Biotechnology and Bioengineering

Cover image for Vol. 114 Issue 1

Edited By: Douglas S. Clark

Impact Factor: 4.243

ISI Journal Citation Reports © Ranking: 2015: 24/161 (Biotechnology & Applied Microbiology)

Online ISSN: 1097-0290

Virtual Issues

Virtual Issue: Klaus Mosbach Tribute

Joint Virtual Issue Celebrating Two Decades of the Metabolic Engineering Conference from B&B and BTJ

Virtual Issue: Production of Biofuels

Virtual Issue: B&B at BME XVIII in Beijing

Virtual Issue: Synthetic Biology

Virtual Issue: Metabolic Engineering

Virtual Issue: B&B and BTJ

Virtual Issue: Biomass to Biofuels

Virtual Issue: Klaus Mosbach Tribute

Edited by Biotechnology & Bioengineering Editor-in-Chief Douglas Clark

Klaus Mosbach has always been a master of synergy. Whether exploiting molecular proximity to increase reactivity, affinity interactions to improve separations, or shape complementarity to enhance recognition, Klaus has devised countless ways to amplify the performance of biomolecular systems beyond the sum of their parts. Likewise, he has amplified the impact of his field (which is partially of his own creation) through the sheer volume and brilliance of his ideas and the inspirational effect they have had on so many others. A few testimonials of his influence are provided in this tribute, and reprints of the 10 articles he published over the years in Biotechnology & Bioengineering can be found in this special Virtual Issue.

Read more in the article “Klaus Mosbach Tribute” by Douglas Clark and coauthors:

Klaus Mosbach Tribute.
Douglas S. Clark, Isao Karube, Koji Sode, Leif Bülow, Oliver Brüggemann, Christopher Lowe, Poul Børge Poulsen and Robert S. Langer (2015)

Selected B&B articles by Klaus Mosbach and coauthors:

Preparation of a NAD(H)-polymer matrix showing coenzymic function of the bound pyridine nucleotide.
Per-Olof Larsson and Klaus Mosbach (1971)

Covalent coupling of pullulanase to an acrylic copolymer using a water soluble carbodi-imide.
Kaj Mårtensson and Klaus Mosbach (1972)

Hydrolysis of β-galactosides using polymer-entrapped lactase. A study towards producing lactose-free milk.
Arne Dahlqvist, Bo Mattiasson and Klaus Mosbach (1973)

Continuous regeneration of NAD(P)+ by flavins covalently bound to sepharose.
Mats-Olle Månsson, Bo Mattiasson, Stina Gestrelius and Klaus Mosbach (1976)

Application of cyanide-metabolizing enzymes to environmental control; enzyme thermistor assay of cyanide using immobilized rhodanese and injectase
Bo Mattiasson, Klaus Mosbach and Anders Svenson (1977)

Steroid transformation by activated living immobilized Arthrobacter simplex cells
Sten Ohlson, Per Olof Larsson and Klaus Mosbach (1978)

Peptide synthesis in aqueous–organic solvent mixtures with α-chymotrypsin immobilized to tresyl chloride-activated agarose
Kurt Nilsson and Klaus Mosbach (1984)

Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails.
Jonas Carlsson, Klaus Mosbach and Leif Bülow (1996)

Use of molecularly imprinted polymers in a biotransformation process
Lei Ye, Olof Ramström, Richard J. Ansell, Mats-Olle Månsson and Klaus Mosbach (1999)

Removal of the fermentation by-product succinyl L-tyrosine from the β-lactamase inhibitor clavulanic acid using a molecularly imprinted polymer.
Yihua Yu, Lei Ye, Vern de Biasi and Klaus Mosbach (2002)


Joint Virtual Issue Celebrating Two Decades of the Metabolic Engineering Conference from B&B and BTJ

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.


