BACKGROUND
pseudomonas putida is a non-pathogenic, soil bacterium that developed a notably versatile metabolism and thus can inhabit several environmental niches.
numerous strains, some of them being solvent tolerant  <cit> , are able to produce efficiently a range of bulk and fine chemicals or, conversely, degrade various substances that are by products or waste of industrial processes  <cit> .
these features, along with the amenability for genetic manipulation and suitability as a host for the expression of heterologous genes have rendered p. putida an attractive object of research for biotechnological applications  <cit> .
the sequencing of the genome of p. putida brought a significant leap in developing its applications by unveiling the metabolic potential encoded in the genome  <cit> .
in an effort to enable the analysis of p. putida kt <dig> from a systems biology perspective and to foster the development of its biotechnological applications, we built a constraint-based metabolic reconstruction  of this strain  <cit> .
although a lot of knowledge pertaining to this strain was collected while building the model, some information had to be approximated from the escherichia coli model  <cit> .
the information approximated included the biomass composition of the bacterium and the energetic expenses related to growth and maintenance of life-functions )  <cit> .
one of the most common assumptions underlying simulations built upon genome-scale constraint-based metabolic reconstructions is that bacteria try to maximize their growth yield.
consequently, growth associated parameters can be specified prior to performing simulations of the growth, which provide a basis for numerous applications of metabolic reconstructions including rational strain design for biotechnological  applications.
furthermore, these parameters influence significantly the outcome of such simulations, as they confine fluxes of important parts of the metabolism  <cit> .
moreover, they may vary significantly not only among organisms, but also among different growth conditions.
thus, for applications of metabolic reconstructions in biotechnology, the knowledge of maintenance coefficients is pivotal as their coefficients determine how much of the supply is “wasted” for the activities not related to the investigated application itself.
to increase the accuracy of the genome-scale metabolic model of p. putida  growth related parameters as well as the macro molecular composition of the biomass were measured carefully during continuous bioreactor cultures at different dilution rates with glucose as sole carbon limited source.
the solution space of the improved model was constrained, based on the newly measured growth parameters and by contrasting model simulations with available 13c flux measurements  <cit> .
the augmented accuracy led to an increased flux for the acetyl-coa production, which anti correlated strongly with the change of biomass yield .
moreover, transcriptomic data with good consistency with the model predictions were used to find inconsistencies within the model by comparing expression levels with simulations  <cit> .
RESULTS
macromolecular components
the molecular composition of the p. putida kt <dig> cell remained fairly stable over the whole measured range of d. the biomass fractions of rna, lipids, water-soluble proteins, and carbohydrates showed no significant dependency on the μ, based on linear regression , whereas dna exhibited an increase .
the standard deviation of these measurements is based on the variability of optical density  measurements.
the macromolecular composition of the biomass is summarized in table  <dig>  about  <dig> %  of the dry cell weight  was identified with the measurements of the macromolecules.
the mass missing in the macromolecular measurements justified the addition of  <dig> % hydrophobic proteins  <cit> .
water-insoluble proteins, peptidoglycan, vitamins and co-factors were not included in the measurements.
by balancing carbon and nitrogen measurements of the continuous fermentations, the average fraction of these compounds in the c
x
was computed to be  <dig> %  and  <dig> % , respectively.
the amino acid composition of the proteins of the cells grown at a dilution rate  of  <dig>  h- <dig> was analyzed .
by dividing the total concentration of amino acids by the measured c
x
the mass of proteins constituted  <dig> %  of the biomass.
when the none-identified amino acid concentrations are included of cysteine, methionine, and tryptophan, based on the molar fractions as in e. coli <cit> , the final protein concentration amounts  <dig> %.
this concentration seems to be very similar to the total protein concentration of the original model  at  <dig> %  and the total protein concentration determined at  <dig>  % .
p. putida
kt <dig> biomass
p. putida
p. putida
.
p. putida
kt <dig> at a dilution rate  of  <dig>  h
-1
concentrations of cysteine, methionine, and tryptophan could not be identified in this assay and were assumed to have identical molar fractions as in the e. coli biomass  <cit> .
asx = asp + asn; glx = glm + gln.
creation of p. putida specific biomass equation
a new biomass equation for the metabolic model  was computed based on the cell composition at a d of  <dig>  h- <dig> and other sources .
this new equation differed only to some extent from the old one, with respect to both macromolecular and elemental composition .
the main difference for the macromolecular composition is the lower fraction of lipids and the higher fraction of rna .
from the in silico biomass equation created in this work and from the in silico biomass equation used in the original model ijp <dig> model   <cit> , the elemental composition of p. putida kt <dig> was computed, which is shown in table  <dig>  this composition corresponded well with the elemental compositions that were calculated from the c and n balances and the values that were reported for the ashing and chemical determination of the biomass from p. putida atcc  <dig> grown in continuous culture on peptone-yeast extract medium  <cit> .
the lower carbon fraction in the in silico biomass and the appropriate nitrogen concentration could suggest that the lipids fraction was underestimated since this macromolecule has a carbon fraction exceeding 50% and contains a relative low concentration of nitrogen  <cit> .
in silico p. putida
in silico
in order to assess the effect of change of the biomass equation on the accuracy of predicted flux values, the flux variability analysis -distances were computed from simulations performed using either biomass equation.
the maximal and minimal fva-distances changed from  <dig> and  <dig> to  <dig> and  <dig>  respectively.
as a result the new biomass equation caused no significant change of the fva-distances, which is in agreement with the small differences between the biomass equations.
growth related factors
not only biomass composition, but also growth-related parameters were determined during the continuous fermentations of p. putida kt <dig> on glucose at different d. the biomass production rate , co <dig> production rate , and glucose uptake rate  increased until d  <dig>  h- <dig>  with the highest measured y
x/s
of  <dig>  gdcw · gglc- <dig>  the maximum growth rate  was determined to be  <dig>  h- <dig> by a wash out experiment .
