On structural identifiability analysis of the cascaded linear dynamic systems in isotopically non-stationary 13C labelling experiments.

The isotopically non-stationary 13C labelling experiments, as an emerging experimental technique, can estimate the intracellular fluxes of the cell culture under an isotopic transient period. However, to the best of our knowledge, the issue of the structural identifiability analysis of non-stationary isotope experiments is not well addressed in the literature. In this work, the local structural identifiability analysis for non-stationary cumomer balance equations is conducted based on the Taylor series approach. The numerical rank of the Jacobian matrices of the finite extended time derivatives of the measured fractions with respect to the free parameters is taken as the criterion. It turns out that only one single time point is necessary to achieve the structural identifiability analysis of the cascaded linear dynamic system of non-stationary isotope experiments. The equivalence between the local structural identifiability of the cascaded linear dynamic systems and the local optimum condition of the nonlinear least squares problem is elucidated in the work. Optimal measurements sets can then be determined for the metabolic network. Two simulated metabolic networks are adopted to demonstrate the utility of the proposed method.

Comprehensive reconstruction and evaluation of Pichia pastoris genome-scale metabolic model that accounts for 1243 ORFs.

BACKGROUND: Pichia pastoris is one of the most important cell factories for production of industrial enzymes and heterogenous proteins. The genome-scale metabolic model of high quality is crucial for comprehensive understanding of the P. pastoris metabolism. METHODS: In this paper, we upgraded P. pastoris genome-scale metabolic model based on the combination of latest genome annotations and literatures. Then the performance of the new model was evaluated using the Cobra Toolbox v2.0. RESULTS: Compared with the recently published model iMT1026, the reaction number in the new model iRY1243 was increased from 2035 to 2407 and the metabolite number was increased from 1018 to 1094. Accordingly, the unique ORF number was increased from 1026 to 1243. To improve the metabolic functions of P. pastoris genome-scale metabolic model, the biosynthesis pathways of vitamins and cofactors were carefully added. iRY1243 showed good performances when predicting the growth capability on most of the reported carbon and nitrogen sources, the metabolic flux distribution with glucose as a sole carbon source, the essential and partially essential genes, and the effects of gene deletion or overexpression on cell growth and S-adenosyl-l-methionine production. CONCLUSION: iRY1243 is an upgraded P. pastoris genome-scale metabolic model with significant improvements in the metabolic coverage and prediction ability, and thus it will be a potential platform for further systematic investigation of P. pastoris metabolism.

Combined 13C-assisted metabolomics and metabolic flux analysis reveals the impacts of glutamate on the central metabolism of high ß-galactosidase-producing Pichia pastoris.

BACKGROUND: Pichia pastoris is a popular recombinant protein expression system for its accessibility of efficient gene manipulation and high protein production. Sufficient supply of precursors, energy, and redox cofactors is crucial for high recombinant protein production. In our present work, we found that the addition of glutamate improved the recombinant ß-galactosidase (ß-gal) production by P. pastoris G1HL. METHODS: To elucidate the impacts of glutamate on the central metabolism in detail, a combined 13C-assisted metabolomics and 13C metabolic flux analysis was conducted based on LC-MS/MS and GC-MS data. RESULTS: The pool sizes of intracellular amino acids were obviously higher on glucose/glutamate (Glc/Glu). The fluxes in EMP entry reaction and in downstream TCA cycle were 50 and 67% higher on Glc/Glu than on Glc, respectively. While the fluxes in upstream TCA cycle kept almost unaltered, the fluxes in PPP oxidative branch decreased. CONCLUSION: The addition of glutamate leads to a remarkable change on the central metabolism of high ß-galactosidase-producing P. pastoris G1HL. To meet the increased demands of redox cofactors and energy for higher ß-galactosidase production on Glc/Glu, P. pastoris G1HL redistributes the fluxes in central metabolism through the inhibitions and/or activation of the enzymes in key nodes together with the energy and redox status.

Impacts of high ß-galactosidase expression on central metabolism of recombinant Pichia pastoris GS115 using glucose as sole carbon source via (13)C metabolic flux analysis.

The yeast Pichia pastoris GS115 is a widely used microbial cell factory for the production of heterologous protein. In order to reveal the impacts of high heterologous protein expression on the central metabolism of Pichia pastoris GS115 using glucose as sole carbon source, we engineered a high ß-galactosidase expression strain P. pastoris G1HL and a low expression control strain P. pastoris GHL through controlling the initiation strength of constitutive promoter pGAP. The carbon flux distributions in these two strains were quantified via (13)C metabolic flux analysis. Compared to the control strain, G1HL showed a lower growth rate, a higher flux through glycolysis pathway, a higher flux through pentose phosphate pathway, and a lower flux through by-products secretion pathway. The metabolic flux redistribution in G1HL was thought to compensate the increased redox cofactors and energy demands caused by the high protein expression. Although the fluxes through Krebs cycle in two engineered strains were almost the same, they were significantly lower than those in wild strain. The enhanced expression of ß-galactosidase by glutamate supplementation demonstrated the potential of P. pastoris GS115 to catabolize more carbon through the Krebs cycle for even higher protein expression. In conclusion, our work indicates that P. pastoris GS115 can readjusts the central metabolism for higher heterologous protein expression and provides strategies for strain development or process optimization for enhancing production of heterologous protein.

Process modeling of comprehensive integrated forest biorefinery--an integrated approach.

The key to expanding the energy supply, increasing energy security, and reducing the dependency on foreign oil is to develop advanced technologies to efficiently transform our renewable bioresources into domestically produced bioenergy and bioproducts. Conventional biorefineries, i.e., forest products industry's pulp and paper mills with long history of sustainable utilization of lignocellulose (wood), offer a suitable platform for being expanded into future integrated forest biorefineries. Due to the pre-existing infrastructure in current forest products operations, this could present a very cost-effective approach to future biorefineries. In order to better understand the overall process, technical, economic, and environmental impacts, a detailed process modeling of the whole integrated forest biorefinery is presented here. This approach uses a combination of Aspen Plus, WinGEMS, and Microsoft Excel to simulate the entire biorefinery in detail with sophisticated communication interface between the three simulations. Preliminary results for a simple case study of an integrated biorefinery show the feasibility of this approach. Further investigations, including additional details, more process options, and complete integration, are currently underway.