Optimization of the ion-exchange analysis of organic acids from fermentation

306

267

Overflow metabolism in the form of aerobic acetate excretion by Escherichia coli is an important physiological characteristic of this common industrial microorganism. Although acetate formation occurs under conditions of high glucose consumption, the genetic mechanisms that trigger this phenomenon are not clearly understood. We report on the role of the NADH/NAD ratio (redox ratio) in overflow metabolism. We modulated the redox ratio in E. coli through the expression of Streptococcus pneumoniae (water-forming) NADH oxidase. Using steady-state chemostat cultures, we demonstrated a strong correlation between acetate formation and this redox ratio. We furthermore completed genome-wide transcription analyses of a control E. coli strain and an E. coli strain overexpressing NADH oxidase. The transcription results showed that in the control strain, several genes involved in the tricarboxylic acid (TCA) cycle and respiration were repressed as the glucose consumption rate increased. Moreover, the relative repression of these genes was alleviated by expression of NADH oxidase and the resulting reduced redox ratio. Analysis of a promoter binding site upstream of the genes which correlated with redox ratio revealed a degenerate sequence with strong homology with the binding site for ArcA. Deletion of arcA resulted in acetate reduction and increased the biomass yield due to the increased capacities of the TCA cycle and respiration. Acetate formation was completely eliminated by reducing the redox ratio through expression of NADH oxidase in the arcA mutant, even at a very high glucose consumption rate. The results provide a basis for studying new regulatory mechanisms prevalent at reduced NADH/NAD ratios, as well as for designing more efficient bioprocesses.Escherichia coli accumulates acetic acid when growing at a high rate of glucose consumption even in the presence of ample oxygen (2,11,30). This phenomenon is known as overflow metabolism. Acetate is generated when carbon flux from acetyl-coenzyme A (CoA) is directed to acetate instead of entering the tricarboxylic acid (TCA) cycle (11). This by-product induces a stress response even at extremely low concentrations (21), hinders growth (26), and reduces the production of recombinant proteins (37). Overflow metabolism has been attributed to an enzymatic limitation in the TCA cycle (27). In E. coli the complete oxidation of 1 mol of glucose in glycolysis and the TCA cycle generates 10 mol of NAD(P)H and 2 mol of FADH 2 (31):If the rate of oxygen utilization is sufficiently high, the reduced cofactors generated by glucose consumption are reoxidized in the electron transport chain, which serves the dual purpose of maintaining an optimal redox environment and generating energy by oxidative phosphorylation. In the absence of oxygen glucose cannot be completely oxidized, and metabolic intermediates accumulate to maintain the redox balance. Even in the presence of oxygen, if the rate of glucose consumption is greater than the capacity to reoxidize the reduced equivalents generated...

Section: Methodsmentioning

confidence: 99%

Overflow Metabolism in Escherichia coli during Steady-State Growth: Transcriptional Regulation and Effect of the Redox Ratio

Vemuri

Altman

Sangurdekar

et al. 2006

306

267

“…OD 600 (UV-650 spectrophotometer; Beckman Instruments, San Jose, CA) was used to monitor cell growth, and this value was correlated with dry cell mass. The final concentrations of soluble organic compounds were determined by liquid chromatography (12). When Ca(OH) 2 was used for pH control, a homogeneous sample was diluted by a factor of 10 to dissolve any calcium lactate prior to analysis.…”