Virtual Issue: Production of Biofuels


Edited by Harvey Blanch


There has been a resurgence of interest in the production of biofuels from renewable resources, driven in part by concerns over the role of CO2 on climate and measures designed to reduce CO2 emissions. In the US, the Renewable Fuels Standard (RFS) has mandated the production of biofuels, providing a cap on corn-derived ethanol and requiring additional biofuels be produced from sources other than corn, such as lignocellulosic biomass. The conversion of biomass to fuels presents formidable technical and economic challenges, including the price and availability of lignocellulosic biomass, the pretreatment of biomass to permit its conversion to monosaccharides by cellulase enzymes and the fermentation of biomass sugars to ethanol and other fuels. Chemical approaches to convert biomass to fuels include biomass pyrolysis and liquefaction, both of which suffer from low yields. B&B has been the home to manuscripts that have focused on biological routes from biomass to fuels, including many groundbreaking early publications from the 1970s and 1980s, when ethanol production from lignocellulosic biomass was first explored.


In this virtual issue, B&B highlights recent publications addressing the important hurdles in biofuels production from a number of approaches. The saccharification of biomass to simple sugars requires pretreatment of the biomass to facilitate enzymatic hydrolysis. Hou and coworkers describe a new approach in which biomass is dissolved in ionic liquids, reporting the development of novel ionic liquids that are renewable and potentially less costly, than imidazolium-based ionic liquids. Bose et al. show that cellulase enzymes can be stable and exhibit significant activity in ionic liquids, which would enable biomass biomass pretreatment and saccharification to be consolidated into one unit operation. Wang et al. highlight the importance of biomass accessibility in enzymatic saccharification, examining cellulase adsorption and hydrolysis in dependence of biomass accessible surface area (effective pore size). The cost of enzymes employed for saccharification remains a significant economic hurdle; Klein-Marcuschamer et al. provide a detailed economic analysis of enzyme cost for biomass conversion to sugars. Despite a number of projections indicating lowered costs, the authors show that enzymes represent the most important operating cost component after that of the biomass itself. Cannella and Jorgensen highlight the importance of the cellulase components in the saccharification of biomass at high solids loadings (reflecting industrially-relevant conditions). Several operating strategies (SSF, SHF) are reviewed, and the significance of the newly-discovered polysaccharide monoxygenases (LPMOs) in certain strategies is apparent. Wu and Arnold demonstrate an approach to enhance the thermal stability of fungal cellulase components (Cel6A and Cel7A), showing that activity can be retained at elevated temperatures. This may impact the saccharification process and reduce microbial contamination. The selection of the organism for production of improved variant enzymes is highlighted by Dana et al., who show that post-translational modification of the N-terminal glutamine of Cel7A by glutamine cyclase is required for full enzyme activity. Fungal hosts perform this modification, while bacteria and yeast typically do not. 


The acetone-butanol-ethanol (ABE) fermentation has been re-examined as a route to ferment hexose, pentose and oligosaccharides to a mixed solvent product. Jang et al. report an approach to develop a high productivity ABE fermentation by random mutagenesis. By using a cell recycle fermentation and the improved strain, exceptional specific and volumetric productivities of ABE could be obtained. Using a solventogenic Clostridium, Richter and coworkers showed the ability of this organism to convert butyric acid to butanol using glucose as a co-substrate to provide reducing equivalents and ATP. 


Products other than fuels are also of interest in biomass conversion to products. Riedel et al. show that palm oil can be converted to polyhydroxyalkanoates by Ralstonia, providing very high titers (139 gm/L) and high volumetric productivities (> 1gm/ These are levels that are commercially attractive. Conversion of glycerol to fuels could provide a means to use this inexpensive substrate in fatty acid ethyl ester production. Yu et al. show a route to accomplish this by incorporation of a bacterial acyltransferase in the yeast S. cerevisiae.




