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1.
Food Microbiol ; 87: 103392, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31948633

RESUMO

Genetic diversity and metabolic properties of Lactococcus lactis subsp. lactis were explored using phylogenetic, pan-genomic and metatranscriptomic analysis. The genomes, used in the current study, were available and downloaded from the GenBank which were primarily related with microorganisms isolated from dairy products and secondarily from other foodstuffs. To study the genetic diversity of the microorganism, various bioinformatics tools were employed such as average nucleotide identity, digital DNA-DNA hybridization, phylogenetic analysis, clusters of orthologous groups analysis, KEGG orthology analysis and pan-genomic analysis. The results showed that Lc. lactis subsp. lactis strains cannot be sufficiently separated into phylogenetic lineages based on the 16S rRNA gene sequences and core genome-based phylogenetic analysis was more appropriate. Pan-genomic analysis of the strains indicated that the core, accessory and unique genome comprised of 1036, 3146 and 1296 genes, respectively. Considering the results of pan-genomic and KEGG orthology analyses, the metabolic network of Lc. lactis subsp. lactis was rebuild regarding its carbohydrates' metabolic capabilities. Based on the metatranscriptomic data during the ripening of the Swiss-type Maasdam cheese at 20 °C and 5 °C, it was shown that the microorganism performed mixed acid fermentation producing lactate, formate, acetate, ethanol and 2,3-butanediol. Mixed acid fermentation was more pronounced at higher ripening temperatures. At lower ripening temperatures, the genes involved in mixed acid fermentation were repressed while lactate production remained unaffected resembling to a homolactic fermentation. Comparative genomics and metatranscriptomic analysis are powerful tools to gain knowledge on the genomic diversity of the lactic acid bacteria used as starter cultures as well as on the metabolic activities occurring in fermented dairy products.


Assuntos
Metabolismo dos Carboidratos , Queijo/microbiologia , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Carboidratos/química , Fermentação , Microbiologia de Alimentos , Variação Genética , Genômica , Lactococcus lactis/classificação , Lactococcus lactis/isolamento & purificação , Filogenia
2.
Microb Cell Fact ; 18(1): 217, 2019 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-31884954

RESUMO

BACKGROUND: Diacetyl provides the buttery aroma in products such as butter and margarine. It can be made via a harsh set of chemical reactions from sugarcane bagasse, however, in dairy products it is normally formed spontaneously from α-acetolactate, a compound generated by selected lactic acid bacteria in the starter culture used. Due to its bacteriostatic properties, it is difficult to achieve high levels of diacetyl by fermentation. Here we present a novel strategy for producing diacetyl based on whole-cell catalysis, which bypasses the toxic effects of diacetyl. RESULTS: By expressing a robust α-acetolactate synthase (ALS) in a metabolically optimized Lactococcus lactis strain we obtained a whole-cell biocatalyst that efficiently converted pyruvate into α-acetolactate. After process optimization, we achieved a titer for α-acetolactate of 172 ± 2 mM. Subsequently we used a two-stage production setup, where pyruvate was produced by an engineered L. lactis strain and subsequently used as the substrate for the biocatalyst. Using this approach, 122 ± 5 mM and 113 ± 3 mM α-acetolactate could be made from glucose or lactose in dairy waste, respectively. The whole-cell biocatalyst was robust and fully active in crude fermentation broth containing pyruvate. CONCLUSIONS: An efficient approach for converting sugar into α-acetolactate, via pyruvate, was developed and tested successfully. Due to the anaerobic conditions used for the biotransformation, little diacetyl was generated, and this allowed for efficient biotransformation of pyruvate into α-acetolactate, with the highest titers reported to date. The use of a two-step procedure for producing α-acetolactate, where non-toxic pyruvate first is formed, and subsequently converted into α-acetolactate, also simplified the process optimization. We conclude that whole cell catalysis is suitable for converting lactose in dairy waste into α-acetolactate, which favors resource utilization.


