An Engineered E. coli Strain for Direct in Vivo Fluorination

Selectively fluorinated compounds are found frequently in pharmaceutical and agrochemical products where currently 25–30 % of optimised compounds emerge from development containing at least one fluorine atom. There are many methods for the site-specific introduction of fluorine, but all are chemical and they often use environmentally challenging reagents. Biochemical processes for C−F bond formation are attractive, but they are extremely rare. In this work, the fluorinase enzyme, originally identified from the actinomycete bacterium Streptomyces cattleya, is engineered into Escherichia coli in such a manner that the organism is able to produce 5′-fluorodeoxyadenosine (5′-FDA) from S-adenosyl-l -methionine (SAM) and fluoride in live E. coli cells. Success required the introduction of a SAM transporter and deletion of the endogenous fluoride efflux capacity in order to generate an E. coli host that has the potential for future engineering of more elaborate fluorometabolites.     

Enhancing and Reversing the Stereoselectivity of Escherichia coli Transketolase via Single‐Point Mutations

Chiral auxiliary methodology and chiral assays have been developed to establish the enantiomeric purities of erythrulose and 1,3‐dihydroxypentan‐2‐one generated using wild‐type (WT) Escherichia coli transketolase (TK). L ‐Erythrulose was formed in 95% ee and (3S)‐1,3‐dihydroxypentan‐2‐one in 58% ee. Since the latter compound was formed in moderate ee, TK libraries were screened to identify higher performing mutants. A colorimetric screen and chiral assay were successfully applied to a 96‐well format, and new active TK mutants were identified, which gave 1,3‐dihydroxypentan‐2‐one in high stereoselectivities. Remarkably, active‐site single‐point mutants were identified that were able to both enhance and reverse the stereoselectivity of TK.     

The Minimum Activation Peptide from ilvH Can Activate the Catalytic Subunit of AHAS from Different Species

Acetohydroxyacid synthases (AHASs), which catalyze the first step in the biosynthesis of branched‐chain amino acids, are composed of a catalytic subunit (CSU) and a regulatory subunit (RSU). The CSU harbors the catalytic site, and the RSU is responsible for the activation and feedback regulation of the CSU. Previous results from Chipman and co‐workers and our lab have shown that heterologous activation can be achieved among isozymes of Escherichia coli AHAS. It would be interesting to find the minimum peptide of ilvH (the RSU of E. coli AHAS III) that could activate other E. coli CSUs, or even those of ## species. In this paper, C‐terminal, N‐terminal, and C‐ and N‐terminal truncation mutants of ilvH were constructed. The minimum peptide to activate ilvI (the CSU of E. coli AHAS III) was found to be Δ_N14–Δ_C89. Moreover, this peptide could not only activate its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana plumbaginifolia. However, this peptide totally lost its ability for feedback regulation by valine, thus suggesting different elements for enzymatic activation and feedback regulation. Additionally, the apparent dissociation constant (K_d) of Δ_N14–Δ_C89 when binding CSUs of different species was found to be 9.3–66.5 μM by using microscale thermophoresis. The ability of this peptide to activate different CSUs does not correlate well with its binding ability (K_d) to these CSUs, thus implying that key interactions by specific residues is more important than binding ability in promoting enzymatic reactions. The high sequence similarity of the peptide Δ_N14–Δ_C89 to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs.     

C-terminal His-tagging results in substrate specificity changes of the thioesterase I from Escherichia coli

