Minimizing acetate formation in <Emphasis Type="Italic">E. coli</Emphasis> fermentations

Escherichia coli remains the best-established production organism in industrial biotechnology. However, when aerobic fermentation runs at high growth rates, considerable amounts of acetate are accumulated as by-product. This by-product has negative effects on growth and protein production. Over the last 20 years, substantial research efforts have been expended on reducing acetate accumulation during aerobic growth of E. coli on glucose. From the onset it was clear that this quest would not be a simple or uncomplicated one. Simple deletion of the acetate pathway reduced the acetate accumulation, but other by-products were formed. This mini review gives a clear outline of these research efforts and their outcome, including bioprocess level approaches and genetic approaches. Recently, the latter seems to have some promising results.     

Characterization and evaluation of a pta (phosphotransacetylase) negative mutant of Escherichia coli HB101 as production host of foreign lipase

In order to evaluate the pta(phosphotransacetylase) (–) mutant of Escherichia coli as a potential host of foreign lipase expression, the pta(–) mutant HB101 was constructed for the purpose of blocking the acetate synthetic pathway. Since acetate is known as a major inhibitory by-product of cell growth and foreign protein production, the growth characteristics and expression kinetics of the microbial lipase of the pta(–) E. coli mutant were investigated. The growth rate was considerably decreased (about 30%) when grown on M9 minimal media containing glucose, mannose or glycerol. Growth retardation was not observed when a gluconeogenic carbon source (acetate, malate or succinate) was utilized. It should be noted that the growth rate of the mutant was enhanced (about 20%) in modified M9 media including a gluconeogenic carbon source and NZ-amine. Growth inhibition of the pta(–) mutant by menadione, a representative redox-cycling drug, was more pronounced than that of the parental type of E. coli. Furthermore, the inhibition effect was more pronounced in glucose minimal medium, whereas the menadione sensitivity was not observed when a gluconeogenic carbon source was used as a sole carbon source or the lactate dehydrogenase gene from Lactobacillus casei was introduced in the pta(–) mutant. Therefore, it is suggested that the growth deficiency of the pta(–) mutant is closely related to the intracellular redox balance. When the pseudomonad lipase was expressed in the pta(–) mutant, a comparable expression rate and yield to the parental type strain was observed. High-cell-density culture if the mutant was easy to achieve even under the fluctuating conditions of residual glucose concentration.     

DNA synthesis by Escherichia coli B/r/l synchronized and grown under conditions of slow growth

Escherichia coli B/r/l was synchronized by a novel method and its growth was followed in a minimal salts medium containing glucose, acetate, aspartate or succinate as the sole carbon source. Thymine incorporation experiments showed agreement with the Cooper-Helmstetter model for DNA synthesis, during the division cycle, both in glucose grown culture with a doubling time 57.5 min and in acetate, aspartate and succinate where the doubling time was extended up to 90 min. The ratio C/C+D was identical or close to that predicted by the model. Prolonged growth of the synchronized cultures prior to each experiment was practised in order to ensure their physiological state without causing any considerable deterioration of synchrony.     

Acetate‐containing substrate mixtures improve recombinant protein secretion in Schizosaccharomyces pombe

Recombinant protein secretion in yeasts poses a burden to the metabolism of the host cell. Consequently, unfavorable cultivation conditions during strain screening or process development can lead to limitations in the energy and carbon metabolism of the cell, constraining the cell's ability to secrete the protein of interest. Recently, we demonstrated that improving cultivation conditions by using substrate mixtures of glycerol and acetate strongly elevated secretion of the homologous model protein maltase in the fission yeast Schizosaccharomyces pombe. In this work, we investigated if these previous findings were also applicable to the expression of recombinant proteins. Strains were constructed secreting either green fluorescent protein or a fluorescent single‐chain antibody fragment. These strains were cultivated under fermentative and respiratory growth conditions on glucose as sole carbon source and on mixtures of glucose/acetate and glycerol/acetate. We observed an increase in the specific secretion of both recombinant proteins by 1.8‐ and 3.8‐fold, respectively. This clearly demonstrates that the proper choice of process conditions and the applied carbon sources have a significant impact on the secretion of at least two recombinant proteins in S. pombe allowing an improved production of the protein of interest.     

