Evolving improved Synechococcus Rubisco functional expression in Escherichia coli

2008 ◽  
Vol 414 (2) ◽  
pp. 205-214 ◽  
Author(s):  
Oliver Mueller-Cajar ◽  
Spencer M. Whitney
Keyword(s):  
Escherichia Coli ◽  
Large Subunit ◽  
Fold Increase ◽  
Functional Expression ◽  
Kinetic Properties ◽  
Selection System ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
E Coli ◽  
Enhanced Expression

The photosynthetic CO2-fixing enzyme Rubisco [ribulose-P2 (D-ribulose-1,5-bisphosphate) carboxylase/oxygenase] has long been a target for engineering kinetic improvements. Towards this goal we used an RDE (Rubisco-dependent Escherichia coli) selection system to evolve Synechococcus PCC6301 Form I Rubisco under different selection pressures. In the fastest growing colonies, the Rubisco L (large) subunit substitutions I174V, Q212L, M262T, F345L or F345I were repeatedly selected and shown to increase functional Rubisco expression 4- to 7-fold in the RDE and 5- to 17-fold when expressed in XL1-Blue E. coli. Introducing the F345I L-subunit substitution into Synechococcus PCC7002 Rubisco improved its functional expression 11-fold in XL1-Blue cells but could not elicit functional Arabidopsis Rubisco expression in the bacterium. The L subunit substitutions L161M and M169L were complementary in improving Rubisco yield 11-fold, whereas individually they improved yield ∼5-fold. In XL1-Blue cells, additional GroE chaperonin enhanced expression of the I174V, Q212L and M262T mutant Rubiscos but engendered little change in the yield of the more assembly-competent F345I or F345L mutants. In contrast, the Rubisco chaperone RbcX stimulated functional assembly of wild-type and mutant Rubiscos. The kinetic properties of the mutated Rubiscos varied with noticeable reductions in carboxylation and oxygenation efficiency accompanying the Q212L mutation and a 2-fold increase in Kribulose-P2 (KM for the substrate ribulose-P2) for the F345L mutant, which was contrary to the ∼30% reductions in Kribulose-P2 for the other mutants. These results confirm the RDE systems versatility for identifying mutations that improve functional Rubisco expression in E. coli and provide an impetus for developing the system to screen for kinetic improvements.

KINETICS OF THREONINE DEAMINASE OF ESCHERICHIA COLI K-12 AND A STREPTOMYCIN-DEPENDENT MUTANT

1967 ◽  
Vol 45 (1) ◽  
pp. 1-10 ◽  
Author(s):  
I. D. Desai ◽  
W. J. Polglase
Keyword(s):  
Escherichia Coli ◽  
Selective Advantage ◽  
Kinetic Properties ◽  
Wild Type ◽  
Threonine Deaminase ◽  
E Coli ◽  
Type K ◽  
Hyperbolic Curve ◽  
K 12 ◽  
Kinetics Of

The relation between threonine deaminase activity and threonine concentration in sonic extracts of wild-type and streptomycin-dependent Escherichia coli K-12 was found to follow a hyperbolic curve. A similar relationship was obtained between enzyme activity and pyridoxal concentration. However, when serine was used as substrate, the activity–concentration curve was sigmoid, suggesting that serine may be a weaker effector of allosteric transition than threonine. The kinetic properties of the (derepressed) threonine deaminase of streptomycin-dependent E. coli K-12 were found to be similar to those of the enzyme of the wild-type K-12.It is postulated that derepression of threonine deaminase in streptomycin-dependent E. coli K-12 provides a selective advantage which permits exponential growth of this mutant in the presence of L-valine, which is an excretory product of streptomycin-dependent microorganisms.

scholarly journals Expression of the Corynebacterium glutamicum panD Gene Encoding l-Aspartate-α-Decarboxylase Leads to Pantothenate Overproduction in Escherichia coli

1999 ◽  
Vol 65 (4) ◽  
pp. 1530-1539 ◽  
Author(s):  
Nicole Dusch ◽  
Alfred Pühler ◽  
Jörn Kalinowski
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Specific Productivity ◽  
Decarboxylase Activity ◽  
Coding Region ◽  
E Coli ◽  
Enhanced Expression

