Methods, reagents and cells for biosynthesizing compounds
An active, hydroxyalkyl technology, applied in the field of recombinant host cells and recombinant hosts
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Embodiment 1
[0368] Enzymatic activity of omega-transaminase using glutaric semialdehyde as a substrate and forming 5-aminovaleric acid
[0369] Nucleotide sequences encoding N-terminal His-tags were added to the genes from Chromobacterium violaceum and Rhodococcus sphaeroides encoding ω-transaminases of SEQ ID NOs: 8 and 10, respectively (see Figure 10 ), allowing the production of N-terminal HIS-tagged omega-aminotransferases. Each resulting modified gene was cloned into the pET21a expression vector under the control of the T7 promoter, and each expression vector was transformed into a BL21[DE3] E. coli host. The resulting recombinant E. coli strains were grown in 250 mL shake flask cultures containing 50 mL LB medium and antibiotic selection pressure at 37°C with shaking at 230 rpm. Each culture was induced overnight at 16°C with 1 mM IPTG.
[0370] Pellets from each induced shake flask culture were harvested by centrifugation. Each pellet was resuspended and lysed by sonication. C...
Embodiment 2
[0376] Enzymatic activity of carboxylic acid reductase using 5-hydroxyvaleric acid as a substrate and forming 5-hydroxyvaleraldehyde
[0377] Nucleotide sequences encoding His-tags were added to M. marinum, M. smegmatis, M. smegmatis, Segniliparus from M. marinum, M. smegmatis, M. smegmatis, Segniliparus encoding carboxylic acid reductases of SEQ ID NOs: 2-4, 6 and 7, respectively Genes of rugosus, M. marseii, and Segniliparus rotundus (GenBank accession numbers ACC40567.1, ABK71854.1, EFV11917.1, EIV11143.1, and ADG98140.1, respectively) (see Figure 10 ), allowing the production of N-terminal HIS-tagged carboxylate reductases. Each modified gene was cloned into the pET Duet expression vector, both under the control of the T7 promoter, along with the sfp gene encoding the His-tagged phosphopantethelin transferase from Bacillus subtilis. Each expression vector was transformed into a BL21[DE3] E. coli host. Each resulting recombinant E. coli strain was grown in a 250 mL shake...
Embodiment 3
[0382] Omega-transaminase activity for 5-aminopentanol to form 5-oxopentanol
[0383] Nucleotide sequences encoding N-terminal His-tags were added to Chromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae, Rhodobacter sphaeroides, Escherichia coli encoding ω-transaminases of SEQ ID NOs: 8-13, respectively Bacillus and Vibrio fluvius genes (see Figure 10 ), allowing the production of N-terminal HIS-tagged omega-aminotransferases. The modified gene was cloned into the pET21a expression vector under the T7 promoter. Each expression vector was transformed into a BL21[DE3] E. coli host. Each resulting recombinant E. coli strain was grown in a 250 mL shake flask containing 50 mL of LB medium and antibiotic selection pressure at 37°C with shaking at 230 rpm. Each culture was induced overnight at 16°C with 1 mM IPTG.
[0384] Pellets from each induced shake flask culture were harvested by centrifugation. Each pellet was resuspended and lysed by sonication. Cell...
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