Engineering of Bacillus subtilis for enhanced total synthesis of folic acid.

We investigated whether the yield of the B vitamin folic acid could be elevated in Bacillus subtilis. Strategies for increasing the folic acid yield were investigated by employing computer-aided flux analysis and mutation. Controlling the activity of the enzyme pyruvate kinase by placing it under inducible control was one strategy devised to elevate yield while insuring that a rapid growth rate results. Other single mutation strategies included amplifying the expression of the genes in the folate operon and overexpressing the Escherichia coli aroH gene, which encodes 2-dehydro-3-deoxyphosphoheptonate aldolase. The latter could conceivably elevate the abundance of the folic acid precursor, para-aminobenzoic acid. Strains that combined two or more mutations were also constructed. Overall, a strain possessing inducible pyruvate kinase, overexpressed aroH, and increased transcription and translation of genes from the folic operon exhibited the best yield. The yield was eightfold higher than that displayed by the parent B. subtilis 168 strain.

A metabolic network analysis & NMR experiment design tool with user interface-driven model construction for depth-first search analysis.

A Windows program for metabolic engineering analysis and experimental design has been developed. A graphical user interface enables the pictorial, "on-screen" construction of a metabolic network. Once a model is composed, balance equations are automatically generated. Model construction, modification and information exchange between different users is thus considerably simplified. For a given model, the program can then be used to predict all the extreme point flux distributions that optimize an objective function while satisfying balances and constraints by using a depth-first search strategy. One can also find the minimum reaction set that satisfies different conditions. Based on the identified flux distributions or linear combinations, the user can simulate the NMR and GC/MS spectra of selected signal molecules. Alternately, spectra vectorization allows for the automated optimization of labeling experiments that are intended to distinguish between different, yet plausible flux extreme point distributions. The example provided entails predicting the flux distributions associated with deleting pyruvate kinase and designing 13C NMR experiments that can maximally discriminate between the flux distributions.

Cell growth and by-product formation in a pyruvate kinase mutant of E. coli.

In this paper, we report on the analysis of acid formation in an E. coli pyk mutant. The results demonstrate that acid formation is insignificant for both the wild-type and the mutant at low glucose concentrations. However, at relatively high glucose concentrations, acid formation remains very low for the mutant but is significant for the wild-type. This substantial reduction in acids is accompanied by an increase in CO(2) production. Moreover, unlike the B. subtilis pyk mutant, the E. coli pyk mutant did not show a substantial increase in the PEP pool.

A MILP-based flux alternative generation and NMR experimental design strategy for metabolic engineering.

A mixed-integer linear program (MILP) is described that can enumerate all the ways fluxes can distribute in a metabolic network while still satisfying the same constraints and objective function. The multiple solutions can be used to (1) generate alternative flux scenarios that can account for limited experimental observations, (2) forecast the potential responses to mutation (e.g., new reaction pathways may be used), and (3) (as illustrated) design (13)C NMR experiments such that different potential flux patterns in a mutant can be distinguished. The experimental design is enabled by using the MILP results as an input to an isotopomer mapping matrices (IMM)-based program, which accounts for the network circulation of (13)C from a precursor such as glucose. The IMM-based program can interface to common plotting programs with the result that the user is provided with predicted NMR spectra that are complete with splittings and Lorentzian line-shape features. The example considered is the trafficking of carbon in an Escherichia coli mutant, which has pyruvate kinase activity deleted for the purpose of eliminating acetate production. Similar yields and extracellular measurements would be manifested by the flux alternatives. The MILP-IMM results suggest how NMR experiments can be designed such that the spectra of glutamate for two flux distribution scenarios differ significantly.

Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant.

Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pyk mutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of the pyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO(2) production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pyk mutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.

Expansion of hematopoietic stem cells in vitro as a model system for human tissue engineering.

The authors have taken a new approach to finding optimal conditions for stimulating conservative division of single isolated CD34(+)lin(-) hematopoietic stem cell candidates from human umbilical cord blood. The approach required the design and development of a novel multi-well single cell combinatorial culture system. This system incorporates the use of a multi-well tissue culture plate in which each well receives a single hematopoietic stem cell candidate. During an experiment lasting several days to weeks, each cell-containing well is moved sequentially and serially to a microscopic imaging system. This movement is facilitated by computer control of a motorized stage and stabilization of the experiment in an environmentally controlled Biobox built on the microscopic stage. New image analysis software facilitates tracking of cell movement, recording the time of cell division, and immunophenotyping of multiple, individual, or recently doubled cells in real time by a robotically controlled pipetting station. The principles of single cell culture should help solve many problems in human hematopoietic stem cell expansion and may be applicable to a wide range of other systems of interest in tissue engineering.

13C NMR evidence for pyruvate kinase flux attenuation underlying suppressed acid formation in Bacillus subtilis.

