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High-Level Production of the Low-Calorie Sugar Sorbitol by Lactobacillus plantarum through Metabolic Engineering

Victor Ladero,^1, Ana Ramos,² Anne Wiersma,³ Philippe Goffin,^1, André Schanck,⁴ Michiel Kleerebezem,³ Jeroen Hugenholtz,³ Eddy J. Smid,³ and Pascal Hols^1*

Unité de Génétique, Institut des Sciences de la Vie, Université catholique de Louvain, 1348 Louvain-la-Neuve, Belgium,¹ Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, and Instituto de Biologia Experimental e Tecnológica, Rua da Quinta Grande, 6, Apt. 127, 2780-156 Oeiras, Portugal,² Wageningen Centre for Food Sciences, NIZO food research, 6710 BA Ede, The Netherlands,³ Laboratoire de Chimie Physique et de Cristallographie, Université catholique de Louvain, Place Louis Pasteur 1, 1348 Louvain-la-Neuve, Belgium⁴

Received 29 September 2006/ Accepted 9 January 2007

Sorbitol is a low-calorie sugar alcohol that is largely used as an ingredient in the food industry, based on its sweetness and its high solubility. Here, we investigated the capacity of Lactobacillus plantarum, a lactic acid bacterium found in many fermented food products and in the gastrointestinal tract of mammals, to produce sorbitol from fructose-6-phosphate by reverting the sorbitol catabolic pathway in a mutant strain deficient for both L- and D-lactate dehydrogenase activities. The two sorbitol-6-phosphate dehydrogenase (Stl6PDH) genes (srlD1 and srlD2) identified in the genome sequence were constitutively expressed at a high level in this mutant strain. Both Stl6PDH enzymes were shown to be active, and high specific activity could be detected in the overexpressing strains. Using resting cells under pH control with glucose as a substrate, both Stl6PDHs were capable of rerouting the glycolytic flux from fructose-6-phosphate toward sorbitol production with a remarkably high efficiency (61 to 65% glucose conversion), which is close to the maximal theoretical value of 67%. Mannitol production was also detected, albeit at a lower level than the control strain (9 to 13% glucose conversion), indicating competition for fructose-6-phosphate rerouting by natively expressed mannitol-1-phosphate dehydrogenase. By analogy, low levels of this enzyme were detected in both the wild-type and the lactate dehydrogenase-deficient strain backgrounds. After optimization, 25% of sugar conversion into sorbitol was achieved with cells grown under pH control. The role of intracellular NADH pools in the determination of the maximal sorbitol production is discussed.

Published ahead of print on 19 January 2007.

Present address: Instituto de Productos Lácteos de Asturias (IPLA, CSIC), Carretera de Infiesto s/n., 33300 Villaviciosa, Spain.

Present address: Wageningen Centre for Food Sciences, NIZO food research, 6710 BA Ede, The Netherlands.