Effect of F209S Mutation
of Escherichia coli AroG on Resistance to
Phenylalanine Feedback Inhibition
JIANG Pei-Hong, SHI Mi, QIAN Zhi-Kang, LI Ning-Jun, HUANG
Wei-Da*
( Department of Biochemistry, School of Life Science, Fudan University,
Shanghai 200433, China )
Abstract In Escherichia
coli, 80% of the 3-deoxy-D-arabino-heptulosonate 7-phosphate(DAHP)
synthase was encoded by aroG gene. The aroG gene was
amplified by polymerase chain reaction(PCR) from strain K-12 and a mutant
strain resistant to phenylalanine analogues. The PCR products were cloned and
subject to DNA sequence analysis. A single base mutation of T→C was detected at
nucleotide 625, which causes a substitution of Phe(209) by Ser in the gene
product. The gene was expressed on pTrc99A in E.coli strain JM105.
Under the induction of IPTG, distinct band with the expected molecule weight
was detected on SDS-polyacrylamide gel electrophoresis and the specific activity
of DAHP of the crude extract of the transformed cells increased by 1.8-fold.
Enzyme activity inhibition analysis revealed the high resistance of mutant AroG
to feedback inhibition by phenylalanine. JM105 cells harboring with mutant aroG
gene showed were able to grow on medium containing higher concentration of
analogues than that carrying normal aroG gene. Discussion was focused
on the varieties of mutations contributing to desensitization of feedback
inhibition.
Key words aroG gene； phenylalanine analogues； point mutation； resistance
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