Response of Fe-S cluster assembly machinery of Escherichia coli to mechanical stress in a model of amino-acid crystal fermentation.

During amino-acid crystal fermentation, mechanical stress on bacterial cells caused by crystal collision often impacts negatively on bacterial growth and amino-acid production. When Escherichia coli cells were cultivated under mechanical stress of polyvinyl chloride particles as a model of the crystal fermentation, activities of iron-sulfur (Fe-S) cluster-containing enzymes were apparently decreased. Based on an assumption that function of Fe-S cluster assembly machinery would be elevated to recover the enzyme activities in such stressed cells, we analyzed levels of various components of Fe-S cluster assembly machinery by western blotting. It was found that the expression of HscA, a chaperon component of the machinery, was up-regulated and that shorter forms of HscA with the N-terminal region truncated were accumulated, suggesting an important role of HscA against the mechanical stress. An overexpression of HscA gene in E. coli cells gave a positive effect on rescue of the stress-induced decrease of the activity of Fe-S cluster-containing enzyme. These results may provide a new strategy to alleviate the mechanical stress during the amino-acid crystal fermentation.

OpenFLUX2: (13)C-MFA modeling software package adjusted for the comprehensive analysis of single and parallel labeling experiments.

BACKGROUND: Steady-state (13)C-based metabolic flux analysis ((13)C-MFA) is the most powerful method available for the quantification of intracellular fluxes. These analyses include concertedly linked experimental and computational stages: (i) assuming the metabolic model and optimizing the experimental design; (ii) feeding the investigated organism using a chosen (13)C-labeled substrate (tracer); (iii) measuring the extracellular effluxes and detecting the (13)C-patterns of intracellular metabolites; and (iv) computing flux parameters that minimize the differences between observed and simulated measurements, followed by evaluating flux statistics. In its early stages, (13)C-MFA was performed on the basis of data obtained in a single labeling experiment (SLE) followed by exploiting the developed high-performance computational software. Recently, the advantages of parallel labeling experiments (PLEs), where several LEs are conducted under the conditions differing only by the tracer(s) choice, were demonstrated, particularly with regard to improving flux precision due to the synergy of complementary information. The availability of an open-source software adjusted for PLE-based (13)C-MFA is an important factor for PLE implementation. RESULTS: The open-source software OpenFLUX, initially developed for the analysis of SLEs, was extended for the computation of PLE data. Using the OpenFLUX2, in silico simulation confirmed that flux precision is improved when (13)C-MFA is implemented by fitting PLE data to the common model compared with SLE-based analysis. Efficient flux resolution could be achieved in the PLE-mediated analysis when the choice of tracer was based on an experimental design computed to minimize the flux variances from different parts of the metabolic network. The analysis provided by OpenFLUX2 mainly includes (i) the optimization of the experimental design, (ii) the computation of the flux parameters from LEs data, (iii) goodness-of-fit testing of the model's adequacy, (iv) drawing conclusions concerning the identifiability of fluxes and construction of a contribution matrix reflecting the relative contribution of the measurement variances to the flux variances, and (v) precise determination of flux confidence intervals using a fine-tunable and convergence-controlled Monte Carlo-based method. CONCLUSIONS: The developed open-source OpenFLUX2 provides a friendly software environment that facilitates beginners and existing OpenFLUX users to implement LEs for steady-state (13)C-MFA including experimental design, quantitative evaluation of flux parameters and statistics.

Mechanical damage to Escherichia coli cells in a model of amino-acid crystal fermentation.

We investigated the mechanical damage to the Escherichia coli cell caused by polyvinyl chloride particles as a model of amino-acid crystal fermentation. Our results indicated that the glucose-consumption rate and the intracellular ATP concentration temporarily increased by the mechanical damage, and decreased after considerable damage had occurred on cell membrane.

Dynamic modeling of Escherichia coli metabolic and regulatory systems for amino-acid production.

