scholarly journals ArcD1 and ArcD2 Arginine/Ornithine Exchangers Encoded in the Arginine Deiminase Pathway Gene Cluster of Lactococcus lactis

2015 ◽  
Vol 197 (22) ◽  
pp. 3545-3553 ◽  
Author(s):  
Elke E. E. Noens ◽  
Michał B. Kaczmarek ◽  
Monika Żygo ◽  
Juke S. Lolkema
Keyword(s):  
Gene Cluster ◽  
Lactococcus Lactis ◽  
Physiological Function ◽  
Arginine Deiminase ◽  
Growth Media ◽  
Growth Enhancement ◽  
Resting Cells ◽  
Gene Encoding ◽  
Content Type ◽  
Pathway Gene

ABSTRACTThe arginine deiminase (ADI) pathway gene cluster inLactococcus lactiscontains two copies of a gene encoding anl-arginine/l-ornithine exchanger, thearcD1andarcD2genes. The physiological function of ArcD1 and ArcD2 was studied by deleting the two genes. Deletion ofarcD1resulted in loss of the growth advantage observed in the presence of highl-arginine in different growth media. Uptake ofl-arginine andl-ornithine by resting cells was reduced to the low level observed for an ArcD1/ArcD2 double deletion mutant. Deletion of thearcD2gene did not affect the growth enhancement, and uptake activities were slightly reduced. Nevertheless, recombinant expression of ArcD2 in the ArcD1/ArcD2 double mutant did recover the growth advantage. Kinetic characterization of ArcD1 and ArcD2 showed high affinities for bothl-arginine andl-ornithine (Kmin the micromolar range). A difference between the two transporters was the significantly lower affinity of ArcD2 for the cationic amino acidsl-ornithine,l-lysine, andl-histidine. In contrast, the affinity of ArcD2 was higher for the neutral amino acidl-alanine. Moreover, ArcD2 efficiently translocatedl-alanine, while ArcD1 did not. Both transporters revealed affinities in the mM range for agmatine, cadaverine, histamine, and putrescine. These amines bind but are not translocated. It is concluded that ArcD1 is the mainl-arginine/l-ornithine exchanger in the ADI pathway and that ArcD2 is not functionally expressed in the media used. ArcD2 is proposed to function together with thearcTgene that encodes a putative transaminase and is found adjacent to thearcD2gene.IMPORTANCEThe arginine deiminase (ADI) pathway gene cluster inLactococcus lactiscontains two copies of a gene encoding anl-arginine/l-ornithine exchanger, thearcD1andarcD2genes. The physiological function of ArcD1 and ArcD2 was studied by deleting the two genes. It is concluded that ArcD1 is the mainl-arginine/l-ornithine exchanger in the ADI pathway. ArcD2 is proposed to function as al-arginine/l-alanine exchanger in a pathway together with thearcTgene, which is found adjacent to thearcD2gene in the ADI gene cluster.

scholarly journals Transcriptional Regulator PhlH Modulates 2,4-Diacetylphloroglucinol Biosynthesis in Response to the Biosynthetic Intermediate and End Product

2017 ◽  
Vol 83 (21) ◽  
Author(s):  
Xu Yan ◽  
Rui Yang ◽  
Rui-Xue Zhao ◽  
Jian-Ting Han ◽  
Wen-Juan Jia ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Gene Cluster ◽  
Plant Pathogens ◽  
Feedback Regulation ◽  
Biosynthetic Gene Cluster ◽  
Sequence Motif ◽  
Target Sequence ◽  
Gene Encoding ◽  
Negative Feedback Regulation ◽  
Content Type