this means that when p. putida kt <dig> was cultivated with the highest d of  <dig>  h- <dig> the specific growth rate  was equal to μ
max
and as a consequence no steady state was reached.
above d of  <dig>  h- <dig> an increasing glucose concentration  was observed .
this is most probably a consequence of approaching the μ
max
, whereas the classical monod-relationship  <cit>  was not observed - <dig> was exponential rather than linear).
the maximal biomass yield  and the maintenance coefficient  of p. putida kt <dig> were determined with high accuracy to be  <dig>  gdcw · gglc- <dig> and  <dig>  gglc · gdcw-1 · h- <dig>  respectively .
the linear regression was very strong, as r2 =  <dig> .
the average concentration of cells was  <dig> e +  <dig>  cells per ml.
the trend was decreasing with an increasing d. on average  <dig> % of the cells were alive  and  <dig> % were dead .
furthermore, the increase of y
x/s
at higher d is caused by a gradual increase of innercomplexity  and an abrupt augmentation of the cell size at a μ of  <dig>  h- <dig> .
this is illustrated by a distinctive trend of forward and side scatter plots, measured by facscalibur flow cytometer  .
the analysis of feed medium by nuclear magnetic resource  showed strong characteristic signals of the α- and β-anomeric forms of d-glucopyranose together with those of the antifoam, as well as very small signals of impurities associated with the technical grade materials used .
during steady state of the fermentation the signals for glucose had completely disappeared and only antifoam and those of the small impurity signals could be detected.
the signal-to-noise ratio was high enough to show that no organic metabolites as e.g.
acetate, proteins or peptides accumulated during the fermentation.
the position of such signals was in regions of the spectrum that were essentially free from overlap.
because no other metabolites were found in the medium by 1h-nmr at a d of  <dig> ,  <dig>  and  <dig>  h- <dig>  it was concluded that there is no accumulation of any intermediates of anabolism or catabolism in the range of d under carbon limited conditions.
new maintenance coefficients
based on the experimental data the y
x/s
max
and m
s
were estimated, and used to fit the growth associated maintenance  and the non-growth associated maintenance  parameters of ijp <dig>  these were computed to be  <dig> mmolatp gdcw- <dig> and  <dig>  mmolatp gdcw-1 · h- <dig> for gam and ngam, respectively.
due to a lower y
x/s
max
than assumed in the original model the gam increased.
the determined y
x/s
max
is  <dig> and  <dig> percent lower than in the original p. putida kt <dig> and e. coli models, respectively.
the ngam was decreased approximately  <cit>  as a consequence of lower value of the m
s
.
the change of gam and ngam coefficients caused larger changes to the flux distribution than the change of biomass equation.
the min fva-distance did decrease by 8% and the maximal fva-coefficient did manifest itself unexpectedly by an increased maximal fva-distance of 10%.
however the numbers were still in the same range.
evaluation of limits within the central metabolism
when inspecting the fva-results for the reactions whose fluxes were derived via 13c-measurements  <cit> , one enzyme was identified that showed a high possible variability of activity, namely malate dehydrogenase .
it was next tested, how the restriction of the flux of the respective reaction, during growth on glucose, would influence the output of fva-simulations.
the reaction was constrained to the experimentally measured flux value based on 13c measurement.
imposing this limit, had a very strong impact on the flux prediction.
the drop in the fva-distance was much higher than implied by the limit on the particular reaction.
this drop was mainly observed for the maximal distance, leading to a residue of 3% of the original solution space.
the minimal solution space was constrained for 10%.
as a consequence, constraining this reaction did partly lead to identification of a solution that is closer to the experimental value by significantly minimizing the minimal distance in the original model, but it mainly excluded futile cycles that are far from the experimental.
the fva predicts in such a case, infinite fluxes.
the limitation of malate dehydrogenase led to a minimal and maximal fva-distance of  <dig> and  <dig>  which are in the same range of values.
isotopic 13c measurements showed that glucose 6-phosphate isomerase  operated in the reverse direction.
fva could not determine such inefficiency, given it is not in line with the evolutionary selection to reach a high y
x/s
max as defined by the function of the model.
influence of growth stoichiometry and maintenance changes on model predictions
the original p. putida model was used to generate hypotheses pertaining to increasing the production of acetyl-coa  <cit> .
this compound is an entry metabolite for the synthesis of polyhydroxyalkanoates – biodegradable plastics of industrial and medical relevance  <cit> .
mutants described by poblete castro et al.
showed an increased acetyl-coa production flux  <cit> .
in order to evaluate, how the changes of biomass composition and maintenance coefficients influenced predictions of the model, the fva-calculation for the previously defined mutant with blocked enzymatic activity for glucose dehydrogenase  was executed for the old and new conditions without any constraints.
compared to the old values, the new values lead to an decrease in the maximum accoa production flux and y
x/s
max
: from  <dig>  to  <dig>   and from  <dig>  to  <dig>  , respectively, which thus narrows down the predictive range.
evaluating the consistency between the transcriptomic data and the model
to further validate the model, the consistency between model predictions and the transcriptomics data, generated at steady-state, was assessed.
the flux balance analysis -predicted fluxes and the levels of respective mrna transcripts showed overall good agreement with each other.
it should be emphasized, however, that measured abundances of mrna transcripts cannot be directly translated into fluxes.
the consistency was evaluated by analyzing the expression levels of: i) genes essential for growth, ii) non-essential genes with low expression that are involved in reactions with non-zero flux, and iii) genes with high expression values that are only involved in the catalysis of the reactions with predicted zero flux.
expression of the genes essential for growth: the first assessment aimed at identifying genes essential for growth in silico that are actually not expressed.