Aerobic and anaerobic ethanol production by<Emphasis Type="Italic"> Mucor circinelloides</Emphasis> during submerged growth

The dimorphic organism Mucor circinelloides is currently being investigated as a potential host for heterologous protein production. The production of ethanol on pentose and hexose sugars was studied in submerged batch cultivations to further the general knowledge of Mucor physiology, with a view to the minimisation or elimination of the by-product ethanol for future process design. Large amounts of ethanol were produced during aerobic growth on glucose under non-oxygen limiting conditions, which is indicative of M. circinelloides being a Crabtree-positive organism. Ethanol production on galactose or xylose was less significant. The response of the organism to increased ethanol concentrations, both as the sole carbon source and in the presence of a sugar, was investigated in terms of biomass formation and morphology.     

Acetyl-CoA synthetase overexpression in Escherichia coli demonstrates more efficient acetate assimilation and lower acetate accumulation: a potential tool in metabolic engineering

The overexpression of acetyl-CoA (CoA) synthetase (ACS) in Escherichia coli showed significant reduction in acetate during glucose fermentation. It also greatly enhanced acetate assimilation when acetate was used as a carbon source. These features are ideal for applications in metabolic engineering. ACS overexpression can be strategically applied to reduce acetate byproduct, recover wasted carbon, and redirect carbon flux toward more favorable pathways. The native acs gene was cloned and overexpressed in E. coli. Studies showed significant effects on acetate production and assimilation in cultures grown in minimal and complex media with glucose or acetate as the carbon source.     

Increased product formation induced by a directed secondary substrate limitation in a batch Hansenula polymorpha culture

By the use of directed limitations of secondary substrates, the metabolic flux should be deflected from biomass production to product formation. In order to study the impact of directed limitations caused by various secondary substrates on the growth and product formation of the methylotrophic yeast Hansenula polymorpha, the cultivation systems respiration activity monitoring system (RAMOS) and BioLector were used in parallel. While the RAMOS device allows the online monitoring of the oxygen transfer rate in shake flasks, the BioLector enables in microtiter plates the monitoring of scattered light and the fluorescence intensity of the green fluorescent protein (GFP). Secondary substrate limitations of phosphate, potassium, and magnesium were analyzed in batch fermentations. The sole carbon source was either 10 g/L glucose or 10 g/L glycerol. The expression of the GFP gene is controlled by the FMD promoter (formate dehydrogenase). In batch cultures with glucose as carbon source, a directed limitation of phosphate increased the GFP production 1.87-fold, compared to phosphate unlimited conditions. Under potassium-limited conditions with glycerol as sole carbon source, the GFP production was 1.41-fold higher compared to unlimited conditions. A limitation of the substrate magnesium resulted in a 1.22-fold increase GFP formation in the case of glycerol as carbon source.     

Cultivation of Escherichia coli with mixtures of 3-phenylpropionic acid and glucose: Dynamics of growth and substrate consumption

In technical as well as natural ecosystems, pollutants are often mineralised in the presence of easily degradable carbon sources. A laboratory model system consisting of Escherichia coli ML 30 growing with mixtures of 3-phenylpropionic acid (3ppa, pollutant) and glucose (easily degradable substrate) was investigated in batch and carbon-limited continuous culture. Untypically, a linear growth pattern was observed during batch cultivation with 3ppa as the only carbon/energy source. When exposed to mixtures of both substrates in batch culture, E. coli utilised the two compounds sequentially. However, 3ppa and glucose were consumed simultaneously in continous culture. Whereas a pulse of excess glucose to a batch culture growing with 3ppa led to the repression of 3ppa utilisation, an excess of glucose added into continuous culture did not inhibit the utilisation of 3ppa. During continuous cultivation the 3ppa-degrading enzyme system operated close to saturation.     

