Enzymatic properties of UDP-glycosyltransferase 89B1 from radish and modulation of enzyme catalytic activity via loop region mutation.

Glycosyltransferases (GTs), crucial enzymes in plants, alter natural substances through glycosylation, a process with extensive applications in pharmaceuticals, food, and cosmetics. This study narrows its focus to GT family 1, specifically UDP-glycosyltransferases (UGTs), which are known for glycosylating small phenolic compounds, especially hydroxybenzoates. We delve into the workings of Raphanus sativus glucosyltransferase (Rs89B1), a homolog of Arabidopsis thaliana UGT89B1, and its mutant to explore their glycosyltransferase activities toward hydroxybenzoates. Our findings reveal that Rs89B1 glycosylates primarily the para-position of mono-, di-, trihydroxy benzoic acids, and its substrate affinity is swayed by the presence and position of the hydroxyl group on the benzene ring of hydroxybenzoate. Moreover, mutations in the loop region of Rs89B1 impact both substrate affinity and catalytic activity. The study demonstrates that insertional/deletional mutations in non-conserved regions, which are distant from the UGT's recognition site, can have an effect on the UGT's substrate recognition site, which in turn affects acceptor substrate selectivity and glycosyltransferase activity. This research uncovers new insights suggesting that mutations in the loop region could potentially fine-tune enzyme properties and enhance its catalytic activity. These findings not only have significant implications for enzyme engineering in biotechnological applications but also contribute to a more profound understanding of this field.

Optimization of tyrosol-producing pathway with tyrosine decarboxylase and tyramine oxidase in high-tyrosine-producing Escherichia coli.

Tyrosol (4-hydroxyphenylethanol) is a phenolic compound used in the pharmaceutical and chemical industries. However, current supply methods, such as extraction from natural resources and chemical synthesis, have disadvantages from the viewpoint of cost and environmental protection. Here, we developed a tyrosol-producing Escherichia coli cell factory from a high-tyrosine-producing strain by expressing selected tyrosine decarboxylase-, tyramine oxidase (TYO)-, and medium-chain dehydrogenase/reductase (YahK)-encoding genes. The genes were controlled by the strong T7 promoter and integrated into the chromosome because of the advantages over plasmid-based systems. The strain produced a melanin-like pigment as a by-product, which is suggested to be formed from 4-hydroxyphenylacetaldehyde (a TYO product/YahK substrate). By using a culture medium containing a high concentration of glycerol, which was reported to enhance NADH supply required for YahK activity, the final titer of tyrosol reached 2.42 g/L in test tube-scale cultivation with a concomitant decrease in the amount of pigment. These results indicate that chromosomally integrated and T7 promoter-controlled gene expression system in E. coli is useful for high production of heterologous enzymes and might be applied for industrial production of useful compounds including tyrosine and tyrosol.

Engineered Escherichia coli platforms for tyrosine-derivative production from phenylalanine using phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system.

