Functional Complementation in Escherichia coli of Different Phytoene Desaturase Genes and Analysis of Accumulated Carotenes

1991 ◽  
Vol 46 (11-12) ◽  
pp. 1045-1051 ◽  
Author(s):  
Hartmut Linden ◽  
Norihiko Misawa ◽  
Daniel Chamovitz ◽  
Iris Pecker ◽  
Joseph Hirschberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rhodobacter Capsulatus ◽  
Phytoene Desaturase ◽  
Reaction Products ◽  
Double Bonds ◽  
E Coli ◽  
Synechococcus Pcc 7942 ◽  
Lycopene Cyclase ◽  
Pcc 7942 ◽  
Cis Isomer

Three different phytoene desaturase genes, from Rhodobacter capsulatus, Erwinia uredovora, and Synechococcus PCC 7942, have been functionally complemented with a gene construct from E. uredovora which encodes all enzymes responsible for formation of 15-cis phytoene in Escherichia coli. As indicated by the contrasting reaction products detected in the pigmented E. coli cells after co-transformation, a wide functional diversity of these three different types of phytoene desaturases can be concluded. The carotenes formed by the phytoene desaturase from R. capsulatus were trans-neurosporene with three additional double bonds and two cis isomers. Furthermore, small amounts of three ζ-carotene isomers (2 double bonds more than phytoene) and phytofluene (15-cis and all-trans with + 1 double bond) were detected as inter- mediates. When the subsequent genes from E. uredovora which encode for lycopene cyclase and β-carotene hydroxylase were present, neurosporene, the phytoene desaturase product of R. capsulatus, was subsequently converted to the monocyclic β-zeacarotene and its mono- hydroxylation product. The most abundant carotene resulting from phytoene desaturation by the E. uredovora enzyme was trans-lycopene together with a cis isomer. In addition, bisdehy-drolycopene was also formed. The reaction products of Synechococcus phytoene desaturase were two cis isomers of ζ-carotene and only small amounts of trans-ζ-carotene including 15-cis. The I50 values for flurtamone and diphenylamine to inhibit phytoene desaturation were determined and differential inhibition was observed for diphenylamine.

scholarly journals The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli

1996 ◽  
Vol 316 (1) ◽  
pp. 251-257 ◽  
Author(s):  
Michinori MUTSUDA ◽  
Takahiro ISHIKAWA ◽  
Toru TAKEDA ◽  
Shigeru SHIGEOKA
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Molecular Mass ◽  
Peroxidase Activity ◽  
Catalase Activity ◽  
Native Enzyme ◽  
Nucleotide Sequence Analysis ◽  
Synechococcus Pcc 7942 ◽  
Catalase Peroxidase ◽  
Pcc 7942

Synechococcus PCC 7942, a cyanobacterium, possesses catalase–peroxidase as the sole hydrogen peroxide-scavenging system. The enzyme has been purified to electrophoretic homogenenity from the cells. The native enzyme had a molecular mass of 150 kDa and was composed of two identical subunits of molecular mass 79 kDa. The apparent Km value of the catalase activity for H2O2 was 4.2±0.27 mM and the kcat value was 2.6×104 s-1. The enzyme contained high catalase activity and an appreciable peroxidase activity with o-dianisidine and pyrogallol. The catalase activity was not inhibited by 3-amino-1,2,4-triazole but by KCN and NaN3 (apparent Ki values 19.3±0.84 and 20.2±0.95 μM respectively). The enzyme showed an absorption spectrum of typical protohaem and contained one protohaem molecule per dimer. The gene encoding catalase–peroxidase was cloned from the chromosomal DNA of Synechococcus PCC 7942. A 2160 bp open reading frame (ORF), coding a catalase–peroxidase of 720 amino acid residues (approx. 79.9 kDa), was observed. The deduced amino acid sequence coincided with that of the N-terminus of the purified enzyme and showed a remarkable similarity to those of a family of catalase–peroxidases of prokaryotic cells. Escherichia coli BL21(DE3)plysS, harbouring a recombinant plasmid containing the catalase–peroxidase gene, produced a large amount of proteins that co-migrated on SDS/PAGE with the native enzyme. The recombinant enzyme showed the same ratio of catalase activity to peroxidase activity with o-dianisidine and the same Km for H2O2 as the native enzyme.

