(2R,3R,4S,5R,6S)-2-(hydroxymethyl)-6-propan-2-ylsulfanyloxane-3,4,5-triol

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

Procedure: coli MBX769 carrying the plasmid pFS16 (FIG. 2), which permitted the expression of the Clostridium kluyveri 4-hydroxybutyryl-CoA transferase, was precultured at 37° C. in 100 mL of LB medium containing 100 μg/mL sodium ampicillin in a 250-mL Erlenmeyer flask with shaking at 200 rpm. The cells were centrifuged at 5000 g for 10 minutes to remove them from the LB medium after 16 hours, and they were resuspended in 100 mL of a medium containing, per liter: 4.1 or 12.4 g sodium 4-hydroxyvalerate (4HV); 5 g/L sodium 4-hydroxybutyrate (4HB); 2 g glucose; 2.5 g LB broth powder (Difco; Detroit, Mich.); 50 mmol potassium phosphate, pH7; 100 μg/mL sodium ampicillin; and 0.1 mmol isopropyl-β-D-thiogalactopyranoside (IPTG). The sodium 4-hydroxyvalerate was obtained by saponification of γ-valerolactone in a solution of sodium hydroxide. The cells were incubated in this medium for 3 days with shaking at 200 rpm at 32° C. in the same flask in which they had been precultured. When 4.1 g/L sodium 4-hydroxyvalerate was present initially, the cells accumulated a polymer to 52.6% of the dry cell weight that consisted of 63.4% 3HB units and 36.6% 4HB units but no 4HV units. was precultured at 37° C. in 100 mL of LB medium to remove them from the LB medium after 16 hours containing, per liter

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

Procedure: Putative nitrilase up-mutants were assayed in triplicate. Each transformant was grown in 5 mL LB (100 μg/mL ampicillin), at 37° C., 220 rpm for 18 h. The overnight culture was diluted 2-fold and nitrilase expression induced at 37° C., 220 rpm with 0.1 mM IPTG for 6 h. Cells were harvested by centrifugation, washed in 100 mM pH 7 sodium phosphate buffer and then re-suspended in 1 mL of 100 mM HGN in 100 mM pH 7 sodium phosphate buffer. Reactions were allowed to proceed for at least 36 h at 22° C. with gentle agitation. Reaction progress was monitored by TLC (1:1 EtOAc:Hexanes, Rf=0.5, nitrile; Rf=0.0, acid). Cells and other debris were removed by centrifugation and the treated with one volume methanol prior to lyophilization. The lyophilizate was re-suspended in methanol and treated with TMS-diazomethane (10 equivalents, 2 M solution in hexanes) until gas evolution ceased and yellow color persisted in order to prepare the methyl ester for GC analysis. Selected nitrilase variants producing (R)-(−)-3-hydroxy-4-cyanobutyric acid of 95% ee or greater were then evaluated for performance at 2.25 M HGN. The overnight culture was diluted 2-fold induced at 37° C. washed in 100 mM pH 7 sodium phosphate buffer to proceed for at least 36 h at 22° C. with gentle agitation Reaction progress Cells and other debris were removed by centrifugation the treated with one volume methanol

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

Procedure: Bacterial cells were harvested by centrifugation and resuspended in SDS sample buffer (0.0625M Tris, pH 6.8, 10% glycerol, 5% mercaptoethanol, 2.3% SDS). The resuspended pellet was boiled for 5 min. and then cleared of insoluble cellular debris by centrifugation. The supernatants obtained from IPTG-induced cultures of GE3-2, GE9-2, GE15-1, GE17-2, GE4-8, EXP3-7, GE1-N and GE57 were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) together with uninduced lysates. The proteins from these gels were then transferred to nitrocellulose filters (i.e., by Western blotting).

