Field Of Metabolic Engineering Research Articles

Expanding the chemical palate of cells by combining systems biology and metabolic engineering

The field of Metabolic Engineering has recently undergone a transformation that has led to a rapid expansion of the chemical palate of cells. Now, it is conceivable to produce nearly any organic molecule of interest using a cellular host. Significant advances have been made in the production of biofuels, biopolymers and precursors, pharmaceuticals and nutraceuticals, and commodity and specialty chemicals. Much of this rapid expansion in the field has been, in part, due to synergies and advances in the area of systems biology. Specifically, the availability of functional genomics, metabolomics and transcriptomics data has resulted in the potential to produce a wealth of new products, both natural and non-natural, in cellular factories. The sheer amount and diversity of this data however, means that uncovering and unlocking novel chemistries and insights is a non-obvious exercise. To address this issue, a number of computational tools and experimental approaches have been developed to help expedite the design process to create new cellular factories. This review will highlight many of the systems biology enabling technologies that have reduced the design cycle for engineered hosts, highlight major advances in the expanded diversity of products that can be synthesized, and conclude with future prospects in the field of metabolic engineering.

Computational tools for metabolic engineering

A great variety of software applications are now employed in the metabolic engineering field. These applications have been created to support a wide range of experimental and analysis techniques. Computational tools are utilized throughout the metabolic engineering workflow to extract and interpret relevant information from large data sets, to present complex models in a more manageable form, and to propose efficient network design strategies. In this review, we present a number of tools that can assist in modifying and understanding cellular metabolic networks. The review covers seven areas of relevance to metabolic engineers. These include metabolic reconstruction efforts, network visualization, nucleic acid and protein engineering, metabolic flux analysis, pathway prospecting, post-structural network analysis and culture optimization. The list of available tools is extensive and we can only highlight a small, representative portion of the tools from each area.

Synthetic regulatory tools for microbial engineering

Microbial engineering requires accurate information about cellular metabolic networks and a set of molecular tools that can be predictably applied to the efficient redesign of such networks. Recent advances in the field of metabolic engineering and synthetic biology, particularly the development of molecular tools for synthetic regulation in the static and dynamic control of gene expression, have increased our ability to efficiently balance the expression of genes in various biological systems. It would accelerate the creation of synthetic pathways and genetic programs capable of adapting to environmental changes in real time to perform the programmed cellular behavior. In this paper, we review current developments in the field of synthetic regulatory tools for static and dynamic control of microbial gene expression.

Genome-wide functional annotation and structural verification of metabolic ORFeome of Chlamydomonas reinhardtii

BackgroundRecent advances in the field of metabolic engineering have been expedited by the availability of genome sequences and metabolic modelling approaches. The complete sequencing of the C. reinhardtii genome has made this unicellular alga a good candidate for metabolic engineering studies; however, the annotation of the relevant genes has not been validated and the much-needed metabolic ORFeome is currently unavailable. We describe our efforts on the functional annotation of the ORF models released by the Joint Genome Institute (JGI), prediction of their subcellular localizations, and experimental verification of their structural annotation at the genome scale.ResultsWe assigned enzymatic functions to the translated JGI ORF models of C. reinhardtii by reciprocal BLAST searches of the putative proteome against the UniProt and AraCyc enzyme databases. The best match for each translated ORF was identified and the EC numbers were transferred onto the ORF models. Enzymatic functional assignment was extended to the paralogs of the ORFs by clustering ORFs using BLASTCLUST.In total, we assigned 911 enzymatic functions, including 886 EC numbers, to 1,427 transcripts. We further annotated the enzymatic ORFs by prediction of their subcellular localization. The majority of the ORFs are predicted to be compartmentalized in the cytosol and chloroplast. We verified the structure of the metabolism-related ORF models by reverse transcription-PCR of the functionally annotated ORFs. Following amplification and cloning, we carried out 454FLX and Sanger sequencing of the ORFs. Based on alignment of the 454FLX reads to the ORF predicted sequences, we obtained more than 90% coverage for more than 80% of the ORFs. In total, 1,087 ORF models were verified by 454 and Sanger sequencing methods. We obtained expression evidence for 98% of the metabolic ORFs in the algal cells grown under constant light in the presence of acetate.ConclusionsWe functionally annotated approximately 1,400 JGI predicted metabolic ORFs that can facilitate the reconstruction and refinement of a genome-scale metabolic network. The unveiling of the metabolic potential of this organism, along with structural verification of the relevant ORFs, facilitates the selection of metabolic engineering targets with applications in bioenergy and biopharmaceuticals. The ORF clones are a resource for downstream studies.