from genes identified by the model as essential those were selected and termed as potentially not expressed that complied with at least one of the following criteria: i) the number of “absent” calls  was higher than  <dig> , or ii) the number of “present” calls was lower than  <dig>   or iii) the mean log <dig> absolute gene expression value was lower than  <dig>  .
out of  <dig> essential genes  <dig> were selected that were potentially not expressed.
they are listed .
inspection of these genes showed that the overlap between the methods is limited.
the “present” call condition is the least restrictive one, while the mean condition is the most restrictive.
many of the genes that fulfilled one of the “call” conditions show log <dig> absolute gene expression values higher than  <dig>  whereas the average over all arrays was  <dig> .
this suggests that the call methods are relatively untrustworthy and that, in many cases, it rather points out the genes for which experimental problems possibly occurred, e.g.
due to cross-hybridization of the respective probes with mrna from some other gene.
a cluster of genes pp0237-pp <dig>  that was originally annotated to be involved in sulfate transport was consistently identified as absent.
after close inspection, these genes appeared to be miss-assigned, since there was a group of other genes that is responsible for sulfate transport .
the model was updated accordingly.
inspecting the genes that complied with at least two of the criteria revealed that there were a number of those involved in biosynthesis of cofactors .
the corresponding reactions usually have a low flux so it may be that low protein levels suffice for the required enzymatic activity.
there are also six genes involved in the synthesis of lps .
the majority of these genes has relatively high expression levels and were selected due to the presence/absence criteria.
lps is needed in rather limited amounts so a low expression may be sufficient for the required enzymatic activity.
furthermore these six genes constitute only around 50% of the genes involved in lps synthesis, so this also suggests that the expression levels are sufficient.
pp <dig>  pp <dig>  and pp <dig>  are genes that were labeled as essential due incompleteness of the metabolic model.
the reason these genes are essential is, that s-adenosyl homocysteine is produced during the synthesis of cyclo-fatty acids.
this compound needs to be converted further.
apparently the only way is to convert it into ribose and adenine which are then phosphorylated to ribose-6-phosphate and amp, respectively.
this route seems, however, not to be the proper one.
expression of non-essential genes required for optimal growth: the second assessment was an extension to the previous one.
here reactions with non-zero minimal fluxes in fva-simulations were identified.
for these reactions the “expression value” was computed and reactions with a log <dig> expression value below  <dig>  were selected.
there were only five such reactions identified.
they were all involved in fatty acid biosynthesis and all catalyzed by the same pair of isozymes  whose mean expression values did both not exceed  <dig> .
highly expressed genes not required for growth: finally, genes with high expression levels were identified that only catalyze reactions with a low flux .
these genes are listed .
several interesting observations could be made out of it.
first, branched-chain amino acids abc transporters constituted a significant fraction of these genes , suggesting that the bacterium is actively searching for alternative nutrition sources.
second, a number of genes are involved in the synthesis of cofactors as pyridoxal phosphate  and vitamins which include biotin , thiamin pyrophosphate , and riboflavin  .
for the last one, only a single gene from its biosynthesis pathway showed high expression and, as riboflavin is included into biomass composition, there is a flux going through the reactions involved in its synthesis.
this flux depends on the share of the riboflavin in the biomass.
the high expression of this gene may suggest that this coefficient should be higher, yet the evidence is weak, as other genes belonging to the pathway did not show high expression.
the other three compounds are not included in the biomass equation, but the expression levels of respective genes suggest that they should be included.
the mechanism  of the synthesis of pyridoxal phosphate in p. putida kt <dig> is known, whereas it is not the case for the other two compounds.
third, reactions involved in rhamnosugars synthesis  are also highly expressed.
these are used for synthesis of rhamnolipids that are a constituent of the bacterial envelope and are not part of the biomass in the current version of the model.
this assay suggests that they should be included.
this requires however the determination of the exact composition of these metabolites and their share in the biomass.
the reason of higher expression of remaining genes could not be elucidated.
discussion
constraint-based modeling provides a valuable framework for navigating microbial metabolic networks and for identification and prediction of intra-cellular flux distributions, thereby aiding the discovery and application of bacteria e.g.
in biotechnology  <cit> .
however, to be reasonably predictive, the metabolic reconstructions created within this framework require proper and accurate input information.
striving to improve the metabolic reconstruction of p. putida kt <dig>  we characterized experimentally the macromolecular composition of the bacterium cultivated continuously on a minimal medium with glucose as the sole carbon and energy source at a range of d .
these measurements showed that the macromolecular biomass composition does not change significantly when the d and thus the μ varies.
the only macromolecule that did change significantly upon increasing d was the dna.
this might have been a consequence of the appearance of a higher number of replication forks.
at a μ of  <dig>  h- <dig> the cell size increases rapidly, which might be related to the increase of genome replication  <cit> .
in other organisms like e. coli k <dig>   <cit> , e. coli b/r   <cit> , and p. denitrificans   <cit> , the dna fraction was observed to decrease at increasing d, or remain stable as in s. cerevisiae   <cit>  and a. calcoaceticus   <cit> .
baart et al.
<cit>  observed however that in n. meningitidis  the dna fraction increases too.
the stability of the rna and protein fractions in p. putida kt <dig> also contradicts to some extent previous findings, as in some other organisms – e. coli and p. denitrificans – these fractions increased and decreased with the d, respectively  <cit> .
however, no significant changes were observed for the rna and protein fractions in e. coli k <dig> at the d range of  <dig> - <dig>  h- <dig> and n. meningitides at the d range of  <dig> - <dig>  h- <dig> <cit> .
in a. calcoaceticus   <cit>  only the rna fraction increased and in s. cerevisiae   <cit>  both the rna and protein fractions increased.
the increase of the rna fraction most probably reflects the extra need for ribosomal rna for the synthesis of proteins  <cit> .
however, besides the increase of the number of ribosomes, the higher need for proteins can be fulfilled by an increase in the efficiency of their synthesis  <cit> .
based on these results and a multitude of different sources, including our amino acid composition measurements, a new p. putida specific biomass equation was created.
the differences between the new and old biomass equation were relatively small, showing that the biomass compositions of p. putida and e. coli do not differ significantly.
this supports the assumption made in many reconstruction efforts that e. coli biomass composition approximates well that of other gram-negative bacteria when grown at the same d. the protein composition is comparable to that determined by sohn et al.
