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1.
PLoS One ; 15(8): e0226235, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32797046

RESUMO

Plant-derived fuels and chemicals from renewable biomass have significant potential to replace reliance on petroleum and improve global carbon balance. However, plant biomass contains significant fractions of oligosaccharides that are not usable natively by many industrial microorganisms, including Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis. Even after chemical or enzymatic hydrolysis, some carbohydrate remains as non-metabolizable oligosaccharides (e.g., cellobiose or longer cellulose-derived oligomers), thus reducing the efficiency of conversion to useful products. To begin to address this problem for Z. mobilis, we engineered a strain (Z. mobilis GH3) that expresses a glycosyl hydrolase (GH) with ß-glucosidase activity from a related α-proteobacterial species, Caulobacter crescentus, and subjected it to an adaptation in cellobiose medium. Growth on cellobiose was achieved after a prolonged lag phase in cellobiose medium that induced changes in gene expression and cell composition, including increased expression and extracellular release of GH. These changes were reversible upon growth in glucose-containing medium, meaning they did not result from genetic mutation but could be retained upon transfer of cells to fresh cellobiose medium. After adaptation to cellobiose, our GH-expressing strain was able to convert about 50% of cellobiose to glucose within 24 h and use it for growth and ethanol production. Alternatively, pre-growth of Z. mobilis GH3 in sucrose medium enabled immediate growth on cellobiose. Proteomic analysis of cellobiose- and sucrose-adapted strains revealed upregulation of secretion-, transport-, and outer membrane-related proteins, which may aid release or surface display of GHs, entry of cellobiose into the periplasm, or both. Our two key findings are that Z. mobilis can be reprogrammed to grow on cellobiose as a sole carbon source and that this reprogramming is related to a natural response of Z. mobilis to sucrose that promotes sucrase production.


Assuntos
Celobiose/metabolismo , Zymomonas/crescimento & desenvolvimento , Zymomonas/metabolismo , Adaptação Fisiológica/fisiologia , Biomassa , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Celulose/metabolismo , Expressão Gênica/genética , Glucose/metabolismo , Hidrolases/metabolismo , Proteômica , Sacarase/metabolismo , Sacarose/metabolismo , Zymomonas/genética , beta-Glucosidase/metabolismo
2.
Chembiochem ; 15(13): 1891-5, 2014 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-25099170

RESUMO

Multienzyme processes represent an important area of biocatalysis. Their efficiency can be enhanced by optimization of the stoichiometry of the biocatalysts. Here we present a workflow for maximizing the efficiency of a three-enzyme system catalyzing a five-step chemical conversion. Kinetic models of pathways with wild-type or engineered enzymes were built, and the enzyme stoichiometry of each pathway was optimized. Mathematical modeling and one-pot multienzyme experiments provided detailed insights into pathway dynamics, enabled the selection of a suitable engineered enzyme, and afforded high efficiency while minimizing biocatalyst loadings. Optimizing the stoichiometry in a pathway with an engineered enzyme reduced the total biocatalyst load by an impressive 56 %. Our new workflow represents a broadly applicable strategy for optimizing multienzyme processes.


Assuntos
Biocatálise , Enzimas/química , Algoritmos , Cinética , Modelos Químicos , Engenharia de Proteínas , Fluxo de Trabalho
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