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PubMed2001

Evolutionary engineering of industrially important microbial phenotypes.

The tremendous complexity of dynamic interactions in cellular systems often impedes practical applications of metabolic engineering that are largely based on available molecular or functional knowledge. In contrast, evolutionary engineering follows nature's 'engineering' principle by variation and selection. Thus, it is a complementary strategy that offers compelling scientific and applied advantages for strain development and process optimization, provided a desired phenotype is amenable to direct or indirect selection. In addition to simple empirical strain development by random mutation and direct selection on plates, evolutionary engineering also encompasses recombination and continuous evolution of large populations over many generations. Two distinct evolutionary engineering applications are likely to gain more relevance in the future: first, as an integral component in metabolic engineering of strains with improved phenotypes, and second, to elucidate the molecular basis of desired phenotypes for subsequent transfer to other hosts. The latter will profit from the broader availability of recently developed methodologies for global response analysis at the genetic and metabolic level. These methodologies facilitate identification of the molecular basis of evolved phenotypes. It is anticipated that, together with novel analytical techniques, bioinformatics, and computer modeling of cellular functions and activities, evolutionary engineering is likely to find its place in the metabolic engineer's toolbox for research and strain development. This review presents evolutionary engineering of whole cells as an emerging methodology that draws on the latest advances from a wide range of scientific and technical disciplines.

Biological Evolution↗

PubMed2001

New directions in carbohydrate engineering: a metabolic substrate-based approach to modify the cell surface display of sialic acids.

This review discusses new directions in the emerging field of carbohydrate engineering. Specifically, it describes substrate-based methodologies that are complementary to the recombinant DNA techniques that now dominate metabolic and cellular engineering endeavors. A substrate-based approach consists of intercepting a biosynthetic pathway with an unnatural analog of a metabolic intermediate. The unnatural compound competes with the endogenous substrate for biosynthetic incorporation into a cellular component by action of the natural enzymes of the cell. The incorporation of the unnatural compound into the cellular architecture can directly modulate cellular properties and biological processes. Alternatively, a molecular handle can be included in the design of the unnatural substrate that allows further elaboration upon reaction with an externally delivered reagent. The sialic acid biosynthetic pathway is presented as a model system to illustrate both the practical aspects and theoretical considerations of a substrate-based cellular engineering approach. Specific applications of carbohydrate-based cell surface engineering include chemical construction of new glycosylation patterns on cells, new approaches to targeting tumor cell with either diagnostic or therapeutic agents, and installation of novel receptors on cells for facilitating viral-mediated gene delivery.

Biotechnology↗

PubMed1994

Alteration of the biochemical valves in the central metabolism of Escherichia coli.

Although E. coli central metabolism has been studied for several decades, many regulatory features are still unknown. To achieve the goal of rational manipulation of cellular metabolism, it is important to understand how E. coli responds to overexpressed enzymes. By studying the biochemical control of fluxes between PEP, pyruvate, and OAA, we have addressed some fundamental questions that may prove to be essential for applications in metabolic engineering. First, we found that simultaneous overexpression of Pck and Ppc, or Pps alone in the presence of glucose leads to phenotypes consistent with futile cycline. In contrast to our expectation, futile cycling per se does not affect the growth rate significantly. However, excessive futile cycling may cause competitive disadvantage in the natural environment. Overexpression of Pck caused growth inhibition but no futile cycling. Therefore, E. coli controls the expression of gluconeogenic enzymes not only to avoid excessive futile cycling, but also to prevent toxicity effects. In metabolic engineering, futile cycling may be used as a strategy to stimulate metabolism for either production of metabolites or digestion of toxic wastes. Second, we found that the expression levels of Pps and Pck in E. coli are not optimal for growth on pyruvate and succinate, respectively. Overexpression of these enzymes increases the growth rate on pyruvate and on succinate, respectively, indicating that the slow growth rates on these substrates are at least partially caused by the insufficient supply of PEP and its derivatives. Moreover, E. coli also has not optimized the Ppc level for optimal growth yield on glucose in uncontrolled batch cultures. These results demonstrate that the central metabolism is not optimized for growth under defined laboratory conditions. Thus, the possibility exists that adjustment of native enzyme levels in the central metabolism can improve bioreactor performance. Third, we found that overexpression of Pck affects the transcriptional levels of unrelated genes. This example indicates that physiological responses to enzyme (over)expression should be interpreted cautiously, as changing the expression level of a specific enzyme may affect many unlinked genes. Similar results have also been obtained by use of two-dimensional electrophoresis of proteins from E. coli. Although more questions remain to be answered, fast progress in the area of metabolic engineering can be expected in the near future.

