Production of Recombinant β-Galactosidase in Lactobacillus plantarum, Using a pSIP-Based Food-Grade Expression System

2014 ◽  
Vol 931-932 ◽  
pp. 1518-1523 ◽  
Author(s):  
Numphon Thaiwong ◽  
Siwatt Thaiudom ◽  
Dietmar Haltrich ◽  
Montarop Yamabhai
Keyword(s):  
Enzyme Production ◽  
Expression Vector ◽  
Expression System ◽  
Selection Marker ◽  
Expression Systems ◽  
Recombinant Enzymes ◽  
Food Grade ◽  
Food Industries ◽  
Specific Enzymatic Activity ◽  
Food Applications

Food-grade expression systems based on using food-grade microorganisms have been developed for the production of recombinant enzymes used in food applications. Lactic acid bacteria (LAB), especially Lactobacilli, have been widely used for various purposes in food and recognized as a promising host of food-grade enzyme production. In this study, the pSIP409 vectors, originally containing the erm gene, were used to replace this selection marker by the alr gene resulting in the production of the pSIP609 expression vector in L. planatarum. This vector could express high amounts of β-galactosidases, showing both high volumetric as well a specific enzymatic activity. Thus, the food-grade recombinant enzyme production in L. planatarum harboring pSIP609 was very fruitful and useful for food industries.

scholarly journals Cell Wall Anchoring of a Bacterial Chitosanase in Lactobacillus plantarum Using a Food-Grade Expression System and Two Versions of an LP × TG Anchor

2020 ◽  
Vol 21 (11) ◽  
pp. 3773 ◽  
Author(s):  
Mai-Lan Pham ◽  
Anh-Minh Tran ◽  
Geir Mathiesen ◽  
Hoang-Minh Nguyen ◽  
Thu-Ha Nguyen
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Lactobacillus Plantarum ◽  
Expression System ◽  
Surface Display ◽  
Cell Surface Display ◽  
Expression Vectors ◽  
Selection Marker ◽  
Food Grade ◽  
Cell Factories

Lactic acid bacteria (LAB) have attracted increasing interest recently as cell factories for the production of proteins as well as a carrier of proteins that are of interest for food and therapeutic applications. In this present study, we exploit a lactobacillal food-grade expression system derived from the pSIP expression vectors using the alr (alanine racemase) gene as the selection marker for the expression and cell-surface display of a chitosanase in Lactobacillus plantarum using two truncated forms of a LP × TG anchor. CsnA, a chitosanase from Bacillus subtilis 168 (ATCC23857), was fused to two different truncated forms (short-S and long-L anchors) of an LP × TG anchor derived from Lp_1229, a key-protein for mannose-specific adhesion in L. plantarum WCFS1. The expression and cell-surface display efficiency driven by the food-grade alr-based system were compared with those obtained from the erm-based pSIP system in terms of enzyme activities and their localisation on L. plantarum cells. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest enzymatic activity of CsnA-displaying cells was obtained from the strain carrying the alr-based expression plasmid with short cell wall anchor S. However, the attachment of chitosanase on L. plantarum cells via the long anchor L was shown to be more stable compared with the short anchor after several repeated reaction cycles. CsnA displayed on L. plantarum cells is catalytically active and can convert chitosan into chito-oligosaccharides, of which chitobiose and chitotriose are the main products.

scholarly journals thyA as a Selection Marker in Construction of Food-Grade Host-Vector and Integration Systems for Streptococcus thermophilus

2004 ◽  
Vol 70 (3) ◽  
pp. 1858-1864 ◽  
Author(s):  
Yasuko Sasaki ◽  
Yoshiyuki Ito ◽  
Takashi Sasaki
Keyword(s):  
Thymidylate Synthase ◽  
Streptococcus Thermophilus ◽  
Lactobacillus Delbrueckii ◽  
Vector System ◽  
Selection Marker ◽  
Temperature Sensitive ◽  
Erythromycin Resistance ◽  
Food Grade ◽  
Food Applications ◽  
Double Crossover Event

ABSTRACT We constructed food-grade host-vector and integration systems for Streptococcus thermophilus by using a thymidylate synthase gene (thyA) as the selection marker. Two thyA genes, thyASt and thyALb , were cloned from S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, respectively. Thymidine-requiring mutants of S. thermophilus were obtained after successive cultures in the presence of trimethoprim, and one of them, TM1-1, was used as the host. Food-grade vectors were constructed by using either thyASt or thyALb as the selection marker. Transformants of TM1-1 created by using these vectors were selected for thymidine autotrophy as efficiently as for erythromycin resistance. By using the host-vector system developed in this way, a foreign amylase gene (amyA) was expressed in TM1-1 and was also integrated into the chromosome by use of a temperature-sensitive integration vector constructed with thyALb as the selection marker via a double-crossover event. The results obtained show that thyA is an efficient and safe selection marker for S. thermophilus that is suitable for food applications.