BACKGROUND: Aromatic compounds derived from tyrosine are important and diverse chemicals that have industrial and commercial applications. Although these aromatic compounds can be obtained by extraction from natural producers, their growth is slow, and their content is low. To overcome these problems, many of them have been chemically synthesized from petroleum-based feedstocks. However, because of the environmental burden and depleting availability of feedstock, microbial cell factories are attracting much attention as sustainable and environmentally friendly processes. RESULTS: To facilitate development of microbial cell factories for producing tyrosine derivatives, we developed simple and convenient tyrosine-producing Escherichia coli platforms with a bacterial phenylalanine hydroxylase, which converted phenylalanine to tyrosine with tetrahydromonapterin as a cofactor, using a synthetic biology approach. By introducing a tetrahydrobiopterin-regeneration system, the tyrosine titer of the plasmid-based engineered strain was 4.63 g/L in a medium supplemented with 5.00 g/L phenylalanine with a test tube. The strains were successfully used to produce industrially attractive compounds, such as tyrosol with a yield of 1.58 g/L by installing a tyrosol-producing module consisting of genes encoding tyrosine decarboxylase and tyramine oxidase on a plasmid. Gene integration into E. coli chromosomes has an advantage over the use of plasmids because it increases genetic stability without antibiotic feeding to the culture media and enables more flexible pathway engineering by accepting more plasmids with artificial pathway genes. Therefore, we constructed a plasmid-free tyrosine-producing platform by integrating five modules, comprising genes encoding the phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system, into the chromosome. The platform strain could produce 1.04 g/L of 3,4-dihydroxyphenylalanine, a drug medicine, by installing a gene encoding tyrosine hydroxylase and the tetrahydrobiopterin-regeneration system on a plasmid. Moreover, by installing the tyrosol-producing module, tyrosol was produced with a yield of 1.28 g/L. CONCLUSIONS: We developed novel E. coli platforms for producing tyrosine from phenylalanine at multi-gram-per-liter levels in test-tube cultivation. The platforms allowed development and evaluation of microbial cell factories installing various designed tyrosine-derivative biosynthetic pathways at multi-grams-per-liter levels in test tubes.

Production of 3-Hydroxytyrosol from Glucose by Chromosomally Engineered Escherichia coli by Fed-Batch Cultivation in a Jar Fermenter.

3-Hydroxytyrosol (HT) is a super antioxidant possessing many physiological advantages for human health. However, the extraction of natural HT from olive (Olea europaea) is expensive, and its chemical synthesis presents an environmental burden. Therefore, microbial production of HT from renewable sources has been investigated over the past decade. In the present study, we modified the chromosome of a phenylalanine-producing strain of Escherichia coli to generate an HT-producing strain. The initial strain showed good HT production in tests performed by test tube cultivation, but this performance did not transfer to jar-fermenter cultivation. To grow well and achieve higher titers, the chromosome was further engineered and the cultivation conditions were further modified. The final strain achieved a higher HT titer (8.8 g/L) and yield (8.7%) from glucose in the defined synthetic medium. These yields are the best reported to date for the biosynthesis of HT from glucose.

Chromosome Engineering To Generate Plasmid-Free Phenylalanine- and Tyrosine-Overproducing Escherichia coli Strains That Can Be Applied in the Generation of Aromatic-Compound-Producing Bacteria.

Many phenylalanine- and tyrosine-producing strains have used plasmid-based overexpression of pathway genes. The resulting strains achieved high titers and yields of phenylalanine and tyrosine. Chromosomally engineered, plasmid-free producers have shown lower titers and yields than plasmid-based strains, but the former are advantageous in terms of cultivation cost and public health/environmental risk. Therefore, we engineered here the Escherichia coli chromosome to create superior phenylalanine- and tyrosine-overproducing strains that did not depend on plasmid-based expression. Integration into the E. coli chromosome of two central metabolic pathway genes (ppsA and tktA) and eight shikimate pathway genes (aroA, aroB, aroC, aroD, aroE, aroGfbr , aroL, and pheAfbr ), controlled by the T7lac promoter, resulted in excellent titers and yields of phenylalanine; the superscript "fbr" indicates that the enzyme encoded by the gene was feedback resistant. The generated strain could be changed to be a superior tyrosine-producing strain by replacing pheAfbr with tyrAfbr A rational approach revealed that integration of seven genes (ppsA, tktA, aroA, aroB, aroC, aroGfbr , and pheAfbr ) was necessary as the minimum gene set for high-yield phenylalanine production in E. coli MG1655 (tyrR, adhE, ldhA, pykF, pflDC, and ascF deletant). The phenylalanine- and tyrosine-producing strains were further applied to generate phenyllactic acid-, 4-hydroxyphenyllactic acid-, tyramine-, and tyrosol-producing strains; yield of these aromatic compounds increased proportionally to the increase in phenylalanine and tyrosine yields.IMPORTANCE Plasmid-free strains for aromatic compound production are desired in the aspect of industrial application. However, the yields of phenylalanine and tyrosine have been considerably lower in plasmid-free strains than in plasmid-based strains. The significance of this research is that we succeeded in generating superior plasmid-free phenylalanine- and tyrosine-producing strains by engineering the E. coli chromosome, which was comparable to that in plasmid-based strains. The generated strains have a potential to generate superior strains for the production of aromatic compounds. Actually, we demonstrated that four kinds of aromatic compounds could be produced from glucose with high yields (e.g., 0.28 g tyrosol/g glucose).