Opportunities for yeast metabolic engineering: Lessons from synthetic biology

Constant progress in genetic engineering has given rise to a number of promising areas of research that facilitated the expansion of industrial biotechnology. The field of metabolic engineering, which utilizes genetic tools to manipulate microbial metabolism to enhance the production of compounds of interest, has had a particularly strong impact by providing new platforms for chemical production. Recent developments in synthetic biology promise to expand the metabolic engineering toolbox further by creating novel biological components for pathway design. The present review addresses some of the recent advances in synthetic biology and how these have the potential to affect metabolic engineering in the yeast Saccharomyces cerevisiae. While S. cerevisiae for years has been a robust industrial organism and the target of multiple metabolic engineering trials, its potential for synthetic biology has remained relatively unexplored and further research in this field could strongly contribute to industrial biotechnology. This review also addresses are general considerations for pathway design, ranging from individual components to regulatory systems, overall pathway considerations and whole-organism engineering, with an emphasis on potential contributions of synthetic biology to these areas. Some examples of applications for yeast synthetic biology and metabolic engineering are also discussed.

A computational tool for the simulation and optimization of microbial strains accounting integrated metabolic/regulatory information

Background and scopeRecently, a number of methods and tools have been proposed to allow the use of genome-scale metabolic models for the phenotype simulation and optimization of microbial strains, within the field of Metabolic Engineering (ME). One of the limitations of most of these algorithms and tools is the fact that only metabolic information is taken into account, disregarding knowledge on regulatory events. Implementation and performancesThis work proposes a novel software tool that implements methods for the phenotype simulation and optimization of microbial strains using integrated models, encompassing both metabolic and regulatory information. This tool is developed as a plug-in that runs over OptFlux, a computational platform that aims to be a reference tool for the ME community. AvailabilityThe plug-in is made available in the OptFlux web site (www.optflux.org) together with examples and documentation.

Genetic and metabolic engineering of isoflavonoid biosynthesis

Isoflavonoids are a diverse group of secondary metabolites derived from the phenylpropanoid pathway. These compounds are distributed predominantly in leguminous plants and play important roles in plant-environment interactions and human health. Consequently, the biosynthetic pathway of isoflavonoid compounds has been widely elucidated in the past decades. Up to now, most of the structural genes and some of the regulatory genes involved in this pathway have been isolated and well characterized. Nowadays, the protective effects of the legume isoflavonoids against hormone dependent cancers, cardiovascular disease, osteoporosis, and menopausal symptoms have generated considerable interest within the genetic and metabolic engineering fields to enhance the dietary intake of these compounds for disease prevention. Subsequently, there are some great progresses in genetic and metabolic engineering to improve their yields in leguminous and non-leguminous plants and/or microorganisms. Because of the field of flavonoid biosynthesis has been reviewed fairly extensively in the past, this review concentrates on the more recent development in the isoflavonoid branch of phenylpropanoid pathway, including gene isolation and characterization. In addition, we describe the state-of-the-art research with respect to genetic and metabolic engineering of isoflavonoid biosynthesis.

Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli

The field of metabolic engineering has the potential to produce a wide variety of chemicals in both an inexpensive and ecologically-friendly manner. Heterologous expression of novel combinations of enzymes promises to provide new or improved synthetic routes towards a substantially increased diversity of small molecules. Recently, we constructed a synthetic pathway to produce d-glucaric acid, a molecule that has been deemed a “top-value added chemical” from biomass, starting from glucose. Limiting flux through the pathway is the second recombinant step, catalyzed by myo-inositol oxygenase (MIOX), whose activity is strongly influenced by the concentration of the myo-inositol substrate. To synthetically increase the effective concentration of myo-inositol, polypeptide scaffolds were built from protein–protein interaction domains to co-localize all three pathway enzymes in a designable complex as previously described ( Dueber et al., 2009). Glucaric acid titer was found to be strongly affected by the number of scaffold interaction domains targeting upstream Ino1 enzymes, whereas the effect of increased numbers of MIOX-targeted domains was much less significant. We determined that the scaffolds directly increased the specific MIOX activity and that glucaric acid titers were strongly correlated with MIOX activity. Overall, we observed an approximately 5-fold improvement in product titers over the non-scaffolded control, and a 50% improvement over the previously reported highest titers. These results further validate the utility of these synthetic scaffolds as a tool for metabolic engineering.

Applications of Synthetic Biology in Microbial Biotechnology

Applications of Synthetic Biology in Microbial Biotechnology

Metabolic flux analysis and pharmaceutical production

Rational engineering of biological systems is an inherently complex process due to their evolved nature. Metabolic engineering emerged and developed over the past 20 years as a field in which methodologies for the rational engineering of biological systems is now being applied to specific industrial, medical, or scientific problems. Of considerable interest is the determination of metabolic fluxes within the cell itself, called metabolic flux analysis. This special issue and this review have a particular interest in the application of metabolic flux analysis for improving the pharmaceutical production process (for both small and large molecules). Though metabolic flux analysis has been somewhat limited in application towards pharmaceutical production, the overall goal is to: (1) have a better understanding of the organism and/or process in question, and (2) provide a rational basis to further engineer (on both metabolic and process scales) improved pharmaceutical production in these organisms. The focus of this review article is to present how experimental and computational methods of metabolic flux analysis have matured, mirroring the maturation of the metabolic engineering field itself, while highlighting some of the successful applications towards both small- and large-molecule pharmaceuticals.

Model-driven evaluation of the production potential for growth-coupled products of Escherichia coli

Integrated approaches utilizing in silico analyses will be necessary to successfully advance the field of metabolic engineering. Here, we present an integrated approach through a systematic model-driven evaluation of the production potential for the bacterial production organism Escherichia coli to produce multiple native products from different representative feedstocks through coupling metabolite production to growth rate. Designs were examined for 11 unique central metabolism and amino acid targets from three different substrates under aerobic and anaerobic conditions. Optimal strain designs were reported for designs which possess maximum yield, substrate-specific productivity, and strength of growth-coupling for up to 10 reaction eliminations (knockouts). In total, growth-coupled designs could be identified for 36 out of the total 54 conditions tested, corresponding to eight out of the 11 targets. There were 17 different substrate/target pairs for which over 80% of the theoretical maximum potential could be achieved. The developed method introduces a new concept of objective function tilting for strain design. This study provides specific metabolic interventions (strain designs) for production strains that can be experimentally implemented, characterizes the potential for E. coli to produce native compounds, and outlines a strain design pipeline that can be utilized to design production strains for additional organisms.

Impact of systems biology on metabolic engineering ofSaccharomyces cerevisiae

Industrial biotechnology is a rapidly growing field. With the increasing shift towards a bio-based economy, there is rising demand for developing efficient cell factories that can produce fuels, chemicals, pharmaceuticals, materials, nutraceuticals, and even food ingredients. The yeast Saccharomyces cerevisiae is extremely well suited for this objective. As one of the most intensely studied eukaryotic model organisms, a rich density of knowledge detailing its genetics, biochemistry, physiology, and large-scale fermentation performance can be capitalized upon to enable a substantial increase in the industrial application of this yeast. Developments in genomics and high-throughput systems biology tools are enhancing one's ability to rapidly characterize cellular behaviour, which is valuable in the field of metabolic engineering where strain characterization is often the bottleneck in strain development programmes. Here, the impact of systems biology on metabolic engineering is reviewed and perspectives on the role of systems biology in the design of cell factories are given.

