Reaktion #794928

Reaktionsgleichung

spectinomycin

IPTG

Glucose

uracil

→

heptadecadiene

Lösungsmittel

methanol

Reaktionsbedingungen

Aufarbeitung

1. 1
   Sonstigewere cultured at 37° C. in an M9 mineral medium
2. 2
   workup.ADDITIONwas added
3. 3
   Sonstigewas shifted to 25° C
4. 4
   Sonstigesonicated for 30 minutes
5. 5
   Extraktionextracted with 4 mL hexane
6. 6
   SonstigeAfter solvent evaporation, samples

Vorschrift

The genomic DNA encoding ATCC 8456_orf880, which was codon-optimized for expression in E. coli, was cloned into vector OP80 (pCL1920 derivative) under the control of a Ptrc promoter, and E. coli MG1655 ΔfadD was transformed with the resulting vector. The E. coli cells were cultured at 37° C. in an M9 mineral medium supplemented with 20 μg/mL uracil and 100 μg/mL spectinomycin. Glucose (1%, w/v) was the only source of carbon and energy. When the culture reached an OD600 of 0.8 to 1.0, IPTG (1 mM) was added and the temperature was shifted to 25° C. After growth for an additional 18 to 24 hours at 25° C., cells from 10 mL of culture were pelleted, resuspended in 1 mL methanol, sonicated for 30 minutes, and extracted with 4 mL hexane. After solvent evaporation, samples were resuspended in 0.1 mL hexane and analyzed by GC-MS. In contrast to the vector-only control, E. coli cells transformed with the orf880-bearing vector produced the α-olefins 1-pentadecene and heptadecadiene. This result indicates that expression of ORF880 confers the ability to biosynthesize α-olefins to E. coli when cultured on glucose, and that the direct precursors are the most abundant fatty acids in E. coli, namely hexadecanoic acid and vaccenic acid (11-cis-octadecenoic acid).

Quelle