Efficient enzymatic production of benzaldehyde from l-phenylalanine with a mutant form of 4-hydroxymandelate synthase.

Benzaldehyde is an organic compound with an almond-like aroma and one of the most important and widely used flavorings in the food industry. To develop an enzymatic process for the production of benzaldehyde from l-phenylalanine, four enzymes were expressed in Escherichia coli; l-amino acid deaminase, 4-hydroxymandelate synthase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase. Although each E. coli strain could be used to synthesize benzaldehyde from l-phenylalanine, the yield was low due to the accumulation of an intermediate, phenylpyruvic acid. We developed a second reaction step by engineering 4-hydroxymandelate synthase of Actinoplanes teichomyceticus. A quadruple mutant of 4-hydroxymandelate synthase (A199V/Q206R/I217V/K337Q) obtained by random and site-directed mutagenesis demonstrated 2.4-fold higher activity than wild type. Furthermore, the mutant-expressing strain was able to produce benzaldehyde from 100 mm l-phenylalanine at a conversion rate of 84% (wild type, 37%). We report the development of an efficient process for benzaldehyde production using l-phenylalanine as a substrate.

Purification, characterization, and gene cloning of a novel aminoacylase from Burkholderia sp. strain LP5_18B that efficiently catalyzes the synthesis of N-lauroyl-l-amino acids.

An N-lauroyl-l-phenylalanine-producing bacterium, identified as Burkholderia sp. strain LP5_18B, was isolated from a soil sample. The enzyme was purified from the cell-free extract of the strain and shown to catalyze degradation and synthesis activities toward various N-acyl-amino acids. N-lauroyl-l-phenylalanine and N-lauroyl-l-arginine were obtained with especially high yields (51% and 89%, respectively) from lauric acid and l-phenylalanine or l-arginine by the purified enzyme in an aqueous system. The gene encoding the novel aminoacylase was cloned from Burkholderia sp. strain LP5_18B and expressed in Escherichia coli. The gene contains an open reading frame of 1,323 nucleotides. The deduced protein sequence encoded by the gene has approximately 80% amino acid identity to several hydratase of Burkholderia. The addition of zinc sulfate increased the aminoacylase activity of the recombinant E. coli strain.

Directed evolution for thermostabilization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus.

To obtain a selection marker gene functional in a thermophilic bacterium, Thermus thermophilus, an in vivo-directed evolutionary strategy was conducted on a hygromycin B phosphotransferase gene (hyg) from Streptomyces hygroscopicus. The expression of wild-type hyg in T. thermophilus provided hygromycin B (HygB) resistance up to 60 °C. Through selection of mutants showing HygB resistance at higher temperatures, eight amino acid substitutions and the duplication of three amino acids were identified. A variant containing seven substitutions and the duplication (HYG10) showed HygB resistance at a highest temperature of 74 °C. Biochemical and biophysical analyses of recombinant HYG and HYG10 revealed that HYG10 was in fact thermostabilized. Modeling of the three-dimensional structure of HYG10 suggests the possible roles of the various substitutions and the duplication on thermostabilization, of which three substitutions and the duplication located at the enzyme surface suggested that these mutations made the enzyme more hydrophilic and provided increased stability in aqueous solution.

Crystal structures of the ternary complex of APH(4)-Ia/Hph with hygromycin B and an ATP analog using a thermostable mutant.

Aminoglycoside 4-phosphotransferase-Ia (APH(4)-Ia)/Hygromycin B phosphotransferase (Hph) inactivates the aminoglycoside antibiotic hygromycin B (hygB) via phosphorylation. The crystal structure of the binary complex of APH(4)-Ia with hygB was recently reported. To characterize substrate recognition by the enzyme, we determined the crystal structure of the ternary complex of non-hydrolyzable ATP analog AMP-PNP and hygB with wild-type, thermostable Hph mutant Hph5, and apo-mutant enzyme forms. The comparison between the ternary complex and apo structures revealed that Hph undergoes domain movement upon binding of AMP-PNP and hygB. This was about half amount of the case of APH(9)-Ia. We also determined the crystal structures of mutants in which the conserved, catalytically important residues Asp198 and Asn203, and the non-conserved Asn202, were converted to Ala, revealing the importance of Asn202 for catalysis. Hph5 contains five amino acid substitutions that alter its thermostability by 16°C; its structure revealed that 4/5 mutations in Hph5 are located in the hydrophobic core and appear to increase thermostability by strengthening hydrophobic interactions.