Bacterial Proteins↗

PubMed1996

Metabolic pathway engineering in cotton: biosynthesis of polyhydroxybutyrate in fiber cells.

Alcaligenes eutrophus genes encoding the enzymes, beta-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-d-(-)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton. Endogenous beta-ketothiolase activity is present in cotton fibers. Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, beta-glucuronidase (GUS), expression. Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes. Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm. High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 x 10(6) to 1.8 x 10(6) Da. The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties. The fibers exhibited better insulating characteristics. The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity. These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources. This is an important step toward producing new generation fibers for the textile industry.

Journal Article↗

PubMedDate not supplied

Engineering the E. coli UDP-glucose synthesis pathway for oligosaccharide synthesis.

A metabolic engineering strategy was successfully applied to engineer the UDP-glucose synthesis pathway in E. coli. Two key enzymes of the pathway, phosphoglucomutase and UDP-glucose pyrophosphorylase, were overexpressed to increase the carbon flux toward UDP-glucose synthesis. When additional enzymes (a UDP-galactose epimerase and a galactosyltransferease) were introduced to the engineered strain, the increased flux to UDP-glucose synthesis led to an enhanced UDP-galactose derived disaccharide synthesis. Specifically, close to 20 mM UDP-galactose derived disaccharides were synthesized in the engineered strain, whereas in the control strain only 2.5 mM products were obtained, indicating that the metabolic engineering strategy was successful in channeling carbon flux (8-fold more) into the UDP-glucose synthesis pathway. UDP-sugar synthesis and oligosaccharide synthesis were shown to increase according to the enzyme expression levels when inducer concentration was between 0 and 0.5 mM. However, this dependence on the enzyme expression stopped when expression level was further increased (IPTG concentration was increased from 0.5 to 1 mM), indicating that other factors emerged as bottlenecks of the synthesis. Several likely bottlenecks and possible engineering strategies to further improve the synthesis are discussed.

Carbon↗

PubMed1995

Subtlety in control--metabolic pathway engineering.

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Biological Products↗

PubMed2003

Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum.

A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.

Cell Division↗

PubMed2005

Genetic engineering of terpenoid metabolism attracts bodyguards to Arabidopsis.

Herbivore-damaged plants release complex mixtures of volatiles that attract natural enemies of the herbivore. To study the relevance of individual components of these mixtures for predator attraction, we manipulated herbivory-induced volatiles through genetic engineering. Metabolic engineering of terpenoids, which dominate the composition of many induced plant volatile bouquets, holds particular promise. By switching the subcellular localization of the introduced sesquiterpene synthase to the mitochondria, we obtained transgenic Arabidopsis thaliana plants emitting two new isoprenoids. These altered plants attracted carnivorous predatory mites (Phytoseiulus persimilis) that aid the plants' defense mechanisms.

Animals↗

PubMed2005

Integrating genomics and metabolomics for engineering plant metabolic pathways.

Plant metabolites are characterized by an enormous chemical diversity, every plant having its own complex set of metabolites. This variety poses analytical challenges, both for profiling multiple metabolites in parallel and for the quantitative analysis of selected metabolites. We are only just starting to understand the roles of these metabolites, many of them being involved in adaptations to specific ecological niches and some finding beneficial use (e.g. as pharmaceuticals). Spectacular advances in plant metabolomics offer new possibilities, together with the aid of systems biology, to explore the extraordinary complexity of the plant biochemical capacity. State-of-the art genomics tools can be combined with metabolic profiling to identify key genes that could be engineered for the production of improved crop plants.

