Advances In Metabolic Engineering Research Articles

Multi‐scale modeling for sustainable chemical production

With recent advances in metabolic engineering, it is now technically possible to produce a wide portfolio of existing petrochemical products from biomass feedstock. In recent years, a number of modeling approaches have been developed to support the engineering and decision-making processes associated with the development and implementation of a sustainable biochemical industry. The temporal and spatial scales of modeling approaches for sustainable chemical production vary greatly, ranging from metabolic models that aid the design of fermentative microbial strains to material and monetary flow models that explore the ecological impacts of all economic activities. Research efforts that attempt to connect the models at different scales have been limited. Here, we review a number of existing modeling approaches and their applications at the scales of metabolism, bioreactor, overall process, chemical industry, economy, and ecosystem. In addition, we propose a multi-scale approach for integrating the existing models into a cohesive framework. The major benefit of this proposed framework is that the design and decision-making at each scale can be informed, guided, and constrained by simulations and predictions at every other scale. In addition, the development of this multi-scale framework would promote cohesive collaborations across multiple traditionally disconnected modeling disciplines to achieve sustainable chemical production.

Transcription factor-based screens and synthetic selections for microbial small-molecule biosynthesis.

Continued advances in metabolic engineering are increasing the number of small molecules being targeted for microbial production. Pathway yields and productivities, however, are often suboptimal, and strain improvement remains a persistent challenge given that the majority of small molecules are difficult to screen for and their biosynthesis does not improve host fitness. In this work, we have developed a generalized approach to screen or select for improved small-molecule biosynthesis using transcription factor-based biosensors. Using a tetracycline resistance gene 3' of a small-molecule inducible promoter, host antibiotic resistance, and hence growth rate, was coupled to either small-molecule concentration in the growth medium or a small-molecule production phenotype. Biosensors were constructed for two important chemical classes, dicarboxylic acids and alcohols, using transcription factor-promoter pairs derived from Pseudomonas putida, Thauera butanivorans, or E. coli. Transcription factors were selected for specific activation by either succinate, adipate, or 1-butanol, and we demonstrate product-dependent growth in E. coli using all three compounds. The 1-butanol biosensor was applied in a proof-of-principle liquid culture screen to optimize 1-butanol biosynthesis in engineered E. coli, identifying a pathway variant yielding a 35% increase in 1-butanol specific productivity through optimization of enzyme expression levels. Lastly, to demonstrate the capacity to select for enzymatic activity, the 1-butanol biosensor was applied as synthetic selection, coupling in vivo 1-butanol biosynthesis to E. coli fitness, and an 120-fold enrichment for a 1-butanol production phenotype was observed following a single round of positive selection.

Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases

Recent advances in metabolic engineering have demonstrated that microbial biosynthesis can provide a viable alternative to chemical synthesis for the production of bulk and fine chemicals. Introduction of a new biosynthetic pathway typically requires the expression of multiple heterologous enzymes in the production host, which can impose stress on the host cell and, thereby, limit performance of the pathway. Unfortunately, analysis and treatment of the host stress response can be difficult, because there are many sources of stress that may interact in complex ways. We use a systems biological approach to analyze the stress imposed by expressing different enzyme variants from a lineage of soluble P450 monooxygenases, previously evolved for heterologous activity in Saccharomyces cerevisiae. Our analysis identifies patterns of stress imposed on the host by heterologous enzyme overexpression that are consistent across the evolutionary lineage, ultimately implicating heme depletion as the major stress. We show that the monooxygenase evolution, starting from conditions of either high or low stress, caused the cellular stress to converge to a common level. Overexpression of rate-limiting enzymes in the endogenous heme biosynthetic pathway alleviates the stress imposed by expression of the P450 monooxygenases and increases the enzymatic activity of the final evolved P450 by an additional 2.3-fold. Heme overexpression also increases the total activity of an endogenous cytosolic heme-containing catalase but not a heterologous P450 that is membrane-associated. This work demonstrates the utility of combining systems and synthetic biology to analyze and optimize heterologous enzyme expression.

