<Emphasis Type="SmallCaps">L</Emphasis>-Tyrosine production by deregulated strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The excretion of the aromatic amino acid l-tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was eliminated by deleting the tyrR gene. The generation of this l-tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing l-tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that l-tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential growth phase. The final l-tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of l-tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively.     

Identification and characterization of endoglucanases for fungicidal activity in <Emphasis Type="Italic">Anabaena laxa</Emphasis> (Cyanobacteria)

A cyanobacterial strain (Anabaena laxa RPAN8) exhibiting fungicidal activity and β-1,3 and 1,4 endoglucanase activities was selected for identifying the gene(s) involved. Functional analyses of the genomic library revealed that four clones (8, 64, 116, and 248) of RPAN8 exhibited fungicidal activity and induced structural deformities in the cell wall of the growing mycelia of Pythium aphanidermatum. Higher expression of fungicidal and β-1,4 endoglucanase activities, along with low expression of β-1,3 endoglucanase activity, was recorded in two E. coli clones (8 and 64). Clones 8 and 64 exhibited identical sequences while clones 116 and 248 were also similar. Bioinformatic analyses were undertaken only for the two non-identical clones 8 and 116 which showed open reading frames (ORFs) of 348 (end 1) and 656 amino acid residues (end 2), respectively. The amino acid sequence analyses revealed that the end 1 encoding endoglucanases belonged to peptidase M20 family while end 2 showed significant similarities with several known genes. The putative promoters and ribosomal binding sites were identified and amino acid exchanges were observed in both end 1 and 2. The presence of signal peptides of 24 and 20 amino acid residues respectively revealed the secretory nature of these proteins.     

Functional Characterization of Trehalose Biosynthesis Genes from <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis>: An Osmolyte Involved in Stress Tolerance

Trehalose (1-α-d-glucopyranosyl-1-α-d-glucopyranoside), a non-reducing disaccharide is a major compatible solute, which maintains fluidity of membranes and protects the biological structure of organisms under stress. In this study, trehalose-6-phosphate synthase (otsA) and trehalose-6-phosphate phosphatase (otsB) genes encoding for trehalose biosynthesis from Escherichia coli was cloned as an operon and expressed in E. coli M15(pREP4). The recombinant E. coli strain showed a threefold increase in the activity of otsBA pathway enzymes, compared to the control strain. The transgenic E. coli accumulated up to 0.86 mg/l of trehalose. The sequence of otsA and otsB genes reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in the altered amino acid sequences of the translated proteins.     

From scratch to value: engineering <Emphasis Type="Italic">Escherichia coli</Emphasis> wild type cells to the production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine and other fine chemicals derived from chorismate

Recombinant strains of Escherichia coli K-12 for the production of the three aromatic amino acids (l-phenylalanine, l-tryptophan, l-tyrosine) have been constructed. The largest demand is for l-phenylalanine (l-Phe), as it can be used as a building block for the low-calorie sweetener, aspartame. Besides l-Phe, an increasing number of shikimic acid pathway intermediates can be produced from appropriate E. coli mutants with blocks in this pathway. The last common intermediate, chorismate, in E. coli not only serves for production of aromatic amino acids but can also be used for high-titer production of non-aromatic compounds, e.g., cyclohexadiene-transdiols. In an approach to diversity-oriented metabolic engineering (metabolic grafting), platform strains with increased flux through the general aromatic pathway were created by suitable gene deletions, additions, or rearrangements. Examples for rational strain constructions for l-phenylalanine and chorismate derivatives are given with emphasis on genetic engineering. As a result, l-phenylalanine producers are available, which were derived through several defined steps from E. coli K-12 wild type. These mutant strains showed l-phenylalanine titers of up to 38 g/l of l-phenylalanine (and up to 45.5 g/l using in situ product recovery). Likewise, two cyclohexadiene-transdiols could be recovered.     

