Biotechnology & Genetic Engineering Reviews

Published by Taylor & Francis
Print ISSN: 0264-8725
The use of plastic produced from non-renewable resources constitutes a major environmental problem of the modern society. Polylactide polymers (PLA) have recently gained enormous attention as one possible substitution of petroleum derived polymers. A prerequisite for high quality PLA production is the provision of optically pure lactic acid, which cannot be obtained by chemical synthesis in an economical way. Microbial fermentation is therefore the commercial option to obtain lactic acid as monomer for PLA production. However, one major economic hurdle for commercial lactic acid production as basis for PLA is the costly separation procedure, which is needed to recover and purify the product from the fermentation broth. Yeasts, such as Saccharomyces cerevisiae (bakers yeast) offer themselves as production organisms because they can tolerate low pH and grow on mineral media what eases the purification of the acid. However, naturally yeasts do not produce lactic acid. By metabolic engineering, ethanol was exchanged with lactic acid as end product of fermentation. A vast amount of effort has been invested into the development of yeasts for lactic acid production since the first paper on this topic by Dequin and Barre appeared 1994. Now yeasts are very close to industrial exploitation - here we summarize the developments in this field.
Polyprotein processing in FMDV. The sites of the primary cleavages and the virus proteins responsible are indicated by the curved arrows. The 18aa long 2A sequence is shown along with the conserved DxExNPGP sequence motif.  
Artificial reporter polyproteins (boxed areas) used to programme in vitro translation systems are shown together with translation profiles obtained from rabbit reticulocyte lysates (right). Analysis of the endogenous processing properties shows the polyprotein constructs without the 2A insertion produces a single uncleaved product whereas polyproteins containing 2A self-cleaves with high efficiency to produce three translational products.  
2A-like sequences in eukaryotic and prokaryotic genomes
Organization of the wild-type PVX; PVX.GFP-CP, PVX modified to express free GFP and PVX. GFP-2A-CP, PVX modified to express the GFP-2A-CP fusion protein. Boxes represent coding sequences. The predicted Mr values of the four viral proteins common to all constructs are indicated.  
Organization of the TCR-CD3 complex and 2A-linked TCR:CD3 constructs used to restore T-cell development and function in CD3-deficient mice (adapted from Call et al., 2002)
The 2A region of the foot-and-mouth disease virus (FMDV) encodes a short sequence that mediates self-processing by a novel translational effect. Translation elongation arrest leads to release of the nascent polypeptide and re-initiation at the next in-frame codon. In this way discrete translation products are derived from a single open reading frame. Active 2A-like sequences have been found in (many) other viruses and trypanosome non-LTR retrotransposons. Exponential growth of 2A technology within the last decade has lead to many biotechnological/biomedical applications including the generation of transgenic plants/animals and genetic manipulation of human embryonic stem cells (hESCs).
The acetone-butanol-ethanol (ABE) fermentation process, using Clostridium acetobutylicum or C. beijerinckii, epitomizes the problems of all fermentation processes vis-a-vis chemical synthesis. The purpose of this chapter is to review some recent developments in fermentation technology as applied to the ABE process. The aim of such technologies is to increase reactor productivity and/or remove product inhibition. In this way, the economics of the overall process can be improved
Gene conversion in recombination-active chicken Ig locus. V(D)J recombination occurs once in one of the two chicken Ig allele to generate the transcription-and recombination-active Ig allele. Using the upstream pseudo V segments (25 segments in the case of IgL) as templates, gene conversion, a type of homologous recombination, occurs unidirectionally and continuously at the recombination-active IgL and IgH V regions. Such gene conversion events create diversity of the IgL and IgH V regions in chicken Bcells. We discovered that TSA treatment of the DT40 cell line enhances the Ig gene conversion in vivo.
