Construction of deoxyriboaldolase-overexpressing Escherichia coli and its application to 2-deoxyribose 5-phosphate synthesis from glucose and acetaldehyde for 2'-deoxyribonucleoside production

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.     

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

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.     

Cloning, expression, and characterization of a new deoxyribose 5-phosphate aldolase from Yersinia sp. EA015

A new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular mass 24.8 kDa. The amino acid sequence was 94% identical to that of DERA from Yersinia intermedia ATCC 29909. DERA was over-expressed in Escherichia coli and purified using Ni–NTA affinity chromatography. The specific activity was 137 μmol/min/mg. The Michaelis constant (k_m value) of DERA was 9.1 mM. DERA was optimally active at pH 6.0 and 50 °C. DERA was tolerant to a high concentration (300 mM) of acetaldehyde.     

Efficient synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose by using new 2-deoxy-d-ribose-5-phosphate aldolase from Streptococcus suis with moderate activity and aldehyde tolerance

The enzyme 2-deoxy-d-ribose-5-phosphate aldolase (DERA) is a useful tool for synthesizing statin side-chain intermediates. In this work, we identified the DERA from Streptococcus suis (SsDERA) by structural and sequence alignment and highly expressed it in Escherichia coli BL21. The recombinant SsDERA had a specific activity of 18.2 U mg⁻¹, K_M of 0.8 mM, and V_max of 32.9 μmol min⁻¹ mg⁻¹ toward 2-deoxy-d-ribose-5-phosphate under the optimal conditions: 40 °C and pH 7.0. The enzyme retained 23.3 % activity after incubation in 200 mM acetaldehyde for 2 h and 58.2 % activity in 100 mM chloroacetaldehyde for 2 h. The enzyme showed moderate activity and aldehyde tolerance compared with reported DERAs. The SsDERA-catalyzed reaction between 200 mM acetaldehyde and 100 mM chloroacetaldehyde generated (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose in 76 % yield in 8 h. This work provides a new DERA for the synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose, which is a potential candidate for the industrial synthesis of statin intermediates.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N backbone and sidechain resonance assignments of a monomeric variant of <Emphasis Type="Italic">E. coli</Emphasis> deoxyribose-5-phosphate aldolase

Deoxyribose-5-phosphate aldolase (DERA) catalyses the reversible conversion of 2-deoxyribose-5-phosphate (dR5P) into glyceraldehyde-3-phosphate (G3P) and acetaldehyde. For industrial applications, this enzyme is used in organic synthesis for aldol reactions between acetaldehyde as a donor and a wide range of aldehydes as acceptors. Here, we present a near complete set of sequence-specific ¹H, ¹³C and ¹⁵N resonance assignments of a 28 kDa monomeric variant of the Escherichia coli DERA. These assignments provide the basis for ongoing structural and dynamic analysis of DERA substrate specificity.     

Sequential aldol condensation catalyzed by hyperthermophilic 2-deoxy-d-ribose-5-phosphate aldolase

Genes encoding 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, were expressed individually in Escherichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E. coli enzyme, even at a low temperature (25 degrees C), although both enzymes showed much less 2-deoxy-d-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25 degrees C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 A. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs.     

One-pot Microbial Synthesis of 2′-deoxyribonucleoside from Glucose, Acetaldehyde, and a Nucleobase

A one-pot enzymatic synthesis of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase was established. Glycolysis by baker’s yeast (Saccharomyces cerevisiae) generated ATP which was used to produce d-glyceraldehyde 3-phosphate production from glucose via fructose 1,6-diphosphate. The d-glyceraldehyde 3-phosphate produced was transformed to 2′-deoxyribonucleoside via 2-deoxyribose 5-phosphate and then 2-deoxyribose 1-phosphate in the presence of acetaldehyde and a nucleobase by deoxyriboaldolase, phosphopentomutase expressed in Escherichia coli, and a commercial nucleoside phosphorylase. About 33 mM 2′-deoxyinosine was produced from 600 mM glucose, 333 mM acetaldehyde and 100 mM adenine in 24 h. 2′-Deoxyinosine was produced from adenine due to the adenosine deaminase activity of E. coli transformants.     