Assuntos
Lactatos/metabolismo , Lactococcus lactis/metabolismo , Ácido Pirúvico/metabolismo , Acetolactato Sintase/genética , Acetolactato Sintase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Laticínios/análise , Fermentação , Glucose/metabolismo , Lactococcus lactis/genética , Lactose/metabolismo , Resíduos
3.
World J Microbiol Biotechnol ; 35(12): 185, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31728760

RESUMO

Glutathione (GSH) and S-adenosyl methionine (SAM) have been applied as liver-protective factors to prevent and treat many different liver damages and diseases. Due to their low stability and short half-life, oral administration of GSH or SAM might be replaced by continuous supplying through living lactic bacteria in yogurt. In this study, Lactococcus lactis was engineered via synthetic biology strategies to produce these two important molecules. The bi-functional GSH synthase gene (gshF) and SAM synthase gene (metK) were transformed into food-grade L. lactis together with an adhesion factor gene (cwaA). The highest accumulation of SAM (9.0 mg/L) and GSH (17.3 mg/L) was achieved after 17 h cultivation of the recombinant L. lactis. Meanwhile, the autoaggregation and hydrophobicity were also improved significantly, which suggested that this engineered L. lactis might have an increased colonization-prone ability in human GI. Our studies demonstrated one potential route to self-produce and deliver the liver-healthy factors within living probiotic bacteria.


Assuntos
Glutationa/metabolismo , Lactococcus lactis/metabolismo , Engenharia Metabólica/métodos , S-Adenosilmetionina/metabolismo , Adesinas Bacterianas/genética , Vias Biossintéticas , Fermentação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Lactococcus lactis/enzimologia , Lactococcus lactis/genética , Lactococcus lactis/crescimento & desenvolvimento , Metionina Adenosiltransferase/genética , Nisina/metabolismo , Probióticos
4.
Nat Commun ; 10(1): 4304, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541094

RESUMO

The widespread Mn2+-sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch aptamer from Xanthomonas oryzae at 2.96 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >100 µs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.


Assuntos
Magnésio/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Riboswitch/fisiologia , Transdução de Sinais , Xanthomonas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Ligantes , Manganês/metabolismo , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Conformação de Ácido Nucleico , RNA Bacteriano/genética
5.
Nat Commun ; 10(1): 3552, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391532

RESUMO

CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Edição de Genes , Microscopia/métodos , Plasmídeos/genética , Imagem Individual de Molécula/métodos , Proteína 9 Associada à CRISPR/genética , Dosagem de Genes , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia/instrumentação , Modelos Genéticos , Método de Monte Carlo , Motivos de Nucleotídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Imagem Individual de Molécula/instrumentação , Fatores de Tempo
6.
Microb Cell Fact ; 18(1): 129, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387603

RESUMO

BACKGROUND: Vitamin K2 (menaquinone, MK-n) is a lipid-soluble vitamin that functions as a carboxylase co-factor for maturation of proteins involved in many vital physiological processes in humans. Notably, long-chain vitamin K2 is produced by bacteria, including some species and strains belonging to the group of lactic acid bacteria (LAB) that play important roles in food fermentation processes. This study was performed to gain insights into the natural long-chain vitamin K2 production capacity of LAB and the factors influencing vitamin K2 production during cultivation, providing a basis for biotechnological production of vitamin K2 and in situ fortification of this vitamin in food products. RESULTS: We observed that six selected Lactococcus lactis strains produced MK-5 to MK-10, with MK-8 and MK-9 as the major MK variant. Significant diversities between strains were observed in terms of specific concentrations and titres of vitamin K2. L. lactis ssp. cremoris MG1363 was selected for more detailed studies of the impact of selected carbon sources tested under different growth conditions [i.e. static fermentation (oxygen absent, heme absent); aerobic fermentation (oxygen present, heme absent) and aerobic respiration (oxygen present, heme present)] on vitamin K2 production in M17 media. Aerobic fermentation with fructose as a carbon source resulted in the highest specific concentration of vitamin K2: 3.7-fold increase compared to static fermentation with glucose, whereas aerobic respiration with trehalose resulted in the highest titre: 5.2-fold increase compared to static fermentation with glucose. When the same strain was applied to quark fermentation, we consistently observed that altered carbon source (fructose) and aerobic cultivation of the pre-culture resulted in efficient vitamin K2 fortification in the quark product. CONCLUSIONS: With this study we demonstrate that certain LAB strains can be employed for efficient production of long-chain vitamin K2. Strain selection and optimisation of growth conditions offer a viable strategy towards natural vitamin K2 enrichment of fermented foods, and to improved biotechnological vitamin K2 production processes.


