scholarly journals Integrative Transformation System for the Metabolic Engineering of the Sphingoid Base-Producing Yeast Pichia ciferrii

2003 ◽  
Vol 69 (2) ◽  
pp. 812-819 ◽  
Author(s):  
Jung-Hoon Bae ◽  
Jung-Hoon Sohn ◽  
Chang-Seo Park ◽  
Joon-Shick Rhee ◽  
Eui-Sung Choi
Keyword(s):  
Metabolic Engineering ◽  
De Novo ◽  
Integration Site ◽  
Fold Increase ◽  
Selection Marker ◽  
Serine Palmitoyltransferase ◽  
Transformation System ◽  
Wild Type ◽  
Gene Encoding ◽  
Integrative Transformation

ABSTRACT We have developed an integrative transformation system for metabolic engineering of the tetraacetyl phytosphingosine (TAPS)-secreting yeast Pichia ciferrii. The system uses (i) a mutagenized ribosomal protein L41 gene of P. ciferrii as a dominant selection marker that confer resistance to the antibiotic cycloheximide and (ii) a ribosomal DNA (rDNA) fragment of P. ciferrii as a target for multicopy gene integration into the chromosome. A locus within the nontranscribed region located between 5S and 26S rDNAs was selected as the integration site. A maximum frequency of integrative transformation of approximately 1,350 transformants/μg of DNA was observed. To improve the de novo synthesis of sphingolipid, the LCB2 gene, encoding a subunit of serine palmitoyltransferase, which catalyzes the first committed step of sphingolipid synthesis, was cloned from P. ciferrii and overexpressed under the control of the P. ciferrii glyceraldehyde-3-phosphate dehydrogenase promoter. After transformation of an LCB2 gene expression cassette, several transformants that contained approximately five to seven copies of transforming DNA in the chromosome and exhibited about 50-fold increase in LCB2 mRNA relative to the wild type were identified. These transformants were observed to produce approximately two times more TAPS than the wild type.

scholarly journals Use of the alr Gene as a Food-Grade Selection Marker in Lactic Acid Bacteria

2002 ◽  
Vol 68 (11) ◽  
pp. 5663-5670 ◽  
Author(s):  
Peter A. Bron ◽  
Marcos G. Benchimol ◽  
Jolanda Lambert ◽  
Emmanuelle Palumbo ◽  
Marie Deghorain ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Competitive Inhibitor ◽  
Selection Marker ◽  
Wild Type ◽  
Gene Encoding ◽  
Food Grade ◽  
Selection For ◽  
Isogenic Mutants ◽  
Type Strains

ABSTRACT Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of d-alanine and l-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Δalr) showed auxotrophy for d-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented d-alanine auxotrophy in the L. plantarum Δalr and L. lactis Δalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to d-cycloserine, a competitive inhibitor of Alr (600 and 200 μg/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that d-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Δalr. The resulting strain could grow in the absence of d-alanine only when expression of the alr gene was induced with nisin.

scholarly journals Involvement of Quinolinate Phosphoribosyl Transferase in Promotion of Potato Growth by a Burkholderia Strain

2006 ◽  
Vol 72 (1) ◽  
pp. 760-768 ◽  
Author(s):  
Keri Wang ◽  
Kenneth Conn ◽  
George Lazarovits
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
De Novo ◽  
Growth Promotion ◽  
Amino Acid Level ◽  
Growth Stimulation ◽  
Wild Type ◽  
Gene Encoding ◽  
Burkholderia Strain ◽  
Potato Growth

