RESUMEN
Fumarate reductase is an enzyme involved in maintaining redox balance through regeneration of reduced cofactors during oxygen deficiency conditions. This work reports the identification and characterization of the gene and its promoter and terminator elements that encodes cytosolic fumarate reductase enzyme in the nonconventional yeast, Arxula adeninivorans. The gene harbours an ORF of 1446 bp, encoding a 482-amino acid protein. The deduced amino acid sequence is similar to those of fumarate reductases from other yeast and fungi, such as the two fumarate reductases of Saccharomyces cerevisiae, Frd1p (44%) and Osm1p (41%). This enzyme is located in the cytosol and has a pH optimum of ca. 7.5 and a Michaelis constant (K(M)) of 2.9 mM with fumarate as the substrate. Expression of AFRD1 is regulated by the cultivation conditions. A shift from NaCl-free to NaCl-supplemented media and aerobic to hypoxic growth conditions leads to reduced AFRD1 transcription levels, but not to alteration in the concentration of Afrd1p. The functional analyses of Afrd1p were performed in A. adeninivorans and S. cerevisiae disruption mutants. The A. adeninivorans fumarate reductase is capable of functional complementation of the missing S. cerevisiae genes during anoxia; however, it is not involved in yeast growth under osmotic stress.
Asunto(s)
Proteínas Fúngicas/metabolismo , Saccharomycetales/enzimología , Saccharomycetales/genética , Succinato Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Hipoxia de la Célula , Clonación Molecular , Medios de Cultivo , ADN de Hongos/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutación , Presión Osmótica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomycetales/crecimiento & desarrollo , Análisis de Secuencia de ADN , Succinato Deshidrogenasa/genéticaRESUMEN
The yeast Arxula adeninivorans provides an attractive expression platform and can be exploited as gene source for biotechnologically interesting proteins. In the following study, a striking example for the combination of both aspects is presented. The transaldolase-encoding A. adeninivorans ATAL gene, including its promoter and terminator elements, was isolated and characterized. The gene includes a coding sequence of 963 bp encoding a putative 321 amino acid protein of 35.0 kDa. The enzyme characteristics analyzed from isolates of native strains and recombinant strains overexpressing the ATAL gene revealed a molecular mass of ca. 140 kDa corresponding to a tetrameric structure, a pH optimum of ca. 5.5, and a temperature optimum of 20 degrees C. The preferred substrates for the enzyme include D-erythrose-4-phosphate and D-fructose-6-phosphate, whereas D-glyceraldehyde is not converted. The ATAL expression level under salt-free conditions was observed to increase in media supplemented with 5% NaCl rendering the ATAL promoter attractive for moderate heterologous gene expression under high-salt conditions. Its suitability was assessed for the expression of a human serum albumin (HSA) reporter gene.