Assuntos
Carbono/metabolismo , Metabolismo Energético , Lactococcus lactis/metabolismo , Oxigênio/metabolismo , Temperatura Ambiente , Vitamina K 2/metabolismo , Meios de Cultura , Fermentação , Glucose/metabolismo , Microbiologia Industrial
7.
Braz J Microbiol ; 50(4): 915-925, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31385286

RESUMO

The production of a highly concentrated probiotic preparation of Lactococcus lactis CECT 539 was studied in both batch and realkalized fed-batch fermentations in diluted whey (DW) media (DW25, DW50, DW75, DW100, and DW125) supplemented with MRS broth nutrients (except glucose and Tween 80) at 25, 50, 75, 100, and 125% of their standard concentrations in the complex medium. The fed-batch culture using DW100 medium provided the highest concentrations of probiotic biomass (5.98 g/L) and nisin (258.47 BU/mL), which were obtained at lower production costs than those estimated for the fed-batch culture in DW medium. The batch and fed-batch cultures reduced the initial chemical oxygen demand (COD) of the media by 29.1-41.7% and 31.2-54.2%, respectively. Graphical abstract.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Lactococcus lactis/crescimento & desenvolvimento , Lactococcus lactis/metabolismo , Nisina/biossíntese , Probióticos/química , Proteínas do Soro do Leite/metabolismo , Biomassa , Meios de Cultura/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Probióticos/metabolismo
8.
Microb Cell Fact ; 18(1): 136, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409416

RESUMO

BACKGROUND: Microbial cell factories are widely used in the production of acidic products such as organic acids and amino acids. However, the metabolic activity of microbial cells and their production efficiency are severely inhibited with the accumulation of intracellular acidic metabolites. Therefore, it remains a key issue to enhance the acid tolerance of microbial cells. In this study, we investigated the effects of four ATP-binding cassette (ABC) transporters on acid stress tolerance in Lactococcus lactis. RESULTS: Overexpressing the rbsA, rbsB, msmK, and dppA genes exhibited 5.8-, 12.2-, 213.7-, and 5.2-fold higher survival rates than the control strain, respectively, after acid shock for 3 h at pH 4.0. Subsequently, transcriptional profile alterations in recombinant strains were analyzed during acid stress. The differentially expressed genes associated with cold-shock proteins (csp), fatty acid biosynthesis (fabH), and coenzyme A biosynthesis (coaD) were up-regulated in the four recombinant strains during acid stress. Additionally, some genes were differentially expressed in specific recombinant strains. For example, in L. lactis (RbsB), genes involved in the pyrimidine biosynthetic pathway (pyrCBDEK) and glycine or betaine transport process (busAA and busAB) were up-regulated during acid stress, and the argG genes showed up-regulations in L. lactis (MsmK). Finally, we found that overexpression of the ABC transporters RbsB and MsmK increased intracellular ATP concentrations to protect cells against acidic damage in the initial stage of acid stress. Furthermore, L. lactis (MsmK) consistently maintained elevated ATP concentrations under acid stress. CONCLUSIONS: This study elucidates the common and specific mechanisms underlying improved acid tolerance by manipulating ABC transporters and provides a further understanding of the role of ABC transporters in acid-stress tolerance.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Ácidos/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Estresse Fisiológico , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Expressão Gênica , Engenharia Genética
9.
J Ind Microbiol Biotechnol ; 46(11): 1621-1629, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31414323