<cit> .
on the contrary, the peptidoglycan concentration reached  <dig> % of the biomass weight in the macro molecular biomass composition.
this concentration was surprisingly high as the sum of the fractions exceeded 100%.
of the determined growth related factors, the specific rate of substrate utilization  increased linearly with the d when the culture was glucose-limited.
this is in agreement with the y
x/s
/m
s
model of pirt  <cit> .
y
x/s
max
and m
s
were used to update the coefficients of gam and ngam in the model.
in contrast to the biomass composition, the new values differed substantially from the old ones.
particularly the y
x/s
max
was significantly lower and, consequently, the gam higher, than assumed before.
this means that the energy required for assembly of a cell of p. putida is much higher than in e. coli, which gam coefficient was used in the original version of the model.
this resulted in less energy being available for biotechnological processes.
on the other hand even at a d of  <dig>  h- <dig>  no overflow metabolism was observed, i.e.
no by-products  were accumulated.
the opposite is the case for many organisms at high μ values and is a result of limitations in the tca cycle or respiratory nadh turnover  <cit> .
for example, in e. coli cultures at d above  <dig>  h- <dig> acetate accumulates in the medium  <cit> .
the lack of overflow metabolism is of great importance as it points out the capacity of p. putida kt <dig> as biocatalyst.
the ability to generate sufficient energy and the availability of nadh and nad + are of importance for applications of a strain in biocatalysis, especially for a cofactor dependent conversion and the primary export of the product out of the cell.
these results confirm earlier presented data that p. putida can meet high energy demands  <cit> .
controlling the availability of the energy equivalent atp at a sufficient level during the production phase, forms an interesting lead to optimize the biocatalyst by using metabolic engineering.
after implementing the new biomass equation and the new coefficients of gam and ngam the model was contrasted with 13c-flux measurement .
the new biomass, gam, and ngam did not largely influence the range of the distances between fva-solutions.
the increased maximum fva-distance after implementing the new gam and ngam can possibly be explained by the fixed glucose uptake instead of additional conversion of glucose to gluconate and 2-ketogluconate.
if we look at the similar fva-distances it may mean that the model itself was not improved , but it can also mean that the fva-distance is not driven by the macromolecular biomass composition and the growth related parameters.
due to the use of measured and exact numbers, the model is more in line with the actual phenotype.
therefore, we think it is important to construct the model using correct data, as it is of meaning for future applications.
essential was that the minimal fva-distance was kept relatively stable at the same range of numbers, even when the maximum fva-distance significantly did decrease after restricting the flux for malate dehydrogenase.
this shows that indeed possibly the best agreement was reached within limitations of constraint-based modeling.
given the fairly stable macro-molecular composition in the measured μ range it can be assumed that the flux distribution does not significantly change in p. putida kt <dig> when the specific growth rate varies.
this is relevant for the phenotype in case the strain is subjected to gene deletion.
the experimental data used for validation encompass only a small part of the metabolic network.
consequently, the validation does not cover metabolic pathways outside the central metabolism.
the single mutant  showed the highest increased synthesis of pha when characterized by poblete-castro et al.
<cit> .
based on the fva-analysis the y
x/s
max
was  <dig>  gdcw·gglc- <dig>  poblete-castro et al.
reported a y
x/s
max
of  <dig>  gdcw · gglc- <dig> for the single mutant, which seems a rather reasonable agreement.
the remaining slight deviation might result from slight differences in the fermentation setup.
the contrast formed with transcriptomic data showed good agreement between the revalidated model and the experimental results.
more than 90% of genes identified by the transcriptomics as potentially not expressed were involved in the catalysis of reactions not required for growth.
for the reactions catalyzed by proteins of which zero-flux was predicted, only a limited number of genes were expressed.
a lot of them are involved in the transport of amino acids, and the synthesis of co-factors, indicating that the bacterium tries to reduce the nutrient limitation.
in general, with transcriptomic assays reproducible results are obtained  <cit> .
yet, the translation of the expression values into fluxes and vice versa needs to be done with care.
for example, some genes might be expressed constitutively, consequently they do not always correspond with metabolic fluxes.
also there is no linear correspondence between the abundance of the mrna transcript, as measured by transcriptomics, and the activity of the enzyme.
this is due to a multitude of factors that influence the efficiency of protein production from mrna as post-transcriptional regulation  <cit> .
CONCLUSIONS
by measuring carefully key biological parameters and contrasting simulation results with available experimental data, this work enabled to explain missing links and inconsistencies in the previously developed genome-scale metabolic model , and improved considerably its accuracy, providing thereby a more solid basis for its use in designing biotechnological metabolic processes.
thus, the application of this approach under various conditions is a useful strategy to reconcile in vivo and in silico analysis and should therefore be incorporated into efficient strain and bioprocess optimization.

