Growth characteristics and metabolic flux analysis of Candida milleri.

The growth characteristics of the sourdough yeast Candida milleri was studied in a carbon-limited aerobic chemostat culture on defined medium. The effect of glucose, xylose, and glucose-xylose mixture on metabolite production and on key enzyme activities was evaluated. Xylose as a sole carbon source was not metabolized by C. milleri. Glucose as a sole carbon source produced only biomass and carbon dioxide. When a glucose-xylose mixture (125:125 C-mM) was used as a carbon source, a small amount of xylose was consumed and a low concentration of xylitol was produced (7.20 C-mM). Enzymatic assays indicated that C. milleri does not possess xylitol dehydrogenase activity and its xylose reductase is exclusively NADPH-dependent. In glucose medium both NAD(+)- and NADP(+)-dependent aldehyde dehydrogenase activities were found, whereas in a glucose-xylose medium only NADP(+)-dependent aldehyde dehydrogenase activity was detected. The developed metabolic flux analysis corresponded well with the experimentally measured values of metabolite production, oxygen consumption (OUR), and carbon dioxide production (CER). Turnover number in generation and consumption of ATP, mitochondrial and cytosolic NADH, and cytosolic NADPH could be calculated and redox balance was achieved. Constraints were imposed on the flux estimates such that the directionality of irreversible reactions is not violated, and cofactor dependence of the measured enzyme activities were taken into account in constructing the metabolic flux network.

Metabolic flux analysis of Escherichia coli expressing the Bacillus subtilis acetolactate synthase in batch and continuous cultures.

Metabolically engineered Escherichia coli expressing the B. subtilis acetolactate synthase has shown to be capable of reducing acetate accumulation. This reduction subsequently led to a significant enhancement in recombinant protein production. The main focus of this study is to systematically examine the effect of ALS in the metabolic patterns of E. coli in batch and continuous culture. The specific acetate production rate of a strain carrying the B. subtilis als gene is 75% lower than that of the control strain (host carrying the control plasmid pACYC184) in batch cultures. The ALS strain is further demonstrated to be capable of maintaining a reduced specific acetate production rate in continuous cultures at dilution rates ranging from 0.1 to 0.4 h-1. In addition, this ALS strain is shown to have a higher ATP yield and lower maintenance coefficient. The metabolic flux analysis of carbon flux distribution of the central metabolic pathways and at the pyruvate branch point reveals that this strain has the ability to channel excess pyruvate to the much less toxic compound, acetoin.

Improvement of biomass yield and recombinant gene expression in Escherichia coli by using fructose as the primary carbon source.

The feasibility of substituting glucose with fructose as a carbon source in Escherichia coli fermentations was investigated. Glucose, the most commonly used sugar in bacterial cultivations, is well-known to pose a number of drawbacks; the most important of which is the Crabtree effect, which results in acidogenesis. Fructose, a glucose structural isomer, offers a reasonable alternative for glucose, since its uptake and utilization are more tightly regulated. Comparative fermentation studies indicate that lower acetate excretion and higher biomass yields were attained in fructose-supplemented growth media compared with those of glucose media. More specifically, cells grown in defined media supplemented with fructose do not excrete detectable amounts of acetate, while about 40 mM of acetate was detected extracellularly in similar glucose cultures. A reduction in the initial growth rate of about 20% was observed with fructose, but final cell densities were about 70% higher compared with glucose supplements. Growth in complex LB media supplemented with fructose again resulted in higher biomass yields (up to 40%) and lower acetate excretion (30-40%) than the comparable glucose media. In bioreactor studies using LB media, acetate levels were reduced from 90 to less than 6 mM, while achieving a 25% improvement in biomass yield. When using richer media, cell densities of more than 40 g L-1 dry cell weight were attained in batch cultivation using fructose compared with 30 g L-1 for glucose. These results have immense applicability in the area of recombinant protein processes. Recombinant E. coli, overexpressing beta-galactosidase under the control of the strong pH-inducible promoter, achieved a volumetric recombinant protein yield of 2.2 million U mL-1 (corresponding to approximately 1.5 g L-1) in batch fructose cultures. This represents a 65% recombinant protein yield enhancement when compared to similar glucose cultivations.

Metabolic flux analysis of Escherichia coli deficient in the acetate production pathway and expressing the Bacillus subtilis acetolactate synthase.

Several approaches to reduce acetate accumulation in Escherichia coli cultures have recently been reported. This reduction subsequently led to a significant enhancement in recombinant protein production. In those studies, metabolically engineered E. coli strains with reduced acetate synthesis rates were constructed through the modification of glucose uptake rate, the elimination of critical enzymes that are involved in the acetate formation pathways, and the redirection of carbon flux toward less inhibitory byproducts. In particular, it has been shown that strains carrying the Bacillus subtilis acetolactate synthase (ALS) gene not only produce less acetate but also have a higher ATP yield. Metabolic flux analysis of carbon flux distribution of the central metabolic pathways and at the pyruvate branch point revealed that this strain has the ability to channel excess pyruvate to the much less toxic compound, acetoin. The main focus of this study is the systematic analysis of the effects of small perturbations in the host's existing pathways on the redistribution of carbon fluxes. Specifically, a mutant with deleted acetate kinase (ACK) and acetyl phosphotransferase (PTA) was constructed and studied. Results from the metabolic analysis of carbon redistribution show the ackA-pta mutation will reduce acetate level at the expense of the growth rate. In addition, in the ackA-pta deficient strain a much higher lactate formation rate with simultaneously lower formate and ethanol synthesis rates was found. Expression of the B. subtilis ALS in ackA-pta mutants further reduces acetate levels while cell density similar to that of the parent strain is attained.

