Table of Contents

Volume 1, Issue 5

  September/October 2010

  Pages 303 - 368

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EXPERT COMMENTARY

Open Access Article

Engineering organisms for industrial fuel production

David A. Berry
Pages 303 - 308
http://dx.doi.org/10.4161/bbug.1.5.12941
Abstract | PDF

Volatile fuel costs, need to reduce greenhouse gas emissions, and fuel security concerns are driving efforts to produce sustainable renewable fuels and chemicals.  Petroleum comes from sunlight, CO₂, and water converted via a biological intermediate into fuel over a several million year timescale.  It stands to reason that using biology to short-circuit this time cycle offers an attractive alternative-but only with relevant products at or below market prices.  The state of the art of biological engineering over the past five years has progressed to allow for market needs to drive innovation rather than trying to adapt existing approaches to the market.  This report describes two innovations using synthetic biology to dis-intermediate fuel production.  LS9 is developing a means to convert biological intermediates such as cellulosic hydrolysates into drop-in hydrocarbon product replacements such as diesel.  Joule Unlimited is pioneering approaches eliminate feedstock dependency by efficiently capturing sunlight, CO₂, and water to produce fuels and chemicals.  The innovations behind these companies are built with the market in mind, focused on low cost biosynthesis of existing products of the petroleum industry.  Through successful deployment of technologies such as those behind LS9 and Joule, alternative sources of petroleum products will mitigate the many of the issues faced with our petroleum-based economy.




COMMENTARY

Open Access Article

Synthetic biology: A foundation for multi-scale molecular biology

Adam G. Bower, Maria K. McClintock and Stephen S. Fong
Pages 309 - 312
http://dx.doi.org/10.4161/bbug.1.5.12391
Abstract | PDF

The field of synthetic biology has made rapid progress in a number of areas including method development, novel applications, and community building.  In seeking to make biology “engineerable,” synthetic biology is increasing the accessibility of biological research to researchers of all experience levels and backgrounds.  One of the underlying strengths of synthetic biology is that it may establish the framework for a rigorous bottom-up approach to studying biology starting at the DNA level.  Building upon the existing framework established largely by the Registry of Standard Biological Parts, careful consideration of future goals may lead to integrated multi-scale approaches to biology.  Here we describe some of the current challenges that need to be addressed or considered in detail to continue the development of synthetic biology.  Specifically, discussion on the areas of elucidating biological principles, computational methods, and experimental construction methodologies are presented.




REVIEWS

Open Access Article

Lactococcus lactis as an adjuvant and delivery vehicle of antigens against pneumococcal respiratory infections

Marcela Susana Medina, Elisa Ofelia Vintiñi, Julio Villena, Raul R. Raya and Susana Gladis Alvarez
Pages 313 - 325
http://dx.doi.org/10.4161/bbug.1.5.12086
Abstract | PDF

Most studies of Lactococcus lactis as delivery vehicles of pneumococcal antigens are
focused on the effectiveness of mucosal recombinant vaccines against Streptococcus
pneumoniae in animal models. At present, there are three types of pneumococcal
vaccines: capsular polysaccharide pneumococcal vaccines (PPV), protein-
polysaccharide conjugate pneumococcal vaccines (PCV) and protein-based
pneumococcal vaccines (PBPV). Only PPV and PCV have been licensed. These
vaccines, however, do not represent a definitive solution. Novel, safe and inexpensive
vaccines are necessary, especially in developing countries. Probiotic microorganisms
such as lactic acid bacteria (LAB) are an interesting alternative for their use as vehicles
in pneumococcal vaccines due to their GRAS (Generally Recognized As Safe) status.
Thus, the adjuvanticity of Lactococcus lactis by itself represents added value over the
use of other bacteria, a question dealt with in this review. In addition, the expression of
different pneumococcal antigens as well as the use of oral and nasal mucosal routes of
administration of lactococcal vaccines is considered. The advantages of nasal live
vaccines are evident; nonetheless, oral vaccines can be a good alternative when the
adequate dose is used. Another point addressed here is the use of live versus inactivated
vaccines. In this sense, few researches have focused on inactivated strains to be used as
vaccines against pneumoccoccus. The immunogenicity of live vaccines is better than the
one afforded by inactivated ones; however, the probiotic-inactivated vaccine
combination has improved this matter considerably. The progress made so far in the
protective immune response induced by recombinant vaccines, the successful trials in
animal models and the safety considerations of their application in humans suggest that
the use of recombinant vaccines represents a good short-term option in the control of
pneumococcal diseases.