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

Procedure: A plasmid (pHuCol-Fl, FIG. 6) encoding the gene sequence of the first 80 amino acids of human Type 1 (α1) collagen (FIG. 5 (Seq. ID No. 2) placed behind the isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible tac promotor and also encoding β-lactamase is transformed into Escherichia coli proline auxotrophic strain NM519 (pro-) by standard heat shock transformation. Transformation cultures are plated on Luria Broth (LB) containing 100 μg/ml ampicillin and after overnight growth a single ampicillin-resistant colony is used to inoculate 5 ml of LB containing 100 μg/ml ampicillin. After growth for 10-16 hours with shaking (225 rpm) at 37° C., this culture is used to inoculate 1 L of M9 minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 μg/ml ampicillin, supplemented with all amino acids at 20 μg/mL except proline which is supplemented at 12.5 mg/L) in a 1.5 L shaker flask. After growth at 37° C., 225 rpm, for 15-20 hours post-inoculation, the optical density at 600 nm is constant at approximately 0.5 OD/ml. The cells are harvested by centrifugation (5000 rpm, 5 minutes), the media decanted, and the cells resuspended in 1 L of M9 minimal media containing 100 μg/ml ampicillin, 0.08M L-hydroxyproline, and 0.5M NaC1. Following growth for 1 hour at 37° C., 225 rpm, IPTG is added to 1 mM and the cultures allowed to grow for an additional 5-15 hours. Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysed by mechanical disruption. The hydroxyproline-containing collagen fragment is purified by ammonium sulfate fractionation and column chromatography. After growth at 37° C., 225 rpm, for 15-20 hours post-inoculation, the optical density at 600 nm is constant at approximately 0.5 OD/ml are harvested by centrifugation (5000 rpm, 5 minutes) the media decanted containing 100 μg/ml ampicillin, 0.08M L-hydroxyproline, and 0.5M NaC1 for 1 hour at 37° C. 225 rpm, IPTG is added to 1 mM to grow for an additional 5-15 hours are harvested by centrifugation (5000 rpm, 10 minutes) The hydroxyproline-containing collagen fragment is purified by ammonium sulfate fractionation and column chromatography

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

Procedure: A plasmid (pHuCol, FIG. 2) encoding the gene sequence of human Type I (α1) collagen (FIGS. 3A and 3B (Seq. ID No. 1placed behind the isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible tac promotor and also encoding β-lactamase is transformed into Escherichia coli proline auxotrophic strain NM519 (pro-) by standard heat shock transformation. Transformation cultures are plated on Luria Broth (LB) containing 100 μg/ml ampicillin and after overnight growth a single ampicillin-resistant colony is used to inoculate 5 ml of LB containing 100 μg/ml ampicillin. After growth for 10-16 hours with shaking (225 rpm) at 37° C., this culture is used to inoculate 1 L of M9 minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 μg/ml ampicillin, supplemented with all amino acids at 20 μg/mL except proline which is supplemented at 12.5 mg/L) in a 1.5 L shaker flask. After growth at 37° C., 225 rpm, for 15-20 hours post-inoculation, the optical density at 600 nm is constant at approximately 0.5 OD/ml. The cells are harvested by centrifugation (5000 rpm, 5 minutes), the media decanted, and the cells resuspended in 1 L of M9 minimal media containing 100 μg/ml ampicillin, 0.08M L-hydroxyproline, and 0.5M NaCI. Following growth for 1 hour at 37° C., 225 rpm, IPTG is added to 1 mM and the cultures allowed to grow for an additional 5-15 hours. Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysed by mechanical disruption. Hydroxyproline-containing collagen is purified by ammonium sulfate fractionation and column chromatography. After growth at 37° C., 225 rpm, for 15-20 hours post-inoculation, the optical density at 600 nm is constant at approximately 0.5 OD/ml are harvested by centrifugation (5000 rpm, 5 minutes) the media decanted containing 100 μg/ml ampicillin, 0.08M L-hydroxyproline, and 0.5M NaCI for 1 hour at 37° C. 225 rpm, IPTG is added to 1 mM to grow for an additional 5-15 hours are harvested by centrifugation (5000 rpm, 10 minutes) Hydroxyproline-containing collagen is purified by ammonium sulfate fractionation and column chromatography

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

Procedure: To test the activity of the XR/Lad1/Alx1 operon, plasmid pATX112 was inserted into strain ZUC99 by electroporation (ZUC99/pATX112). The strain was inoculated from a single colony into 3 mL LB media supplemented with ampicillin (200 mg/L) and incubated overnight at 30° C. with shaking (250 RPM). After incubation, an aliquot of the culture was diluted 100-fold into 20 mL of fresh LB media contain L-arabinose (1% w/v) and ampicillin (200 mg/L) and incubated (30° C., 250 RPM) for 2 hrs. The culture was induced with isopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM). The culture was incubated at 30° C. with shaking (250 RPM) and aliquots were removed at various time points after the IPTG induction. The samples were monitored for xylitol formation from L-arabinose by HPLC analysis using an Aminex HPX-87P column (BioRad, USA). After 24 hrs post induction, the XR/Lad1/Alx1 operon strain (ZUC99/pATX112) displayed a 2% conversion of L-arabinose to xylitol, showing that the recombinant bacterium acquires the ability to produce xylitol from L-arabinose (Table 6). After incubation, an aliquot of the culture was diluted 100-fold into 20 mL of fresh LB media (30° C., 250 RPM) for 2 hrs was incubated at 30° C. with shaking (250 RPM) and aliquots were removed at various time points after the IPTG induction

