scholarly journals Engineering central pathways for industrial-level (3R)-acetoin biosynthesis in Corynebacterium glutamicum

2020 ◽  
Author(s):  
Lingxue Lu ◽  
Yufeng Mao ◽  
Mengyun Kou ◽  
Zhenzhen Cui ◽  
Biao Jin ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Citrate Synthase ◽  
Optical Purity ◽  
Value Added ◽  
Tca Cycle ◽  
Product Yield ◽  
The Tca Cycle ◽  
Acetoin Production ◽  
Industrial Level ◽  
Optically Pure

Abstract Background: Acetoin, especially the optically pure (3S)- or (3R)-enantiomer, is a high-value-added bio-based platform chemical and important potential pharmaceutical intermediate. Over the past decades, intense efforts have been devoted to the production of acetoin through green biotechniques. However, efficient and economical methods for the production of optically pure acetoin enantiomers are rarely reported. Previously, we systematically engineered the GRAS microorganism Corynebacterium glutamicum to efficiently produce (3R)-acetoin from glucose. Nevertheless, its yield and average productivity were still unsatisfactory for industrial bioprocesses.Results: In this study, cellular carbon fluxes in the acetoin producer CGR6 were further redirected toward acetoin synthesis using several metabolic engineering strategies, including blocking anaplerotic pathways, attenuating key genes of the TCA cycle and integrating additional copies of the alsSD operon into the genome. Among them, the combination of attenuation of citrate synthase and inactivation of phosphoenolpyruvate carboxylase showed a significant synergistic effect on acetoin production. Finally, the optimal engineered strain CGS11 produced a titer of 102.45 g/L acetoin with a yield of 0.419 g/g glucose at a rate of 1.86 g/L/h in a 5 L fermenter. The optical purity of the resulting (3R)-acetoin surpassed 95%.Conclusion: To the best of our knowledge, this is the highest titer of highly enantiomerically enriched (3R)-acetoin, together with a competitive product yield and productivity, achieved in a simple, green processes without expensive additives or substrates. This process therefore opens the possibility to achieve easy, efficient, economical and environmentally-friendly production of (3R)-acetoin via microbial fermentation in the near future.

scholarly journals Engineering central pathways for industrial-level D-(-)-acetoin biosynthesis in Corynebacterium glutamicum

2020 ◽  
Author(s):  
Lingxue Lu ◽  
Yufeng Mao ◽  
Mengyun Kou ◽  
Zhenzhen Cui ◽  
Biao Jin ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Citrate Synthase ◽  
Value Added ◽  
Environmentally Friendly ◽  
Product Yield ◽  
Microbial Fermentation ◽  
Near Future ◽  
Optically Pure ◽  
Environmentally Friendly Production ◽  
Competitive Product

Abstract Background: Acetoin, especially the optically pure L-(+)- or D-(-)-enantiomer, is a high-value-added bio-based platform chemical and important potential pharmaceutical intermediate. Over the past decades, intense efforts have been devoted to the production of acetoin through green biotechniques. However, efficient and economical methods for the production of optically pure acetoin enantiomers are rarely reported. Previously, we systematically engineered the GRAS microorganism Corynebacterium glutamicum to efficiently produce D-(-)-acetoin from glucose. Nevertheless, its yield and average productivity were still unsatisfactory for industrial bioprocesses. Results: In this study, cellular carbon fluxes in the acetoin producer CGR6 were further redirected toward acetoin synthesis using several metabolic engineering strategies, including blocking anaplerotic pathways, attenuating key genes of the TCA cycle and integrating additional copies of the alsSD operon into the genome. Among them, the combination of attenuation of citrate synthase and inactivation of phosphoenolpyruvate carboxylase showed a significant synergistic effect on acetoin production. Finally, the optimal engineered strain CGS11 produced a titer of 102.45 g/L acetoin with a yield of 0.419 g/g glucose at a rate of 1.86 g/L/h in a 5 L fermenter. The optical purity of the resulting D-(-)-acetoin surpassed 95%. To the best of our knowledge, this is the highest titer of highly enantiomerically enriched D-(-)-acetoin, together with a competitive product yield and productivity, achieved in a simple, green process without expensive additives or substrates. This process therefore opens the possibility to achieve easy, efficient, economical and environmentally-friendly production of D-(-)-acetoin via microbial fermentation in the near future. Conclusion: To the best of our knowledge, this is the highest titer of highly enantiomerically enriched D-(-)-acetoin, together with a competitive product yield and productivity, achieved in a simple, green processes without expensive additives or substrates. This process therefore opens the possibility to achieve easy, efficient, economical and environmentally-friendly production of D-(-)-acetoin via microbial fermentation in the near future.