ABSTRACT Certain strains of biocontrol bacterium Pseudomonas fluorescens produce the secondary metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) to antagonize soilborne phytopathogens in the rhizosphere. The gene cluster responsible for the biosynthesis of 2,4-DAPG is named phlACBDEFGH and it is still unclear how the pathway-specific regulator phlH within this gene cluster regulates the metabolism of 2,4-DAPG. Here, we found that PhlH in Pseudomonas fluorescens strain 2P24 represses the expression of the phlG gene encoding the 2,4-DAPG hydrolase by binding to a sequence motif overlapping with the −35 site recognized by σ70 factors. Through biochemical screening of PhlH ligands we identified the end product 2,4-DAPG and its biosynthetic intermediate monoacetylphloroglucinol (MAPG), which can act as signaling molecules to modulate the binding of PhlH to the target sequence and activate the expression of phlG. Comparison of 2,4-DAPG production between the ΔphlH, ΔphlG, and ΔphlHG mutants confirmed that phlH and phlG impose negative feedback regulation over 2,4-DAPG biosynthesis. It was further demonstrated that the 2,4-DAPG degradation catalyzed by PhlG plays an insignificant role in 2,4-DAPG tolerance but contributes to bacterial growth advantages under carbon/nitrogen starvation conditions. Taken together, our data suggest that by monitoring and down-tuning in situ levels of 2,4-DAPG, the phlHG genes could dynamically modulate the metabolic loads attributed to 2,4-DAPG production and potentially contribute to rhizosphere adaptation. IMPORTANCE 2,4-DAPG, which is synthesized by biocontrol pseudomonad bacteria, is a broad-spectrum antibiotic against bacteria, fungi, oomycetes, and nematodes and plays an important role in suppressing soilborne plant pathogens. Although most of the genes in the 2,4-DAPG biosynthetic gene cluster (phl) have been characterized, it is still not clear how the pathway-specific regulator phlH is involved in 2,4-DAPG metabolism. This work revealed the role of PhlH in modulating 2,4-DAPG levels by controlling the expression of 2,4-DAPG hydrolase PhlG in response to 2,4-DAPG and MAPG. Since 2,4-DAPG biosynthesis imposes a metabolic burden on biocontrol pseudomonads, it is expected that the fine regulation of phlG by PhlH offers a way to dynamically modulate the metabolic loads attributed to 2,4-DAPG production.

scholarly journals Lactococcus lactis Metabolism and Gene Expression during Growth on Plant Tissues

2014 ◽  
Vol 197 (2) ◽  
pp. 371-381 ◽  
Author(s):  
Benjamin L. Golomb ◽  
Maria L. Marco
Keyword(s):  
Lactococcus Lactis ◽  
Gene Knockout ◽  
Leaf Tissue ◽  
Transcriptome Profiling ◽  
Laboratory Culture ◽  
Arginine Deiminase ◽  
Plant Tissues ◽  
Xylose Metabolism ◽  
Plant Materials ◽  
Content Type

Lactic acid bacteria have been isolated from living, harvested, and fermented plant materials; however, the adaptations these bacteria possess for growth on plant tissues are largely unknown. In this study, we investigated plant habitat-specific traits ofLactococcus lactisduring growth in anArabidopsis thalianaleaf tissue lysate (ATL).L. lactisKF147, a strain originally isolated from plants, exhibited a higher growth rate and reached 7.9-fold-greater cell densities during growth in ATL than the dairy-associated strainL. lactisIL1403. Transcriptome profiling (RNA-seq) of KF147 identified 853 induced and 264 repressed genes during growth in ATL compared to that in GM17 laboratory culture medium. Genes induced in ATL included those involved in the arginine deiminase pathway and a total of 140 carbohydrate transport and metabolism genes, many of which are involved in xylose, arabinose, cellobiose, and hemicellulose metabolism. The induction of those genes corresponded withL. lactisKF147 nutrient consumption and production of metabolic end products in ATL as measured by gas chromatography-time of flight mass spectrometry (GC-TOF/MS) untargeted metabolomic profiling. To assess the importance of specific plant-inducible genes forL. lactisgrowth in ATL, xylose metabolism was targeted for gene knockout mutagenesis. Wild-typeL. lactisstrain KF147 but not anxylAdeletion mutant was able to grow using xylose as the sole carbon source. However, both strains grew to similarly high levels in ATL, indicating redundancy inL. lactiscarbohydrate metabolism on plant tissues. These findings show that certain strains ofL. lactisare well adapted for growth on plants and possess specific traits relevant for plant-based food, fuel, and feed fermentations.