Promoted performance of microbial fuel cells using Escherichia coli cells with multiple-knockout of central metabolism genes.

The effect of central metabolic activity of Escherichia coli cells acting as biocatalysts on the performance of microbial fuel cells (MFCs) was studied with glucose used as the energy source. Milliliter-scale two-chambered MFCs were used with 2-hydroxy-1,4-naphthoquinone (HNQ) as an electron mediator. Among the single-gene deletions examined, frdA, pdhR, ldhA, and adhE increased the average power output of the constructed MFC. Next, multiple-gene knockout mutants were constructed using P1 transduction. The Δ5 (ΔfrdAΔpdhRΔldhAΔadhEΔpta) strain showed the highest ave. power output (1.82 mW) and coulombic efficiency (21.3%). Our results show that the combination of multiple-gene knockout in E. coli cells leads to the development of an excellent catalyst for MFCs. Finally, preventing a decrease in the pH of the anodic solution was a key factor for improving the power output of the Δ5 strain, and a maximum ave. power output of 2.21 mW was achieved with 5% NaHCO3 in the buffer. The ave. power density of the constructed MFC was 0.27 mW/cm3, which is comparable to an enzymatic fuel cell of a Milliliter-scale using glucose dehydrogenase.

Identifying membrane-bound quinoprotein glucose dehydrogenase from acetic acid bacteria that produce lactobionic and cellobionic acids.

Acetic acid bacteria are used in the commercial production of lactobionic acid (LacA). However, the lactose-oxidizing enzyme of these bacteria remains unidentified. Lactose-oxidizing activity has been detected in bacterial membrane fractions and is strongly inhibited by d-glucose, suggesting that the enzyme was a membrane-bound quinoprotein glucose dehydrogenase, but these dehydrogenases have been reported to be incapable of oxidizing lactose. Thus, we generated m-GDH-overexpressing and -deficient strains of Komagataeibacter medellinensis NBRC3288 and investigated their lactose-oxidizing activities. Whereas the overexpressing variants produced ~2-5-fold higher amounts of LacA than the wild-type strains, the deficient variant produced no LacA or d-gluconic acid. Our results indicate that the lactose-oxidizing enzyme from acetic acid bacteria is membrane-bound quinoprotein glucose dehydrogenase. Abbreviations: LacA: lactobionic acid; AAB: acetic acid bacterium; m-GDH: membrane-bound quinoprotein glucose dehydrogenase; DCIP: 2,6-dichlorophenolindophenol; HPAEC-PAD: high-performance anion-exchange chromatography with pulsed amperometric detection.

Escherichia coli chromosome-based T7-dependent constitutive overexpression system and its application to generating a phenylalanine producing strain.

Many metabolic engineering approaches have been attempted to generate strains capable of producing valuable compounds. One of main goals is industrial application of these strains. Integration of synthetic pathway genes into the Escherichia coli chromosome enables generation of a plasmid-free strain that is stable and useful for industrial applications. Strains that do not require induction are advantageous in terms of cost. In the present study, we constructed a constitutive overexpression system in E. coli to generate plasmid-free and inducer-free strains. The T7 RNA polymerase/T7 promoter overexpression system, which is an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible gene overexpression system (T7-dependent inducible overexpression system), was modified to be a constitutive overexpression system. The constructed overexpression system, a "chromosome-based T7-dependent constitutive overexpression system", was applied in a metabolic engineering study to generate a plasmid-free and inducer-free phenylalanine producing strain of E. coli.

