(2R,3S,4S,5R,6R)-2-(aminomethyl)-6-[(1R,2R,3S,4R,6S)-4,6-diamino-3-[(2S,3R,4S,5S,6R)-4-amino-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxyoxane-3,4,5-triol

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of 125 ml of dichloromethane was added 1 gram of kanamycin sulfate in 10 ml of aqueous solution. To the resulting solution was added 20 ml of soy lecithin (Alcolec S). The solution was mixed by shaking. Four ml of the solution was applied to each side of a 1"×1" foam wound dressing, which was then allowed to dry. These materials were placed in foil heat sealed bags and subjected to sterilization by use of cobalt irradiation (3.2 megarads). The materials were assayed for antibiotic activity as described in Example 12. The eluent contained fully active entrapped antibiotic after irradiation by using standard antimicrobial assay techniques. The solution was mixed to dry heat sealed bags subjected to sterilization by use of cobalt irradiation (3.2 megarads) after irradiation

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The cell-suspension was transferred onto a minimum medium prepared by adding 5 μg/dl kanamycin, 10 mg/dl L-leucine, 10 mg/dl L-leucine, 10 mg/dl L-arginine, 100 mg/dl D, L-m-fluorophenylalanine and 2 g/dl agar to a basal minimum medium of pH 7.2 containing 0.6 g/dl KH2PO4, 1.4 g/dl K2HPO4, 0.2 g/dl (NH4)2SO4, 0.1 g/dl sodium citrate, 0.02 g/dl MgSO4.7H2O, and 0.5 g/dl glucose. After 3 days cultivation at 37° C., two colonies appeared on the agar-medium. prepared

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The cell-suspension was transferred onto a minimum medium prepared by adding 5 μg/dl kanamycin, 10 mg/dl L-leucine, 10 mg/dl L-leucine, 10 mg/dl L-arginine, 100 mg/dl D, L-m-fluorophenylalanine and 2 g/dl agar to a basal minimum medium of pH 7.2 containing 0.6 g/dl KH2PO4, 1.4 g/dl K2HPO4, 0.2 g/dl (NH4)2SO4, 0.1 g/dl sodium citrate, 0.02 g/dl MgSO4.7H2O, and 0.5 g/dl glucose. After 3 days cultivation at 37° C., two colonies appeared on the agar-medium. prepared

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: L-tyrosine production experiments were performed at 37° C. with 225 rpm orbital shaking in 50 ml MOPS minimal medium (Teknova) (Neidhardt, Bloch et al. 1974) cultures supplemented with 5 g/l glucose and an additional 4 g/l NH4Cl. All liquid cultivations were conducted in at least triplicates. When appropriate, antibiotics were added in the following concentrations: 34 μg/ml chloramphenicol for maintenance of pHACM-derived plasmids and 20 μg/ml kanamycin for maintenance of pZE-derived plasmids. L-phenylalanine auxotrophic (ΔpheA) cultures were additionally provided with L-phenylalanine (Sigma) at a concentration of 0.35 mM. were performed at 37° C. with 225 rpm When appropriate, antibiotics were added in the following concentrations

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: pAET7 was introduced into V12Sα obtained in Example 16, and the pMIV-FRTKmFRT-EABDAS#10 plasmid was also introduced to obtain the V12Sα strains where the lysE, dapA, dapB, asd, lysA, and ddh genes were introduced into the chromosome by the method described in Example 16. Randomly selected 200 strains were cultured in an SEII production medium containing 50 mg/L streptomycin and 20 mg/L kanamycin to select a strain which produced the most L-lysine, and was designated as Vmac1. A streptomycin-sensitive strain was selected form the Vmac1 strains to obtain a strain where pAET7 was eliminated. The pFLP31 plasmid shown in Example 9 was introduced into the strain, and a kanamycin-resistant gene region was eliminated from the mini-Mu cassette on the chromosome by the method described in Example 9. The kanamycin-sensitive strains obtained by the procedure were designated as Vmac1S strain. A streptomycin-sensitive strain was selected from the Vmac1S strain to obtain a strain where the pFLP31 plasmid was eliminated. The pAET7 plasmid was introduced again into this strain, and the plasmid pMIV-FRTKmFRT-EABDAS#10 was also introduced again. Then, the strain which produced the most L-lysine was selected and designated as the Vmac2 strain. In the same way as described above, the Vmac2S strain, Vmac3 strain, and Vmac3S strain were obtained. The amount of Lys produced by V12 was defined as 100, and the relative value of the amount of Lys produced by the Vmac3S strain was calculated and shown in Table 10. asd, lysA, and ddh genes were introduced into the chromosome by the method

