scholarly journals High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered Escherichia coli Strain

2008 ◽  
Vol 74 (21) ◽  
pp. 6649-6655 ◽  
Author(s):  
Yihui Zhu ◽  
Mark A. Eiteman ◽  
Ronni Altman ◽  
Elliot Altman
Keyword(s):  
Escherichia Coli ◽  
Nadh Oxidase ◽  
Pyruvate Dehydrogenase Complex ◽  
Formation Rate ◽  
Batch Process ◽  
Defined Medium ◽  
Coli Strain ◽  
Glycolytic Flux ◽  
Exponential Feeding ◽  
Phosphoenolpyruvate Synthase

ABSTRACT We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h−1. Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g � h), pyruvate formation rate (1.11 g/g � h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g � h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h−1 achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter � h and yield of 0.68 g/g.

scholarly journals Homolactate Fermentation by Metabolically Engineered Escherichia coli Strains

2006 ◽  
Vol 73 (2) ◽  
pp. 456-464 ◽  
Author(s):  
Y. Zhu ◽  
M. A. Eiteman ◽  
K. DeWitt ◽  
E. Altman
Keyword(s):  
Escherichia Coli ◽  
Tricarboxylic Acid Cycle ◽  
Pyruvate Dehydrogenase Complex ◽  
Ph Control ◽  
Defined Medium ◽  
Nuclear Magnetic Resonance Analysis ◽  
Aerobic Growth ◽  
Resonance Analysis ◽  
Phosphoenolpyruvate Synthase ◽  
Anaerobic Production

ABSTRACT We report the homofermentative production of lactate in Escherichia coli strains containing mutations in the aceEF, pfl, poxB, and pps genes, which encode the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, and phosphoenolpyruvate synthase, respectively. The process uses a defined medium and two distinct fermentation phases: aerobic growth to an optical density of about 30, followed by nongrowth, anaerobic production. Strain YYC202 (aceEF pfl poxB pps) generated 90 g/liter lactate in 16 h during the anaerobic phase (with a yield of 0.95 g/g and a productivity of 5.6 g/liter · h). Ca(OH)2 was found to be superior to NaOH for pH control, and interestingly, significant succinate also accumulated (over 7 g/liter) despite the use of N2 for maintaining anaerobic conditions. Strain ALS961 (YYC202 ppc) prevented succinate accumulation, but growth was very poor. Strain ALS974 (YYC202 frdABCD) reduced succinate formation by 70% to less than 3 g/liter. 13C nuclear magnetic resonance analysis using uniformly labeled acetate demonstrated that succinate formation by ALS974 was biochemically derived from acetate in the medium. The absence of uniformly labeled succinate, however, demonstrated that glyoxylate did not reenter the tricarboxylic acid cycle via oxaloacetate. By minimizing the residual acetate at the time that the production phase commenced, the process with ALS974 achieved 138 g/liter lactate (1.55 M, 97% of the carbon products), with a yield of 0.99 g/g and a productivity of 6.3 g/liter · h during the anaerobic phase.

Inhibition of oxidative metabolism in Escherichia coli by d-camphor and restoration of oxidase activity by quinones

1975 ◽  
Vol 21 (9) ◽  
pp. 1357-1361 ◽  
Author(s):  
Maria A. Cardullo ◽  
James J. Gilroy
Keyword(s):  
Escherichia Coli ◽  
Vitamin K ◽  
Oxidative Metabolism ◽  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Substrate Inhibition ◽  
Lower Surface ◽  
Coli Strain ◽  
Cell Permeability ◽  
Alumina Powder