The biochemical properties of Escherichia coli thioesterase I, His-tagged (HT) on the C-terminal, were systematically analyzed and compared with that without the His-tag (WT). These two types of enzymes exhibit similar optimal temperature and pH dependence, but subtle differences were detected. Kinetic studies revealed that the k _car/JK _m values of the HT enzyme for the substrates palmitoyl-CoA and p-nitrophenyl dodecanoate were 36- and 10-fold lower than those of the WT, respectively. In contrast, HT had a fivefold increased catalytic efficiency for p-nitrophenyl acetate, and up to fourfold increases toward phenylalanine- and tyrosine-derived ester substrates, l-NBPNPE (N-carbobenzoxy-l-phenylalanine p-nitrophenyl ester) and l-NBTNPE (N-carbobenzoxyl-l-tyrosine p-nitrophenyl ester), respectively. For l-NBPNPE and l-NBTNPE, the increases were attributed to the higher k _cat values with little changes in K _m, whereas the increase for p-nitrophenyl acetate was mainly attributed to the lower K _m value. It is concluded that addition of six hydrophilic histidine residues on the C-terminus resulted in a change in substrate specificity of E. coli thioesterase I toward more hydrophilic substrates.     

Biotransformation of medium-chain alkanes using recombinant P450 monooxygenase from <Emphasis Type="Italic">Alcanivorax borkumensis</Emphasis> SK2 expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A bioconversion system for medium-chain alkanes was constructed by using a recombinant Escherichia coli whole-cell biocatalyst expressing P450 monooxygenase genes, ferredoxin, and ferredoxin reductase cloned from Alcanivorax borkumensis as an operon. The recombinant E. coli harboring the P450 gene and two related expression component enzymes, ferredoxin and ferredoxin reductase, was constructed in a single vector pET21(a) and successfully expressed in E. coli BL21(DE3) as a soluble form, showing a molecular weight of 53 kDa on 10% SDS-PAGE. When the cell-free extract of E. coli BL21 expressing p450 monooxygenase was subjected to reduced CO difference spectral analysis, a soret band near 450 nm appeared indicating that the cloned P450 was expressed as a functionally active enzyme. The E. coli cells harboring the expressed P450 gene were able to convert n-octane and 1-decene, producing approximately 450 μg/ml of n-octanol and 290 μg/ml of 1,2-epoxydecane, respectively, at pH 7.0 and 30 °C. However, the recombinant E. coli cells were not able to convert the branched alkane, 2,6,10,14-tetramethylpentadecane (C19).     

Expression and Characterization of a New Self-Sufficient P450 Monooxygenase (P450 AZC1) from Azorhizobium caulinodans

Oxyfunctionalization of non-activated carbon bonds by P450 monooxygenases has drawn great industrial attraction. Self-sufficient P450s containing catalytic heme and reductase domains in a single polypeptide chain offer many advantages since they do not require external electron transfer partners. Here, we report the first P450 enzyme identified and expressed from Azorhizobium caulinodans. Firstly, expression conditions of P450 AZC1 were optimized for enhanced expression in E.coli. The highest P450 content was obtained in E.coli Rosetta DE3 plysS when it was incubated in TB media supplemented with 0.75 mM IPTG, 0.5 mM ALA, and 0.75 mM FeCl₃ at 25 °C for 24 hours. Subsequently, the purified enzyme showed a broad substrate spectrum including fatty acids, linear and cyclic alkanes, aromatics, and pharmaceuticals. Finally, P450 AZC1 showed optimal activity at pH 6.0 and 40 °C and a broad pH and temperature profile, making it a promising candidate for industrial applications.     

Effects of Genetically Engineered Stem Cells Expressing Cytosine Deaminase and Interferon-Beta or Carboxyl Esterase on the Growth of LNCaP Prostate Cancer Cells

The risk of prostate cancer has been increasing in men by degrees. To develop a new prostate cancer therapy, we used a stem cell-derived gene directed prodrug enzyme system using human neural stem cells (hNSCs) that have a tumor-tropic effect. These hNSCs were transduced with the therapeutic genes for bacterial cytosine deaminase (CD), alone or in combination with the one encoding human interferon-beta (IFN-β) or rabbit carboxyl esterase (CE) to generate HB1.F3.CD, HB1.F3.CD.IFN-β, and HB1.F3.CE cells, respectively. CD enzyme can convert the prodrug 5-fluorocytosine (5-FC) into the activated form 5-fluorouracil (5-FU). In addition, CE enzyme can convert the prodrug CPT-11 into a toxic agent, SN-38. In our study, the human stem cells were found to migrate toward LNCaP human prostate cancer cells rather than primary cells. This phenomenon may be due to interactions between chemoattractant ligands and receptors, such as VEGF/VEGFR2 and SCF/c-Kit, expressed as cancer and stem cells, respectively. The HB1.F3.CE, HB.F3.CD, or HB1.F3.CD.IFN-β cells significantly reduced the LNCaP cell viability in the presence of the prodrugs 5-FC or CPT-11. These results indicate that stem cells expressing therapeutic genes can be used to develop a new strategy for selectively treating human prostate cancer.     