scholarly journals Development of a new gene expression vector for Thermus thermophilus using a silica-inducible promoter

2020 ◽  
Author(s):  
Yasuhiro Fujino ◽  
Shuichiro Goda ◽  
Yuri Suematsu ◽  
Katsumi Doi
Keyword(s):  
Gene Expression ◽  
Expression Vector ◽  
Heterologous Gene Expression ◽  
Thermus Thermophilus ◽  
Expression System ◽  
Active Form ◽  
Inducible Promoter ◽  
Additional Treatment ◽  
Selection Marker ◽  
Thermostable Enzymes

Abstract Background Thermostable enzymes are commonly produced in mesophilic hosts for research and bioengineering purposes. However, these hosts do not overexpress the active forms of some biologically functional thermoenzymes. Therefore, an efficient thermophilic expression system is needed. Thermus thermophilus contains an easily manipulable genome and is therefore among the best candidate microbes for a “hot” expression system. We previously identified a strong and inducible promoter that was active in T. thermophilus under supersaturated silica conditions. Here, we report a new heterologous gene expression system based on a silica-inducible promoter in T. thermophilus.Results A Thermus sp. A4 gene encoding thermostable β-galactosidase was cloned as a reporter gene into the expression vector pSix1, which contains a selection marker that confers thermostable resistance to hygromycin and a 600-bps DNA region containing a putative silica-inducible promoter. β-Galactosidase activity was 11-fold higher in the presence than in the absence of 10 mM silicic acid. SDS-PAGE revealed a prominent band corresponding to β-galactosidase, and this enzyme was expressed as an active and soluble protein (yield: 27 mg/L) in Thermus but as an inclusion body in Escherichia coli. Deletion of the promoter region improved the yield of the target protein, possibly by avoiding plasmid instability due to homologous recombination. Finally, we developed an expression vector containing the pSix1 backbone and a 100-bps DNA region corresponding to the silica-inducible promoter. We used this vector to successfully express the active form of glutamate dehydrogenase from Pyrobaculum islandicum (PisGDH) without additional treatment (yield: 9.5 mg/L), whereas the expression of active PisGDH in E. coli required heat treatment.Conclusion We successfully expressed the thermoenzymes β-galactosidase and PisGDH in T. thermophilus and achieved the highest known protein expression levels in this species. These thermoenzymes were expressed in active and soluble forms. Our results validate the use of our silica-inducible expression system as a novel strategy for the intracellular overexpression of thermostable proteins.

A food-grade fimbrial adhesin FaeG expression system inLactococcus lactisandLactobacillus casei

2016 ◽  
Vol 62 (3) ◽  
pp. 241-248 ◽  
Author(s):  
W.W. Lu ◽  
T. Wang ◽  
Y. Wang ◽  
M. Xin ◽  
J. Kong
Keyword(s):  
Genetically Modified ◽  
Expression System ◽  
Enterotoxigenic Escherichia Coli ◽  
Selection Marker ◽  
Delivery Vehicle ◽  
Food Grade ◽  
Neonatal Piglets ◽  
Extracellular Secretion ◽  
Etec Infection ◽  
Fimbrial Adhesin

Enterotoxigenic Escherichia coli (ETEC) infection is the major cause of diarrhea in neonatal piglets. The fimbriae as colonizing factor in the pathogenesis of ETEC constitute a primary target for vaccination against ETEC. Lactic acid bacteria (LAB) are attractive tools to deliver antigens at the mucosal level. With the safety of genetically modified LAB in mind, a food-grade secretion vector (pALRc or pALRb) was constructed with DNA entirely from LAB, including the replicon, promoter, signal peptide, and selection marker alanine racemase gene (alr). To evaluate the feasibility of the system, the nuclease gene (nuc) from Staphylococcus aureus was used as a reporter to be expressed in both Lactococcus lactis and Lactobacillus casei. Subsequently, the extracellular secretion of the fimbrial adhesin FaeG of ETEC was confirmed by Western blot analysis. These results showed that this food-grade expression system has potential as the delivery vehicle for the safe use of genetically modified LAB for the development of vaccines against ETEC infection.