Higher histone acetylation at recombination-active V region. A , Schematic diagrams of recombination-active and -inactive Ig alleles. B , Chromatin immunoprecipitation assay using anti-acetylated histones reveals that the recombination-active V region exhibits higher histone H4 levels as compared to the recombination inactive Ig and ovalbumin alleles. C , quantification of results in B . The vertical axis represents the relative efficiency of chromatin immunoprecipitation (precipitated DNA vs. total input DNA) with anti acetylated histone H4. D , TSA dose dependency of histone acetylation. TSA induces histone acetylation of both Ig alleles, although transcription and gene conversion occurs only at the active allele possibly because it lacks a silencer for transcription that is located in the inactive allele (within a region shown in the thick line in A ). Error bars (s.d.) in C and D are based on two independent experiments. Modified from the previous report (Seo et al ., 2005). 
Treatment of DT40 with TSA enhances the Ig gene conversion. Prolonged TSA treatment causes autonomous diversification of both IgH and IgL V regions. The figure illustrates diversification of IgL V regions. Data are from Seo et al ., 2005. A , Time course experiments of gene conversion monitored by FACS. The DT40 CL18 clone has a frameshift mutation in the complementarity-determining region-1 (CDR1), therefore no surface IgM is produced. When some gene conversion events rescue this frameshift mutation, CL18 can produce surface IgM, which can be detected by FACS with fluorescein-conjugated anti-IgM antibodies. Note that most of the cells are converted to surface IgM positive in the presence of at least 1.25 ng/ml TSA, but little conversion occurs without TSA. If TSA treatment is terminated (1.25 ng/ml to 0 ng/ml), we detect a marked slowdown of the rate at which the population of surface IgM positive cells increases. This suggests that the action of TSA may be reversible. B , DNA sequence analysis of the diversified IgL V region. In the left panel, thick horizontal bars, open boxes, filled and open circles, and an open triangle represent DNA sequence alterations attributed as gene conversion, deletion, single mutation and deletion, and insertion events, respectively. The numbers above the bars represent the possible donor pseudo gene number. The right bar graph indicates the proportion of each sequence alteration with indication of numbers for each pattern. 
Schematic of the ADLib system. Once an ADLib has been prepared, screening steps require about one week, since the doubling time of DT40 is very short (7-8 days).
Since monoclonal antibodies (MAbs) were developed more than three decades ago, their importance has been increasing in various fields of biological research, clinical diagnosis and therapy. MAbs enable us to supply antibodies of uniform quality that can be produced without limit. In addition, each particular MAb recognizes a single epitope on the antigen of interest. Therefore, MAbs are suitable for medical purposes such as diagnostic reagents and antibody-based medicines, which require rigorous quality controls and a stable and sustainable supply. Currently, MAbs are actively utilized as antibody medicines against cancer and some chronic diseases (reviewed by Adams and Weiner, 2005). Antibody molecules have a long lifespan in the human body and they cause few side effects, since they originally function as a “natural molecule-targeted medicine” in the humoral immune systems of vertebrates evolved later than cyclostomes. One of the most successful antibody medicines is Herceptin (Trastuzumab), which targets the extracellular segment of the HER2/erbB2 receptor. Herceptin is used for the therapy of a certain type of metastatic breast cancer for patients whose tumors overexpress this receptor. It has been reported that the combination of Herceptin with chemotherapy greatly improves both survival and response rate of the patients. Owing to the success of these pioneer antibody medicines, many pharmaceutical companies are actively involved in the development of MAbs for antibody medicines. However, the current standard system of monoclonal antibody production presents several technical bottlenecks that remain unsolved. The monoclonal antibody system of Köhler and Milstein is based on the cell fusion of myeloma and B cells obtained from spleens of immunized mice (Köhler and Milstein, 1975). The procedure includes steps for immunization of animals, selection of hybridoma cells, multiple rounds of limited dilution, screening of positive clones producing appropriate MAbs, and clonal expansion for antibody preparation. Thus, the whole process of MAb preparation using this method is laborious and often very time-consuming (e.g., several months). In addition, the initial immunization process poses the fundamental problem of “immune tolerance” for external antigens. In the thymus, the adaptive immune system excludes lymphocytes that are involved in the immune response to autoantigens, as well as to evolutionally conserved proteins. Hence, raising antibodies against autoantigens and highly conserved antigens needs additional elaboration. Small molecules and saccharide chains are also known to be tough antigens; preparing antibodies that show high affinity and specificity against them is very challenging. To overcome such problems, and especially to bypass the immune tolerance bottleneck, an in vitro-based system for antibody or protein design was developed (reviewed in Rothe et al., 2006). In the middle of 1980, Smith’s group established a system called “phage display” (Griffiths and Duncan, 1998; Smith, 1985; Winter et al., 1994), with which one can screen polypeptides interacting with any antigen of interest from a library of bacteriophages displaying foreign proteins fused with the phage coat protein. Alternative in vitro systems are called polysome display, ribosome display, or in vitro virus system (Mattheakis, et al., 1994; Hanes and Plückthun., 1997; Gersuk et al., 1997; Nemoto et al., 1997; Hanes et al., 1998). For example, ribosome display and in vitro virus systems are based on the formation of stable antibody-ribosome-mRNA complexes in which the antibody protein is directly linked to its encoding DNA sequence. Binz and coworkers have reported another in vitro based system for high-affinity binder selection using ankyrin repeat protein libraries (Binz et al., 2004). Although these in vitro-based methods have made great progress, they still require additional preparation time after screening in order to produce full antibodies by the use of recombinant DNA techniques. Here we describe an ex vivo MAb design system using a chicken B-cell derived DT40 cell line which undergoes an enhanced genetic rearrangement at the immunoglobulin (Ig) locus. The system is based on our previous findings on the regulatory role of histone acetylation in yeast homologous recombination during meiosis. In the next section, we briefly describe the background of this system.
Meal replacements and viscous soluble fibre represent safe and sustainable aids for weight loss. Our purpose was to determine if PGX® meal replacements and PGX(®) fibre complex in combination with a calorie-restricted diet would aid in weight loss in a clinical setting. Fifty-two overweight and obese participants (49 women, 3 men; average age 47.1 years) with a mean body mass index (BMI) of 33.8 ± 6.4 kg/m(2) consumed 57 g of proprietary PGX® meal replacement product at breakfast and another 57 g at lunch for 12 weeks. In addition to the meal replacements, they were also asked to consume 5 g/day of PGX® fibre in the form of granules, powder or capsules together with 250 mlwater. A registered dietician recommended low-fat, low-glycaemic-index foods for snacks and the dinner menus such that each volunteer was consuming a total of 1200 kcal/day. All participants (n = 52) lost a significant amount of weight from baseline (-4.69 ± 3.73 kg), which was further reflected in the reductions in their waist (-7.11 ± 6.35 cm) and hip circumference (-5.59 ± 3.58 cm) over the 12-week study (p < 0.0001). BMI scores (n = 51) were reduced by 1.6 ± 1.4 kg/m(2). The use of PGX® meal replacements and PGX(®) fibre along with a controlled dietary caloric intake is of benefit for short-term weight loss.
The use of site-directed mutagenesis (Smith, 1985) to replace amino acids at any chosen position in a protein, coupled with advances in analytical procedures, has greatly advanced our understanding of biological structure-function relationships in recent years. The only limitation of conventional site-directed mutagenesis is that substitutions are restricted to the 20 naturally occurring amino acids. However, the discovery of a 21st amino acid, selenocysteine, and the development of novel in vitro translation techniques have demonstrated that considerably more site-specific replacements are possible during protein engineering. These techniques have already found a wide range of applications and have shown that the translational machinery is able to accommodate an enormously divergent range of aminoacylated tRNAs. Although these techniques are mainly restricted to in vitro systems, recent progress in our understanding of aminoacyl-tRNA synthetase-catalyzed tRNA charging suggests that it may ultimately be possible to extend this technique to growing cells.
Overview of major insertions and deletions in family 19 chitinases. 
Overview of major insertions and deletions in family 19 chitinases. 
