Vera Georgievna Doroshenko

Pho regulon promoter-mediated transcription of the key pathway gene aroGFbr improves the performance of an l-phenylalanine-producing Escherichia coli strain

DAHP synthase (EC 4.1.2.15) is one of the key enzymes involved in aromatic amino acid biosynthesis in Escherichia coli. An approximately twofold decrease in DAHP synthase activity level was detected in the late growth phase of the l-phenylalanine (Phe)-producing E. coli strain, in which this enzyme encoded by aroG4 is resistant to feedback inhibition. An additional copy of aroG4 that is controlled by promoters of E. coli phoA or pstS genes was integrated into the chromosome of the Phe producer. The choice of promoter was based on the detected activation of the Pho regulon that occurs in response to the depletion of soluble inorganic orthophosphate (Pi) in the medium, provided that the optical density of the Phe-producing culture did not exceed 70% of its maximum value. Pho-mediated aroG4 transcription increased both the accumulation of Phe and the level of DAHP synthase activity in the late stage of batch cultivation on glucose in Pi-limited conditions. Disruption of rpoS led to the improved performance of a PphoA-aroG4 strain. The pstS promoter that is recognized by the σ70/σS-associated core RNA polymerase resulted in the stable maintenance of DAHP synthase activity during long-drawn fed-batch cultivation of the RpoS+ strain carrying the PpstS-aroG4.

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 ΔtyrA. 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 TyrAwt strain.

Membrane Topology Analysis of the Escherichia coli Aromatic Amino Acid Efflux Protein YddG

YddG is an inner membrane protein (IMP) that exports aromatic amino acids in Escherichia coli. Topology models of YddG produced by sequence-based analysis in silico have predicted the presence of 9 or 10 potential transmembrane segments. To experimentally analyze the membrane topology of YddG, we used randomly created fusions to β-lactamase (BlaM) as a reporter. The selection of such fusions under 50 µg/ml of ampicillin had to fit with the periplasmic location of the BlaM domain. Five periplasmic loops of YddG predicted by the 10-transmembrane (TM) helices model were identified via the characterization of 12 unique in-frame fusions distributed along the yddG coding region. To confirm the 10-TM helices model further, cytoplasmic regions of YddG were identified with the help of ZsGreen fluorescent protein as a reporter. The presence of four cytoplasmic regions and the cytoplasmic localization of the C-terminus were revealed. Therefore, a 10-TM helices topology with cytoplasmic locations of the N- and C-termini is supported. The present data confirm the ‘positive-inside rule’ for IMPs and the early results of other workers regarding the cytoplasmic location of the C-terminus of YddG. The pole-specific localization of YddG-ZsGreen in E. coli cells was detected by fluorescence microscopy.

Metabolic engineering of Escherichia coli for the production of phenylalanine and related compounds

In this review, the metabolic engineering approaches including those used by the authors in creating phenylalanine producers based on Escherichia coli were systematized. Optimization of the amino acid biosynthesis was conducted in order to obtain significant quantities of phenylalanine and to ensure the availability of its direct precursors in cell metabolism—erythrose 4-phosphate and phosphoenolpyruvic acid. The possibility of altering global regulation mechanisms was investigated for a full reorientation of the metabolism to phenylalanine synthesis with the use of Csr (carbon storage regulator). The identification of the aromatic amino acids exporter YddG is associated with the use of the phenylalanine producer as a test-system. Novel approaches to phenylalanine producer construction (use of the “leaky” allele tyrA—ssrA, promoters of the phosphate regulon), as well as new methods of obtaining producers of similar amino acid tyrosine, were discussed. Examples of the synthesis of useful aromatic compounds from phenylalanine or its precursor, phenylpyruvic acid, with E. coli as the ecipient for foreign gene expression were examined.

2D [1H,13C] NMR study of carbon fluxes during glucose utilization by Escherichia coli MG1655

Carbon fluxes through main pathways of glucose utilization in Escherichia coli cells-glycolysis, pentose phosphate pathway (PPP), and Enther-Doudoroff pathway (EDP)—were studied. Their ratios were analyzed in E. coli strains MG1655, MG1655Δ(edd-eda), MG1655Δ(zwf, edd-eda), and MG1655Δ(pgi, edd-eda). It was shown that the carbon flux through glycolysis was the main route of glucose utilization, averaging ca. 80%. Inactivation of EDP did not affect growth parameters. Nevertheless, it altered carbon fluxes through the tricarboxylic acid cycles and energy metabolism in the cell. Inactivation of PPP decreased growth rate to a lesser degree than glycolysis inactivation.

Contents Vol. 19, 2010

F. Barras, Marseille D.H. Bartlett, San Diego, Calif. P. Beguin, Paris I. Booth, Aberdeen L.J. Brady, Gainesville, Fla. Y.J. Chung, Jeonju P. Courvalin, Paris C.J. Dorman, Dublin J.S. Edwards, Albuquerque, N. Mex. M.Y. Galperin, Bethesda, Md. A. Goff eau, Louvain-la-Neuve C.F. Gonzalez, Gainesville, Fla. M. Gribskov, West Lafayette, Ind. G.F. Hatfull, Pittsburgh, Pa. P.J.F. Henderson, Leeds S. Horinouchi, Tokyo G. Klug, Giessen G.L. Lorca, Gainesville, Fla. J. Lutkenhaus, Kansas City, Kans. W. Qin, Th under Bay, Ont. M. Schaechter, San Diego, Calif. K.C. Schuster, Lenzing R. Skurray, Sydney F. Titgemeyer, Münster R. Vazquez-Duhalt, Cuernavaca H. Wolf-Watz, Umea X. Zhou, Pullman, Wash. Vol. 19, 2010