Production of xylitol from D-xylose by recombinant Lactococcus lactis.

The D-xylose reductase from Pichia stipitis CBS 5773 and the xylose transporter from Lactobacillus brevis ATCC 8287 were expressed in active form in Lactococcus lactis NZ9800. Xylitol production was investigated using non-growing recombinant cells in high cell-density under microaerobic conditions in the presence of xylose and glucose. Besides xylose, the recombinant strain with xylose reductase activity reduced l-arabinose and D-ribose in significant extent to the corresponding pentitols. The ratio of xylitol produced per glucose consumed was almost 10-fold higher under glucose limitation than the ratio in the presence of excess initial glucose. The co-expression of the xylose transporter with the xylose reductase did not increase the efficiency of xylitol production appreciably when compared to the strain in which only the xylose reductase gene was expressed. A fed-batch experiment with high initial xylose concentration (160 gl(-1)) under glucose limitation was carried out using the strain co-expressing xylose reductase and xylose transporter genes. The xylitol yield from xylose was 1.0 mol mol(-1) and the ratio of xylitol produced per glucose consumed was 2.5 mol mol(-1). The volumetric productivity was 2.72 gl(-1)h(-1) at 20 h. Of the xylose initially present, 34% was consumed. Analysis of the fermentation metabolites revealed a shift from homolactic to mixed acid fermentation at early stages of the experiment.

[1]  C. Batt,et al.  Dissolution of Xylose Metabolism inLactococcus lactis , 2000, Applied and Environmental Microbiology.

[2]  R. Albert,et al.  Die katalytische Herstellung von Zuckeralkoholen und deren Verwendung , 1980 .

[3]  Jin-Ho Seo,et al.  Production of xylitol in cell recycle fermentations of Candida tropicalis , 2000, Biotechnology Letters.

[4]  M. Rizzi,et al.  Xylose fermentation by yeasts , 1984, Biotechnology Letters.

[5]  A. Scheinin.  Turku sugar studies I-XXI , 1975 .

[6]  J. Yoshitake,et al.  Xylitol Production by an Enterobacter Species , 1973 .

[7]  José Manuel Domínguez,et al.  Biotechnological production of xylitol. Part 1: Interest of xylitol and fundamentals of its biosynthesis , 1998 .

[8]  T. Granström.  BIOTECHNOLOGICAL PRODUCTION OF XYLITOL WITH CANDIDA YEASTS , 2002 .

[9]  C. Hollenberg,et al.  Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase-encoding gene (XYL1) from the xylose-assimilating yeast Pichia stipitis. , 1991, Gene.

[10]  M. Leisola,et al.  Actinopolyspora halophila has two separate pathways for betaine synthesis , 2001, Archives of Microbiology.

[11]  J. Parajó,et al.  Biotechnological production of xylitol. Part 2 : Operation in culture media made with commercial sugars , 1998 .

[12]  P. Postma,et al.  Molecular Cloning and Functional Expression inLactobacillus plantarum 80 of xylT, Encoding thed-Xylose–H+ Symporter ofLactobacillus brevis , 1998, Applied and Environmental Microbiology.

[13]  J. Villadsen,et al.  The level of pyruvate-formate lyase controls the shift from homolactic to mixed-acid product formation in Lactococcus lactis , 2002, Applied Microbiology and Biotechnology.

[14]  A. Driessen,et al.  Characteristics and osmoregulatory roles of uptake systems for proline and glycine betaine in Lactococcus lactis , 1993, Journal of bacteriology.

[15]  J. Domínguez,et al.  Production of Xylitol from D-xylose by Debaryomyces hansenii. , 1997 .

[16]  J. Yoshitake,et al.  Production of Polyalcohol by a Corynebacterium sp , 1971 .

[17]  A. Emodi,et al.  Xylitol: its properties and food applications. , 1978 .

[18]  V. Crow,et al.  Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis , 1984, Applied and environmental microbiology.

[19]  J. Domínguez,et al.  Xylitol production from hardwood hemicellulose hydrolysates by Pachysolen tannophilus, Debaryomyces hansenii, and Candida guilliermondii , 1999 .

[20]  S. Salminen.  Lactic acid bacteria: microbiology and functional aspects. , 1998 .

[21]  Jin-Ho Seo,et al.  Characterization of two-substrate fermentation processes for xylitol production using recombinant Saccharomyces cerevisiae containing xylose reductase gene , 2000 .

[22]  I. Nes,et al.  High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media , 1989, Applied and environmental microbiology.

[23]  M. Rizzi,et al.  Xylose fermentation by yeasts , 1988, Applied Microbiology and Biotechnology.

[24]  F. Agblevor,et al.  Screening of facultative anaerobic bacteria utilizing D-xylose for xylitol production , 2002, Applied Microbiology and Biotechnology.

[25]  Henk,et al.  Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis. , 1985, The Biochemical journal.

[26]  Jacky L. Snoep,et al.  Effect of the energy source on the NADH/NAD ratio and on pyruvate catabolism in anaerobic chemostat cultures of Enterococcus faecalis NCTC 775 , 1991 .

[27]  A. Afschar,et al.  Microbial production of xylitol from D-xylose using Candida tropicalis , 1994 .

[28]  W. V. van Zyl,et al.  Xylitol production by recombinant Saccharomyces cerevisiae expressing the Pichia stipitis and Candida shehatae XYL1 genes , 2001, Applied Microbiology and Biotechnology.

[29]  N. Meinander,et al.  Fed-batch xylitol production with two recombinant Saccharomyces cerevisiae strains expressing XYL1 at different levels, using glucose as a cosubstrate: a comparison of production parameters and strain stability. , 1997, Biotechnology and bioengineering.

[30]  W. D. de Vos,et al.  Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin , 1996, Applied and environmental microbiology.

[31]  Eleonora Winkelhausen,et al.  Microbial conversion of d-xylose to xylitol , 1998 .

[32]  E. Galinski.  Compatible solutes of halophilic eubacteria: molecular principles, water-solute interaction, stress protection , 1993, Experientia.

[33]  S. Yokoyama,et al.  Expression of xyrA gene encoding for D-Xylose reductase of Candida tropicalis and production of xylitol in Escherichia coli. , 1999, Journal of bioscience and bioengineering.

[34]  Seppo Salminen,et al.  Lactic acid bacteria: classification and physiology. , 1993 .

[35]  S. Fridkin,et al.  Epidemiology of nosocomial fungal infections , 1996, Clinical microbiology reviews.

[36]  M. Kleerebezem,et al.  Metabolic engineering of lactic acid bacteria: overview of the approaches and results of pathway rerouting involved in food fermentations. , 1999, Current opinion in biotechnology.

[37]  K. Kiviharju,et al.  High-level production of D-mannitol with membrane cell-recycle bioreactor , 2002, Journal of Industrial Microbiology and Biotechnology.

[38]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.