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PubMed2000

The role of metabolic engineering in the improvement of Saccharomyces cerevisiae: utilization of industrial media.

Metabolic engineering has become a very important approach to strain improvement in parallel with classical strain development. Although Saccharomyces cerevisiae has been domesticated for ethanol and bread production, there are still some fundamental problems associated with its industrial use. The industrially used carbon sources often consist of a sugar mixture, and due to glucose repression these sugars are utilized sequentially, resulting in prolonged production time. In this article we discuss the application of metabolic engineering for construction of glucose-derepressed strains and specify advantages as well as difficulties associated with this approach.

Journal Article↗

PubMed2004

The yeast Zygosaccharomyces bailii: a new host for heterologous protein production, secretion and for metabolic engineering applications.

Molecular tools for the production of heterologous proteins and metabolic engineering applications of the non-conventional yeast Zygosaccharomyces bailii were developed. The combination of Z. bailii's resistance to relatively high temperature, osmotic pressure and low pH values, with a high specific growth rate renders this yeast potentially interesting for exploitation for biotechnological purposes as well as for the understanding of the biological phenomena and mechanisms underlying the respective resistances. Looking forward to these potential applications, here we present the tools required for the production and the secretion of different heterologous proteins, and one example of a metabolic engineering application of this non-conventional yeast, employing the newly developed molecular tools.

Cloning, Molecular↗

PubMedDate not supplied

Metabolic engineering of propanediol pathways.

Microbial fermentation is an important technology for the conversion of renewable resources to chemicals. In this paper, we describe the application of metabolic engineering for the development of two new fermentation processes: the microbial conversion of sugars to 1,3-propanediol (1,3-PD) and 1,2-propanediol (1,2-PD). A variety of naturally occurring organisms ferment glycerol to 1,3-PD, but no natural organisms ferment sugars directly to 1,3-PD. We first describe the fed-batch fermentation of glycerol to 1,3-PD by Klebsiella pneumoniae. We then present various approaches for the conversion of sugars to 1,3-PD, including mixed-culture fermentation, cofermentation of glycerol and glucose, and metabolic engineering of a "sugars to 1,3-PD" pathway in a single organism. Initial results are reported for the expression of genes from the K. pneumoniae 1,3-PD pathway in Saccharomyces cerevisiae. The best naturally occurring organism for the fermentation of sugars to 1,2-PD is Thermoanaerobacterium thermosaccharolyticum. We describe the fermentation of several different sugars to 1,2-PD by this organism in batch and continuous culture. We report that Escherichia coli strains engineered to express either aldose reductase or glycerol dehydrogenase convert glucose to (R)-1,2-PD. We then analyze the ultimate potential of fermentation processes for the production of propanediols. Linear optimization studies indicate that, under aerobic conditions, propanediol yields that approach the theoretical maximum are possible and CO2 is the primary coproduct. Without the need to produce acetate, final product titers in the range of 100 g/L should be possible; the high titers and low coproduct levels should make product recovery and purification straightforward. The examples given in this paper illustrate the importance of metabolic engineering for fermentation process development in general.

Biotechnology↗

PubMed2005

Production of vanillin by metabolically engineered Escherichia coli.

E. coli was metabolically engineered to produce vanillin by expression of the fcs and ech genes from Amycolatopsis sp. encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively. Vanillin production was optimized by leaky expression of the genes, under the IPTG-inducible trc promoter, in complex 2YT medium. Supplementation with glucose, fructose, galactose, arabinose or glycerol severely decreased vanillin production. The highest vanillin production of 1.1 g l(-1) was obtained with cultivation for 48 h in 2YT medium with 0.2% (w/v) ferulate, without IPTG and no supplementation of carbon sources.

Actinomycetales↗

PubMed2005

Improvement of xylose uptake and ethanol production in recombinant Saccharomyces cerevisiae through an inverse metabolic engineering approach.