Metabolic engineering of microorganisms for the synthesis of plant natural products

Of more than 200,000 plant natural products known to date, many demonstrate important pharmacological activities or are of biotechnological significance. However, isolation from natural sources is usually limited by low abundance and environmental, seasonal as well as regional variation, whereas total chemical synthesis is typically commercially unfeasible considering the complex structures of most plant natural products. With advances in DNA sequencing and recombinant DNA technology many of the biosynthetic pathways responsible for the production of these valuable compounds have been elucidated, offering the opportunity of a functional integration of biosynthetic pathways in suitable microorganisms. This approach offers promise to provide sufficient quantities of the desired plant natural products from inexpensive renewable resources. This review covers recent advancements in the metabolic engineering of microorganisms for the production of plant natural products such as isoprenoids, phenylpropanoids and alkaloids, and highlights general approaches and strategies to gain access to the rich biochemical diversity of plants by employing the biosynthetic power of microorganisms.

Synergistic Optimal Integration of Continuous and Fed-Batch Reactors for Enhanced Productivity of Lignocellulosic Bioethanol

With significant technological advances in metabolic engineering, there are currently available efficient recombinant yeast strains capable of fermenting lignocellulosic sugars (i.e., glucose and xylose) to bioethanol. Conventional batch fermentation, preferred in most bioprocesses, may not provide the most appropriate environments for those engineered strains to perform their best. In this article, we consider new reactor configurations integrating different types of reactors to examine their maximal productivity of lignocellulosic bioethanol. Among various possible scenarios, the highest performance was acquired from a synergistic operation of continuous and fed-batch reactors. In a chemostat, glucose is fermented alone by the hexose-only fermenting (wild-type) yeast, and unconverted xylose is fed to a batch reactor where mixed sugars are fermented by recombinant yeast. Theoptimization ofthe feed rate is acritical issue inorder tomaximizethe productivity ina fed-batch reactor. Itis shown that theproposedideaisabletoincreasethebioethanolproductivitybyupto50%incomparisontoasimplebatchoperation.Obviously, these considerations must be integrated with a more comprehensive costbenefit analysis before a clear choice of reactor configuration can emerge.

Engineering antibiotic production and overcoming bacterial resistance

Progress in DNA technology, analytical methods and computational tools is leading to new developments in synthetic biology and metabolic engineering, enabling new ways to produce molecules of industrial and therapeutic interest. Here, we review recent progress in both antibiotic production and strategies to counteract bacterial resistance to antibiotics. Advances in sequencing and cloning are increasingly enabling the characterization of antibiotic biosynthesis pathways, and new systematic methods for de novo biosynthetic pathway prediction are allowing the exploration of the metabolic chemical space beyond metabolic engineering. Moreover, we survey the computer-assisted design of modular assembly lines in polyketide synthases and non-ribosomal peptide synthases for the development of tailor-made antibiotics. Nowadays, production of novel antibiotic can be tranferred into any chosen chassis by optimizing a host factory through specific strain modifications. These advances in metabolic engineering and synthetic biology are leading to novel strategies for engineering antimicrobial agents with desired specificities.

Advanced Stoichiometric Analysis of Metabolic Networks of Mammalian Systems

Metabolic engineering tools have been widely applied to living organisms to gain a comprehensive understanding about cellular networks and to improve cellular properties. Metabolic flux analysis (MFA), flux balance analysis (FBA), and metabolic pathway analysis (MPA) are among the most popular tools in stoichiometric network analysis. Although application of these tools into well-known microbial systems is extensive in the literature, various barriers prevent them from being utilized in mammalian cells. Limited experimental data, complex regulatory mechanisms, and the requirement of more complex nutrient media are some major obstacles in mammalian cell systems. However, mammalian cells have been used to produce therapeutic proteins, to characterize disease states or related abnormal metabolic conditions, and to analyze the toxicological effects of some medicinally important drugs. Therefore, there is a growing need for extending metabolic engineering principles to mammalian cells in order to understand their underlying metabolic functions. In this review article, advanced metabolic engineering tools developed for stoichiometric analysis including MFA, FBA, and MPA are described. Applications of these tools in mammalian cells are discussed in detail, and the challenges and opportunities are highlighted.