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

Procedure: Bacterial cells were harvested by centrifugation and resuspended in SDS sample buffer (0.0625M Tris, pH 6.8, 10% glycerol, 5% mercaptoethanol, 2.3% SDS). The resuspended pellet was boiled for 5 min. and then cleared of insoluble cellular debris by centrifugation. The supernatants obtained from IPTG-induced cultures of GE3-2, GE9-2, GE15-1, GE17-2, GE4-8, EXP3-7, GE1-N and GE57 were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) together with uninduced lysates. The proteins from these gels were then transferred to nitrocellulose filters (i.e., by Western blotting).

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

Procedure: Bacterial cells were harvested by centrifugation and resuspended in SDS sample buffer (0.0625M Tris, pH 6.8, 10% glycerol, 5% mercaptoethanol, 2.3% SDS). The resuspended pellet was boiled for 5 min. and then cleared of insoluble cellular debris by centrifugation. The supernatants obtained from IPTG-induced cultures of GE3-2, GE9-2, GE15-1, GE17-2, GE4-8, EXP3-7, GE1-N and GE57 were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) together with uninduced lysates. The proteins from these gels were then transferred to nitrocellulose filters (i.e., by Western blotting).

Conditions: temp:28.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: BL21 Star™ (DE3) E. coli cells (Invitrogen) were co-transformed with the plasmids pACYCDuet-4506 and Ct94-pETDuet and transformed cells were selected on carbenicillin (50 μg/ml) chloramphenicol (34 μg/ml) LB-agarose plates. Single colonies were used to inoculate 5 mL LB medium with 50 μg/ml carbenicilin and 34 μg/ml chloramphenicol. The culture was incubated overnight at 37° C. The next day 2 mL of TB medium supplemented with the same antibiotics were inoculated with 0.2 mL of the overnight culture. After 6 hours incubation at 37° C., the culture was cooled down to 28° C. and 1 mM IPTG, 2 mg/mL mevalonate (prepared by dissolving mevalonolactone (Sigma) in 0.5N NaOH at a concentration of 1 g/mL and incubating the solution for 30 minutes at 37° C.) and 0.2 mL decane were added to each tube. The cultures were incubated for 48 hours at 28° C. The cultures were then extracted twice with 2 volumes of ethyl acetate, the organic phase was concentrated to 500 μL and analyzed by GC-MS as described above in Example 3. In these conditions sesquiterpene production above 200 mg/L was routinely achieved. Beta-santalene was produced. were inoculated with 0.2 mL of the overnight culture After 6 hours incubation at 37° C. for 30 minutes at 37° C. were added to each tube The cultures were incubated for 48 hours at 28° C The cultures were then extracted twice with 2 volumes of ethyl acetate the organic phase was concentrated to 500 μL

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

Procedure: The coding sequence of the VHH was subcloned in vector pET22 using the NcoI and NotI restriction sites according to the manufacturer's instructions (Novagen, Darmstadt, Germany). Transformed E. coli BL 21 (DE3) cells expressed VHHs in the periplasm after induction by IPTG 1 mM for 18 hours at 15° C. Periplasmic extracts were obtained by spheroplasting cells, suspended in 50 mM sodium phosphate buffer pH 8 containing 20% sucrose and 1 mM EDTA, and hydrolysing the peptidoglycan with 5 mg/ml lysozyme for 20 min at 4° C., in the presence of protease inhibitors (Complete™, Boehringer Mannheim, Germany). The suspension was then centrifuged 2 min at 10,000 rpm. The supernatant corresponding to the periplasmic extract was kept at 4° C. Purified VHHs were obtained by IMAC using a chelating agarose column charged with Ni2+ (Superflow Ni-NTA, Qiagen Ltd, UK) according to manufacturer's instructions. Purified VHH were dialysed against PBS and the protein content was measured using the Bradford reagent. The purity of the final preparation was evaluated by SDS-PAGE with Coomassie staining and by Western blot. for 18 hours at 15° C Periplasmic extracts were obtained The suspension was then centrifuged 2 min at 10,000 rpm The supernatant corresponding to the periplasmic extract was kept at 4° C Purified VHHs