When batch and continuous Bacillus subtilis cultures are provided with a small amount of citrate, acid production ceases, carbon yield increases by more than 2-fold, and the productivity of recombinant protein increases. It has been hypothesized that pyruvate kinase activity is attenuated, which in turn lowers glucose flux and minimizes the acid overflow prompted by low Krebs cycle capacity. To complement existing enzyme activity, linear programming, and metabolite pool studies, (13)C NMR studies were performed. Atom mapping and isotopomer mapping matrix methods were used to select the best glucose label. "Best" was defined such that the NMR spectra of glutamate associated with metabolizing labeled glucose via the different candidate metabolic trafficking scenarios would differ considerably in fine structure (e.g., relative singlet intensities). When experiments were performed with 1-(13)C glucose, the observed NMR spectra corresponded well to the one predicted to arise when the metabolic trafficking occurs according to a pyruvate kinase attenuation scenario. This evidence further fortifies the prospects for successfully basing a metabolic engineering strategy on reducing pyruvate kinase activity to better match glycolytic and Krebs cycle capacities.

Computer model for glucose-limited growth of a single cell of Escherichia coli B/r-A. Reprinted from Biotechnology and Bioengineering, Vol. 26, Issue 3, Pp 203-216 (1984).

A computer model is described which is capable of predicting changes in cell composition, cell size, cell shape, and the timing of chromosome synthesis in response to changes in external glucose limitation. The model is constructed primarily from information on unrestricted growth in glucose minimal medium. The ability of the model to make reasonable quantitative predictions under glucose-limitation is a test of the plausibility of the basic biochemical mechanisms included in the model. Such a model should be of use in differentiating among competing hypotheses for biological mechanisms and in suggesting as yet unobserved phenomena. The last two points are illustrated with the testing of a mechanism for the control of the initiation of DNA synthesis and predictions on cell-width variations during the division cycle.

Expansion of hematopoietic stem cells in vitro as a model system for human tissue engineering.

The authors have taken a new approach to finding optimal conditions for stimulating conservative division of single isolated CD34 + lin hematopoietic stem cell candidates from human umbilical cord blood. The approach required the design and development of a novel multi-well single cell combinatorial culture system. This system incorporates the use of a multi-well tissue culture plate in which each well can receive a single hematopoietic stem cell candidate. Sequential movement of each cell-containing well to a microscopic imaging system, serially over a several-day to several-week experiment, is facilitated by computer control of a motorized stage and stabilization of the experiment in an environmentally controlled Bio-box built on the microscope stage. New image analysis software facilitates in the tracking of cell movement, recording of the time of cell division, and immunophenotyping of each of multiple individual or recently doubled cells in real time by a robotically controlled pipetting station. The principles of single cell culture should help solve many problems in human hematopoietic stem cell expansion and also may be applicable to a wide range of other systems of interest in tissue engineering.

Metabolic fluxes, pools, and enzyme measurements suggest a tighter coupling of energetics and biosynthetic reactions associated with reduced pyruvate kinase flux.

In this study, it is found that, for Bacillus subtilis, citrate-glucose cometabolism leads to zero acid production over a wide range of growth rates and nearly theoretical carbon yield. Experimental results are presented that point to pyruvate kinase (PYK) as a site of citrate-mediated glycolytic flux attenuation. First, the measured fluxes show that, compared with cultures grown on glucose, the PYK flux drops by more than tenfold when citrate is added. Second, relative to cultures metabolizing glucose, the phosphoenolpyruvate (PEP) pool elevates substantially, whereas the pyruvate pool drops, when citrate is present. Finally, our modeling results indicate that maximizing carbon yield corresponds to nearly eliminating pyruvate kinase (PYK) flux and that the pyruvate supplied by the PEP-consuming glucose transport system can supply the biosynthetic requirements. A literature review suggests some mechanisms for how PYK attenuation by citrate addition can occur. At this juncture, we hypothesize that direct PYK inhibition occurs which, in turn, also leads to phosphofructokinase inhibition via the elevated PEP pool. These two inhibition events combine to throttle glycolytic flux; minimize acid formation; and substantially increase cellular, product, and energetic yields.

Performance trade-offs in in situ chemostat NMR.