Biochemical Characterization of the Lysine Acetylation of Tyrosyl‐tRNA Synthetase in Escherichia coli

Aminoacyl‐tRNA synthetases (aaRSs) play essential roles in protein synthesis. As a member of the aaRS family, the tyrosyl‐tRNA synthetase (TyrRS) in Escherichia coli has been shown in proteomic studies to be acetylated at multiple lysine residues. However, these putative acetylation targets have not yet been biochemically characterized. In this study, we applied a genetic‐code‐expansion strategy to site‐specifically incorporate N^?‐acetyl‐l ‐lysine into selected positions of TyrRS for in vitro characterization. Enzyme assays demonstrated that acetylation at K85, K235, and K238 could impair the enzyme activity. In vitro deacetylation experiments showed that most acetylated lysine residues in TyrRS were sensitive to the E. coli deacetylase CobB but not YcgC. In vitro acetylation assays indicated that 25 members of the Gcn5‐related N‐acetyltransferase family in E. coli, including YfiQ, could not acetylate TyrRS efficiently, whereas TyrRS could be acetylated chemically by acetyl‐CoA or acetyl‐phosphate (AcP) only. Our in vitro characterization experiments indicated that lysine acetylation could be a possible mechanism for modulating aaRS enzyme activities, thus affecting translation.     

Identification of a Novel Inhibition Site in Translocase MraY Based upon the Site of Interaction with Lysis Protein E from Bacteriophage ϕX174

Translocase MraY is the site of action of lysis protein E from bacteriophage ?X174. Previous genetic studies have shown that mutation F288L in transmembrane helix 9 of E. coli MraY confers resistance to protein E. Construction of a helical wheel model for transmembrane helix 9 of MraY and the transmembrane domain of protein E enabled the identification of an Arg‐Trp‐x‐x‐Trp (RWxxW) motif in protein E that might interact with Phe288 of MraY and the neighbouring Glu287. This motif is also found in a number of cationic antimicrobial peptide sequences. Synthetic dipeptides and pentapeptides based on the RWxxW consensus sequence showed inhibition of particulate E. coli MraY activity (IC₅₀ 200–600 μM ), and demonstrated antimicrobial activity against E. coli (MIC 31–125 μg mL^?1). Cationic antimicrobial peptides at a concentration of 100 μg mL^?1 containing Arg‐Trp sequences also showed 30–60 % inhibition of E. coli MraY activity. Assay of the synthetic peptide inhibitors against recombinant MraY enzymes from Bacillus subtilis, Pseudomonas aeruginosa, and Micrococcus flavus (all of which lack Phe288) showed reduced levels of enzyme inhibition, and assay against recombinant E. coli MraY F288L and an E287A mutant demonstrated either reduced or no detectable enzyme inhibition, thus indicating that these peptides interact at this site. The MIC of Arg‐Trp‐octyl ester against E. coli was increased eightfold by overexpression of mraY, and was further increased by overexpression of the mraY mutant F288L, also consistent with inhibition at the RWxxW site. As this site is on the exterior face of the cytoplasmic membrane, it constitutes a potential new site for antimicrobial action, and provides a new cellular target for cationic antimicrobial peptides.     

