Phenylalanine production by metabolically engineered Corynebacterium glutamicum with the pheA gene of Escherichia coli

Abstract

The bifunctional enzyme chorismate mutase (CM)-prephenate dehydratase (PD), which is encoded by the pheA gene of Escherichia coli, catalyses the two consecutive key steps in phenylalanine biosynthesis. To utilize the enzyme for metabolic engineering of phenylalanine-producing Corynebacterium glutamicum KY10694, the intact gene was cloned on a multicopy vector to yield pEA11.C. glutamicum cells transformed with pEA11 exhibited a more than tenfold increase in CM and PD activities relative to the host cells. Moreover, the level of pheA expression was further elevated a fewfold when cells were starved of phenylalanine, suggesting that the attenuation regulation of pheA expression functions in heterogeneous C. glutamicum. Plasmid pEA11 encoding the wild-type enzyme was mutated to yield pEA22, which specified CM-PD exhibiting almost complete resistance to end-product inhibition. When pEA22 was introduced into KY10694, both the activities of CM and PD were highly maintained throughout the cultivation, thus leading to a 35% increased production (23 g/l) of phenylalanine.

Towards bacterial strains overproducing l-tryptophan and other aromatics by metabolic engineering

“…For the L-phenylalanine production processes, it is desirable to minimize by-production of L-tyrosine to a level at which supplementary purification of L-phenylalanine is not necessary. In this sense, the pheA expression has an additional merit because of its effect on diminished by-production of L-tyrosine (Ikeda et al 1993). Metabolic conversion from L-tryptophan production to L-phenylalanine production by amplifying the genes for the deregulated branch-point enzymes, that is, aro II (NCgl2098), csm (NCgl0819), and pheA (NCgl2799), was also reported (Fig.…”

“…Likewise, large increases in L-phenylalanine production have been achieved by amplifying the deregulated enzyme(s) in existing C. glutamicum producers (Ozaki et al 1985;Ikeda et al 1993;Ito et al 1900bIto et al , 1990c. Primary targets for engineering have been the three key enzymes DS, CM, and PD (Fig.…”

“…1). While most cases have used endogenous enzymes from the organisms themselves, the desired metabolic engineering could be attained by the use of enzymes from heterologous organisms, as exemplified by a study with a recombinant C. glutamicum strain overexpressing the E. coli pheA gene that encodes the bifunctional key enzymes CM and PD (Ikeda et al 1993). For the L-phenylalanine production processes, it is desirable to minimize by-production of L-tyrosine to a level at which supplementary purification of L-phenylalanine is not necessary.…”

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

“…Due to extended research, complex and efficient [41,42]. A similar complex picture is yielded for biosynthesis of the aromatic amino acids, where enzyme inhibition by feedback regulation and negative transcriptional control are involved [6,43].…”

“…Effective approaches comprised the modification of terminal pathways leading to removal of undesired control mechanisms, engineering of the PPP for increased supply of precursors, and transport engineering leading to reduced intracellular pools. Among the most striking findings was the overexpression of the tkt gene in the PPP encoding transketolase which strongly enhanced tryptophan production by improved supply of the precursor E4P [154,155]. To avoid accumulation of indole, the last intermediate in the tryptophan pathway, due 40 C. Wittmann to a limiting supply of L-serine, the availability of L-serine was improved by amplification of the serA gene for 3-phosphoglycerate dehydrogenase, the key enzyme in the L-serine biosynthesis [156].…”