scholarly journals Enhanced 3-Hydroxypropionic Acid Production From Acetate via the Malonyl-CoA Pathway in Corynebacterium glutamicum

2022 ◽  
Vol 9 ◽  
Author(s):  
Zhishuai Chang ◽  
Wei Dai ◽  
Yufeng Mao ◽  
Zhenzhen Cui ◽  
Zhidan Zhang ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Citrate Synthase ◽  
Tca Cycle ◽  
Acid Synthesis ◽  
Acetyl Coa ◽  
The Tca Cycle ◽  
Malonyl Coa ◽  
Alternative Carbon Source ◽  
Coa Reductase ◽  
Hydroxypropionic Acid

Acetate is an economical and environmental-friendly alternative carbon source. Herein, the potential of harnessing Corynebacterium glutamicum as a host to produce 3-hydroxypropionic acid (3-HP) from acetate was explored. First, the expression level of malonyl-CoA reductase from Chloroflexus aurantiacus was optimized through several strategies, strain Cgz2/sod-N-C* showed an MCR enzyme activity of 63 nmol/mg/min and a 3-HP titer of 0.66 g/L in flasks. Next, the expression of citrate synthase in Cgz2/sod-N-C* was weakened to reduce the acetyl-CoA consumption in the TCA cycle, and the resulting strain Cgz12/sod-N-C* produced 2.39 g/L 3-HP from 9.32 g/L acetate. However, the subsequent deregulation of the expression of acetyl-CoA carboxylase genes in Cgz12/sod-N-C* resulted in an increased accumulation of intracellular fatty acids, instead of 3-HP. Accordingly, cerulenin was used to inhibit fatty acid synthesis in Cgz14/sod-N-C*, and its 3-HP titer was further increased to 4.26 g/L, with a yield of 0.50 g 3-HP/g-acetate. Finally, the engineered strain accumulated 17.1 g/L 3-HP in a bioreactor without cerulenin addition, representing the highest titer achieved using acetate as substrate. The results demonstrated that Corynebacterium glutamicum is a promising host for 3-HP production from acetate.

Systematic metabolic engineering of Corynebacterium glutamicum for the industrial-level production of optically pure d-(−)-acetoin

2017 ◽  
Vol 19 (23) ◽  
pp. 5691-5702 ◽  
Author(s):  
Yufeng Mao ◽  
Jing Fu ◽  
Ran Tao ◽  
Can Huang ◽  
Zhiwen Wang ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Corynebacterium Glutamicum ◽  
Value Added ◽  
Industrial Product ◽  
Platform Chemical ◽  
Industrial Level ◽  
Optically Pure ◽  
Level Production

Acetoin is a high-value-added industrial product and a promising bio-based platform chemical.

scholarly journals RSM–GA Based Optimization of Bacterial PHA Production and In Silico Modulation of Citrate Synthase for Enhancing PHA Production

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 872 ◽  
Author(s):  
Apoorva Rao ◽  
Shafiul Haque ◽  
Hesham A. El-Enshasy ◽  
Vineeta Singh ◽  
Bhartendu Nath Mishra
Keyword(s):  
Molecular Docking ◽  
Nitrogen Source ◽  
In Silico ◽  
Citrate Synthase ◽  
Tca Cycle ◽  
Management Approach ◽  
Carbon And Nitrogen ◽  
The Tca Cycle ◽  
Different Types ◽  
Pha Production

The inexhaustible nature and biodegradability of bioplastics like polyhydroxyalkanoates (PHAs) make them suitable assets to replace synthetic plastics. The eventual fate of these eco-friendly and non-toxic bioplastics relies upon the endeavors towards satisfying cost and, in addition, execution necessity. In this study, we utilized and statistically optimized different food (kitchen-/agro-) waste as a sole carbon/nitrogen source for the production of PHA at a reduced cost, indicating a proficient waste administration procedure. Seven different types of kitchen-/agro-waste were used as unique carbon source and four different types of nitrogen source were used to study their impact on PHA production by Bacillus subtilis MTCC 144. Among four different studied production media, mineral salt medium (MSM) (biomass: 37.7 g/L; cell dry weight: 1.8 g/L; and PHA: 1.54 g/L) was found most suitable for PHA production. Further, carbon and nitrogen components of MSM were optimized using one-factor-at-a-time experiments, and found that watermelon rind (PHA = 12.97 g/L) and pulse peel (PHA = 13.5 g/L) were the most suitable carbon and nitrogen sources, respectively, in terms of PHA (78.60%) recovery. The concentrations of these factors (sources) were statistically optimized using response surface methodology coupled with the genetic algorithm approach. Additionally, in order to enhance microbial PHA production, the interaction of citrate synthase, a key enzyme in the TCA cycle, with different known inhibitors was studied using in silico molecular docking approach. The inhibition of citrate synthase induces the blockage of the tricarboxylic cycle (TCA), thereby increasing the concentration of acetyl-CoA that helps in enhanced PHA production. Molecular docking of citrate synthase with different inhibitors of PubChem database revealed that hesperidin (PubChem compound CID ID 10621), generally present in citrus fruits, is the most efficient inhibitor of the TCA cycle with the binding score of –11.4 and warrants experimental validation. Overall, this study provides an efficient food waste management approach by reducing the production cost and enhancing the production of PHA, thereby lessening our reliance on petroleum-based plastics.