L-phenylalanine production by auxotrophic regulatory mutants ofEscherichia coli—L-phenylalanine production by mutants ofE. coli

Various regulatory mutants ofEscherichia coli have been isolated using phenylalanine and tyrosine analogues. It has been found that the growth of wild type strain ofE. coli W3110 was strongly inhibited by phenylalanine analogues. Regulatory mutants resistant to phenylalanine analogue could accumulate b-phenylalanine at concentrations of 5–6 g/l. However, L-phenylalanine accumulation was increased significantly up to 11.4 g/1 using a tyrosine auxotrophic mutant resistant to phenylalanine analogue such as β-2-thienyl-DL-alanine.     

Identification of Fluorinases from Streptomyces sp MA37, Norcardia brasiliensis,and Actinoplanes sp N902‐109 by Genome Mining

The fluorinase is an enzyme that catalyses the combination of S‐adenosyl‐L ‐methionine (SAM) and a fluoride ion to generate 5′‐fluorodeoxy adenosine (FDA) and L ‐methionine through a nucleophilic substitution reaction with a fluoride ion as the nucleophile. It is the only native fluorination enzyme that has been characterised. The fluorinase was isolated in 2002 from Streptomyces cattleya, and, to date, this has been the only source of the fluorinase enzyme. Herein, we report three new fluorinase isolates that have been identified by genome mining. The novel fluorinases from Streptomyces sp. MA37, Nocardia brasiliensis, and an Actinoplanes sp. have high homology (80–87 % identity) to the original S. cattleya enzyme. They all possess a characteristic 21‐residue loop. The three newly identified genes were overexpressed in E. coli and shown to be fluorination enzymes. An X‐ray crystallographic study of the Streptomyces sp. MA37 enzyme demonstrated that it is almost identical in structure to the original fluorinase. Culturing of the Streptomyces sp. MA37 strain demonstrated that it not only also elaborates the fluorometabolites, fluoroacetate and 4‐fluorothreonine, similar to S. cattleya, but this strain also produces a range of unidentified fluorometabolites. These are the first new fluorinases to be reported since the first isolate, over a decade ago, and their identification extends the range of fluorination genes available for fluorination biotechnology.     

Asymmetric Synthesis and Biological Evaluation of Glycosidic Prodrugs for a Selective Cancer Therapy

A severe limitation in cancer therapy is the often insufficient differentiation between malign and benign tissue using known chemotherapeutics. One approach to decrease side effects is antibody‐directed enzyme prodrug therapy (ADEPT). We have developed new glycosidic prodrugs such as (?)‐(1S)‐ 26 b based on the antibiotic (+)‐duocarmycin SA ((+)‐ 1 ) with a QIC₅₀ value of 3500 (QIC₅₀=IC₅₀ of prodrug/IC₅₀ of prodrug+enzyme) and an IC₅₀ value for the corresponding drug (prodrug+enzyme) of 16 pM . The asymmetric synthesis of the precursor (?)‐(1S)‐ 19 was performed by arylation of the enantiomerically pure epoxide (+)‐(S)‐ 29 (≥98 % ee).     

RadA,a Key Gene of the Circadian Rhythm of Escherichia coli

Circadian rhythms are present in almost all living organisms, and their activity relies on molecular clocks. In prokaryotes, a functional molecular clock has been defined only in cyanobacteria. Here, we investigated the presence of circadian rhythms in non-cyanobacterial prokaryotes. The bioinformatic approach was used to identify a homologue of KaiC (circadian gene in cyanobacteria) in Escherichia coli. Then, strains of E. coli (wild type and mutants) were grown on blood agar, and sampling was made every 3 h for 24 h at constant conditions. Gene expression was determined by qRT-PCR, and the rhythmicity was analyzed using the Cosinor model. We identified RadA as a KaiC homologue in E. coli. Expression of radA showed a circadian rhythm persisting at least 3 days, with a peak in the morning. The circadian expression of other E. coli genes was also observed. Gene circadian oscillations were lost in radA mutants of E. coli. This study provides evidence of molecular clock gene expression in E. coli with a circadian rhythm. Such a finding paves the way for new perspectives in antibacterial treatment.     

