Abstract:The use of the filamentous fungus, Ashbya gossypii, to improve riboflavin production at an industrial scale is described in this paper. A riboflavin overproducing strain was isolated by ultraviolet irradiation. Ten minutes after spore suspensions of A. gossypii were irradiated by ultraviolet light, a survival rate of 5.5% spores was observed, with 10% of the surviving spores giving rise to riboflavin-overproducing mutants. At this time point, a stable mutant of the wild strain was isolated. Riboflavin producti… Show more
“…The medium pH quickly dropped from pH 6.0 on the first day to pH 2.0 at the end of incubation, which had a negative effect on riboflavin production. At pH 2.0 the riboflavin production starts to decrease until has become negligible or absent after 72 h of the incubation time 38 . Figure 4 Growth and riboflavin production of G.oxydans FBFS97 in fermentation media, the results represent mean values for three shake flasks.…”
Section: Resultsmentioning
confidence: 98%
“…The medium pH quickly dropped from pH 6.0 on the first day to pH 2.0 at the end of incubation, which had a negative effect on riboflavin production. At pH 2.0 the riboflavin production starts to decrease until has become negligible or absent after 72 h of the incubation time 38 .…”
Section: Genome Features and Identification Of Riboflavin In The G Omentioning
A novel bacterial strain of acetic acid bacteria capable of producing riboflavin was isolated from the soil sample collected in Wuhan, China. The isolated strain was identified as Gluconobacter oxydans FBFS97 based on several phenotype characteristics, biochemicals tests, and 16S rRNA gene sequence conducted. Furthermore, the complete genome sequencing of the isolated strain has showed that it contains a complete operon for the biosynthesis of riboflavin. In order to obtain the maximum concentration of riboflavin production, Gluconobacter oxydans FBFS97 was optimized in shake flask cultures through response surface methodology employing Plackett-Burman design (PBD), and Central composite design (CCD). The results of the pre-experiments displayed that fructose and tryptone were found to be the most suitable sources of carbon and nitrogen for riboflavin production. Then, PBD was conducted for initial screening of eleven minerals (FeSO 4 , FeCl 3 , KH 2 po 4 , K 2 HPO 4 , MgSO 4 , ZnSO 4 , NaCl, CaCl 2 , KCl, ZnCl 2 , and AlCl 3 .6H 2 O) for their significances on riboflavin production by Gluconobacter oxydans strain FBFS97. The most significant variables affecting on riboflavin production are K 2 HPO 4 and cacl 2 , the interaction affects and levels of these variables were optimized by CCD. After optimization of the medium compositions for riboflavin production were determined as follows: fructose 25 g/L, tryptone 12.5 g/L, K 2 HPO 4 9 g/L, and CaCl 2 0.06 g/L with maximum riboflavin production 23.24 mg/L. Riboflavin or the so-called vitamin B2 is a water-soluble vitamin that belongs to the B vitamins complex group, a vital component of the energy metabolism, and a bioactive molecule that has an important role in various cellular functions 1,2. In addition, to its role as the precursor of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the major products of riboflavin that act a fundamental role in metabolism, acting as cofactors for a wide variety of enzymes intermediating many redox reactions in the cell 3,4. In contrast, to many microorganisms and plants, humans and animals cannot synthesize this vitamin, and thus need to supply their diet with external riboflavin to meet their nutritional requirements 5. Riboflavin is used on a large scale as food and feed additives, food colorant, and pharmaceutical preparations. The commercial production of this vitamin can be accomplished by chemical synthesis or biological synthesis, yet in recent times the chemical synthesis has totally replaced to the microbial fermentation because of its cost-effectiveness, reduction in waste and energy requirements, and the use of renewable resources 6. At present, several species of bacteria and fungi are harnessed
“…The medium pH quickly dropped from pH 6.0 on the first day to pH 2.0 at the end of incubation, which had a negative effect on riboflavin production. At pH 2.0 the riboflavin production starts to decrease until has become negligible or absent after 72 h of the incubation time 38 . Figure 4 Growth and riboflavin production of G.oxydans FBFS97 in fermentation media, the results represent mean values for three shake flasks.…”
Section: Resultsmentioning
confidence: 98%
“…The medium pH quickly dropped from pH 6.0 on the first day to pH 2.0 at the end of incubation, which had a negative effect on riboflavin production. At pH 2.0 the riboflavin production starts to decrease until has become negligible or absent after 72 h of the incubation time 38 .…”
Section: Genome Features and Identification Of Riboflavin In The G Omentioning