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni²⁺, Ba²⁺ and Fe²⁺. The apparent K _m and V _max values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min^?1 mg^?1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min^?1 mg^?1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.     

Molecular Cloning,Sequencing, and Expression of a <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine <Emphasis Type="SmallCaps">d</Emphasis>-Fructose 6-Phosphate Amidotransferase Gene from <Emphasis Type="Italic">Volvariella volvacea</Emphasis>

Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K _m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N ³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.     

Structure-based mutagenesis approaches toward expanding the substrate specificity of D-2-deoxyribose-5-phosphate aldolase

2-Deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) catalyzes the reversible aldol reaction between acetaldehyde and D-glyceraldehyde-3-phosphate to generate D-2-deoxyribose-5-phosphate. It is unique among the aldolases as it catalyzes the reversible asymmetric aldol addition reaction of two aldehydes. In order to expand the substrate scope and stereoselectivity of DERA, structure-based substrate design as well as site-specific mutation has been investigated. Using the 1.05 A crystal structure of DERA in complex with its natural substrate as a guide, five site-directed mutants were designed in order to improve its activity with the unnatural nonphosphorylated substrate, D-2-deoxyribose. Of these, the S238D variant exhibited a 2.5-fold improvement over the wild-type enzyme in the retroaldol reaction of 2-deoxyribose. Interestingly, this S238D mutant enzyme was shown to accept 3-azidopropinaldehyde as a substrate in a sequential asymmetric aldol reaction to form a deoxy-azidoethyl pyranose, which is a precursor to the corresponding lactone and the cholesterol-lowering agent Lipitor. This azidoaldehyde is not a substrate for the wild-type enzyme. Another structure-based design of new nonphosphorylated substrates was focused on the aldol reaction with inversion in enantioselectivity using the wild type or the S238D variant as the catalyst and 2-methyl-substituted aldehydes as substrates. An example was demonstrated in the asymmetric synthesis of a deoxypyranose as a new effective synthon for the total synthesis of epothilones. In addition, to facilitate the discovery of new enzymatic reactions, the engineered E. coli strain SELECT (Deltaace, adhC, DE3) was developed to be used in the future for selection of DERA variants with novel nonphosphorylated acceptor specificity.     

2-Deoxy-d-ribose-5-phosphate aldolase (DERA): applications and modifications

2-Deoxy-d-ribose-5-phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C–C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.

     

Biochemical retrosynthesis of 2′-deoxyribonucleosides from glucose, acetaldehyde, and a nucleobase

2′-Deoxyribonucleosides are important as building blocks for the synthesis of antisense drugs, antiviral nucleosides, and 2′-deoxyribonucleotides for polymerase chain reaction. The microbial production of 2′-deoxyribonucleosides from simple materials, glucose, acetaldehyde, and a nucleobase, through the reverse reactions of 2′-deoxyribonucleoside degradation and the glycolytic pathway, was investigated. The glycolytic pathway of baker’s yeast yielded fructose 1,6-diphosphate from glucose using the energy of adenosine 5′-triphosphate generated from adenosine 5′-monophosphate through alcoholic fermentation with the yeast. Fructose 1,6-diphosphate was further transformed to 2-deoxyribose 5-phosphate in the presence of acetaldehyde by deoxyriboaldolase-expressing Escherichia coli cells via d-glyceraldehyde 3-phosphate. E. coli transformants expressing phosphopentomutase and nucleoside phosphorylase produced 2′-deoxyribonucleosides from 2-deoxyribose 5-phosphate and a nucleobase via 2-deoxyribose 1-phosphate through the reverse reactions of 2′-deoxyribonucleoside degradation. Coupling of the glycolytic pathway and deoxyriboaldolase-catalyzing reaction efficiently supplied 2-deoxyribose 5-phosphate, which is a key intermediate for 2′-deoxyribonucleoside synthesis. 2′-Deoxyinosine (9.9 mM) was produced from glucose, acetaldehyde, and adenine through three-step reactions via fructose 1,6-diphosphate and then 2-deoxyribose 5-phosphate, the molar yield as to glucose being 17.8%.     