Investigating cell cultures with NMR requires high cell densities to provide adequate signal-to-noise, or scans must be summed over long time periods and short-term events are lost. The mixing within a chemostat can be used to shorten the time required to acquire informative in situ NMR spectra from cell cultures. However, performance trade-offs can occur between net signal, spectral resolution, and oxygenation due to sampling volume, conductivity, gas bubble, and fluid flow effects. These trade-offs and the effect of different mixing regimes were theoretically analyzed to quantify how device design decisions impact performance. The results were found to concur with data from cell-free NMR experiments performed in 18 mS/cm conductivity medium. The results also guided the redesign of an NMR bioreactor in terms of relative radio frequency (rf) coil and sample dimensions and other factors. The design, which entails using chemostat mixing to shunt sample through a rf coil in ca. 0.4 s, provides adequate oxygenation for the 4-16% (v/v) cell suspensions examined. Gains realized include lower conductive losses, better magnetic field homogeneity, and the exclusion of gas bubbles from the sampling zone. These gains enable the acquistion of spectra from dilute (3-4% v/v) Saccharomyces cerevisiae chemostat cultures in 6.9 min with high resolution in both the orthophosphate and the beta-NTP regions. Samples with 16% (v/v) cells also yield useful spectra within 0.5-1.0 min.

NMR-Observed phosphate trafficking and polyphosphate dynamics in wild-type and vph1-1 mutant Saccharomyces cerevisae in response to stresses.

The phosphagenic, osmotic, and metabolic roles of polyphosphate in chemostat-cultivated yeast were investigated with a new NMR cultivator. Wild-type yeast and a vacuolar vph1-1 mutant, which lacks polyphosphate, were subjected to different stimuli. Starved wild-type yeast exclusively directed phosphate to vacuoles despite other competing sinks. After DNP or iodoacetate exposure, which significantly affected cytosolic pH or ATP metabolism, polyphosphate hydrolysis did not occur, which casts doubt on the phosphagen function of vacuolar polyphosphate. It took about 1 h for Mn2+ to traffic to vacuoles, and some evidence was obtained for polyphosphate responding to osmotic challenges. Fast NMR scans show that rapid polyphosphate hydrolysis to small polymers follows alkalinization. The small polymers then degrade to orthophosphate, which coincides with sugar phosphates increasing and subsequent reacidification. In contrast, when vph1-1 mutants were subjected to alkalinization, the absence of a vacuolar source of phosphate slowed reacidification. Based on known yeast physiology and observed sugar phosphate dynamics, polyphosphate degradation may enable rapid glycogen mobilization to glycolysis for considerable acid and ATP production. Overall, maintaining both polyphosphate and carbohydrate reserves may endow yeast with the ability to rapidly manage the extracellular environment.

Supply-Side Analysis of Growth of Bacillus subtilis on Glucose-Citrate Medium: Feasible Network Alternatives and Yield Optimality.

Volume 63, no. 2, p. 714, Figure 2: panel b should appear as shown below. FIG. 2b [This corrects the article on p. 710 in vol. 63.].

Supply-Side Analysis of Growth of Bacillus subtilis on Glucose-Citrate Medium: Feasible Network Alternatives and Yield Optimality.

Our prior work revealed that compared to the case for glucose metabolism, increased carbon yield and nil acid formation result when Bacillus subtilis grows on glucose medium containing citrate. To scrutinize further how citrate addition may alter metabolic flux regulation and the degree that the observed carbon yield corresponds to the maximal value, experimental (by least-squares analysis) and optimal (by linear programming) fluxes and yields were contrasted. Networks with differing reaction routes, directionality constraints, and transhydrogenase activities were examined. To attain an elevated carbon yield, citrate-glucose utilization need not alleviate any stoichiometric constraints that can sometimes interfere with the attainment of network objectives. Rather, the high carbon yield and nil acid formation attained may be linked to restriction of glycolytic capacity, particularly at the level of pyruvate kinase, which is consistent with a hypothesized effect of coupled metal-citrate uptake. Allowing for malic enzyme activity, hexose monophosphate pathway cycling, and transhydrogenase activity may also lead to the flux distributions underlying the high carbon yield observed. Finally, the observed carbon yield corresponded well to the maximum yield provided by all the network alternatives examined. Collectively, these results suggest that (i) the observed carbon yield is essentially equal to the maximal values associated with plausible networks and (ii), as suggested by others, nonoptimal flux regulation may contribute significantly to apparent cellular maintenance requirements.

In situ 31P nuclear magnetic resonance for observation of polyphosphate and catabolite responses of chemostat-cultivated Saccharomyces cerevisiae after alkalinization.

The proposed pH buffering and phosphagenic functions of polyphosphate were investigated by subjecting chemostat-cultivated Saccharomyces cerevisiae to alkalinization (NaOH addition) and anaerobiosis. The subsequent changes in intracellular phosphate-containing species were observed in situ by nuclear magnetic resonance (NMR) spectroscopy by using the NMR cultivator we developed. For the alkalinization experiments, changes in catabolite secretion were also measured in parallel experiments. Additionally, a range of potential neutralization capacity was investigated: a dilute culture and concentrated cultures with low or high polyphosphate content. The concentrated cultures displayed increased cytosolic pH and rapid polyphosphate degradation to small chains. The pH changes and extent of polyphosphate degradation depended inversely on initial polyphosphate content. The dilute culture restored extracellular pH rapidly and secreted acetate. The concentrated culture with low polyphosphate reserves also secreted acetate. In contrast to the alkalinization-induced polyphosphate dynamics, anaerobiosis resulted in the complete hydrolysis of polyphosphate to P(i), as opposed to small chains, and reduced cytosolic pH. The results and calculations suggest that the bulk of NMR-observable polyphosphate (vacuolar) degradation to short polymers conceivably contributes to neutralizing added alkalinity. In other circumstances, such as anaerobiosis, degradation serves other functions, such as phosphorylation potential regulation.