A novel bacterial strain of acetic acid bacteria capable of producing riboflavin was isolated from the soil sample collected in Wuhan, China. The isolated strain was identified as Gluconobacter oxydans FBFS97 based on several phenotype characteristics, biochemicals tests, and 16S rRNA gene sequence conducted. Furthermore, the complete genome sequencing of the isolated strain has showed that it contains a complete operon for the biosynthesis of riboflavin. In order to obtain the maximum concentration of riboflavin production, Gluconobacter oxydans FBFS97 was optimized in shake flask cultures through response surface methodology employing Plackett-Burman design (PBD), and Central composite design (CCD). The results of the pre-experiments displayed that fructose and tryptone were found to be the most suitable sources of carbon and nitrogen for riboflavin production. Then, PBD was conducted for initial screening of eleven minerals (FeSO 4 , FeCl 3 , KH 2 po 4 , K 2 HPO 4 , MgSO 4 , ZnSO 4 , NaCl, CaCl 2 , KCl, ZnCl 2 , and AlCl 3 .6H 2 O) for their significances on riboflavin production by Gluconobacter oxydans strain FBFS97. The most significant variables affecting on riboflavin production are K 2 HPO 4 and cacl 2 , the interaction affects and levels of these variables were optimized by CCD. After optimization of the medium compositions for riboflavin production were determined as follows: fructose 25 g/L, tryptone 12.5 g/L, K 2 HPO 4 9 g/L, and CaCl 2 0.06 g/L with maximum riboflavin production 23.24 mg/L. Riboflavin or the so-called vitamin B2 is a water-soluble vitamin that belongs to the B vitamins complex group, a vital component of the energy metabolism, and a bioactive molecule that has an important role in various cellular functions 1,2. In addition, to its role as the precursor of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the major products of riboflavin that act a fundamental role in metabolism, acting as cofactors for a wide variety of enzymes intermediating many redox reactions in the cell 3,4. In contrast, to many microorganisms and plants, humans and animals cannot synthesize this vitamin, and thus need to supply their diet with external riboflavin to meet their nutritional requirements 5. Riboflavin is used on a large scale as food and feed additives, food colorant, and pharmaceutical preparations. The commercial production of this vitamin can be accomplished by chemical synthesis or biological synthesis, yet in recent times the chemical synthesis has totally replaced to the microbial fermentation because of its cost-effectiveness, reduction in waste and energy requirements, and the use of renewable resources 6. At present, several species of bacteria and fungi are harnessed
“…The production levels of these bioproducts by other microorganisms are also presented for comparison. likely to be contaminated or degraded by native proteins; 15,16 (d) high genetic tractability, with a rich molecular toolbox available for its manipulation; 2,25 (e) remarkable genomic similarities with the budding yeast S. cerevisiae, which facilitates the transfer of accumulated know-how on this model yeast genetics and cell biology to A. gossypii; 2 (f) ability to grow in cheap waste-derived substrates to high cell densities; [26][27] (g) and demonstrated suitability for use in large-scale industrial fermentation processes. 1,3 While the first heterologous proteins secreted by A. gossypii (endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from Trichoderma reesei) were produced at very low yields, 16 latter on it was found that the expression vector and promoter (from ScPGK1) initially used were inefficient in promoting gene overexpression in A. gossypii.…”
Section: Recombinant Protein Productionmentioning
confidence: 99%
“…10 On the other hand, one important way to lower production costs is by using inexpensive carbon sources and nutrients, preferably from widely available industrial residues. In this sense, the known ability of A. gossypii to grow in some industrial by-products and low-cost oils 2,26,27 may be easily explored. Additionally, the expansion of the carbon source range that A. gossypii can utilize by metabolic engineering (e.g., xylose utilization) may open new perspectives in terms of the raw substrates that may be used in these processes (such as lignocellulosic biomass).…”
Section: Optimization Of Production Conditionsmentioning
The filamentous fungus Ashbya gossypii has long been considered a paradigm of the White Biotechnology in what concerns riboflavin production. Its industrial relevance led to the development of a significant molecular and in silico modeling toolbox for its manipulation. This, together with the increasing knowledge of its genome and metabolism has helped designing effective metabolic engineering strategies for optimizing riboflavin production, but also for developing new A. gossypii strains for novel biotechnological applications, such as production of recombinant proteins, single cell oils (SCOs), and flavour compounds. With the recent availability of its genome-scale metabolic model, the exploration of the full biotechnological potential of A. gossypii is now in the spotlight. Here, we will discuss some of the challenges that these emerging A. gossypii applications still need to overcome to become economically attractive and will present future perspectives for these and other possible biotechnological applications for A. gossypii.