Application of chromosomal gene insertion into Escherichia coli for expression of recombinant proteins.

Escherichia coli is the most popular organism used for producing recombinant proteins. However, the expression of recombinant proteins in E. coli sometimes results in the aggregation of proteins as an inclusion body in host cells. In such cases, it is necessary to optimize the refolding conditions to obtain the recombinant protein in its native form. Several techniques, such as reducing the concentration of the induction reagent during E. coli cultivation, have been developed to prevent the formation of inclusion bodies by controlling protein expression levels. In this study, we inserted one copy of a target gene under the control of T7 promoter into the E. coli chromosome using the Red-mediated recombination system. This system enabled soluble expression of the putative d-aminoacylase from Pyrococcus abyssi, which is expressed in an insoluble form following the use of conventional plasmid-based T7 promoter/polymerase systems. The relationship between the number of inserted gene copies and amount of soluble recombinant protein produced was evaluated by multiple insertions of the eGFP gene into the E. coli chromosome. The results revealed that the total expression from the insertion of one copy was around 1/5 that of the pET plasmid system and that expression increased as the inserted gene copy number increased up to five copies.

Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway.

Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress-responsive control. Production levels dropped dramatically relative to plasmid-mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR-Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid-optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering.

Production of P-aminobenzoic acid by metabolically engineered escherichia coli.

The production of chemical compounds from renewable resources is an important issue in building a sustainable society. In this study, Escherichia coli was metabolically engineered by introducing T7lac promoter-controlled aroF(fbr), pabA, pabB, and pabC genes into the chromosome to overproduce para-aminobenzoic acid (PABA) from glucose. Elevating the copy number of chromosomal PT7lac-pabA-pabB distinctly increased the PABA titer, indicating that elevation of 4-amino-4-deoxychorismic acid synthesis is a significant factor in PABA production. The introduction of a counterpart derived from Corynebacterium efficiens, pabAB (ce), encoding a fused PabA and PabB protein, resulted in a considerable increase in the PABA titer. The introduction of more than two copies of PT7lac-pabAB (ce-mod), a codon-optimized pabAB (ce), into the chromosome of a strain that simultaneously overexpressed aroF(fbr) and pabC resulted in 5.1 mM PABA from 55.6 mM glucose (yield 9.2%). The generated strain produced 35 mM (4.8 g L(-1)) PABA from 167 mM glucose (yield 21.0%) in fed-batch culture.

Expression from engineered Escherichia coli chromosome and crystallographic study of archaeal N,N'-diacetylchitobiose deacetylase.

UNLABELLED: In order to develop a structure-based understanding of the chitinolytic pathway in hyperthermophilic Pyrococcus species, we performed crystallographic studies on N,N'-diacetylchitobiose deacetylases (Dacs) from Pyrococcus horikoshii (Ph-Dac) and Pyrococcus furiosus (Pf-Dac). Neither Ph-Dac nor Pf-Dac was expressed in the soluble fraction of Escherichia coli harboring the expression plasmid. However, insertion of the target genes into the chromosome of E. coli yielded the soluble recombinant protein. The purified Pyrococcus Dacs were active and thermostable up to 85 °C. The crystal structures of Ph-Dac and Pf-Dac were determined at resolutions of 2.0 Å and 1.54 Å, respectively. The Pyrococcus Dac forms a hexamer composed of two trimers. These Dacs are characterized by an intermolecular cleft, which is formed by two polypeptides in the trimeric assembly. In Ph-Dac, catalytic Zn situated at the end of the cleft is coordinated by three side chain ligands from His44, Asp47, and His155, and by a phosphate ion derived from the crystallization reservoir solution. We considered that the bound phosphate mimicked the tetrahedral oxyanion, which is an intermediate of hydrolysis of the N-acetyl group, and proposed an appropriate reaction mechanism. In the proposed mechanism, the N(ε) atom of His264 (from the adjacent polypeptide in the Ph-Dac sequence) is directly involved in the stabilization of the oxyanion intermediate. Mutation analysis also indicated that His264 was essential to the catalysis. These factors give the archaeal Dacs an unprecedented active site architecture a Zn-dependent deacetylases. DATABASE: Structural data are available in the Protein Data Bank database under accession numbers 3WL3, 3WL4, and 3WE7.

