title: Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris.
abstract: Enzymes for use in the sugar industry are preferred to be thermotolerant. In this study, a synthetic codon-optimized gene encoding a highly thermostable β-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima was expressed in the yeast Pichia pastoris. The gradual increase of the transgene dosage from one to four copies under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter had an additive effect on BfrA yield without causing cell toxicity. Maximal values of cell biomass (115 g/l, dry weight) and overall invertase activity (241 U/ml) were reached at 72 h in fed-batch fermentations using cane sugar as the main carbon source for growth. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (44 %) and extracellular release (56 %) of BfrA. The presence of N-linked oligosaccharides did not influence the optimal activity, thermal stability, kinetic properties, substrate specificity, and exo-type action mode of the yeast-secreted BfrA in comparison to the native unglycosylated enzyme. Complete inversion of cane sugar at initial concentration of 60 % (w/v) was achieved by periplasmic BfrA in undisrupted cells reacting at pH 5.5 and 70 °C, with average productivity of 4.4 g of substrate hydrolyzed per grams of biomass (wet weight) per hour. The high yield of fully active glycosylated BfrA here attained by recombinant P. pastoris in a low-cost fermentation process appears to be attractive for the large-scale production of this thermostable enzyme useful for the manufacture of inverted sugar syrup.
authors: Plant-Microbe Interactions Laboratory, Center for Genetic Engineering and Biotechnology (CIGB), Ave 31 entre 158 y 190, Apartado Postal 6162, Havana, 10600, Cuba. carmen.menendez@cigb.edu.cu
authors: Carmen
authors: C
authors: Menéndez
authors: Duniesky
authors: D
authors: Martínez
authors: Luis E
authors: LE
authors: Trujillo
authors: Yuliet
authors: Y
authors: Mazola
authors: Ernesto
authors: E
authors: González
authors: Enrique R
authors: ER
authors: Pérez
authors: Lázaro
authors: L
authors: Hernández
mesh_terms: D018533:Biomass; D050260:Carbohydrate Metabolism; D002244:Carbon / Q000378:metabolism; D003062:Codon; D004795:Enzyme Stability; D005285:Fermentation; D018628:Gene Dosage; D015870:Gene Expression; D007700:Kinetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D043324:beta-Fructofuranosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D002244:Carbon; D043324:beta-Fructofuranosidase