Engineering Corynebacterium glutamicum for the production of pyruvate

Wieschalka, Stefan; Blombach, Bastian; Eikmanns, Bernhard J.

doi:10.1007/s00253-011-3843-9

Cited by 105 publications

(77 citation statements)

References 59 publications

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“…Consistent with previous reports, synthesis of lactate was greatly reduced from 0.85 to 0.05% by the deletion of ldh (Wieschalka et al, 2012). Moreover, the deletion of ldh was not the limiting step in the metabolic flow, since the growth rate and the Lleucine concentration of the ldh deletion strains were higher than the control.…”

Section: Effect Of Gene Deletions On Cell Growth and L-leucine Producsupporting

confidence: 91%

Metabolic engineering of Corynebacterium glutamicum to enhance L-leucine production

Huang¹,

Liang²,

Wu³

et al. 2017

Afr. J. Biotechnol.

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This work aimed to develop an efficient L-leucine industrial production strain of Corynebacterium glutamicum by using metabolic engineering. A recombinant C. glutamicum strain was constructed by expressing a feedback-resistant leuA-encoded 2-isopropylmalate synthase (IPMS) that carries three amino acid exchanges (R529H, G532D and L535V) from the mutant strain C. glutamicum ML1-9 which was obtained by screening for structural analogues. In order to improve the expression of IPMS, a strong promoter (tac promoter) was used to ensure efficient expression of the rate-limiting enzyme. In addition, reasonable metabolic modifications on the central carbon metabolic pathway and competitive metabolic pathways to optimize the L-leucine biosynthesis pathway by redistribution of various types of precursors and repression of negative regulation were used aimed for increased L-leucine production. The modifications involved (1) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (2) deletion of the gene encoding the AlaT to decrease the concentration of extracellular L-alanine, and increased availability of pyruvate for L-leucine formation, (3) deletion of the gene encoding the threonine dehydratase to abolish L-isoleucine synthesis and to eliminate the intermediate precursor of L-isoleucine biosynthesis competing with L-leucine biosynthesis, (4) inactivation of the pantothenate synthetase to increase α-ketoisovalerate formation, and to enable its further conversion to L-leucine, and (5) inactivation of lactate dehydrogenase to decrease lactate production and its pyruvate consumption, concomitant to decreased glucose consumption rates and prevention of lactic acid to restrict cell growth. The production performance of the engineered strain MDLeu-19/pZ8-1leuA r was characterized with cultivations in a bioreactor. Under fed-batch conditions in a 50-L automated fermentor, the best producer strain accumulated 38.1 g L -1 of L-leucine; the molar product yield being 0.42 mol L-leucine per mole of glucose (glucose conversion rate attained 26.4%). Moreover, during large-scale fermentation using a 150-m 3 fermentor, this strain produced more than 37.5 g L -1 L-leucine and the glucose conversion rate was 25.8%, making this process potentially viable for industrial production.

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Section: Effect Of Gene Deletions On Cell Growth and L-leucine Producsupporting

confidence: 91%

Metabolic engineering of Corynebacterium glutamicum to enhance L-leucine production

Huang¹,

Liang²,

Wu³

et al. 2017

Afr. J. Biotechnol.

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“…These approaches included (i) inactivating enzymes of D-pantothenate synthesis to limit CoA availability for the PDHC reaction (22,30,46), (ii) applying anaerobic conditions to abolish/reduce oxidative tricarboxylic acid (TCA) flux (47-49), (iii) using an ATPase-defective mutant leading to increased pyruvate availability (50,51), and (iv) deleting the aceE gene, encoding the E1p subunit of the PDHC (11,15,16,(25)(26)(27)(28). However, all these approaches either resulted TABLE 3 Substrate-specific biomass yield (Y X/S ), final titer, substrate-specific L-lysine yield (Y P/S ), and biomass-specific production rate (q P ) of C. glutamicum L-lysine producers cultivated in CGXII medium with 4% (wt/vol) glucose and 0.5% (wt/vol) BHI in shake flasks (DM1800) or in bioreactors (DM1933) in a requirement for D-pantothenate, ethanol, or acetate, did not allow an adequate adjustment of the PDHC activity and the carbon flux into the TCA cycle, and/or required a cleverly devised redox state of the cell (under anaerobic conditions).…”

