scholarly journals Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli

1997 ◽  
Vol 324 (2) ◽  
pp. 421-426 ◽  
Author(s):  
Susumu KAJIWARA ◽  
Paul D. FRASER ◽  
Keiji KONDO ◽  
Norihiko MISAWA
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Genomic Library ◽  
Haematococcus Pluvialis ◽  
Isoprenoid Biosynthesis ◽  
Nucleotide Sequence Analysis ◽  
Efficient Production ◽  
Isopentenyl Diphosphate ◽  
E Coli ◽  
Β Carotene

Escherichia coli expressing the Erwinia carotenoid biosynthesis genes, crtE, crtB, crtI and crtY, form yellow-coloured colonies due to the presence of β-carotene. This host was used as a visible marker for evaluating regulatory systems operating in isoprenoid biosynthesis of E. coli. cDNAs enhancing carotenoid levels were isolated from the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. Nucleotide sequence analysis indicated that they coded for proteins similar to isopentenyl diphosphate (IPP) isomerase of the yeast Saccharomyces cerevisiae. Determination of enzymic activity confirmed the identity of the gene products as IPP isomerases. The corresponding gene was isolated from the genomic library of S. cerevisiae based on its nucleotide sequence, and was confirmed to have the same effect as the above two IPP isomerase genes when introduced into the E. coli transformant accumulating β-carotene. In the three E. coli strains carrying the individual exogenous IPP isomerase genes, the increases in carotenoid levels are comparable to the increases in IPP isomerase enzyme activity with reference to control strains possessing the endogenous gene alone. These results imply that IPP isomerase forms an influential step in isoprenoid biosynthesis of the prokaryote E. coli, with potential for the efficient production of industrially useful isoprenoids by metabolic engineering.

scholarly journals Cloning, Nucleotide Sequence, and Expression in Escherichia coli of Levansucrase Genes from the Plant PathogensPseudomonas syringae pv. glycinea and P. syringaepv. phaseolicola

1998 ◽  
Vol 64 (9) ◽  
pp. 3180-3187 ◽  
Author(s):  
Ursula Hettwer ◽  
Frank R. Jaeckel ◽  
Jens Boch ◽  
Manfred Meyer ◽  
Klaus Rudolph ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Pseudomonas Syringae ◽  
Pathogenic Bacteria ◽  
Genomic Library ◽  
Bacterial Species ◽  
Extracellular Polysaccharides ◽  
Nucleotide Sequence Analysis ◽  
Pcr Screening ◽  
E Coli

Plant-pathogenic bacteria produce various extracellular polysaccharides (EPSs) which may function as virulence factors in diseases caused by these bacteria. The EPS levan is synthesized by the extracellular enzyme levansucrase in Pseudomonas syringae,Erwinia amylovora, and other bacterial species. Thelsc genes encoding levansucrase from P. syringae pv. glycinea PG4180 and P. syringae pv. phaseolicola NCPPB 1321 were cloned, and their nucleotide sequences were determined. Heterologous expression of the lsc gene inEscherichia coli was found in four and two genomic library clones of strains PG4180 and NCPPB 1321, respectively. A 3.0-kbPstI fragment common to all six clones conferred levan synthesis on E. coli when further subcloned. Nucleotide sequence analysis revealed a 1,248-bp open reading frame (ORF) derived from PG4180 and a 1,296-bp ORF derived from NCPPB 1321, which were both designated lsc. Both ORFs showed high homology to theE. amylovora and Zymomonas mobilis lsc genes at the nucleic acid and deduced amino acid sequence levels. Levansucrase was not secreted into the supernatant but was located in the periplasmic fraction of E. coli harboring thelsc gene. Expression of lsc was found to be dependent on the vector-based P lac promoter, indicating that the native promoter of lsc was not functional in E. coli. Insertion of an antibiotic resistance cassette in the lsc gene abolished levan synthesis in E. coli. A PCR screening with primers derived from lsc of P. syringae pv. glycinea PG4180 allowed the detection of this gene in a number of related bacteria.

scholarly journals Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide

2004 ◽  
Vol 70 (3) ◽  
pp. 1570-1575 ◽  
Author(s):  
Dae Heoun Baek ◽  
Jae Jun Song ◽  
Seok-Joon Kwon ◽  
Chung Park ◽  
Chang-Min Jung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Reading Frame ◽  
E Coli ◽  
Specificity Constant ◽  
Optimal Ph

ABSTRACT A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k cat /Km ) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 � 2.2 [E = (k cat /Km ) l,d /(k cat /Km ) d,d ], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 � 0.5 [E = (k cat /Km ) l,d /(k cat /Km ) l,l ] at 55�C. The enzyme was stable up to 55�C, and the optimal pH and temperature were 8.5 and 65�C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.

scholarly journals Cloning of the groE operon of the marine bacterium Vibrio harveyi using a lambda vector.