Enhancing survival of <Emphasis Type="Italic">Escherichia coli</Emphasis> by increasing the periplasmic expression of Cu,Zn superoxide dismutase from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The gene for the Cu,Zn superoxide dismutase (Cu,ZnSOD) from Saccharomyces cerevisiae was cloned and expressed in Escherichia coli LMG194. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the E. coli periplasmic expression vector pBAD/gIIIA, yielding pBAD-1. E. coli was transformed using the constructed plasmid pBAD-1 and induced by adding 0.02% l-arabinose to express Cu,ZnSOD protein. The results indicated that Cu,ZnSOD enzyme activity in the periplasmic space was about fivefold to sixfold higher in the recombinant E. coli strains bearing the sod gene than in the control strains. The yields of Cu,ZnSOD were about threefold higher at 48 h than at 24 h in the recombinant E. coli cells. Significantly higher survival of strains was obtained in cells bearing the sod gene than in the control cells when the cells were treated by heat shock and superoxide-generating agents, such as paraquat and menadione.     

Cloning and Characterization of Two Novel Crystal Protein Genes, <Emphasis Type="Italic">cry54Aa1</Emphasis> and <Emphasis Type="Italic">cry30Fa1</Emphasis>, from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain BtMC28

Bacillus thuringiensis strain BtMC28 was isolated from the soil sample in China. Two novel crystal protein genes were found by using the PCR-RFLP method. Moreover, the full-length sequences of two novel genes were obtained by a single oligonucleotide nested (SON)-PCR upstream and downstream strategy. Sequence analysis revealed that one gene encoded a polypeptide of 673 amino acid residues with a molecular mass of 76.3 kDa, 38% identical to Cry10Aa, and the other encoded a polypeptide of 687 amino acid residues with a molecular mass of 77.1 kDa, 74% identical to Cry30Aa. These two novel crystal protein genes were designated as cry54Aa1 and cry30Fa1 by Bt Insecticidal Crystal Proteins Nomenclature Committee, respectively. The Cry54Aa1 and Cry30Fa1 proteins retained five conserved regions commonly found in the existing Cry proteins. Cry54Aa1 protein exhibited insecticidal activities against Laphygma exigua (Lepidoptera), Helicoverpa armigera (Lepidoptera), and Aedes aegypti (Diptera) when its encoding gene was expressed in an Escherichia coli host strain. The authors, Furong Tan and Jun Zhu contributed equally to this work.     

Distinct substrate specificities and unusual substrate flexibilities of two hydroxycinnamoyltransferases,rosmarinic acid synthase and hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl-transferase,from <Emphasis Type="Italic">Coleus blumei</Emphasis> Benth.

cDNAs and genes encoding a hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase (CbRAS; rosmarinic acid synthase) and a hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (CbHST) were isolated from Coleus blumei Benth. (syn. Solenostemon scutellarioides (L.) Codd; Lamiaceae). The proteins were expressed in E. coli and the substrate specificity of both enzymes was tested. CbRAS accepted several CoA-activated phenylpropenoic acids as donor substrates and d-(hydroxy)phenyllactates as acceptors resulting in ester formation while shikimate and quinate were not accepted. Unexpectedly, amino acids (d-phenylalanine, d-tyrosine, d-DOPA) also yielded products, showing that RAS can putatively catalyze amide formation. CbHST was able to transfer cinnamic, 4-coumaric, caffeic, ferulic as well as sinapic acid from CoA to shikimate but not to quinate or acceptor substrates utilized by CbRAS. In addition, 3-hydroxyanthranilate, 3-hydroxybenzoate and 2,3-dihydroxybenzoate were used as acceptor substrates. The reaction product with 3-aminobenzoate putatively is an amide. For both enzymes, structural requirements for donor and acceptor substrates were deduced. The acceptance of unusual acceptor substrates by CbRAS and CbHST resulted in the formation of novel compounds. The rather relaxed substrate as well as reaction specificity of both hydroxycinnamoyltransferases opens up possibilities for the evolution of novel enzymes forming novel secondary metabolites in plants and for the in vitro formation of new compounds with putatively interesting biological activities.     