Structures of ChiA and ChiB from S. marcescens. The figure shows the structure of ChiB (upper panel, cyan, PDB: le6n) and ChiA (lower panel, magenta, PDB: lehn) in complex with (G1cNAc)5 and (G1cNAc)8, respectively (orange). In ChiB, the chitin-binding domain (to the right) extends the substrate binding cleft in the reducing end direction, whereas in ChiA the chitin-binding domain (to the left) extends towards the non-reducing end. The substrate-binding groove and the surfaces of the chitin binding domains are lined with aromatic side chains (yellow). Note that the catalytic domains have approximately similar orientations in both pictures; the substrates bind to subsites-2 to +3 in ChiB and subsites-6 to +2 in ChiA. The pictures were made with PYMOL (DeLano 2004). The structures are described in Papanikolau et al., 2001 (ChiA) and Van Aalten et al., 2001 (ChiB). 
Location of active site residues in equivalent positions in the conserved structural cores of enzymes in the lysozyme superfamily. GH19, 23, 24 and 46 enzymes act by an inverting mechanism, whereas GH22 acts by a retaining mechanism. This table is adapted from (Lacombe-Harvey et al. 
Enzymatic conversions of chitin and its soluble, partially deacetylated derivative chitosan are of great interest. Firstly, chitin metabolism is an important process in fungi, insects and crustaceans. Secondly, such enzymatic conversions may be used to transform an abundant biomass to useful products such as bioactive chito-oligosaccharides. Enzymes acting on chitin and chitosan are abundant in nature. Here we review current knowledge on the structure and function of enzymes involved in the conversion of these polymeric substrates: chitinases (glycoside hydrolase families 18 & 19), chitosanases (glycoside hydrolase families 8, 46, 75 & 80) and chitin deacetylases (carbohydrate esterase family 4).
Metagenomics is a relatively new but fast growing field within environmental biology directed at obtaining knowledge on genomes of environmental microbes as well as of entire microbial communities. With the sequencing technologies improving steadily, generating large amounts of sequence is becoming routine. However, it remains difficult to connect specific microbial phyla to specific functions in the environment. A number of 'functional metagenomics' approaches have been implemented in the recent years that allow high-resolution genomic analysis of uncultivated microbes, connecting them to specific functions in the environment. These include analysis of niche-specialized low complexity communities, reactor enrichments, and the use labeling technologies. Metatranscriptomics and metaproteomics are the newest sub-disciplines within the metagenomics field that provide further levels of resolution for functional analysis of uncultivated microbes and communities. The recent emergence of new (next generation) sequencing technologies, resulting in higher sequence output and dramatic drop in the price of sequencing, will be defining a new era in metagenomics. At this time the sequencing effort will be taken to a new level to allow addressing new, previously unattainable biological questions as well as accelerating genome-based discovery for medical and biotechnological applications.
Lipid membranes are versatile and convenient alternatives to study the properties of natural cell membranes. Self-assembled, artificial, substrate-supported lipid membranes have taken a central role in membrane research due to a combination of factors such as ease of creation, control over complexity, stability and the applicability of a large range of different analytical techniques. While supported lipid bilayers have been investigated for several decades, recent advances in the understanding of the assembly of such membranes from liposomes have spawned a renaissance in the field. Supported lipid bilayers are a highly promising tool to study transmembrane proteins in their native state, an application that could have tremendous impact on, e.g. drug discovery, development of biointerfaces and as platforms for glycomics and probing of multivalent binding which requires ligand mobility. Parallel advances in microfluidics, biosensor design, micro- and nanofabrication have converged to bring self-assembled supported lipid bilayers closer to a versatile and easy to use research tool as well as closer to industrial applications. The field of supported lipid bilayer research and application is thus rapidly expanding and diversifying with new platforms continuously being proposed and developed. In order to use supported lipid bilayers for such applications several advances have to be made: decoupling of the membrane from the support while maintaining it close to the surface, making use of biologically relevant lipid compositions, patterning of lipid membranes into arrays, and application to nanostructured substrates and sensors. This review summarizes recent advances in the field which addresses these challenges.
