Metabolic engineering of the L-valine biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation

Holátko, Jiří; Elišáková, Veronika; Prouza, Marek; Sobotka, M.; Nešvera, Jan; Pátek, Miroslav

doi:10.1016/j.jbiotec.2008.12.005

Cited by 86 publications

(45 citation statements)

References 40 publications

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“…On the other hand, these transcripts were detected in the sigH mutant as well as in the wild type under the nonstress conditions, indicating that these genes can also be expressed in a H -independent manner. In this context, a A -dependent promoter of the ilvD gene has been reported previously (45), and a A -dependent promoter-like sequence was found upstream of the TSP at the start codon of lipA in this study ( Fig. 1C and F).…”

Section: Resultssupporting

confidence: 86%

“…The transcriptional start points (TSPs) (ϩ1) and the Ϫ35 and Ϫ10 regions of the A -and H -dependent promoters are indicated in boldface. The TSP from the A -dependent promoter of ilvD has been previously determined (45). The terminal and start codons of cgR_1345 and ilvD (C) and those of lipB and lipA (F) are indicated with asterisks and arrows, respectively.…”

Section: Resultsmentioning

confidence: 99%

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Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σ^Hin Corynebacterium glutamicum

et al. 2014

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bThe extracytoplasmic function sigma factor H is responsible for the heat and oxidative stress response in Corynebacterium glutamicum. Due to the hierarchical nature of the regulatory network, previous transcriptome analyses have not been able to discriminate between direct and indirect targets of H . Here, we determined the direct genome-wide targets of H using chromatin immunoprecipitation with microarray technology (ChIP-chip) for analysis of a deletion mutant of rshA, encoding an antifactor of H . Seventy-five H -dependent promoters, including 39 new ones, were identified. H -dependent, heat-inducible transcripts for several of the new targets, including ilvD encoding a labile Fe-S cluster enzyme, dihydroxy-acid dehydratase, were detected, and their 5= ends were mapped to the H -dependent promoters identified. Interestingly, functional internal H -dependent promoters were found in operon-like gene clusters involved in the pentose phosphate pathway, riboflavin biosynthesis, and Zn uptake. Accordingly, deletion of rshA resulted in hyperproduction of riboflavin and affected expression of Zn-responsive genes, possibly through intracellular Zn overload, indicating new physiological roles of H . Furthermore, sigA encoding the primary factor was identified as a new target of H . Reporter assays demonstrated that the H -dependent promoter upstream of sigA was highly heat inducible but much weaker than the known A -dependent one. Our ChIP-chip analysis also detected the H -dependent promoters upstream of rshA within the sigH-rshA operon and of sigB encoding a group 2 factor, supporting the previous findings of their H -dependent expression. Taken together, these results reveal an additional layer of the sigma factor regulatory network in C. glutamicum.

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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σ^Hin Corynebacterium glutamicum

et al. 2014

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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%

“…Holátko et al (30) reduced activity of the ilvA (encoding threonine deaminase) and leuA (encoding isopropylmalate synthase) promoters and increased the activities of the ilvD and ilvE promoters by site-directed mutagenesis of the respective (extended) Ϫ10 regions. These modifications in combination with the deletion of panB (see above) and expression of ilvBN alleles encoding a feedback-resistant variant of the AHAS resulted in an L-isoleucine bradytrophy and improved production strain, which is, however, still auxotrophic for D-pantothenate (30). To improve L-lysine production with C. glutamicum, several studies employed the strong promoters of the superoxide dismutase gene or of the elongation factor TU gene to replace the native chromosomal promoters of target genes (31)(32)(33).…”

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confidence: 99%

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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“…An intermediate of L-Val synthesis, 2-oxovalerate, is a precursor for L-Leu synthesis. These mechanisms have been genetically manipulated to breed bacteria producing branched-chain amino acids, which are of industrial use in nutritional and health supplements and in chemical synthesis (1,17). However, the mechanism of eukaryotic microbes, especially the hypoxic response of the mechanism, has been relatively less characterized.…”

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confidence: 99%

Mechanism of De Novo Branched-Chain Amino Acid Synthesis as an Alternative Electron Sink in Hypoxic Aspergillus nidulans Cells

Shimizu

Fujii

Masuo

et al. 2010

Appl Environ Microbiol

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Although branched-chain amino acids are synthesized as building blocks of proteins, we found that the fungus Aspergillus nidulans excretes them into the culture medium under hypoxia. The transcription of predicted genes for synthesizing branched-chain amino acids was upregulated by hypoxia. A knockout strain of the gene encoding the large subunit of acetohydroxy acid synthase (AHAS), which catalyzes the initial reaction of the synthesis, required branched-chain amino acids for growth and excreted very little of them. Pyruvate, a substrate for AHAS, increased the amount of hypoxic excretion in the wild-type strain. These results indicated that the fungus responds to hypoxia by synthesizing branched-chain amino acids via a de novo mechanism. We also found that the small subunit of AHAS regulated hypoxic branched-chain amino acid production as well as cellular AHAS activity. The AHAS knockout resulted in higher ratios of NADH/NAD ؉ and NADPH/NADP ؉ under hypoxia, indicating that the branched-chain amino acid synthesis contributed to NAD ؉ and NADP ؉ regeneration. The production of branched-chain amino acids and the hypoxic induction of involved genes were partly repressed in the presence of glucose, where cells produced ethanol and lactate and increased levels of lactate dehydrogenase activity. These indicated that hypoxic branched-chain amino acid synthesis is a unique alternative mechanism that functions in the absence of glucose-to-ethanol/lactate fermentation and oxygen respiration.

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Metabolic engineering of the L-valine biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation

Cited by 86 publications

References 40 publications

Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σ^Hin Corynebacterium glutamicum

Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σ^Hin Corynebacterium glutamicum

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

Mechanism of De Novo Branched-Chain Amino Acid Synthesis as an Alternative Electron Sink in Hypoxic Aspergillus nidulans Cells

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Metabolic engineering of the L-valine biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation

Cited by 86 publications

References 40 publications

Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σHin Corynebacterium glutamicum

Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σHin Corynebacterium glutamicum

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

Mechanism of De Novo Branched-Chain Amino Acid Synthesis as an Alternative Electron Sink in Hypoxic Aspergillus nidulans Cells

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Product

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Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σ^Hin Corynebacterium glutamicum

Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σ^Hin Corynebacterium glutamicum