Arabidopsis↗

PubMed2003

[Quantitation & optimization of guanosine fermentation process: prevention of NH4+ accumulation increases guanosine production by 70%].

Metabolic engineering has become a powerful tool for optimization of industrial fermentation processes. Metabolic engineering usually undergoes three steps: construction of a recombinant strain with improved properties, genetic and biochemical analysis of the strain, and identification of target for further improvement. Metabolic fluxes analysis is an important part of the biochemical analysis. Based on the law of mass conservation and assuming pseudo-steady-state for the intermediates in the metabolic pathways, we have quantitatively analyzed the time course of the flux distribution in Bacillus subtilis and used the data to reveal the nature of the so-called "40 hour" phenomenon in fermentation of guanosine, a key raw material for the synthesis of additives for human consumption and animal feeds. The phenomenon refers to the observation that guanosine production, which proceeds at high rate from 12 hour on, declines around 40 hour while consumption of glucose keeps increasing, leading to the lower yield of the nucleoside. Equations based upon the metabolic network of Bacillus subtilis consisted of EMP pathway, HMP pathway, TCA cycle, oxidative phosphorylation pathway and others reactions of the intermediates, was constructed. The equations were solved by using the quantitative data obtained in this study. The air flow and volume, concentration of oxygen and carbon dioxide in the exit-gas were monitored online; the concentration of biomass, glucose and guanosine was analyzed manually; and the concentration of acetate, citric acid, pyruvate, and 17 amino acids were HPLC quantified. The solutions of the equation were proved to be valid, as the experimental data on oxygen consumption agrees with that of predicted form the equation. The results indicated that at 40h of the fermentation process the flux of HMP pathway, which provides the precursor of the nucleoside, decreased while that of EMP pathway and the pathways that generate amino acids and organic acids increased. The shift correlated with the accumulation of NH4+ in the broth. The assimilation of NH4+ is an energy consuming process and could shift the metabolism to the energy generating EMP pathway. Accordingly, measures were taken to prevent the accumulation of NH4+. The interference indeed stopped the metabolism shift and boosted the guanosine production at 30 g/L, 70% higher than the level reported in literature.

Bacillus subtilis↗

PubMed2006

In vivo engineering of metabolically active hepatic tissues in a neovascularized subcutaneous cavity.

Recent success in clinical hepatocyte transplantation therapy has encouraged further investigation into bioengineering hepatic tissues in vivo. Engineering tissues in the subcutaneous space is an attractive method; however, hepatocyte survival has been transient due to insufficient vascular network formation. To establish a vascularized cavity, we created a polyethylene terephthalate mesh device coated with poly(vinylalcohol) that allowed for the gradual release of basic fibroblast growth factor (bFGF), a potent angiogenic factor. The efficacy of the bFGF-releasing device in inducing vascular network formation in the subcutaneous space was observed in mouse and rat studies. Isolated mouse hepatocytes transplanted into newly vascularized subcutaneous cavities allowed for persistent survival up to 120 days. In the absence of a vascularized compartment, the survival of the transplanted hepatocytes was markedly diminished. Functional maintenance of the engineered hepatic tissues was confirmed by high expression of liver-specific mRNAs and proteins. These engineered hepatic tissues have the ability to take up inoculated compounds and express strong induction of drug-metabolizing enzymes, demonstrating functional relevance as a metabolic tissue. In conclusion, we have created a novel technology to engineer functionally active hepatic tissues in the subcutaneous space, which will likely facilitate hepatocyte-based therapies.

Animals↗

PubMed1998

Antisense strategies for glycosylation engineering of Chinese hamster ovary (CHO) cells.

