scholarly journals Development of Fatty Acid-Producing Corynebacterium glutamicum Strains

2013 ◽  
Vol 79 (21) ◽  
pp. 6776-6783 ◽  
Author(s):  
Seiki Takeno ◽  
Manami Takasaki ◽  
Akinobu Urabayashi ◽  
Akinori Mimura ◽  
Tetsuhiro Muramatsu ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Fatty Acid ◽  
Palmitic Acid ◽  
Acid Production ◽  
Type Strain ◽  
Wild Type Strain ◽  
The Other ◽  
Pcr Analysis ◽  
Wild Type ◽  
Selection Of

ABSTRACTTo date, no information has been made available on the genetic traits that lead to increased carbon flow into the fatty acid biosynthetic pathway ofCorynebacterium glutamicum. To develop basic technologies for engineering, we employed an approach that begins by isolating a fatty acid-secreting mutant without depending on mutagenic treatment. This was followed by genome analysis to characterize its genetic background. The selection of spontaneous mutants resistant to the palmitic acid ester surfactant Tween 40 resulted in the isolation of a desired mutant that produced oleic acid, suggesting that a single mutation would cause increased carbon flow down the pathway and subsequent excretion of the oversupplied fatty acid into the medium. Two additional rounds of selection of spontaneous cerulenin-resistant mutants led to increased production of the fatty acid in a stepwise manner. Whole-genome sequencing of the resulting best strain identified three specific mutations (fasR20,fasA63up, andfasA2623). Allele-specific PCR analysis showed that the mutations arose in that order. Reconstitution experiments with these mutations revealed that onlyfasR20gave rise to oleic acid production in the wild-type strain. The other two mutations contributed to an increase in oleic acid production. Deletion offasRfrom the wild-type strain led to oleic acid production as well. Reverse transcription-quantitative PCR analysis revealed that thefasR20mutation brought about upregulation of thefasAandfasBgenes encoding fatty acid synthases IA and IB, respectively, by 1.31-fold ± 0.11-fold and 1.29-fold ± 0.12-fold, respectively, and of theaccD1gene encoding the β-subunit of acetyl-CoA carboxylase by 3.56-fold ± 0.97-fold. On the other hand, thefasA63upmutation upregulated thefasAgene by 2.67-fold ± 0.16-fold. In flask cultivation with 1% glucose, thefasR20 fasA63upfasA2623triple mutant produced approximately 280 mg of fatty acids/liter, which consisted mainly of oleic acid (208 mg/liter) and palmitic acid (47 mg/liter).

scholarly journals Enterococcus faecalisEncodes an Atypical Auxiliary Acyl Carrier Protein Required for Efficient Regulation of Fatty Acid Synthesis by Exogenous Fatty Acids

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Lei Zhu ◽  
Qi Zou ◽  
Xinyun Cao ◽  
John E. Cronan
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Type Strain ◽  
Wild Type Strain ◽  
De Novo ◽  
Acid Synthesis ◽  
Wild Type ◽  
Content Type

ABSTRACTAcyl carrier proteins (ACPs) play essential roles in the synthesis of fatty acids and transfer of long fatty acyl chains into complex lipids. TheEnterococcus faecalisgenome contains two annotatedacpgenes, calledacpAandacpB. AcpA is encoded within the fatty acid synthesis (fab) operon and appears essential. In contrast, AcpB is an atypical ACP, having only 30% residue identity with AcpA, and is not essential. Deletion ofacpBhas no effect onE. faecalisgrowth orde novofatty acid synthesis in media lacking fatty acids. However, unlike the wild-type strain, where growth with oleic acid resulted in almost complete blockage ofde novofatty acid synthesis, theΔacpBstrain largely continuedde novofatty acid synthesis under these conditions. Blockage in the wild-type strain is due to repression offaboperon transcription, leading to levels of fatty acid synthetic proteins (including AcpA) that are insufficient to supportde novosynthesis. Transcription of thefaboperon is regulated by FabT, a repressor protein that binds DNA only when it is bound to an acyl-ACP ligand. Since AcpA is encoded in thefaboperon, its synthesis is blocked when the operon is repressed andacpAthus cannot provide a stable supply of ACP for synthesis of the acyl-ACP ligand required for DNA binding by FabT. In contrast to AcpA,acpBtranscription is unaffected by growth with exogenous fatty acids and thus provides a stable supply of ACP for conversion to the acyl-ACP ligand required for repression by FabT. Indeed,ΔacpBandΔfabTstrains have essentially the samede novofatty acid synthesis phenotype in oleic acid-grown cultures, which argues that neither strain can form the FabT-acyl-ACP repression complex. Finally, acylated derivatives of both AcpB and AcpA were substrates for theE. faecalisenoyl-ACP reductases and forE. faecalisPlsX (acyl-ACP; phosphate acyltransferase).IMPORTANCEAcpB homologs are encoded by many, but not all, lactic acid bacteria (Lactobacillales), including many members of the human microbiome. The mechanisms regulating fatty acid synthesis by exogenous fatty acids play a key role in resistance of these bacteria to those antimicrobials targeted at fatty acid synthesis enzymes. Defective regulation can increase resistance to such inhibitors and also reduce pathogenesis.