Cloning and Sequencing Analysis of Alginate Lyase Genes from the Marine Bacterium Vibrio sp. O2

We isolated a new marine bacteria, which displayed alginate-depolymerizing activity in plate assays, from seawater in Mihonoseki Harbor, Japan. Analysis of the 16S ribosomal RNA gene sequence of one of the isolates proved that this alginate-depolymerizing bacterium belonged to the genus Vibrio and it was named Vibrio sp. O2. The alginate lyase genes of Vibrio sp. O2 were cloned and expressed in Escherichia coli. Two alginate lyase-producing clones, pVOA-A4 and pVOA-B5, were obtained. The alginate lyase gene alyVOA from pVOA-A4 was composed of an 858-bp open reading frame (ORF) encoding 285 amino acid residues, while alyVOB from pVOA-B5 was composed of an 828-bp ORF encoding 275 amino acid residues. The degree of identity between the deduced amino acid sequences of AlyVOA or AlyVOB and Photobacterium sp. ATCC43367 alginate poly(ManA)lyase AlxM was 92.3% or 32.6%, respectively. Alginate lyase consensus regions corresponding to the sequences YFKAGXYXQ and RXELR were observed in all three of these sequences. AlyVOA and AlyVOB both degraded polymannuronate in plate assays and were therefore confirmed to be poly(β-D-mannuronate)lyases.     

Analogue of aspartate and glutamate active at synapses are attractants forEscherichia coli

Summary 1. This research was carried out to compareEscherichia coli bacteria with animals in their response tol-aspartate andl-glutamate and their analogues.2. Various analogues of aspartate and glutamate known to be neurotransmitters at synapses were shown to be attractants forE. coli.3. The amino acid sequences of the animal receptors and the bacterial receptor, however, have no detectable relationship. Based on the amino acid sequence, evolutionarily the two systems appear not to be related.     

Two isoforms of peroxiredoxin 6 of <Emphasis Type="Italic">Xenopus laevis</Emphasis>

The genome of Xenopus laevis codes for two genes of peroxiredoxin 6, i.e., xen1 (Acc. no. EMBL Data Bank-BCO54278) and xen2 (Acc. no. EMBL Data Bank-BCO54309). Both the genes were cloned and expressed in Escherichia coli. The amino acid sequences of Xen1 and Xen2 enzymes are identical by 95%, and they possess the same peroxidase activity as well as similar optimums of temperature, pH, and thermostability. The genes of peroxiredoxin 6 of Xenopus laevis considerably differ in the period of expression during ontogenesis; i.e., xen2 is expressed during every stage of development, somewhat more intensively after stages 0–5; the expression of xen1 is initiated later, i.e., during the developmental stages of 47–48 h. Expression of xen2 increases after the incubation of embryos in a medium with hydrogen peroxide. Comparison of the amino acid sequences of Xen1 and Xen2 proteins shows that only Xen2 can possess phospholipase activity because its amino acid sequence contain residues of the phospholipase A2 active center: Ser31, His25, and Asp139.     

Cloning, expression and characterization of l-aspartate β-decarboxylase gene from Alcaligenes faecalis CCRC 11585

l -Aspartate β-decarboxylase (Asd) is an important enzyme to produce l-alanine and d-aspartate. The genomic library of Alcaligenes faecalis CCRC 11585 was cloned into pBK-CMV and transformed into Escherichia coli. One clone, which carried the asd gene and expressed Asd activity, was isolated and chosen for further study. PBK-asdAE1 was subcloned and its sequence analysis revealed an open reading frame, consisting of 1599 bp, that encodes a 533-amino-acid polypeptide. The nucleotide sequence of the asd gene from A. faecalis CCRC 11585 (asdA) showed 84% identity with that from Pseudomonas dacunhae CCRC 12623, and the amino acid sequence showed 93% identity. The amino acid sequence of the AsdA showed 51–58% homology with various aminotransferases. Alignment of the AsdA with several aspartate or tyrosine aminotransferases revealed 17 conserved amino acids that appeared in most of the conserved amino acid residues within the pyridoxal-5′-phosphate (PLP) binding domains of aminotransferases. Furthermore, the asdA gene was cloned into expression vector pET-21a and transformed into E. coli BL21(DE3). A protein band sized at 61 kDa is present on the SDS-PAGE gel from the intracellular soluble form of E. coli BL21(DE3)/pET-asdA. The specific activities of the pET-AsdA purified by using His-Bind chromatography is 215 U/mg at 45°C and pH 5.0, which is 1000-fold higher than that of the A. faecalis crude extract. This is the first report of an asdA gene sequence from A. faecalis and represents the potential application of a recombinant AsdA for production of l-alanine or d-aspartic acid. Journal of Industrial Microbiology & Biotechnology (2000) 25, 132–140. Received 02 November 1999/ Accepted in revised form 23 June 2000     

