Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria

Multicopy plasmids are often chosen for the expression of recombinant genes in Escherichia coli. The high copy number is generally desired for maximum gene expression; however, the metabolic burden effects that usually result from multiple plasmid copies could prove to be detrimental for maximum productivity in certain metabolic engineering applications. In this study, low-copy mini-F plasmids were compared to high-copy pMB1-based plasmids for production of two metabolites in E. coli: polyphosphate (polyP) and lycopene derived from isopentenyl diphosphate (IPP). The stationary-phase accumulation of polyP on a per cell basis was enhanced approximately 80% when either high- or low-copy plasmids were used, from 120 micromol/g DCW without augmented polyP kinase (PPK) activity to approximately 220 micromol/g DCW. The cell density of the high-copy plasmid-containing culture at stationary phase was approximately 24% lower than the low-copy culture and 30% lower than the control culture. This difference in cell density is likely a metabolic burden effect and resulted in a lower overall product concentration for the high-copy culture (approximately 130 micromol/L culture) relative to the low-copy culture (approximately 160 micromol/L culture). When the gene for DXP (1-deoxy-D-xylulose 5-phosphate) synthase, the first enzyme in the IPP mevalonate-independent biosynthetic pathway, was expressed from the tac promoter on multicopy and low-copy plasmids, lycopene production was enhanced two- to threefold over that found in cells expressing the chromosomal copy only. Cell growth and lycopene production decreased substantially when isopropyl beta-D-thiogalactosidase (IPTG) was added to the high-copy plasmid-containing culture, suggesting that overexpression of DXP synthase was a significant metabolic burden. In the low-copy plasmid-containing culture, no differences in cell growth or lycopene production were observed with any IPTG concentrations. When dxs was placed under the control of the arabinose-inducible promoter (P(BAD)) on the low-copy plasmid, the amount of lycopene produced was proportional to the arabinose concentration and no significant changes in cell growth resulted. These results suggest that low-copy plasmids may be useful in metabolic engineering applications, particularly when one or more of the substrates used in the recombinant pathway are required for normal cellular metabolism.     

Modification of central metabolic pathway in escherichia coli to reduce acetate accumulation by heterologous expression of the bacillus subtilis acetolactate synthase gene

A novel metabolic engineering technique involving the redirection ofcellular carbon fluxes was employed to reduce acetate production in an Escherichia coli culture. Metabolic engineering was achieved by cloning E. coli the gene for the Bacillus subtilis acetolactate synthase (ALS), an enzyme capable of catalyzing the conversion of pyruvate to nonacidic and less harmful species. The heterologous expression of the ALS catabolic enzyme in Escherichia coli drastically modified the cellular glycolytic fluxes. In particular, acetate excretion, which is a common characteristic of E. coli, as well as a physiological burden, was minimized. The residual acetate level was kept under control and maintained at a level that was below the toxic threshold. The expression of the biologically active ALS enzyme in E. coli did not result in any detectable changes on either cell growth rate or cell yields. The alternative product, acetoin, was shown to be 50 times less harmful than acetate. Similarities in the growth pattern of two different E. coli strains, RR1 and GJT001, under all cultivation conditions suggested that the ability of ALS to reduce acetate accumulation is generic and not strain-specific. (c) 1994 John Wiley & Sons, Inc.     

Mediators of galactose sensitivity in UDP-galactose 4'-epimerase-impaired mammalian cells

