Effect of transketolase modifications on carbon flow to the purine-nucleotide pathway in Corynebacterium ammoniagenes

Abstract

Transketolase, one of the enzymes in the nonoxidative branch of the pentose phosphate pathway, operates to shuttle ribose 5-phosphate and glycolytic intermediates together with transaldolase, and might be involved in the availability of ribose 5-phosphate, a precursor of nucleotide biosynthesis. The tkt and tal genes encoding transketolase and transaldolase, respectively, were cloned from the typical nucleotide- and nucleoside-producing organism Corynebacterium ammoniagenes by a PCR approach using oligonucleotide primers derived from conserved regions of each amino acid sequence from other organisms. Enzymatic and molecular analyses revealed that the two genes were clustered on the genome together with the glucose 6-phosphate dehydrogenase gene (zwf). The effect of transketolase modifications on the production of inosine and 5'-xanthylic acid was investigated in industrial strains of C. ammoniagenes. Multiple copies of plasmid-borne tkt caused about tenfold increases in transketolase activity and resulted in 10-20% decreased yields of products relative to the parents. In contrast, site-specific disruption of tkt enabled both producers to accumulate 10-30% more products concurrently with a complete loss of transketolase activity and the expected phenotype of shikimate auxotrophy. These results indicate that transketolase normally shunts ribose 5-phosphate back into glycolysis in these biosynthetic processes and interception of this shunt allows cells to redirect carbon flux through the oxidative pentose pathway from the intermediate towards the purine-nucleotide pathway.

Amplified Expression of Fructose 1,6-Bisphosphatase in Corynebacterium glutamicum Increases In Vivo Flux through the Pentose Phosphate Pathway and Lysine Production on Different Carbon Sources

“…Comparable results were previously obtained for the wild-type strain C. glutamicum ATCC 13032 grown on glucose, gluconate, ribose, citrate, pyruvate, or lactate (23). Due to a strong metabolic regulation, this enzyme is supposed to exhibit negligible in vivo activity during growth on sugars (10,32). The metabolic regulation of FBPase was also observed in mutants with an overexpressed fbp gene, since the expression level, but not the properties, of the enzyme were changed.…”

“…In this regard, the redirection of flux toward the PPP resulting from overexpression of fructose 1,6-bisphosphatase should also be of interest for the production of other NADPH-demanding compounds, such as methionine (14), isoleucine (18), and fatty acids (6), as well as for products directly stemming from the PPP, such as vitamin B 2 (25), nucleotides (1,10), and aromatic amino acids (7,8). Due to negative metabolic regulation of fructose 1,6-bisphosphatase, only a certain fraction of the enzyme is probably active in vivo, so that high expression levels are required to achieve significant metabolic effects.…”

“…The strain contains a feedback-resistant aspartokinase. Fluxes were estimated from the best fit to the experimental results using a comprehensive approach of combined metabolite balancing and isotopomer modeling for a 13 C tracer experiment during growth on [1][2][3][4][5][6][7][8][9][10][11][12][13] C]glucose and measurement of labeling of amino acids from the cell protein and of trehalose from the culture supernatant by GC-MS. Net fluxes are given in square symbols; for reversible reactions the direction of the net flux is indicated by an arrow beside the corresponding black box. Numbers in brackets below the fluxes of transaldolase, transketolase, and glucose 6-phosphate isomerase indicate flux reversibility.…”

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

“…Reviewing the strategies, it is found that increasing the availability of PEP and E4P has attracted much attention as a common strategy in metabolic engineering for all aromatic production, leading to several different approaches. However, in accessing the central metabolism, we should note that previous attempts to drastically redirect carbon flux into a desired pathway resulted in growth impairment and/or formation of unwanted by-products (Miller et al 1987;Mascarenhas et al 1991;Kamada et al 2001;Marx et al 2003). The junction between glycolysis and TCA cycle, in particular, must be given special attention because the junction consists of several enzyme reactions carrying fluxes that connect PEP with oxaloacetate or pyruvate, pyruvate with oxaloacetate, and pyruvate with malate, and are thus important for sharing metabolic fluxes with not only aromatic biosynthesis but also various biosynthetic pathways.…”

Effects ofeddandpgiDisruptions on Inosine Accumulation inEscherichia coli

“…26,27) According to them, the non-oxidative route alone cannot provide sufficient ribose-5-phosphate for higher purine nucleotide production in C. ammoniagenes. Similarly to our result, encouragement of the oxidative pentose phosphate pathway was more effective for inosine production in E. coli.…”