Low-specificity l-threonine aldolase of Pseudomonas sp. NCIMB 10558: purification, characterization and its application to β-hydroxy-α-amino acid synthesis

Low-specificity l-threonine aldolase, catalyzing the reversible cleavage/condensation reaction between l-threonine/l-allo-threonine and glycine plus acetaldehyde, was purified to homogeneity from Pseudomonas sp. NCIMB 10558. The enzyme has an apparent molecular mass of approximately 145 kDa and consists of four identical subunits with a molecular mass of 38 kDa. The enzyme, requiring pyridoxal- 5′-phosphate as a coenzyme, is strictly l-specific at the α position, whereas it can not distinguish between threo and erythro forms at the β position. Besides the reversible cleavage/condensation of threonine, the enzyme also catalyzes the reversible interconversion between glycine plus various aldehydes and l-β-hydroxy-α-amino acids, including l-β-(3,4-dihydroxyphenyl)serine, l-β-(3,4-met‐hylenedioxyphenyl)serine and l-β-phenylserine, providing a new route for the industrial production of these important amino acids. Received: 10 November 1997 / Received revision: 7 January 1998 / Accepted 30 January 1998     

Cloning and characterisation of a new 2-deoxy-D-ribose-5-phosphate aldolase from Rhodococcus erythropolis

A new 2-deoxy-D-ribose-5-phoshate aldolase (DERA) gene was cloned from Rhodococcus erythropolis strain DSM 311, recombinantly expressed in Escherichia coli, and purified via affinity chromatography which yielded a homo-dimeric enzyme of 44.3 kDa as apparent by size exclusion chromatography. To characterise the enzyme, investigations about pH and temperature tolerance, stability, as well as analyses on resistance to organic solvents and acetaldehyde were performed. In addition, kinetic constants of the new DERA(RE) were compared to respective values of the DERA from E. coli (DERA(EC)). Stability of DERA(RE) turned out to be a crucial factor: The pH for optimal DERA(RE) activity was determined to be 7.0, whereas the highest stability was achieved at pH 9.0 with a half-life of approximately 20 days. The optimal temperature for DERA(RE) activity was 65 °C, but coupled with a rather low stability (half-life of 2 min). The highest stability was achieved at 25 °C. The new enzyme exhibits high resistance to organic solvents and acetaldehyde with a half-life being 2.5× higher compared to DERA(EC) under the exposure of 300 mM acetaldehyde. Hence it has the potential as a new promising biocatalyst with applications in organic synthesis.     

Covalent immobilization of recombinant Citrobacter koseri transaminase onto epoxy resins for consecutive asymmetric synthesis of L-phosphinothricin

Transaminase responsible for alienating prochiral ketone compound is applicable to asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). In this work, the covalent immobilization of recombinant transaminase from Citrobacter koseri (CkTA) was investigated on different epoxy resins. Using optimum ES-105 support, a higher immobilized activity was obtained via optimizing immobilization process in terms of enzyme loading, coupling time and initial PLP concentration. Crucially, due to blocking unreacted epoxy groups on support surface with amino acids, the reaction temperature of blocked immobilized biocatalyst was enhanced from 37 to 57 °C. Its thermostability at 57 °C was also found to be superior to that of free CkTA. The K_m value was shifted from 36.75 mM of free CkTA to 39.87 mM of blocked immobilized biocatalyst, demonstrating that the affinity of enzyme to the substrate has not been apparently altered. Accordingly, the biocatalyst performed the consecutive synthesis of L-PPT for 11 cycles (yields>91%) with retaining more than 91.13% of the initial activity. The seemingly the highest reusability demonstrates this biocatalyst has prospective for reducing the costs of consecutive synthesis of L-PPT with high conversion.