UDP-galactose 4'-epimerase (GALE) catalyzes the final step in the Leloir pathway of galactose metabolism, interconverting UDP-galactose and UDP-glucose. Unlike its Escherichia coli counterpart, mammalian GALE also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. Considering the key roles played by all four of these UDP-sugars in glycosylation, human GALE therefore not only contributes to the Leloir pathway, but also functions as a gatekeeper overseeing the ratios of important substrate pools required for the synthesis of glycosylated macromolecules. Defects in human GALE result in the disorder epimerase-deficiency galactosemia. To explore the relationship among GALE activity, substrate specificity, metabolic balance, and galactose sensitivity in mammalian cells, we employed a previously described GALE-null line of Chinese hamster ovary cells, ldlD. Using a transfection protocol, we generated ldlD derivative cell lines that expressed different levels of wild-type human GALE or E. coli GALE and compared the phenotypes and metabolic profiles of these lines cultured in the presence versus absence of galactose. We found that GALE-null cells accumulated abnormally high levels of Gal-1-P and UDP-Gal and abnormally low levels of UDP-Glc and UDP-GlcNAc in the presence of galactose and that human GALE expression corrected each of these defects. Comparing the human GALE- and E. coli GALE-expressing cells, we found that although GALE activity toward both substrates was required to restore metabolic balance, UDP-GalNAc activity was not required for cell proliferation in the presence of otherwise cytostatic concentrations of galactose. Finally, we found that uridine supplementation, which essentially corrected UDP-Glc and, to a lesser extent UDP-GlcNAc depletion, enabled ldlD cells to proliferate in the presence of galactose despite the continued accumulation of Gal-1-P and UDP-Gal. These data offer important insights into the mechanism of galactose sensitivity in epimerase-impaired cells and suggest a potential novel therapy for patients with epimerase-deficiency galactosemia.     

Comparative metabolic analysis of lactate for CHO cells in glucose and galactose

t-PA producing CHO cells have been shown to undergo a metabolic shift when the culture medium is supplemented with a mixture of glucose and galactose. This metabolic change is characterized by the reincorporation of lactate and its use as an additional carbon source. The aim of this work is to understand lactate metabolism. To do so, Chinese hamster ovary cells were grown in batch cultures in four different conditions consisting in different combinations of glucose and galactose. In experiments supplemented with glucose, only lactate production was observed. Cultures with glucose and galactose consumed glucose first and produced lactate at the same time, after glucose depletion galactose consumption began and lactate uptake was observed. Comparison of the metabolic state of cells with and without the shift by metabolic flux analysis show that the metabolic fluxes distribution changes mostly in the reactions involving pyruvate metabolism. When not enough pyruvate is being produced for cells to support their energy requirements, lactate dehydrogenase complex changes the direction of the reaction yielding pyruvate to feed the TCA cycle. The slow change from high fluxes during glucose consumption to low fluxes in galactose consumption generates intracellular conditions that allow the influx of lactate. Lactate consumption is possible in cell cultures supplemented with glucose and galactose due to the low rates at which galactose is consumed. Evidence suggests that an excessive production and accumulation of pyruvate during glucose consumption leads to lactate production and accumulation inside the cell. Other internal conditions such as a decrease in internal pH, forces the flow of lactate outside the cell. After metabolic shift the intracellular pool of pyruvate, lactate and H⁺ drops permitting the reversal of the monocarboxylate transporter direction, therefore leading to lactate uptake. Metabolic analysis comparing glucose and galactose consumption indicates that after metabolic shift not enough pyruvate is produced to supply energy metabolism and lactate is used for pyruvate synthesis. In addition, MFA indicates that most carbon consumed during low carbon flux is directed towards maintaining energy metabolism.     

Synthesis and posttranslational regulation of pyruvate formate-lyase in Lactococcus lactis

The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L. lactis PFL. It was demonstrated that activated L. lactis PFL was sensitive to oxygen, as in E. coli, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions. The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose. This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively. During anaerobic growth in defined medium, complete activation of PFL was observed. Strikingly, although no formate was produced during aerobic growth of L. lactis, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen. In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium. This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation. In E. coli, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE. It was shown that in L. lactis, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed. Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in E. coli and L. lactis.     