Global physiological understanding and metabolic engineering of microorganisms based on omics studies

Through metabolic engineering, scientists seek to modify the metabolic pathways of living organisms to facilitate optimized, efficient production of target biomolecules. During the past decade, we have seen notable improvements in biotechnology, many of which have been based on metabolically engineered microorganisms. Recent developments in the fields of functional genomics, transcriptomics, proteomics, and metabolomics have changed metabolic engineering strategies from the local pathway level to the whole system level. This article focuses on recent advances in the field of metabolic engineering, which have been powered by the combined approaches of the various "omics" that allow us to understand the microbial metabolism at a global scale and to develop more effectively redesigned metabolic pathways for the enhanced production of target bioproducts.

Phenotypic characterization of glucose repression mutants of Saccharomyces cerevisiae using experiments with 13C-labelled glucose.

In the field of metabolic engineering and functional genomics, methods for analysis of metabolic fluxes in the cell are attractive as they give an overview of the phenotypic response of the cells at the level of the active metabolic network. This is unlike several other high-throughput experimental techniques, which do not provide information about the integrated response a specific genetic modification has on the cellular function. In this study we have performed phenotypic characterization of several mutants of the yeast Saccharomyces cerevisiae through the use of experiments with (13)C-labelled glucose. Through GC-MS analysis of the (13)C incorporated into the amino acids of cellular proteins, it was possible to obtain quantitative information on the function of the central carbon metabolism in the different mutants. Traditionally, such labelling data have been used to quantify metabolic fluxes through the use of a suitable mathematical model, but here we show that the raw labelling data may also be used directly for phenotypic characterization of different mutant strains. Different glucose derepressed strains investigated employed are the disruption mutants reg1, hxk2, grr1, mig1 and mig1mig2 and the reference strain CEN.PK113-7D. Principal components analysis of the summed fractional labelling data show that deleting the genes HXK2 and GRR1 results in similar phenotype at the fluxome level, with a partial alleviation of glucose repression on the respiratory metabolism. Furthermore, deletion of the genes MIG1, MIG1/MIG2 and REG1 did not result in a significant change in the phenotype at the fluxome level.

Metabolic engineering: advances in modeling and intervention in health and disease.

The field of metabolic engineering encompasses a powerful set of tools that can be divided into (a) methods to model complex metabolic pathways and (b) techniques to manipulate these pathways for a desired metabolic outcome. These tools have recently seen increased utility in the medical arena, and this paper aims to review significant accomplishments made using these approaches. The modeling of metabolic pathways has been applied to better understand disease-state physiology in a variety of cellar, subcellular, and organ systems, including the liver, heart, mitochondria, and cancerous cells. Metabolic pathway engineering has been used to generate cells with novel biochemical functions for therapeutic use, and specific examples are provided in the areas of glycosylation engineering and dopamine-replacement therapy. In order to document the potential of applying both metabolic modeling and pathway manipulation, we describe pertinent advances in the field of diabetes research. Undoubtedly, as the field of metabolic engineering matures and is applied to a wider array of problems, new advances and therapeutic strategies will follow.

Metabolic engineering--a genetic toolbox for small molecule organic synthesis.

Metabolic engineering is an emerging field that exploits biosynthetic machinery as a means to genetically design small molecule production within heterologous host organisms. Molecular design and synthesis with biological tools has lagged behind total synthesis technology for about seventy-five years and owes its existence to relatively new molecular biology techniques. Here the field of metabolic engineering is explained as a comparison to total organic synthesis, including the sequence of scientific events leading up to successful implementation and future goals in the field. It is expected that metabolic engineering will take a place alongside traditional organic synthesis as a powerful means to design and create small organic molecules.

Engineering of coordinated up- and down-regulation of two glycosyltransferases of the O-glycosylation pathway in Chinese hamster ovary (CHO) cells.