scholarly journals Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

2012 ◽  
Vol 56 (8) ◽  
pp. 4175-4183 ◽  
Author(s):  
Seung Young Kim ◽  
Kou-San Ju ◽  
William W. Metcalf ◽  
Bradley S. Evans ◽  
Tomohisa Kuzuyama ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Citrate Synthase ◽  
Wide Spectrum ◽  
The United States ◽  
Biosynthetic Gene Cluster ◽  
Gene Encoding ◽  
Acute Cystitis ◽  
Streptomyces Species ◽  
Content Type

ABSTRACTFosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production inPseudomonas syringaePB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that inStreptomyces fradiaeandStreptomyces wedmorensis. The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome ofP. syringaePB-5123 lacks a gene encoding the PnPy decarboxylase found in theStreptomycesstrains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin inPseudomonas aeruginosafrom a fosmid encoding the fosfomycin biosynthetic cluster fromP. syringaePB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent.

scholarly journals Novel Aggregative Adherence Fimbria Variant of Enteroaggregative Escherichia coli

2015 ◽  
Vol 83 (4) ◽  
pp. 1396-1405 ◽  
Author(s):  
Rie Jønsson ◽  
Carsten Struve ◽  
Nadia Boisen ◽  
Ramona Valentina Mateiu ◽  
Araceli E. Santiago ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Gene Encoding ◽  
Content Type ◽  
Fimbrial Subunit ◽  
Eaec Strain ◽  
Aggregative Adherence ◽  
In The Beginning ◽  
Important Variant ◽  
Pcr Targeting

EnteroaggregativeEscherichia coli(EAEC) organisms belong to a diarrheagenic pathotype known to cause diarrhea and can be characterized by distinct aggregative adherence (AA) in a stacked-brick pattern to cultured epithelial cells. In this study, we investigated 118 EAEC strains isolated from the stools of Danish adults with traveler's diarrhea. We evaluated the presence of the aggregative adherence fimbriae (AAFs) by a multiplex PCR, targeting the four known major subunit variants as well as their usher-encoding genes. Almost one-half (49/118) of the clinical isolates did not possess any known AAF major fimbrial subunit, despite the presence of other AggR-related loci. Further investigation revealed the presence of an AAF-related gene encoding a yet-uncharacterized adhesin, termedagg5A. The sequence of theagg5DCBAgene cluster shared fimbrial accessory genes (usher, chaperone, and minor pilin subunit genes) with AAF/III, as well as the signal peptide present in the beginning of theagg3Agene. The completeagg5DCBAgene cluster from a clinical isolate, EAEC strain C338-14, with the typical stacked-brick binding pattern was cloned, and deletion of the cluster was performed. Transformation to a nonadherentE. coliHB101 and complementation of the nonadherent C338-14 mutant with the complete gene cluster restored the AA adhesion. Overall, we found theagg5Agene in 12% of the 118 strains isolated from Denmark, suggesting that this novel adhesin represents an important variant.

scholarly journals The carB Gene Encoding the Large Subunit of Carbamoylphosphate Synthetase from Lactococcus lactis Is Transcribed Monocistronically

1998 ◽  
Vol 180 (17) ◽  
pp. 4380-4386 ◽  
Author(s):  
Jan Martinussen ◽  
Karin Hammer
Keyword(s):  
Lactococcus Lactis ◽  
Large Subunit ◽  
Arginine Deiminase ◽  
Gene Encoding ◽  
Pyrimidine Nucleotides ◽  
Reading Frame ◽  
Glutathione Peroxidases ◽  
Carbamoylphosphate Synthetase ◽  
Genes Encoding ◽  
High Degree