Novel renewable ionic liquids as highly effective solvents for pretreatment of rice straw biomass by selective removal of lignin

Xue-Dan Hou, Thomas J. Smith, Ning Li and Min-Hua Zong


Enhanced stability and activity of cellulase in an ionic liquid and the effect of pretreatment on cellulose hydrolysis

Sayantan Bose, Charles A. Barnes andJacob W. Petrich


Evaluations of cellulose accessibilities of lignocelluloses by solute exclusion and protein adsorption techniques

Q.Q.Wang, Z. He, Z. Zhu, Y.-H. P. Zhang, Y. Ni, X.L. Luo and J.Y. Zhu


The challenge of enzyme cost in the production of lignocellulosic biofuels

Daniel Klein-Marcuschamer, Piotr Oleskowicz-Popiel, Blake A. Simmons and Harvey W. Blanch


Do new cellulolytic enzyme preparations affect the industrial strategies for high solids lignocellulosic ethanol production?

David Cannella and Henning Jørgensen


Engineered thermostable fungal Cel6A and Cel7A cellobiohydrolases hydrolyze cellulose efficiently at elevated temperatures

Indira Wu and Francis H. Arnold


The importance of pyroglutamate in cellulase Cel7A 

Craig M. Dana, Alexandra Dotson-Fagerstrom, Christine M. Roche, Sarala M. Kal, Harshal A. Chokhawala, Harvey W. Blanch and Douglas S. Clark


Acetone–butanol–ethanol production with high productivity using Clostridium acetobutylicum BKM19

Yu-Sin Jang, Alok Malaviya and Sang Yup Lee


Prolonged conversion of n-butyrate to n-butanol with Clostridium saccharoperbutylacetonicum in a two-stage continuous culture with in-situ product removal

Hanno Richter, Nasib Qureshi, Sebastian Heger, Bruce Dien, Michael A. Cotta and Largus T. Angenent


Synthesis of FAEEs from glycerol in engineered Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression of an unspecific bacterial acyltransferase

Kyung Ok Yu, Ju Jung, Seung Wook Kim, Chul Hwan Park and Sung Ok Han



Virtual Issue: B&B at BME XVIII in Beijing

This collection of articles celebrates our authors who are also participating as speakers at Biochemical and Molecular Engineering XVIII in Beijing, China, June 16-20, 2013.  The selections highlight both the topical and East Meets West themes of the conference. 


In the foundational technologies for synthetic biology area, Wang et al (2012) use global regulator engineering to identify furfural tolerant irrE mutants to improve biofuel production from lignocellulosic biomass. Blazeck et al (2012) describe a synthetic hybrid promoter approach.  They built strong promoter libraries in the model yeast Saccharomyces cerevisiae and illustrated the utility of their approach with three case studies.  An optimized yeast strain is employed by Fukutani et al (2012) to produce an application for a biomimetic odor sensor via a bioluminescence-based signaling assay system.  The review by Sun and Zhao (2013) delineates recent developments in the genome editing tools area, highlighting the power and possible future developments of transcription activator-like effector nucleases (TALENs).


Strain development for (bacterial) biopolymer polyhydroxybutyrate (PHB) production in Saccharomyces cerevisiae is described by Kocharin, Siewers, and Nielsen (2013) whereas Jang, Malaviya, and Lee (2013) provide an improved Clostridium acetobutylicum strain for acetone–butanol–ethanol (ABE) fermentation, generating the highest productivities to date. Woodruff et al (2013) add to that mix with their call for a metabolic engineering strain commons—a set of well-characterized platform strains to apply new metabolic engineering strategies—and provide an example using ethanol production in an  Escherichia coli strain.  A new in silico model, thermodynamic optimum searching (TOS), was developed by Zhu et al (2013) to aid in understanding and optimizing the engineering of microbial cell factories.