Characterization of a pH-inducible promoter system for high-level expression of recombinant proteins in Escherichia coli.

A pH-inducible promoter system was characterized and its potential applicability in recombinant protein production was evaluated using a plasmid construct, pSM552-545C(-), in which the promoter and activator coding sequences of the cad operon were inserted into the upstream region of a lacZ' reporter gene. Graded gene expression levels with respect to culture pH between 8.0 and 5.5 were observed and the induction range can be as high as 200-fold. The effects of several cultivation parameters, including pH, temperature, induction cell density, and inoculum size, were systematically examined. The practical application of this expression system to high level production of recombinant proteins was successfully demonstrated using a rich medium, superbroth. An extremely high recombinant protein productivity at a value of approximately 1.4 g/L with a specific expression level as high as 35% of total cellular protein can be obtained in a simple batch cultivation. The behavior of this expression system was further investigated using chemostat cultures. An uncommon relationship between the volumetric or specific recombinant protein activity and the dilution rate, with a maximal activity at a dilution rate of approximately 0.4 h(-1)was observed. (c) 1995 John Wiley & Sons, Inc.

Metabolic engineering of Escherichia coli to enhance recombinant protein production through acetate reduction.

Genetic and metabolic engineering provide powerful and effective tools for the systematic manipulation and fine tuning of cellular metabolic activities. In this study, successful application of such techniques to enhance recombinant protein production by reducing acetate accumulation in Escherichia coli is presented. The alsS gene from Bacillus subtilis encoding the enzyme acetolactate synthase was introduced into E. coli cells using a multicopy plasmid. This newly introduced heterologous enzyme modifies the glycolytic fluxes by redirecting excess pyruvate away from acetate to acetolactate. Acetolactate is then converted to a nonacidic and less harmful byproduct acetoin, which appears in the broth. Furthermore, comparative fermentation studies show that the reduction in acetate accumulation leads to a significant improvement of recombinant protein production. The expression of a model recombinant CadA/beta-galactosidase fusion protein, under the control of a strong pH-regulated promoter, was found to increase by about 60% for the specific protein activity (to a level of 30% of total cellular protein) and 50% in terms of the volumetric activity in a batch fermenter. In fed-batch cultivation, the engineered strain achieved a volumetric recombinant protein yield of 1.6 million units/mL (about 1.1 g/L of beta-galactosidase), which represented a 220% enhancement over the control strain. In the meantime, acetate excretion was maintained below 20 mM compared with 80 mM for the control, and the final cell density was improved by 35%.

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.

Strategies in high-level expression of recombinant protein in Escherichia coli.

The accumulation of acetate is one of the most commonly encountered problems in attaining high levels of recombinant protein production using E. coli. Two different approaches are examined to reduce the rate of acetate formation. The effects of reduced acetate accumulation on recombinant protein production were also investigated. In the first approach, E. coli mutant strains deficient in enzymes involved in the acetate synthesis pathways were isolated and characterized. The level of specific production of beta-galactosidase by the mutant strain is three times higher than its parent strain. In another approach, metabolic engineering techniques were employed to fine-tune the central metabolic pathways to reduce the amount of acetate formation. The resulting strain, which carries the acetolactase synthase gene from B. subtilis, is successful in maintaining a very low level of acetate accumulation. The ALS-containing strain is also capable of producing higher levels of recombinant protein than its parent strain.

An optimization study of a pH-inducible promoter system for high-level recombinant protein production in Escherichia coli.

The unique properties exhibited by the pH-inducible promoter system are clearly demonstrated by the plasmid construct, pSM552-545C-. Step changes of pH substantially increase the expression of beta-galactosidase. Very high expression, a level of around 40% of total cellular protein, can be achieved with superbroth. The high level of induction in rich media, typical of those commonly used to achieve high cell density, suggests the system is versatile enough to be adapted to many specific situations. The variable degree of induction by pH within the range of 8.0 and 5.5 makes possible a degree of expression control not easily accomplished with the existing systems. By precise monitoring of induction pH, a "fine tuning" of foreign gene expression and growth rate to optimum levels is possible. The effect of several operating parameters on recombinant protein production are evaluated. Our results show that operating environments play an extremely important role in achieving high recombinant protein expression levels in a dense culture. Under suboptimal conditions, as are shown in this study, only moderately high levels can be obtained. Even for suboptimal cases, an expression level of about 10 to 15% of total cellular protein while achieving an optical density higher than 25 is routinely obtained. Our results also show that a proper balance between cell growth and recombinant protein synthesis processes are critical in maintaining high expression levels in a dense culture. Any imbalance will most likely lead to more cell growth and poorer protein productivity. We have also demonstrated that reactor operating temperature can be a useful parameter to fine-tune this balance, resulting in significantly improved results.