Development of a fed-batch cultivation for antibody-producing cells based on combined feeding strategy of glucose and galactose

In order to achieve enhanced cell mass and productivity with less lactate accumulation, a fed-batch culture based on a combined feeding strategy of glucose and galactose was developed. Cell performance was first examined with feeding of galactose alone. While cell growth was improved compared with glucose-feeding culture, cell maintenance was inefficient with rapid lactate depletion and considerable ammonium accumulation. Subsequently, to improve cell maintenance, a combined feeding strategy of glucose and galactose was proposed focusing on optimizing the ratio of glucose to galactose and feeding time. In addition, the compositions of amino acids and vitamins in feeding medium were refined for balanced supply of nutrients. With the combined feeding strategy, the metabolic shift of lactate from production to consumption occurred, but not accompanied by rapid lactate depletion and ammonium production. Furthermore, energy metabolism was more efficient and better utilization of carbon sources was achieved. Compared with the glucose-feeding culture in bioreactor, maximum lactate concentration was reduced by 55%; IVCC and the specific production rate of antibody were increased by 45% and 143%, respectively.     

Relationship between UDP-galactose 4'-epimerase activity and galactose sensitivity in yeast

UDP-galactose 4'-epimerase (GALE) catalyzes the final step of the highly conserved Leloir pathway of galactose metabolism. Loss of GALE in humans results in a variant form of the metabolic disorder, galactosemia. Loss of GALE in yeast results in galactose-dependent growth arrest. Although the role of GALE in galactose metabolism has been recognized for decades, the precise relationship between GALE activity and galactose sensitivity has remained unclear. Here we have explored this relationship by asking the following. 1) Is GALE rate-limiting for galactose metabolism in yeast? 2) What is the relationship between GALE activity and galactose-dependent growth arrest in yeast? 3) What is the relationship between GALE activity and the abnormal accumulation of galactose metabolites in yeast? To answer these questions we engineered a strain of yeast in which GALE was doxycycline-repressible and studied these cells under conditions of intermediate GALE expression. Our results demonstrated a smooth linear relationship between galactose metabolism and GALE activity over a range from 0 to approximately 5% but a steep threshold relationship between growth rate in galactose and GALE activity over the same range. The relationship between abnormal accumulation of metabolites and GALE activity was also linear over the range from 0 to approximately 5%, suggesting that if the abnormal accumulation of metabolites underlies galactose-dependent growth-arrest in GALE-impaired yeast, either the impact of individual metabolites must be synergistic and/or the threshold of sensitivity must be very steep. Together these data reveal important points of similarity and contrast between the roles of GALE and galactose-1-phosphate uridylyltransferase in galactose metabolism in yeast and provide a framework for future studies in mammalian systems.     

Evaluating metabolic stress and plasmid stability in plasmid DNA production by Escherichia coli

In the context of recombinant DNA technology, the development of feasible and high-yielding plasmid DNA production processes has regained attention as more evidence for its efficacy as vectors for gene therapy and DNA vaccination arise. When producing plasmid DNA in Escherichia coli, a number of biological restraints, triggered by plasmid maintenance and replication as well as culture conditions are responsible for limiting final biomass and product yields. This termed "metabolic burden" can also cause detrimental effects on plasmid stability and quality, since the cell machinery is no longer capable of maintaining an active metabolism towards plasmid synthesis and the stress responses elicited by plasmid maintenance can also cause increased plasmid instability. The optimization of plasmid DNA production bioprocesses is still hindered by the lack of information on the host metabolic responses as well as information on plasmid instability. Therefore, systematic and on-line approaches are required not only to characterise this "metabolic burden" and plasmid stability but also for the design of appropriate metabolic engineering and culture strategies. The monitoring tools described to date rapidly evolve from laborious, off-line and at-line monitoring to online monitoring, at a time-scale that enables researchers to solve these bioprocessing problems as they occur. This review highlights major E. coli biological alterations caused by plasmid maintenance and replication, possible causes for plasmid instability and discusses the ability of currently employed bioprocess monitoring techniques to provide information in order to circumvent metabolic burden and plasmid instability, pointing out the possible evolution of these methods towards online bioprocess monitoring.     