ABSTRACT Burkholderia sp. strain PsJN stimulates root growth of potato explants compared to uninoculated controls under gnotobiotic conditions. In order to determine the mechanism by which this growth stimulation occurs, we used Tn5 mutagenesis to produce a mutant, H41, which exhibited no growth-promoting activity but was able to colonize potato plants as well as the wild-type strain. The gene associated with the loss of growth promotion in H41 was shown to exhibit 65% identity at the amino acid level to the nadC gene encoding quinolinate phosphoribosyltransferase (QAPRTase) in Ralstonia solanacearum. Complementation of H41 with QAPRTase restored growth promotion of potato explants by this mutant. Expression of the gene identified in Escherichia coli yielded a protein with QAPRTase activities that catalyzed the de novo formation of nicotinic acid mononucleotide (NaMN). Two other genes involved in the same enzymatic pathway, nadA and nadB, were physically linked to nadC. The nadA gene was cotranscribed with nadC as an operon in wild-type strain PsJN, while the nadB gene was located downstream of the nadA-nadC operon. Growth promotion by H41 was fully restored by addition of NaMN to the tissue culture medium. These data suggested that QAPRTase may play a role in the signal pathway for promotion of plant growth by PsJN.

scholarly journals A Strong Negative Transcriptional Regulatory Region between the Human Cytomegalovirus UL127 Gene and the Major Immediate-Early Enhancer

1999 ◽  
Vol 73 (11) ◽  
pp. 9039-9052 ◽  
Author(s):  
Christopher A. Lundquist ◽  
Jeffrey L. Meier ◽  
Mark F. Stinski
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
De Novo ◽  
Human Fibroblasts ◽  
Regulatory Region ◽  
Fold Increase ◽  
Immediate Early ◽  
Wild Type ◽  
Viral Protein Synthesis ◽  
Unique Region

ABSTRACT The region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region. DNase I protection analysis with human cell nuclear extracts demonstrated multiple protein binding sites in this region of the viral genome (P. Ghazal, H. Lubon, C. Reynolds-Kohler, L. Hennighausen, and J. A. Nelson, Virology 174:18–25, 1990). However, the function of this region in the context of the viral genome is not known. In wild-type human CMV-infected human fibroblasts, cells permissive for viral replication, there is little to no transcription from UL127. We determined that the unique region prevented transcription from the UL127 promoter but had no effect on the divergent MIE promoter. In transient-transfection assays, the basal level of expression from the UL127 promoter increased significantly when the wild-type unique sequences were mutated. In recombinant viruses with similar mutations in the unique region, expression from the UL127 promoter occurred only after de novo viral protein synthesis, typical of an early viral promoter. A 111-bp deletion-substitution of the unique sequence caused approximately a 20-fold increase in the steady-state level of RNA from the UL127 promoter and a 245-fold increase in the expression of a downstream indicator gene. This viral negative regulatory region was also mutated at approximately 50-bp regions proximal and distal to the UL127 promoter. Although some repressive effects were detected in the distal region, mutations of the region proximal to the UL127 promoter had the most significant effects on transcription. Within the proximal and distal regions, there are potential cis sites for known eucaryotic transcriptional repressor proteins. This region may also bind unknown viral proteins. We propose that the unique region upstream of the UL127 promoter and the MIE enhancer negatively regulates the expression from the UL127 promoter in permissive human fibroblast cells. This region may be a regulatory boundary preventing the effects of the very strong MIE enhancer on this promoter.

Development of an integrative transformation system for the opportunistic pathogenic yeastCandida lusitaniae usingURA3 as a selection marker

Yeast ◽  
2004 ◽  
Vol 21 (2) ◽  
pp. 95-106 ◽  
Author(s):  
Fabienne François ◽  
Florence Chapeland-Leclerc ◽  
Jean Villard ◽  
Thierry Noël
Keyword(s):  
Selection Marker ◽  
Transformation System ◽  
Integrative Transformation ◽  
Opportunistic Pathogenic

scholarly journals Enhanced pyruvate metabolism in plastids by overexpression of plastidial pyruvate transporter in Phaeodactylum tricornutum