 pseudomonas putida is a non-pathogenic, soil bacterium that developed a notably versatile metabolism and thus can inhabit several environmental niches. numerous strains, some of them being solvent tolerant, are able to produce efficiently a range of bulk and fine chemicals or, conversely, degrade various substances that are by products or waste of industrial processes. these features, along with the amenability for genetic manipulation and suitability as a host for the expression of heterologous genes have rendered p. putida an attractive object of research for biotechnological applications. the sequencing of the genome of p. putida brought a significant leap in developing its applications by unveiling the metabolic potential encoded in the genome. in an effort to enable the analysis of p. putida kt from a systems biology perspective and to foster the development of its biotechnological applications, we built a constraint-based metabolic reconstruction of this strain. although a lot of knowledge pertaining to this strain was collected while building the model, some information had to be approximated from the escherichia coli model. the information approximated included the biomass composition of the bacterium and the energetic expenses related to growth and maintenance of life-functions ). one of the most common assumptions underlying simulations built upon genome-scale constraint-based metabolic reconstructions is that bacteria try to maximize their growth yield. consequently, growth associated parameters can be specified prior to performing simulations of the growth, which provide a basis for numerous applications of metabolic reconstructions including rational strain design for biotechnological applications. furthermore, these parameters influence significantly the outcome of such simulations, as they confine fluxes of important parts of the metabolism. moreover, they may vary significantly not only among organisms, but also among different growth conditions. thus, for applications of metabolic reconstructions in biotechnology, the knowledge of maintenance coefficients is pivotal as their coefficients determine how much of the supply is “wasted” for the activities not related to the investigated application itself. to increase the accuracy of the genome-scale metabolic model of p. putida growth related parameters as well as the macro molecular composition of the biomass were measured carefully during continuous bioreactor cultures at different dilution rates with glucose as sole carbon limited source. the solution space of the improved model was constrained, based on the newly measured growth parameters and by contrasting model simulations with available 13c flux measurements. the augmented accuracy led to an increased flux for the acetyl-coa production, which anti correlated strongly with the change of biomass yield. moreover, transcriptomic data with good consistency with the model predictions were used to find inconsistencies within the model by comparing expression levels with simulations.  macromolecular components the molecular composition of the p. putida kt cell remained fairly stable over the whole measured range of d. the biomass fractions of rna, lipids, water-soluble proteins, and carbohydrates showed no significant dependency on the μ, based on linear regression, whereas dna exhibited an increase. the standard deviation of these measurements is based on the variability of optical density measurements. the macromolecular composition of the biomass is summarized in table  about % of the dry cell weight was identified with the measurements of the macromolecules. the mass missing in the macromolecular measurements justified the addition of % hydrophobic proteins. water-insoluble proteins, peptidoglycan, vitamins and co-factors were not included in the measurements. by balancing carbon and nitrogen measurements of the continuous fermentations, the average fraction of these compounds in the c x was computed to be % and %, respectively. the amino acid composition of the proteins of the cells grown at a dilution rate of  h- was analyzed. by dividing the total concentration of amino acids by the measured c x the mass of proteins constituted % of the biomass. when the none-identified amino acid concentrations are included of cysteine, methionine, and tryptophan, based on the molar fractions as in e. coli, the final protein concentration amounts %. this concentration seems to be very similar to the total protein concentration of the original model at % and the total protein concentration determined at %. p. putida kt biomass p. putida p. putida. p. putida kt at a dilution rate of  h -1 concentrations of cysteine, methionine, and tryptophan could not be identified in this assay and were assumed to have identical molar fractions as in the e. coli biomass. asx = asp + asn; glx = glm + gln. creation of p. putida specific biomass equation a new biomass equation for the metabolic model was computed based on the cell composition at a d of  h- and other sources. this new equation differed only to some extent from the old one, with respect to both macromolecular and elemental composition. the main difference for the macromolecular composition is the lower fraction of lipids and the higher fraction of rna. from the in silico biomass equation created in this work and from the in silico biomass equation used in the original model ijp model, the elemental composition of p. putida kt was computed, which is shown in table  this composition corresponded well with the elemental compositions that were calculated from the c and n balances and the values that were reported for the ashing and chemical determination of the biomass from p. putida atcc grown in continuous culture on peptone-yeast extract medium. the lower carbon fraction in the in silico biomass and the appropriate nitrogen concentration could suggest that the lipids fraction was underestimated since this macromolecule has a carbon fraction exceeding 50% and contains a relative low concentration of nitrogen. in silico p. putida in silico in order to assess the effect of change of the biomass equation on the accuracy of predicted flux values, the flux variability analysis -distances were computed from simulations performed using either biomass equation. the maximal and minimal fva-distances changed from and to and respectively. as a result the new biomass equation caused no significant change of the fva-distances, which is in agreement with the small differences between the biomass equations. growth related factors not only biomass composition, but also growth-related parameters were determined during the continuous fermentations of p. putida kt on glucose at different d. the biomass production rate, co production rate, and glucose uptake rate increased until d  h- with the highest measured y x/s of gdcw · gglc- the maximum growth rate was determined to be  h- by a wash out experiment. this means that when p. putida kt was cultivated with the highest d of  h- the specific growth rate was equal to μ max and as a consequence no steady state was reached. above d of  h- an increasing glucose concentration was observed. this is most probably a consequence of approaching the μ max, whereas the classical monod-relationship was not observed - was exponential rather than linear). the maximal biomass yield and the maintenance coefficient of p. putida kt were determined with high accuracy to be gdcw · gglc- and gglc · gdcw-1 · h- respectively. the linear regression was very strong, as r2 = . the average concentration of cells was e +  cells per ml. the trend was decreasing with an increasing d. on average % of the cells were alive and % were dead. furthermore, the increase of y x/s at higher d is caused