We used an inverse metabolic engineering approach to identify gene targets for improved xylose assimilation in recombinant Saccharomyces cerevisiae. Specifically, we created a genomic fragment library from Pichia stipitis and introduced it into recombinant S. cerevisiae expressing XYL1 and XYL2. Through serial subculturing enrichment of the transformant library, 16 transformants were identified and confirmed to have a higher growth rate on xylose. Sequencing of the 16 plasmids isolated from these transformants revealed that the majority of the inserts (10 of 16) contained the XYL3 gene, thus confirming the previous finding that XYL3 is the consensus target for increasing xylose assimilation. Following a sequential search for gene targets, we repeated the complementation enrichment process in a XYL1 XYL2 XYL3 background and identified 15 fast-growing transformants, all of which harbored the same plasmid. This plasmid contained an open reading frame (ORF) designated PsTAL1 based on a high level of homology with S. cerevisiae TAL1. To further investigate whether the newly identified PsTAL1 ORF is responsible for the enhanced-growth phenotype, we constructed an expression cassette containing the PsTAL1 ORF under the control of a constitutive promoter and transformed it into an S. cerevisiae recombinant expressing XYL1, XYL2, and XYL3. The resulting recombinant strain exhibited a 100% increase in the growth rate and a 70% increase in ethanol production (0.033 versus 0.019 g ethanol/g cells . h) on xylose compared to the parental strain. Interestingly, overexpression of PsTAL1 did not cause growth inhibition when cells were grown on glucose, unlike overexpression of the ScTAL1 gene. These results suggest that PsTAL1 is a better gene target for engineering of the pentose phosphate pathway in recombinant S. cerevisiae.

Base Sequence↗

PubMed2002

Metabolic engineering of amino acids and storage proteins in plants.

Amino acid pathways are important targets for plant metabolic engineering. Since plants represent the major global food supply, large efforts are devoted to increasing the content of "essential" amino acids, which are absolutely required in human foods and animal feeds. Engineering of amino acids is also undertaken to improve plant growth and stress tolerance. Many of the pathways of amino acid metabolism in plants have been elucidated, and genes encoding most of the enzymes are now available. The expression of recombinant genes in transgenic plants, coupled with genetic and biochemical approaches, has contributed significantly to the understanding of regulatory networks of the metabolism of amino acids and their incorporation into proteins. This knowledge is now being extensively applied to metabolic engineering of crops, and this is reflected by a large patent literature. The problems of engineering plant amino acid metabolism, and ways to solve them, are discussed using the essential amino acids lysine and methionine as examples.

Amino Acids, Essential↗

PubMed2002

Optimizing bioconversion pathways through systems analysis and metabolic engineering.

We demonstrate a general approach for metabolic engineering of biocatalytic systems comprising the uses of a chemostat for strain improvement and radioisotopic tracers for the quantification of pathway fluxes. Flux determination allows the identification of target pathways for modification as validated by subsequent overexpression of the corresponding gene. We demonstrate this method in the indene bioconversion network of Rhodococcus modified for the overproduction of 1,2-indandiol, a key precursor for the AIDS drug Crixivan.

Biomedical Engineering↗

PubMedDate not supplied

[Metabolic engineering in design of cellular elements of biosensors].

An extended definition of the term "metabolic engineering" is given and main spheres of its using in fundamental studies and modern biotechnology are discussed in this article. Emphasis is made on specific using the approaches of metabolic engineering in construction of the cell elements of sensors based on the use of mutant and chemically modified cells of methylotrophic yeasts. This investigation is designed in the laboratory of Biochemical Genetics of the Division of Cell Regulatory Systems, A. V. Palladin Institute of Biochemistry. Genetic and chemical modifications have allowed to provide some directed changes in cell sensoring output toward methanol, ethanol and formaldehyde that result in enhanced selectivity and shortened time-output of the corresponding potentiometric and amperometric sensors.