RESUMO

During fermentation, acid stress caused by the accumulation of acidic metabolites seriously affects the metabolic activity and production capacity of microbial cells. To elucidate the acid stress-tolerance mechanisms of microbial cells, we performed genome mutagenesis combined with high-throughput technologies to screen acid stress-tolerant strains. Mutant strain Lactococcus lactis WH101 showed a 16,000-fold higher survival rate than that of the parent strain after 5 h of acid shock at pH 4.0 and maintained higher ATP, NH4+, and intracellular pH (pHi) levels during acid stress. Additionally, comparative transcriptomics analysis revealed enhanced regulation of carbohydrate metabolism and sugar transport to provide additional energy, amino acid metabolism and transport to maintain pHi homeostasis and ATP generation, and fatty acid metabolism to enhance cellular acid tolerance. Moreover, overexpression of identified components resulted in 12.6- and 12.9-fold higher survival rates after acid shock for 3 h at pH 4.0 in L. lactis (ArcB) and L. lactis (MalQ) compared to the control strain, respectively. These findings provide valuable insight into the acid stress-response mechanisms of L. lactis and promote the further development of robust industrial strains.


Assuntos
Lactococcus lactis/metabolismo , Ácidos/farmacologia , Fermentação , Homeostase , Concentração de Íons de Hidrogênio , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/genética , Estresse Fisiológico , Transcriptoma
10.
Nature ; 572(7771): 665-669, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31435014

RESUMO

Intestinal commensal bacteria can inhibit dense colonization of the gut by vancomycin-resistant Enterococcus faecium (VRE), a leading cause of hospital-acquired infections1,2. A four-strained consortium of commensal bacteria that contains Blautia producta BPSCSK can reverse antibiotic-induced susceptibility to VRE infection3. Here we show that BPSCSK reduces growth of VRE by secreting a lantibiotic that is similar to the nisin-A produced by Lactococcus lactis. Although the growth of VRE is inhibited by BPSCSK and L. lactis in vitro, only BPSCSK colonizes the colon and reduces VRE density in vivo. In comparison to nisin-A, the BPSCSK lantibiotic has reduced activity against intestinal commensal bacteria. In patients at high risk of VRE infection, high abundance of the lantibiotic gene is associated with reduced density of E. faecium. In germ-free mice transplanted with patient-derived faeces, resistance to VRE colonization correlates with abundance of the lantibiotic gene. Lantibiotic-producing commensal strains of the gastrointestinal tract reduce colonization by VRE and represent potential probiotic agents to re-establish resistance to VRE.


Assuntos
Bacteriocinas/metabolismo , Bacteriocinas/farmacologia , Enterococcus faecium/efeitos dos fármacos , Lactococcus lactis/metabolismo , Probióticos , Resistência a Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Animais , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacteriocinas/genética , Bacteriocinas/isolamento & purificação , Enterococcus faecium/crescimento & desenvolvimento , Enterococcus faecium/isolamento & purificação , Fezes/microbiologia , Feminino , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Vida Livre de Germes , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Humanos , Lactococcus lactis/química , Lactococcus lactis/crescimento & desenvolvimento , Lactococcus lactis/fisiologia , Camundongos , Testes de Sensibilidade Microbiana , Microbiota/genética , Nisina/química , Nisina/farmacologia , Simbiose/efeitos dos fármacos , Vancomicina/farmacologia , Enterococos Resistentes à Vancomicina/crescimento & desenvolvimento , Enterococos Resistentes à Vancomicina/isolamento & purificação
11.
Appl Microbiol Biotechnol ; 103(18): 7567-7581, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31367857

RESUMO

The molecular weight (Mw) of hyaluronic acid (HA) determines its suitability for medical and cosmetic applications. Here, we characterize in vitro and in vivo HA synthesis of streptococcal HA synthases (HASs) with a special focus on HA Mw. To date, four streptococcal HA producers are described (Streptococcus equi subsp. equi, S. equi subsp. zooepidemicus, S. pyogenes, and S. uberis). We identified two more potential HA producers in this study: S. iniae and S. parauberis. Indeed, the HA Mw produced by the different streptococcal HASs differs in vitro. To exploit these different HA Mw synthesis capacities, Lactococcus lactis strains expressing the streptococcal HASs were constructed. HA of different Mw was also produced in vivo by these engineered strains, strongly suggesting that the protein sequences of the HASs influence HA Mw. Since the HA Mw in vivo is also influenced by metabolic factors like specific growth rate and HA precursor availability, these were also determined. In summary, the maximal Mw of HA synthesized is specific for the individual synthase, while any decrease from the maximal HA Mw is influenced by physiological and metabolic factors. The results open new avenues for Mw-tailored HA synthesis.


