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

Abstract

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.

[Retracted] Effects of Trace Irrigation at Different Depths on Transcriptome Expression Pattern in Cotton (G. hirsutum L.) Leaves

“…We also found that some genes related to metabolism and growth in plants were downregulated by the D50 irrigation regime, including CAD1 , UGT90A1 , UGT74E2 , UGT92A1 , GAPN , PsbW , and PSB28. UGT74E2 was involved in water stress response in Arabidopsis plant through its activity to auxin indole-3-butyric acid ( IBA ) [ 63 ]; GapN catalyzes the oxidation of glyceraldehyde 3-phosphate to 3-phosphoglycerate and is an alternative NADPH source [ 64 , 65 ]; PsbW and PSB28 are involved in the stabilization and recovery of photosystem II complexes, respectively [ 66 , 67 ]. The downregulation of these genes in response to the D50 irrigation regime compared with the D30 irrigation regime might reveal the disturbance of photosystem II systems and the energy source by the D50 regime.…”

Lysine production from the sugar alcohol mannitol: Design of the cell factory Corynebacterium glutamicum SEA-3 through integrated analysis and engineering of metabolic pathway fluxes

“…GapN was also valuable for increasing lysine production from glucose in C. glutamicum because it provided an extra source of NADPH (Takeno et al, 2010). More recently, it was discovered that GapN could even compensate for the absence of the oxidative PP pathway, which is normally the major source of NADPH in C. glutamicum (Takeno et al, 2016). A similar benefit was seen with mannitol as the substrate.…”

Equilibrium of the intracellular redox state for improving cell growth and l-lysine yield of Corynebacterium glutamicum by optimal cofactor swapping

“…Therefore, balancing the oxidation–reduction state of NAD(H/ + ) and NADP(H/ + ) is crucial for both catabolism and anabolism. Some examples have illustrated the importance of redox balance and availability for producing the fine chemicals including amino acids [7–9], vitamins [10, 11] and lipids [12, 13]. l -Lysine, as an essential amino acid for animals and humans, is mainly produced by C. glutamicum or its derivatives, which were created by traditional mutagenesis-screening techniques or by genetic engineering [14, 15].…”

“…l -Lysine, as an essential amino acid for animals and humans, is mainly produced by C. glutamicum or its derivatives, which were created by traditional mutagenesis-screening techniques or by genetic engineering [14, 15]. Notably, 4 mol of NADPH must be supplied for 1 mol of l -lysine biosynthesis from oxaloacetate, and thus numerous studies have focused on engineering the metabolism of NADPH for improving l -lysine producing strains [9, 16–19]. For example, genetic modification of the native glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been proven to improve the l -lysine production because of the increase of NADPH availability [17, 19, 20].…”