by a gradual increase of innercomplexity and an abrupt augmentation of the cell size at a μ of  h-. this is illustrated by a distinctive trend of forward and side scatter plots, measured by facscalibur flow cytometer. the analysis of feed medium by nuclear magnetic resource showed strong characteristic signals of the α- and β-anomeric forms of d-glucopyranose together with those of the antifoam, as well as very small signals of impurities associated with the technical grade materials used. during steady state of the fermentation the signals for glucose had completely disappeared and only antifoam and those of the small impurity signals could be detected. the signal-to-noise ratio was high enough to show that no organic metabolites as e.g. acetate, proteins or peptides accumulated during the fermentation. the position of such signals was in regions of the spectrum that were essentially free from overlap. because no other metabolites were found in the medium by 1h-nmr at a d of, and  h- it was concluded that there is no accumulation of any intermediates of anabolism or catabolism in the range of d under carbon limited conditions. new maintenance coefficients based on the experimental data the y x/s max and m s were estimated, and used to fit the growth associated maintenance and the non-growth associated maintenance parameters of ijp these were computed to be mmolatp gdcw- and mmolatp gdcw-1 · h- for gam and ngam, respectively. due to a lower y x/s max than assumed in the original model the gam increased. the determined y x/s max is and percent lower than in the original p. putida kt and e. coli models, respectively. the ngam was decreased approximately as a consequence of lower value of the m s. the change of gam and ngam coefficients caused larger changes to the flux distribution than the change of biomass equation. the min fva-distance did decrease by 8% and the maximal fva-coefficient did manifest itself unexpectedly by an increased maximal fva-distance of 10%. however the numbers were still in the same range. evaluation of limits within the central metabolism when inspecting the fva-results for the reactions whose fluxes were derived via 13c-measurements, one enzyme was identified that showed a high possible variability of activity, namely malate dehydrogenase. it was next tested, how the restriction of the flux of the respective reaction, during growth on glucose, would influence the output of fva-simulations. the reaction was constrained to the experimentally measured flux value based on 13c measurement. imposing this limit, had a very strong impact on the flux prediction. the drop in the fva-distance was much higher than implied by the limit on the particular reaction. this drop was mainly observed for the maximal distance, leading to a residue of 3% of the original solution space. the minimal solution space was constrained for 10%. as a consequence, constraining this reaction did partly lead to identification of a solution that is closer to the experimental value by significantly minimizing the minimal distance in the original model, but it mainly excluded futile cycles that are far from the experimental. the fva predicts in such a case, infinite fluxes. the limitation of malate dehydrogenase led to a minimal and maximal fva-distance of and which are in the same range of values. isotopic 13c measurements showed that glucose 6-phosphate isomerase operated in the reverse direction. fva could not determine such inefficiency, given it is not in line with the evolutionary selection to reach a high y x/s max as defined by the function of the model. influence of growth stoichiometry and maintenance changes on model predictions the original p. putida model was used to generate hypotheses pertaining to increasing the production of acetyl-coa. this compound is an entry metabolite for the synthesis of polyhydroxyalkanoates – biodegradable plastics of industrial and medical relevance. mutants described by poblete castro et al. showed an increased acetyl-coa production flux. in order to evaluate, how the changes of biomass composition and maintenance coefficients influenced predictions of the model, the fva-calculation for the previously defined mutant with blocked enzymatic activity for glucose dehydrogenase was executed for the old and new conditions without any constraints. compared to the old values, the new values lead to an decrease in the maximum accoa production flux and y x/s max: from to and from to, respectively, which thus narrows down the predictive range. evaluating the consistency between the transcriptomic data and the model to further validate the model, the consistency between model predictions and the transcriptomics data, generated at steady-state, was assessed. the flux balance analysis -predicted fluxes and the levels of respective mrna transcripts showed overall good agreement with each other. it should be emphasized, however, that measured abundances of mrna transcripts cannot be directly translated into fluxes. the consistency was evaluated by analyzing the expression levels of: i) genes essential for growth, ii) non-essential genes with low expression that are involved in reactions with non-zero flux, and iii) genes with high expression values that are only involved in the catalysis of the reactions with predicted zero flux. expression of the genes essential for growth: the first assessment aimed at identifying genes essential for growth in silico that are actually not expressed. from genes identified by the model as essential those were selected and termed as potentially not expressed that complied with at least one of the following criteria: i) the number of “absent” calls was higher than, or ii) the number of “present” calls was lower than or iii) the mean log absolute gene expression value was lower than. out of essential genes were selected that were potentially not expressed. they are listed. inspection of these genes showed that the overlap between the methods is limited. the “present” call condition is the least restrictive one, while the mean condition is the most restrictive. many of the genes that fulfilled one of the “call” conditions show log absolute gene expression values higher than whereas the average over all arrays was. this suggests that the call methods are relatively untrustworthy and that, in many cases, it rather points out the genes for which experimental problems possibly occurred, e.g. due to cross-hybridization of the respective probes with mrna from some other gene. a cluster of genes pp0237-pp that was originally annotated to be involved in sulfate transport was consistently identified as absent. after close inspection, these genes appeared to be miss-assigned, since there was a group of other genes that is responsible for sulfate transport. the model was updated accordingly. inspecting the genes that complied with at least two of the criteria revealed that there were a number of those involved in biosynthesis of cofactors. the corresponding reactions usually have a low flux so it may be that low protein levels suffice for the required enzymatic activity. there are also six genes involved in the synthesis of lps. the majority of these genes has relatively high expression levels and were selected due to the presence/absence criteria. lps is needed in rather limited amounts so a low expression may be sufficient for the required enzymatic activity. furthermore these six genes constitute only around 50% of the genes involved in lps synthesis, so this also suggests that the expression levels are sufficient. pp pp and pp are genes that were labeled as essential due incompleteness of the metabolic model. the reason these genes are essential is, that s-adenosyl homocysteine is produced during the synthesis