In the industrial bioprocess realm, Xu, Xia, and Zhong (2013) describe how sodium ion addition to fermentation improves production of ganoderic acid, one of the key biologically active components of Ganoderma lucidum.  In the CHO cell culture area, an application of global protein sequencing using mass spectrometry was championed by Qiu et al (2013) to identify contaminating virus particles in bioreactor fluids.  To improve timelines and flexibility in the manufacturing process, Seth et al (2013) developed a method to cryopreserve CHO cells in large volume bags and compared their results with existing protocols.  Martinez et al (2013) apply directed evolution to the Bacillus gibsonii Alkaline Protease (BgAP) to improve its temperature properties for an industrial application in the detergent industry. Finally, protein aggregation is addressed by Reza et al (2012) by using polyphenolic disaccharides as an additive to prevent this phenomenon.


Rounding out the series, at the bio-nano interface, Fischback et al (2012) describe a direct enzyme immobilization technique to improve the power density of enzymatic biofuel cells (BFCs).

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

Kanokarn Kocharin, Verena Siewers, and Jens Nielsen


Transcription activator-like effector nucleases (TALENs): A highly efficient and versatile tool for genome editing

Ning Sun and Huimin Zhao


Induced effect of Na+ on ganoderic acid biosynthesis in static liquid culture of Ganoderma lucidum via calcineurin signal transduction

Yi-Ning Xu, Xiao-Xia Xia, and Jian-Jiang Zhong


Acetone–butanol–ethanol production with high productivity using Clostridium acetobutylicum BKM19

Yu-Sin Jang, Alok Malaviya, and Sang Yup Lee


Identification and quantitation of vesivirus 2117 particles in bioreactor fluids from infected Chinese hamster ovary cell cultures

Yongchang Qiu, Nathan Jones, Michelle Busch, Peng Pan, Jesse Keegan, Weichang Zhou, Mark Plavsic, Michael Hayes, John M. McPherson, Tim Edmunds, Kate Zhang, and Robert J. Mattaliano


Development of a new bioprocess scheme using frozen seed train intermediates to initiate CHO cell culture manufacturing campaigns

Gargi Seth, Robert W. Hamilton, Thomas R. Stapp, Lisa Zheng, Angela Meier, Krista Petty, Stephenie Leung, and Srikanth Chary


Towards a metabolic engineering strain “commons”: An Escherichia coli platform strain for ethanol production

Lauren B.A. Woodruff, Brian L. May, Joseph R. Warner, and Ryan T. Gill


Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution

Ronny Martinez, Felix Jakob, Ran Tu, Petra Siegert, Karl-Heinz Maurer, and Ulrich Schwaneberg


Development of thermodynamic optimum searching (TOS) to improve the prediction accuracy of flux balance analysis

Yan Zhu, Jiangning Song, Zixiang Xu, Jibin Sun, Yanping Zhang, Yin Li, and Yanhe Ma


Global regulator engineering significantly improved Escherichia coli tolerances toward inhibitors of lignocellulosic hydrolysates

Jianqing Wang, Yan Zhang, Yilu Chen, Min Lin, and Zhanglin Lin


An improved bioluminescence-based signaling assay for odor sensing with a yeast expressing a chimeric olfactory receptor

Yosuke Fukutani, Jun Ishii, Keiichi Noguchi, Akihiko Kondo, and Masafumi Yohda


Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters

John Blazeck, Rishi Garg, Ben Reed, and Hal S. Alper


Polyphenolic disaccharides endow proteins with unusual resistance to aggregation

Ali Reza A. Ladiwala, Joseph M. Perchiacca, Zachary S. Fishman, Moumita Bhattacharya, Anne Marie Hickey, Bonnie G. Domigan, Jonathan S. Dordick, and Peter M. Tessier


Enzyme precipitate coatings of glucose oxidase onto carbon paper for biofuel cell applications

Mike Fischback, Ki Young Kwon, Inseon Lee, Su Jeong Shin, Hyun Gyu Park, Byoung Chan Kim, Yongchai Kwon, Hee-Tae Jung, Jungbae Kim, and Su Ha


Virtual Issue: Synthetic Biology

Edited by Ryan Gill

Synthetic biology has progressed from the domain of a visionary set of scientists to an increasingly mainstream aspect of modern biotechnology and bioengineering. B&B has been a leader in the publication of synthetic biology research; both indirectly through a longstanding emphasis on protein, pathway, and metabolic engineering, as well as via the publication of a range of recent efforts of direct synthetic biology relevance.

