require Se as essential element, but is able to metabolise and accumulate Se. This work shows a study of Se-metabolism in yeast that lead to the definition of a metabolic engineering strategy and to an optimized bioprocess to increase the levels of beneficial Se-compounds in yeast. After a preliminary study on Se uptake dynamics and Se-metabolite profile in yeast, a recombinant yeast strain was generated, by introducing heterologous genes from plants belonging to Brassicaceae and Fabaceae families. Due to the delicate balance between toxic and beneficial effects of Se, the cultivation process was carefully optimized in terms of medium composition and Se feeding. The established bioprocess allowed minimizing the toxicity of Se and redirecting Se fluxes towards the biosynthesis of SeMCys at the same time.