Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus
Mohedano, M.L. ; Garcia-Cayuela, T. ; Perez-Ramos, A. ; Gaiser, R.A. ; Requena, T. ; Lopez, P. - \ 2015
Journal of Industrial Microbiology and Biotechnology 42 (2015)2. - ISSN 1367-5435 - p. 247 - 253.
lactic-acid bacteria - controlled gene-expression - streptococcus-pneumoniae - lactococcus-lactis - plasmid - cloning
Lactobacilli are widespread in natural environments and are increasingly being investigated as potential health modulators. In this study, we have adapted the broad-host-range vector pNZ8048 to express the mCherry protein (pRCR) to expand the usage of the mCherry protein for analysis of gene expression in Lactobacillus. This vector is also able to replicate in Streptococcus pneumoniae and Escherichia coli. The usage of pRCR as a promoter probe was validated in Lactobacillus acidophilus by characterizing the regulation of lactacin B expression. The results show that the regulation is exerted at the transcriptional level, with lbaB gene expression being specifically induced by co-culture of the L. acidophilus bacteriocin producer and the S. thermophilus STY-31 inducer bacterium.
Indigenous and environmental modulation of mutation frequencies in Lactobacillus plantarum
Machielsen, M.P. ; Alen-Boerrigter, I.J. van; Koole, L.A. ; Bongers, R.S. ; Kleerebezem, M. ; Hylckama-Vlieg, J.E.T. van - \ 2010
Applied and Environmental Microbiology 76 (2010)5. - ISSN 0099-2240 - p. 1587 - 1595.
controlled gene-expression - lactic-acid bacteria - site-specific recombination - repair protein muts - lactococcus-lactis - escherichia-coli - mismatch-repair - streptococcus-lactis - hydrogen-peroxide - oxidative stress
The reliability of microbial (starter) strains in terms of quality, functional properties, growth performance and robustness is essential for industrial applications. In an industrial fermentation process, the bacterium should be able to successfully withstand various adverse conditions during processing such as acid, osmotic, temperature, and oxidative stress. Besides the evolved defense mechanisms, stress-induced mutations participate in adaptive evolution towards survival under these stress conditions. However, this may lead to the accumulation of mutant strains, which may be accompanied by loss of desired functional properties. Defining the effect of specific fermentation or processing conditions on the mutation frequencies is an important step towards preventing loss of genome integrity and maintaining the productivity of industrial strains. Therefore, a set of Lactobacillus plantarum mutator reporter strains suitable for qualitative and quantitative analysis of low-frequency mutation events was developed. The mutation reporter system constructed was validated by using chemical mutagenesis (NTG) and by controlled expression of endogenous candidate mutator genes (e.g. truncated derivative of the L. plantarum hexA gene). Growth at different temperatures, in low pH conditions, at high salt concentrations or in starvation conditions did not result in a significant effect on the mutation frequency. However, incubation with sublethal levels of hydrogen peroxide showed a 100-fold increase of the mutation frequency when compared to the background mutation frequency. Importantly, when cells of L. plantarum were adapted to 42 degrees C, prior to the treatment with sublethal levels of hydrogen peroxide, this induced a 10-fold increase in peroxide treatment survival, with a concomitant 50-fold decrease of the mutation frequency. These results show that specific environmental conditions encountered by bacteria may significantly influence the genetic stability of strains, while protection against mutagenic conditions may be achieved by pre-treatment of cultures with other, non-mutagenic stress conditions
Safe use of genetically modified lactic acid bacteria in food. Bridging the gap between consumers, green groups, and industry
Sybesma, W. ; Hugenholtz, J. ; Vos, W.M. de; Smid, E.J. - \ 2006
Electronic Journal of Biotechnology 9 (2006)4. - ISSN 0717-3458 - p. 424 - 448.