The completion of the WHO Schistosoma Genome Project in 2008, although not fully annotated, provides a golden opportunity to actively pursue fundamental research on the parasites genome. This analysis will aid identification of targets for drugs, vaccines and markers for diagnostic tools as well as for studying the biological basis of drug resistance, infectivity and pathology. For the validation of drug and vaccine targets, the genomic sequence data is only of use if functional analyses can be conducted (in the parasite itself). Until recently, gene manipulation approaches had not been seriously addressed. This situation is now changing and rapid advances have been made in gene silencing and transgenesis of schistosomes.
Environmental stresses - especially drought and salinity - and iron limitation are the primary causes of crop yield losses. Therefore, improvement of plant stress tolerance has paramount relevance for agriculture, and vigorous efforts are underway to design stress-tolerant crops. Three aspects of this ongoing research are reviewed here. First, attempts have been made to strengthen endogenous plant defences, which are characterised by intertwined, hierarchical gene networks involved in stress perception, signalling, regulation and expression of effector proteins, enzymes and metabolites. The multigenic nature of this response requires detailed knowledge of the many actors and interactions involved in order to identify proper intervention points, followed by significant engineering of the prospective genes to prevent undesired side-effects. A second important aspect refers to the effect of concurrent stresses as plants normally meet in the field (e.g., heat and drought). Recent findings indicate that plant responses to combined environmental hardships are somehow unique and cannot be predicted from the addition of the individual stresses, underscoring the importance of programming research within this conceptual framework. Finally, the photosynthetic microorganisms from which plants evolved (i.e., algae and cyanobacteria) deploy a totally different strategy to acquire stress tolerance, based on the substitution of stress-vulnerable targets by resistant isofunctional proteins that could take over the lost functions under adverse conditions. Reintroduction of these ancient traits in model and crop plants has resulted in increased tolerance to environmental hardships and iron starvation, opening a new field of opportunities to increase the endurance of crops growing under suboptimal conditions.
Heterodimeric cytokines belonging to IL-12 family: structural and functional characteristics. Naturally occurring IL-12, IL-23, IL-27, and IL-35 are heterodimeric cytokines with a high sharing rate. The p40 β-chain is shared by IL-12 and IL-23, EBI3 β-chain is shared by IL-27 and IL-35, p35 α-chain is shared by IL-12 and IL-35, whereas IL-23 and IL-27 have unique α-chains, p19 and p28, respectively. Only IL-12 and IL-23 are disulfide-linked heterodimers. The receptor complexes of heterodimeric cytokines also include shared subunits; IL-12Rβ1 is present in receptors for IL-12 and IL-23, IL-12Rβ2 in those for IL-12 and IL35, and gp130 is common to IL-27 and IL35 receptor complexes. Jak-STAT signaling partners of these receptors are indicated, as well as the functional effects of related cytokines. 
General architecture of constructs encoding for engineered single-chain cytokines of the IL-12 family. Schematic diagrams of the DNA constructs for single-chain IL-12, IL-23, IL-27, and IL-35 fusion proteins are reported, showing that the β chain (p40 and EBI3 subunits) and the α chain (p35, p19 and p28 subunits) can be sequentially and genetically fused with a DNA linker (L). Resulting constructs can be further fused with an appropriate tag sequence (mainly consisting of Fc of IgG and 3ÂFLAG). 
The interleukin 12 (IL-12) family comprises a group of heterodimeric cytokines that can cope with a great variety of immune conditions as the microenvironment demands. By sharing cytokine and receptor subunits, IL-12 (comprised of p40/p35 subunits), IL-23 (p40/p19), IL-27 (p28/EBI3), and IL-35 (p35/EBI3) represent, as a whole, a highly versatile system participating in controlling the continuum from inflammation to tolerance. Promiscuity, a peculiar feature of those cytokines, is a powerful and economic means of producing individual factors with distinct activities via different combinations of a single set of subunits. Whereas IL-12 and IL-23 have a clearly dominant immunostimulatory functional profile and IL-35 is a potent immunosuppressive agent, IL-27 can exert both adjuvant and regulatory effects, depending on the cytokine milieu. Promiscuity itself, however, may significantly hamper the therapeutic use of heterodimeric cytokines. The subunits of a recombinant cytokine, when administered in its native form, will rapidly dissociate in vivo and reassociate with alternative partners, thus generating different heterodimeric or even homodimeric molecules (i.e., p40/p40) with unwanted effects. As in other areas, bioengineering has provided a formidable tool to overcome the constraints associated with the potential use of IL-12 family cytokines. The generation of several gene constructs expressing IL-12, IL-23, IL-27, IL-35, or even the homodimer p40/p40, in their monomerized, single-chain form has allowed us to unveil the efficacy of those molecules in several experimental settings, including neoplasia, viral infection, chronic inflammation, allergy and autoimmunity. Although work is still needed to obtain an overall picture of therapeutic vs. adverse effects of individual molecules before any use in humans, the new frontiers of bioengineering are now driving the production of completely new combinations of cytokine subunits that may further extend the potential clinical use of such eclectic proteins.