ABSTRACT The Corynebacterium glutamicum panD gene was identified by functional complementation of an Escherichia coli panDmutant strain. Sequence analysis revealed that the coding region ofpanD comprises 411 bp and specifies a protein of 136 amino acid residues with a deduced molecular mass of 14.1 kDa. A definedC. glutamicum panD mutant completely lackedl-aspartate-α-decarboxylase activity and exhibited β-alanine auxotrophy. The C. glutamicum panD(panDC.g. ) as well as the E. coli panD (panDE.c. ) genes were cloned into a bifunctional expression plasmid to allow gene analysis in C. glutamicum as well as in E. coli. The enhanced expression of panDC.g. in C. glutamicum resulted in the formation of two distinct proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, leading to the assumption that the panDC.g. gene product is proteolytically processed into two subunits. By increased expression of panDC.g. in C. glutamicum, the activity of l-aspartate-α-decarboxylase was 288-fold increased, whereas the panDE.c. gene resulted only in a 4-fold enhancement. The similar experiment performed inE. coli revealed that panDC.g. achieved a 41-fold increase and that panDE.c. achieved a 3-fold increase of enzyme activity. The effect of thepanDC.g. and panDE.c. gene expression in E. coli was studied with a view to pantothenate accumulation. Only by expression of thepanDC.g. gene was sufficient β-alanine produced to abolish its limiting effect on pantothenate production. In cultures expressing the panDE.c. gene, the maximal pantothenate production was still dependent on external β-alanine supplementation. The enhanced expression ofpanDC.g. in E. coli yielded the highest amount of pantothenate in the culture medium, with a specific productivity of 140 ng of pantothenate mg (dry weight)−1h−1.

scholarly journals Pinpointing Synechococcus Rubisco Large Subunit Sections Involved in Heterologous Holoenzyme Formation in Escherichia Coli

2021 ◽  
Author(s):  
Wei Chi Ong ◽  
Moh Lan Yap ◽  
Hann Ling Wong ◽  
Boon Hoe Lim
Keyword(s):  
Escherichia Coli ◽  
Large Subunit ◽  
Synechococcus Elongatus ◽  
Bisphosphate Carboxylase ◽  
E Coli ◽  
Successful Expression

Abstract Background: Heterologous holoenzyme formation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been a challenge due to limited understanding of its biogenesis. Unlike bacterial Rubiscos, eukaryotic Rubiscos are incompatible with the Escherichia coli chaperone system to fold and assemble into the functional hexadecameric conformation (L8S8), which comprise eight large subunits (RbcL) and eight small subunits (RbcS). Our previous study reported three sections (residues 248-297, 348-397 and 398-447) within the RbcL of Synechococcus elongatus PCC6301 may be important for formation of L8S8 in E. coli. Present study further examined these three sections separately, by dividing them into six sections of 25 residues (i.e. residues 248-272, 273-297, 348-372, 373-397, 398-422 and 423-447). Methods and Results: Six chimeric Rubiscos with each section within the RbcL from Synechococcus replaced by their respective counterpart sequence from Chlamydomonas reinhardtii were constructed and checked for their effect on holoenzyme formation in E. coli. Present study shows that Section 1 (residues 248-272; section of Synechococcus RbcL replaced by corresponding Chlamydomonas sequence), Section 2 (residues 273-297), Section 3 (residues 348-372) and Section 6 (residues 423-447) chimeras failed to fold and/or assemble despite successful expression of both RbcL and RbcS. Only Section 4 (residues 373-397) and 5 (residues 398-422) chimeras could form L8S8 in E. coli. Conclusion: As GroEL chaperonin mediates folding of bacterial RbcL in E. coli, residues 248-297, 348-372 and 423-447 of Synechococcus RbcL may be important for interacting with the GroEL chaperonin for successful holoenzyme formation in E. coli.