2020 ◽  
Author(s):  
Seungbeom Seo ◽  
Joon Kim ◽  
Jun-Woo Lee ◽  
Onyou Nam ◽  
Kwang Suk Chang ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Phaeodactylum Tricornutum ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Biofuel Production ◽  
Wild Type ◽  
Strain Development ◽  
Acid Biosynthesis ◽  
Pyruvate Metabolism ◽  
Lipid Contents

Abstract BackgroundThe development of microalgal strains for enhanced biomass and biofuel production has received increased attention. Moreover, strain development via metabolic engineering for commercial production is being considered as the most efficient strategy. Pyruvate is an essential metabolite in the cells and plays an essential role in amino acid biosynthesis and de novo fatty acid biosynthesis in plastids. Although pyruvate can be a valuable target for metabolic engineering, its transporters have rarely been studied in microalgae. In this study, we aimed to identify the plastidial pyruvate transporter of Phaeodactylum tricornutum and utilize it for strain development.ResultsWe identified pyruvate transporter localized in the plastid membrane of Phaeodactylum tricornutum. Transformants overexpressing the pyruvate transporter were generated to increase the influx of pyruvate into plastids. Overexpression of a plastidial pyruvate transporter in P. tricornutum resulted in enhanced biomass (13.6% to 21.9%), lipid contents (11% to 30%), and growth (3.3% to 8.0%) compared to those of wild type during one-stage cultivation.ConclusionTo regulate the pyruvate influx and its metabolism in plastids, we generated transformants overexpressing the plastidial pyruvate transporter in P. tricornutum. They showed that compartmentalizing pyruvate in plastids could be an attractive strategy for the effective production of biomass and lipids with better growth, via enhanced pyruvate metabolism in plastids.

Metabolic engineering of capsular polysaccharides

2018 ◽  
Vol 2 (3) ◽  
pp. 337-348 ◽  
Author(s):  
Asher Williams ◽  
Robert J. Linhardt ◽  
Mattheos A.G. Koffas
Keyword(s):  
Metabolic Engineering ◽  
De Novo ◽  
Gene Knockout ◽  
Polysialic Acid ◽  
Genetically Engineered ◽  
Capsular Polysaccharides ◽  
Wild Type ◽  
Level Control ◽  
Biotechnological Processes ◽  
Type Strains

With rising concerns about sustainable practices, environmental complications, and declining resources, metabolic engineers are transforming microorganisms into cellular factories for producing capsular polysaccharides (CPSs). This review provides an overview of strategies employed for the metabolic engineering of heparosan, chondroitin, hyaluronan, and polysialic acid — four CPSs that are of interest for manufacturing a variety of biomedical applications. Methods described include the exploitation of wild-type and engineered native CPS producers, as well as genetically engineered heterologous hosts developed through the improvement of naturally existing pathways or newly (de novo) designed ones. The implementation of methodologies like gene knockout, promoter engineering, and gene expression level control has resulted in multiple-fold improvements in CPS fermentation titers compared with wild-type strains, and substantial increases in productivity, reaching as high as 100% in some cases. Optimization of these biotechnological processes can permit the adoption of industrially competitive engineered microorganisms to replace traditional sources that are generally toxic, unreliable, and inconsistent in product quality.

scholarly journals Blockade of Hemichannels Normalizes the Differentiation Fate of Myoblasts and Features of Skeletal Muscles from Dysferlin-Deficient Mice

2020 ◽  
Vol 21 (17) ◽  
pp. 6025
Author(s):  
Luis A. Cea ◽  
Gabriela Fernández ◽  
Guisselle Arias-Bravo ◽  
Mario Castillo-Ruiz ◽  
Rosalba Escamilla ◽  
...  
Keyword(s):  
Skeletal Muscles ◽  
De Novo ◽  
Similar Amount ◽  
Wild Type ◽  
Membrane Repair ◽  
Gene Encoding ◽  
Multinucleated Cells ◽  
Therapeutic Opportunity ◽  
And Function ◽  
Connexin Hemichannels