Assuntos
Proteínas de Bactérias/metabolismo , Hialuronan Sintases/metabolismo , Ácido Hialurônico/biossíntese , Streptococcus/enzimologia , Proteínas de Bactérias/genética , Hialuronan Sintases/genética , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Peso Molecular , Streptococcus/genética
12.
J Dairy Sci ; 102(11): 9763-9766, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31447143

RESUMO

The effects of unfavorable conditions responsible for the viability loss of Lactococcus cells during spray drying can be minimized by the application of dairy matrices as encapsulating materials. This study aimed to evaluate the use of dairy matrices with different lactose contents on the survival of Lactococcus lactis during drying and storage. The use of hydrolyzed-lactose milk resulted in notable loss of cell viability (3.90 log cycles). However, milk enriched with lactose or without fat showed better protection (viability loss between 0.26 and 1.41 log cfu/g) and greater cell survival during storage at room temperature. The enrichment of milk with lactose seems to be ideal for the drying of heat-sensitive bacteria.


Assuntos
Dessecação/métodos , Lactococcus lactis/metabolismo , Lactose/metabolismo , Leite/metabolismo , Análise de Variância , Animais , Temperatura Alta , Hidrólise , Leite/química
13.
Appl Microbiol Biotechnol ; 103(19): 7931-7941, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31456001

RESUMO

Ulcerative colitis (UC) is one of the two major forms of inflammatory bowel disease (IBD) characterized by superficial mucosal inflammation, rectal bleeding, diarrhea, and abdominal pain. Anti-inflammatory and immunosuppressive drugs have been used in the therapy of human UC. Interleukin (IL)-35, which functions as an anti-inflammatory cytokine, has been shown to play a potential therapeutic role in a UC-like mouse colitis induced by dextran sodium sulfate (DSS). However, the contribution of IL-35 via oral administration to colitis prevention has not been determined. In order to explore its preventative potentiality, a dairy Lactococcus lactis NZ9000 strain was engineered to express murine IL-35 (NZ9000/IL-35), and this recombinant bacteria was applied to prevent and limit the development of DSS-induced mouse colitis. We found that oral administration of NZ9000/IL-35 induced the accumulation of IL-35 in the gut lumen of normal mice. When administrated preventatively, NZ9000/IL-35-gavaged mice exhibited decreased weight loss, DAI score, colon shortening as well as colitis-associated histopathological changes in colon, indicating that the oral administration of NZ9000/35 contributed to the suppression of DSS-induced colitis progression. Moreover, much less Th17 cells and higher level of Treg cells in lamina propria, as well as increased colon and serum levels of IL-10 with a concomitant reduced pro-inflammatory cytokines, IL-6, IL-17A, IFN-γ, and TNF-α were apparently regulated by NZ9000/IL-35 in colitis mice. Together, we put forward direct evidence pinpointing the effectiveness of NZ9000/IL-35 in preventing UC-like mouse colitis, implying a potential candidate of this recombinant Lactococcus lactis that prevent the progression of IBD.