Conditions: temp:37.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: First, the pAET7 plasmid described in Example 1 was introduced into a wild-type strain of M. methylotrophus AS1 (NCIMB10515), and a transformant was selected in an SEII agar medium containing 50 mg/L streptomycin. The pMIV-FRTKmFRT-EA plasmid was introduced into the strain by electroporation in accordance with the method described in Example 2, and strains to which the mini-Mu cassette (EA unit) including the lysE24+dapA* genes was transferred to the chromosome were selected in an SEII agar medium containing 50 mg/L streptomycin and 20 mg/L kanamycin. From the strains, 200 strains were selected randomly and inoculated on SEII plates containing 50 mg/L streptomycin and 20 mg/L kanamycin, followed by culturing at 37° C. overnight. Then, bacterial cells present on the medium surface of about 0.3 square centimeters were scraped off and inoculated into an SEII production medium (5 ml) containing 50 mg/l streptomycin and 20 mg/L kanamycin, and the cells were cultured with shaking at 37° C. for 34 hours. After completion of culture, the bacterial cells were removed by centrifugation, and the concentration of L-lysine in each culture supernatant was measured using a Biotech-analyzer AS-210 (manufactured by Sakura Seiki Co., Ltd.) to select the strain having the highest concentration of L-lysine. The strain was designated as V1. Transfer was repeated 12 times in the same way as Example 3 to obtain the V12 strain. The amount of Lys produced by V1 was defined as 100, and the relative value of the amount of Lys produced by the V12 strain was calculated and shown in Table 9. was introduced into a wild-type strain of M After completion of culture, the bacterial cells were removed by centrifugation to obtain the V12 strain

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Ten colonies of the transformed Agrobacteria containing a promoter construct of interest were picked from a fresh plate and inoculated into 10 ml YEB, containing 50 mg/l rifampicin, and 50 mg/l kanamycin, in a 50 ml flask. Cell cultures were incubated overnight at 28° C. with shaking. Approximately 1 ml of overnight culture was taken. Cultures were diluted with 50 ml YEB plus antibiotics so that the OD600 was approximately 0.3. Agrobacteria were grown at 28° C. with shaking until the OD600 was approximately 0.6. The cells were then pelleted by centrifugation. Supernatants were removed and Agrobacteria were resuspended in 40 ml fresh YEB by pipetting with a 1 ml tip. Wild type tobacco leaves were cut into small pieces under sterile conditions and put in a 50 ml beaker. Agrobacteria solution was poured onto the tobacco leaves and the mixture was swirled together and left for 5 min for the Agrobacteria to infect the leaves. Infected leaves were put on MS plates with filter paper (to limit Agrobacteria grow). The MS plates did not contain antibiotics. Plates were left for 2 days in the dark. After 2 days the tobacco pieces were transferred onto MS plates containing 1 mg/l 6 BA, 500 mg/l cefotaxin and 100 mg/l kanamycin. Samples were left in the light at 28° C. After 1 month small transgenic tobacco plants were visible. The plants were transferred to pots with ½ MS medium with 150 mg/l kanamycin. Several week old plants were transplanted into soil and grown under standard greenhouse conditions. Seeds from primary transformants were collected and germinated on ½ MS medium with 150 mg/l kanamycin and 500 mg/l cefotaxin. Transgenic seedlings were tested for promoter activity. Ten colonies of the transformed Agrobacteria containing a promoter Approximately 1 ml of overnight culture Cultures were diluted with 50 ml YEB plus antibiotics so that the OD600 were grown at 28° C. with shaking until the OD600 Supernatants were removed Wild type tobacco leaves Agrobacteria solution was poured onto the tobacco leaves left for 5 min for the Agrobacteria Infected leaves with filter paper (to limit Agrobacteria grow) Plates were left for 2 days in the dark After 2 days the tobacco pieces were transferred onto MS plates containing 1 mg/l 6 BA, 500 mg/l cefotaxin and 100 mg/l kanamycin Samples were left in the light at 28° C After 1 month small transgenic tobacco plants were visible Several week old plants Seeds from primary transformants were collected