The ability to genetically modify pigs has enabled scientists to create pigs that are beneficial to humans in ways that were previously unimaginable. Improvements in the methods to make genetic modifications have opened up the possibilities of introducing transgenes, knock-outs and knock-ins with precision. The benefits to medicine include the production of pharmaceuticals, the provision of organs for xenotransplantation into humans, and the development of models of human diseases. The benefits to agriculture include resistance to disease, altering the carcass composition such that it is healthier to consume, improving the pig's resistance to heat stress, and protecting the environment. Additional types of genetic modifications will likely provide animals with characteristics that will benefit humans in currently unimagined ways.
Genetic instability is very common in Streptomyces species, but only affects specific genes in any one strain. It sometimes occurs at high frequency spontaneously, but may be stimulated by treatments such as UV irradiation or intercalating agents. Deletion of genes occurs and may be accompanied by DNA amplifications. It is unlikely that there is plasmid involvement in most cases. Little is yet known about the molecular mechanisms of deletion and DNA amplification. Genetic instability can be a problem during commercial antibiotic production. DNA amplification of cloned genes is potentially useful for achieving both stability and high gene dosage.
Sulfate-reducing bacteria (SRB) are an environmentallysignificant group belonging to the anaerobic delta-Proteobacteria thatrespire sulfate for growth. From an industrial stand point, SRB pose athreat through corrosion of ferrous metals and production of toxicsulfides. The more positive aspects of the metabolism of the SRB includea robust but poorly understood hydrogen metabolism that is of interest toalternative energy studies. SRB also immobilize a number of heavy metalsthrough sulfide precipitation or through changing the redox state of themetal and thus its solubility. When metals are made less soluble, as isthe case with chromium (Cr(VI) to Cr(III)) or uranium (U(VI) to U(IV)),toxicity is reduced by limiting biological availability. Despite theeconomic and environmental impacts associated with SRB activities, ourcurrent knowledge of their metabolism is inadequate. Among the SRB,members of the Desulfovibrio genus have received most attention becausethese strains are most readily grown in pure culture. Therefore,Desulfovibrio strains have been the focus of biochemical and biophysicalanalyses, however, genetic studies have been more difficult. Over thelast 15 years, progress has been made in developing techniques for DNAtransformation, gene mutagenesis and over expression, and proteintagging. Since the last genetics of SRB review by van Dongen, 10 yearshave passed (van Dongen, 1995) and the complete genome sequences of a fewstrains are now available (Heidelberg, et al., 2004). This reviewhighlights the current advances in the genetic manipulation ofDesulfovibrio species and the potential use of these tools inunderstanding the metabolism of sulfate reducers for biotechnologicalpurposes.
This invited review considers the contribution sedimentation velocity, sedimentation equilibrium and density gradient analytical ultracentrifugation have made to our understanding of not only the genetically engineered molecules themselves, but also those macromolecules associated with the engineering process: nucleic acids and nucleic acid binding proteins
Top-cited authors
Keith L. Williams
  • BKG Group Sydney Australia
Jean-Charles Sanchez
  • University of Geneva
Ron D Appel
  • Swiss Institute of Bioinformatics
Andrew Arthur Gooley
  • Macquarie University
Ian Humphery-Smith