Expression of an Aspergillus niger Glucose Oxidase in Saccharomyces cereWisiae and Its Use to Optimize Fructo-oligosaccharides Synthesis Magdalena Valdivieso-Ugarte, Carmen Ronchel, Oscar Ban ˜ uelos, Javier Velasco, and Jose ´ L. Adrio* Department of Biotechnology, Puleva Biotech, S.A., Camino de Purchil, 66, 18004 Granada, Spain Fructo-oligosaccharides (FOS) represent the most abundantly supplied and utilized group of nondigestible oligosaccharides as food ingredients. These prebiotics can be produced from sucrose using the transglycosylating activity of -fructofuranosidases (EC 3.2.1.26) at high concentrations of the starting material. The main problem during FOS synthesis is that the activity of the enzyme is inhibited by the glucose generated during the reaction, and therefore the maximum FOS content in commercial products reaches up to 60% on a dry substance basis. The glucose oxidase (gox) gene from Aspergillus niger BT18 was cloned and integrated, as part of an expression cassette, into the ribosomal DNA of a Saccharomyces cereVisiae host strain. One of the recombinant strains with a high copy number of the gox gene and showing a high GOX specific activity was used to produce the enzyme. Addition of the extracellular glucose oxidase to the FOS synthesis reaction helped to remove the glucose generated, avoiding the inhibition of the fungal -fructofuranosidase. As a result, a final syrup containing up to 90% of FOS was obtained. Introduction The most abundantly supplied and utilized group of non- digestible oligosaccharides as food ingredients are fructo- oligosaccharides (FOS). In addition to their health benefits as prebiotics, FOS possess a number of other interesting properties such as having low calorie content, being safe for diabetics, having lower sweetness intensity than sucrose, and being noncariogenic (1, 2). The FDA (Food and Drug Administration) has designated FOS as a safe substance (GRAS Notice no. GRN 000044), and in 2001, the Scientific Committee on Food of the European Union approved the use of FOS and GOS (galacto- oligosaccharides) in infant formulas in concentrations up to 0.08 g/L of product. The industrial production of FOS is carried out by the transglycosylation activity of fructosyltransferases (also called -fructofuranosidases, EC 3.2.1.26) at high sucrose concentra- tions (3). In the process of FOS production, the main problem is that the activity of the enzyme is severely inhibited by glucose, which is generated as a byproduct. As a result, most commercial FOS products are a mixture of FOS, sucrose, and glucose. The maximum FOS content, on a dry substance basis, obtained using -fructofuranosidases alone is known to be 55-60% (4). A good chance to enhance the production of FOS is to eliminate the glucose by enzymatic reactions using glucose isomerase or glucose oxidase. The latter seems to have potential advantages (5) and could lead to mixtures with a FOS content up to 98%. Glucose oxidase (GOX, -D-glucose:oxygen 1-oxidoreduc- tase, EC 1.1.3.4.) catalyzes the oxidation of glucose to glucono- 1,5-lactone and the concomitant reduction of molecular oxygen to hydrogen peroxide (6). GOX has been purified from Aspergillus and Penicillium species (7), and crude or partially purified preparations of this enzyme have been used in many different industrial applications such as determination of glucose in solution, as a source of hydrogen peroxide for food preserva- tion, or for the production of gluconic acid (8). However, some of those applications have been hampered by the high cost of Aspergillus preparations due to the presence of other concomi- tant enzymes such as cellulase or catalase. GOX has been successfully expressed in yeasts such as Saccharomyces cereVisiae, Hansenula polymorpha, and Pichia pastoris (9-11). However, in the first two hosts, expression was accomplished using episomal vectors that are known to show low stability during cultivation. On the other hand, one of the major drawbacks for both Hansenula and Pichia, two excellent expression systems, is that both are not recognized as safe hosts for the production of healthcare and, especially, food products (non-GRAS status). To make industrial production of a protein economically feasible, two important criteria must be fulfilled: a high copy number of the gene introduced into the host organism, and high mitotic stability of that gene since the production of proteins often requires long-term fermentations in nonselective media. Because of its FDA GRAS status, S. cereVisiae is one of the host systems used for the production of food and health-care products. To meet the requirements mentioned above, a good approach is to target the integration into the ribosomal DNA, which encompasses between 100 and 200 copies of a 9.1 kb unit repeated in tandem on chromosome XII (12, 13), using defective selection markers (14, 15). In this paper we report the integration of several copies of an expression cassette containing the gox gene from an A. niger strain into the S. cereVisiae rDNA. The amounts of extracellular GOX obtained allowed us to add this enzyme into FOS synthesis reactions, leading to a final product with high concentrations of FOS (up to 90%). Materials and Methods Microorganisms and Media. Escherichia coli TOP 10 F′: F′{lacI q , Tn 10(Tet R )}, mcr A ∆(mrr-hsdRMS-mcrBC), Φ * To whom correspondence should be addressed. Ph: +34 958 24 02 27. Fax: +34 958 24 01 60. E-mail: jladrio@pulevabiotech.es. 1096 Biotechnol. Prog. 2006, 22, 1096-1101 10.1021/bp060076k CCC: $33.50 © 2006 American Chemical Society and American Institute of Chemical Engineers Published on Web 07/04/2006