scholarly journals Enhanced Glucose Consumption and Organic Acid Production by Engineered Corynebacterium glutamicum Based on Analysis of a pfkB1 Deletion Mutant

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Satoshi Hasegawa ◽  
Yuya Tanaka ◽  
Masako Suda ◽  
Toru Jojima ◽  
Masayuki Inui
Keyword(s):  
Organic Acid ◽  
Corynebacterium Glutamicum ◽  
Acid Production ◽  
Deletion Mutant ◽  
Glucose Consumption ◽  
Oxygen Deprivation ◽  
Glycolytic Flux ◽  
Wild Type ◽  
Organic Acid Production ◽  
Content Type

ABSTRACT In the analysis of a carbohydrate metabolite pathway, we found interesting phenotypes in a mutant strain of Corynebacterium glutamicum deficient in pfkB1, which encodes fructose-1-phosphate kinase. After being aerobically cultivated with fructose as a carbon source, this mutant consumed glucose and produced organic acid, predominantly l-lactate, at a level more than 2-fold higher than that of the wild-type grown with glucose under conditions of oxygen deprivation. This considerably higher fermentation capacity was unique for the combination of pfkB1 deletion and cultivation with fructose. In the metabolome and transcriptome analyses of this strain, marked intracellular accumulation of fructose-1-phosphate and significant upregulation of several genes related to the phosphoenolpyruvate:carbohydrate phosphotransferase system, glycolysis, and organic acid synthesis were identified. We then examined strains overexpressing several of the identified genes and demonstrated enhanced glucose consumption and organic acid production by these engineered strains, whose values were found to be comparable to those of the model pfkB1 deletion mutant grown with fructose. l-Lactate production by the ppc deletion mutant of the engineered strain was 2,390 mM (i.e., 215 g/liter) after 48 h under oxygen deprivation, which was a 2.7-fold increase over that of the wild-type strain with a deletion of ppc. IMPORTANCE Enhancement of glycolytic flux is important for improving microbiological production of chemicals, but overexpression of glycolytic enzymes has often resulted in little positive effect. That is presumably because the central carbon metabolism is under the complex and strict regulation not only transcriptionally but also posttranscriptionally, for example, by the ATP/ADP ratio. In contrast, we studied a mutant strain of Corynebacterium glutamicum that showed markedly enhanced glucose consumption and organic acid production and, based on the findings, identified several genes whose overexpression was effective in enhancing glycolytic flux under conditions of oxygen deprivation. These results will further understanding of the regulatory mechanisms of glycolytic flux and can be widely applied to the improvement of the microbial production of useful chemicals.

Aerobiosis–anaerobiosis transition has a significant impact on organic acid production by Corynebacterium glutamicum

2017 ◽  
Vol 52 ◽  
pp. 10-21 ◽  
Author(s):  
Abdoul-Karim Kaboré ◽  
Eric Olmos ◽  
Michel Fick ◽  
Fabrice Blanchard ◽  
Emmanuel Guedon ◽  
...  
Keyword(s):  
Organic Acid ◽  
Corynebacterium Glutamicum ◽  
Acid Production ◽  
Organic Acid Production

scholarly journals Overexpression of Genes Encoding Glycolytic Enzymes in Corynebacterium glutamicum Enhances Glucose Metabolism and Alanine Production under Oxygen Deprivation Conditions

2012 ◽  
Vol 78 (12) ◽  
pp. 4447-4457 ◽  
Author(s):  
Shogo Yamamoto ◽  
Wataru Gunji ◽  
Hiroaki Suzuki ◽  
Hiroshi Toda ◽  
Masako Suda ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Corynebacterium Glutamicum ◽  
Glucose Consumption ◽  
Glycolytic Enzyme ◽  
Oxygen Deprivation ◽  
Glycolytic Enzymes ◽  
Content Type ◽  
Extracellular Metabolites ◽  
High Copy Number Plasmid ◽  
Genes Encoding

ABSTRACTWe previously reported thatCorynebacterium glutamicumstrain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encodinggapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159–165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements inC. glutamicumglucose metabolism under oxygen deprivation. In addition togapA, overexpressing pyruvate kinase-encodingpykand phosphofructokinase-encodingpfkenabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encodinggpiin strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed thatgapAoverexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD+ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.

scholarly journals Improvement of the Redox Balance Increases l-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions

2011 ◽  
Vol 78 (3) ◽  
pp. 865-875 ◽  
Author(s):  
Satoshi Hasegawa ◽  
Kimio Uematsu ◽  
Yumi Natsuma ◽  
Masako Suda ◽  
Kazumi Hiraga ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Glucose Consumption ◽  
Redox Balance ◽  
Oxygen Deprivation ◽  
Limiting Factor ◽  
Acetohydroxy Acid Synthase ◽  
Content Type ◽  
Production And Consumption ◽  
Dehydrogenase Gene ◽  
Valine Biosynthesis

ABSTRACTProduction ofl-valine under oxygen deprivation conditions byCorynebacterium glutamicumlacking the lactate dehydrogenase geneldhAand overexpressing thel-valine biosynthesis genesilvBNCDEwas repressed. This was attributed to imbalanced cofactor production and consumption in the overalll-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole ofl-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement forl-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded byilvCand introduction ofLysinibacillus sphaericusleucine dehydrogenase in place of endogenous transaminase B, encoded byilvE. The intracellular NADH/NAD+ratio significantly decreased, and glucose consumption andl-valine production drastically improved. Moreover,l-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD+ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor forl-valine production under oxygen deprivation conditions. Thel-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded byilvBN. The resultant strain produced 1,470 mMl-valine after 24 h with a yield of 0.63 mol mol of glucose−1, and thel-valine productivity reached 1,940 mM after 48 h.

scholarly journals Engineering of Corynebacterium glutamicum for High-Yield l-Valine Production under Oxygen Deprivation Conditions

2012 ◽  
Vol 79 (4) ◽  
pp. 1250-1257 ◽  
Author(s):  
Satoshi Hasegawa ◽  
Masako Suda ◽  
Kimio Uematsu ◽  
Yumi Natsuma ◽  
Kazumi Hiraga ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Glucose Consumption ◽  
Resistant Mutant ◽  
Oxygen Deprivation ◽  
High Yield ◽  
Acetohydroxy Acid Synthase ◽  
Succinate Production ◽  
Content Type ◽  
High Productivity ◽  
By Products

ABSTRACTWe previously demonstrated efficientl-valine production by metabolically engineeredCorynebacterium glutamicumunder oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis ofl-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase fromLysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase geneldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress thel-valine yield. Eliminating these by-products therefore was deemed key to improving thel-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase geneppc, succinate production was effectively suppressed, but both glucose consumption andl-valine production dropped considerably due to the severely elevated intracellular NADH/NAD+ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, includinggapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higherl-valine yield and productivity. Deleting the alanine transaminase geneavtAsuppressed alanine production. The resultant strain produced 1,280 mMl-valine at a yield of 88% mol mol of glucose−1after 24 h under oxygen deprivation, a vastly improved yield over our previous best.

scholarly journals CgMED3 Changes Membrane Sterol Composition To Help Candida glabrata Tolerate Low-pH Stress

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Xiaobao Lin ◽  
Yanli Qi ◽  
Dongni Yan ◽  
Hui Liu ◽  
Xiulai Chen ◽  
...  
Keyword(s):  
Acid Production ◽  
Candida Glabrata ◽  
Sterol Composition ◽  
Low Ph ◽  
Mediator Complex ◽  
Wild Type ◽  
Organic Acid Production ◽  
Content Type ◽  
Ph Stress

ABSTRACT Candida glabrata is a promising microorganism for organic acid production. The present study aimed to investigate the role of C. glabrata Mediator complex subunit 3 (CgMed3p) in protecting C. glabrata under low-pH conditions. To this end, genes CgMED3A and CgMED3B were deleted, resulting in the double-deletion Cgmed3ABΔ strain. The final biomass and cell viability levels of Cgmed3ABΔ decreased by 64.5% and 35.8%, respectively, compared to the wild-type strain results at pH 2.0. In addition, lack of CgMed3ABp resulted in selective repression of a subset of genes in the lipid biosynthesis and metabolism pathways. Furthermore, C18:1, lanosterol, zymosterol, fecosterol, and ergosterol were 13.2%, 80.4%, 40.4%, 78.1%, and 70.4% less abundant, respectively, in the Cgmed3ABΔ strain. In contrast, the concentration of squalene increased by about 44.6-fold. As a result, membrane integrity, rigidity, and H+-ATPase activity in the Cgmed3ABΔ strain were reduced by 62.7%, 13.0%, and 50.3%, respectively. In contrast, overexpression of CgMED3AB increased the levels of C18:0, C18:1, and ergosterol by 113.2%, 5.9%, and 26.4%, respectively. Moreover, compared to the wild-type results, dry cell weight and pyruvate production increased, irrespective of pH buffering. These results suggest that CgMED3AB regulates membrane composition, which in turn enables cells to tolerate low-pH stress. We propose that regulation of CgMed3ABp may provide a novel strategy for enhancing low-pH tolerance and increasing organic acid production by C. glabrata. IMPORTANCE The objective of this study was to investigate the role of Candida glabrata Mediator complex subunit 3 (CgMed3ABp) and its regulation of gene expression at low pH in C. glabrata. We found that CgMed3ABp was critical for cellular survival and pyruvate production during low-pH stress. Measures of the levels of plasma membrane fatty acids and sterol composition indicated that CgMed3ABp could play an important role in regulating homeostasis in C. glabrata. We propose that controlling membrane lipid composition may enhance the robustness of C. glabrata for the production of organic acids.