controlled gene-expression - alpha-acetolactate decarboxylase - beta-galactosidase gene - lactococcus-lactis - streptococcus-thermophilus - lactobacillus-plantarum - subsp lactis - postmarketing surveillance - molecular characterization - acetaldehyde production
Within the European Union (EU), the use of genetically modified organisms (GMOs) in food production is not widely applied and accepted. In contrast to the United States of America, the current EU legislation limits the introduction of functional foods derived from GMOs that may bring a clear benefit to the consumer. Genetically modified lactic acid bacteria (GM-LAB) can be considered as a different class of GMOs, and the European Union is preparing regulations for the risk assessment of genetically modified microorganisms. Since these procedures are not yet implemented, the current risk assessment procedure is shared for GMOs derived from micro organisms, plants, or animals. At present, the use of organisms in food production that have uncontrolled genetic alterations made through random mutagenesis, is permitted, while similar applications with organisms that have controlled genetic alterations are not allowed. The current paper reviews the opportunities that genetically modified lactic acid bacteria may offer the food industry and the consumer. An objective risk profile is described for the use of GM-LAB in food production. To enhance the introduction of functional foods with proven health claims it is proposed to adapt the current safety assessment procedures for (GM)-LAB and suggestions are made for the related cost accountability. A qualified presumption of safety as proposed by SANCO (EU SANCO 2003), based on taxonomy and on the history of safe use of LAB applied in food, could in the near future be applied to any kind of LAB or GM-LAB provided that a series of modern profiling methods are used to verify the absence of unintended effects of altered LAB that may cause harm to the health of the consumer.
Identification cloning and characterization of a branched-chain alpha-keto acid decarboxylase from Lactococcus lactis, involved in flavour formation
Smit, B.A. ; Meijer, L. ; Engels, W.J.M. ; Wouters, J.T.M. ; Smit, G. - \ 2005
Applied and Environmental Microbiology 71 (2005)1. - ISSN 0099-2240 - p. 303 - 311.
controlled gene-expression - complete genome sequence - gram-positive bacteria - indolepyruvate decarboxylase - pyruvate decarboxylase - enterobacter-cloacae - escherichia-coli - indole-3-acetic-acid biosynthesis - saccharomyces-cerevisiae - amino-acids
The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain ¿-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3¿ terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain ¿-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.
Overproduction of heterologous mannitol 1-phosphatase : a key factor for engineering mannitol production by Lactococcus lactis
Wisselink, H.W. ; Moers, A.P.H.A. ; Mars, A.E. ; Hoefnagel, M.H.N. ; Vos, W.M. de; Hugenholtz, J. - \ 2005
Applied and Environmental Microbiology 71 (2005)3. - ISSN 0099-2240 - p. 1507 - 1514.
controlled gene-expression - complete genome sequence - acid bacteria - lactate-dehydrogenase - lactobacillus-plantarum - leuconostoc - deficient - protects - glucose - mesenteroides
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.
Engineering metabolic highways in Lactococci and other lactic acid bacteria
Vos, W.M. de; Hugenholtz, J. - \ 2004
Trends in Biotechnology 22 (2004)2. - ISSN 0167-7799 - p. 72 - 79.
controlled gene-expression - complete genome sequence - exopolysaccharide production - lactate-dehydrogenase - sugar catabolism - streptococcus-thermophilus - molecular characterization - functional-analysis - diacetyl production - zymomonas-mobilis
Lactic acid bacteria (LAB) are widely used in industrial food fermentations and are receiving increased attention for use as cell factories for the production of food and pharmaceutical products. Glycolytic conversion of sugars into lactic acid is the main metabolic highway in these Gram-positive bacteria and Lactococcus lactis has become the model organism because of its small genome, genetic accessibility and simple metabolism. Here we discuss the metabolic engineering of L. lactis and the value of metabolic models compared with other LAB, with a particular focus on the food-grade production of metabolites involved in flavour, texture and health.
Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-rhamnose biosynthesis
Boels, I.C. ; Beerthuyzen, M.M. ; Kosters, M.H. ; Kaauwen, M.P.W. van; Kleerebezem, M. ; Vos, W.M. de - \ 2004
Journal of Bacteriology 186 (2004)5. - ISSN 0021-9193 - p. 1239 - 1248.
controlled gene-expression - gram-positive bacteria - subsp cremoris - capsular polysaccharide - streptococcus-pneumoniae - acid bacteria - cell-wall - o-antigen - exopolysaccharide production - escherichia-coli
dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis. We cloned the rfbACBD operon from L. lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli, the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in L. lactis resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L. lactis. Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.
Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of the spa-box in subtilin-responsive promoters
Kleerebezem, M. ; Bongers, R. ; Rutten, G. ; Vos, W.M. de; Kuipers, O.P. - \ 2004
Peptides 25 (2004)9. - ISSN 0196-9781 - p. 1415 - 1424.
gram-positive bacteria - lactic-acid bacteria - controlled gene-expression - nisin-controlled expression - lantibiotic subtilin - lactococcus-lactis - signal-transduction - transcriptional activation - dependent regulation - immunity
The production of the type 1 antimicrobial peptide (AMP) subtilin by Bacillus subtilis is regulated in a cell-density-dependent manner [Kleerebezem M, de Vos WM, Kuipers OP. The lantibiotics nisin and subtilin act as extracellular regulators of their own biosynthesis. In: Dunny GM, Winans SC, editors. Cell-cell signaling in bacteria. Washington, D.C., USA: ASM Press; 1999. p. 159-74; Stein T, Borchert S, Kiesau P. Heinzmann S, Moss S, Klein C, Helfrich M, Entian KD. Dual control of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2002;44:403-16; Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627-36]. Three subtilin-responsive promoter elements within the spaBTCSIFEGRK are controlled by the specific cis-acting sequence element called the spa-box, which represents the binding site of the subtilin regulator SpaR [Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627-36]. Here, we describe the functional characterization of the spaB, spaS and spaI promoters by transcriptional fusion with a promoterless P-glucuronidase encoding gusA gene. Within these gusA fusion constructs, transcription initiation start sites of the spaS and spa1 promoters were mapped to be located downstream of the spa-box, which is in contrast to previous reports [Banerjee S, Hansen JN. Structure and expression of a gene encoding the precursor of subtilin, a small protein antibiotic. J Biol Chem 1988;263:9508-14; Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627-36]. Nevertheless, all spa-promoters displayed typical cell-density-dependent activity in a subtilin-producing strain B. subtilis ATCC6633. Moreover, analysis of P-glucuronidase activities in a spaB mutant of B. subtilis ATCC6633 and a derivative of strain 168 that harbors the spaRK genes integrated in the chromosomal amyE locus, confirmed that these promoters are activated by subtilin-triggered, SpaRK-mediated, quorum-sensing control. Quantitative analysis showed that the spaS promoter strength at a given subtilin concentration appeared to be approximately five-fold higher than the spaB promoter, which in turn is approximately two-fold higher than the spa1 promoter. Finally, it is shown that the elementary components involved in subtilin-mediated regulation are the two-component system, SpaRK, and a spa-box containing promoter. (C) 2004, Elsevier Inc. All rights reserved.
Engineering of carbon distribution between glycolysis and sugar nucleobiosynthesis in Lactococcus lactis
Boels, I.C. ; Kleerebezem, M. ; Vos, W.M. de - \ 2003
Applied and Environmental Microbiology 69 (2003). - ISSN 0099-2240 - p. 1129 - 1135.