Efficient Nepsilon-lauroyl-L-lysine production by recombinant epsilon-lysine acylase from Streptomyces mobaraensis.

epsilon-Lysine acylase from Streptomyces mobaraensis (Sm-ELA), which specifically catalyzes hydrolysis of the epsilon-amide bond in various Nepsilon-acyl-L-lysines, was cloned and sequenced. The Sm-ELA gene consists of a 1617-bp open reading frame that encodes a 538-amino acid protein with a molecular mass of 55,816Da. An NCBI protein-protein BLAST search revealed that the enzyme belongs to the YtcJ-like metal-dependent amidohydrolase family, which is further characterized as the metallo-dependent hydrolase superfamily. The Sm-ELA gene was ligated into a pUC702 vector for expression in Streptomyces lividans TK24. Expression of recombinant Sm-ELA in S. lividans was approximately 300-fold higher than that in wild-type S. mobaraensis. The recombinant Sm-ELAs from the cell-free extract and culture supernatant were purified to homogeneity. The specific activities of the purified Sm-ELAs were 2500-2800U/mg, which were similar to that obtained for the wild-type Sm-ELA. Using the cell-free extract of the recombinant S. lividans cells, Nepsilon-lauroyl-L-lysine was synthesized from 500mM L-lysine hydrochloride and 50, 100, or 250mM lauric acid in an aqueous buffer solution at 37 degrees C. The yields were close to 100% after 6 and 9h of reaction for 50 and 100mM lauric acid, respectively, and 90% after 24h for 250mM lauric acid.

Enzymatic analysis of a thermostabilized mutant of an Escherichia coli hygromycin B phosphotransferase.

An Escherichia coli hygromycin B phosphotransferase (HPH) and its thermostabilized mutant protein, HPH5, containing five amino acid substitutions, D20G, A118V, S225P, Q226L, and T246A (Nakamura et al., J. Biosci. Bioeng., 100, 158-163 (2005)), obtained by an in vivo directed evolution procedure in Thermus thermophilus, were produced and purified from E. coli recombinants, and enzymatic comparisons were performed. The optimum temperatures for enzyme activity were 50 and 55 degrees C for HPH and HPH5 respectively, but the thermal stability of the enzyme activity and the temperature for protein denaturation of HPH5 increased, from 36 and 37.2 degrees C of HPH to 53 and 58.8 degrees C respectively. Specific activities and steady-state kinetics measured at 25 degrees C showed only slight differences between the two enzymes. From these results we concluded that HPH5 was thermostabilized at the protein level, and that the mutations introduced did not affect its enzyme activity, at least under the assay conditions.

Crystallization and preliminary crystallographic analysis of hygromycin B phosphotransferase from Escherichia coli.

Aminoglycoside antibiotics, such as hygromycin, kanamycin, neomycin, spectinomycin and streptomycin, inhibit protein synthesis by acting on bacterial and eukaryotic ribosomes. Hygromycin B phosphotransferase (Hph; EC 2.7.1.119) converts hygromycin B to 7''-O-phosphohygromycin using a phosphate moiety from ATP, resulting in the loss of its cell-killing activity. The Hph protein has been crystallized for the first time using a thermostable mutant and the hanging-drop vapour-diffusion method. The crystal provided diffraction data to a resolution of 2.1 A and belongs to space group P3(2)21, with unit-cell parameters a = b = 71.0, c = 125.0 A. Crystals of complexes of Hph with hygromycin B and AMP-PNP or ADP have also been obtained in the same crystal form as that of the apoprotein.

In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus.

An in vivo-directed evolutionary strategy was used to obtain a thermostabilized Escherichia coli hygromycin B phosphotransferase, using a host-vector system of Thermus thermophilus. Introduction of the mutant gene containing two amino acid substitutions, S52T and W238C, which was previously reported by Cannio et al. [J. Bacteriol., 180, 3237-3240 (1998)], did not confer hygromycin resistance on T. thermophilus cells at 55 degrees C; however, five spontaneously-generated independent mutants were obtained by selection of the transformants at this temperature. Each mutant gene contained one amino acid substitution of either A118V or T246A. Further selection with increasing temperature, at 58 degrees C and then 61 degrees C, led to acquisition of three more substitutions: D20G, S225P and Q226L. These mutations cumulatively influenced the maximum growth temperature of the T. thermophilus transformants in the presence of hygromycin; T. thermophilus carrying a mutant gene containing all the five substitutions was able to grow at up to 67 degrees C. This mutant gene, hph5, proved useful as a selection marker in the T. thermophilus host-vector system, either on the plasmid or by genome integration, at temperatures up to 65 degrees C.