Biomedical Engineering↗

PubMed1998

Quantification of metabolites in the indole alkaloid pathways of catharanthus roseus: implications for metabolic engineering

In this article, we present a review of the current state of metabolic engineering in Catharanthus roseus. A significant amount of research has contributed to characterization of several individual steps in the biosynthetic pathway of medicinally valuable alkaloids. However, knowledge of the regulation of these pathways is still sparse. Using hairy root cultures, we studied the responses of alkaloid metabolism to environmental stimulation such as light and elicitation. Through precursor feeding studies, the putative rate-limiting steps of the terpenoid pathway in hairy root cultures also have been examined. Relating this knowledge to specific events at the molecular level, and the cloning of corresponding genes are the next key steps in metabolic engineering of the C. roseus alkaloids. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗

PubMed2001

Metabolic engineering of Saccharomyces cerevisiae for xylose utilization.

Metabolic engineering of Saccharomyces cerevisiae for ethanolic fermentation of xylose is summarized with emphasis on progress made during the last decade. Advances in xylose transport, initial xylose metabolism, selection of host strains, transformation and classical breeding techniques applied to industrial polyploid strains as well as modeling of xylose metabolism are discussed. The production and composition of the substrates--lignocellulosic hydrolysates--is briefly summarized. In a future outlook iterative strategies involving the techniques of classical breeding, quantitative physiology, proteomics, DNA micro arrays, and genetic engineering are proposed for the development of efficient xylose-fermenting recombinant strains of S. cerevisiae.

Biological Transport↗

PubMed2005

Metabolic engineering in the -omics era: elucidating and modulating regulatory networks.

The importance of regulatory control in metabolic processes is widely acknowledged, and several enquiries (both local and global) are being made in understanding regulation at various levels of the metabolic hierarchy. The wealth of biological information has enabled identifying the individual components (genes, proteins, and metabolites) of a biological system, and we are now in a position to understand the interactions between these components. Since phenotype is the net result of these interactions, it is immensely important to elucidate them not only for an integrated understanding of physiology, but also for practical applications of using biological systems as cell factories. We present some of the recent "-omics" approaches that have expanded our understanding of regulation at the gene, protein, and metabolite level, followed by analysis of the impact of this progress on the advancement of metabolic engineering. Although this review is by no means exhaustive, we attempt to convey our ideology that combining global information from various levels of metabolic hierarchy is absolutely essential in understanding and subsequently predicting the relationship between changes in gene expression and the resulting phenotype. The ultimate aim of this review is to provide metabolic engineers with an overview of recent advances in complementary aspects of regulation at the gene, protein, and metabolite level and those involved in fundamental research with potential hurdles in the path to implementing their discoveries in practical applications.

Bacteria↗

PubMed2002

Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase.

Metabolic engineering studies have generally focused on manipulating enzyme levels through either the amplification, addition, or deletion of a particular pathway. However, with cofactor-dependent production systems, once the enzyme levels are no longer limiting, cofactor availability and the ratio of the reduced to oxidized form of the cofactor can become limiting. Under these situations, cofactor manipulation may become crucial in order to further increase system productivity. Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. However, cofactor manipulations can potentially become a powerful tool for metabolic engineering. Nicotinamide adenine dinucleotide (NAD) functions as a cofactor in over 300 oxidation-reduction reactions and regulates various enzymes and genetic processes. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. This paper investigates a genetic means of manipulating the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase. More specifically, it explores the effect on the metabolic patterns in Escherichia coli under anaerobic and aerobic conditions of substituting the native cofactor-independent formate dehydrogenase (FDH) by and NAD(+)-dependent FDH from Candida boidinii. The over-expression of the NAD(+)-dependent FDH doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed, increased the final cell density, and provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically. Under anaerobic conditions, the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol-to-acetate ratio. Even more interesting is the observation that during aerobic growth, the increased availability of NADH induced a shift to fermentation even in the presence of oxygen by stimulating pathways that are normally inactive under these conditions.

Aerobiosis↗

PubMed2002

Characterization of heterologous and native enzyme activity profiles in metabolically engineered Zymomonas mobilis strains during batch fermentation of glucose and xylose mixtures.

