scholarly journals Harnessing Adaptive Evolution to Achieve Superior Mannitol Production by Lactococcus lactis Using Its Native Metabolism

2020 ◽  
Vol 68 (17) ◽  
pp. 4912-4921
Author(s):  
Hang Xiao ◽  
Qi Wang ◽  
Claus Heiner Bang-Berthelsen ◽  
Peter Ruhdal Jensen ◽  
Christian Solem
Keyword(s):  
Lactococcus Lactis ◽  
Adaptive Evolution ◽  
Mannitol Production

scholarly journals Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds

2004 ◽  
Vol 70 (7) ◽  
pp. 4286-4292 ◽  
Author(s):  
H. Wouter Wisselink ◽  
Astrid E. Mars ◽  
Pieter van der Meer ◽  
Gerrit Eggink ◽  
Jeroen Hugenholtz
Keyword(s):  
Metabolic Engineering ◽  
Lactococcus Lactis ◽  
High Performance ◽  
Nuclear Magnetic Resonance Analysis ◽  
Resting Cells ◽  
Resonance Analysis ◽  
Glucose Depletion ◽  
Dehydrogenase Gene ◽  
Mannitol Production ◽  
Phosphofructokinase Activity

ABSTRACT To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and 13C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.

scholarly journals Deciphering the regulation of the mannitol operon paves the way for efficient production of mannitol in L. lactis

2021 ◽  
Author(s):  
Hang Xiao ◽  
Claus Heiner Bang-Berthelsen ◽  
Peter Ruhdal Jensen ◽  
Christian Solem
Keyword(s):  
Stationary Phase ◽  
Lactococcus Lactis ◽  
Transcriptional Activator ◽  
High Yield ◽  
Efficient Production ◽  
Phosphotransferase System ◽  
Transcriptional Terminator ◽  
Mannitol Production ◽  
Transcriptional Units ◽  
Foreign Genes

Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated, and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positives, mtlF and mtlD , encoding the Enzyme IIA component (EIIA mtl ) of the mannitol phosphotransferase system (PTS), and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA , encoding the Enzyme IIBC component (EIIBC mtl ) of the mannitol PTS, mtlR , encoding a transcriptional activator, and mtlF , and a separately expressed mtlD . The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive elements ( cre ). Presence of carbon catabolite repression was demonstrated, and was shown to be relieved in stationary phase cells. The transcriptional activator MtlR ( mtlR ), in some Gram-positives, is repressed by phosphorylation by EIIA mtl , and when we knocked-out mtlF we indeed observed enhanced expression from the two promotors, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary phase cells as biocatalysts, we attained 10.1 g/L mannitol with a 55% yield, which is the highest titer ever reported for L. lactis . Summing up, the results of our study should be useful for improving the mannitol producing capacity of this important industrial organism. Importance Lactococcus lactis is the most studied species of the Lactic Acid Bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis into producing mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis , with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated, and demonstrate how this impacts on mannitol production capability. We harness this information and manage to establish efficient mannitol production, without introducing foreign genes.

scholarly journals Adaptive Evolution of Industrial Lactococcus lactis Under Cell Envelope Stress Provides Phenotypic Diversity

2018 ◽  
Vol 9 ◽  
Author(s):  
María Jesús López-González ◽  
Susana Escobedo ◽  
Ana Rodríguez ◽  
A. Rute Neves ◽  
Thomas Janzen ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Adaptive Evolution ◽  
Phenotypic Diversity ◽  
Cell Envelope ◽  
Envelope Stress

scholarly journals Overproduction of Heterologous Mannitol 1-Phosphatase: a Key Factor for Engineering Mannitol Production by Lactococcus lactis

2005 ◽  
Vol 71 (3) ◽  
pp. 1507-1514 ◽  
Author(s):  
H. Wouter Wisselink ◽  
Antoine P. H. A. Moers ◽  
Astrid E. Mars ◽  
Marcel H. N. Hoefnagel ◽  
Willem M. de Vos ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Lactate Dehydrogenase ◽  
Lactococcus Lactis ◽  
Clear Correlation ◽  
Dehydrogenase Gene ◽  
Mannitol Production ◽  
Key Factor ◽  
Phosphofructokinase Activity ◽  
Overexpression System ◽  
Substrate Concentrations

ABSTRACT To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisin-dependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.

scholarly journals Engineering Lactococcus lactis for Production of Mannitol: High Yields from Food-Grade Strains Deficient in Lactate Dehydrogenase and the Mannitol Transport System

2004 ◽  
Vol 70 (3) ◽  
pp. 1466-1474 ◽  
Author(s):  
Paula Gaspar ◽  
Ana Rute Neves ◽  
Ana Ramos ◽  
Michael J. Gasson ◽  
Claire A. Shearman ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Lactococcus Lactis ◽  
Selection Marker ◽  
Phosphotransferase System ◽  
Acid Fermentation ◽  
Food Grade ◽  
Mixed Acid ◽  
Glucose Depletion ◽  
Mannitol Production

ABSTRACT Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTSMtl). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (ΔldhΔmtlA and ΔldhΔmtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo 13C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTSMtl. Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.

Exportation de protéines hétérologues chez Lactococcus lactis : développement d'outils moléculaires et applications

2000 ◽  
Vol 20 (1) ◽  
pp. 135-142
Author(s):  
Philippe Langella ◽  
Yves Le Loir ◽  
Jean-Christophe Piard ◽  
Isabelle Pocquet ◽  
Alexandra Gruss
Keyword(s):  
Lactococcus Lactis
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