Biotechnology and Bioengineering

Cover image for Vol. 112 Issue 8

Edited By: Douglas S. Clark

Impact Factor: 4.126

ISI Journal Citation Reports © Ranking: 2014: 24/162 (Biotechnology & Applied Microbiology)

Online ISSN: 1097-0290

Metabolic Engineering - Joint Virtual Issue B&B and Biotechnology Journal

Virtual Issue Celebrating Two Decades of the Metabolic Engineering Conference

Edited by Biotechnology and Bioengineering Editor-in-Chief Dr. Douglas S. Clark and Biotechnology Journal Editor-in-Chief Dr. Sang Yup Lee 

The International Metabolic Engineering Conference held every two years is celebrating its 10th gathering in Vancouver, Canada. This conference has become not only the flagship conference in metabolic engineering, but also a “must-attend” conference for biotechnologists from academia and industry alike. Biotechnology Journal and Biotechnology and Bioengineering would like to celebrate this unique conference’s 20 years of leadership in providing a platform for sharing state-of-the-art developments as well as effective communication and enjoyable interaction among its many participants. Indeed, over the past two decades metabolic engineering has become an increasingly important and vibrant discipline. In recognition of this exciting milestone, we selected 10 recent articles in the field of metabolic engineering, five from each journal, to share with metabolic engineers – all of these articles are freely accessible online. The articles encompass different aspects of metabolic engineering and illustrate the considerable scope and sophistication of its current state. 

Throughout the post-genomic era of the past decade metabolic engineering has been taking more systems-level approaches. One important strategy of metabolic engineering is genome-scale modeling and simulation for deciphering global metabolic characteristics and also for identifying targets to be manipulated. The most popular flux balance analysis, however, does not allow determination of dynamic metabolic responses triggered by various perturbations. Hatzimanikatis and colleagues report construction of a large-scale mechanistic kinetic model of optimally grown Escherichia coli using the optimization and risk analysis of complex living entities (ORACLE) framework. Hatzimanikatis and colleagues also investigated the complex interplay between stoichiometry, thermodynamics, and kinetics in determining the flexibility and capabilities of metabolism, which is of great interest to researchers in the field for genome-scale dynamic simulation. Cintolesi and colleagues applied kinetic modeling and metabolic control analysis (MCA) to analyze the fermentative metabolism of glycerol in Escherichia coli. Based on this comprehensive analysis they identified two enzymes − glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM) − that almost exclusively control glycerol metabolism in E. coli, and report a production of 20 g/L ethanol from waste glycerol as the result of over expressing the two enzymes.

Liao, Maranas and colleagues report a novel methodology for the optimization-driven identification of genetic perturbations for the accelerated convergence of model parameters in ensemble modeling of metabolic networks. Interestingly, the results showed that optimal perturbations are not always located close to reactions whose fluxes are measured. Also, there seems to be a maximum number of simultaneous perturbations beyond which no appreciable increase in the divergence of flux predictions is achieved. Pfleger and colleagues report the development of a plasmid-free strain of E. coli TY05 that produces C12 and C14 free fatty acid (FFA) species at levels comparable to antibiotic-resistant strains expressing a thioesterase from a plasmid. Pfleger and colleagues devised a kinetic model and estimated optimal culture parameters for the effective FFA production based on the data from continuous cultures of E. coli TY05, leading to a subsequent 37% increase in the FFA yields over batch cultures in the same medium. Toxicity of the FFA overproduction in cells and increased maintenance energy requirements of TYO5 are two major issues the authors intend to address in future studies.

Isotope-labeling experiments based on 13C-carbon have long been important for the accurate determination of metabolic fluxes. However, the mathematical models that have been developed have commonly neglected the influence of kinetic isotope effects on the distribution of 13C-label in intracellular metabolites. Stephanopoulos and his colleague performed rigorous experiments on the fractionation at the pyruvate node as an example, and report that the kinetic isotope effects must be considered in the assessment of errors in 13C-labeling data, fitting between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions.

Recombineering at the genome level has become another important tool for the metabolic engineering of microorganisms. Gill and co-workers report the results of experiments that address the problem of multiple chromosomes, calculations predicting how many generations are needed to obtain a pure colony, and how changes in experimental procedure or genetic background can minimize the effect of multiple chromosomes.