In this virtual issue, B&B is pleased to highlight several of such recent publications starting with research directed at the development of basic synthetic biology capabilities and extending through to the construction of complex circuits and pathways. In Hartenbach and Fussenegger (2006) as well as Du et al. (2009) we see the engineering of basic genetic parts (a mammalian promoter or transcriptional terminators), which enable the construction of synthetic circuits in various cell types. Weiss et al. (2008) provide an example of a simple circuit that allows the direct observation of communication between two engineered strains (a sender and a receiver strain). While Keum et al. (2009) report on the use of an in vitro system to investigate basic genetic circuits involving anti-sense based regulation. We also highlight two papers out of the Shuler group (Echtenkamp et al., 2009; Foley and Shuler, 2010) that describe efforts directed at the larger synthetic biology goal of designing and constructing complete genomes and synthetic cells.

In addition to efforts directed at the development of foundational technologies, B&B has also led in the publication of applications of such technologies to dramatically speed the engineering of production strains (Qian et al., 2009; Wang et al., 2010). Finally, we include two articles (Bagh et al., 2011; Clarke and Voigt, 2011) that demonstrate the potential of this growing field to address problems of broader societal relevance.

A novel synthetic mammalian promoter derived from an internal ribosome entry site, Shizuka Hartenbach and Martin Fussenegger

Engineering motility as a phenotypic response to LuxI/R-dependent quorum sensing in Escherichia coli, Lucien E. Weiss, Jonathan P. Badalamenti, Lane J. Weaver, Anthony R. Tascone, Paul S. Weiss, Tom L. Richard, and Patrick C. Cirino

Combinatorial, selective and reversible control of gene expression using oligodeoxynucleotides in a cell-free protein synthesis system, Jung-Won Keum, Jin-Ho Ahn, Taek Jin Kang, and Dong-Myung Kim

Cell cycle progression in Escherichia coli B/r affects transcription of certain genes: Implications for synthetic genome design, Patricia L. Echtenkamp, David B. Wilson, and Michael L. Shuler

Metabolic engineering of Escherichia coli for the production of putrescine: a four carbon diamine, Zhi-Gang Qian, Xiao-Xia Xia and Sang Yup Lee

Engineering multigene expression in vitro and in vivo with small terminators for T7 RNA polymerase,Liping Du, Rong Gao, and Anthony C. Forster

Considerations for the design and construction of a synthetic platform cell for biotechnological applications, Patricia L. Foley and Michael L. Shuler

Farnesol production from Escherichia coli by harnessing the exogenous mevalonate pathway, Chonglong Wang, Sang-Hwal Yoon, Asad Ali Shah, Young-Ryun Chung, Jae-Yean Kim, Eu--Sung Choi, Jay D. Keasling, and Seon-Won Kim

An active intracellular device to prevent lethal disease outcomes in virus-infected bacterial cells, Sangram Bagh, Mahuya Mandal, Jordan Ang and David R. McMillen

Characterization of combinatorial patterns generated by multiple two-component sensors in E. coli that respond to many stimuli, Elizabeth J. Clark and Christopher A. Voight


Virtual Issue: Metabolic Engineering

Edited by Jens Nielsen

Metabolic engineering is the enabling science associated with the design and development of efficient cell factories that can be used for sustainable, bio-based production of fuels, chemicals and materials. Due to the substantial engineering component of metabolic engineering, B&B has been a natural home for many published papers on metabolic engineering since its emergence as a research field in the early 1990's.