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Ten colonies of the transformed Agrobacteria containing a promoter construct of interest were picked from a fresh plate and inoculated into 10 ml YEB, containing 50 mg/l rifampicin, and 50 mg/l kanamycin, in a 50 ml flask. Cell cultures were incubated overnight at 28° C. with shaking. Approximately 1 ml of overnight culture was taken. Cultures were diluted with 50 ml YEB plus antibiotics so that the OD600 was approximately 0.3. Agrobacteria were grown at 28° C. with shaking until the OD600 was approximately 0.6. The cells were then pelleted by centrifugation. Supernatants were removed and Agrobacteria were resuspended in 40 ml fresh YEB by pipetting with a 1 ml tip. Wild type tobacco leaves were cut into small pieces under sterile conditions and put in a 50 ml beaker. Agrobacteria solution was poured onto the tobacco leaves and the mixture was swirled together and left for 5 min for the Agrobacteria to infect the leaves. Infected leaves were put on MS plates with filter paper (to limit Agrobacteria grow). The MS plates did not contain antibiotics. Plates were left for 2 days in the dark. After 2 days the tobacco pieces were transferred onto MS plates containing 1 mg/l 6 BA, 500 mg/l cefotaxin and 100 mg/l kanamycin. Samples were left in the light at 28° C. After 1 month small transgenic tobacco plants were visible. The plants were transferred to pots with ½ MS medium with 150 mg/l kanamycin. Several week old plants were transplanted into soil and grown under standard greenhouse conditions. Seeds from primary transformants were collected and germinated on ½ MS medium with 150 mg/l kanamycin and 500 mg/l cefotaxin. Transgenic seedlings were tested for promoter activity. Ten colonies of the transformed Agrobacteria containing a promoter Approximately 1 ml of overnight culture Cultures were diluted with 50 ml YEB plus antibiotics so that the OD600 were grown at 28° C. with shaking until the OD600 Supernatants were removed Wild type tobacco leaves Agrobacteria solution was poured onto the tobacco leaves left for 5 min for the Agrobacteria Infected leaves with filter paper (to limit Agrobacteria grow) Plates were left for 2 days in the dark After 2 days the tobacco pieces were transferred onto MS plates containing 1 mg/l 6 BA, 500 mg/l cefotaxin and 100 mg/l kanamycin Samples were left in the light at 28° C After 1 month small transgenic tobacco plants were visible Several week old plants Seeds from primary transformants were collected

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: An overnight culture with the pET30Trp racemase construct was subcultured into fresh LB medium with the appropriate antibiotics (50 μg/mL, kanamycin and 20 μg/mL chloramphenicol) and grown to an OD600 ˜0.6 (37° C. with aeration). Expression was induced with 100 μM IPTG and incubation was continued at 37° C. with aeration for 2 hours. The cells were harvested by centrifugation and stored at −80° C. until use. The cell pellet was thawed on ice and cells were lysed using BugBuster Primary Amine Free Cell Lysis Reagent and Benzonase Nuclease (Novagen. Madison, Wis.). Cell debris was removed by centrifugation and the supernatant was used as the crude protein extract. The crude protein extract was filtered using a 0.45 μm syringe filter and applied to a HisBind column (Novagen, Madison, Wis.) that had been pre-equilibrated according to the manufacturer's instructions. The column was washed and the protein was eluted as directed in the manufacturer's protocol. The purified protein was desalted with a PD-10 column (GE Healthcare, Piscataway, N.J.) using 50 mM potassium phosphate pH 8.0, 10 μM pyridoxal-5′-phosphate (“PLP”) as the eluent. The desalted protein was concentrated using Amicon centrifugal concentrators (Millipore, Billerica, Mass.). Wild-type alanine racemase was purified as described above. An overnight culture grown to an OD600 ˜0.6 (37° C. with aeration) stored at −80° C. until use Cell debris was removed by centrifugation extract The crude protein extract was filtered filter The column was washed the protein was eluted The desalted protein was concentrated Wild-type alanine racemase was purified