ABSTRACT The biosynthesis of carbamoylphosphate is catalyzed by the heterodimeric enzyme carbamoylphosphate synthetase. The genes encoding the two subunits of this enzyme in procaryotes are normally transcribed as an operon, but the gene encoding the large subunit (carB) in Lactococcus lactis is shown to be transcribed as an isolated unit. Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine. By mutant analysis,L. lactis is shown to possess only onecarB gene; the same gene product is thus required for both biosynthetic pathways. Furthermore, arginine may satisfy the requirement for carbamoylphosphate in pyrimidine biosynthesis through degradation by means of the arginine deiminase pathway. The expression of the carB gene is subject to regulation at the level of transcription by pyrimidines, most probably by an attenuator mechanism. Upstream of the carB gene, an open reading frame showing a high degree of similarity to those of glutathione peroxidases from other organisms was identified.

scholarly journals Evolution of Chemical Diversity in Echinocandin Lipopeptide Antifungal Metabolites

2015 ◽  
Vol 14 (7) ◽  
pp. 698-718 ◽  
Author(s):  
Qun Yue ◽  
Li Chen ◽  
Xiaoling Zhang ◽  
Kuan Li ◽  
Jingzu Sun ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Incomplete Lineage Sorting ◽  
Gene Clusters ◽  
Antifungal Drugs ◽  
Chemical Diversity ◽  
Specific Gene ◽  
Gene Trees ◽  
Content Type ◽  
Antifungal Metabolites ◽  
Pathway Gene

ABSTRACTThe echinocandins are a class of antifungal drugs that includes caspofungin, micafungin, and anidulafungin. Gene clusters encoding most of the structural complexity of the echinocandins provided a framework for hypotheses about the evolutionary history and chemical logic of echinocandin biosynthesis. Gene orthologs among echinocandin-producing fungi were identified. Pathway genes, including the nonribosomal peptide synthetases (NRPSs), were analyzed phylogenetically to address the hypothesis that these pathways represent descent from a common ancestor. The clusters share cooperative gene contents and linkages among the different strains. Individual pathway genes analyzed in the context of similar genes formed unique echinocandin-exclusive phylogenetic lineages. The echinocandin NRPSs, along with the NRPS from theinpgene cluster inAspergillus nidulansand its orthologs, comprise a novel lineage among fungal NRPSs. NRPS adenylation domains from different species exhibited a one-to-one correspondence between modules and amino acid specificity that is consistent with models of tandem duplication and subfunctionalization. Pathway gene trees and Ascomycota phylogenies are congruent and consistent with the hypothesis that the echinocandin gene clusters have a common origin. The disjunct Eurotiomycete-Leotiomycete distribution appears to be consistent with a scenario of vertical descent accompanied by incomplete lineage sorting and loss of the clusters from most lineages of theAscomycota. We present evidence for a single evolutionary origin of the echinocandin family of gene clusters and a progression of structural diversification in two fungal classes that diverged approximately 290 to 390 million years ago. Lineage-specific gene cluster evolution driven by selection of new chemotypes contributed to diversification of the molecular functionalities.

scholarly journals Characterization of a Novel cis-3-Hydroxy-l-Proline Dehydratase and a trans-3-Hydroxy-l-Proline Dehydratase from Bacteria

2017 ◽  
Vol 199 (16) ◽  
Author(s):  
Seiya Watanabe ◽  
Fumiyasu Fukumori ◽  
Mao Miyazaki ◽  
Shinya Tagami ◽  
Yasuo Watanabe
Keyword(s):  
Gene Cluster ◽  
Active Sites ◽  
Plant Cell Wall ◽  
Physiological Role ◽  
Type I ◽  
Peptide Antibiotics ◽  
Gene Encoding ◽  
Content Type ◽  
Metabolic Genes ◽  
Enzyme Functions