It is notable that more and more chemicals and materials produced by the petrochemical industry can now be generated by biorefineries. Zhao and colleagues report 4.0− and 4.4−fold increases in the cellobiose uptake rate and ethanol production, respectively, in Saccharomyces cerevisiae under anaerobic conditions by the directed evolution of cellodextrin transporter 2 (CDT2). Zhao and colleagues utilized higher energetic benefits of CDT2 as a sugar facilitator over a more efficient transporter CDT1 by modifying the CDT2 substrate specificity and expression levels in the HTT mutant, which significantly enhanced its ability to produce biofuel from cellobiose under anaerobic conditions. Lee and colleagues [8] report the development of metabolically engineered E. coli for the production of phenol from glucose. Theysimultaneously engineered 18 E. coli strains for the production of phenol using synthetic regulatory small RNA (sRNA) technology and introducing the tyrosine phenol-lyase reaction. As phenol is toxic to cells, Lee and colleagues used biphasic fed-batch fermentation, with glycerol tributyrate as an extractant of phenol. The concentration of phenol in the glycerol tributyrate phase and fermentation broth reached 9.84 and 0.3 g/L, respectively, in 21 hours, which translates into the final phenol titer and productivity of 3.79 g/L and 0.18 g/L/h, respectively. 2, 3-Butanediol is an important compound for the production of platform chemicals and biofuels. However, 2, 3-butanediol is traditionally produced in pathogenic microorganisms, such as Klebsiella pneumonia and Klebsiella oxytoca, which only ferment sugars at 37°C, far below the 50-55°C threshold for optimal sugar sacharification and fermentation (SSF). Chamu and colleagues report the creation of the first non-pathogenic Bacillus licheniformis BL5 and BL8 strains that are capable of the improved production of high purity D-(-) 2,3-butanediol (98% optical purity) from lignocellulosic biomass at 50°C. Finally, Nielsen and colleagues investigated the effect of the polyhydroxybutyrate (PHB) precursor acetyl-CoA, and the cofactor NADPH, on PHB biosynthesis in S. cerevisiae by engineering either or both of them in different combinations. Interestingly, the best performing strains over-expressed either acetyl-CoA or NADPH individually, but not simultaneously, with the highest PHB yields detected in the strain SCKK032, which utilizes the engineered phosphoketolase pathway to overproduce acetyl-CoA and NADPH. These results reveal a more important role of precursor in the improved PHB production.

Metabolic engineering is crucial for the advancement of the bio-based economies that are the strategic development objectives of many companies and governments across the globe – at the current pace of development, we are confident that a bio-based economy is a slogan that will become more a part of everyday vocabulary in the future.

Towards kinetic modeling of genome-scale metabolic networks without sacrificing stoichiometric, thermodynamic and physiological constraints.

Chakrabarti, A., Miskovic, L., Soh, K. C. and Hatzimanikatis, V. (2013),

Biotechnology Journal.

Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli.

Cintolesi, A., Clomburg, J. M., Rigou, V., Zygourakis, K. and Gonzalez, R. (2012),

Biotechnology and Bioengineering.

Optimization-driven identification of genetic perturbations accelerates the convergence of model parameters in ensemble modeling of metabolic networks.

Zomorrodi, A. R., Lafontaine Rivera, J. G., Liao, J. C. and Maranas, C. D. (2013),

Biotechnology Journal.

Kinetic modeling of free fatty acid production in Escherichia coli based on continuous cultivation of a plasmid free strain.

Youngquist, J. T., Lennen, R. M., Ranatunga, D. R., Bothfeld, W. H., II, W. D. M. and Pfleger, B. F. (2012),

Biotechnology and Bioengineering.

Kinetic isotope effects significantly influence intracellular metabolite 13C labeling patterns and flux determination

Wasylenko, T. M. and Stephanopoulos, G. (2013),

Biotechnology Journal.

Recombineering to homogeneity: extension of multiplex recombineering to large-scale genome editing.

Boyle, N. R., Reynolds, T. S., Evans, R., Lynch, M. and Gill, R. T. (2013),

Biotechnology Journal.

Directed evolution of a cellodextrin transporter for improved biofuel production under anaerobic conditions in Saccharomyces cerevisiae.

Lian, J., Li, Y., HamediRad, M. and Zhao, H. (2014),

Biotechnology and Bioengineering.

Metabolic engineering of Escherichia coli for the production of phenol from glucose.

Kim, B., Park, H., Na, D. and Lee, S. Y. (2013),

Biotechnology Journal.

Metabolic engineering of thermophilic Bacillus licheniformis for chiral pure D-2,3-butanediol production

Wang, Q., Chen, T., Zhao, X. and Chamu, J. (2012),

Biotechnology and Bioengineering.

Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway.

Kocharin, K., Siewers, V. and Nielsen, J. (2013),

Biotechnology and Bioengineering.