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

scholarly journals Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii

2007 ◽  
Vol 73 (23) ◽  
pp. 7542-7547 ◽  
Author(s):  
Dag Anders Brede ◽  
Sheba Lothe ◽  
Zhian Salehian ◽  
Therese Faye ◽  
Ingolf F. Nes
Keyword(s):  
Selection Marker ◽  
Multiple Cloning Site ◽  
Propionibacterium Freudenreichii ◽  
Expression Plasmid ◽  
Physiochemical Properties ◽  
Food Grade ◽  
Cloning System ◽  
Immunity Gene ◽  
Membrane Bound ◽  
High Level

ABSTRACT This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 107 transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive PpampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ∼91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.

scholarly journals Research Advances on Health Effects of Edible Artemisia Species and Some Sesquiterpene Lactones Constituents

Foods ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 65
Author(s):  
Antoaneta Trendafilova ◽  
Laila M. Moujir ◽  
Pedro M. C. Sousa ◽  
Ana M. L. Seca
Keyword(s):  
Adverse Effects ◽  
Health Effects ◽  
Functional Foods ◽  
Sesquiterpene Lactones ◽  
Therapeutic Effects ◽  
Nutritional Properties ◽  
Food Industries ◽  
Food Applications ◽  
Artemisia Species

The genus Artemisia, often known collectively as “wormwood”, has aroused great interest in the scientific community, pharmaceutical and food industries, generating many studies on the most varied aspects of these plants. In this review, the most recent evidence on health effects of edible Artemisia species and some of its constituents are presented and discussed, based on studies published until 2020, available in the Scopus, Web of Sciences and PubMed databases, related to food applications, nutritional and sesquiterpene lactones composition, and their therapeutic effects supported by in vivo and clinical studies. The analysis of more than 300 selected articles highlights the beneficial effect on health and the high clinical relevance of several Artemisia species besides some sesquiterpene lactones constituents and their derivatives. From an integrated perspective, as it includes therapeutic and nutritional properties, without ignoring some adverse effects described in the literature, this review shows the great potential of Artemisia plants and some of their constituents as dietary supplements, functional foods and as the source of new, more efficient, and safe medicines. Despite all the benefits demonstrated, some gaps need to be filled, mainly related to the use of raw Artemisia extracts, such as its standardization and clinical trials on adverse effects and its health care efficacy.

scholarly journals In Vitro Reconstitution of an NADPH-Dependent Superoxide Reduction Pathway from Pyrococcus furiosus

2005 ◽  
Vol 71 (3) ◽  
pp. 1522-1530 ◽  
Author(s):  
Amy M. Grunden ◽  
Francis E. Jenney ◽  
Kesen Ma ◽  
Mikyoung Ji ◽  
Michael V. Weinberg ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Pyrococcus Furiosus ◽  
Expression System ◽  
Anaerobic Bacteria ◽  
Flavin Mononucleotide ◽  
Superoxide Reductase ◽  
Recombinant Enzymes ◽  
Significant Similarity ◽  
Midpoint Potential

ABSTRACT A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H:rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of −173 mV at 23°C (−193 mV at 80°C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80°C) and low (23°C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway.

scholarly journals Encapsulation Through The Use Of Emulsified Microemulsions

2021 ◽  
Author(s):  
Ruby R. Rafanan
Keyword(s):  
Controlled Release ◽  
Continuous Phase ◽  
Pharmaceutical Products ◽  
Water Soluble ◽  
Scanning Calorimetry ◽  
Food Grade ◽  
X Ray ◽  
X Ray Scattering ◽  
Food Applications ◽  
Glycerol Monooleate

Emulsified microemulsions (EMEs), first described in detail in 2005 by the group of Garti, consist of a thermodynamically stable water-in-oil microemulsion phase (w1/o) further dispersed within an aqueous continuous phase (w2). These internally-structured w1/o/w2 dispersions are promising controlled release vehicles for water-soluble flavouring compounds, drugs and nutraceuticals. With a stable internal droplet structure, storage stability is improved over non-thermodynamically stable structured emulsions and may exhibit unique controlled release behaviour. Use of food-grade components allows for wider and safer applications in food and pharmaceutical products. In this thesis, a food-grade w1/o microemulsion consisting of glycerol monooleate, tricaprylin and water was dispersed in an aqueous (w2) phase by membrane emulsification and stabilized by a caseinate-pectin complex to produce w1/o/w2 EMEs. The resulting EME showed no signs of phase separation for weeks at room temperature. The microemulsion and EME were characterized by differential scanning calorimetry (DSC), cryo-TEM and small angle x-ray scattering (SAXS) to determine whether the microemulsion’s internal structure was maintained after emulsification. It was shown that EME droplets displayed ordering around the periphery consistent with some loss of microemulsion structure, but maintained the characteristic disordered microemulsion structure at the droplet core. Overall, this research demonstrated the feasibility of developing EME for possible applications in food and non-food applications.

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