Dysferlinopathies are muscle dystrophies caused by mutations in the gene encoding dysferlin, a relevant protein for membrane repair and trafficking. These diseases are untreatable, possibly due to the poor knowledge of relevant molecular targets. Previously, we have shown that human myofibers from patient biopsies as well as myotubes derived from immortalized human myoblasts carrying a mutated form of dysferlin express connexin proteins, but their relevance in myoblasts fate and function remained unknown. In the present work, we found that numerous myoblasts bearing a mutated dysferlin when induced to acquire myogenic commitment express PPARγ, revealing adipogenic instead of myogenic commitment. These cell cultures presented many mononucleated cells with fat accumulation and within 48 h of differentiation formed fewer multinucleated cells. In contrast, dysferlin deficient myoblasts treated with boldine, a connexin hemichannels blocker, neither expressed PPARγ, nor accumulated fat and formed similar amount of multinucleated cells as wild type precursor cells. We recently demonstrated that myofibers of skeletal muscles from blAJ mice (an animal model of dysferlinopathies) express three connexins (Cx39, Cx43, and Cx45) that form functional hemichannels (HCs) in the sarcolemma. In symptomatic blAJ mice, we now show that eight-week treatment with a daily dose of boldine showed a progressive recovery of motor activity reaching normality. At the end of this treatment, skeletal muscles were comparable to those of wild type mice and presented normal CK activity in serum. Myofibers of boldine-treated blAJ mice also showed strong dysferlin-like immunoreactivity. These findings reveal that muscle dysfunction results from a pathophysiologic mechanism triggered by mutated dysferlin and downstream connexin hemichannels expressed de novo lead to a drastic reduction of myogenesis and favor muscle damage. Thus, boldine could represent a therapeutic opportunity to treat dysfernilopathies.

scholarly journals Virulence of a Phosphoribosylaminoimidazole Carboxylase-DeficientCandidaalbicans Strain in an Immunosuppressed Murine Model of Systemic Candidiasis

2001 ◽  
Vol 69 (4) ◽  
pp. 2542-2548 ◽  
Author(s):  
Matthew Donovan ◽  
Jon J. Schumuke ◽  
William A. Fonzi ◽  
Sheri L. Bonar ◽  
Karen Gheesling-Mullis ◽  
...  
Keyword(s):  
Parent Strain ◽  
De Novo ◽  
Synthetic Medium ◽  
Wild Type ◽  
Gene Encoding ◽  
Site Specific ◽  
De Novo Purine Biosynthesis ◽  
Exogenous Addition ◽  
Novel Target ◽  
Immunosuppressed Mice

ABSTRACT The relative pathogenicities of three Candida albicans strains differing in the function ofADE2 (the gene encoding phosphoribosylaminoimidazole carboxylase) were evaluated in a murine candidiasis model. C. albicans strain CAI7 (ade2/ade2), previously constructed by site-specific recombination, was avirulent in immunosuppressed mice compared to the parent strain, CAF2-1, and a heterozygous ADE2/ade2 strain obtained by transforming CAI7 with a wild-type allele. The reduced virulence of CAI7 was correlated with the inability to proliferate in either synthetic medium or serum without the exogenous addition of >10 μg of adenine/ml. The loss of virulence upon site-specific disruption of the ade2 locus, and the restoration of wild-type virulence with the repair of just one ade2 allele, confirmed that the ADE2 gene and de novo purine biosynthesis were required for Candida pathogenicity. The potential of the phosphoribosylaminoimidazole carboxylase enzyme as a novel target for antifungal drug discovery is discussed.

scholarly journals Increased Production of Zeaxanthin and Other Pigments by Application of Genetic Engineering Techniques toSynechocystis sp. Strain PCC 6803