     

Stable and efficient immobilization technique of aldolase under consecutive microwave irradiation at low temperature

To establish a stable and efficient immobilization technique under microwave irradiation, a focused microwave reaction system was used, where the temperature was set appropriately in the microwave system and cooling module to produce consecutive microwave irradiation. 2-Deoxy-D-ribose-5-phosphate aldolase (DERA) was rapidly and efficiently immobilized in mesocellular siliceous foams (MCFs) under microwave irradiation. When the output power in the microwave system was set to 30 W, after 3 min, 88.4% of the enzyme protein was coupled to the wall of the support pores and the specific activity of the immobilized enzyme was 2.24 U mg⁻¹, 149.2% higher than that of the free enzyme and 157.0% higher than that of the non-microwave-assisted immobilized enzyme. In catalysis, microwave-assisted immobilized DERA tolerated a wider range of both pH and temperature than other DERA preparations. The thermal and storage stabilities were also significantly improved. This focused; microwave-assisted immobilization technique has proven to be simple, stable and highly efficient. This technique could also be applied to other enzyme immobilizations.     

Characterization of a novel dye-linked <Emphasis Type="SmallCaps">l</Emphasis>-proline dehydrogenase from an aerobic hyperthermophilic archaeon, <Emphasis Type="Italic">Pyrobaculum calidifontis</Emphasis>

The activity of a dye-linked l-proline dehydrogenase (dye-l-proDH) was found in the crude extract of an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis JCM 11548, and was purified 163-fold through four sequential chromatography steps. The enzyme has a molecular mass of about 108 kDa and is a homodimer with a subunit molecular mass of about 46 kDa. The enzyme retained more than 90% of its activity after incubation at 100 °C for 120 min (pH 7.5) or after incubation at pHs 4.5–9.0 for 30 min at 50 °C. The enzyme catalyzed l-proline dehydrogenation to Δ¹-pyroline-5-carboxylate using 2,6-dichloroindophenol (DCIP) as the electron acceptor and the Michaelis constants for l-proline and DCIP were 1.67 and 0.026 mM, respectively. The prosthetic group on the enzyme was identified as flavin adenine dinucleotide by high-performance liquid chromatography. The subunit N-terminal amino acid sequence was MYDYVVVGAG. Using that sequence and previously reported genome information, the gene encoding the enzyme (Pcal_1655) was identified. The gene was then cloned and expressed in Escherichia coli and found to encode a polypeptide of 415 amino acids with a calculated molecular weight of 46,259. The dye-l-proDH gene cluster in P. calidifontis inherently differs from those in the other hyperthermophiles reported so far.     

Amino acid racemase with broad substrate specificity,its properties and use in phenylalanine racemization

Summary Broad substrate specificity amino acid racemase (EC 5.1.1.10) was purified from a crude extract of Pseudomonas putida SCRC-744 to near homogeneity. The enzyme has an isoelectric point of 7.6 and a molecular weight of 62,000–65,000. The enzyme showed a broad substrate specificity toward amino acids, utilizing d-glutamine as the best substrate. d-Phenylalanine acted as a substrate to 1% the velocity for d-glutamine. Maximal reaction velocities were observed at 50°–60°C and around pH 8. The apparent K_m values for d-glutamine and d-phenylalanine were 7.8 mM and 25.7 mM, respectively. Both enantiomers of phenylalanine were efficiently racemized by acetone-dried cells of P. putida SCRC-744.     

Thin layer chromatographical detection of tyrosine produced from <Emphasis Type="SmallCaps">l</Emphasis>-[U-<Superscript>14</Superscript>C]phenylalanine by ruminal microbes

Summary.  Thin layer chromatographical detection of tyrosine (Tyr) synthesized from l-[U-¹⁴C]phenylalanine (Phe) (1 mM) by rumen bacteria (B) and protozoa (P) collected from fistulated Japanese Goat was carried out. About 16 and 12% of the added Phe was converted to Tyr by B and P, respectively. Large amount of radioactivity in ether fractions indicated an abundant production of aromatic acids from Phe. Small amount of radioactivity found in CO₂ fractions implied an occurrence of considerable decarboxylation reaction(s) by rumen bacteria and protozoa. Received July 18, 2001 Accepted December 3, 2001