Zymomonas mobilis has been metabolically engineered to broaden its substrate utilization range to include D-xylose and L-arabinose. Both genomically integrated and plasmid-bearing Z. mobilis strains that are capable of fermenting the pentose D-xylose have been created by incorporating four genes: two genes encoding xylose utilization metabolic enzymes (xylA/xylB) and two genes encoding pentose phosphate pathway enzymes (talB/tktA). We have characterized the activities of the four newly introduced enzymes for xylose metabolism, along with those of three native glycolytic enzymes, in two different xylose-fermenting Z. mobilis strains. These strains were grown on glucose-xylose mixtures in computer-controlled fermentors. Samples were collected and analyzed to determine extracellular metabolite concentrations as well as the activities of several intracellular enzymes in the xylose and glucose uptake and catabolism pathways. These measurements provide new insights on the possible bottlenecks in the engineered metabolic pathways and suggest methods for further improving the efficiency of xylose fermentation.

Acetates↗

PubMed2005

Metabolic engineering of new fatty acids in plants.

Metabolic engineering of plants to express high levels of new fatty acids that are of nutritional and industrial importance has proven to be highly challenging. Significant advances have been made recently, however, particularly in the development of the first plant oils to contain long-chain polyunsaturated fatty acids, such as arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid. Methods of increasing the accumulation of Delta12-modified fatty acids synthesized by transgenically expressed FAD2-like enzymes have also been investigated. Biochemical analyses of plants that express these introduced fatty-acid metabolic pathways have highlighted the central importance of ensuring the removal of novel fatty acids from their site of synthesis on phosphatidylcholine to enable their further modification, exclusion from membrane lipids and accumulation in seed triacylglycerols.

Fatty Acids, Nonesterified↗

PubMed1997

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.

Journal Article↗

PubMed2006

Metabolic engineering under uncertainty. I: framework development.

Standard bioprocess conditions have been widely applied for the microbial conversion of raw material to essential industrial products. Successful metabolic engineering (ME) strategies require a comprehensive framework to manage the complexity embedded in cellular metabolism, to explore the impacts of bioprocess conditions on the cellular responses, and to deal with the uncertainty of the physiochemical parameters. We have recently developed a computational and statistical framework that is based on Metabolic Control Analysis and uses a Monte Carlo method to simulate the uncertainty in the values of the system parameters [Wang, L., Birol, I., Hatzimanikatis, V., 2004. Metabolic control analysis under uncertainty: framework development and case studies. Biophys. J. 87(6), 3750-3763]. In this work, we generalize this framework to incorporate the central cellular processes, such as cell growth, and different bioprocess conditions, such as different types of bioreactors. The framework provides the mathematical basis for the quantification of the interactions between intracellular metabolism and extracellular conditions, and it is readily applicable to the identification of optimal ME targets for the improvement of industrial processes [Wang, L., Hatzimanikatis, V., 2005. Metabolic engineering under uncertainty. II: analysis of yeast metabolism. Submitted].

Bioreactors↗

PubMed2001

Metabolic engineering as an integrating platform for strain development.

Integration of the analytical framework and experimental tools of metabolic engineering with emerging technologies such as DNA microarrays and directed evolution stands to dramatically improve the approaches by which strain improvement and biocatalyst design are pursued in the future. Progress in genomics and applied molecular biology, together with increasing emphasis on renewable resource utilization for chemical production, has advanced metabolic engineering to the forefront of biotechnological interest.

Industrial Microbiology↗

PubMed2002

Nuclear magnetic resonance and plant metabolic engineering.

Nuclear magnetic resonance (NMR) can be used to measure metabolite levels and metabolic fluxes, to probe the intracellular environment, and to follow transport and energetics nondestructively. NMR methods are therefore powerful aids to understanding plant metabolism and physiology. Both spectroscopy and imaging can help overcome the unique challenges that plants present to the metabolic engineer by detecting, identifying, quantifying, and localizing novel metabolites in vivo and in extracts; revealing the composition and physical state of cell wall and other polymers; allowing the identification of active pathways; providing quantitative measures of metabolic flux; and testing hypotheses about the effects of engineered traits on plant physiological function. The aim of this review is to highlight recent studies in which NMR has contributed to metabolic engineering of plants and to illustrate the unique characteristics of NMR measurements that give it the potential to make greater contributions in the future.

Biopolymers↗