Temporal accumulation of mannitol and arabitol in Geotrichum candidum

The temporal depletion and accumulation of polyols were investigated in the fungus Geotrichum candidum. The major intracellular polyols were tentatively identified by paper chromatography as mannitol and arabitol. Inositol was also present in small quantities, and trehalose was also detected in appreciable concentrations.Germination and vegetative growth depended on the type and concentration of the sole exogenous carbon source. Mannitol occurred in arthrospores at 9.4% of the dry weight after several days growth in 2% (w/v) glucose solid medium, and became depleted during germination and vegetative growth in liquid medium containing 2% (w/v) glucose, 2% (w/v) sodium acetate or 25% (w/v) glucose as sole carbon source. This hexitol latter accumulated during arthrosporulation. The depletion and accumulation of ethanol-soluble carbohydrate believed to be primarily trehalose was temporally similar to that of mannitol. Arabitol accumulated intracellularly during germination and vegetative growth in sodium acetate medium and 25% glucose medium. This pentitol was not detected intracellularly at any culture age during growth in 2% glucose medium.Prolonged incubation of the culture in 25% glucose medium after stationary phase was reached resulted in the gradual disappearance of arabitol from the arthrospores simultaneously with an increase in intracellular mannitol. In comparison, ethanol-soluble carbohydrate did not change with prolonged incubation in this medium.     

The utilisation of glucose/acetate mixtures by Escherichia coli W3110 under aerobic growth conditions

Byproduct acetate is of major concern when considering the growth of Escherichia coli on glucose. Besides the fact that acetate production detracts from the overall yield, acetate itself is also a growth inhibitor. To further complicate matters, E. coli is capable of growth on acetate via the glyoxylate bypass. In an effort to evaluate the influence of acetate on the growth of E. coli, mixed glucose/acetate substrate batch and continuous cultivations have been carried out. The results have been examined in the context of their implications for industrial-scale bioreactors. It was found that acetate was growth inhibitory at relatively low concentrations <1.25 g/l. A batch culture supplied with both glucose and acetate utilised glucose in preference to acetate. Simultaneous utilisation of substrates was observed in continuous cultures at low imposed growth rates, i.e., below ca. 0.30 h^у.     

Differential quantitative proteome analysis of Escherichia coli grown on acetate versus glucose

Relative protein abundances of Escherichia coli MG1655 growing exponentially on minimal medium with acetate or glucose as the sole carbon source were investigated in a quantitative shotgun proteome analysis with TMT6‐plex isobaric tags. Peptides were separated by high resolution high/low pH 2D‐LC, using an optimized fraction pooling scheme followed by mass spectrometric analysis. Quantitative data were acquired for 2099 proteins covering 49% of the predicted E. coli proteins, showing system‐wide effects of growth conditions. In total, 507 proteins showed a fold change of at least 1.5 and 205 proteins changed by more than twofold. Significant differences in abundance were observed for most of the proteins in the central carbon metabolism and in proteins relevant for amino acid and protein synthesis, processing of environmental information and scavenging of a variety of alternate carbon sources. Periplasmic‐binding proteins were also more abundant on acetate, especially proteins involved in scavenging extracellular resources such as sugars. All MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD003863).     

Evaluation of small organic acids present in fast pyrolysis bio‐oil from lignocellulose as feedstocks for bacterial bioconversion

Small organic acids derived from fast pyrolysis of lignocellulosic biomass represent a significant proportion of microbially accessible carbon in bio‐oil. However, using bio‐oil for microbial cultivation is a highly challenging task due to its strong adverse effects on microbial growth as well as its complex composition. In this study, the main small organic acids present in bio‐oil as acetate, formate and propionate were evaluated with respect to their suitability as feedstocks for bacterial growth. For this purpose, the growth behavior of four biotechnological production hosts—Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Corynebacterium glutamicum—was quantified and compared. The bacteria were cultivated on single acids and mixtures of acids in different concentrations and evaluated using common biotechnological efficiency parameters. In addition, cultivation experiments on pretreated fast pyrolysis‐derived bio‐oil fractions were performed with respect to the suitability of the bacterial strains to tolerate inhibitory substances. Results suggest that both P. putida and C. glutamicum metabolize acetate—the major small organic acid generated during fast pyrolysis of lignocellulosic biomass—as sole carbon source over a wide concentration range, are able to grow on mixtures of small organic acids present in bio‐oil and can, to a limited extent, tolerate the highly toxic inhibitory substances within bio‐oil. This work provides an important step in search of suitable bacterial strains for bioconversion of lignocellulosic‐based feedstocks and thus contributes to establishing efficient bioprocesses within a future bioeconomy.     