1998 ◽  
Vol 45 (1) ◽  
pp. 261-270 ◽  
Author(s):  
D Kuchanny ◽  
G Klein ◽  
J Krzewska ◽  
A Czyz ◽  
B Lipińska
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Marine Bacterium ◽  
Southern Hybridization ◽  
Genomic Library ◽  
Vibrio Harveyi ◽  
Bacteriophage Lambda ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Dna Fragment

groES and groEL genes encode two co-operating proteins GroES and GroEL, belonging to a class of chaperone proteins highly conserved during evolution. The GroE chaperones are indispensable for the growth of bacteriophage lambda in Escherichia coli cells. In order to clone the groEL and groES genes of the marine bacterium Vibrio harveyi, we constructed the V. harveyi genomic library in the lambdaEMBL1 vector, and selected clones which were able to complement mutations in both groE genes of E. coli for bacteriophage lambda growth. Using Southern hybridization, in one of these clones we identified a DNA fragment homologous to the E. coli groE region. Analysis of the nucleotide sequence of this fragment showed that the cloned region contained a sequence in 71.7% homologous to the 3' end of the groEL gene of E. coli. This confirmed that the lambda clone indeed carries the groE region of V. harveyi. The positive result of our strategy of cloning with the use of the genomic library in lambda vector suggests that the same method might be useful in the isolation of the groE homologues from other bacteria. The V. harveyi cloned groE genes did not suppress thermosensitivity of the E. coli groE mutants.

scholarly journals The lytB Gene of Escherichia coli Is Essential and Specifies a Product Needed for Isoprenoid Biosynthesis

2001 ◽  
Vol 183 (24) ◽  
pp. 7403-7407 ◽  
Author(s):  
Sean McAteer ◽  
Andrew Coulson ◽  
Neil McLennan ◽  
Millicent Masters
Keyword(s):  
Escherichia Coli ◽  
Protein Structure ◽  
Cell Lysis ◽  
Isoprenoid Biosynthesis ◽  
Globular Protein ◽  
Limited Growth ◽  
E Coli ◽  
Nonmevalonate Pathway ◽  
Tim Barrel

ABSTRACT LytB and GcpE, because they are codistributed with other pathway enzymes, have been predicted to catalyze unknown steps in the nonmevalonate pathway for isoprenoid biosynthesis. We constructed a conditional Escherichia coli lytB mutant and found that LytB is essential for survival and that depletion of LytB results in cell lysis, which is consistent with a role for this protein in isoprenoid biosynthesis. Alcohols which can be converted to pathway intermediates beyond the hypothesized LytB step(s) support limited growth of E. coli lytB mutants. An informatic analysis of protein structure suggested that GcpE is a globular protein of the TIM barrel class and that LytB is also a globular protein. Possible biochemical roles for LytB and GcpE are suggested.

scholarly journals Metabolic engineering of Escherichia coli W for isobutanol production on chemically defined medium and cheese whey as alternative raw material

2020 ◽  
Vol 47 (12) ◽  
pp. 1117-1132
Author(s):  
Katharina Novak ◽  
Juliane Baar ◽  
Philipp Freitag ◽  
Stefan Pflügl
Keyword(s):  
Escherichia Coli ◽  
Cheese Whey ◽  
Strain Improvement ◽  
Defined Medium ◽  
Raw Material ◽  
Efficient Production ◽  
Substrate Uptake ◽  
Chemically Defined Medium ◽  
E Coli ◽  
Plasmid Library