of cyclo-fatty acids. this compound needs to be converted further. apparently the only way is to convert it into ribose and adenine which are then phosphorylated to ribose-6-phosphate and amp, respectively. this route seems, however, not to be the proper one. expression of non-essential genes required for optimal growth: the second assessment was an extension to the previous one. here reactions with non-zero minimal fluxes in fva-simulations were identified. for these reactions the “expression value” was computed and reactions with a log expression value below were selected. there were only five such reactions identified. they were all involved in fatty acid biosynthesis and all catalyzed by the same pair of isozymes whose mean expression values did both not exceed. highly expressed genes not required for growth: finally, genes with high expression levels were identified that only catalyze reactions with a low flux. these genes are listed. several interesting observations could be made out of it. first, branched-chain amino acids abc transporters constituted a significant fraction of these genes, suggesting that the bacterium is actively searching for alternative nutrition sources. second, a number of genes are involved in the synthesis of cofactors as pyridoxal phosphate and vitamins which include biotin, thiamin pyrophosphate, and riboflavin. for the last one, only a single gene from its biosynthesis pathway showed high expression and, as riboflavin is included into biomass composition, there is a flux going through the reactions involved in its synthesis. this flux depends on the share of the riboflavin in the biomass. the high expression of this gene may suggest that this coefficient should be higher, yet the evidence is weak, as other genes belonging to the pathway did not show high expression. the other three compounds are not included in the biomass equation, but the expression levels of respective genes suggest that they should be included. the mechanism of the synthesis of pyridoxal phosphate in p. putida kt is known, whereas it is not the case for the other two compounds. third, reactions involved in rhamnosugars synthesis are also highly expressed. these are used for synthesis of rhamnolipids that are a constituent of the bacterial envelope and are not part of the biomass in the current version of the model. this assay suggests that they should be included. this requires however the determination of the exact composition of these metabolites and their share in the biomass. the reason of higher expression of remaining genes could not be elucidated. discussion constraint-based modeling provides a valuable framework for navigating microbial metabolic networks and for identification and prediction of intra-cellular flux distributions, thereby aiding the discovery and application of bacteria e.g. in biotechnology. however, to be reasonably predictive, the metabolic reconstructions created within this framework require proper and accurate input information. striving to improve the metabolic reconstruction of p. putida kt we characterized experimentally the macromolecular composition of the bacterium cultivated continuously on a minimal medium with glucose as the sole carbon and energy source at a range of d. these measurements showed that the macromolecular biomass composition does not change significantly when the d and thus the μ varies. the only macromolecule that did change significantly upon increasing d was the dna. this might have been a consequence of the appearance of a higher number of replication forks. at a μ of  h- the cell size increases rapidly, which might be related to the increase of genome replication. in other organisms like e. coli k, e. coli b/r, and p. denitrificans, the dna fraction was observed to decrease at increasing d, or remain stable as in s. cerevisiae and a. calcoaceticus. baart et al. observed however that in n. meningitidis the dna fraction increases too. the stability of the rna and protein fractions in p. putida kt also contradicts to some extent previous findings, as in some other organisms – e. coli and p. denitrificans – these fractions increased and decreased with the d, respectively. however, no significant changes were observed for the rna and protein fractions in e. coli k at the d range of -  h- and n. meningitides at the d range of -  h-. in a. calcoaceticus only the rna fraction increased and in s. cerevisiae both the rna and protein fractions increased. the increase of the rna fraction most probably reflects the extra need for ribosomal rna for the synthesis of proteins. however, besides the increase of the number of ribosomes, the higher need for proteins can be fulfilled by an increase in the efficiency of their synthesis. based on these results and a multitude of different sources, including our amino acid composition measurements, a new p. putida specific biomass equation was created. the differences between the new and old biomass equation were relatively small, showing that the biomass compositions of p. putida and e. coli do not differ significantly. this supports the assumption made in many reconstruction efforts that e. coli biomass composition approximates well that of other gram-negative bacteria when grown at the same d. the protein composition is comparable to that determined by sohn et al.. on the contrary, the peptidoglycan concentration reached % of the biomass weight in the macro molecular biomass composition. this concentration was surprisingly high as the sum of the fractions exceeded 100%. of the determined growth related factors, the specific rate of substrate utilization increased linearly with the d when the culture was glucose-limited. this is in agreement with the y x/s /m s model of pirt. y x/s max and m s were used to update the coefficients of gam and ngam in the model. in contrast to the biomass composition, the new values differed substantially from the old ones. particularly the y x/s max was significantly lower and, consequently, the gam higher, than assumed before. this means that the energy required for assembly of a cell of p. putida is much higher than in e. coli, which gam coefficient was used in the original version of the model. this resulted in less energy being available for biotechnological processes. on the other hand even at a d of  h- no overflow metabolism was observed, i.e. no by-products were accumulated. the opposite is the case for many organisms at high μ values and is a result of limitations in the tca cycle or respiratory nadh turnover. for example, in e. coli cultures at d above  h- acetate accumulates in the medium. the lack of overflow metabolism is of great importance as it points out the capacity of p. putida kt as biocatalyst. the ability to generate sufficient energy and the availability of nadh and nad + are of importance for applications of a strain in biocatalysis, especially for a cofactor dependent conversion and the primary export of the product out of the cell. these results confirm earlier presented data that p. putida can meet high energy demands. controlling the availability of the energy equivalent atp at a sufficient level during the production phase, forms an interesting lead to optimize the biocatalyst by using metabolic engineering. after implementing the new biomass equation and the new coefficients of gam and ngam the model was contrasted with 13c-flux measurement. the new biomass, gam, and ngam did not largely influence the range of the distances between fva-solutions. the increased maximum fva-distance after implementing the new gam and ngam can possibly be explained by the fixed glucose uptake instead of additional conversion of glucose to gluconate and 2-ketogluconate. if we look at the similar fva-distances