scholarly journals Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

2021 ◽  
Vol 8 ◽  
Author(s):  
Feiyan Xue ◽  
Zihui Liu ◽  
Yue Yu ◽  
Yangjie Wu ◽  
Yuxin Jin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Specific Activity ◽  
Codon Optimization ◽  
Rhodotorula Glutinis ◽  
E Coli ◽  
Physical Conditions ◽  
Acid Protein ◽  
Heterogeneous Expression

PAL (phenylalanine ammonia lyase) is important for secondary metabolite production in plants and microorganisms. There is broad interest in engineering PAL for its biocatalytic applications in industry, agriculture, and medicine. The production of quantities of high-activity enzymes has been explored by gene cloning and heterogeneous expression of the corresponding protein. Here, we cloned the cDNA of Rhodotorula glutinis PAL (RgPAL) and introduced codon optimization to improve protein expression in Escherichia coli and enzyme activities in vitro. The RgPAL gene was cloned by reverse transcription and named pal-wt. It had a full-length of 2,121 bp and encoded a 706-amino-acid protein. The pal-wt was inefficiently expressed in E. coli, even when the expression host and physical conditions were optimized. Therefore, codon optimization was used to obtain the corresponding gene sequence, named pal-opt, in order to encode the same amino acid for the RgPAL protein. The recombinant protein encoded by pal-opt, named PAL-opt, was successfully expressed in E. coli and then purified to detect its enzymatic activity in vitro. Consequently, 55.33 ± 0.88 mg/L of PAL-opt protein with a specific activity of 1,219 ± 147 U/mg and Km value of 609 μM for substrate L-phenylalanine was easily obtained. The enzyme protein also displayed tyrosine ammonia lyase (TAL)–specific activity of 80 ± 2 U/mg and Km value of 13.3 μM for substrate L-tyrosine. The bifunctional enzyme RgPAL/TAL (PAL-opt) and its easy expression advantage will provide an important basis for further applications.

scholarly journals Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4891-4896 ◽  
Author(s):  
Ji Qiu ◽  
James R. Swartz ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
Plasminogen Activator ◽  
Disulfide Bonds ◽  
Specific Activity ◽  
Active Form ◽  
Tissue Type ◽  
Heterologous Proteins ◽  
E Coli ◽  
Disulfide Isomerases

ABSTRACT The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins inE. coli without the need for in vitro refolding.

scholarly journals Cloning, Nucleotide Sequence, and Overexpression of the l-Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli

2004 ◽  
Vol 70 (6) ◽  
pp. 3298-3304 ◽  
Author(s):  
Khim Leang ◽  
Goro Takada ◽  
Akihiro Ishimura ◽  
Masashi Okita ◽  
Ken Izumori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Pseudomonas Stutzeri ◽  
Fold Increase ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Volumetric Yield

ABSTRACT The gene encoding l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the l-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of l-RhI from E. coli are conserved in that from P. stutzeri. The l-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of l-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant l-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant l-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60�C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

scholarly journals Characterization of an Atypical Superoxide Dismutase from Sinorhizobium meliloti

1999 ◽  
Vol 181 (15) ◽  
pp. 4509-4516 ◽  
Author(s):  
Renata Santos ◽  
Stephane Bocquet ◽  
Alain Puppo ◽  
Danièle Touati
Keyword(s):  
Superoxide Dismutase ◽  
Amino Acid ◽  
Sinorhizobium Meliloti ◽  
Genomic Library ◽  
Specific Activity ◽  
Growth Conditions ◽  
Aerobic Bacterium ◽  
Degenerate Primers ◽  
E Coli ◽  
Acid Protein