2000 ◽  
Vol 66 (1) ◽  
pp. 64-72 ◽  
Author(s):  
Delphine Lagarde ◽  
Laurent Beuf ◽  
Wim Vermaas
Keyword(s):  
Mutant Strain ◽  
Fold Increase ◽  
Carotenoid Biosynthesis ◽  
Carotenoid Content ◽  
Wild Type ◽  
Gene Encoding ◽  
Genes Encoding ◽  
In The Wild ◽  
Β Carotene ◽  
Pcc 6803

ABSTRACT The psbAII locus was used as an integration platform to overexpress genes involved in carotenoid biosynthesis inSynechocystis sp. strain PCC 6803 under the control of the strong psbAII promoter. The sequences of the genes encoding the yeast isopentenyl diphosphate isomerase (ipi) and theSynechocystis β-carotene hydroxylase (crtR) and the linked Synechocystis genes coding for phytoene desaturase and phytoene synthase (crtP andcrtB, respectively) were introduced intoSynechocystis, replacing the psbAII coding sequence. Expression of ipi, crtR, andcrtP and crtB led to a large increase in the corresponding transcript levels in the mutant strains, showing that the psbAII promoter can be used to drive transcription and to overexpress various genes in Synechocystis. Overexpression of crtP and crtB led to a 50% increase in the myxoxanthophyll and zeaxanthin contents in the mutant strain, whereas the β-carotene and echinenone contents remained unchanged. Overexpression of crtR induced a 2.5-fold increase in zeaxanthin accumulation in the corresponding overexpressing mutant compared to that in the wild-type strain. In this mutant strain, zeaxanthin becomes the major pigment (more than half the total amount of carotenoid) and the β-carotene and echinenone amounts are reduced by a factor of 2. However, overexpression of ipi did not result in a change in the carotenoid content of the mutant. To further alter the carotenoid content of Synechocystis, the crtOgene, encoding β-carotene ketolase, which converts β-carotene to echinenone, was disrupted in the wild type and in the overexpressing strains so that they no longer produced echinenone. In this way, by a combination of overexpression and deletion of particular genes, the carotenoid content of cyanobacteria can be altered significantly.

scholarly journals clk1, a Serine/Threonine Protein Kinase-Encoding Gene, Is Involved in Pathogenicity of Colletotrichum lindemuthianum on Common Bean

1998 ◽  
Vol 11 (2) ◽  
pp. 99-108 ◽  
Author(s):  
Marie Dufresne ◽  
John A. Bailey ◽  
Michel Dron ◽  
Thierry Langin
Keyword(s):  
Protein Kinase ◽  
Common Bean ◽  
Insertional Mutagenesis ◽  
Integration Site ◽  
Gene Replacement ◽  
Infection Process ◽  
Colletotrichum Lindemuthianum ◽  
Selection Marker ◽  
Wild Type ◽  
Kinase Gene

A random insertional mutagenesis in Colletotrichum lindemuthianum, the causal agent of common bean anthracnose, generated four mutants that showed altered pathogenicity when tested on intact seedlings, excised leaves, and/or excised hypocotyls. One of these mutants, H290, produced very few lesions on bean leaves and appeared affected in its ability to penetrate the leaf cuticle. Molecular analyses showed that the border sequences of the unique integration site of the disrupting pAN7-1 plasmid in the mutant exhibited homology with conserved domains of serine/threonine protein kinases. The corresponding wild-type sequences were cloned and a gene replacement vector with a mutated copy harboring a selection marker constructed. Transformation of the wild-type pathogen produced a strain with a phenotype identical to the original mutant. Genomic and cDNA sequences indicated that the disrupted gene is a member of the serine/threonine protein kinase family. The gene, called clk1 (Colletotrichum lindemuthianum kinase 1), was weakly expressed in the mycelium of the wild-type strain grown on rich and minimal synthetic media but was undetectable during the infection even when a sensitive reverse transcriptase-polymerase chain reaction methodology was used. This study represents the first characterization of altered pathogenicity mutants in C. lindemuthianum produced by random mutagenesis and demonstrates the involvement of a member of the serine/threonine kinase gene family in the early steps of the infection process.

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