scholarly journals The existence of a nonclassical TCA cycle in the nucleus that wires the metabolic-epigenetic circuitry

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Xujun Liu ◽  
Wenzhe Si ◽  
Lin He ◽  
Jianguo Yang ◽  
Yani Peng ◽  
...  
Keyword(s):  
Epigenetic Regulation ◽  
Citrate Synthase ◽  
Fumarate Hydratase ◽  
Tca Cycle ◽  
Chromatin Dynamics ◽  
Metabolic Intermediates ◽  
The Tca Cycle ◽  
Transport Chain ◽  
Outstanding Question ◽  
Aconitase 2

AbstractThe scope and variety of the metabolic intermediates from the mitochondrial tricarboxylic acid (TCA) cycle that are engaged in epigenetic regulation of the chromatin function in the nucleus raise an outstanding question about how timely and precise supply/consumption of these metabolites is achieved in the nucleus. We report here the identification of a nonclassical TCA cycle in the nucleus (nTCA cycle). We found that all the TCA cycle-associated enzymes including citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 3 (IDH3), oxoglutarate dehydrogenase (OGDH), succinyl-CoA synthetase (SCS), fumarate hydratase (FH), and malate dehydrogenase 2 (MDH2), except for succinate dehydrogenase (SDH), a component of electron transport chain for generating ATP, exist in the nucleus. We showed that these nuclear enzymes catalyze an incomplete TCA cycle similar to that found in cyanobacteria. We propose that the nTCA cycle is implemented mainly to generate/consume metabolic intermediates, not for energy production. We demonstrated that the nTCA cycle is intrinsically linked to chromatin dynamics and transcription regulation. Together, our study uncovers the existence of a nonclassical TCA cycle in the nucleus that links the metabolic pathway to epigenetic regulation.

scholarly journals Metabolic Engineering of the Tricarboxylic Acid Cycle for Improved Lysine Production by Corynebacterium glutamicum

2009 ◽  
Vol 75 (24) ◽  
pp. 7866-7869 ◽  
Author(s):  
Judith Becker ◽  
Corinna Klopprogge ◽  
Hartwig Schröder ◽  
Christoph Wittmann
Keyword(s):  
Metabolic Engineering ◽  
Corynebacterium Glutamicum ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Start Codon ◽  
Flux Analysis ◽  
Lysine Production ◽  
Acid Cycle ◽  
The Tca Cycle

ABSTRACT In the present work, lysine production by Corynebacterium glutamicum was improved by metabolic engineering of the tricarboxylic acid (TCA) cycle. The 70% decreased activity of isocitrate dehydrogenase, achieved by start codon exchange, resulted in a >40% improved lysine production. By flux analysis, this could be correlated to a flux shift from the TCA cycle toward anaplerotic carboxylation.

scholarly journals Structural and biochemical characterization of mitochondrial citrate synthase 4 from Arabidopsis thaliana

2020 ◽  
Vol 76 (3) ◽  
pp. 109-115
Author(s):  
Kazuya Nishio ◽  
Tsunehiro Mizushima
Keyword(s):  
Arabidopsis Thaliana ◽  
Coenzyme A ◽  
Biochemical Characterization ◽  
Citrate Synthase ◽  
Tca Cycle ◽  
Folded Structure ◽  
The Tca Cycle ◽  
High Level ◽  
Inhibition Analysis

Citrate synthase (CS) catalyzes the conversion of oxaloacetate and acetyl coenzyme A into citrate and coenzyme A in the mitochondrial tricarboxylic acid (TCA) cycle. In plants, mitochondrial metabolism, including the TCA cycle, occurs in interaction with photosynthetic metabolism. The controlled regulation of several enzymes in the TCA cycle, such as CS, is important in plants. Here, the first crystal structure of a plant mitochondrial CS, CSY4 from Arabidopsis thaliana (AtCSY4), has been determined. Structural comparison of AtCSY4 with mitochondrial CSs revealed a high level of similarity. Inhibition analysis showed a similar manner of inhibition as in mitochondrial CSs. The effect of oxidation on one of a pair of cysteine residues in AtCSY4 was speculated upon based on the folded structure.

