RESUMO
AIM: Aspergillus terreus produces itaconic acid at low pH but lovastatin and other secondary metabolites at higher pH in the fermentation. The utilization of glucose as a carbon substrate was investigated for secondary metabolite production by A. terreus. METHODS AND RESULTS: With a starting pH of 6.5, glucose was rapidly metabolized to gluconic acid by the wild-type strain and by transformants harbouring Aspergillus niger genes encoding 6-phosphofructo-1-kinases with superior kinetic and regulatory properties for bioproduction of metabolites from glucose. On exhaustion of the glucose in batch fermentations, the accumulated gluconic acid was utilized as a carbon source. CONCLUSIONS: A novel pathway of glucose catabolism was demonstrated in A. terreus, a species whose wild type is, without any strain development, capable of producing gluconic acid at high molar conversion efficiency (up to 0.7 mol mol(-1) glucose consumed). SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus terreus is a potential novel producer organism for gluconic acid, a compound with many uses as a bulk chemical. With a new knowledge of glucose catabolism by A. terreus, fermentation strategies for secondary metabolite production can be devised with glucose feeding using feedback regulation by pH.
Assuntos
Aspergillus/metabolismo , Gluconatos/metabolismo , Biotecnologia/métodos , Carbono/metabolismo , Meios de Cultura/química , Fermentação , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Fosfofrutoquinase-1/genética , Fosfofrutoquinase-1/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Cell lines derived from human squamous cell (EPCL), large cell (LCLC), and small cell lung cancer (SCLC) lines were investigated for the expression of cathepsin B (Cat B) and cysteine proteinase inhibitors (CPIs). The EPLC and LCLC lines expressed 5- to 50-fold more Cat B activity and contained more mature Cat B of M(r) 27-29 kDa (> 2.5 microg/mg total protein) than the SCLC lines (< 1.0 microg/mg total protein). The LPLC lines also secreted the highest amounts of Cat B precursor of M(r) about 46 kDa. Inhibitory activities against Cat B and papain were associated with high molecular mass (HMM) and low molecular mass (LMM) inhibitory proteins, both in cell extracts and in media. About 75% of the inhibitory activity was associated with HMM inhibitors, the majority of which were kininogens (M(r) > or = 67 kDa). The LMM inhibitors of M(r) 10-15 kDa were cystatin C and stefins A and B, which were quantitated by ELISA: stefins A and B were present in cell extracts and medium in similar concentrations (5-200 ng/10(6) cells), while 80-99% of the cystatin C was released in the medium (10-195 ng/10(6) cells). Phorbol ester (PMA), which induces protein-kinase C mediated signal transduction and enhances cellular differentiation in many non-small cell lung cancer (NSCLC) cell lines, increased intracellular Cat B activity and Cat B protein as well as its secretion in some cell lines but not in others, regardless of their histological type. PMA significantly (P < 0.049) decreased intracellular stefin A concentrations in two EPLC lines and non-significantly in two LCLC lines. PMA decreased secretion of stefin A in all EPLC lines, but not in LCLC lines, while IGF-I significantly increased stefin B secretion in both SCLC lines. These data showed that lung tumor cells produce both cysteine proteinases and cystatins. As the antagonistic molecules are regulated differently in histologically different types of lung tumor cells, it is possible that an imbalance between the proteinases and their specific inhibitors plays a role in progression of certain types of lung tumors in vivo.
Assuntos
Catepsina B/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Neoplasias Pulmonares/metabolismo , Carcinógenos/farmacologia , Carcinoma de Células Grandes/tratamento farmacológico , Carcinoma de Células Grandes/metabolismo , Carcinoma de Células Grandes/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Cistatina A , Cistatina B , Cistatina C , Cistatinas/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Focalização Isoelétrica , Isoenzimas , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Peso Molecular , Proteína Quinase C/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The change from pentose phosphate pathway to glycolysis plays a significant role in the physiology of Aspergillus niger during the induction of citric acid accumulation. Evidence is shown for the importance of 6-phosphofructo-1-kinase in this process since it is activated by phosphorylation. By incubating a purified active form of the enzyme together with commercially available alkaline phosphatase, 6-phosphofructo-1-kinase activity was lost after a certain time suggesting that the enzyme was dephosphorylated. Inactive 6-phosphofructo-1-kinase could be isolated from the cells in the early stage of growth in a high citric acid yielding medium. The enzyme was "in vitro" activated by isolated protein kinase in the presence of cAMP, ATP and Mg2+ ions. Additional evidence for covalent phosphorylation of inactive 6-phosphofructo-1-kinase was obtained by incubating both enzymes together with labelled [gamma-32P]ATP. The activating enzyme was partially purified from A. niger mycelium.
Assuntos
Aspergillus niger/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosfofrutoquinase-1/metabolismo , Fosfatase Alcalina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/isolamento & purificação , Ativação Enzimática , Fosfofrutoquinase-1/isolamento & purificação , FosforilaçãoRESUMO
The transcriptional regulation of the pkaC gene, encoding the catalytic subunit of cAMP-dependent protein kinase from Aspergillus niger, was analysed under different environmental conditions. Quantitative determination of pkaC transcript showed a significant decrease in concentration of specific mRNA immediately after a temperature, hypoosmotic and hyperosmotic shock followed by stimulated synthesis. The amount of pkaC mRNA as well as PKA enzymatic activity steadily decreased during the initial phase of growth in 15% sucrose medium while a slight increase was observed at the time of a change in morphology from bulbous cells to filamentous growth. Transcriptional alternation might be mediated by multiple putative stress elements in the promoter region of pkaC gene.