Open Access Article

The bacterial ghost platform system: Production and applications

Timo Langemann, Verena Juliana Koller, Abbas Muhammad, Pavol Kudela, Ulrike Beate Mayr and Werner Lubitz
Pages 326 - 336
http://dx.doi.org/10.4161/bbug.1.5.12540
Abstract | PDF

The Bacterial Ghost (BG) platform technology is an innovative system for vaccine, drug or active substance delivery and for technical applications in white biotechnology. BGs are cell envelopes derived from  Gram-negative bacteria. BGs are devoid of all cytoplasmic content but have a preserved cellular morphology including all cell surface structures. Using BGs as delivery vehicles for subunit or DNA-vaccines the particle structure and surface properties of BGs are targeting the carrier itself to primary antigen-presenting cells. Furthermore, BGs exhibit intrinsic adjuvant properties and trigger an enhanced humoral and cellular immune response to the target antigen. Multiple antigens of the native BG envelope and recombinant protein or DNA antigens can be combined in a single type of BG. Antigens can be presented on the inner or outer membrane of the BG as well as in the periplasm that is sealed during BG formation. Drugs or supplements can also be loaded to the internal lumen or periplasmic space of the carrier. BGs are produced by batch fermentation with subsequent product recovery and purification via tangential flow filtration. For safety reasons all residual bacterial DNA is inactivated during the BG production process by the use of staphylococcal nuclease A and/or the treatment with b-propiolactone. After purification BGs can be stored long-term at ambient room temperature as lyophilized product. The production cycle from the inoculation of the pre-culture to the purified BG concentrate ready for lyophilization does not take longer than a day and thus meets modern criteria of rapid vaccine production rather than keeping large stocks of vaccines. The broad spectrum of possible applications in combination with the comparably low production costs make the BG platform technology a safe and sophisticated product for the targeted delivery of vaccines and active agents as well as carrier of immobilized enzymes for applications in white biotechnology.




REPORTS

Open Access Article

Rapid generation of random mutant libraries

Mary Abou-Nader and Michael J. Benedik
Pages 337 - 340
http://dx.doi.org/10.4161/bbug.1.5.12942
Abstract | PDF

A simple and efficient method utilizing in vivo recombination to create recombinant libraries incorporating the products of PCR amplification is described. This will be especially useful for generating large pools of randomly mutagenized clones after error-prone PCR mutagenesis. Here we investigate various parameters to optimize this approach and we demonstrate that as little as1 pmole of PCR fragment can generate a library with greater than 10⁴ clones in a single transformation without ligation.

Open Access Article

Transgenic organisms expressing genes from Bacillus thuringiensis to combat insect pests

Arieh Zaritsky, Eitan Ben-Dov, Dov Borovsky, Sammy Boussiba, Monica Einav, Galina Gindin, A. Rami Horowitz, Mikhail Kolot, Olga Melnikov, Zvi Mendel and Ezra Yagil
Pages 341 - 344
http://dx.doi.org/10.4161/bbug.1.5.13087
Abstract | PDF

Various subspecies (spp.) of Bacillus thuringiensis (Bt) are considered the best agents known so far to control insects, being highly specific and safe, easily mass produced and with long shelf life¹. The para-crystalline body that is produced during sporulation in the exosporium includes polypeptides named d-endotoxins, each killing a specific set of insects. The different entomopathogenic toxins of various Bt ssp. can be manipulated genetically in an educated way to construct more efficient transgenic bacteria or plants that express combinations of toxin genes to control pests². Joint research projects in our respective laboratories during the last decade demonstrate what can be done by implementing certain ideas using molecular biology with Bt ssp. israelensis (Bti) as a model system. Here, we describe our progress achieved with Gram-negative bacterial species, including cyanobacteria, and some preliminary experiments to form transgenic plants, mainly to control mosquitoes (Diptera), but also a particular Lepidopteran and Coleopteran pest species. In addition, a system is described by which environment-damaging genes can be removed from the recombinants thus alleviating procedures for obtaining permits to release them in nature.




ADDENDA

Open Access Article

Investigating the importance of charged residues in lantibiotics

Srinivas Suda, Colin Hill, Paul D. Cotter and R. Paul Ross
Pages 345 - 351
http://dx.doi.org/10.4161/bbug.1.5.12353
Abstract | PDF

Lantibiotics are antimicrobial peptides which can have a broad spectrum activity against many Gram positive pathogens. Many of these peptides contain charged amino acids which may be of critical importance with respect to antimicrobial activity. We have recently carried out an in-depth bioengineering based investigation of the importance of charged residues in a representative two peptide lantibiotic, lacticin 3147, and here we discuss the significance of these findings in the context of other lantibiotics and cationic antimicrobial peptides.