Novel glycoproteins, inaccessible by other techniques, can be obtained by metabolic engineering of the oligosaccharide biosynthesis pathway. Furthermore, alteration of cell-surface oligosaccharides can change the properties of receptors involved in cell-cell adhesion. Sialyl Lewis X (sLex) is a cell-surface oligosaccharide determinant which is specifically expressed on granulocytes and monocytes and which interacts with selectins to influence leukocyte trafficking, thrombosis, inflammation, and cancer. Antisense technology targeting fucosyltransferase VI (Fuc-TVI), an enzyme necessary for the synthesis of the sLex in engineered Chinese hamster ovary (CHO) cells, has reduced Fuc-TVI activity, sLex synthesis, and adhesion to endothelial cells. Antisense methodology to reduce targeted activity in oligosaccharide biosynthesis or other pathways is an important addition to CHO cell metabolic engineering capabilities.

Animals↗

PubMed2006

Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells.

Rational engineering of complicated metabolic networks involved in the production of biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Whereas comprehensive genome-wide functional genomics approaches can be successfully applied to analyze a select number of model plants, these holistic approaches are not yet available for the study of nonmodel plants that include most, if not all, medicinal plants. We report here a comprehensive profiling analysis of the Madagascar periwinkle (Catharanthus roseus), a source of the anticancer drugs vinblastine and vincristine. Genome-wide transcript profiling by cDNA-amplified fragment-length polymorphism combined with metabolic profiling of elicited C. roseus cell cultures yielded a collection of known and previously undescribed transcript tags and metabolites associated with terpenoid indole alkaloids. Previously undescribed gene-to-gene and gene-to-metabolite networks were drawn up by searching for correlations between the expression profiles of 417 gene tags and the accumulation profiles of 178 metabolite peaks. These networks revealed that the different branches of terpenoid indole alkaloid biosynthesis and various other metabolic pathways are subject to differing hormonal regulation. These networks also served to identify a select number of genes and metabolites likely to be involved in the biosynthesis of terpenoid indole alkaloids. This study provides the basis for a better understanding of periwinkle secondary metabolism and increases the practical potential of metabolic engineering of this important medicinal plant.

Catharanthus↗

PubMed2001

Retrobiosynthetic nuclear magnetic resonance analysis of amino acid biosynthesis and intermediary metabolism. Metabolic flux in developing maize kernels.

Information on metabolic networks could provide the basis for the design of targets for metabolic engineering. To study metabolic flux in cereals, developing maize (Zea mays) kernels were grown in sterile culture on medium containing [U-(13)C(6)]glucose or [1,2-(13)C(2)]acetate. After growth, amino acids, lipids, and sitosterol were isolated from kernels as well as from the cobs, and their (13)C isotopomer compositions were determined by quantitative nuclear magnetic resonance spectroscopy. The highly specific labeling patterns were used to analyze the metabolic pathways leading to amino acids and the triterpene on a quantitative basis. The data show that serine is generated from phosphoglycerate, as well as from glycine. Lysine is formed entirely via the diaminopimelate pathway and sitosterol is synthesized entirely via the mevalonate route. The labeling data of amino acids and sitosterol were used to reconstruct the labeling patterns of key metabolic intermediates (e.g. acetyl-coenzyme A, pyruvate, phosphoenolpyruvate, erythrose 4-phosphate, and Rib 5-phosphate) that revealed quantitative information about carbon flux in the intermediary metabolism of developing maize kernels. Exogenous acetate served as an efficient precursor of sitosterol, as well as of amino acids of the aspartate and glutamate family; in comparison, metabolites formed in the plastidic compartments showed low acetate incorporation.

Amino Acids↗

PubMed2004

Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast.

The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc(-)) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc(-) S. cerevisiae strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C(2) compound, these strains cannot grow in batch cultures on synthetic medium with glucose. We used two subsequent phenotypic selection strategies to obtain a Pdc(-) strain without these growth defects. An acetate-independent Pdc(-) mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively lowering the acetate content in the feed. Transcriptome analysis did not reveal the mechanisms behind the C(2) independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc(-) S. cerevisiae mutant (TAM) that could grow in batch cultures ( micro (max) = 0.20 h(-1)) on synthetic medium, with glucose as the sole carbon source. Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucose-repressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose. In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate. By repeated glucose feeding, a pyruvate concentration of 135 g liter(-1) was obtained, with a specific pyruvate production rate of 6 to 7 mmol g of biomass(-1) h(-1) during the exponential-growth phase and an overall yield of 0.54 g of pyruvate g of glucose(-1).