ABSTRACT Sinorhizobium meliloti Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants. A single detectable superoxide dismutase (SOD) was found in free-living growth conditions. The corresponding gene was isolated from a genomic library by using a sod fragment amplified by PCR from degenerate primers as a probe. ThesodA gene was located in the chromosome. It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical M r of 22,430 and pI of 5.8.S. meliloti SOD complemented a deficient E. coli mutant, restoring aerobic growth of a sodA sodB recA strain, when the gene was expressed from the synthetictac promoter but not from its own promoter. Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H2O2. The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg. Despite its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of monomer). The apoenzyme was prepared from crude extracts of S. meliloti. Activity was restored by dialysis against either MnCl2 or Fe(NH4)2(SO4)2, demonstrating the cambialistic nature of the S. melilotiSOD. The recovered activity with manganese was sevenfold higher than with iron. Both reconstituted enzymes were resistant to H2O2. Sequence comparison with 70 FeSODs and MnSODs indicates that S. meliloti SOD contains several atypical residues at specific sites that might account for the activation by manganese and resistance to H2O2of this unusual Fe-type SOD.

scholarly journals Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

2003 ◽  
Vol 69 (7) ◽  
pp. 3791-3797 ◽  
Author(s):  
Nobuyuki Horinouchi ◽  
Jun Ogawa ◽  
Takafumi Sakai ◽  
Takako Kawano ◽  
Seiichiro Matsumoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Specific Activity ◽  
Cell Extract ◽  
Predicted Amino Acid Sequence ◽  
Enzymatic Reactions ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli

ABSTRACT The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2′-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.

scholarly journals Correlation between Quinolone Susceptibility Patterns and Sequences in the A and B Subunits of DNA Gyrase in Mycobacteria

1998 ◽  
Vol 42 (8) ◽  
pp. 2084-2088 ◽  
Author(s):  
Isabelle Guillemin ◽  
Vincent Jarlier ◽  
Emmanuelle Cambau
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Acquired Resistance ◽  
Dna Gyrase ◽  
Mycobacterial Species ◽  
Numbering System ◽  
E Coli ◽  
B Subunits ◽  
Susceptibility Patterns

ABSTRACT The in vitro activities of seven quinolones and the sequences of the quinolone resistance-determining regions (QRDR) in the A and B subunits of DNA gyrase were determined for 14 mycobacterial species. On the basis of quinolone activity, quinolones were arranged from that with the greatest to that with the least activity as follows: sparfloxacin, levofloxacin, ciprofloxacin, ofloxacin, pefloxacin, flumequine, and nalidixic acid. Based on MICs, the species could be organized into three groups: resistant (Mycobacterium avium, M. intracellulare, M. marinum,M. chelonae, M. abscessus [ofloxacin MICs, ≥8 μg/ml]), moderately susceptible (M. tuberculosis,M. bovis BCG, M. kansasii, M. leprae, M. fortuitum third biovariant, M. smegmatis [ofloxacin MICs, 0.5 to 1 μg/ml]), and susceptible (M. fortuitum, M. peregrinum, M. aurum [ofloxacin MICs, ≤0.25 μg/ml]). Peptide sequences of the QRDR of GyrB were identical in all the species, including the amino acids at the three positions known to be involved in acquired resistance to quinolone, i.e., 426 (Asp), 447 (Arg), and 464 (Asn) (numbering system used for Escherichia coli). The last two residues could be involved in the overall low level of susceptibility of mycobacteria to quinolones since they differ from those found in the very susceptible E. coli (Lys-447 and Ser-464) but are identical to those found in the less susceptible Staphylococcus aureus and Streptococcus pneumoniae. Peptide sequences of the QRDR of GyrA were identical in all the species, except for the amino acid at position 83, which was an alanine in the two less susceptible groups and a serine in the most susceptible one, as inE. coli, suggesting that this amino acid is involved in the observed differences of quinolone susceptibility within theMycobacterium genus.

scholarly journals Purification and characterization of a diptericin homologue from Sarcophaga peregrina (flesh fly)

1992 ◽  
Vol 287 (2) ◽  
pp. 573-578 ◽  
Author(s):  
M Ishikawa ◽  
T Kubo ◽  
S Natori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Bactericidal Activity ◽  
Fat Body ◽  
Amino Acid Sequencing ◽  
Purification And Characterization ◽  
E Coli ◽  
Escherichia Coli Cells