scholarly journals Carbon Flux Analysis by13C Nuclear Magnetic Resonance To Determine the Effect of CO2on Anaerobic Succinate Production by Corynebacterium glutamicum

2014 ◽  
Vol 80 (10) ◽  
pp. 3015-3024 ◽  
Author(s):  
Dušica Radoš ◽  
David L. Turner ◽  
Luís L. Fonseca ◽  
Ana Lúcia Carvalho ◽  
Bastian Blombach ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Organic Acid ◽  
Corynebacterium Glutamicum ◽  
Tricarboxylic Acid Cycle ◽  
Fold Increase ◽  
Flux Analysis ◽  
Organic Acid Production ◽  
Content Type ◽  
Nuclear Magnetic

ABSTRACTWild-typeCorynebacterium glutamicumproduces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO2on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO2caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense ofl-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2was estimated by using13C-labeled glucose and13C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (∼5%) regardless of the presence or absence of CO2. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured byin vivoNMR, and the stoichiometry (H+:organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate withC. glutamicumwithout genetic manipulation.

scholarly journals Biochar Enhances Aspergillus niger Rock Phosphate Solubilization by Increasing Organic Acid Production and Alleviating Fluoride Toxicity

2014 ◽  
Vol 80 (10) ◽  
pp. 3081-3085 ◽  
Author(s):  
Gilberto de Oliveira Mendes ◽  
David Lopez Zafra ◽  
Nikolay Bojkov Vassilev ◽  
Ivo Ribeiro Silva ◽  
José Ivo Ribeiro ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Organic Acid ◽  
Acid Production ◽  
Rock Phosphate ◽  
Phosphate Solubilization ◽  
Activated Alumina ◽  
Organic Acid Production ◽  
Content Type ◽  
Soluble P ◽  
High Concentrations

ABSTRACTDuring fungal rock phosphate (RP) solubilization, a significant quantity of fluoride (F−) is released together with phosphorus (P), strongly inhibiting the process. In the present study, the effect of two F−adsorbents [activated alumina (Al2O3) and biochar] on RP solubilization byAspergillus nigerwas examined. Al2O3adsorbed part of the F−released but also adsorbed soluble P, which makes it inappropriate for microbial RP solubilization systems. In contrast, biochar adsorbed only F−while enhancing phosphate solubilization 3-fold, leading to the accumulation of up to 160 mg of P per liter. By comparing the values of F−measured in solution at the end of incubation and those from a predictive model, it was estimated that up to 19 mg of F−per liter can be removed from solution by biochar when added at 3 g liter−1to the culture medium. Thus, biochar acted as an F−sink during RP solubilization and led to an F−concentration in solution that was less inhibitory to the process. In the presence of biochar,A. nigerproduced larger amounts of citric, gluconic, and oxalic acids, whether RP was present or not. Our results show that biochar enhances RP solubilization through two interrelated processes: partial removal of the released F−and increased organic acid production. Given the importance of organic acids for P solubilization and that most of the RPs contain high concentrations of F−, the proposed solubilization system offers an important technological improvement for the microbial production of soluble P fertilizers from RP.

Sites of Organic Acid Production and Patterns of Digesta Movement in the Gastrointestinal Tract of Dogs

1979 ◽  
Vol 109 (9) ◽  
pp. 1592-1600 ◽  
Author(s):  
Charles A. Banta ◽  
Edgar T. Clemens ◽  
Mary M. Krinsky ◽  
Ben E. Sheffy
Keyword(s):  
Gastrointestinal Tract ◽  
Organic Acid ◽  
Acid Production ◽  
Organic Acid Production

Microbial Applications in Organic Acid Production

2021 ◽  
pp. 104-124
Author(s):  
Jyoti Singh Jadaun ◽  
Amit K. Rai ◽  
Sudhir P. Singh
Keyword(s):  
Organic Acid ◽  
Acid Production ◽  
Organic Acid Production
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