Cloning, Expression, and Characterization of a Methionyl Aminopeptidase from a Hyperthermophilic Archaeon Thermococcus sp. NA1

Genomic analysis of a hyperthermophilic archaeon Thermococcus sp. NA1 revealed the presence of an 885-bp open reading frame encoding a protein of 295 amino acids with a calculated molecular mass of 32,981 Da. Analysis of the deduced amino acid sequence showed that amino acid residues important for catalytic activity and the metal binding ligands conserved in all of methionyl aminopeptidases (MetAP) were also conserved and belonged to type IIa MetAP. The protein, designated TNA1_MetAP (Thermococcus sp. NA1 MetAP), was cloned and expressed in Escherichia coli. The recombinant enzyme was a Mn²⁺-, Ni²⁺-, Fe²⁺-, or Co²⁺-dependent metallopeptidase. Optimal MetAP activity against l-methionine p-nitroanilide (Met-pNA) (K _m = 0.68 mM) occurred at pH 7.0 and 80 to 90°C. The MetAP was very unstable compared to Pyrococcus furiosus MetAP, which was completely inactivated by heating at 80°C for 5 min. It seemed likely that the cysteine residue (Cys53) played a critical role in regulating the thermostability of TNA1_MetAP.     

Probing the antigenicity of E. coli l-asparaginase by mutational analysis

A strategy, termed alanine-scanning mutagenesis, was used to identify the amino acid residues which are critical to the antigenicity of Escherichia coli l-asparaginase (l-ASP). Three continuous alkaline residues, 195RKH197, were mutated to Ala selectively. Four mutant recombinant l-ASPs were constructed and expressed in E. coli, and then purified. The purified mutants showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were more than 95% pure by reverse high-perfomance liquid chromatography. The activities of wild-type and m l-ASPs in the fermentative medium were all about 130 U/mL. The change from 195RKH 197 to 195AAA 197 reduced the antigenicity ofhe enzyme greatly as shown in competition enzyme-linked immunosorbent assay using polyclonal antibodies raised against the wild-type l-ASP from rabbits. The results show that residues 195RKH197 of E. coli l-ASP are critical to its antigenicity. These authors contributed equally to this work.     

Cloning,sequencing, overexpression and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-rhamnose isomerase from <Emphasis Type="Italic">Bacillus pallidus</Emphasis> Y25 for rare sugar production

The l-rhamnose isomerase gene (L -rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L -rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60°C for 60 min. The apparent affinity (K _m) and catalytic efficiency (k _cat/K _m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 10⁵ M⁻¹ min⁻¹, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.     

Cloning and overexpression of ketopantoic acid reductase gene from Stenotrophomonas maltophilia and its application to stereospecific production of D-pantoic acid

Ketopantoic acid (KPA) reductase catalyzes the stereospecific reduction of ketopantoic acid to d-pantoic acid. Based on the N-terminal amino acid sequence of KPA reductase from Stenotrophomonas maltophilia 845, the KPA reductase gene was cloned from S. maltophilia NBRC14161 and sequenced. This gene contains an open reading frame of 777 bp encoding 258 amino acid residues, and the deduced amino acid sequence showed high similarity to the SDR superfamily proteins. An expression vector, pETSmKPR, containing the full KPA reductase gene was constructed and introduced into Escherichia coli BL21 (DE3) to overexpress the enzyme. Bioreduction of KPA using E. coli transformant cells coexpressing KPA reductase together with cofactor regeneration enzyme gene was also performed. The conversion yield of KPA to d-pantoic acid reached over 88% with a substrate concentration up to 1.17 M.     

