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1.
Biotechnol Bioeng ; 112(6): 1132-45, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25546846

RESUMO

An unstructured macrokinetic model for heterologous production of Rhizopus oryzae lipase (ROL) by a Pichia pastoris PAOX1 based system was developed. Mean specific rates and state variables for various fed-batch cultures, under methanol limited and non-limited conditions were used for modeling. The most representative kinetic functions (viz., Monod, Haldane, Pirt and Luedeking-Piret) were used for the specific rates of cell growth (µ), substrate consumption (qS ) and product formation (qP ). The performance of two different process models was assessed via simulation tests, using an overall validation procedure. The best model describes the cellular growth by a non-monotonic substrate function, substrate uptake via Pirt's equation and product formation through a Luedeking-Piret equation. A comparative meta-analysis of heterologous protein production of various target proteins by Pichia pastoris under AOX1 promoter was conducted and a general strategy for improving protein production from process kinetics was developed as a key to bioprocess optimization.


Assuntos
Aldeído Oxidase/genética , Expressão Gênica , Lipase/biossíntese , Lipase/genética , Pichia/genética , Pichia/metabolismo , Regiões Promotoras Genéticas , Meios de Cultura/química , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Rhizopus/enzimologia
2.
Microb Cell Fact ; 13(1): 36, 2014 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-24606982

RESUMO

BACKGROUND: In Pichia pastoris bioprocess engineering, classic approaches for clone selection and bioprocess optimization at small/micro scale using the promoter of the alcohol oxidase 1 gene (PAOX1), induced by methanol, present low reproducibility leading to high time and resource consumption. RESULTS: An automated microfermentation platform (RoboLector) was successfully tested to overcome the chronic problems of clone selection and optimization of fed-batch strategies. Different clones from Mut+P. pastoris phenotype strains expressing heterologous Rhizopus oryzae lipase (ROL), including a subset also overexpressing the transcription factor HAC1, were tested to select the most promising clones.The RoboLector showed high performance for the selection and optimization of cultivation media with minimal cost and time. Syn6 medium was better than conventional YNB medium in terms of production of heterologous protein.The RoboLector microbioreactor was also tested for different fed-batch strategies with three clones producing different lipase levels. Two mixed substrates fed-batch strategies were evaluated. The first strategy was the enzymatic release of glucose from a soluble glucose polymer by a glucosidase, and methanol addition every 24 hours. The second strategy used glycerol as co-substrate jointly with methanol at two different feeding rates. The implementation of these simple fed-batch strategies increased the levels of lipolytic activity 80-fold compared to classical batch strategies used in clone selection. Thus, these strategies minimize the risk of errors in the clone selection and increase the detection level of the desired product.Finally, the performance of two fed-batch strategies was compared for lipase production between the RoboLector microbioreactor and 5 liter stirred tank bioreactor for three selected clones. In both scales, the same clone ranking was achieved. CONCLUSION: The RoboLector showed excellent performance in clone selection of P. pastoris Mut+ phenotype. The use of fed-batch strategies using mixed substrate feeds resulted in increased biomass and lipolytic activity. The automated processing of fed-batch strategies by the RoboLector considerably facilitates the operation of fermentation processes, while reducing error-prone clone selection by increasing product titers.The scale-up from microbioreactor to lab scale stirred tank bioreactor showed an excellent correlation, validating the use of microbioreactor as a powerful tool for evaluating fed-batch operational strategies.


Assuntos
Proteínas Fúngicas/metabolismo , Lipase/metabolismo , Pichia/metabolismo , Rhizopus/enzimologia , Técnicas de Cultura Celular por Lotes , Biomassa , Reatores Biológicos , Proteínas Fúngicas/genética , Vetores Genéticos/metabolismo , Glicerol/metabolismo , Lipase/genética , Metanol/metabolismo , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
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