Oxidative metabolism in whole cells of Escherichia coli strain 82/r was inhibited by d-camphor when glucose, pyruvate, or succinate was used as substrate. Inhibition was not due to lower surface tension in d-camphor-treated cell suspensions nor was it a function of cell permeability. Succinic, lactic, and NADH-oxidase activities were inhibited in alumina powder cell-free extracts (80 μg of protein/ml) by d-camphor (1100 μg/ml). NADH: and succinic: DCPIP oxidoreductase enzymes were unaffected by d-camphor. Menadione (vitamin K3) restored succinic, lactic, and NADH-oxidase activities in d-camphor-inhibited cell-free extracts. Concentrations of menadione used to restore succinic and NADH oxidase activities were not stimulatory in non-camphor-treated extracts. Succinic oxidase activity in d-camphor-inhibited cell-free extracts was also restored by ubiquinone (Q6) but not by vitamin K1. These results are interpreted to indicate that d-camphor may affect quinone function in E. coli.

scholarly journals Engineering of a Highly Efficient Escherichia coli Strain for Mevalonate Fermentation through Chromosomal Integration

2016 ◽  
Vol 82 (24) ◽  
pp. 7176-7184 ◽  
Author(s):  
Jilong Wang ◽  
Suthamat Niyompanich ◽  
Yi-Shu Tai ◽  
Jingyu Wang ◽  
Wenqin Bai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shake Flask ◽  
Mevalonate Pathway ◽  
High Titer ◽  
Scale Up ◽  
Coli Strain ◽  
High Yield ◽  
Glycolytic Flux ◽  
Chromosomal Integration ◽  
Theoretical Yield

ABSTRACTChromosomal integration of heterologous metabolic pathways is optimal for industrially relevant fermentation, as plasmid-based fermentation causes extra metabolic burden and genetic instabilities. In this work, chromosomal integration was adapted for the production of mevalonate, which can be readily converted into β-methyl-δ-valerolactone, a monomer for the production of mechanically tunable polyesters. The mevalonate pathway, driven by a constitutive promoter, was integrated into the chromosome ofEscherichia colito replace the native fermentation geneadhEorldhA. The engineered strains (CMEV-1 and CMEV-2) did not require inducer or antibiotic and showed slightly higher maximal productivities (0.38 to ∼0.43 g/liter/h) and yields (67.8 to ∼71.4% of the maximum theoretical yield) than those of the plasmid-based fermentation. Since the glycolysis pathway is the first module for mevalonate synthesis,atpFHdeletion was employed to improve the glycolytic rate and the production rate of mevalonate. Shake flask fermentation results showed that the deletion ofatpFHin CMEV-1 resulted in a 2.1-fold increase in the maximum productivity. Furthermore, enhancement of the downstream pathway by integrating two copies of the mevalonate pathway genes into the chromosome further improved the mevalonate yield. Finally, our fed-batch fermentation showed that, with deletion of theatpFHandsucAgenes and integration of two copies of the mevalonate pathway genes into the chromosome, the engineered strain CMEV-7 exhibited both high maximal productivity (∼1.01 g/liter/h) and high yield (86.1% of the maximum theoretical yield, 30 g/liter mevalonate from 61 g/liter glucose after 48 h in a shake flask).IMPORTANCEMetabolic engineering has succeeded in producing various chemicals. However, few of these chemicals are commercially competitive with the conventional petroleum-derived materials. In this work, chromosomal integration of the heterologous pathway and subsequent optimization strategies ensure stable and efficient (i.e., high-titer, high-yield, and high-productivity) production of mevalonate, which demonstrates the potential for scale-up fermentation. Among the optimization strategies, we demonstrated that enhancement of the glycolytic flux significantly improved the productivity. This result provides an example of how to tune the carbon flux for the optimal production of exogenous chemicals.

scholarly journals Alterations of Cellular Physiology in Escherichia coli in Response to Oxidative Phosphorylation Impaired by Defective F1-ATPase

2006 ◽  
Vol 188 (19) ◽  
pp. 6869-6876 ◽  
Author(s):  
Sakiko Noda ◽  
Yuji Takezawa ◽  
Tomohiko Mizutani ◽  
Tomoaki Asakura ◽  
Eiichiro Nishiumi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Citrate Synthase ◽  
Pyruvate Dehydrogenase Complex ◽  
Growth Yield ◽  
Glucose Consumption ◽  
Tca Cycle ◽  
Total Activity ◽  
Glycolytic Flux ◽  
Flux Analysis ◽  
Physiological Benefits

