Sarcosine sensitivity in Escherichia coli is mediated by activation of the glycine cleavage system.

Corynebacterium glutamicum AJ1511 and Escherichia coli BW25113 strains were compared in terms of resistance to sarcosine (N-methylglycine). The E. coli strain was more sensitive to sarcosine than C. glutamicum, especially when grown in minimal medium. Growth inhibition of the BW25113 strain in minimal M9 medium containing 0.5 m sarcosine was overcome by the addition of glycine. Inactivation of the glycine cleavage (GCV) system (∆gcvP) as well as the removal of its activator (∆gcvA) in BW25113 cells increased the threshold for sarcosine inhibition up to 0.75 m. Activation of the promoter of the E. coli gcvTHP operon by 0.1-0.4 m sarcosine added to M9 medium was demonstrated in vivo using dasherGFP as the reporter. Sensitivity to sarcosine on glucose minimal medium is suggested to be a characteristic of Gram-negative bacteria with GcvA/GcvR regulation of the GCV system.

Metabolic engineering of Escherichia coli to produce (2S, 3R, 4S)-4-hydroxyisoleucine.

The stereo-specific L-isoleucine-4-hydroxylase (L-isoleucine dioxygenase (IDO)) was cloned and expressed in an Escherichia coli 2Δ strain lacking the activities of α-ketoglutarate dehydrogenase (EC 1.2.4.2), isocitrate liase (EC 4.1.3.1), and isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5). The 2Δ strain could not grow in a minimal-salt/glucose/glycerol medium due to the blockage of TCA during succinate synthesis. The IDO activity in the 2Δ strain was able to "shunt" destroyed TCA, thereby coupling L-isoleucine hydroxylation and cell growth. Using this strain, we performed the direct biotransformation of L-isoleucine into 4-HIL with an 82% yield.

Construction of an L-phenylalanine-producing tyrosine-prototrophic Escherichia coli strain using tyrA ssrA-like tagged alleles.

To construct a Phe-producing Tyr(+) Escherichia coli strain, TyrA (chorismate mutase/prephenate dehydrogenase) activity was varied by engineering a proteolytically unstable protein. The tyrA in the E. coli BW25113 was altered to include ssrA-like tags. The tagged tyrA genes, which ensured different growth rates in M9 medium, were introduced into a Phe-producing strain to replace DeltatyrA. Strains with unstable TyrA-(A)ANDENYALAA proteins had a lower biomass yield and a higher Phe accumulation than strains generating the more stable TyrA-(A)ANDENYALDD. The Tyr/Phe ratio produced by the TyrA-tag strains was 10-fold less than that produced by the TyrA(wt) strain.