In this virtual issue, B&B highlights several recent publications in the field of metabolic engineering. These include advances in metabolic modeling, a key component of cell factory design, novel molecular biology tools for rapid construction of novel cell factories and implementation of metabolic engineering strategies, i.e. the development and evaluation of cell factories that are producing valuable chemicals. In Song and Ramkrishna (2010) an approach is presented to improve the prediction from limited experimental data through the use of mathematical modeling and Brunk et al. (2011) presents a way to retrofit enzymes to specific biosynthetic pathways using computational methods. Among modeling tools there is also a paper by Colletti et al. (2011) that presents an evaluation of factors that influences overall yields based on statistical analysis of historical data.

In metabolic engineering there is a constant need for advancing our ability to rapidly implement genetic engineering strategies, and even though many advanced methods have been developed for Escherichia coli and Saccharomyces cerevisiae, two widely used cell factories, there is still need for new experimental techniques for these and other organisms. Several new interesting techniques are presented in this virtual issue. These include an accumulative site-specific integration system for CHO cells described by Kameyama et al. (2010), a set of broad-host range vectors for protein expression across different Gram negative bacteria described by Prior et al. (2010), and a genetic reporter system to evaluate cell proliferation rate of mammalian cells presented by Ryan et al. (2011).

The virtual issue also collects several papers on design and implementation of improved cell factories, including yeast improved for production of isoprenoids by Asadollahi et al. (2010) and Fischer et al. (2011), E. coli producing amino acids by Park et al. (2011) and Jung et al. (2010), 1,2-propanediol production by E. coli presented by Clomburg and Gonzalez (2011) and E. coli producing polyketides presented by Boghigian et al. (2011). In recent years adaptive evolution has also shown to be a powerful tool in metabolic engineering and two papers address this issue for improving sugar uptake rate, i.e. galactose by yeast by Lee et al. (2011) and xylose by Zymomonas mobilis by Agrawal et al. (2011).

Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers
Yu Kyung Jung, Tae Yong Kim, Si Jae Park, and Sang Yup Lee

An accumulative site-specific gene integration system using cre recombinase-mediated cassette exchange
Yujiro Kameyama, Yoshinori Kawabe, Akira Ito, and Masamichi Kamihira

Enhancement of farnesyl diphosphate pool as direct precursor of sesquiterpenes through metabolic engineering of the mevalonate pathway in Saccharomyces cerevisiae
Mohammad A. Asadollahi, Jérôme Maury, Michel Schalk, Anthony Clark, and Jens Nielsen

Prediction of metabolic function from limited data: Lumped hybrid cybernetic modeling (L-HCM)
Hyun-Seob Song and Doraiswami Ramkrishna

Broad-host-range vectors for protein expression across gram negative hosts
Jamie E. Prior, Michael D. Lynch, and Ryan T. Gill

Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering
Ki-Sung Lee, Min-Eui Hong, Suk-Chae Jung, Suk-Jin Ha, Byung Jo Yu, Hyun Min Koo, Sung Min Park, Jin-Ho Seo, Dae-Hyuk Kweon, Jae Chan Park, and Yong-Su Jin

Adaptation yields a highly efficient xylose-fermenting Zymomonas mobilis strain
Manoj Agrawal, Zichao Mao, and Rachel Ruizhen Chen

Metabolic engineering of Escherichia coli for the production of 1,2-propanediol from glycerol
James M. Clomburg and Ramon Gonzalez

Evaluating factors that influence microbial synthesis yields by linear regression with numerical and ordinal variables
Peter F. Colletti, Yogesh Goyal, Arul M. Varman, Xueyang Feng, Bing Wu, and Yinjie J. Tang

Escherichia coli W as a new platform strain for the enhanced production of L-valine by systems metabolic engineering
Jin Hwan Park, Yu-Sin Jang, Jeong Wook Lee, and Sang Yup Lee