Considerations on the lactate consumption by CHO cells in the presence of galactose

A CHO cell line producing t-PA was cultured using glutamate and glucose or galactose to decrease the formation of metabolic end-products and therefore improving the process. In batch cultures using glutamate (6 mM) with glucose at two different levels (5 and 20 mM) or with glucose and galactose (5 and 20 mM, respectively) a remarkable difference in cell culture parameters was evidenced. For 20 mM glucose, a usual cell pattern was observed with lactate built-up in the medium. For 5 mM glucose, cell growth was arrested due to glucose depletion and only a limited use of the excreted lactate could be observed, not supporting cell growth sufficiently. However, when glucose 5 mM and galactose 20 mM were used together, cells consumed the glucose first and, interestingly, in a second phase they continued growing on galactose with the simultaneous consumption of the endogenous lactate. Under these conditions, cell growth was even improved with respect to growth on 20 mM glucose, used as a control. This metabolic behavior is further investigated by using metabolic flux analysis, suggesting that the lactate produced is not used in the oxidative metabolism through the TCA cycle. Metabolic fate of the lactate consumed is discussed.     

Mutants in transmission of chemotactic signals from two independent receptors of E. coli.

We have characterized chemotactic mutants of E. coli that appear to be defective in a common linkage of two independent receptors to the central chemotactic components. The mutants do not respond to gradients of ribose or galactose and thus are called trg (taxis to ribose and galactose), after Ordal and Adler (1974b). These trg mutants are indistinguishable from their parent in tactic response to other attractants, swimming pattern, growth rates, and transport of ribose and galactose. The mutant cells contain the usual amounts of ribose and galactose receptors, and those proteins function normally in their other role, transport of their respective ligands. The mutations, generated by insertion of translocatable drug-resistance elements (transposons)8 are located near 31 min on the map of the E. coli chromosome, a locus far removed from the genes coding for the ribose and galactose receptors. Trg mutants do not resemble either specific receptor mutants or che mutants. The nature of the requirement for the trg product in the response to ribose and galactose is not defined, but evidence for interference of tactic signals from the ribose and galactose receptors (Strange and Koshland, 1976) supports the idea that the product functions directly in the transmission of tactic signals from the two receptors to the flagella.     

Decoupling cell growth and product formation in Chinese hamster ovary cells through metabolic control.

The development of a strategy for the culture of Chinese hamster ovary (CHO) cells producing tissue plasminogen activator (t-PA) is investigated. This strategy is based on the replacement of the main carbon source, glucose, by another compound that is slowly metabolizable, particularly galactose. The introduction of this change allows for acute change in cell behavior at various levels. Cell growth is stopped after this nutrient shift, and the cells can be kept in long-duration culture at a low growth rate and high viability as compared with a culture strategy based solely on glucose utilization. Moreover, the capability of cells to produce recombinant proteins (t-PA in this work) can be maintained over the entire period of galactose feeding. From the metabolic point of view, use of a slowly metabolizable carbon source (galactose) introduces important changes in the production of lactate, ammonia, and some amino acids. The use of this metabolic shift enables the generation of biphasic processes, with a first phase with cell growth on glucose and a second stationary phase on galactose, which is particularly suited to perfusion systems.     

Genetic reconstruction of the aerobic central metabolism in Escherichia coli for the absolute aerobic production of succinate

Most reported efforts to enhance production of the industrially valuable specialty chemical succinate have been done under anaerobic conditions, where E. coli undergoes mixed-acid fermentation. These efforts have often been hampered by the limitations of NADH availability, poor cell growth, and slow production. An aerobic succinate production system was strategically designed that allows E. coli to produce and accumulate succinate efficiently and substantially as a product under absolute aerobic conditions. Mutations in the tricarboxylic acid cycle (sdhAB, icd, iclR) and acetate pathways (poxB, ackA-pta) of E. coli were created to construct the glyoxylate cycle for aerobic succinate production. Experiments in flask studies showed that 14.28 mM of succinate could be produced aerobically with a yield of 0.344 mole/mole using 55 mM glucose. In aerobic batch reactor studies, succinate production rate was faster, reaching 0.5 mole/mole in 24 h with a concentration of 22.12 mM; further cultivation showed that succinate production reached 43 mM with a yield of 0.7. There was also substantial pyruvate and TCA cycle C(6) intermediate accumulation in the mutant. The results suggest that more metabolic engineering improvements can be made to this system to make aerobic succinate production more efficient. Nevertheless, this aerobic succinate production system provides the first platform for enhancing succinate production aerobically in E. coli based on the creation of a new aerobic central metabolic network.     