Assuntos
Colite Ulcerativa/prevenção & controle , Fatores Imunológicos/metabolismo , Interleucinas/metabolismo , Lactococcus lactis/metabolismo , Administração Oral , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/patologia , Colo/patologia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Fatores Imunológicos/genética , Interleucinas/genética , Mucosa Intestinal/patologia , Lactococcus lactis/genética , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Resultado do Tratamento
14.
Tuberculosis (Edinb) ; 117: 24-30, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31378264

RESUMO

The aim of this study was to determine the reliability of lactic acid bacteria (LAB) as heterologous hosts for the expression of MPB70 and MPB83, two Mycobacterium bovis antigens that possess diagnostics and immunogenic properties, respectively. We therefore generated recombinant cells of Lactococcus lactis and Lactobacillus plantarum that carried hybrid genes encoding MPB70 and MPB83 fused to signal peptides that are specifically recognized by LAB. Only L. lactis was able to secrete MPB70 using the L. lactis signal peptide Usp45, and to produce MPB83 as an immunogenic membrane protein following its expression with the signal peptide of the L. plantarum lipoprotein prsA. Inactivated cells of MPB83-expressing L. lactis cultures enhanced NF-κB activation in macrophages. Our results show that L. lactis is a reliable host for the secretion and functional expression of antigens that are naturally produced by M. bovis, the causative agent of bovine tuberculosis (bTB). This represents the first step on a long process to establishing whether recombinant LAB could serve as a food-grade platform for potential diagnostic tools and/or vaccine interventions for use against bTB, a chronic disease that primarily affects cattle but also humans and a wide range of domestic and wild animals.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Lactobacillales/metabolismo , Proteínas de Membrana/metabolismo , Mycobacterium bovis/imunologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Células Cultivadas , DNA Bacteriano/biossíntese , Expressão Gênica , Vetores Genéticos/imunologia , Humanos , Lactobacillales/genética , Lactobacillus plantarum/genética , Lactobacillus plantarum/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/genética , NF-kappa B/metabolismo , Recombinação Genética
15.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31298208

RESUMO

Lactococcus lactis is the best characterized species among the lactococci, and among the most consumed food-fermenting bacteria worldwide. Thanks to their importance in industrialized food production, lactococci are among the lead bacteria understood for fundamental metabolic pathways that dictate growth and survival properties. Interestingly, lactococci belong to the Streptococcaceae family, which includes food, commensal and virulent species. As basic metabolic pathways (e.g., respiration, metal homeostasis, nucleotide metabolism) are now understood to underlie virulence, processes elucidated in lactococci could be important for understanding pathogen fitness and synergy between bacteria. This chapter highlights major findings in lactococci and related bacteria, and covers five themes: distinguishing features of lactococci, metabolic capacities including the less known respiration metabolism in Streptococcaceae, factors and pathways modulating stress response and fitness, interbacterial dialogue via metabolites, and novel applications in health and biotechnology.


Assuntos
Lactococcus lactis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fermentação , Lactococcus lactis/classificação , Lactococcus lactis/metabolismo , Redes e Vias Metabólicas
16.
Appl Microbiol Biotechnol ; 103(17): 6989-7001, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31267232

RESUMO

The molecular weight of hyaluronic acid (HA) is a critical property which determines its usage in various biomedical applications. This study investigates the correlation between the availability of a critical cofactor, acetyl-CoA, the concentration of a limiting precursor, UDP-N-acetylglucosamine (UDP-GlcNAc), and the molecular weight of HA (MWHA) produced by recombinant Lactococcus lactis MKG6 cultures. This strain expressed three heterologous HA-pathway genes obtained from the has operon of Streptococcus zooepidemicus in an ldh-mutant host strain, L. lactis NZ9020. A flux balance analysis, performed using the L. lactis genome-scale metabolic network, showed a positive correlation of acetyl-CoA flux with the UDP-GlcNAc flux and the experimental data on HA productivity. To increase the intracellular levels of acetyl-CoA, acetate was supplemented as a pulse feed in anaerobic batch cultures. However, acetate is effectively utilized only in the presence of glucose and exhaustion of glucose resulted in decreasing the final MWHA (1.5 MDa). Co-supplementation of acetate resulted in enhancing the acetyl-CoA and UDP-GlcNAc levels as well as the MWHA to 2.5 MDa. This logic was extended to fed-batch cultures, designed with a pH-based feedback control of glucose feeding and pulse acetate supplementation. When the glucose feed concentration was optimally adjusted to prevent glucose exhaustion or accumulation, the acetate utilization was found to be high, resulting in significantly enhanced levels of acetyl-CoA and UDP-GlcNAc as well as a MWHA of 3.4 MDa, which was sustained at this value throughout the process. This study provides the possibility of commercially producing high MWHA using recombinant L. lactis strains.