Pathogenic and virulence characterization of colonial mutants of Nocardia asteroides GUH-2

1983 ◽  
Vol 29 (9) ◽  
pp. 1126-1135 ◽  
Author(s):  
Cynthia A. Vistica ◽  
Blaine L. Beaman
Keyword(s):  
Fatty Acid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Growth Curves ◽  
The Other ◽  
Nocardia Asteroides ◽  
Growth Stages ◽  
Fatty Acid Profiles ◽  
Wild Type ◽  
Colonial Morphology

The pathogenicities in mice (comparing LD50 determinations) of two mutant strains and one wild-type strain of Nocardia asteroides GUH-2, each possessing a colonial morphology distinct from the other, were compared at respective stages of growth. Despite the three strains' colinear growth curves and similar physiological properties, unique patterns of pathogenicity emerged for each strain upon analysis. Ultrastructural and fatty acid profiles of cultures at the various growth stages were monitored. The mutant strain SCII-A1 was consistently less virulent than the other strains N. asteroides GUH-2 (SCII-P and SCII-C). Further, its fatty acid profiles as well as the shape and consistency of its colonies differed greatly from those of the wild-type strain. The fatty acid composition and the colonial morphology of strain SCII-C more closely resembled those of the parent, although its virulence was both greater than (before 28 h of growth) and less than the parent's depending upon the specific stage of growth. The comparative degrees of cellular fragmentation and complexity, as determined by scanning and transmission electron microscopy, were found to coincide with changes in relative degrees of pathogenicity.

scholarly journals Role Of Selected Fimbrial Adheins In Pathogenesis Of Acid-Induced Eneterohemorrhagic Escherichia Coli 0157:H7

2021 ◽  
Author(s):  
Shahnaz Haque
Keyword(s):  
Fatty Acid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Short Chain Fatty Acid ◽  
Acid Stress ◽  
Chain Fatty Acid ◽  
Short Chain ◽  
Wild Type ◽  
Food Borne

Enterohemorrhagic Escherichia coli (EHEC) 0157:H7 is a food-borne pathogen that causes hemolytic uremic syndrome and hemorrhagic colitis. The mechanisms underlying the adhesion of EHEC 0157:H7 to intestinal epithelial cells are not well understood. Like other food-borne pathogens, ECEC 0157:H7 must survive the acid stress of the gastric juice in the stomach and short chain fatty acid in the intestine in order to colonize the large intestine. We have found that acid stress and short chain fatty acid stress significantly enhance host-adhesion of EHEC 0157:H7 and also upregulates expression of EHEC fimbrial genes, lpfA1, lpfA2 and yagZ, as demonstrated by our DNA microarray. We now report that disruption of the yagZ (also known as the E. coli common pilus A) gene results in loss of the acid-induced and short chain fatty acid-induced adhesion increase seen for the wild type strain. When the yagZ mutant is complemented with yagZ, the sress-induced and short chain fatty acid-induced adhesion increase seen for the wild type strain. When the yagZ mutant is complemented with yagZ, the stress-induced adhesion pehnotype is restored, confirming the role of yagZ in the acid as well as short chain fatty acid induced adhesion to HEp-2 cells. On the other hand, neither disruption in the long polar fimbria genes lpfA1 or lpfA2 in the wild type showed any effect in adherence to HEp-2 cells; rather displaying a hyperadherant phenotype to HEp-2 cells after acid-induced or short chain fatty acid-induced stress. The results also indicate that acid or short chain fatty acid stress, which is a part of the host's natural defense mechanism against pathogens, may regulate virulence factors resulting in enhanced bacteria-host attachment during colonization in the human or bovine host.