scholarly journals Mutagenesis of Escherichia coli acetohydroxyacid synthase isoenzyme II and characterization of three herbicide-insensitive forms

1998 ◽  
Vol 335 (3) ◽  
pp. 653-661 ◽  
Author(s):  
Craig M. HILL ◽  
Ronald G. DUGGLEBY
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Molecular Model ◽  
Small Subunit ◽  
Acetohydroxyacid Synthase ◽  
Kinetic Properties ◽  
Wild Type ◽  
Thiamin Diphosphate ◽  
E Coli ◽  
Branched Chain

Sulphonylurea and imidazolinone herbicides act by inhibiting acetohydroxyacid synthase (AHAS; EC 4.1.3.18), the enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids. AHAS requires as cofactors thiamin diphosphate, a bivalent metal ion and, usually, FAD. Escherichia coli contains three isoenzymes and this study concerns isoenzyme II, the most herbicide-sensitive of the E. coli forms. A plasmid containing the large and small subunit genes of AHAS II was mutagenized using hydroxylamine and clones resistant to the sulphonylurea chlorimuron ethyl were selected. Three mutants were isolated; A26V, V99M and A108V. A26V has been described previously whereas the equivalent mutation of A108V has been reported in a herbicide-insensitive variant of yeast AHAS. The V99M mutation has not been discovered previously in AHAS from any source. The mutants were each over-expressed in E. coli, and the enzymes were purified to homogeneity. Some differences from wild type in the kinetic properties (kcat, Km and cofactor affinities) were observed, most notably a 28-fold decrease in the affinity for thiamin diphosphate of V99M. None of the mutants shows marked changes from the wild type in sensitivity to three imidazolinones, with the largest increase in the apparent inhibition constant being a factor of approximately 5. The A26V mutant is weakly resistant (6- to 20-fold) to six sulphonylureas, whereas stronger resistance is seen in V99M (20- to 250-fold) and A108V (35- to 420-fold). Resistance as a result of these mutations is consistent with a molecular model of the herbicide-binding site, which predicts that mutation of G249 might also confer herbicide insensitivity. Three G249 mutants were constructed, expressed and purified but all are inactive, apparently because they cannot bind FAD.

scholarly journals Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA.

1996 ◽  
Vol 40 (6) ◽  
pp. 1426-1431 ◽  
Author(s):  
R Frothingham ◽  
W A Meeker-O'Connell ◽  
E A Talbot ◽  
J W George ◽  
K N Kreuzer
Keyword(s):  
Escherichia Coli ◽  
Genetic Locus ◽  
Fold Increase ◽  
Pyridine Nucleotide ◽  
Cloning And Expression ◽  
Insertion Mutant ◽  
Activity Levels ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
E Coli

Pyrazinamide (PZA) is one of the three most important drugs for treatment of Mycobacterium tuberculosis infections. The antibacterial activity of PZA requires a bacterial enzyme, pyrazinamidase (PZAase), which hydrolyzes PZA to form pyrazinoic acid and ammonia. Most PZA-resistant clinical M. tuberculosis isolates lack PZAase activity. With the goal of eventually identifying and characterizing the M.tuberculosis PZAase gene, we began with the more tractable organism, Escherichia coli, which also has PZAase activity. We screened a transposon-generated E. coli insertion mutant library, using a qualitative PZAase assay. Two PZAase-negative mutants out of 4,000 colonies screened were identified. In each mutant, the transposon interrupted the same 639-bp open reading frame (ORF), ORF1. The expression of ORF1 on a multicopy plasmid complemented a PZAase-negative mutant, leading to PZAase activity levels approximately 10-fold greater than those of the wild type. PZA has a structure similar to that of nicotinamide, a pyridine nucleotide cycle intermediate, so we tested our strains for nicotinamidase activity (EC 3.5.1.19) (genetic locus pncA). The construct with multiple plasmid copies of ORF1 had an approximately 10-fold increase in levels of nicotinamidase activity. This overexpressing strain could utilize nicotinamide as a sole nitrogen source, through wild-type E. coli cannot. We conclude that a single E. coli enzyme accounts for both PZAase and nicotinamidase activities and that ORF1 is the E.coli PZAase and nicotinamidase gene, pncA.

