Biotechnology and Bioengineering

Cover image for Vol. 112 Issue 6

Edited By: Douglas S. Clark

Impact Factor: 4.164

ISI Journal Citation Reports © Ranking: 2013: 26/165 (Biotechnology & Applied Microbiology)

Online ISSN: 1097-0290

Virtual Issues

Biotechnology & Bioengineering Virtual Issues

Virtual Issues from Biotechnology & 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: 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: 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


Biotechnology & Bioengineering and Biotechnology Journal

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.


Biomass to Biofuel

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