“…Dentre os fungos,Cheng et al (2011), estudaram a produção de riboflavina por Eremothecium ashbyii, avaliando os efeitos de diferentes fontes de carbono alcançando uma produção máxima de 3107,59 mg/L de riboflavina. Já Suzuki, Fleuri e Macedo (2012) avaliaram a produção de riboflavina por Candida sp, estudando os efeitos de diferentes fontes de carbono e nitrogênio, resultando em uma produção de 105,7 mg/mL de riboflavina Wei et al (2012). estudaram o isolamento de Ashbya gossypii com ultravioleta, gerando uma linhagem mutante capaz de produzir 8,12 g/L de riboflavina, com potencial para aplicação na produção em escala industrial dessa vitamina.A produção microbiológica de biotina foi estudada por El-Refai et al (2010) que demonstraram a eficiência do delineamento experimental multifatorial na produção de biotina por Rhizopus nigricans.…”
Assinatura do orientador (a) CAMPINAS 2014 iv v BANCA EXAMINADORA PROF.ª DR.ª GABRIELA ALVES MACEDO ORIENTADORA PROF.ª DR.ª DERLENE ATTILI DE ANGELIS CPQBA/UNESP (MEMBRO TITULAR) PROF.ª DR.ª MARTA CRISTINA TEIXEIRA DUARTE CPQBA/UNICAMP (MEMBRO TITULAR) PROF.ª DR.ª JULIANA AZEVEDO LIMA PALLONE FEA/UNICAMP (MEMBRO SUPLENTE) PROF. DR. REINALDO GASPAR BASTOS UFSCar/ARARAS (MEMBRO SUPLENTE) vi RESUMO A bioprospecção de micro-organismos representa um grande potencial tecnológico para produção de biocompostos de interesse comercial. Dentre estes compostos estão as enzimas lipolíticas, como as lipases (triacilglicerol acil-hidrolases EC 3.1.1.3) que apresentam um enorme potencial para aplicações biotecnológicas. Outro tipo de biocomposto de relevante interesse comercial são as vitaminas do complexo B, as quais possuem função essencial na atividade de enzimas que regulam reações do metabolismo. Desta forma, o objetivo desse trabalho foi isolar e selecionar linhagens de leveduras do solo de diferentes biomas do Estado de São Paulo com potencial para produção de lipase e de vitaminas do complexo B (biotina e riboflavina). Para tanto,foram isoladas 132 leveduras de amostras de solos provenientes da Mata Atlântica e da região de transição (Mata Atlântica e Cerrado). Essas linhagens foram depositadas na coleção de micro-organismos do laboratório de Bioquímica de Alimentos da FEA-UNICAMP, que já contava com 300 leveduras provenientes da região de Cerrado. As 432 linhagens presentes na coleção foram então avaliadas quanto ao potencial para a produção de lipase extracelular através de seleção em meio sólido diferencial e cultivo em meio líquido, sendo a atividade de lipase determinada no sobrenadante por titulometria de neutralização. O potencial para a produção de biotina e riboflavina extracelular foi avaliada por meio de seleção em meio de cultivo líquido, sendo as concentrações das vitaminas determinadas no sobrenadante por espectrofotometria e fluorimetria. Desta forma 33 leveduras apresentaram potencial para produção de lipase alcançando valores de atividade enzimática que variaram de 6,51 U/mL a 21,44 U/mL. Em relação à produção das vitaminas do complexo B, 38 linhagens apresentaram concentrações de biotina no sobrenadante do cultivo que variaram de 0,28 µg/mL a 18,61 µg/mL e 64 linhagens apresentaram concentrações de riboflavina que variaram de 0,03 µg/mL a 0,77 µg/mL. A linhagem RP.C153 apresentou potencial para produção de lipase e riboflavina, enquanto a linhagem RP.J1308 para produção de lipase e biotina. A linhagem RP.C153 foi classificada como Pichia caribbica e a linhagem RP.J1308 como Candida oleophila.
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