Metabolic Engineering of Escherichia coli for l -Tyrosine Production by Expression of Genes Coding for the Chorismate Mutase Domain of the Native Chorismate Mutase-Prephenate Dehydratase and a Cyclohexadienyl Dehydrogenase from Zymomonas mobilis

Abstract

The expression of the feedback inhibition-insensitive enzyme cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase domain from native chorismate mutase-prephenate dehydratase (PheA CM ) from Escherichia coli was compared to the expression of native feedback inhibition-sensitive chorismate mutase-prephenate dehydrogenase (CM-TyrA p ) with regard to the capacity to produce L-tyrosine in E. coli strains modified to increase the carbon flow to chorismate. Shake flask experiments showed that TyrC increased the yield of L-tyrosine from glucose (Y L-Tyr/Glc ) by 6.8-fold compared to the yield obtained with CM-TyrA p . In bioreactor experiments, a strain expressing both TyrC and PheA CM produced 3 g/liter of L-tyrosine with a Y L-Tyr/Glc of 66 mg/g. These values are 46 and 48% higher than the values for a strain expressing only TyrC. The results show that the feedback inhibition-insensitive enzymes can be employed for strain development as part of a metabolic engineering strategy for L-tyrosine production.

Genetic and biochemical identification of the chorismate mutase from Corynebacterium glutamicum

“…Chorismate mutases (CMs, EC 5.4.99.5) are of interest for bioindustry and medicine, because the enzyme is the target to improve production of aromatic amino acids such as Lphenylalanine (Phe) and L-tyrosine (Tyr) (Chavez-Bejar et al, 2008;Yakandawala et al, 2008) as well as being a putative new drug target against deadly diseases such as tuberculosis (Qamra et al, 2006;Schneider et al, 2008). CMs catalyse the conversion of chorismate to prephenate, a reaction also known as the Claisen rearrangement, and the only naturally occurring Claisen reaction in the primary metabolism of living organisms (Krappmann et al, 1999;Sugimoto & Shiio, 1980a).…”

Computational identification of altered metabolism using gene expression and metabolic pathways

“…Understanding altered metabolism is an important issue because altered metabolism is often revealed as a cause or an effect in pathogenesis (Dakubo et al, 2006;Eapen, 2007;Lohmander, 2004;Picton, 1998), and it is also a crucial factor when manipulating an organism's metabolism in metabolic engineering (Chavez-Bejar et al, 2008;Covert et al, 2004;Keasling and Chou, 2008;Kim et al, 2008). With the development of high-throughput technologies, deeper understanding of biological processes has arisen through the integration of data from genomics, proteomics, and metabolomics.…”

Engineering

“…Introduction of plasmid-encoded copies of aroF fbr and aroG fbr combined with additional plasmid-cloned gene tktA, or their chromosomal integrations in gene clusters, have resulted in increased carbon flow from the CCM to the SHK pathway for the production of L-PHE [11,55,56], L-TYR [5,57,58] and L-TRP [59][60][61]. Positive results were also obtained with the insertion of an aroG fbr gene into the chromosome of an L-PHE producing strain while being controlled by a promoter that is active during late cultivation stages, in order to counteract the fall of DAHPS activity in stationary phase [62].…”

“…Feedback-resistant mutants of TyrA and PheA E. coli enzymes have been used for the efficient overproduction of L-TYR [11,17,57,67] and L-PHE [55,76] in combination with some of the previously described alterations in CCM and the SHK pathway (Table 1). An alternative approach to take advantage of the natural feedback-resistant diversity in the TyrA enzyme family was the expression of the TyrC fbr enzyme (cyclohexadienyl dehydrogenase) from Z. mobilis and the CHA mutase domain of native PheA from E. coli, relieving rate-limiting steps and increasing the carbon flux towards L-TYR [57].…”