RESUMO
BACKGROUND: Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. RESULTS: This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) - 1 , the lowest rate of 0.3 g (l h) - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in ß -glucosidase activity compared to batch processes using the same type and amount of carbon sources. CONCLUSION: We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology.
Assuntos
Celulase , Hypocreales , Trichoderma , Celulase/metabolismo , Lactose/metabolismo , Reprodutibilidade dos Testes , Biotecnologia , Trichoderma/metabolismoRESUMO
IMPORTANCE: Investigating fundamental aspects of metabolism is vital for advancing our understanding of the diverse biochemical capabilities and biotechnological applications of bacteria. The origin of the essential thymidylate kinase function in the model bacterium Pseudomonas putida KT2440, seemingly interrupted due to the presence of a large genomic island that disrupts the cognate gene, eluded a satisfactory explanation thus far. This is a first-case example of an essential metabolic function, likely acquired by horizontal gene transfer, which "landed" in a locus encoding the same activity. As such, foreign DNA encoding an essential dNMPK could immediately adjust to the recipient host-instead of long-term accommodation and adaptation. Understanding how these functions evolve is a major biological question, and the work presented here is a decisive step toward this direction. Furthermore, identifying essential and accessory genes facilitates removing those deemed irrelevant in industrial settings-yielding genome-reduced cell factories with enhanced properties and genetic stability.
Assuntos
Pseudomonas putida , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Ilhas Genômicas , BiotecnologiaRESUMO
Sweeteners improve the dietary properties of many foods. A candidate for a new natural sweetener is 5-ketofructose. In this study a fed-batch process for the production of 5-ketofructose was developed. A Gluconobacter oxydans strain overexpressing a fructose dehydrogenase from G. japonicus was used and the sensory properties of 5-ketofructose were analyzed. The compound showed an identical sweet taste quality as fructose and a similar intrinsic sweet threshold concentration of 16.4â¯mmol/L. The production of 5-ketofructose was characterized online by monitoring of the respiration activity in shake flasks. Pulsed and continuous fructose feeding was realized in 2â¯L stirred tank reactors and maximum fructose consumption rates were determined. 5-Ketofructose concentrations of up to 489â¯g/L, product yields up to 0.98â¯g5-KF/gfructose and space time yields up to 8.2â¯g/L/h were reached highlighting the potential of the presented process.
Assuntos
Frutose , Gluconobacter oxydans , Edulcorantes , Fermentação , Frutose/análogos & derivados , OxirredutasesRESUMO
The synthesis of amphiphilic polyaza crown ether monomers X (palmityl-substituted), Y (cholesteryl-substituted) and Z (dipalmityl-subtituted) and their incorporation into oligonucleotides are described. Their effects on thermal duplex stability were investigated by UV melting curve analysis. Thermal denaturation experiments showed remarkable stabilization of dsDNA by polyaza crown ether monomers when incorporated in opposite positions. The series of polyaza crown ether monomers (X, Y, and Z) with different lipophilicity showed a trend of increased stability of the corresponding dsDNA with increasing lipophilicity of the polyaza crown ether monomer.
Assuntos
Éteres de Coroa/química , DNA/química , Oligonucleotídeos/química , Éteres de Coroa/síntese química , Temperatura Alta , Desnaturação de Ácido Nucleico , Nucleosídeos/químicaRESUMO
A number of functionalized polyaza crown ether building blocks have been incorporated into DNA-conjugates as catalytic Cu(2+) binding sites. The effect of the DNA-conjugate catalyst on the stereochemical outcome of a Cu(2+)-catalyzed Diels-Alder reaction will be presented.
Assuntos
Cobre/química , Éteres de Coroa/química , DNA de Cadeia Simples/química , Oligonucleotídeos/química , Domínio Catalítico , EstereoisomerismoRESUMO
The synthesis of amphiphilic polyaza crown ether monomers X (benzyl-substituted), Y (palmityl-substituted) and Z (cholesteryl-substituted) and their incorporation into oligonucleotides are described. Their effects on thermal duplex stability were investigated by UV melting curve analysis in different alkaline metal buffer solutions. Thermal-denaturation experiments showed remarkable stabilization of dsDNA by polyaza crown ether monomers when incorporated in opposite positions. The series of polyaza crown ether monomers (X, Y and Z) with different overall lipophilicities showed a trend of increased stability of the corresponding dsDNA with increasing lipophilicity of the polyaza crown ether monomer. Multiple incorporations of benzyl-substituted polyaza crown ether monomer X as dangling ends on both sides of dsDNA resulted in strongly increased stability of the corresponding duplex.
Assuntos
Compostos Aza/química , Éteres de Coroa/química , DNA/química , DNA/síntese química , Ressonância Magnética Nuclear Biomolecular , Desnaturação de Ácido Nucleico , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Crown ether 4 as a receptor core for protonated primary amines such as amino acids has been synthesized and incorporated into oligodeoxynucleotides as dangling ends.
Assuntos
Compostos Aza/síntese química , Éteres de Coroa/síntese química , DNA/química , Oligodesoxirribonucleotídeos/síntese química , Amidas/química , Aminoácidos/química , Compostos Aza/química , Sítios de Ligação , Éteres de Coroa/química , Metais/química , Oligodesoxirribonucleotídeos/química , Ácidos Fosfóricos/químicaRESUMO
Synthesis of an asymmetrically substituted triaza crown ether, its incorporation into the 3'-end and 5'-end of ninemer oligonucleotides, and the influence of various alkanediamine ligands on duplex thermostabilities are reported.