Isolation of telomere DNA from Neurospora crassa

1987 ◽  
Vol 7 (9) ◽  
pp. 3168-3177
Author(s):  
M G Schechtman
Keyword(s):  
Linkage Group ◽  
Neurospora Crassa ◽  
Genetic Background ◽  
Type Strain ◽  
Wild Type Strain ◽  
The Other ◽  
Wild Type ◽  
Oak Ridge ◽  
Entire Chromosome ◽  
Different Genetic Background

The most distal known gene on Neurospora crassa linkage group VR, his-6, was cloned. A genomic walk resulted in isolation of the telomere at VR. It was obtained from a library in which the endmost nucleotides of the chromosome had not been removed by nuclease treatment before being cloned, and mapping indicates that the entire chromosome end has probably been cloned. Sequences homologous to the terminal 2.5 kilobases of DNA from VR from these Oak Ridge N. crassa strains are found at other sites in the genome. To characterize these sites, I crossed an Oak Ridge-derived his-6 strain with a wild-type strain of different genetic background (Mauriceville) and characterized the hybridization patterns seen in the progeny. It appears that the sequences homologous to the VR terminus are found at genetically different sites in the two parental strains, and no hybridization to the VR telomere from Mauriceville was detected. The other genomic copies identified in the Oak Ridge parent were not telomeres. I suggest that any repeating sequence blocks found immediately adjacent to the VR terminus in Oak Ridge strains must be small and that the repeating element identified in that background may be an N. crassa transposable element integrated near the the chromosome end at VR.

scholarly journals Glycerol Utilization Gene Cluster in Streptomyces clavuligerus

2009 ◽  
Vol 75 (9) ◽  
pp. 2991-2995 ◽  
Author(s):  
Sonia Baños ◽  
Rosario Pérez-Redondo ◽  
Bert Koekman ◽  
Paloma Liras
Keyword(s):  
Clavulanic Acid ◽  
Gene Cluster ◽  
Acid Production ◽  
Type Strain ◽  
Wild Type Strain ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Glycerol Utilization ◽  
In The Wild

ABSTRACT The Streptomyces clavuligerus ATCC 27064 glycerol cluster gylR-glpF1K1D1 is induced by glycerol but is not affected by glucose. S. clavuligerus growth and clavulanic acid production are stimulated by glycerol, but this does not occur in a glpK1-deleted mutant. Amplification of glpK1D1 results in transformants yielding larger amounts of clavulanic acid in the wild-type strain and in overproducer S. clavuligerus Gap15-7-30 or S. clavuligerus ΔrelA strains.

scholarly journals Comparative analysis and mutation effects of fpp2–fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1196-1204 ◽  
Author(s):  
David Pérez-Pascual ◽  
Esther Gómez ◽  
Beatriz Álvarez ◽  
Jessica Méndez ◽  
Pilar Reimundo ◽  
...  
Keyword(s):  
Gene Function ◽  
Type Strain ◽  
Wild Type Strain ◽  
Proteolytic Enzymes ◽  
Function Analysis ◽  
Phenotypic Characterization ◽  
Pcr Analysis ◽  
Wild Type ◽  
Flavobacterium Psychrophilum ◽  
Genes Encoding

Flavobacterium psychrophilum is a very significant fish pathogen that secretes two biochemically characterized extracellular proteolytic enzymes, Fpp1 and Fpp2. The genes encoding these enzymes are organized as an fpp2–fpp1 tandem in the genome of strain F. psychrophilum THC02/90. Analysis of the corresponding encoded proteins showed that they belong to two different protease families. For gene function analysis, new genetic tools were developed in F. psychrophilum by constructing stable isogenic fpp1 and fpp2 mutants via single-crossover homologous recombination. RT-PCR analysis of wild-type and mutant strains suggested that both genes are transcribed as a single mRNA from the promoter located upstream of the fpp2 gene. Phenotypic characterization of the fpp2 mutant showed lack of caseinolytic activity and higher colony spreading compared with the wild-type strain. Both characteristics were recovered in the complemented strain. One objective of this work was to assess the contribution to virulence of these proteolytic enzymes. LD50 experiments using the wild-type strain and mutants showed no significant differences in virulence in a rainbow trout challenge model, suggesting instead a possible nutritional role. The gene disruption procedure developed in this work, together with the knowledge of the complete genome sequence of F. psychrophilum, open new perspectives for the study of gene function in this bacterium.

scholarly journals Engineered Fatty Acid Biosynthesis inStreptomyces by Altered Catalytic Function of β-Ketoacyl-Acyl Carrier Protein Synthase III

2001 ◽  
Vol 183 (7) ◽  
pp. 2335-2342 ◽  
Author(s):  
Natalya Smirnova ◽  
Kevin A. Reynolds
Keyword(s):  
Fatty Acid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Chain Fatty Acid ◽  
Wild Type ◽  
Acid Biosynthesis ◽  
Branched Chain