Assuntos
Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Ácido Hialurônico/biossíntese , Ácido Hialurônico/química , Lactococcus lactis/metabolismo , Acetatos/análise , Proteínas de Bactérias/genética , Técnicas de Cultura Celular por Lotes , Meios de Cultura/química , Meios de Cultura/metabolismo , Glucose/análise , Glucose/metabolismo , Lactococcus lactis/genética , Análise do Fluxo Metabólico , Redes e Vias Metabólicas/genética , Peso Molecular , Proteínas Recombinantes/genética , Streptococcus equi/genética , Uridina Difosfato N-Acetilglicosamina/metabolismo
17.
Curr Microbiol ; 76(10): 1186-1192, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31302724

RESUMO

Bioprocess development is a current requirement to enhance the global production of D-lactic acid. Herein, we report a new bioprocess for D-lactic acid production directly from starch using engineered Lactococcus lactis NZ9000. To modify L. lactis as a D-lactic acid producer, its major endogenous L-lactate dehydrogenase (L-Ldh) gene was replaced with a heterologous D-Ldh gene from Lactobacillus delbrueckii subsp. lactis JCM 1107. The resulting strain AH1 showed a somewhat slower growth rate but similar lactic acid production compared to those of the intact strain when cultivated with glucose as a carbon source. The chemical purity of D-lactic acid produced by L. lactis AH1 was 93.8%, and the enzymatic activities of D- and L-Ldh in AH1 were 1.54 U/mL and 0.05 U/mL, respectively. Next, a heterologous α-amylase gene from Streptococcus bovis NRIC 1535 cloned into an expression vector pNZ8048 was introduced into AH1. The resulting strain AH2 showed an amylolytic activity of 0.26 U/mL in the culture supernatant. Direct production of D-lactic acid from starch as the carbon source was demonstrated using L. lactis AH2, resulting in D-lactic acid production at a concentration of 15.0 g/L after 24 h cultivation. To our knowledge, this is the first report on D-lactic acid production in engineered L. lactis.


Assuntos
Engenharia Genética , Ácido Láctico/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Amido/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fermentação , Glucose/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenases/genética , Lactato Desidrogenases/metabolismo , Lactobacillus delbrueckii/genética , Lactococcus lactis/enzimologia , Lactococcus lactis/crescimento & desenvolvimento , Streptococcus bovis/genética , alfa-Amilases/genética , alfa-Amilases/metabolismo
18.
PLoS One ; 14(6): e0218508, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31220124

RESUMO

Small regulatory RNAs (sRNAs) and their enormous potential and versatility have provided us with an astounding insight in the complexity of bacterial transcriptomes. sRNAs have been shown to be involved in a variety of cellular processes that range from stress to general metabolism. Here we report that the gene encoding the transcriptional regulator ArgR is immediately followed by the gene of the small regulatory RNA ArgX. The latter is transcribed from its own promoter. The production of ArgX is induced by increasing arginine concentrations and repressed by CcpA. Previously, ArgR was shown to act as a transcriptional repressor of the catabolic arginine deiminase pathway (arc operon) by binding in the promoter region of arcA. Here we demonstrate that ArgX downregulates arc mRNA levels. Furthermore, ArgX putatively blocks the translation of one of the genes in the operon, arcC1, a process that would redirect an intermediate in arginine degradation, carbamoyl phosphate, towards pyrimidine synthesis. Our findings exemplify, for the first time, the combinatorial power of a transcription factor and a small regulatory RNA derived from the 3'-UTR region. The regulators ArgR and ArgX share a common target, but act on transcription and on RNA level, respectively.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/genética , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Regiões 3' não Traduzidas , Arginina/metabolismo , Proteínas de Bactérias/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Lactococcus lactis/metabolismo , Óperon , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo
19.
Lett Appl Microbiol ; 69(3): 181-189, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31220356