Metabolism Is Not Boring

![Figure][1] CREDIT: PAINTING: DAVID S. GOODSELL/SCIENTIFIC DESIGN: ZHOU DU AND DANIEL J. KLIONSKY So investigator Steven McKnight admonished his audience last spring after a presentation at a conference (on metabolism and cancer, no less). He may have been “preaching to the choir” in this case, but what led him to issue such a blunt reminder? It seems that for a generation of biological scientists, metabolism was an area of biochemistry to be mastered and then put aside. Metabolism was always there in the background, providing the cell with the energy and resources to do what was required, but was rarely recognized to determinedly influence, and be influenced by, the physiological state of the cell. This special issue of Science celebrates a resurgence of interest in metabolism and an appreciation of its central role in disparate areas of cell biology, physiology, medicine, and synthetic biology ([www.sciencemag.org/special/metabolism/][2]). McKnight's Perspective (p. [1338][3]) takes a broad look at the field and justifies his excitement that recognizing the reciprocal regulation of metabolism and other cellular processes promises to advance our understanding of complex physiology. There is renewed interest in the metabolism of cancer cells and its potential as a therapeutic target. Levine and Puzio-Kuter (p. [1340][4]) review metabolic changes in cancer cells, as well as the recent suggestion that alterations in a metabolic enzyme can lead to the production of an “oncometabolite” that supports cancer cell growth. When energy sources are limited, cells use a process known as autophagy for breaking down cellular components to provide substrates for metabolism. Rabinowitz and White's Review (p. [1344][5]) discusses roles of autophagy in metabolism, its regulation, and its implications for cancer and degenerative diseases. Of course not all fields have neglected metabolism over the years, and an enormous literature describes the role of insulin and related hormones in controlling cellular metabolism. In a Focus Issue of Science Signaling , a Research Article and Perspective describe an unexpected signal from the insulin receptor that confers sensitivity to cell death when insulin is not present. Also highlighted are a role for lipids as cellular sensors of glucose metabolism and a mechanism by which cells can survive oxidative stress by shifting the activity of the cell's protein degradation complex, the proteasome. In Science Translational Medicine , Vallerie and Hotamisligil review how in a strategy to combat obesity and insulin resistance, a therapeutic inhibitor of cell signaling pathways must have coordinated actions in multiple cell types. Back in Science , Bass and Takahashi (p. [1349][6]) review new insights into the interaction of metabolism with circadian clocks. Displacement of eating and periods of activity away from the normal light-dark cycle, as experienced in jet lag or shift work, have marked effects on metabolic diseases. Cellular metabolic pathways, particularly in yeast or bacteria, can be exploited to synthesize compounds that are difficult or expensive to produce by other means. Keasling (p. [1355][7]) reviews advances in metabolic engineering and looks forward to a future in which customized microbes made by computer-aided design can efficiently produce desired chemicals, ranging from fuels to pharmaceuticals. See? It's not boring at all! [1]: pending:yes [2]: http://www.sciencemag.org/special/metabolism/ [3]: /lookup/doi/10.1126/science.1199908 [4]: /lookup/volpage/330/1340?iss=6009 [5]: /lookup/doi/10.1126/science.1193497 [6]: /lookup/volpage/330/1349?iss=6009 [7]: /lookup/doi/10.1126/science.1193990

Metabolically engineered Escherichia coli for biotechnological production of four-carbon 1,4-dicarboxylic acids

Confronted with inescapable exhaustion of the earth's fossil energy resources, the bio-based process to produce industrial chemicals is receiving significant interest. Biotechnological production of four-carbon 1,4-dicarboxylic acids (C4 diacids) from renewable plant biomass is a promising and attractive alternative to conventional chemistry routes. Although the C4 diacids pathway is well characterized and microorganisms able to convert biomass to these acids have been isolated and described, much still has to be done to make this process economically feasible. Metabolically engineered Escherichia coli has been developed as a biocatalyst to provide new processes for the biosynthesis of many valuable chemicals. However, E. coli does not naturally produce C4 diacids in large quantities. Rational strain development by metabolic engineering based on efficient genetic tools and detailed knowledge of metabolic pathways are crucial to successful production of these compounds. This review summarizes recent efforts and experiences devoted to metabolic engineering of the industrial model bacteria E. coli that led to efficient recombinant biocatalysts for the production of C4 diacids, including succinate, fumarate, malate, oxaloacetate, and aspartate, as well as the key limitations and challenges. Continued advancements in metabolic engineering will help to improve the titers, yields, and productivities of the C4 diacids discussed here.