ABSTRACT The Streptomyces glaucescens β-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers. This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process. Three mutants (Cys122Ala, Cys122Ser, Cys122Gln) were created by site-directed mutagenesis. Plasmid-based expression of these mutants in S. glaucescens resulted in strains which generated 75 (Cys122Ala) to 500% (Cys122Gln) more straight-chain fatty acids (SCFA) than the corresponding wild-type strain. In contrast, plasmid-based expression of wild-type KASIII had no effect on fatty acid profiles. These observations are attributed to an uncoupling of the condensation and decarboxylation activities in these mutants (malonyl-ACP is thus converted to acetyl-ACP, a SCFA precursor). Incorporation experiments with perdeuterated acetic acid demonstrated that 9% of the palmitate pool of the wild-type strain was generated from an intact D3 acetyl-CoA starter unit, compared to 3% in a strain expressing the Cys122Gln KASIII. These observations support the intermediacy of malonyl-ACP in generating the SCFA precursor in a strain expressing this mutant. To study malonyl-ACP decarboxylase activity in vitro, the KASIII mutants were expressed and purified as His-tagged proteins in Escherichia coli and assayed. In the absence of the acyl-CoA substrate the Cys122Gln mutant and wild-type KASIII were shown to have comparable decarboxylase activities in vitro. The Cys122Ala mutant exhibited higher activity. This activity was inhibited for all enzymes by the presence of high concentrations of isobutyryl-CoA (>100 μM), a branched-chain fatty acid biosynthetic precursor. Under these conditions the mutant enzymes had no activity, while the wild-type enzyme functioned as a ketoacyl synthase. These observations indicate the likely upper and lower limits of isobutyryl-CoA and related acyl-CoA concentrations within S. glaucescens.

Enzymes of agmatine degradation and the control of their synthesis in Streptococcus faecalis

1982 ◽  
Vol 152 (2) ◽  
pp. 676-681
Author(s):  
J P Simon ◽  
V Stalon
Keyword(s):  
Energy Source ◽  
Catabolite Repression ◽  
Type Strain ◽  
Wild Type Strain ◽  
Arginine Deiminase ◽  
The Other ◽  
Wild Type ◽  
Streptococcus Faecalis ◽  
Agmatine Deiminase ◽  
Sole Energy

Streptococcus faecalis ATCC 11700 uses agmatine as its sole energy source for growth. Agmatine deiminase and putrescine carbamoyltransferase are coinduced by growth on agmatine. Glucose and arginine were found to exert catabolite repression on the agmatine deiminase pathway. Four mutants unable to utilize agmatine as an energy source, isolated from the wild-type strain, exhibited three distinct phenotypes. Two of these strains showed essentially no agmatine deiminase, one mutant showed negligible activity of putrescine carbamoyltransferase, and one mutant was defective in both activities. Two carbamate kinases are present in S. faecalis, one belonging to the arginine deiminase pathway, the other being induced by growth on agmatine. These two enzymes have the same molecular weight, 82,000, and seem quite different in size from the kinases isolated from other streptococci.

scholarly journals Interrelationships in trace-element metabolism in metal toxicities in nickel-resistant strains of Neurospora crassa

1983 ◽  
Vol 212 (1) ◽  
pp. 205-210 ◽  
Author(s):  
P Maruthi Mohan ◽  
K Sivarama Sastry
Keyword(s):  
Neurospora Crassa ◽  
Metal Ion ◽  
Type Strain ◽  
Heavy Metal Ions ◽  
Wild Type Strain ◽  
The Other ◽  
Wild Type ◽  
Resistant Strains ◽  
Trace Element Metabolism ◽  
Very High

Three different Ni2+-resistant strains of Neurospora crassa (NiR1, NiR2 and NiR3) have been isolated. All are stable mutants and are fourfold more resistant to Ni2+ than the parent wild-type strain. NiR1 and NiR2 are also sixfold more resistant to Co2+, whereas NiR3 is only twice as resistant to Co2+; the former two are also twofold more resistant to Zn2+, but NiR3 is not. These three strains also differ in sensitivity to Cu2+. Toxicities and concomitant accumulation patterns of Ni2+, Co2+ and Cu2+ have been examined in these strains. NiR1 and NiR2, despite quantitative individual differences, generally accumulate very high amounts of Ni2+ and Co2+, and Mg2+ reverses the toxicities of these two ions by different mechanisms; Ni2+ uptake is suppressed, but not that of Co2+. In NiR3, Mg2+ controls uptake of both Ni2+ and Co2+. Studies indicate that two kinds of Ni2+-resistant strains of N. crassa exist; one kind is resistant because it can tolerate high intracellular concentrations of heavy-metal ions, whereas the other is resistant because it can control metal-ion accumulation.

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