Open Access Article

Improving yield of industrial biomass propagation by increasing the Trx2p dosage

Rocío Gómez-Pastor, Roberto Pérez-Torrado and Emilia Matallana
Pages 352 - 353
http://dx.doi.org/10.4161/bbug.1.5.12384
Abstract | PDF

The beneficial effect of improving yeast redox response by increasing
thioredoxin levels has been shown. Decreased lipid and protein oxidation is reflected in
an increased biomass yield. In addition, increased redox defenses like glutathione and
ROS scavenging enzymes are observed. Furthermore, the wine produced with the
modified strain presented more aromatic compounds than the control strain, and its
organoleptic properties increased. Here, we hypothesize that reduced glycolytic enzyme
carbonylation can increase not only the glycolytic flux but also, and consequently, the
biomass yield in the industrial biomass propagation process. The commercial use of the
thioredoxin bioengineered yeast as an antioxidant dietetic supplement is also discussed.

Open Access Article

Gene knockdown by ihpRNA-triggering in the ectomycorrhizal basidiomycete fungus Laccaria bicolor

Minna J. Kemppainen and Alejandro G. Pardo
Pages 354 - 358
http://dx.doi.org/10.4161/bbug.1.5.12385
Abstract | PDF

Ectomycorrhiza (ECM) is a mutualistic association between fungi and the roots of the vast majority of trees. These include numerous ecologically and economically relevant species and the participating fungal symbionts are predominantly filamentous basidiomycetes. In natural ecosystems the plant nutrient uptake from soil takes place via the extra-radical mycelia of these ECM mycosimbionts as a trade for plant photosyntates. The symbiotic phase in the life cycle of ECM basidiomycetes is the dikaryotic hyphae. Therefore, studies on symbiotic relevant gene functions require the inactivation of both gene copies in these dikaryotic fungi. RNA silencing is a eukaryotic sequence homology-dependent degradation of target RNAs which is believed to have evolved as a protection mechanism against invading nucleic acids. In different eukaryotic organisms, including fungi, the RNA silencing pathway can be artificially triggered to target and degrade gene transcripts of interest, resulting in gene knock-down. Most importantly, RNA silencing can act at the cytosolic level affecting mRNAs originating from several gene copies and different nuclei thus offering an efficient means of altering gene expression in dikaryotic organisms. Therefore, the pHg/pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII-promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy two-step PCR-cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300-based binary vector carrying a hygromycin resistance cassette, makes the pHg/pSILBAγ plasmid compatible with Agrobacterium-mediated transformation. The pHg/pSILBAγ-system results in predominantly single integrations of RNA silencing triggering T-DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. Besides the optimized use in L. bicolor, general consideration was taken to build a vector system with maximum compatibility with other homobasidiomycetes and different transformation techniques.

Open Access Article

Microbial renewable feedstock utilization: A substrate-oriented approach

Karl Rumbold, Hugo J. J. van Buijsen, Vincent M. Gray, Johan W. van Groenestijn, Karin M. Overkamp, Ronald S. Slomp, Mariët J. van der Werf and Peter J. Punt
Pages 359 - 366
http://dx.doi.org/10.4161/bbug.1.5.12389
Abstract | PDF

Increasingly lignocellulosic biomass hydrolysates are used as the feedstock for industrial fermentations. These biomass hydrolysates consist of complex mixtures of different fermentable sugars, but also contain inhibitors and salts that affect the performance of the product-generating microbes. The performance of six industrially relevant microorganisms, i.e. two bacteria (Escherichia coli and Corynebacterium glutamicum ), two yeasts ( Saccharomyces cerevisiae and Pichia stipitis ) and two fungi ( Aspergillus niger and Trichoderma reesei ) were compared for their ability to utilize and grow on different feedstock hydrolysates (corn stover, wheat straw, sugar cane bagasse and willow wood). Moreover, the ability of the selected hosts to utilize waste glycerol from the biodiesel industry was evaluated. P. stipitis and A. niger were found to be the most versatile and C. glutamicum, and S. cerevisiae were shown to be the least adapted to renewable feedstocks. Clear differences in the utilization of the more abundant carbon sources in these feedstocks were observed between the different species. Moreover, in a species-specific way the production of various metabolites, in particular polyols, alcohols and organic acids was observed during fermentation. Based on the results obtained we conclude that a substrate-oriented instead of the more commonly used product oriented approach towards the selection of a microbial production host will avoid the requirement for extensive metabolic engineering. Instead of introducing multiple substrate utilization and detoxification routes to efficiently utilize lignocellulosic hydrolysates only one biosynthesis route forming the product of interest has to be engineered.




EXTRA VIEW

Open Access Article

BT-engineered bugs versus insect pests

Alejandro G. Pardo
Pages 367 - 368
http://dx.doi.org/10.4161/bbug.1.5.13656
Abstract | PDF

Insects are one of the most significant causes of vector-transmitted human diseases and agricultural pests. In fact, dengue, yellow fever and malaria transmitted by mosquitoes are still among the most serious public health problems in many tropical and subtropical regions of the world, mostly in poor and in socially underdeveloped countries.