Metabolic flux engineering of l-lysine production in Corynebacterium glutamicum—over expression and modification of G6P dehydrogenase

Becker, Jörg; Klopprogge, Corinna; Herold, Andrea; Zelder, Oskar; Bolten, Christoph J.; Wittmann, Christoph

doi:10.1016/j.jbiotec.2007.05.026

Cited by 162 publications

(99 citation statements)

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“…It has been reported in this organism that NADPH is generated mainly through the oxidative pentose phosphate pathway (PPP) (Marx et al, 1996). Several reports have shown that redirection of carbon from glycolysis toward the PPP leads to a significant improvement in L-lysine production (Becker et al, 2011(Becker et al, , 2007(Becker et al, , 2005; Marx et al, 2003;Ohnishi et al, 2005). Despite this positive effect, relying on the PPP is disadvantageous in terms of carbon yield, because the pathway inevitably involves the release of 1 mol of carbon dioxide accompanied by the oxidation of 1 mol of hexose (Contador et al, 2009).…”

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confidence: 99%

l -Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

Takeno

Hori

Ohtani

et al. 2016

Metabolic Engineering

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We have recently developed a Corynebacterium glutamicum strain that generates NADPH via the glycolytic pathway by replacing endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GapA) with a nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans. Strain RE2, a suppressor mutant spontaneously isolated for its improved growth on glucose from the engineered strain, was proven to be a high-potential host for L-lysine production (Takeno et al., 2010). In this study, the suppressor mutation was identified to be a point mutation in rho encoding the transcription termination factor Rho. Strain RE2 still showed retarded growth despite the mutation rho696. Our strategy for reconciling improved growth with a high level of L-lysine production was to use GapA together with GapN only in the early growth phase, and subsequently shift this combination-type glycolysis to one that depends only on GapN in the rest of the growth phase. To achieve this, we expressed gapA under the 2 myo-inositol-inducible promoter of iolT1 encoding a myo-inositol transporter in strain RE2. The resulting strain RE2A iol was engineered into an L-lysine producer by introduction of a plasmid carrying the desensitized lysC, followed by examination for culture conditions with myo-inositol supplementation. We found that as a higher concentration of myo-inositol was added to the seed culture, the following fermentation period became shorter while maintaining a high level of L-lysine production. This finally reached a fermentation period comparable to that of the control GapA strain, and yielded a 1.5-fold higher production rate compared with strain RE2. The transcript level of gapA, as well as the GapA activity, in the early growth phase increased in proportion to the myo-inositol concentration and then fell to low levels in the subsequent growth phase, indicating that improved growth was a result of increased GapA activity, especially in the early growth phase. Moreover, blockade of the pentose phosphate pathway through a defect in glucose 6-phosphate dehydrogenase did not significantly affect L-lysine production in the engineered GapN strains, while a drastic decrease in L-lysine production was observed for the control GapA strain. Determination of the intracellular NADPH/NADP + ratios revealed that the ratios in the engineered strains were significantly higher than the ratio of the control GapA strain irrespective of the pentose phosphate pathway. These results demonstrate that our strain engineering strategy allows efficient L-lysine production independent of the oxidative pentose phosphate pathway.

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confidence: 99%

l -Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

Takeno

Hori

Ohtani

et al. 2016

Metabolic Engineering

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“…In C. glutamicum, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate 13 C flux analysis: [112] (white circles), [125] (gray circles), [126] (black circles), [127] (white squares), [111] (gray squares), [111] (black squares), [128] (white triangles), [116] (gray triangles), [26] (black triangles), [129] (white diamonds), [115] (gray diamonds), [120] (black diamonds), [76] (white hexagons), and [118] (gray hexagons). The flux reversibility at the pyruvate node is defined as the ratio of the back flux to the anaplerotic net flux [130] 36 C. Wittmann dehydrogenase, and malic enzyme catalyze NADPH generating reactions, whereas NADPH consuming reactions comprise growth with a stoichiometric demand of 16.4 mmol NADPH (g biomass) À1 [34] and overproduction.…”