ABSTRACT Hydroxyprolines, such as trans-4-hydroxy-l-proline (T4LHyp), trans-3-hydroxy-l-proline (T3LHyp), and cis-3-hydroxy-l-proline (C3LHyp), are present in some proteins including collagen, plant cell wall, and several peptide antibiotics. In bacteria, genes involved in the degradation of hydroxyproline are often clustered on the genome (l-Hyp gene cluster). We recently reported that an aconitase X (AcnX)-like hypI gene from an l-Hyp gene cluster functions as a monomeric C3LHyp dehydratase (AcnXType I). However, the physiological role of C3LHyp dehydratase remained unclear. We here demonstrate that Azospirillum brasilense NBRC 102289, an aerobic nitrogen-fixing bacterium, robustly grows using not only T4LHyp and T3LHyp but also C3LHyp as the sole carbon source. The small and large subunits of the hypI gene (hypI S and hypI L, respectively) from A. brasilense NBRC 102289 are located separately from the l-Hyp gene cluster and encode a C3LHyp dehydratase with a novel heterodimeric structure (AcnXType IIa). A strain disrupted in the hypI S gene did not grow on C3LHyp, suggesting its involvement in C3LHyp metabolism. Furthermore, C3LHyp induced transcription of not only the hypI genes but also the hypK gene encoding Δ1-pyrroline-2-carboxylate reductase, which is involved in T3LHyp, d-proline, and d-lysine metabolism. On the other hand, the l-Hyp gene cluster of some other bacteria contained not only the AcnXType IIa gene but also two putative proline racemase-like genes (hypA1 and hypA2). Despite having the same active sites (a pair of Cys/Cys) as hydroxyproline 2-epimerase, which is involved in the metabolism of T4LHyp, the dominant reaction by HypA2 was clearly the dehydration of T3LHyp, a novel type of T3LHyp dehydratase that differed from the known enzyme (Cys/Thr). IMPORTANCE More than 50 years after the discovery of trans-4-hydroxy-l-proline (generally called l-hydroxyproline) degradation in aerobic bacteria, its genetic and molecular information has only recently been elucidated. l-Hydroxyproline metabolic genes are often clustered on bacterial genomes. These loci frequently contain a hypothetical gene(s), whose novel enzyme functions are related to the metabolism of trans-3-hydroxyl-proline and/or cis-3-hydroxyl-proline, a relatively rare l-hydroxyproline in nature. Several l-hydroxyproline metabolic enzymes show no sequential similarities, suggesting their emergence by convergent evolution. Furthermore, transcriptional regulation by trans-4-hydroxy-l-proline, trans-3-hydroxy-l-proline, and/or cis-3-hydroxy-l-proline significantly differs between bacteria. The results of the present study show that several l-hydroxyprolines are available for bacteria as carbon and energy sources and may contribute to the discovery of potential metabolic pathways of another hydroxyproline(s).

scholarly journals Molecular Basis for Mycophenolic Acid Biosynthesis in Penicillium brevicompactum

2011 ◽  
Vol 77 (9) ◽  
pp. 3035-3043 ◽  
Author(s):  
Torsten Bak Regueira ◽  
Kanchana Rueksomtawin Kildegaard ◽  
Bjarne Gram Hansen ◽  
Uffe H. Mortensen ◽  
Christian Hertweck ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Mycophenolic Acid ◽  
Molecular Basis ◽  
Polyketide Synthase ◽  
Functional Characterization ◽  
Gene Encoding ◽  
Biosynthesis Gene Cluster ◽  
Content Type ◽  
Polyketide Synthase Gene ◽  
Penicillium Brevicompactum

ABSTRACTMycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production inPenicillium brevicompactum. mpaCresides in what most likely is a 25-kb gene cluster in the genome ofPenicillium brevicompactum. The gene cluster was successfully localized by targeting putative resistance genes, in this case an additional copy of the gene encoding IMP dehydrogenase (IMPDH). We report the cloning, sequencing, and the functional characterization of the MPA biosynthesis gene cluster by deletion of the polyketide synthase genempaCofP. brevicompactumand bioinformatic analyses. As expected, the gene deletion completely abolished MPA production as well as production of several other metabolites derived from the MPA biosynthesis pathway ofP. brevicompactum. Our work sets the stage for engineering the production of MPA and analogues through metabolic engineering.