Tricarboxylic acid cycle enzymes of a pseudophyllid cestode Penetrocephalus ganapatii

1990 ◽  
Vol 64 (1) ◽  
pp. 51-53
Author(s):  
S. Dhandayuthapani ◽  
K. Nellaiappan
Keyword(s):  
Isocitrate Dehydrogenase ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Reverse Direction ◽  
Acid Cycle ◽  
Tca Cycle Enzymes ◽  
The Tca Cycle ◽  
Atp Generation

ABSTRACTStudies on the tricarboxylic acid cycle (TCA cycle) enzymes of Penetrocephalus ganapatii reveal that the TCA cycle is only partially operative, as some of the enzymes at the start of the cycle viz. citrate synthase, aconitase and isocitrate dehydrogenase are found to be low in their activities. The high activities of malate dehydrogenase and fumarase, showing affinity towards a reverse direction, indicate that the TCA cycle operates in the reverse direction resulting in the formation of fumarate. The low succinate dehydrogenase/fumarate reductase ratio suggests that ATP generation may occur at site I of the respiratory chain during the reduction of fumarate into succinate.

scholarly journals Metabolic Fate of the Increased Yeast Amino Acid Uptake Subsequent to Catabolite Derepression

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
John S. Hothersall ◽  
Aamir Ahmed
Keyword(s):  
Amino Acid ◽  
Protein Expression ◽  
Citrate Synthase ◽  
Tca Cycle ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Leucine Oxidation ◽  
The Tca Cycle ◽  
Branched Chain ◽  
Ketoacid Dehydrogenase

Catabolite repression (CCR) regulates amino acid permeases in Saccharomyces cerevisiae via a TOR-kinase mediated mechanism. When glucose, the preferred fuel in S. cerevisiae, is substituted by galactose, amino acid uptake is increased. Here we have assessed the contribution and metabolic significance of this surfeit of amino acid in yeast undergoing catabolite derepression (CDR). L-[U-14C]leucine oxidation was increased 15 ± 1 fold in wild type (WT) strain grown in galactose compared to glucose. Under CDR, leucine oxidation was (i) proportional to uptake, as demonstrated by decreased uptake and oxidation of leucine in strains deleted of major leucine permeases and (ii) entirely dependent upon the TCA cycle, as cytochrome c1 (Cyt1) deleted strains could not grow in galactose. A regulator of amino acid carbon entry into the TCA cycle, branched chain ketoacid dehydrogenase, was also increased 29 ± 3 fold under CCR in WT strain. Protein expression of key TCA cycle enzymes, citrate synthase (Cs), and Cyt1 was increased during CDR. In summary, CDR upregulation of amino acid uptake is accompanied by increased utilization of amino acids for yeast growth. The mechanism for this is likely to be an increase in protein expression of key regulators of the TCA cycle.

scholarly journals Genetic and Biochemical Interactions Involving Tricarboxylic Acid Cycle (TCA) Function Using a Collection of Mutants Defective in All TCA Cycle Genes

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 153-166 ◽  
Author(s):  
Beata Przybyla-Zawislak ◽  
Devi M Gadde ◽  
Kurt Ducharme ◽  
Mark T McCammon
Keyword(s):  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Gene Encoding ◽  
Unique Role ◽  
Yeast Strains ◽  
The Tca Cycle ◽  
Mutant Collection

Abstract The eight enzymes of the tricarboxylic acid (TCA) cycle are encoded by at least 15 different nuclear genes in Saccharomyces cerevisiae. We have constructed a set of yeast strains defective in these genes as part of a comprehensive analysis of the interactions among the TCA cycle proteins. The 15 major TCA cycle genes can be sorted into five phenotypic categories on the basis of their growth on nonfermentable carbon sources. We have previously reported a novel phenotype associated with mutants defective in the IDH2 gene encoding the Idh2p subunit of the NAD+-dependent isocitrate dehydrogenase (NAD-IDH). Null and nonsense idh2 mutants grow poorly on glycerol, but growth can be enhanced by extragenic mutations, termed glycerol suppressors, in the CIT1 gene encoding the TCA cycle citrate synthase and in other genes of oxidative metabolism. The TCA cycle mutant collection was utilized to search for other genes that can suppress idh2 mutants and to identify TCA cycle genes that display a similar suppressible growth phenotype on glycerol. Mutations in 7 TCA cycle genes were capable of functioning as suppressors for growth of idh2 mutants on glycerol. The only other TCA cycle gene to display the glycerol-suppressor-accumulation phenotype was IDH1, which encodes the companion Idh1p subunit of NAD-IDH. These results provide genetic evidence that NAD-IDH plays a unique role in TCA cycle function.

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