Expression and characterization of a thermostable acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis involved in the degradation of insoluble cellulose acetate.

A thermostable acetylxylan esterase gene, TTE0866, which catalyzes the deacetylation of cellulose acetate, was cloned from the genome of Caldanaerobacter subterraneus subsp. tengcongensis. The pH and temperature optima were 8.0 and 60 °C. The esterase was inhibited by phenylmethylsulfonyl fluoride. A mixture of the esterase and cellulolytic enzymes efficiently degraded insoluble cellulose acetate with a higher degree of substitution.

Production of aromatic compounds by metabolically engineered Escherichia coli with an expanded shikimate pathway.

Escherichia coli was metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) from Cupriavidus necator was introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) from Azospirillum brasilense and the phenylacetaldehyde dehydrogenase gene (feaB) from E. coli were introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereas ipdC and the alcohol dehydrogenase gene (adhC) from Lactobacillus brevis were introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases in E. coli encoded by the yqhD, yjgB, and yahK genes. Cointroduction and cooverexpression of each gene with ipdC in the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of the yahK gene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by-products were observed. Deletion of feaB, pheA, and/or tyrA genes from the chromosomes of the constructed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six constructed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aromatic compounds are able to be produced from renewable resources without supplementing with expensive precursors.

Antimicrobial action of a unique phosphorus-adsorbent additive for resin, and the mechanism of its antimicrobial effect.

We found that an additive for a resin, which was comprised of collagen and aluminum (Al), showed a strong and stable antibacterial effect against various bacterium under certain conditions. We tried to clarify its mechanism of action, and investigated optimum conditions for its effects. This additive (Al cross-linked collagen powder: Al-COL) absorbed phosphorus in LB medium, gradually released aluminum in the phosphorus-reduced LB medium, and exhibited a bactericidal effect. Allophane was very suitable as the control subject, because it did not release Al in the medium, decreased phosphorus levels in the medium, and the phosphorus decrease led to a reduction in bacterial growth, though not to a bactericidal effect. On the other hand, the addition of Al to the phosphorus-reduced solution led to a bactericidal effect. These results suggested that Al can exert a strong antibacterial effect in the absence of phosphorus. This phenomenon was confirmed using film-shaped test items mixed with Al-COL powder. Furthermore, the reduction of phosphorus also synergistically led to the enhancement of the antibacterial effect of silver (Ag). The phosphorous absorption promoted the antibacterial action of Al and Ag, and Al, which has seldom been used as an antimicrobial agent, is available as an antibacterial agent in the absence of phosphorus.

A convenient method for multiple insertions of desired genes into target loci on the Escherichia coli chromosome.

We developed a method to insert multiple desired genes into target loci on the Escherichia coli chromosome. The method was based on Red-mediated recombination, flippase and the flippase recognition target recombination, and P1 transduction. Using this method, six copies of the lacZ gene could be simultaneously inserted into different loci on the E. coli chromosome. The inserted lacZ genes were functionally expressed, and ß-galactosidase activity increased in proportion to the number of inserted lacZ genes. This method was also used for metabolic engineering to generate overproducers of aromatic compounds. Important genes of the shikimate pathway (aroF (fbr) and tyrA (fbr) or aroF (fbr) and pheA (fbr)) were introduced into the chromosome to generate a tyrosine or a phenylalanine overproducer. Moreover, a heterologous decarboxylase gene was introduced into the chromosome of the tyrosine or phenylalanine overproducer to generate a tyramine or a phenethylamine overproducer, respectively. The resultant strains selectively overproduced the target aromatic compounds. Thus, the developed method is a convenient tool for the metabolic engineering of E. coli for the production of valuable compounds.