Section: Discussionmentioning

confidence: 99%

“…Recent studies also showed the successful employment of C. glutamicum for the production of the diamines putrescine and cadaverine (5)(6)(7)(8)(9)(10), the organic acids D-lactate, succinate, 2-ketoisovalerate, and pyruvate (11)(12)(13)(14)(15), the biofuels ethanol and isobutanol (16)(17)(18), xylitol (19), and heterologous proteins (20,21).…”

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confidence: 99%

“…In fed-batch fermentations at high cell densities, C. glutamicum ⌬aceE ⌬pqo ⌬ilvE (pJC4 ilvBNCD) produced up to 188 mM KIV and showed a volumetric productivity of about 4.6 mM KIV per h in the overall production phase (11). In further approaches, we used the PDHC-deficient C. glutamicum strain as a platform for the efficient production of about 500 mM pyruvate (i.e., 45 g/liter) or 175 mM isobutanol (i.e., 13 g/liter) and also to improve L-lysine production with C. glutamicum (15,16,28).…”

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confidence: 99%

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Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l -Lysine, l -Valine, and 2-Ketoisovalerate

Buchholz

Schwentner

Brunnenkan

et al. 2013

Appl Environ Microbiol

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Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the L-valine biosynthetic genes ilvBNCE, all strains produced L-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 ⌬pqo ⌬ppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) L-valine with an overall yield (Y P/S ) of 0.36 mol per mol of glucose and a volumetric productivity (Q P ) of 13.6 mM per h [1.6 g/(liter ؋ h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the L-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a Y P/S of 0.24 mol per mol of glucose and a Q P of 6.9 mM per h [0.8 g/(liter ؋ h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum L-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

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“…A subsequent study examined the additional deletion of pyruvate oxidase (pqo, or poxB gene) which improved L-valine production and also introduced an acetate growth requirement, suggesting that in C. glutamicum the combination of pyruvate oxidase, acetate kinase and phosphotransacetylase can bypass pyruvate dehydrogenase to allow acetyl CoA generation [72]. Further knockouts in the genes for lactate dehydrogenase (ldhA), two transaminases (alaT and avtA), and the replacement of native acetohydroxyacid synthase with an attenuated variant (C-T ilvN) led to 46 g/L pyruvate from glucose and acetate in 105 h at a yield of 0.48 g/g [73]. It should be noted that C. glutamicum and various yeasts including S. cerevisiae and C. glabrata have an active pyruvate carboxylase whereas E. coli do not.…”

Section: Metabolic Engineering Of Pathwaysmentioning

confidence: 99%

Recent Progress in the Microbial Production of Pyruvic Acid

Maleki

Eiteman

2017

Fermentation

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Pyruvic acid (pyruvate) is a cellular metabolite found at the biochemical junction of glycolysis and the tricarboxylic acid cycle. Pyruvate is used in food, cosmetics, pharmaceutical and agricultural applications. Microbial production of pyruvate from either yeast or bacteria relies on restricting the natural catabolism of pyruvate, while also limiting the accumulation of the numerous potential by-products. In this review we describe research to improve pyruvate formation which has targeted both strain development and process development. Strain development requires an understanding of carbohydrate metabolism and the many competing enzymes which use pyruvate as a substrate, and it often combines classical mutation/isolation approaches with modern metabolic engineering strategies. Process development requires an understanding of operational modes and their differing effects on microbial growth and product formation.

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Engineering Corynebacterium glutamicum for the production of pyruvate

Cited by 105 publications

References 59 publications

Metabolic engineering of Corynebacterium glutamicum to enhance L-leucine production

Metabolic engineering of Corynebacterium glutamicum to enhance L-leucine production

Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l -Lysine, l -Valine, and 2-Ketoisovalerate

Recent Progress in the Microbial Production of Pyruvic Acid

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