Substrate specificity of a peptidyl-aminoacyl-<Emphasis Type="SmallCaps">l</Emphasis>/<Emphasis Type="SmallCaps">d</Emphasis>-isomerase from frog skin

In the skin of fire-bellied toads (Bombina species), an aminoacyl-l/d-isomerase activity is present which catalyses the post-translational isomerization of the l- to the d-form of the second residue of its substrate peptides. Previously, this new type of enzyme was studied in some detail and genes potentially coding for similar polypeptides were found to exist in several vertebrate species including man. Here, we present our studies to the substrate specificity of this isomerase using fluorescence-labeled variants of the natural substrate bombinin H with different amino acids at positions 1, 2 or 3. Surprisingly, this enzyme has a rather low selectivity for residues at position 2 where the change of chirality at the alpha-carbon takes place. In contrast, a hydrophobic amino acid at position 1 and a small one at position 3 of the substrate are essential. Interestingly, some peptides containing a Phe at position 3 also were substrates. Furthermore, we investigated the role of the amino-terminus for substrate recognition. In view of the rather broad specificity of the frog isomerase, we made a databank search for potential substrates of such an enzyme. Indeed, numerous peptides of amphibia and mammals were found which fulfill the requirements determined in this study. Expression of isomerases with similar characteristics in other species can therefore be expected to catalyze the formation of peptides containing d-amino acids.     

Quorum sensing inhibitors from the red alga,<Emphasis Type="Italic">Ahnfeltiopsis flabelliformis</Emphasis>

Three new acylhomoserine lactone (AHL) inhibitors have been isolated from the Korean red alga.Ahnfeltiopsis flabelliformis, via bioactivity-guided fractionation using the recombinantAgrobacterium tumefaciens liquid culture bioassay. Unlike the majority of AHL inhibitors reported to date, these compounds were α-d-galactopyranosyl-(1→2)-glycerol (floridoside) (1), betonicine (2), and isethionic acid (3), all of which are structurally unrelated to AHLs.     

Probing the catalytically essential residues of a recombinant dipeptidyl carboxypeptidase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

In studying the structure and function of Escherichia coli dipeptidyl carboxypeptidase (EcDCP), we have employed in vitro mutagenesis and subsequent protein expression to genetically dissect the enzyme in order to gain insight into the catalytic mechanism. Comparison of the amino acid sequence of EcDCP with other homologues indicates that the active site of the enzyme exhibits an HEXXH motif, a common feature of zinc metalloenzymes. The third metal binding ligand, presumed to coordinate directly to the active-site zinc ion in concert with His470 and His474 has been proposed as Glu499. Alterations to these residues completely abolished the catalytic activity against N-benzoyl-l-glycyl-l-histidyl-l-leucine. A significant loss of the enzymatic activity was also observed in F472V and F500V mutant enzymes. Intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in all mutant enzymes, whereas circular dichroism spectra were nearly identical for the tested proteins. Computer modeling suggests that residues His470, Glu471, His474, Glu499, and Phe500 are essential for EcDCP in maintaining the stable active-site environment. Taken together, these studies contribute to a more comprehensive understanding of the catalytic mechanism of the enzyme.     

High-level expression of the angiotensin-converting-enzyme-inhibiting peptide, YG-1, as tandem multimers in Escherichia coli

To produce a large quantity of the angiotensin-converting-enzyme(ACE)-inhibiting peptide YG-1, which consists of ten amino acids derived from yeast glyceraldehyde-3-phosphate dehydrogenase, a high-level expression was explored with tandem multimers of the YG-1 gene in Escherichia coli. The genes encoding YG-1 were tandemly multimerized to 9-mers, 18-mers and 27-mers, in which each of the repeating units in the tandem multimers was connected to the neighboring genes by a DNA linker encoding Pro-Gly-Arg for the cleavage of multimers by clostripain. The multimers were cloned into the expression vector pET-21b, and expressed in E. coli BL21(DE3) with isopropyl β-d-thiogalactopyranoside induction. The expressed multimeric peptides encoded by the 9-mer, 18-mer and 27-mer accumulated intracellularly as inclusion bodies and comprised about 67%, 25% and 15% of the total proteins in E. coli respectively. The multimeric peptides expressed as inclusion bodies were cleaved with clostripain, and active monomers were purified to homogeneity by reversed-phase high-performance liquid chromatography. In total, 105 mg pure recombinant YG-1 was obtained from 1 l E. coli culture harboring pETYG9, which contained the 9-mer of the YG-1 gene. The recombinant YG-1 was identical to the natural YG-1 in molecular mass, amino acid sequence and ACE-inhibiting activity. Received: 6 January 1998 / Received revision: 23 February 1998 / Accepted: 24 February 1998