Inhibitory Activity of Cheese Whey Fermented with Kefir Grains

2011 ◽  
Vol 74 (1) ◽  
pp. 94-100 ◽  
Author(s):  
A. LONDERO ◽  
R. QUINTA ◽  
A. G. ABRAHAM ◽  
R. SERENO ◽  
G. DE ANTONI ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactic Acid ◽  
Acid Production ◽  
Cheese Whey ◽  
Lactic Acid Production ◽  
Reaction Products ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Fermentation Products ◽  
E Coli ◽  
Kefir Grains

We investigated the chemical and microbiological compositions of three types of whey to be used for kefir fermentation as well as the inhibitory capacity of their subsequent fermentation products against 100 Salmonella sp. and 100 Escherichia coli pathogenic isolates. All the wheys after fermentation with 10% (wt/vol) kefir grains showed inhibition against all 200 isolates. The content of lactic acid bacteria in fermented whey ranged from 1.04 × 107 to 1.17 × 107 CFU/ml and the level of yeasts from 2.05 × 106 to 4.23 × 106 CFU/ml. The main changes in the chemical composition during fermentation were a decrease in lactose content by 41 to 48% along with a corresponding lactic acid production to a final level of 0.84 to 1.20% of the total reaction products. The MIC was a 30% dilution of the fermentation products for most of the isolates, while the MBC varied between 40 and 70%, depending on the isolate. The pathogenic isolates Salmonella enterica serovar Enteritidis 2713 and E. coli 2710 in the fermented whey lost their viability after 2 to 7 h of incubation. When pathogens were deliberately inoculated into whey before fermentation, the CFU were reduced by 2 log cycles for E. coli and 4 log cycles for Salmonella sp. after 24 h of incubation. The inhibition was mainly related to lactic acid production. This work demonstrated the possibility of using kefir grains to ferment an industrial by-product in order to obtain a natural acidic preparation with strong bacterial inhibitory properties that also contains potentially probiotic microorganisms.

scholarly journals Purification and reactivation of recombinant Synechococcus phytoene desaturase from an overexpressing strain of Escherichia coli

1993 ◽  
Vol 291 (3) ◽  
pp. 687-692 ◽  
Author(s):  
P D Fraser ◽  
H Linden ◽  
G Sandmann
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Deae Cellulose ◽  
Cellular Protein ◽  
Phytoene Desaturase ◽  
E Coli ◽  
Sds Page ◽  
Purification Scheme ◽  
Overexpressing Strain ◽  
Cellulose Column

The Synechococcus phytoene desaturase has been isolated from an overexpressing strain of Escherichia coli. The plasma pPDSde135 mediated the overexpression of the full-length polypeptide directly. The recombinant protein comprised 5% of the total cellular protein and was found predominantly in the inclusion body fraction. Urea was used to solubilize the recombinant protein from the inclusion fraction and the protein was subsequently purified to homogeneity on a DEAE-cellulose column. The purification scheme yielded 4.0 mg of homogeneous desaturase protein after a 20-fold purification, recovering 40% of the original protein from a 100 ml suspension culture of E. coli. The recombinant desaturase had an apparent molecular mass of 53 kDa on SDS/PAGE and crossreacted with an antiserum raised against the expressed protein. Desaturase activity was restored upon the removal of urea. The enzyme catalysed the conversion of phytoene to zeta-carotene via phytofluene. These products of the desaturase reaction existed predominantly in a cis configuration. Lipid replenishment enhanced activity. NAD+ and NADP+ were observed to be involved, whilst FAD was an ineffective electron acceptor.

scholarly journals Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

2018 ◽  
Author(s):  
Huan Fang ◽  
Dong Li ◽  
Jie Kang ◽  
Pingtao Jiang ◽  
Jibin Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Biosynthetic Pathway ◽  
Rhodobacter Capsulatus ◽  
De Novo ◽  
Vitamin B ◽  
E Coli ◽  
De Novo Biosynthesis ◽  
Optimization Of Fermentation ◽  
Aerobic And Anaerobic

ABSTRACTThe only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea, and the only unknown step in its biosynthesis is the production of the intermediate adenosylcobinamide phosphate. Here, using genetic and metabolic engineering, we generated an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. Excitingly, the BluE and CobC enzymes from Rhodobacter capsulatus transform L-threonine into (R)-1-Amino-2-propanol O-2-Phosphate, which is then condensed with adenosylcobyric acid to yield adenosylcobinamide phosphate by either CobD from the aeroic R. capsulatus or CbiB from the anerobic Salmonella typhimurium. These findings suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increased the vitamin B12 yield of a recombinant E. coli strain by more than ∼250-fold to 307.00 µg/g DCW via metabolic engineering and optimization of fermentation conditions. Beyond our scientific insights about the aerobic and anaerobic pathways and our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.

scholarly journals CorA Affects Tolerance of Escherichia coli and Salmonella enterica Serovar Typhimurium to the Lactoperoxidase Enzyme System but Not to Other Forms of Oxidative Stress

2005 ◽  
Vol 71 (11) ◽  
pp. 6515-6523 ◽  
Author(s):  
Jan Sermon ◽  
Eva M.-R. P. Wevers ◽  
Leentje Jansen ◽  
Philipp De Spiegeleer ◽  
Kristof Vanoirbeek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Stress Responses ◽  
Salmonella Enterica ◽  
Specific Inhibitor ◽  
Open Reading Frames ◽  
Reaction Products ◽  
E Coli ◽  
Lactoperoxidase System ◽  
Serovar Typhimurium

ABSTRACT The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg2+ transporter and at least three other inducible open reading frames belonged to the Mg2+ regulon, repression of the Mg stimulon by Mg2+ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni2+, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni2+-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.