Culture Media↗

PubMed1998

Engineering secondary metabolism in maize cells by ectopic expression of transcription factors.

Manipulation of plant natural product biosynthesis through genetic engineering is an attractive but technically challenging goal. Here, we demonstrate that different secondary metabolites can be produced in cultured maize cells by ectopic expression of the appropriate regulatory genes. Cell lines engineered to express the maize transcriptional activators C1 and R accumulate two cyanidin derivatives, which are similar to the predominant anthocyanin found in differentiated plant tissues. In contrast, cell lines that express P accumulate various 3-deoxy flavonoids. Unexpectedly, P-expressing cells in culture also accumulate phenylpropanoids and green fluorescent compounds that are targeted to different subcellular compartments. Two endogenous biosynthetic genes (c2 and a1, encoding chalcone synthase and flavanone/dihydroflavonol reductase, respectively) are independently activated by ectopic expression of either P or C1/R, and there is a dose-response relationship between the transcript level of P and the degree to which c2 or a1 is expressed. Our results support a simple model showing how the gene encoding P may act as a quantitative trait locus controlling insecticidal C-glycosyl flavone level in maize silks, and they suggest how p1 might confer a selective advantage against insect predation in maize.

Journal Article↗

PubMed2005

Modeling Lactococcus lactis using a genome-scale flux model.

BACKGROUND: Genome-scale flux models are useful tools to represent and analyze microbial metabolism. In this work we reconstructed the metabolic network of the lactic acid bacteria Lactococcus lactis and developed a genome-scale flux model able to simulate and analyze network capabilities and whole-cell function under aerobic and anaerobic continuous cultures. Flux balance analysis (FBA) and minimization of metabolic adjustment (MOMA) were used as modeling frameworks. RESULTS: The metabolic network was reconstructed using the annotated genome sequence from L. lactis ssp. lactis IL1403 together with physiological and biochemical information. The established network comprised a total of 621 reactions and 509 metabolites, representing the overall metabolism of L. lactis. Experimental data reported in the literature was used to fit the model to phenotypic observations. Regulatory constraints had to be included to simulate certain metabolic features, such as the shift from homo to heterolactic fermentation. A minimal medium for in silico growth was identified, indicating the requirement of four amino acids in addition to a sugar. Remarkably, de novo biosynthesis of four other amino acids was observed even when all amino acids were supplied, which is in good agreement with experimental observations. Additionally, enhanced metabolic engineering strategies for improved diacetyl producing strains were designed. CONCLUSION: The L. lactis metabolic network can now be used for a better understanding of lactococcal metabolic capabilities and potential, for the design of enhanced metabolic engineering strategies and for integration with other types of 'omic' data, to assist in finding new information on cellular organization and function.

Bacteria, Anaerobic↗

PubMed2004

A genome-wide transcription analysis of a fungal riboflavin overproducer.

The production of many fine chemicals such as vitamins and amino acids is carried out in bioreactors using microorganisms. Usually, these strains are developed from wild-type organisms by classical mutation and selection. After several generations of strain improvement, no further enhancement can be achieved. Therefore, metabolic engineering (ME) is a rational approach to optimise such producer organisms beyond this point, or for starting all over from the beginning. Metabolic Engineering involves detailed analysis of the organism's metabolic and genetic properties, leading to the identification of new target genes. The fungal riboflavin overproducer Ashbya gossypii converts vegetable oil to vitamin B2 in a "one-step reaction". The productivity and selectivity of this microorganism have been optimised significantly over the years, first following a classical approach and now a rational one. The improvement is based on our understanding of vitamin B2 metabolism. We have been able to selectively enhance the pathways that are necessary for the formation of riboflavin and to inhibit those leading to unwanted side products. New targets for further improvements of this process have been found using a genome-wide transcript expression analysis; namely massive parallel signature sequencing (MPSS). With this analysis even completely unknown genes can be used for strain improvement.

Bioreactors↗