AbstractThe aim of this study was to establish isobutanol production on chemically defined medium in Escherichia coli. By individually expressing each gene of the pathway, we constructed a plasmid library for isobutanol production. Strain screening on chemically defined medium showed successful production in the robust E. coli W strain, and expression vector IB 4 was selected as the most promising construct due to its high isobutanol yields and efficient substrate uptake. The investigation of different aeration strategies in combination with strain improvement and the implementation of a pulsed fed-batch were key for the development of an efficient production process. E. coli W ΔldhA ΔadhE Δpta ΔfrdA enabled aerobic isobutanol production at 38% of the theoretical maximum. Use of cheese whey as raw material resulted in longer process stability, which allowed production of 20 g l−1 isobutanol. Demonstrating isobutanol production on both chemically defined medium and a residual waste stream, this study provides valuable information for further development of industrially relevant isobutanol production processes.

scholarly journals Efficient production of (S)-1-phenyl-1, 2-ethanediol using xylan as co-substrate by a coupled multi-enzyme Escherichia coli system

2020 ◽  
Author(s):  
Junchao Rao ◽  
Rongzhen Zhang ◽  
Guanyu Xu ◽  
Lihong Li ◽  
Yan Xu
Keyword(s):  
Escherichia Coli ◽  
Glucose Dehydrogenase ◽  
Optical Purity ◽  
Carbonyl Reductase ◽  
Cofactor Regeneration ◽  
Efficient Production ◽  
Chiral Synthesis ◽  
E Coli ◽  
Concomitant Reduction ◽  
Different Strains

Abstract Background: ( S )-1-phenyl-1,2-ethanediol is an important chiral intermediate in the synthesis of liquid crystals and chiral biphosphines.(S)-carbonyl reductase II from Candida parapsilosis catalyzes the conversion of 2-hydroxyacetophenone to ( S )-1-phenyl-1,2-ethanediol with NADPH as a cofactor. Glucose dehydrogenase with a Ala258Phe mutation is able to catalyze the oxidation of xylose with concomitant reduction of NADP + to NADPH, while endo-β-1,4-xylanase 2 catalyzes the conversion of xylan to xylose. In the present work, the Ala258Phe glucose dehydrogenase mutant and endo-β-1,4-xylanase 2 were introduced into the ( S )-carbonyl reductase II-mediated chiral pathway to strengthen cofactor regeneration by using xylan as a naturally abundant co-substrate. Results: We constructed several coupled multi-enzyme systems by introducing ( S )-carbonyl reductase II, the A258F glucose dehydrogenase mutant and endo-β-1,4-xylanase 2 into Escherichia coli . Different strains were produced by altering the location of the encoding genes on the plasmid. Only recombinant E. coli /pET-G-S-2 expressed all three enzymes, and this strain produced ( S )-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone as a substrate and xylan as a co-substrate. The optical purity was 100% and the yield was 98.3% (6 g/L 2-HAP) under optimal conditions of 35°C, pH 6.5 and a 2:1 substrate-co-substrate ratio. The introduction of A258F glucose dehydrogenase and endo-β-1,4-xylanase 2 into the ( S )-carbonyl reductase II-mediated chiral pathway caused a 54.6% increase in yield, and simultaneously reduced the reaction time from 48 h to 28 h. Conclusions: This study demonstrates efficient chiral synthesis using a pentose as a co-substrate to enhance cofactor regeneration. This provides a new approach for enantiomeric catalysis through the inclusion of naturally abundant materials.

Evaluation of Structurally Different Carotenoids inEscherichia coli Transformants as Protectants against UV-B Radiation

1998 ◽  
Vol 64 (5) ◽  
pp. 1972-1974 ◽  
Author(s):  
Gerhard Sandmann ◽  
Silvia Kuhn ◽  
Peter B�ger
Keyword(s):  
Escherichia Coli ◽  
Inescherichia Coli ◽  
Short Term ◽  
E Coli ◽  
Carotenogenic Genes ◽  
Β Carotene

ABSTRACT Escherichia coli cells transformed with several carotenogenic genes to mediate the formation of ζ-carotene, neurosporene, lycopene, β-carotene, and zeaxanthin were exposed to UV-B radiation. Short-term kinetics revealed that endogenous levels of neurosporene and β-carotene protected E. coli against irradiation with UV-B. Zeaxanthin protected against only the photosensitized UV-B treatment. All other carotenoids were ineffective.