RESUMO

Clovamide and its analogues are N-hydroxycinnamoyl-L-amino acids (HAA) that exhibit antioxidant activities. For environmental and economic reasons, biological synthesis of these plant-derived metabolites has garnered interest. In this study, we exploited HDT1, a BAHD acyltransferase recently isolated from red clover, for the production of clovamide and derivatives in S. cerevisiae and L. lactis. HDT1 catalyses the transfer of hydroxycinnamoyl-coenzyme A (CoA) onto aromatic amino acids. Therefore, by heterologously co-expressing HDT1 with 4-coumarate:CoA ligase (4CL), we succeeded in the biological production of clovamide and more than 20 other HAA, including halogenated ones, upon feeding the engineered micro-organisms with various combinations of cinnamates and amino acids. To the best of our knowledge, this is the first report on the biological synthesis of HAA and, more generally, on the synthesis of plant-derived antioxidant phenolic compounds in L. lactis. The production of these health beneficial metabolites in Generally Recognized As Safe (GRAS) micro-organisms such as S. cerevisiae and L. lactis provides new options for their delivery as therapeutics. SIGNIFICANCE AND IMPACT OF THE STUDY: N-hydroxycinnamoyl-L-amino acids such as clovamide are bioactive plant-derived phenolic compounds with health beneficial effects. Relying on chemical synthesis or direct extraction from plant sources for the supply of these valuable molecules poses challenges to environmental sustainability. As an alternative route, this work demonstrates the potential for biological synthesis of N-hydroxycinnamoyl-L-amino acids using engineered microbial hosts such as Saccharomyces cerevisiae and Lactococcus lactis. Besides being more eco-friendly, this approach should also provide more structurally diverse compounds and offer new methods for their delivery to the human body.


Assuntos
Lactococcus lactis/metabolismo , Saccharomyces cerevisiae/metabolismo , Tirosina/análogos & derivados , Aciltransferases/metabolismo , Antioxidantes , Humanos , Tirosina/biossíntese
20.
BMC Biotechnol ; 19(1): 38, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31238939

RESUMO

BACKGROUND: Anti-Tumor Necrosis Factor-alpha therapy has become clinically important for treating inflammatory bowel disease. However, the use of conventional immunotherapy requires a systemic exposure of patients and collateral side effects. Lactic acid bacteria have been shown to be effective as mucosal delivering system for cytokine and single domain antibodies, and it is amenable to clinical purposes. Therefore, lactic acid bacteria may function as vehicles for delivery of therapeutic antibodies molecules to the gastrointestinal tract restricting the pharmacological effect towards the gut. Here, we use the mucosal delivery of Lactococcus lactis carrying an anti-TNFα scFv expression plasmid on a DSS-induced colitis model in mice. RESULTS: Experimental colitis was induced with DSS administered in drinking water. L. lactis carrying the scFv expression vector was introduced by gavage. After four days of treatment, animals showed a significant improvement in histological score and disease activity index compared to those of untreated animals. Moreover, treated mice display IL-6, IL17A, IL1ß, IL10 and FOXP3 mRNA levels similar to health control mice. Therefore, morphological and molecular markers suggest amelioration of the experimentally induced colitis. CONCLUSION: These results provide evidence for the use of this alternative system for delivering therapeutic biopharmaceuticals in loco for treating inflammatory bowel disease, paving the way for a novel low-cost and site-specific biotechnological route for the treatment of inflammatory disorders.


Assuntos
Colite/terapia , Citocinas/metabolismo , Vetores Genéticos/administração & dosagem , Lactococcus lactis/imunologia , Administração Oral , Animais , Anticorpos/genética , Anticorpos/imunologia , Anticorpos/metabolismo , Colite/induzido quimicamente , Colite/imunologia , Citocinas/genética , Citocinas/imunologia , Sulfato de Dextrana , Modelos Animais de Doenças , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Camundongos Endogâmicos C57BL , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Fator de Necrose Tumoral alfa/imunologia
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