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: An overnight culture with the pET30Trp racemase construct was subcultured into fresh LB medium with the appropriate antibiotics (50 μg/mL, kanamycin and 20 μg/mL chloramphenicol) and grown to an OD600 ˜0.6 (37° C. with aeration). Expression was induced with 100 μM IPTG and incubation was continued at 37° C. with aeration for 2 hours. The cells were harvested by centrifugation and stored at −80° C. until use. The cell pellet was thawed on ice and cells were lysed using BugBuster Primary Amine Free Cell Lysis Reagent and Benzonase Nuclease (Novagen. Madison, Wis.). Cell debris was removed by centrifugation and the supernatant was used as the crude protein extract. The crude protein extract was filtered using a 0.45 μm syringe filter and applied to a HisBind column (Novagen, Madison, Wis.) that had been pre-equilibrated according to the manufacturer's instructions. The column was washed and the protein was eluted as directed in the manufacturer's protocol. The purified protein was desalted with a PD-10 column (GE Healthcare, Piscataway, N.J.) using 50 mM potassium phosphate pH 8.0, 10 μM pyridoxal-5′-phosphate (“PLP”) as the eluent. The desalted protein was concentrated using Amicon centrifugal concentrators (Millipore, Billerica, Mass.). Wild-type alanine racemase was purified as described above. An overnight culture grown to an OD600 ˜0.6 (37° C. with aeration) stored at −80° C. until use Cell debris was removed by centrifugation extract The crude protein extract was filtered filter The column was washed the protein was eluted The desalted protein was concentrated Wild-type alanine racemase was purified

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: An overnight culture with the pET30Trp racemase construct was subcultured into fresh LB medium with the appropriate antibiotics (50 μg/ml kanamycin and 20 μg/ml chloramphenicol) and grown to an OD600 ˜0.6 (37° C. with aeration). Expression was induced with 100 μM IPTG and incubation was continued at 37° C. with aeration for 2 hours. The cells were harvested by centrifugation and stored at −80° C. until use. The cell pellet was thawed on ice and cells were lysed using BugBuster Primary Amine Free Cell Lysis Reagent and Benzoase Nuclease (Novagen, Madison, Wis.). Cell debris was removed by centrifugation and the supernatant was used as the crude protein extract. The crude protein extract was filtered using a 0.45 μm syringe filter and applied to a HisBind column (Novagen, Madison, Wis.) that had been pre-equilibrated according to the manufacturer's instructions. The column was washed and the protein was eluted as directed in the manufacturer's protocol. The purified protein was desalted with a PD-10 column (GE Healthcare, Piscataway, N.J.) using 50 mM potassium phosphate pH 8.0, 10 μM pyridoxal-5′-phosphate (“PLP”) as the eluent. The desalted protein was concentrated using Amicon centrifugal concentrators (Millipore, Billerica, Mass.). Wild-type alanine racemase was purified as described above. An overnight culture grown to an OD600 ˜0.6 (37° C. with aeration) stored at −80° C. until use Cell debris was removed by centrifugation extract The crude protein extract was filtered filter The column was washed the protein was eluted The desalted protein was concentrated Wild-type alanine racemase was purified

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: An overnight culture with the pET30Trp racemase construct was subcultured into fresh LB medium with the appropriate antibiotics (50 μg/ml kanamycin and 20 μg/ml chloramphenicol) and grown to an OD600 ˜0.6 (37° C. with aeration). Expression was induced with 100 μM IPTG and incubation was continued at 37° C. with aeration for 2 hours. The cells were harvested by centrifugation and stored at −80° C. until use. The cell pellet was thawed on ice and cells were lysed using BugBuster Primary Amine Free Cell Lysis Reagent and Benzoase Nuclease (Novagen, Madison, Wis.). Cell debris was removed by centrifugation and the supernatant was used as the crude protein extract. The crude protein extract was filtered using a 0.45 μm syringe filter and applied to a HisBind column (Novagen, Madison, Wis.) that had been pre-equilibrated according to the manufacturer's instructions. The column was washed and the protein was eluted as directed in the manufacturer's protocol. The purified protein was desalted with a PD-10 column (GE Healthcare, Piscataway, N.J.) using 50 mM potassium phosphate pH 8.0, 10 μM pyridoxal-5′-phosphate (“PLP”) as the eluent. The desalted protein was concentrated using Amicon centrifugal concentrators (Millipore, Billerica, Mass.). Wild-type alanine racemase was purified as described above. An overnight culture grown to an OD600 ˜0.6 (37° C. with aeration) stored at −80° C. until use Cell debris was removed by centrifugation extract The crude protein extract was filtered filter The column was washed the protein was eluted The desalted protein was concentrated Wild-type alanine racemase was purified