scholarly journals LanI-Mediated Lantibiotic Immunity inBacillus subtilis: Functional Analysis

2019 ◽  
Vol 85 (11) ◽  
Author(s):  
Christoph Geiger ◽  
Sophie Marianne Korn ◽  
Michael Häsler ◽  
Oliver Peetz ◽  
Janosch Martin ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Lactococcus Lactis ◽  
Abc Transporters ◽  
Extracellular Space ◽  
Pore Formation ◽  
Physiological Function ◽  
Cellular Membrane ◽  
Physical Interaction ◽  
Terminal Part ◽  
Content Type

ABSTRACTLantibiotics subtilin and nisin are produced byBacillus subtilisandLactococcus lactis, respectively. To prevent toxicity of their own lantibiotic, both bacteria express specific immunity proteins, called SpaI and NisI. In addition, ABC transporters SpaFEG and NisFEG prevent lantibiotic toxicity by transporting the respective peptides to the extracellular space. Although the three-dimensional structures of SpaI and NisI have been solved, very little is known about the molecular function of either lipoprotein. Using laser-induced liquid bead ion desorption (LILBID)–mass spectrometry, we show here that subtilin interacts with SpaI monomers. The expression of either SpaI or NisI in a subtilin-nonproducingB. subtilisstrain resulted in the respective strain being more resistant against either subtilin or nisin. Furthermore, pore formation provided by subtilin and nisin was prevented specifically upon the expression of either SpaI or NisI. As shown with a nisin-subtilin hybrid molecule, the C-terminal part of subtilin but not any particular lanthionine ring was needed for SpaI-mediated immunity. With respect to growth, SpaI provided less immunity against subtilin than is provided by the ABC transporter SpaFEG. However, SpaI prevented pore formation much more efficiently than SpaFEG. Taken together, our data show the physiological function of SpaI as a fast immune response to protect the cellular membrane.IMPORTANCEThe two lantibiotics nisin and subtilin are produced byLactococcus lactisandBacillus subtilis, respectively. Both peptides have strong antimicrobial activity against Gram-positive bacteria, and therefore, appropriate protection mechanisms are required for the producing strains. To prevent toxicity of their own lantibiotic, both bacteria express immunity proteins, called SpaI and NisI, and in addition, ABC transporters SpaFEG and NisFEG. Whereas it has been shown that the ABC transporters protect the producing strains by transporting the toxic peptides to the extracellular space, the exact mode of action and the physiological function of the lipoproteins during immunity are still unknown. Understanding the exact role of lantibiotic immunity proteins is of major importance for improving production rates and for the design of newly engineered peptide antibiotics. Here, we show (i) the specificity of each lipoprotein for its own lantibiotic, (ii) the specific physical interaction of subtilin with its lipoprotein SpaI, (iii) the physiological function of SpaI in protecting the cellular membrane, and (iv) the importance of the C-terminal part of subtilin for its interaction with SpaI.

scholarly journals Engineering Trehalose Synthesis in Lactococcus lactis for Improved Stress Tolerance

2011 ◽  
Vol 77 (12) ◽  
pp. 4189-4199 ◽  
Author(s):  
Ana Lúcia Carvalho ◽  
Filipa S. Cardoso ◽  
Andreas Bohn ◽  
Ana Rute Neves ◽  
Helena Santos
Keyword(s):  
Lactococcus Lactis ◽  
Oral Drug Delivery ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Ph Control ◽  
Oral Drug ◽  
Resting Cells ◽  
Content Type ◽  
Food Grade ◽  
Cell Defense

ABSTRACTTrehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose inPropionibacterium freudenreichiiin response to acid stress, a result that led to the idea that endowingLactococcus lactiswith the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway ofL. lactis, i.e.,trePPandpgmB, encoding trehalose 6-phosphate phosphorylase and β-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given thatL. lactislacks trehalose 6-phosphate phosphatase, the respective gene,otsB, from the food-grade organismP. freudenreichiiwas used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4°C); there was also a strong improvement in cell survival in response to heat shock (45°C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery.

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