Engineering the productivity of recombinant Escherichia coli for limonene formation from glycerol in minimal media

The efficiency and productivity of cellular biocatalysts play a key role in the industrial synthesis of fine and bulk chemicals. This study focuses on optimizing the synthesis of (S)‐limonene from glycerol and glucose as carbon sources using recombinant Escherichia coli. The cyclic monoterpene limonene is extensively used in the fragrance, food, and cosmetic industries. Recently, limonene also gained interest as alternative jet fuel of biological origin. Key parameters that limit the (S)‐limonene yield, related to genetics, physiology, and reaction engineering, were identified. The growth‐dependent production of (S)‐limonene was shown for the first time in minimal media. E. coli BL21 (DE3) was chosen as the preferred host strain, as it showed low acetate formation, fast growth, and high productivity. A two‐liquid phase fed‐batch fermentation with glucose as the sole carbon and energy source resulted in the formation of 700 mg L_org^–1 (S)‐limonene. Specific activities of 75 mU g_cdw^–1 were reached, but decreased relatively quickly. The use of glycerol as a carbon source resulted in a prolonged growth and production phase (specific activities of ≥50 mU g_cdw^–1) leading to a final (S)‐limonene concentration of 2,700 mg L_org^–1. Although geranyl diphosphate (GPP) synthase had a low solubility, its availability appeared not to limit (S)‐limonene formation in vivo under the conditions investigated. GPP rerouting towards endogenous farnesyl diphosphate (FPP) formation also did not limit (S)‐limonene production. The two‐liquid phase fed‐batch setup led to the highest monoterpene concentration obtained with a recombinant microbial biocatalyst to date.     

Kinetic Studies and Biochemical Pathway Analysis of Anaerobic Poly-(R)-3-Hydroxybutyric Acid Synthesis in Escherichia coli

Poly-(R)-3-hydroxybutyric acid (PHB) was synthesized anaerobically in recombinant Escherichia coli. The host anaerobically accumulated PHB to more than 50% of its cell dry weight during cultivation in either growth or nongrowth medium. The maximum specific PHB production rate during growth-associated synthesis was approximately 2.3 ± 0.2 mmol of PHB/g of residual cell dry weight/h. The by-product secretion profiles differed significantly between the PHB-synthesizing strain and the control strain. PHB production decreased acetate accumulation for both growth and nongrowth-associated PHB synthesis. For instance under nongrowth cultivation, the PHB-synthesizing culture produced approximately 66% less acetate on a glucose yield basis as compared to a control culture. A theoretical biochemical network model was used to provide a rational basis to interpret the experimental results like the fermentation product secretion profiles and to study E. coli network capabilities under anaerobic conditions. For example, the maximum theoretical carbon yield for anaerobic PHB synthesis in E. coli is 0.8. The presented study is expected to be generally useful for analyzing, interpreting, and engineering cellular metabolisms.     

Fed‐Batch Process for Pyruvate Production by Recombinant Escherichia coli YYC202 Strain

Fed‐batch fermentation was applied to the production of pyruvate by using a recombinant Escherichia coli YYC202 strain. This strain is completely blocked in its ability to convert pyruvate into acetyl‐CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. By controlling acetate and glucose feed rate, a series of lab‐scale fed‐batch experiments were performed at pH 7 and 37 °C. CO₂ production rate (CTR) was used for on‐line regulation of the acetate feed rate. The correlation between CTR and acetate consumption rate (ACR) was determined experimentally. At optimal process conditions a final pyruvate concentration higher than 62 g/L, a space‐time yield of up to 42 g/L/d and pyruvate/glucose molar yield of 1.11 mol/mol were achieved. Experimental evidence was gathered that pyruvate export is active.     

Variability in cell yield for heterogeneous microbial populations of sewage origin grown on glucose

Values of cell yield collected over a period of eight years for heterogeneous populations of sewage origin acclimated to glucose in both batch and continuous culture were subjected to statistical analysis. The cell yield for this sole source of carbon (glucose) ranged from 36 to 88 per cent in batch culture, and 32 to 69 per cent in continuous culture. Because experimental conditions were known and well defined, the variability in cell yield is attributable to the ecological variation inherent in a heterogeneous population. The data presented demonstrate the futility of attempts to define Y for such populations as a precise theoretical constant dependent upon thermodynamic properties of the substrate.     