Multi-factorial engineering of heterologous polyketide production in Escherichia coli reveals complex pathway interactions
Brett A. Boghigian, Haoran Zhang, and Blaine A. Pfeifer

Metabolic engineering of monoterpene synthesis in yeast
Marc J. C. Fischer, Sophie Meyer, Patricia Claudel, Marc Bergdoll, and Francis Karst

A genetic reporter system to gauge cell proliferation rate
Ryan W.S. Peacock and Clifford L. Wang

Integrating computational methods to retrofit enzymes to synthetic pathways
Elizabeth Brunk, Marilisa Neri, Ivano Tavernelli, Vassily Hatzimanikatis, and Ursula Rothlisberger


Virtual Issue: B&B and BTJ

Biotechnology and Bioengineering and Biotechnology Journal present a joint Virtual Issue for the occasion of the "Biochemical and Molecular Engineering XVII - Emerging Frontiers" conference, featuring articles from representative sessions and invited speakers.

Systematizing the generation of missing metabolic knowledge
Orth JD, Palsson BØ.

Advanced biofuel production in microbes
Peralta-Yahya PP, Keasling JD.

Biofabrication of antibodies and antigens via IgG-binding domain engineered with activatable pentatyrosine pro-tag
Wu HC, Shi XW, Tsao CY, Lewandowski AT, Fernandes R, Hung CW, DeShong P, Kobatake E, Valdes JJ, Payne GF, Bentley WE.

Metabolic pathways and fermentative production of L-aspartate family amino acids
Park JH, Lee SY.

Three-dimensional cell culture microarray for high-throughput studies of stem cell fate
Fernandes TG, Kwon SJ, Bale SS, Lee MY, Diogo MM, Clark DS, Cabral JM, Dordick JS.

Tissue engineering for clinical applications
Bhatia SK.

Modeling the competition between aggregation and self-assembly during virus-like particle processing
Ding Y, Chuan YP, He L, Middelberg AP

Production of biopharmaceuticals and vaccines in plants via the chloroplast genome
Daniell H.

Microscale to manufacturing scale-up of cell-free cytokine production—a new approach for shortening protein production development timelines
Zawada JF, Yin G, Steiner AR, Yang J, Naresh A, Roy SM, Gold DS, Heinsohn HG, Murray CJ.

A microwell platform for the scale-up of a multistep bioconversion to bench-scale reactors: sitosterol side-chain cleavage
Marques MP, Cabral JM, Fernandes P.

Reaching the depth of the Chinese hamster ovary cell transcriptome
Jacob NM, Kantardjieff A, Yusufi FN, Retzel EF, Mulukutla BC, Chuah SH, Yap M, Hu WS.

CHO-K1 host cells adapted to growth in glutamine-free medium by FACS-assisted evolution
Bort JA, Stern B, Borth N.


Virtual Issue: Biomass to Biofuels

Gleaning a viable, renewable energy source from biomass, in its many guises, has been a source of active investigation for decades. Inherent in this collection of previous papers from B&B is a continuing exploration and refinement of enzyme development and bioprocessing aspects related to the ethanol-from-lignocellulose approach to liquid biofuel. Articles in this virtual issue provide a snapshot of the field, from the 1970's through the first decade of the 21st century, documenting the importance of physicochemical properties, various production strains, pretreatment technologies, and by-product inhibitors on fermentation processes related to ethanol production.