A protein with a molecular mass of 8 kDa was found to be synthesized specifically when the fat-body from injured Sarcophaga peregrina larvae was cultured in vitro. This protein was purified from the haemolymph of the injured larvae to near-homogeneity. Partial amino acid sequencing revealed that this protein is a diptericin homologue. It showed bactericidal activity on growing, but not resting Escherichia coli cells. E. coli cells become elongated on treatment with this protein.

scholarly journals The Escherichia coli Starvation GenecstC Is Involved in Amino Acid Catabolism

1998 ◽  
Vol 180 (16) ◽  
pp. 4287-4290 ◽  
Author(s):  
C. D. Fraley ◽  
J. H. Kim ◽  
M. P. McCann ◽  
A. Matin
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nitrogen Sources ◽  
Upstream Region ◽  
Amino Acid Catabolism ◽  
Mineral Salts ◽  
Consensus Sequences ◽  
E Coli ◽  
Acid Protein ◽  
Starvation Survival

ABSTRACT Escherichia coli strains mutant in the starvation genecstC grow normally in a mineral salts medium but are impaired in utilizing amino acids as nitrogen sources. They are also compromised in starvation survival, where amino acid catabolism is important. The cstC gene encodes a 406-amino-acid protein that closely resembles the E. coli ArgD protein, which is involved in arginine biosynthesis. We postulate that CstC is a counterpart of ArgD in an amino acid catabolic pathway. ThecstC upstream region contains several regulatory consensus sequences. Both ςS and ς54 promoters are probably involved in cstC transcription and appear to compete with each other, presumably to match cstCexpression to the cellular amino acid catabolic needs.

scholarly journals tRNA-dependent cleavage of the ColE1 plasmid-encoded RNA I

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3467-3476 ◽  
Author(s):  
Zhijun Wang ◽  
Zhenghong Yuan ◽  
Li Xiang ◽  
Junjie Shao ◽  
Grzegorz Węgrzyn
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Plasmid Dna ◽  
Copy Number ◽  
E Coli ◽  
Cole1 Plasmid ◽  
Specific Degradation ◽  
Rna I

Effects of tRNAAla(UGC) and its derivative devoid of the 3′-ACCA motif [tRNAAla(UGC)ΔACCA] on the cleavage of the ColE1-like plasmid-derived RNA I were analysed in vivo and in vitro. In an amino-acid-starved relA mutant, in which uncharged tRNAs occur in large amounts, three products of specific cleavage of RNA I were observed, in contrast to an otherwise isogenic relA + host. Overexpression of tRNAAla(UGC), which under such conditions occurs in Escherichia coli mostly in an uncharged form, induced RNA I cleavage and resulted in an increase in ColE1-like plasmid DNA copy number. Such effects were not observed during overexpression of the 3′-ACCA-truncated tRNAAla(UGC). Moreover, tRNAAla(UGC), but not tRNAAla(UGC)ΔACCA, caused RNA I cleavage in vitro in the presence of MgCl2. These results strongly suggest that tRNA-dependent RNA I cleavage occurs in ColE1-like plasmid-bearing E. coli, and demonstrate that tRNAAla(UGC) participates in specific degradation of RNA I in vivo and in vitro. This reaction is dependent on the presence of the 3′-ACCA motif of tRNAAla(UGC).

scholarly journals High-Level Formation of Active Pseudomonas cepaciaLipase after Heterologous Expression of the Encoding Gene and Its Modified Chaperone in Escherichia coli and Rapid In Vitro Refolding

1999 ◽  
Vol 65 (2) ◽  
pp. 787-794 ◽  
Author(s):  
Dinh Thi Quyen ◽  
Claudia Schmidt-Dannert ◽  
Rolf D. Schmid
Keyword(s):  
Escherichia Coli ◽  
Burkholderia Cepacia ◽  
Specific Activity ◽  
Gc Content ◽  
Pseudomonas Cepacia ◽  
Membrane Anchor ◽  
E Coli ◽  
Highly Active ◽  
High Level