Section: Methodsmentioning

confidence: 99%

Homolactate Fermentation by Metabolically Engineered Escherichia coli Strains

Zhu

Eiteman

DeWitt

et al. 2007

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 N 2 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. 13 C 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.Lactic acid (lactate) and its derivatives have a wide range of applications in the food, pharmaceutical, leather, and textile industries (13,27). Recently, polylactic acid has been developed as a renewable, biodegradable, and environmentally friendly polymer (16,28). An advantage of a biological process over a chemical process for the production of lactate is the prospect of generating optically pure lactate (2, 26, 28), which is an important characteristic for many of its end uses (2, 10, 26).Although several microorganisms can produce lactate by fermentation of glucose and other renewable resources (13), Escherichia coli has many advantages, including rapid growth and simple nutritional requirements. Moreover, the ease of genetic manipulation of E. coli makes possible metabolic engineering strategies for improving lactate accumulation in E. coli (6). Several E. coli strains have been studied for lactate production. For example, a pfl ldhA mutant (FBR11) containing a plasmid with the ldh gene from Streptococcus bovis (Llactate dehydrogenase [L-LDH]) produced L-lactic acid anaerobically on complex media (11). Using glucose, a concentration of 73 g/liter was obtained with a yield of 0.93 g/g and a productivity of 2.3 g/liter · h. A small amount of succinate was also generated (0.9 to 2.2 g/liter). A pta ppc mutant (JP203) first grown aerobically in complex media to 10 g/liter dry cell weight (DCW) generated 62 g/liter of D-lactate under subsequent anaerobic conditions, with ...

“…The presence of numerous soluble organic compounds (e.g., pyruvate, acetate, glucose, malate, ␣-ketoglutarate, citrate, glucuronate, isocitrate, tartrate, malonate, glyoxylate, glycerate, sorbitol, succinate, glycolate, methylglyoxal, fumarate, lactate, formate, glycerol, dihydroxyacetone, acetoacetate, diacetyl, 1,2-propanediol, 1,3-propanediol, propionate, acetoin, acetaldehyde, ethanol, and 2,3-butanediol) was determined above a quantification limit of about 0.05 g/liter by liquid chromatography using a Coregel 64H ion exclusion column (Interaction Chromatography, San Jose, CA) with refractive index detection (14). The concentrations of oxygen and CO 2 in the off-gas were measured (Ultramat 23 gas analyzer, Siemens, Germany).…”

Section: Ggccatcgcgtttgtcctgttttgagaagcacacggtcaca3јmentioning

confidence: 99%

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

Zhu

Eiteman

Altman

et al. 2008

105

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.Pyruvic acid (pyruvate) is widely used in food, chemicals, and pharmaceuticals. The chemical is a precursor for the enzymatic production of L-tryptophan, L-tyrosine, D-/L-alanine, and L-dihydroxyphenylalanine (22), and it also serves in several health-related roles, including weight loss (25,33,34), exercise endurance (32), cholesterol reduction (35), and acne treatment (10). Recently, pyruvate has been used as the key metabolic precursor to the second-generation biofuels isobutanol and 3-methyl-1-butanol (2). By applying metabolic engineering strategies to them, microorganisms such as Escherichia coli and yeasts can be used to produce significant quantities of pyruvate from glucose and other renewable resources (22). In general, such approaches must delete or repress pathways which metabolize pyruvate. For example, pyruvate accumulates readily in E. coli strains having mutations in aceEF, encoding components of the pyruvate dehydrogenase complex (37). Additional mutations, of the ldhA, poxB, pfl, and pps genes, further improve pyruvate formation (48). Figure 1 shows the principal metabolic pathways and enzymes involved in the formation of pyruvate.Because pyruvate resides biochemically at the end of glycolysis, pyruvate production is directly related to the glycolytic flux. Metabolic engineering strategies to form pyruvate therefore also aim to enhance glycolysis (8,45,46). Glycolysis is not transcriptionally limited, and control principally resides outside the pathway in cellular demand for global cofactors, such as ATP and NADH (19,23,40). Glycolytic flux is substantially increased by disrupting oxidative phosphorylation or by increasing ATP hydrolysis (8,19). Increased glycolytic flux assists pyruvate accumulation: for example, an F 1 -ATPase-defective mutant (E. coli lipA2 bgl ϩ atpA401) generated pyruvate more quickly than its parent (45,46). Similarly, E. coli atpFH (strain TC44), defi...