Enzymatic hydrolysis of waste cellulose
Mary Mandels, Lloyd Hontz, John Nystrom

Competition for mixed substrates by microbial populations
H. Yoon, G. Klinzing, H. W. Blanch

Enhanced production of cellulase, hemicellulase, and β-glucosidase by Trichoderma reesei (Rut C-30)
S. Kishen Tangnu, Harvey W. Blanch, Charles R. Wilke

Enzymatic hydrolysis of cellulose and various pretreated wood fractions
J. N. Saddler, H. H. Brownell, L. P. Clermont, N. Levitin

Adsorption of cellulase on cellulose: Effect of physicochemical properties of cellulose on adsorption and rate of hydrolysis
Sun Bok Lee, H. S. Shin, Dewey D. Y. Ryu, M. Mandels

Conversion of pentoses by yeasts
Cheng-Shung Gong, Tanya A. Claypool, Linda D. McCracken, Christine M. Maun, Pear P. Ueng, George T. Tsao

By-product inhibition effects on ethanolic fermentation by Saccharomyces cerevisiae
Brian Maiorella, Harvey W. Blanch, Charles R. Wilke

Competitive adsorption of cellulase components and its significance in a synergistic mechanism
Dewey D. Y. Ryu, Cheol Kim, M. Mandels

The effect of organosolv pretreatment on the enzymatic hydrolysis of poplar
Mark T. Holtzapple, Arthur E. Humphrey

Economic evaluation of alternative ethanol fermentation processes
B. L. Maiorella, H. W. Blanch, C. R. Wilke

Enzymatic hydrolysis and recrystallization behavior of initially amorphous cellulose
Maria Silvia Bertran, Bruce E. Dale

Enzymatic hydrolysis of cellulose in aqueous two-phase systems. I. partition of cellulases from Trichoderma reesei
Folke Tjerneld, Ingrid Persson, Per-Âke Albertsson, Bärbel Hahn-Hägerdal

Study of the enzymatic hydrolysis of cellulose for production of fuel ethanol by the simultaneous saccharification and fermentation process
George P. Philippidis, Tammy K. Smith, Charles E. Wyman

Activity studies of eight purified cellulases: Specificity, synergism, and binding domain effects
Diana C. Irwin, Michael Spezio, Larry P. Walker, David B. Wilson

Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts
Eva Palmqvist, Halfdan Grage, Nina Q. Meinander, Bärbel Hahn-Hägerdal

Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli
Jesus Zaldivar, Alfredo Martinez, Lonnie O. Ingram

Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae
Márk Gárdonyi, Marie Jeppsson, Gunnar Lidén, Marie F. Gorwa-Grauslund, Bärbel Hahn-Hägerdal

Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains
Marco Sonderegger, Marie Jeppsson, Christer Larsson, Marie-Françoise Gorwa-Grauslund, Eckhard Boles, Lisbeth Olsson, Isabel Spencer-Martins, Bärbel Hahn-Hägerdal, Uwe Sauer

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems
Yi-Heng Percival Zhang, Lee R. Lynd

A functionally based model for hydrolysis of cellulose by fungal cellulase
Y.-H. Percival Zhang, Lee R. Lynd

Liquefaction of lignocellulose at high-solids concentrations
Meijuan Zeng, Nathan S. Mosier, Chia-Ping Huang, Debra M. Sherman, Michael R. Ladisch

Fractionating recalcitrant lignocellulose at modest reaction conditions
Henning Jørgensen, Jakob Vibe-Pedersen, Jan Larsen, Claus Felby

Microscopic examination of changes of plant cell structure in corn stover due to hot water pretreatment and enzymatic hydrolysis
Yi-Heng Percival Zhang, Shi-You Ding, Jonathan R. Mielenz, Jing-Biao Cui, Richard T. Elander, Mark Laser, Michael E. Himmel, James R. McMillan, Lee R. Lynd

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility
Tina Jeoh, Claudia I. Ishizawa, Mark F. Davis, Michael E. Himmel, William S. Adney, David K. Johnson

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases
Sanni P. Voutilainen, Terhi Puranen, Matti Siika-aho, Arja Lappalainen, Marika Alapuranen, Jarno Kallio, Satu Hooman, Liisa Viikari, Jari Vehmaanperä, Anu Koivula

Visualization of biomass solubilization and cellulose regeneration during ionic liquid pretreatment of switchgrass
Seema Singh, Blake A. Simmons, Kenneth P. Vogel