it may mean that the model itself was not improved, but it can also mean that the fva-distance is not driven by the macromolecular biomass composition and the growth related parameters. due to the use of measured and exact numbers, the model is more in line with the actual phenotype. therefore, we think it is important to construct the model using correct data, as it is of meaning for future applications. essential was that the minimal fva-distance was kept relatively stable at the same range of numbers, even when the maximum fva-distance significantly did decrease after restricting the flux for malate dehydrogenase. this shows that indeed possibly the best agreement was reached within limitations of constraint-based modeling. given the fairly stable macro-molecular composition in the measured μ range it can be assumed that the flux distribution does not significantly change in p. putida kt when the specific growth rate varies. this is relevant for the phenotype in case the strain is subjected to gene deletion. the experimental data used for validation encompass only a small part of the metabolic network. consequently, the validation does not cover metabolic pathways outside the central metabolism. the single mutant showed the highest increased synthesis of pha when characterized by poblete-castro et al.. based on the fva-analysis the y x/s max was gdcw·gglc- poblete-castro et al. reported a y x/s max of gdcw · gglc- for the single mutant, which seems a rather reasonable agreement. the remaining slight deviation might result from slight differences in the fermentation setup. the contrast formed with transcriptomic data showed good agreement between the revalidated model and the experimental results. more than 90% of genes identified by the transcriptomics as potentially not expressed were involved in the catalysis of reactions not required for growth. for the reactions catalyzed by proteins of which zero-flux was predicted, only a limited number of genes were expressed. a lot of them are involved in the transport of amino acids, and the synthesis of co-factors, indicating that the bacterium tries to reduce the nutrient limitation. in general, with transcriptomic assays reproducible results are obtained. yet, the translation of the expression values into fluxes and vice versa needs to be done with care. for example, some genes might be expressed constitutively, consequently they do not always correspond with metabolic fluxes. also there is no linear correspondence between the abundance of the mrna transcript, as measured by transcriptomics, and the activity of the enzyme. this is due to a multitude of factors that influence the efficiency of protein production from mrna as post-transcriptional regulation.  by measuring carefully key biological parameters and contrasting simulation results with available experimental data, this work enabled to explain missing links and inconsistencies in the previously developed genome-scale metabolic model, and improved considerably its accuracy, providing thereby a more solid basis for its use in designing biotechnological metabolic processes. thus, the application of this approach under various conditions is a useful strategy to reconcile in vivo and in silico analysis and should therefore be incorporated into efficient strain and bioprocess optimization. 
to substantially improve the accuracy of the genome scale model, continuous bioreactor cultures on a mineral medium with glucose as a sole carbon source were carried out at different dilution rates, which covered pulling analysis of the macromolecular composition of the biomass. however, in most reconstructions, due to the lack of appropriate measurements, experimentally determined growth parameters are simply taken from literature including other organisms, which reduces the usefulness and suitability of these models. only the dna fraction increased with the specific growth rate. contrasting already published 13c flux measurements and the improved model allowed for constraining the solution space, by eliminating futile cycles. furthermore, the model predictions were compared with transcriptomic data at overall good consistency, which helped to identify missing links. pseudomonas putida kt is an outstanding biocatalyst given its versatile metabolism, its ability to generate sufficient energy and turnover of nadh and nad. by careful interpretation of growth stoichiometry and kinetics when grown in the presence of glucose, this work reports on an accurate genome scale metabolic model of pseudomonas putida, providing a solid basis for its use in designing superior strains for biocatalysis. to apply this strain optimally in industrial production, a previously developed genome-scale metabolic model was experimentally assessed and streamlined to enable accurate predictions of the outcome of metabolic engineering approaches. besides, the maximum biomass yield of gdcw · gglc- the maintenance coefficient of gglc · gdcw-1 · h- and the maximum specific growth rate of  h- were determined. this resulted in reliable estimation for the growth-associated maintenance of mmolatp · gdcw- and the non growth-associated maintenance of mmolatp · gdcw-1 · h- both values were found significantly different from previous assignment as a consequence of a lower yield and higher maintenance coefficient than originally assumed.
 genome scale metabolic reconstructions are developed to efficiently engineer biocatalysts and bioprocesses based on a rational approach. however, in most reconstructions, due to the lack of appropriate measurements, experimentally determined growth parameters are simply taken from literature including other organisms, which reduces the usefulness and suitability of these models. pseudomonas putida kt is an outstanding biocatalyst given its versatile metabolism, its ability to generate sufficient energy and turnover of nadh and nad. to apply this strain optimally in industrial production, a previously developed genome-scale metabolic model was experimentally assessed and streamlined to enable accurate predictions of the outcome of metabolic engineering approaches.  to substantially improve the accuracy of the genome scale model, continuous bioreactor cultures on a mineral medium with glucose as a sole carbon source were carried out at different dilution rates, which covered pulling analysis of the macromolecular composition of the biomass. besides, the maximum biomass yield of gdcw · gglc- the maintenance coefficient of gglc · gdcw-1 · h- and the maximum specific growth rate of  h- were determined. only the dna fraction increased with the specific growth rate. this resulted in reliable estimation for the growth-associated maintenance of mmolatp · gdcw- and the non growth-associated maintenance of mmolatp · gdcw-1 · h- both values were found significantly different from previous assignment as a consequence of a lower yield and higher maintenance coefficient than originally assumed. contrasting already published 13c flux measurements and the improved model allowed for constraining the solution space, by eliminating futile cycles. furthermore, the model predictions were compared with transcriptomic data at overall good consistency, which helped to identify missing links.  by careful interpretation of growth stoichiometry and kinetics when grown in the presence of glucose, this work reports on an accurate genome scale metabolic model of pseudomonas putida, providing a solid basis for its use in designing superior strains for biocatalysis. by consideration of substrate specific variation in stoichiometry and kinetics, it can be extended to other substrates and new mutants. continuous cultivationp. putida kt2440glucosemetabolic modelingbiomass compositiontranscriptomics 