Production of O-linked oligosaccharides that interact with selectins to mediate cell-cell adhesion occurs in one segment of a branched glycan biosynthesis network. Prior efforts to direct the branched pathway towards selectin-binding oligosaccharides by amplifying enzymes in this branch of the network have had limited success, suggesting that metabolic engineering to simultaneously inhibit the competing pathway may also be required. We report here the partial cloning of the CMP-sialic, acid:Galbeta1,3GalNAcalpha2,3-sialyltransferase (ST3Gal I) gene from Chinese hamster ovary (CHO) cells and the simultaneous inhibition of expression of CHO cell ST3Gal I gene and overexpression of the human UDP-GlcNAc:Galbeta1,3GalNAc-R beta1,6-N-acetylglucosaminyltransferase (C2GnT) gene. A tetracycline-regulated system adjoined to tricistronic expression technology allowed "one-step" transient manipulation of multiple enzyme activities in the O-glycosylation pathway of a previously established CHO cell line already engineered to express alpha1,3-fucosyltransferase VI (alpha1,3-Fuc-TVI). Tetracycline-regulated co-expression of a ST3Gal I fragment, cloned in the antisense orientation, and of C2GnT cDNA resulted in inhibition of the ST3Gal I enzymatic activity and increase in C2GnT activity which varied depending on the extent of tetracycline reduction in the cell culture medium. This simultaneous regulated inhibition and activation of the two key enzyme activities in the O-glycosylation pathway of mammalian cells is an important addition to the metabolic engineering field.

A priori analysis of metabolic flux identifiability from (13)C-labeling data.

The (13)C-labeling technique was introduced in the field of metabolic engineering as a tool for determining fluxes that could not be found using the 'classical' method of flux balancing. An a priori flux identifiability analysis is required in order to determine whether a (13)C-labeling experiment allows the identification of all the fluxes. In this article, we propose a method for identifiability analysis that is based on the recently introduced 'cumomer' concept. The method improves upon previous identifiability methods in that it provides a way of systematically reducing the metabolic network on the basis of structural elements that constitute a network and to use the implicit function theorem to analytically determine whether the fluxes in the reduced network are theoretically identifiable for various types of real measurement data. Application of the method to a realistic flux identification problem shows both the potential of the method in yielding new, interesting conclusions regarding the identifiability and its practical limitations that are caused by the fact that symbolic calculations grow fast with the dimension of the studied system.

Chromosomal editing in Escherichia coli. Vectors for DNA integration and excision.

Chromosomal editing constitutes the direct and specific modification of the genetic information present in the chromosome. In the bacterium Escherichia coli, strategies were originally developed for the production of specific proteins, the genotypic improvement of strains, and the analysis of regulation of gene expression. However, with the emerging field of metabolic engineering and genomics, efficient means of targeting specific genetic mutations into the chromosome are most useful. In this review, a summary of the systems currently available to generate insertions and deletions in the chromosome of E. coli are presented, as well as the current knowledge about the genetic mechanisms responsible for these processes.

Engineering of coordinated up- and down-regulation of two glycosyltransferases of the o-glycosylation pathway in Chinese hamster ovary (CHO) cells

Production of O-linked oligosaccharides that interact with selectins to mediate cell-cell adhesion occurs in one segment of a branched glycan biosynthesis network. Prior efforts to direct the branched pathway towards selectin-binding oligosaccharides by amplifying enzymes in this branch of the network have had limited success, suggesting that metabolic engineering to simultaneously inhibit the competing pathway may also be required. We report here the partial cloning of the CMP-sialic acid:Galbeta1,3GalNAcalpha2, 3-sialyltransferase (ST3Gal I) gene from Chinese hamster ovary (CHO) cells and the simultaneous inhibition of expression of CHO cell ST3Gal I gene and overexpression of the human UDP-GlcNAc:Galbeta1, 3GalNAc-R beta1,6-N-acetylglucosaminyltransferase (C2GnT) gene. A tetracycline-regulated system adjoined to tricistronic expression technology allowed "one-step" transient manipulation of multiple enzyme activities in the O-glycosylation pathway of a previously established CHO cell line already engineered to express alpha1, 3-fucosyltransferase VI (alpha1,3-Fuc-TVI). Tetracycline-regulated co-expression of a ST3Gal I fragment, cloned in the antisense orientation, and of C2GnT cDNA resulted in inhibition of the ST3Gal I enzymatic activity and increase in C2GnT activity which varied depending on the extent of tetracycline reduction in the cell culture medium. This simultaneous regulated inhibition and activation of the two key enzyme activities in the O-glycosylation pathway of mammalian cells is an important addition to the metabolic engineering field.