Capture of Uropathogenic E. coli by Using Synthetic Glycan Ligands Specific for the Pap‐Pilus

Biotinylated mono‐ and biantennary di‐/trisaccharides were synthesized to evaluate their ability to capture E. coli strains that express pilus types with different receptor specificities. The synthesized biotinylated di‐/trisaccharides contain Galα(1→4)Gal, Galα(1→4)GalNHAc, GalNHAcα(1→4)Gal, Galα(1→4)Galβ(1→4)Glc and GalNHAcα(1→4)Galβ(1→4)Glc as carbohydrate epitopes. These biotinylated oligosaccharides were immobilized on streptavidin‐coated magnetic beads, and incubated with different strains of live E. coli. Capturing ability was assessed by using a luciferase assay that detects bacterial ATP. The trisaccharides containing Galα(1→4)Galβ(1→4)Glc and the disaccharides containing Galα(1→4)Gal as the epitopes exhibited strong capturing ability for uropathogenic E. coli strains with the pap pilus genotype, including CFT073, J96 and J96 pilE. The same ligands failed to capture E. coli strains with fim, prs, or foc genotypes. Uropathogenic CFT073 was also captured moderately by biantennary disaccharides containing a GalNHAc moiety at the reducing end; however, other saccharides containing GalNHAc at the nonreducing end did not capture the CFT073 strain. These synthetic glycoconjugates could potentially be adapted as rapid diagnostic agents to differentiate between different E. coli pathovars.     

A Comparison of the Binding of the Ligand Trimethoprim to Bacterial and Vertebrate Dihydrofolate Reductases

In this paper, we report on studies of ligand binding to the enzyme dihydrofolate reductase (DHFR). Energy minimizations of four complexes of DHFR with the inhibitor trimethoprim, an antibiotic, and the cofactor NADPH have been carried out in order to investigate the energetics responsible for the 100,000-fold increase in binding affinity of trimethoprim to E. coli DHFR compared with chicken liver DHFR. Several factors suggested to be responsible for the enhanced binding in bacterial DHFR's were investigated in terms of intermolecular and intramolecular energetics. The strain energies of trimethoprim in the four complexes were calculated and found to be about 6 kcal mol⁻¹ in all complexes of the two species. In the binary complex of chicken liver DHFR, where the largest variation was observed, 2 kcal mol⁻¹ higher than in the other complexes, it was found that this increase was compensated for by the slightly more favorable intermolecular interaction of the trimethoxyphenyl moiety with the protein. Comparison of the minimized binary and ternary complexes of E. coli allowed us to investigate the cooperativity in the binding of trimethoprim and NADPH in the bacterial enzyme in terms of the underlying intermolecular forces. This cooperativity was found to be due to a direct trimethoprim - NADPH interaction in the E. coli enzyme rather than enhanced protein-inhibitor interactions induced upon binding of the cofactor. These interactions are not as favorable in the vertebrate enzyme, consistent with the significantly diminished cooperativity observed in this enzyme.     

Ligand- and Structure-Based Approaches of Escherichia coli FabI Inhibition by Triclosan Derivatives: From Chemical Similarity to Protein Dynamics Influence

Enoyl-acyl carrier protein reductase (FabI) is the limiting step to complete the elongation cycle in type II fatty acid synthase (FAS) systems and is a relevant target for antibacterial drugs. E. coli FabI has been employed as a model to develop new inhibitors against FAS, especially triclosan and diphenyl ether derivatives. Chemical similarity models (CSM) were used to understand which features were relevant for FabI inhibition. Exhaustive screening of different CSM parameter combinations featured chemical groups, such as the hydroxy group, as relevant to distinguish between active/decoy compounds. Those chemical features can interact with the catalytic Tyr156. Further molecular dynamics simulation of FabI revealed the ionization state as a relevant for ligand stability. Also, our models point the balance between potency and the occupancy of the hydrophobic pocket. This work discusses the strengths and weak points of each technique, highlighting the importance of complementarity among approaches to elucidate EcFabI inhibitor's binding mode and offers insights for future drug discovery.     

Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel

Lipase from Burkholderia cepacia (BCL) has proven to be a very useful biocatalyst for the resolution of 2‐substituted racemic acid derivatives, which are important chiral building blocks. Our previous work showed that enantioselectivity of the wild‐type BCL could be improved by chemical engineering of the substrate's molecular structure. From this earlier study, three amino acids (L17, V266 and L287) were proposed as targets for mutagenesis aimed at tailoring enzyme enantioselectivity. In the present work, a small library of 57 BCL single mutants targeted on these three residues was constructed and screened for enantioselectivity towards (R,S)‐2‐chloro ethyl 2‐bromophenylacetate. This led to the fast isolation of three single mutants with a remarkable tenfold enhanced or reversed enantioselectivity. Analysis of substrate docking and access trajectories in the active site was then performed. From this analysis, the construction of 13 double mutants was proposed. Among them, an outstanding improved mutant of BCL was isolated that showed an E value of 178 and a 15‐fold enhanced specific activity compared to the parental enzyme; thus, this study demonstrates the efficiency of the semirational engineering strategy.     

Catalysis of an Essential Step in Vitamin B2 Biosynthesis by a Consortium of Broad Spectrum Hydrolases

An enzyme catalysing the essential dephosphorylation of the riboflavin precursor, 5‐amino‐6‐ribitylamino‐2,4(1H,3H)‐pyrimidinedione 5′‐phosphate ( 6 ), was purified about 800‐fold from a riboflavin‐producing Bacillus subtilis strain, and was assigned as the translation product of the ycsE gene by mass spectrometry. YcsE is a member of the large haloacid dehalogenase (HAD) superfamily. The recombinant protein was expressed in Escherichia coli. It catalyses the hydrolysis of 6 (v_max, 12 μmol mg^?1 min^?1; K_M, 54 μm ) and of FMN (v_max, 25 μmol mg^?1 min^?1; K_M, 135 μm ). A ycsE deletion mutant of B. subtilis was not riboflavin dependent. Two additional proteins (YwtE, YitU) that catalyse the hydrolysis of 6 at appreciable rates were identified by screening 13 putative HAD superfamily members from B. subtilis. The evolutionary processes that have resulted in the handling of an essential step in the biosynthesis of an essential cofactor by a consortium of promiscuous enzymes require further analysis.     

Sterol mutants ofSaccharomyces cerevisiae: Chromatographic analyses

The sterols accumulated by ergosterol deficient mutants of the geneserg6, erg2, erg3, anderg5 (formerlypo11, po12, po13, andpo15) have been analyzed by gas liquid chromatography. Together with pure sterols obtained from the mutants, they were characterized on SE-30, OV-17, and OV-225. The effects of molecular structure on the retention characteristics of a range of C₂₈ ergostane sterols have been studied. The double mutants obtained by crossing the single mutants were also analyzed and their sterols identified where possible. The effects of theerg mutations on the control of sterol biosynthesis in yeast are discussed.     

CYP109E1 from Bacillus megaterium Acts as a 24- and 25-Hydroxylase for Cholesterol

In this study, the ability of CYP109E1 from Bacillus megaterium DSM319 to metabolize cholesterol was investigated. This steroid was identified as a new substrate to be converted by CYP109E1 with adrenodoxin and adrenodoxin reductase as redox partners in vitro. The biotransformation was successfully reproduced in vivo by using Bacillus megaterium cells that overexpressed CYP109E1. To enhance the production of cholesterol derivatives, an Escherichia coli based whole-cell system that harbored CYP109E1 was established. This novel system showed a 3.3-fold higher activity than that of the B. megaterium system, yielding about 45 mg L⁻¹ of these products. Finally, the reaction products were isolated and identified to be the highly important cholesterol derivatives 24(S)- and 25-hydroxycholesterol.