Section: Metabolic Fluxes Of Nadph Metabolismmentioning

confidence: 99%

“…Lysine production flux (%) Fig. 6 Metabolic flux analysis linking lysine production with NADPH metabolism involving the pentose phosphate pathway (a) and isocitrate dehydrogenase (b) in different C. glutamicum strains investigated under various cultivation conditions by 13 C flux analysis: [125] (white circles), [129] (gray circles), [126] (black circles), [127] (white squares), [112] (gray squares), [126] (black squares), [128] (white triangles), [116] (gray triangles), [26,76] (black triangles), [115] (white diamonds), and [120] (gray diamonds)…”

Section: Metabolic Fluxes Of Nadph Metabolismmentioning

confidence: 99%

“…A second major target approached was glucose 6-phosphate dehydrogenase. Overexpression of the encoding zwf gene in the feedback-deregulated lysine producing strain C. glutamicum ATCC13032 lysC fbr resulted in increased lysine production on different carbon sources including the two major industrial sugars, glucose and sucrose [116]. In further successful examples, an increased PPP flux was achieved by modifying the regulatory properties of enzymes in the PPP, i.e., partially releasing glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from allosteric inhibition.…”

Section: Metabolic Engineering Of Nadph Supplymentioning

confidence: 99%

“…In further successful examples, an increased PPP flux was achieved by modifying the regulatory properties of enzymes in the PPP, i.e., partially releasing glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from allosteric inhibition. The substitution A243T in the zwf gene encoding for glucose 6-phosphate dehydrogenase [116,153] and the substitution S361F in the gnd gene encoding for 6-phosphogluconate dehydrogenase [30] both led to significantly increased lysine titer. The modifications cause an increase in the PPP flux which probably resulted from positively changed kinetics of the enzymes.…”

Section: Metabolic Engineering Of Nadph Supplymentioning

confidence: 99%

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Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

Wittmann

2010

Biosystems Engineering I

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The Gram-positive soil bacterium Corynebacterium glutamicum was discovered as a natural overproducer of glutamate about 50 years ago. Linked to the steadily increasing economical importance of this microorganism for production of glutamate and other amino acids, the quest for efficient production strains has been an intense area of research during the past few decades. Efficient production strains were created by applying classical mutagenesis and selection and especially metabolic engineering strategies with the advent of recombinant DNA technology. Hereby experimental and computational approaches have provided fascinating insights into the metabolism of this microorganism and directed strain engineering. Today, C. glutamicum is applied to the industrial production of more than 2 million tons of amino acids per year. The huge achievements in recent years, including the sequencing of the complete genome and efficient post genomic approaches, now provide the basis for a new, fascinating era of research -analysis of metabolic and regulatory properties of C. glutamicum on a global scale towards novel and superior bioprocesses.

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Amino Acid Production:L‐Lysine

Nagai

Ito

Yasueda

2009

Encyclopedia of Industrial Biotechnology

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L ‐Lysine is an essential amino acid for humans and animals and is exclusively used as a feed additive for swine and poultry throughout the world, since most grains used for the feed are deficient in L ‐lysine in nutritional amino acid balance. This amino acid has been extensively produced by fermentation using gram‐positive coryneform bacteria including Corynebacterium glutamicum . To date, many studies on strain improvement have been carried out using various methods including conventional mutagenesis and screening, genetic engineering, and metabolic engineering. Recently, the complete genome sequence of C. glutamicum has been determined, and the genetic information is very valuable for strain development for higher performance, in coordination with various “omics” analyses. Besides C. glutamicum , other bacteria including Escherichia coli have also been considered as promising producers of amino acids with high interest. Together with strain development, the improvements of fermentation and purification technologies also contribute to the success of the industrial production of L ‐lysine. It is estimated that the current annual production of L ‐lysine is more than 850,000 metric tons throughout the world.

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Metabolic flux engineering of l-lysine production in Corynebacterium glutamicum—over expression and modification of G6P dehydrogenase

Cited by 162 publications

References 26 publications

l -Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

l -Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

Amino Acid Production:L‐Lysine

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