Functional analysis of the carbohydrate-binding module of an esterase from Neisseria sicca SB involved in the degradation of cellulose acetate.

An esterase gene from Neisseria sicca SB encoding CaeA, which catalyzes the deacetylation of cellulose acetate, was cloned. CaeA contained a putative catalytic domain of carbohydrate esterase family 1 and a carbohydrate-binding module (CBM) family 2. We constructed two derivatives, with and without the CBM of CaeA. Binding assay indicated that the CBM of CaeA had an affinity for cellulose.

Two groups of thermophilic amino acid aminotransferases exhibiting broad substrate specificities for the synthesis of phenylglycine derivatives.

Thirty two thermophilic amino acid aminotransferases (AATs) were expressed in Escherichia coli as soluble and active proteins. Based on their primary structures, the 32 AATs were divided into four phylogenetic groups (classes I, II, IV, and V). The substrate specificities of these AATs were examined, and 12 AATs were found capable of synthesizing ring-substituted phenylglycine derivatives such as hydroxyl-, methoxy-, and fluorophenylglycines. Eleven out of the 12 AATs were enzymes belonging to two phylogenetic groups namely, one subgroup of the class I family and the class IV family. AATs in these two groups may thus be useful for the synthesis of a variety of ring-substituted phenylglycine derivatives.

Phylogenetic analysis of long-chain hydrocarbon-degrading bacteria and evaluation of their hydrocarbon-degradation by the 2,6-DCPIP assay.

Thirty-six bacteria that degraded long-chain hydrocarbons were isolated from natural environments using long-chain hydrocarbons (waste car engine oil, base oil or the c-alkane fraction of base oil) as the sole carbon and energy source. A phylogenetic tree of the isolates constructed using their 16S rDNA sequences revealed that the isolates were divided into six genera plus one family (Acinetobacter, Rhodococcus, Gordonia, Pseudomonas, Ralstonia, Bacillus and Alcaligenaceae, respectively). Furthermore, most of the isolates (27 of 36) were classified into the genera Acinetobacter, Rhodococcus or Gordonia. The hydrocarbon-degradation similarity in each strain was confirmed by the 2,6-dichlorophenol indophenol (2,6-DCPIP) assay. Isolates belonging to the genus Acinetobacter degraded long-chain normal alkanes (n-alkanes) but did not degrade short-chain n-alkanes or cyclic alkanes (c-alkanes), while isolates belonging to the genera Rhodococcus and Gordonia degraded both long-chain n-alkanes and c-alkanes.

A high-throughput and generic assay method for the determination of substrate specificities of thermophilic alpha-aminotransferases.

For the determination of substrate specificities of thermophilic alpha-aminotransferases (AATs), a novel high-throughput assay method was developed. In this method, a thermophilic omega-aminotransferase (OAT) and a thermophilic aldehyde dehydrogenase (ALDH) are coupled to the AAT reaction. Glutamic acid is used as an amino group donor for the AAT reaction yielding 2-oxoglutalic acid. 2-Oxoglutalic acid produced by the AAT reaction is used as an amino group acceptor in the OAT reaction regenerating glutamic acid. The amino group donor of the OAT reaction is 5-aminopentanoic acid yielding pentanedioic acid semialdehyde which is oxidized by ALDH to pentanedioic acid with concomitant reduction of NADP(+) to NADPH. NADPH thus produced then reduces colorless tetrazolium salt into colored formazan. To construct such a reaction system, the genes for a thermophilic AAT, a thermophilic OAT and a thermophilic ALDH were cloned and expressed in Escherichia coli. These enzymes were subsequently purified and used to determine the activities of AAT for the synthesis of unnatural amino acids. This method allowed the clear detection of the AAT activities as it measures the increase in the absorbance on a low background absorbance reading.