Carotenoid analysis of sweetpotato Ipomoea batatas and functional identification of its lycopene β- and ε-cyclase genes

2016 ◽  
Vol 71 (9-10) ◽  
pp. 313-322 ◽  
Author(s):  
Muhammad Zubair Khan ◽  
Miho Takemura ◽  
Takashi Maoka ◽  
Motoyasu Otani ◽  
Norihiko Misawa
Keyword(s):  
Ipomoea Batatas ◽  
Hplc Analysis ◽  
Gene Sequence ◽  
Efficient Production ◽  
Functional Identification ◽  
Rt Pcr ◽  
Pantoea Ananatis ◽  
E Coli ◽  
White Star ◽  
Β Carotene

Abstract Sweetpotato Ipomoea batatas is known as a hexaploid species. Here, we analyzed carotenoids contained in the leaves and tubers of sweetpotato cultivars ‘White Star’ (WS) and W71. These cultivars were found to contain several carotenoids unique to sweetpotato tubers such as β-carotene-5,6,5′,8′-diepoxide and β-carotene-5,8-epoxide. Next, we isolated two kinds of carotene cyclase genes that encode lycopene β- and ε-cyclases from the WS and W71 leaves, by RT-PCR and subsequent RACE. Two and three lycopene β-cyclase gene sequences were, respectively, isolated from WS, named IbLCYb1, 2, and from W71, IbLCYb3, 4, 5. Meanwhile, only a single lycopene ε-cyclase gene sequence, designated IbLCYe, was isolated from both WS and W71. These genes were separately introduced into a lycopene-synthesizing Escherichia coli transformed with the Pantoea ananatis crtE, crtB and crtI genes, followed by HPLC analysis. β-Carotene was detected in E. coli cells that carried IbLCYb1-4, indicating that the IbLCYb1-4 genes encode lycopene β-cyclase. Meanwhile, the introduction of IbLCYe into the lycopene-synthesizing E. coli led to efficient production of δ-carotene with a monocyclic ε-ring, providing evidence that the IbLCYe gene codes for lycopene ε-(mono)cyclase. Expression of the β- and ε-cyclase genes was analyzed as well.

scholarly journals Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate

1993 ◽  
Vol 295 (2) ◽  
pp. 517-524 ◽  
Author(s):  
M Rohmer ◽  
M Knani ◽  
P Simonin ◽  
B Sutter ◽  
H Sahm
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Carbonyl Group ◽  
Zymomonas Mobilis ◽  
Biosynthetic Pathway ◽  
Isoprenoid Biosynthesis ◽  
Dihydroxyacetone Phosphate ◽  
Isopentenyl Diphosphate ◽  
Valine Biosynthesis

Incorporation of 13C-labelled glucose, acetate, pyruvate or erythrose allowed the determination of the origin of the carbon atoms of triterpenoids of the hopane series and/or of the ubiquinones from several bacteria (Zymomonas mobilis, Methylobacterium fujisawaense, Escherichia coli and Alicyclobacillus acidoterrestris) confirmed our earlier results obtained by incorporation of 13C-labelled acetate into the hopanoids of other bacteria and led to the identification of a novel biosynthetic route for the early steps of isoprenoid biosynthesis. The C5 framework of isoprenic units results most probably (i) from the condensation of a C2 unit derived from pyruvate decarboxylation (e.g. thiamine-activated acetaldehyde) on the C-2 carbonyl group of a triose phosphate derivative issued probably from dihydroxyacetone phosphate and not from pyruvate and (ii) from a transposition step. Although this hypothetical biosynthetic pathway resembles that of L-valine biosynthesis, this amino acid or its C5 precursors could be excluded as intermediates in the formation of isoprenic units.

scholarly journals Improved Squalene Production via Modulation of the Methylerythritol 4-Phosphate Pathway and Heterologous Expression of Genes from Streptomyces peucetius ATCC 27952 in Escherichia coli

2009 ◽  
Vol 75 (22) ◽  
pp. 7291-7293 ◽  
Author(s):  
Gopal Prasad Ghimire ◽  
Hei Chan Lee ◽  
Jae Kyung Sohng
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Isopentenyl Diphosphate ◽  
Isopentenyl Diphosphate Isomerase ◽  
Streptomyces Peucetius ◽  
E Coli ◽  
Expression Of Genes ◽  
Genes Encoding

ABSTRACT Putative hopanoid genes from Streptomyces peucetius were introduced into Escherichia coli to improve the production of squalene, an industrially important compound. High expression of hopA and hopB (encoding squalene/phytoene synthases) together with hopD (encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of dxs and idi, E. coli genes encoding 1-deoxy-d-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.

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