Using differential scanning calorimetry to elucidate metal-protein binding sites in alpha- and gamma-chymotrypsin.

Crystalline alpha-chymotrypsin preparations are contaminated by the post translational variant, gamma-chymotrypsin. The contaminant can account for 5-50 weight percent of the preparation based on thermal analysis. Such contamination can be problematic because this serine protease has both commercial and deactivation model system utility, and the presence of the contaminant may not be detectable by activity assays. Prior work has shown that simple pH gradient elution can separate the two chymotrypsins when loaded to a Cu(2+)-IMAC column; gamma-chymotrypsin eluted first indicating that its interaction with immobilized Cu2+ is weaker. The molecular features that endow these serine proteases with metal affinity has been investigated further by performing differential scanning calorimetry (DSC) studies in the presence and absence of Cu2+, and at different pH values. The dependence of thermostability on pH for fixed metal concentration reveals an interplay between stabilizing and destabilizing metal binding events. The results are consistent with Cu(2+)-chymotrypsin interaction occurring, in part, through binding to a glutamate- or aspartate-containing chelation site. The strength of this site may differ in the two chymotrypsins.

Sustained and constitutive high levels of protein production in continuous cultures of bacillus subtilis.

The feasibility of continuous production of proteins in chemostat cultures of Bacillus subtilis was investigated. An expression system consisting of the bacterium B. subtilis BR151 carrying plasmid p602/19 was used. The plasmid contains the cat (chioramphenicol acetyltrans-ferase) gene downstream of a strong vegetative T5 promoter. It was found that, at a dilution rate of 0.2 h(-1) production of relatively high levels of CAT protein (about 4% ofcellular protein) can be sustained. But, experiments at a higher dilution rate of 0.4 h(-1) were unproductive because of high acidformation and washout. Combination of low cell yield, which results from excessive acid formation, and low dilution rate led to a low volumetric CAT productivity. Our recent work with the nonrecombinant cells, has demonstrated that uptake of small amounts of citrate significantly reduces or entirelyeliminates the acid formation. This superior performance in the presence ofcitrate was hypothesized, based on strong experimental evidence, to be the result of a reduction in glycolysis flux through a sequence of events leading to a reduction in pyruvate kinase and phosphof- ructokinase activities, the regulatory enzymes of glycol-ysis. In this study, it is demonstrated that cofeeding of glucose and citrate substantially reduces theorganic acid formation and significantly increases the recombinant culture productivity. The combination of high specific CAT activity and cell density resulted in a total of six- to tenfold higher culture productivitywhen citrate and glucose were cometabolized than when glucose was the only carbon source. (c) 1995 John Wiley & Sons Inc.

Suppressed acid formation by cofeeding of glucose and citrate in Bacillus cultures: emergence of pyruvate kinase as a potential metabolic engineering site.

Microbial cultures typically produce acids when metabolizing the common carbon source, glucose. Acid production not only represents a waste of carbon, but its accumulation can limit cell concentration and culture stability, thereby reducing productivity. On the basis of prior work, acid production was attributed to be due to a mismatch between glycolytic and tricarboxylic acid (TCA) cycle capacities. To suppress acid production, a strategy entailing adding citrate to glucose minimal medium proved extremely effective. The effect of citrate on in-vivo flux distribution was quantified using a detailed flux-model. When the molar glucose-citrate ratio was varied between 3 and 6, a significant reduction in glycolytic flux and essentially complete suppression of acid formation was found as compared to chemostat cultures grown solely on glucose. Adding other biosynthetic precursors such as glutamine did not invoke the same suppression, thus indicating that citrate's effect is at the regulatory level. We hypothesized that the reduction of glycolytic flux in the presence of citrate results from its transport being coupled with the uptake of divalent metal ions. Citrate transport alters the intracellular balance of metal ions which in turn could trigger a sophisticated series of metabolic events leading to reduction of the activities of the pyruvate kinase and phosphofructokinase (PFK), the regulatory enzymes of glycolysis. On the basis of this scenario and other regulatory information, pyruvate kinase has emerged as a potential metabolic engineering site. It's deactivation in Bacillus subtilis or Escherichia coli strains is expected to yield constructs with a much lower tendency for making acid byproducts.