ABSTRACT The physiological changes in an F1-ATPase-defective mutant of Escherichia coli W1485 growing in a glucose-limited chemostat included a decreased growth yield (60%) and increased specific rates of both glucose consumption (168%) and respiration (171%). Flux analysis revealed that the mutant showed approximately twice as much flow in glycolysis but only an 18% increase in the tricarboxylic acid (TCA) cycle, owing to the excretion of acetate, where most of the increased glycolytic flux was directed. Genetic and biochemical analyses of the mutant revealed the downregulation of many TCA cycle enzymes, including citrate synthase, and the upregulation of the pyruvate dehydrogenase complex in both transcription and enzyme activities. These changes seemed to contribute to acetate excretion in the mutant. No transcriptional changes were observed in the glycolytic enzymes, despite the enhanced glycolysis. The most significant alterations were found in the respiratory-chain components. The total activity of NADH dehydrogenases (NDHs) and terminal oxidases increased about twofold in the mutant, which accounted for its higher respiration rate. These changes arose primarily from the increased (3.7-fold) enzyme activity of NDH-2 and an increased amount of cytochrome bd in the mutant. Transcriptional upregulation appeared to be involved in these phenomena. As NDH-2 cannot generate an electrochemical gradient of protons and as cytochrome bd is inferior to cytochrome bo 3 in this ability, the mutant was able to recycle NADH at a higher rate than the parent and avoid generating an excess proton-motive force. We discuss the physiological benefits of the alterations in the mutant.

scholarly journals Biocatalytic Conversion of Short-Chain Fatty Acids to Corresponding Alcohols in Escherichia coli

Processes ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 973
Author(s):  
Li-Jen Lin ◽  
Mukesh Saini ◽  
Chung-Jen Chiang ◽  
Yun-Peng Chao
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Clostridium Acetobutylicum ◽  
Short Chain Fatty Acids ◽  
Coli Strain ◽  
Short Chain ◽  
Glycolytic Flux ◽  
Pentanoic Acid ◽  
Biocatalytic Production ◽  
Do So

Advanced biofuels possess superior characteristics to serve for gasoline substitutes. In this study, a whole cell biocatalysis system was employed for production of short-chain alcohols from corresponding fatty acids. To do so, Escherichia coli strain was equipped with a biocatalytic pathway consisting of endogenous atoDA and Clostridium acetobutylicum adhE2. The strain was further reprogrammed to improve its biocatalytic activity by direction the glycolytic flux to acetyl-CoA and recycling acetate. The production of 1-propanol and n-pentanol were exemplified with the engineered strain. By substrate (glucose and propionate) feeding, the strain enabled production of 5.4 g/L 1-propanol with productivity reaching 0.15 g/L/h. In addition, the strain with a heavy inoculum was implemented for the n-pentanol production from n-pentanoic acid. The production titer and productivity finally attained 4.3 g/L and 0.86 g/L/h, respectively. Overall, the result indicates that this developed system is useful and effective for biocatalytic production of short-chain alcohols.

scholarly journals L-Alanine Prototrophic Suppressors Emerge from L-Alanine Auxotroph through Stress-Induced Mutagenesis in Escherichia coli

2021 ◽  
Vol 9 (3) ◽  
pp. 472
Author(s):  
Harutaka Mishima ◽  
Hirokazu Watanabe ◽  
Kei Uchigasaki ◽  
So Shimoda ◽  
Shota Seki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Spontaneous Mutation ◽  
Point Mutations ◽  
Whole Genome Analysis ◽  
E Coli ◽  
Clone Pair ◽  
Induced Mutagenesis ◽  
Isogenic Mutants

In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.

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