scholarly journals Artificially evolved Synechococcus PCC6301 Rubisco variants exhibit improvements in folding and catalytic efficiency

2007 ◽  
Vol 404 (3) ◽  
pp. 517-524 ◽  
Author(s):  
Dina N. Greene ◽  
Spencer M. Whitney ◽  
Ichiro Matsumura
Keyword(s):  
Large Subunit ◽  
Calvin Cycle ◽  
Catalytic Efficiency ◽  
Trigger Factor ◽  
Growth Media ◽  
Kinetic Properties ◽  
Evolutionary Potential ◽  
Bisphosphate Carboxylase ◽  
E Coli

The photosynthetic CO2-fixing enzyme, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), is responsible for most of the world's biomass, but is a slow non-specific catalyst. We seek to identify and overcome the chemical and biological constraints that limit the evolutionary potential of Rubisco in Nature. Recently, the horizontal transfer of Calvin cycle genes (rbcL, rbcS and prkA) from cyanobacteria (Synechococcus PCC6301) to γ-proteobacteria (Escherichia coli) was emulated in the laboratory. Three unique Rubisco variants containing single (M259T) and double (M259T/A8S, M259T/F342S) amino acid substitutions in the L (large) subunit were identified after three rounds of random mutagenesis and selection in E. coli. Here we show that the M259T mutation did not increase steady-state levels of rbcL mRNA or L protein. It instead improved the yield of properly folded L subunit in E. coli 4–9-fold by decreasing its natural propensity to misfold in vivo and/or by enhancing its interaction with the GroES–GroEL chaperonins. The addition of osmolites to the growth media enhanced productive folding of the M259T L subunit relative to the wild-type L subunit, while overexpression of the trigger factor and DnaK/DnaJ/GrpE chaperones impeded Rubisco assembly. The evolved enzymes showed improvement in their kinetic properties with the M259T variant showing a 12% increase in carboxylation turnover rate (kccat), a 15% improvement in its KM for CO2 and no change in its KM for ribulose-1,5-bisphosphate or its CO2/O2 selectivity. The results of the present study show that the directed evolution of the Synechococcus Rubisco in E. coli can elicit improvements in folding and catalytic efficiency.

scholarly journals Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain

1994 ◽  
Vol 300 (1) ◽  
pp. 111-115 ◽  
Author(s):  
M H Rider ◽  
K M Crepin ◽  
M De Cloedt ◽  
L Bertrand ◽  
L Hue
Keyword(s):  
Escherichia Coli ◽  
Skeletal Muscle ◽  
Fold Increase ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Wild Type ◽  
Fluorescence Measurements

Asp-130 of the recombinant skeletal-muscle 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase was mutated into Ala in order to study its role in catalysis and/or substrate binding. The D130A mutant displayed a 30- to 140-fold decreased 2-kinase Vmax, depending on the pH, and a 30- and 60-fold increase in Km for MgATP and Fru-6-P respectively at pH 8.5 compared with the wild-type. Mutagenesis of Asp-130 to Ala had no effect on the 2-phosphatase activity, and fluorescence measurements indicated that the changes in kinetic properties of PFK-2 in the D130A mutant were not due to instability. The role of Asp-130 in the 2-kinase reaction is discussed and compared with that of Asp-103 of 6-phosphofructo-1-kinase from Escherichia coli, which binds Mg2+.

scholarly journals Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

1989 ◽  
Vol 9 (4) ◽  
pp. 1507-1512 ◽  
Author(s):  
H Zhu ◽  
H Conrad-Webb ◽  
X S Liao ◽  
P S Perlman ◽  
R A Butow
Keyword(s):  
Genetic Analysis ◽  
Rna Processing ◽  
Fold Increase ◽  
Functional Expression ◽  
Rrna Gene ◽  
Yeast Mitochondria ◽  
Wild Type ◽  
Site Specific ◽  
Var1 Gene ◽  
A Site

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

scholarly journals degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

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