Metabolic engineering of Escherichia coli for biotechnological production of high-value organic acids and alcohols

Confronted with the gradual and inescapable exhaustion of the earth's fossil energy resources, the bio-based process to produce platform chemicals from renewable carbohydrates is attracting growing interest. Escherichia coli has been chosen as a workhouse for the production of many valuable chemicals due to its clear genetic background, convenient to be genetically modified and good growth properties with low nutrient requirements. Rational strain development of E. coli achieved by metabolic engineering strategies has provided new processes for efficiently biotechnological production of various high-value chemical building blocks. Compared to previous reviews, this review focuses on recent advances in metabolic engineering of the industrial model bacteria E. coli that lead to efficient recombinant biocatalysts for the production of high-value organic acids like succinic acid, lactic acid, 3-hydroxypropanoic acid and glucaric acid as well as alcohols like 1,3-propanediol, xylitol, mannitol, and glycerol with the discussion of the future research in this area. Besides, this review also discusses several platform chemicals, including fumaric acid, aspartic acid, glutamic acid, sorbitol, itaconic acid, and 2,5-furan dicarboxylic acid, which have not been produced by E. coli until now.

Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology

The microbial production of biofuels is a promising avenue for the development of viable processes for the generation of fuels from sustainable resources. In order to become cost and energy effective, these processes must utilize organisms that can be optimized to efficiently produce candidate fuels from a variety of feedstocks. Escherichia coli has become a promising host organism for the microbial production of biofuels in part due to the ease at which this organism can be manipulated. Advancements in metabolic engineering and synthetic biology have led to the ability to efficiently engineer E. coli as a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. This review focuses on recent efforts devoted to engineering E. coli for the production of biofuels, with emphasis on the key aspects of both the utilization of a variety of substrates as well as the synthesis of several promising biofuels. Strategies for the efficient utilization of carbohydrates, carbohydrate mixtures, and noncarbohydrate carbon sources will be discussed along with engineering efforts for the exploitation of both fermentative and nonfermentative pathways for the production of candidate biofuels such as alcohols and higher carbon biofuels derived from fatty acid and isoprenoid pathways. Continued advancements in metabolic engineering and synthetic biology will help improve not only the titers, yields, and productivities of biofuels discussed herein, but also increase the potential range of compounds that can be produced.

A Genome May Reduce Your Carbon Footprint

A Genome May Reduce Your Carbon Footprint

Aldehyde–alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations

Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity.

Total synthesis approaches to natural product derivatives based on the combination of chemical synthesis and metabolic engineering

Secondary metabolites are an extremely diverse and important group of natural products with industrial and biomedical implications. Advances in metabolic engineering of both native and heterologous secondary metabolite producing organisms have allowed the directed synthesis of desired novel products by exploiting their biosynthetic potentials. Metabolic engineering utilises knowledge of cellular metabolism to alter biosynthetic pathways. An important technique that combines chemical synthesis with metabolic engineering is mutasynthesis (mutational biosynthesis; MBS), which advanced from precursor-directed biosynthesis (PDB). Both techniques are based on the cellular uptake of modified biosynthetic intermediates and their incorporation into complex secondary metabolites. Mutasynthesis utilises genetically engineered organisms in conjunction with feeding of chemically modified intermediates. From a synthetic chemist's point of view the concept of mutasynthesis is highly attractive, as the method combines chemical expertise with Nature's synthetic machinery and thus can be exploited to rapidly create small libraries of secondary metabolites. However, in each case, the method has to be critically compared with semi- and total synthesis in terms of practicability and efficiency. Recent developments in metabolic engineering promise to further broaden the scope of outsourcing chemically demanding steps to biological systems.

Optimal genetic manipulations in batch bioreactor control

Advances in metabolic engineering have enabled bioprocess optimization at the genetic level. Large-scale systematic models are now available at a genome level for many biological processes. There is, thus, a motivation to develop advanced control algorithms, using these complex models, to identify optimal performance strategies both at the genetic and bioreactor level. In the present paper, the bilevel optimization framework previously developed by the authors is coupled with control algorithms to determine the genetic manipulation strategies in practical bioprocess applications. The bilevel optimization includes a linear programming problem in the inner level and a nonlinear optimization problem in the outer level. Both gradient-based and stochastic methods are used to solve the nonlinear optimization problem. Ethanol production in an anaerobic batch fermentation of Escherichia coli is considered in case studies that demonstrate optimization of ethanol production, batch time, and multi-batch scheduling.