Properties of isocitrate lyase fromEscherichia coli K12 grown on acetate or glycolate

Properties of isocitrate lyase fromEscherichia coli, the first enzyme of the glyoxylate bypass, have been compared from cells grown on either acetate or glycolate as the sole carbon source. Michaelis constants for isocitrate, isoelectric points, native and subunit molecular weights, antigenic properties, peptide mapping with V-8 or trypsin, and several other properties were examined. Our data suggest that only one isocitrate lyase form exists inE. coli regardless of carbon source used for growth.     

Enhancement of pyruvate production byTorulopsis glabrata through supplement of oxaloacetate as carbon source

The capability of utilizing a TCA cycle intermediates as the sole carbon source by the multi-vitamin auxotrophic yeastTorulopsis glabrata CCTCC M202019 was demonstrated with plate count method. It is indicated thatT. glabrata could grew on a medium with one of the TCA cycle intermediates as the sole carbon source, but more colonies were observed when glucose, acetate and one of the TCA cycle intermediates coexisted in the medium. Among the intermediates of the TCA cycle examined in this study, cell growth was improved by supplementing oxaloacetate. Further investigation showed that the presence of acetate was necessary when oxaloacetate was supplemented. By supplementing with 10 g/L of oxaloacetate in pyruvate batch fermentation, dry cell weight increased from 11.8 g/L to 13.6 g/L, and pyruvate productivity was enhanced from 0.96 gL⁻¹h⁻¹ to 1.19 gL⁻¹h⁻¹ after cultivation of 56 h. The yield of pyruvate to glucose was also improved from 0.63 g/g to 0.66 g/g. These results indicate that under vitamins limitation, the productivity and yield of pyruvate could be enhancedvia an increase of cell growth by the supplementation of oxaloacetate.     

The use of multi-parameter flow cytometry to compare the physiological response of Escherichia coli W3110 to glucose limitation during batch, fed-batch and continuous culture cultivations.

Multi-parameter flow cytometric techniques have been developed for the 'at-line' study of bacterial cultivations. Using a mixture of specific fluorescent stains it is possible to resolve an individual cells physiological state beyond culturability, based on the presence or absence of an intact polarised cytoplasmic membrane, enabling assessment of population heterogeneity. It has been shown that during the latter stages of small-scale (5 l), well mixed fed-batch cultivations there is a considerable drop in cell viability, about 17%, as characterised by cytoplasmic membrane depolarisation and permeability. These phenomena are thought to be due to the severe and steadily increasing stress associated with glucose limitation at high cell densities, during the fed-batch process. Such effects were not found in either batch or continuous culture cultivations. The possibility of using these findings for improved process control using 'on-line' flow cytometry are discussed.     

Acetate formation during recombinant protein production in Escherichia coli K‐12 with an elevated NAD(H) pool

Acetate formation is a disadvantage in the use of Escherichia coli for recombinant protein production, and many studies have focused on optimizing fermentation processes or altering metabolism to eliminate acetate accumulation. In this study, E. coli MEC697 (MG1655 nadR nudC mazG) maintained a larger pool of NAD(H) compared to the wild‐type control, and also accumulated lower concentrations of acetate when grown in batch culture on glucose. In steady‐state cultures, the elevated total NAD(H) found in MEC697 delayed the threshold dilution rate for acetate formation to a growth rate of 0.27 h^?1. Batch and fed‐batch processes using MEC697 were examined for the production of β‐galactosidase as a model recombinant protein. Fed‐batch culture of MEC697/pTrc99A‐lacZ compared to MG1655/pTrc99A‐lacZ at a growth rate of 0.22 h^?1 showed only a modest increase of protein formation. However, 1 L batch growth of MEC697/pTrc99A‐lacZ resulted in 50% lower acetate formation compared to MG1655/pTrc99A‐lacZ and a two‐fold increase in recombinant protein production.