ABSTRACT The lipase from Pseudomonas cepacia ATCC 21808 (recently reclassified as Burkholderia cepacia) is widely used by organic chemists for enantioselective synthesis and is manufactured from recombinant P. cepacia harboring on a plasmid the clustered genes for lipase and its chaperone. High levels of expression of inactive lipase (40%) in Escherichia coli were achieved with pCYTEXP1 under the control of the strong, temperature-inducible λPRL promoter. However, no overexpression of the lipase chaperone was achieved in E. coli. Thus, chemical refolding of inactive lipase in the absence of its chaperone yielded only 25 U/mg, compared to 3,470 U of the purified lipase secreted by recombinant P. cepacia per mg. Sequence analysis of the chaperone revealed a high GC content (>90%) in the 5′ region of the gene and the presence of a putative membrane anchor at the N terminus. Hence, the 5′ region of the gene was replaced by a synthetic fragment, and the putative membrane anchor was removed by deletion of the first 34 or 70 N-terminal amino acids. Only truncation of the gene led to overexpression of the chaperone (up to 60%) in E. coli. With this chaperone, it was possible to obtain for the first time in a simple refolding procedure a highly active Pseudomonas lipase (classes I and II) expressed inE. coli with a specific activity of up to 4,850 U/mg and a yield of 314,000 U/g of E. coli wet cells.

scholarly journals Activity of cefepime/zidebactam against MDR Escherichia coli isolates harbouring a novel mechanism of resistance based on four-amino-acid inserts in PBP3

2020 ◽  
Vol 75 (12) ◽  
pp. 3563-3567 ◽  
Author(s):  
Sachin S Bhagwat ◽  
Periasamy Hariharan ◽  
Prashant R Joshi ◽  
Snehal R Palwe ◽  
Rahul Shrivastava ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Tertiary Care ◽  
Resistance Mechanism ◽  
Resistance Mechanisms ◽  
Care Hospital ◽  
E Coli ◽  
Microdilution Method ◽  
Broth Microdilution Method

Abstract Background Recent reports reveal the emergence of Escherichia coli isolates harbouring a novel resistance mechanism based on four-amino-acid inserts in PBP3. These organisms concomitantly expressed ESBLs or/and serine-/metallo-carbapenemases and were phenotypically detected by elevated aztreonam/avibactam MICs. Objectives The in vitro activities of the investigational antibiotic cefepime/zidebactam and approved antibiotics (ceftazidime/avibactam, ceftolozane/tazobactam, imipenem/relebactam and others) were determined against E. coli isolates harbouring four-amino-acid inserts in PBP3. Methods Whole-genome sequenced E. coli isolates (n = 89) collected from a large tertiary care hospital in Southern India (n = 64) and from 12 tertiary care hospitals located across India (n = 25) during 2016–18, showing aztreonam/avibactam MICs ≥1 mg/L (≥4 times the aztreonam epidemiological cut-off) were included in this study. The MICs of antibiotics were determined using the reference broth microdilution method. Results Four-amino-acid inserts [YRIK (n = 30) and YRIN (n = 53)] were found in 83/89 isolates. Among 83 isolates, 65 carried carbapenemase genes [blaNDM (n = 39), blaOXA-48-like (n = 11) and blaNDM + blaOXA-48-like (n = 15)] and 18 isolates produced ESBLs/class C β-lactamases only. At least 16 unique STs were noted. Cefepime/zidebactam demonstrated potent activity, with all isolates inhibited at ≤1 mg/L. Comparator antibiotics including ceftazidime/avibactam and imipenem/relebactam showed limited activities. Conclusions E. coli isolates concurrently harbouring four-amino-acid inserts in PBP3 and NDM are an emerging therapeutic challenge. Assisted by the PBP2-binding action of zidebactam, the cefepime/zidebactam combination overcomes both target modification (PBP3 insert)- and carbapenemase (NDM)-mediated resistance mechanisms in E. coli.

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