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

Procedure: A plasmid (pHuCol-Fl, FIG. 6) encoding the gene sequence of the first 80 amino acids of human Type 1 (α1) collagen (FIG. 5) (SEQ. ID. NO. 2) placed behind the isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible tac promotor and also encoding β-lactamase is transformed into Escherichia coli proline auxotrophic strain NM519 (pro−) by standard heat shock transformation. Transformation cultures are plated on Luria Broth (LB) containing 100 μg/mL ampicillin and after overnight growth a single ampicillin-resistant colony is used to inoculate 5 mL of LB containing 100 μg/mL ampicillin. After growth for 10-16 hours with shaking (225 rpm) at 37° C., this culture is used to inoculate 1 L of M9 minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 μg/mL ampicillin, supplemented with all amino acids at 20 μg/mL except proline which is supplemented at 12.5 mg/L) in a 1.5 L shaker flask. After growth at 37° C., 225 rpm, for 15-20 hours post-inoculation, the optical density at 600 nm is constant at approximately 0.5 OD/mL. The cells are harvested by centrifugation (5000 rpm, 5 minutes), the media decanted, and the cells resuspended in 1 L of M9 minimal media containing 100 μg/mL ampicillin, 0.08M L-hydroxyproline, and 0.5M NaCl. Following growth for 1 hour at 37° C., 225 rpm, IPTG is added to 1 mM and the cultures allowed to grow for an additional 5-15 hours. Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysed by mechanical disruption. The hydroxyproline-containing collagen fragment is purified by ammonium sulfate fractionation and column chromatography. After growth at 37° C., 225 rpm, for 15-20 hours post-inoculation, the optical density at 600 nm is constant at approximately 0.5 OD/mL are harvested by centrifugation (5000 rpm, 5 minutes) the media decanted containing 100 μg/mL ampicillin, 0.08M L-hydroxyproline, and 0.5M NaCl for 1 hour at 37° C. 225 rpm, IPTG is added to 1 mM to grow for an additional 5-15 hours are harvested by centrifugation (5000 rpm, 10 minutes) The hydroxyproline-containing collagen fragment is purified by ammonium sulfate fractionation and column chromatography

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

Procedure: A plasmid (pHuCol, FIG. 4) encoding the gene sequence of human Type I (α1) collagen (FIGS. 3A and 3B) (SEQ. ID. NO. 1) placed behind the isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible tac promotor and also encoding β-lactamase is transformed into Escherichia coli proline auxotrophic strain NM519 (pro−) by standard heat shock transformation. Transformation cultures are plated on Luria Broth (LB) containing 100 μg/ml ampicillin and after overnight growth a single ampicillin-resistant colony is used to inoculate 5 ml of LB containing 100 μg/ml ampicillin. After growth for 10-16 hours with shaking (225 rpm) at 37° C., this culture is used to inoculate 1 L of M9 minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 μg/mL ampicillin, supplemented with all amino acids at 20 μg/mL except proline which is supplemented at 12.5 mg/L) in a 1.5 L shaker flask. After growth at 37° C., 225 rpm, for 15-20 hours post-inoculation, the optical density at 600 nm is constant at approximately 0.5 OD/mL. The cells are harvested by centrifugation (5000 rpm, 5 minutes), the media decanted, and the cells resuspended in 1 L of M9 minimal media containing 100 μg/ml ampicillin, 0.08M L-hydroxyproline, and 0.5M NaCl. Following growth for 1 hour at 37° C., 225 rpm, IPTG is added to 1 mM and the cultures allowed to grow for an additional 5-15 hours. Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysed by mechanical disruption. Hydroxyproline-containing collagen is purified by ammonium sulfate fractionation and column chromatography. After growth at 37° C., 225 rpm, for 15-20 hours post-inoculation, the optical density at 600 nm is constant at approximately 0.5 OD/mL are harvested by centrifugation (5000 rpm, 5 minutes) the media decanted containing 100 μg/ml ampicillin, 0.08M L-hydroxyproline, and 0.5M NaCl for 1 hour at 37° C. 225 rpm, IPTG is added to 1 mM to grow for an additional 5-15 hours are harvested by centrifugation (5000 rpm, 10 minutes) Hydroxyproline-containing collagen is purified by ammonium sulfate fractionation and column chromatography