Catabolite inhibition: a general phenomenon in the control of carbohydrate utilization

When Escherichia coli is grown in synthetic medium with radioactive galactose or lactose as the carbon source, the addition of glucose rapidly inhibited utilization of the radioactive substrate, whether the formation of (14)CO(2) or acid-insoluble products was measured. The inhibition was reversed after the removal of glucose. Experiments with mutants blocked in subsequent steps of galactose and lactose metabolism demonstrated that the inhibition occurs prior to the formation of the first metabolic product. The utilization of a variety of sugars, including maltose, lactose, mannose, galactose, l-arabinose, xylose, and glycerol was inhibited by glucose. Of a number of carbohydrates tested as potential inhibitors, only glucose and, to a lesser extent, glucose-6-phosphate (G-6-P) were capable of inhibiting the utilization of all of the substrates. Glucose did not inhibit G-6-P utilization but G-6-P inhibited glucose utilization. With all substrates, except glycerol, there was a delay before the onset of inhibition by G-6-P. We conclude that E. coli has a general regulatory mechanism, termed catabolite inhibition, which controls the activity of early reactions in carbohydrate metabolism, allowing certain substrates to be utilized preferentially.     

Galactose inhibition of citric acid production from glucose by Aspergillus niger

Summary Previous work in this laboratory has demonstrated that although Aspergillus niger can readily utilize galactose, no citric acid is produced from this carbon source (Hossain et al. 1984). Experiments were now conducted where galactose was added at various concentrations to synthetic growth medium containing glucose as carbon source, so that the effect of galactose on citric acid production from glucose could be observed. The results showed that the presence of galactose or a product of galactose metabolism caused inhibition of citric acid production, and also reduced the rate of glucose utilization. Enzyme analyses using mycelial cell-free extracts indicated that galactose interfered with the glucose-repression of the key enzyme 2-oxoglutarate dehydrogenase.     

Plasmid maintenance and physiology of a genetically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> strain during continuous <Emphasis Type="SmallCaps">l</Emphasis>-carnitine production

The effect of immobilization on cell physiology and how this determines cell metabolic performance is an important concern for developing bioprocess. This is particularly true for genetically modified microorganisms and their genetic stability. For this reason the stability and physiological state of plasmid-bearing E. coli cells were ascertained by flow cytometry. Differences in the cellular DNA and protein content (15-20%) permit discrimination of control and plasmid-bearing cells, as well as adaptation to continuous cultivation conditions in both freely suspended and immobilized states to be monitored. Moreover, the observed metabolic burden due to maintenance and over-expression of plasmid-coded genetic material and slow cell growth in poorly-viable immobilized cells were found to be the main factors contributing to strain stabilization.     

Characterization of the acetate-producing pathways in Escherichia coli

Although the bacterium E. coli is chosen as the host in many bioprocesses, the accumulation of a common byproduct, acetate, is often problematic. Acetate, when present at high levels, will inhibit both cell growth and recombinant protein productivity. In addition, products derived from the central aerobic metabolic pathway often compete with the acetate-producing pathways poxB and ackA-pta for glucose as the substrate. As such, a significant portion of the glucose may be excreted as acetate, wasting substrate that otherwise could have been used for the desired product. We have created mutant E. coli strains with a deletion of either the poxB or the ackA-pta pathway. These two strains, along with the wild-type strain, have been studied in batch reactors over a 12 h time period, at pH 7.0 and 6.0. The wild-type strain has also been studied using glucose as the carbon source. Data were collected to correlate cellular growth, extracellular metabolite production, enzyme activity, and gene expression. Results show that the ackA-pta pathway dominates in exponential phase, and the poxB pathway dominates in stationary phase. The ackA-pta pathway is repressed in acidic environments, whereas the poxB pathway is activated.     

GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae.

The gene encoding the galactose permease of Saccharomyces cerevisiae (GAL2) was cloned. The clone restores galactose permease activity to gal2 yeasts and is regulated by galactose in a manner similar to other GAL gene products (GAL1, -7, and -10). Experiments with temperature-conditional secretory mutants indicated that transport of the GAL2 gene product to the cell surface requires a functional secretory pathway. In addition, gene fusions were constructed between the GAL2 gene and the Escherichia coli lacZ gene. The GAL2-lacZ gene fusions code for galactose-regulated beta-galactosidase activity in yeasts. The beta-galactosidase activity was found to be membrane bound.     

In vivo dynamics of galactose metabolism in Saccharomyces cerevisiae: metabolic fluxes and metabolite levels

The dynamics of galactose metabolism in Saccharomyces cerevisiae was studied by analyzing the metabolic response of the CEN.PK 113-7D wild-type strain when exposed to a galactose pulse during aerobic growth in a galactose-limited steady-state cultivation at a dilution rate of 0.097 h(-1). A fast sampling technique and subsequent methanol-chloroform/solid phase extractions were applied for in vivo measurements of the dynamic changes of the AMP, ADP, ATP levels and the sugar phosphates of the Leloir pathway. The ATP level was found to be significantly lower for yeast growing under galactose limitation (0.37 +/- 0.05 micromol/g CDW) than what has been reported for growth under glucose limitation. The galactose pulse of 5.58 mM was consumed within 40 min (t = 40) and 7 min after the pulse was added cell growth stopped. Subsequently, the cells started to grow and at t = 30 the specific growth rate had recovered to half the steady-state growth rate (0.047 h(-1)). To evaluate the change in flux distribution at steady state and during the galactose transient, a stoichiometric model describing the aerobic metabolism of S. cerevisiae was set up for quantification of the metabolic fluxes. At t = 7 the flux entering the TCA cycle was low and acetate and ethanol started to be excreted to the extracellular medium. During recovery of cell growth the flux entering the TCA cycle increased again, and at t = 30 this flux exceeded the corresponding steady-state flux. During the pulse an enhanced level of Gal-1P was measured, which may be responsible for a toxic metabolic response in S. cerevisiae. The increase in the Gal-1P concentration is intensified by the low affinity of Gal7 towards Gal-1P and, hence, under the physiological conditions examined Gal7 seems to exert control over flux through the Leloir pathway.     

Galactose can be an inducer for production of therapeutic proteins by auto-induction using E. coli BL21 strains

Recently lactose mediated auto-induction in Escherichia coli has gained a lot of interest because higher protein titer could be achieved without the need to monitor growth and add inducer at the proper time. In this study a high level therapeutic protein production by auto-induction was observed in E. coli BL21 using either T7 or tac promoters in the modified Luria Bertani (mLB) medium containing soy peptone instead of tryptone in Luria Bertani (LB) medium. Based on medium analysis and spiking experiments it was found that 0.4 mM galactose from the soy peptone caused the auto-induction. E. coli cultures induced by galactose can saturate at considerably higher density than cultures induced by IPTG. Galactose is not consumed by E. coli BL21. Finally it has been demonstrated that auto-induction can be effectively used in fed-batch fermentation for the industrial production of a therapeutic protein. The principle of galactose mediated auto-induction should be able to apply to high throughput microplates, shake flasks and fed-batch fermentors for clone screening and therapeutic protein expression in E. coli gal(-) strains such as most commonly used BL21.