Transformation of Tobacco with a Mutated Cyanobacterial Phytoene Desaturase Gene Confers Resistance to Bleaching Herbicides

2002 ◽  
Vol 57 (7-8) ◽  
pp. 671-679 ◽  
Author(s):  
Tobias Wagner ◽  
Ute Windhövel ◽  
Susanne Römer
Keyword(s):  
Genetic Manipulation ◽  
Enzymatic Reaction ◽  
Photosynthetic Apparatus ◽  
D1 Protein ◽  
Carotenoid Biosynthesis ◽  
Phytoene Desaturase ◽  
Desaturase Gene ◽  
Synechococcus Pcc 7942 ◽  
Pcc 7942 ◽  
Transformed Tobacco

Carotenoids are constituents of the photosynthetic apparatus and essential for plant survival because of their involvement in protection of chlorophylls against photooxidation. Certain classes of herbicides are interfering with carotenoid biosynthesis leading to pigment destruction and a bleached plant phenotype. One important target site for bleaching herbicides is the enzyme phytoene desaturase catalysing the desaturation of phytoene in ζ-carotene. This enzymatic reaction can be inhibited by norflurazon or fluridone. We have transformed tobacco with a mutated cyanobacterial phytoene desaturase gene (pds) derived from the Synechococcus PCC 7942 mutant NFZ4. Characterization of the resulting transformants revealed an up to 58 fold higher norflurazon resistance in comparison to wild type controls. The tolerance for fluridone was also increased 3 fold in the transgenics. Furthermore, the transformed tobacco maintained a higher level of D1 protein of photosystem II indicating a lower susceptibility to photooxidative damage in the presence of norflurazon. In contrast, the genetic manipulation did not confer herbicide resistance against ζ-carotene desaturase inhibitors.

scholarly journals Heterologous Carotenoid-Biosynthetic Enzymes: Functional Complementation and Effects on Carotenoid Profiles in Escherichia coli

2012 ◽  
Vol 79 (2) ◽  
pp. 610-618 ◽  
Author(s):  
Gyu Hyeon Song ◽  
Se Hyeuk Kim ◽  
Bo Hyun Choi ◽  
Se Jong Han ◽  
Pyung Cheon Lee
Keyword(s):  
Escherichia Coli ◽  
Rhodobacter Capsulatus ◽  
Carotenoid Biosynthesis ◽  
Functional Complementation ◽  
Content Type ◽  
Brevibacterium Linens ◽  
E Coli ◽  
Carotenoid Biosynthesis Pathway ◽  
Different Sources ◽  
Carotenoid Pathway

ABSTRACTA limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous hostEscherichia coli. In order to systematically investigate the functionality of carotenoid pathway enzymes inE. coli, the pathway genes of carotenogenic microorganisms (Brevibacterium linens,Corynebacterium glutamicum,Rhodobacter sphaeroides,Rhodobacter capsulatus,Rhodopirellula baltica, andPantoea ananatis) were modified to form synthetic expression modules and then were complemented withPantoea agglomeranspathway enzymes (CrtE, CrtB, CrtI, CrtY, and CrtZ). The carotenogenic pathway enzymes in the synthetic modules showed unusual activities when complemented withE. coli. For example, the expression of heterologous CrtEs ofB. linens,C. glutamicum, andR. balticainfluencedP. agglomeransCrtI to convert its substrate phytoene into a rare product—3,4,3′,4′-tetradehydrolycopene—along with lycopene, which was an expected product, indicating that CrtE, the first enzyme in the carotenoid biosynthesis pathway, can influence carotenoid profiles. In addition, CrtIs ofR. sphaeroidesandR. capsulatusconverted phytoene into an unusual lycopene as well as into neurosporene. Thus, this study shows that the functional complementation of pathway enzymes from different sources is a useful methodology for diversifying biosynthesis as nature does.

scholarly journals Expression in Escherichia coli and characterization of a recombinant 7Fe ferredoxin of Rhodobacter capsulatus

1992 ◽  
Vol 286 (1) ◽  
pp. 269-273 ◽  
Author(s):  
Y Jouanneau ◽  
C Duport ◽  
C Meyer ◽  
J Gaillard
Keyword(s):  
Escherichia Coli ◽  
Rhodobacter Capsulatus ◽  
Spectroscopic Properties ◽  
Coli Cell ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Gene Coding ◽  
Lac Promoter

The 7Fe ferredoxin of Rhodobacter capsulatus (FdII) could be expressed in Escherichia coli by cloning the fdxA gene coding for FdII downstream from the lac promoter. The expressed recombinant ferredoxin appeared as a brown protein which was specifically recognized in E. coli cell-free extracts by anti-FdII serum. The purified recombinant ferredoxin was indistinguishable from R. capsulatus FdII on the basis of its molecular, redox and spectroscopic properties. These results indicate that the [3Fe-4S] and [4Fe-4S] clusters were correctly inserted into the recombinant ferredoxin.

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