controlled gene-expression - exopolysaccharide biosynthesis - streptococcus-thermophilus - subsp cremoris - cellular phosphoglucomutase - polysaccharide formation - rheological properties - escherichia-coli - acid bacteria - cloning
We describe the effects of modulating the activities of glucokinase, phosphofructokinase, and phosphoglucomutase on the branching point between sugar degradation and the biosynthesis of sugar nucleotides involved in the production of exopolysaccharide biosynthesis by Lactococcus lactis. This was realized by using a described isogenic L. lactis mutant with reduced enzyme activities or by controlled expression of the well-characterized genes for phosphoglucomutase or glucokinase from Escherichia coli or Bacillus subtilis, respectively. The role of decreased metabolic flux was studied in L. lactis strains with decreased phosphofructokinase activities. The concomitant reduction of the activities of phosphofructokinase and other enzymes encoded by the las operon (lactate dehydrogenase and pyruvate kinase) resulted in significant changes in the concentrations of sugar-phosphates. In contrast, a >25-fold overproduction of glucokinase resulted in 7-fold-increased fructose-6-phosphate levels and 2-fold-reduced glucose-1-phosphate and glucose-6-phosphate levels. However, these increased sugar-phosphate concentrations did not affect the levels of sugar nucleotides. Finally, an similar to100-fold overproduction of phosphoglucomutase resulted in 5-fold-increased levels of both UDP-glucose and UDPgalactose. While the increased concentrations of sugar-phosphates or sugar nucleotides did not significantly affect the production of exopolysaccharides, they demonstrate the metabolic flexibility of L. lactis.
Increased production of folate by metabolic engineering of Lactococcus lactis
Sybesma, W.F.H. ; Starrenburg, M. ; Kleerebezem, M. ; Mierau, I. ; Vos, W.M. de; Hugenholtz, J. - \ 2003
Applied and Environmental Microbiology 69 (2003). - ISSN 0099-2240 - p. 3069 - 3076.
controlled gene-expression - folic-acid - streptococcus-thermophilus - dihydropteroate synthase - subsp cremoris - cloning - bacteria - disease - homocysteine - inhibition
The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates large amounts of folate, predominantly in the polyglutamyl form. Only small amounts of the produced folate are released in the extracellular medium. Five genes involved in folate biosynthesis were identified in a folate gene cluster in L. lactis MG1363: folA, folB, folKE, folP, and folC. The gene folKE encodes the biprotein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I. The overexpression of folKE in L. lactis was found to increase the extracellular folate production almost 10-fold, while the total folate production increased almost 3-fold. The controlled combined overexpression of folKE and folC, encoding polyglutamyl folate synthetase, increased the retention of folate in the cell. The cloning and overexpression of folA, encoding dihydrofolate reductase, decreased the folate production twofold, suggesting a feedback inhibition of reduced folates on folate biosynthesis.
Metabolic engineering of lactic acid bacteria for the production of nutraceuticals
Hugenholtz, J. ; Sybesma, W. ; Groot, M.N. ; Wisselink, W. ; Ladero, V. ; Burgess, K. ; Sinderen, D. van; Piard, J.C. ; Eggink, G. ; Smid, E.J. ; Savoy, G. ; Sesma, F. ; Jansen, T. ; Hols, P. ; Kleerebezem, M. - \ 2002
Antonie van Leeuwenhoek: : Nederlandsch tijdschrift voor hygiëne, microbiologie en serologie 82 (2002)1-4. - ISSN 0003-6072 - p. 217 - 235.
controlled gene-expression - lactococcus-lactis - exopolysaccharide biosynthesis - streptococcus-thermophilus - lactobacillus-plantarum - glycosyltransferase genes - nucleotide-sequence - diacetyl production - functional-analysis - nmr-spectroscopy
Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal sugar metabolism to nutritional end-products other than lactic acid such as L-alanine, several low-calorie sugars and oligosaccharides or to enhancement of sugar metabolism for complete removal of (undesirable) sugars from food materials. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to the production of the B-vitamins folate and riboflavin. An overview of these metabolic engineering activities can be found on the website of the Nutra Cells 5th Framework EU-project (www.nutracells.com). Finally, the impact of the developments in the area of genomics and corresponding high-throughput technologies on nutraceutical production will be discussed