Towards bacterial strains overproducing l-tryptophan and other aromatics by metabolic engineering

The aromatic amino acids, L-tryptophan, L-phenylalanine, and L-tyrosine, can be manufactured by bacterial fermentation. Until recently, production efficiency of classical aromatic amino-acid-producing mutants had not yet reached a high level enough to make the fermentation method the most economic. With the introduction of recombinant DNA technology, it has become possible to apply more rational approaches to strain improvement. Many recent activities in this metabolic engineering have led to several effective approaches, which include modification of terminal pathways leading to removal of bottleneck or metabolic conversion, engineering of central carbon metabolism leading to increased supply of precursors, and transport engineering leading to reduced intracellular pool of the aromatic amino acids. In this review, advances in metabolic engineering for the production of the aromatic amino acids and useful aromatic intermediates are described with particular emphasis on two representative producer organisms, Corynebacterium glutamicum and Escherichia coli.

Glycopeptides: Update on an old successful antibiotic class

The natural product glycopeptides vancomycin and teicoplanin have come to play a significant role in the therapy for Gram-positive bacterial infections. In particular vancomycin is the choice for empiric therapy of these infections primarily due to its activity against and the significance of methicillin-resistant Staphylococcus aureus. While high-level problematic glycopeptide resistance among enterococci was observed initially and continues to increase, the slow creep of vancomycin intermediate susceptibility and the fear of frank resistance among the staphylococci have precipitated increasing work leading to creation of new semisynthetic analogs. These new agents, including dalbavancin and telavancin, are within 1–2 years availability in the clinic. Interestingly, chemical modifications resulting in these second-generation analogs and additional characterization have revealed new mechanisms of antibacterial action, and plasticity regarding additional properties including pharmacokinetics for the drug candidates. The unique beneficial properties of the near term vancomycin replacements, semisynthesis of additional important analogs, and advances in metabolic engineering resulting in novel scaffolds signal a new era for the glycopeptide antibiotics.

Production of useful secondary metabolites in plants: Functional genomics approaches

The paradigm of biological research has been changed by recent developments in genomics, high-throughput biology, and bioinformatics. Conventional biology often was based on empirical, labor-intensive, and time-consuming methods. In the new paradigm, biological research e is driven by a holistic approach on the basis of rational, automatic, and high-throughput methods. New functional compounds can be discovered by using high-throughput screening systems. Secondary metabolite pathways and the genes involved in those pathways are then determined by studying functional genomics in conjunction with the data-mining tools of bioinformatics. In addition, these advances in metabolic engineering enable researchers to confer new secondary metabolic pathways to crops by transferring three to five, or more, heterologous genes taken from various other species. In the future, engineering for the production of useful compounds will be designed by a set of software tools that allows the user to specify a cell’s genes, proteins, and other molecules, as well as their individual interactions.

Dynamic Optimal Control of Homeostasis: An Integrative System Approach for Modeling of the Central Nitrogen Metabolism in Saccharomyces cerevisiae

The theory of dynamic optimal metabolic control (DOMC), as developed by Giuseppin and Van Riel (Metab. Eng., 2000), is applied to model the central nitrogen metabolism (CNM) in Saccharomyces cerevisiae. The CNM represents a typical system encountered in advanced metabolic engineering. The CNM is the source of the cellular amino acids and proteins, including flavors and potentially valuable biomolecules; therefore, it is also of industrial interest. In the DOMC approach the cell is regarded as an optimally controlled system. Given the metabolic genotype, the cell faces a control problem to maintain an optimal flux distribution in a changing environment. The regulation is based on strategies and balances feedback control of homeostasis and feedforward regulation for adaptation. The DOMC approach is an integrative, holistic approach, not based on mechanistic descriptions and (therefore) not biased by the variation present in biochemical and molecular biological data. It is an effective tool to structure the rapidly increasing amount of data on the function of genes and pathways. The DOMC model is used successfully to predict the responses of pulses of ammonia and glutamine to nitrogen-limited continuous cultures of a wild-type strain and a glutamine synthetase-negative mutant. The simulation results are validated with experimental data.

Developments in metabolic engineering

The complete sequencing of several microbial genomes has resulted in the increased availability of genes for metabolic engineering. The number of databases and computational tools to deal with this information has also increased. This development has stimulated, and will continue to stimulate, advances in metabolic engineering. Specific recent advances include improvement of pathways for aromatic metabolites, the development of a more complete understanding of the effect of bacterial hemoglobin on cell performance, the development of NMR-based methods for the monitoring of intracellular metabolites and metabolic flux, and the application of metabolic control analysis and metabolic flux analysis to a variety of systems.