Our aim is to construct a practical dynamic-simulation system that can model the metabolic and regulatory processes involved in the production of primary metabolites, such as amino acids. We have simulated the production of glutamate by transient batch-cultivation using a model of Escherichia coli central metabolism. Kinetic data were used to produce both the metabolic parts of the model, including the phosphotransferase system, glycolysis, the pentose-phosphate pathway, the tricarboxylic acid cycle, the glyoxylate shunt, and the anaplerotic pathways, and the regulatory parts of the model, including regulation by transcription factors, cyclic AMP receptor protein (CRP), making large colonies protein (Mlc), catabolite repressor/activator (Cra), pyruvate dehydrogenase complex repressor (PdhR), and acetate operon repressor (IclR). RNA polymerase and ribosome concentrations were expressed as a function of the specific growth rate, mu, corresponding to the changes in the growth rate during batch cultivation. Parameter fitting was performed using both extracellular concentration measurements and in vivo enzyme activities determined by (13)C flux analysis. By manual adjustment of the parameters, we simulated the batch fermentation of glucose or fructose by a wild-type strain (MG1655) and a glutamate-producing strain (MG1655 Delta sucA). The differences caused by the carbon source, and by wild-type and glutamate-producing strains, were clearly shown by the simulation. A sensitivity analysis revealed the factors that could be altered to improve the production process. Furthermore, an in silico deletion experiments could suggested the existence of uncharacterized regulation. We concluded that our simulation model could function as a new tool for the rational improvement and design of metabolic and regulatory networks.

Introduction of a stress-responsive gene, yggG, enhances the yield of L-phenylalanine with decreased acetic acid production in a recombinant Escherichia coli.

A stress-responsive gene, yggG, was introduced into an L-phenylalanine producer, Escherichia coli AJ12741. In shake-flask culture, the yggG-containing recombinant strain (named AJ12741/pHYGG) produced 6.4 g L-phenylalanine l(-1) at the end of culture and its yield on glucose was 0.16 g L-phenylalanine g glucose(-1). These values are much higher than those of the original AJ12741 strain (3.7 g L-phenylalanine l(-1) and 0.09 g L-phenylalanine g glucose(-1), respectively). On the other hand, AJ12741/pHYGG strain produced only 4.5 g acetic acid l(-1) and its yield on glucose was about a half of that of the AJ12741 culture. Analysis of gene expression revealed that in late growth phase, the expression levels of genes involved in acetic acid production (pta, ackA, and poxB) were relatively low in AJ12741/pHYGG cells. In particular, the level of poxB expression in AJ12741/pHYGG strains was one-seventh of that of the original strain. These results suggest that the formation of a bottleneck for acetic acid production brings about a metabolic flow favorable to L-phenylalanine synthesis in the recombinant strain over-expressing the yggG gene.

Metabolic flux analysis in biotechnology processes.

Metabolic flux analysis (MFA) has become a fundamental tool of metabolic engineering to elucidate the metabolic state of the cell and has been applied to various biotechnological processes. In recent years, considerable technical advances have been made. Developments of analytical instruments allow us to determine (13)C labeling distribution of intracellular metabolites with high accuracy and sensitivity. Moreover, kinetic information of intracellular label distribution during isotopic instationary enables us to calculate metabolic fluxes with shortened experimental time and decreased amount of labeled substrate. The (13)C MFA may be one of the most promising approaches for the target estimation to improve strain performances and production processes.

Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS.

Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.

Theoretical analysis of amino acid-producing Escherichia coli using a stoichiometric model and multivariate linear regression.

This work demonstrates a novel computational approach combining flux balance modeling with statistical methods to identify correlations among fluxes in a metabolic network, providing insight as to how the fluxes should be redirected to achieve maximum product yield. The procedure is demonstrated using the example of amino acid production from an industrial Escherichia coli production strain and a hypothetical engineered strain overexpressing two heterologous genes. Regression analysis based on a random sampling of 5,000 points within the feasible solution space of the E. coli stoichiometric network suggested that increased activity of the glyoxylate cycle or PEP carboxylase and elimination of malic enzyme will improve lysine and arginine synthesis.