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PubMed2002

Engineering deoxynivalenol metabolism in wheat through the expression of a fungal trichothecene acetyltransferase gene.

Fusarium head blight occurs in cereals throughout the world and is especially important in humid growing regions. Fusarium head blight (FHB) has re-emerged as a major disease of wheat and barley in the U.S. and Canada since 1993. The primary causal agents of FHB, Fusarium graminearum and Fusarium culmorum, can produce deoxynivalenol (DON), a trichothecene mycotoxin that enhances disease severity and poses a health hazard to humans and monogastric animals. To reduce the effects of DON on wheat, we have introduced FsTRI101, a Fusarium sporotrichioides gene formerly known as TriR, into the regenerable cultivar Bobwhite. TRI101 encodes an enzyme that transfers an acetyl moiety to the C3 hydroxyl group of trichothecenes. Four different transgenic plants carrying the FsTRI101 gene were identified. Although expression levels varied among the four lines, all of them accumulated FsTRI101 transcripts in endosperm and glume. TRI101-encoded acetyltransferase activity was detected in endosperm extracts of a single plant that accumulated FsTRI101 mRNA. Greenhouse resistance tests indicated that the accumulation of FsTRI101-encoded acetyltransferase in this plant confers partial protection against the spread of F. graminearum in inoculated wheat heads (spikes).

Acetyltransferases↗

PubMed2003

Antisense-mediated down-regulation of putrescine N-methyltransferase activity in transgenic Nicotiana tabacum L. can lead to elevated levels of anatabine at the expense of nicotine.

Nicotiana tabacum L. produces a number of pyridine alkaloids, with nicotine representing the major component and anatabine comprising most of the remainder of the alkaloid fraction. An antisense approach was used here to down-regulate activity of the important enzyme putrescine N-methyltransferase (PMT) in transformed roots of this species to determine effects upon alkaloid metabolism. Transformed root lines were produced that contained markedly reduced PMT activity, with a concomitant reduction in nicotine content compared to controls. No negative effects upon growth were observed. Several antisense-PMT transformed root lines, and also leaf tissues of regenerated transformed plants, showed a substantial increase in anatabine content relative to controls. Northern hybridization experiments indicated that the antisense-PMT manipulation had little or no effect upon the transcript levels of other genes encoding enzymes involved in alkaloid metabolism, including quinolinate acid phosphoribosyltransferase (QPT). The latter enzyme plays a key role in regulating the synthesis of nicotinic acid which supplies the pyridine ring necessary for both nicotine and anatabine synthesis. We suggest that elevated anatabine levels in antisense-PMT lines are a direct consequence of a relative oversupply of nicotinic acid which, in the absence of adequate levels of 1-methyl-delta(1)-pyrrolinium cation (the ultimate product of PMT activity), is used to synthesise anatabine directly. As is discussed, no naturally occurring species or varieties of Nicotiana are known that typically produce high levels of anatabine in root or leaf tissues, meaning that the antisense PMT transgenics produced in this study have no natural counterpart. These experiments thus represent an example of metabolic engineering of plant pyridine metabolism, via antisense down-regulation of gene expression in a contributing pathway leading to secondary metabolite biosynthesis.

Alkaloids↗

PubMed2006

Metabolic pathway engineering for complex polyketide biosynthesis in Saccharomyces cerevisiae.

Polyketides are a diverse group of natural products with significance in human and veterinary medicine. Because polyketides are structurally complex molecules and fermentation is the most commercially viable route of production, a generic heterologous host system for high-level polyketide production is desirable. Saccharomyces cerevisiae has been shown to be an excellent production host for a simple polyketide, yielding 1.7 g of 6-methylsalicylic acid per liter of culture in un-optimized shake-flask fermentations. However, a barrier to the heterologous production of more complex 'modular' polyketides in S. cerevisiae is the lack of required polyketide precursor pathways. In this work, we describe the introduction into S. cerevisiae of pathways for the production of methylmalonyl-coenzyme A (CoA), a precursor for complex polyketides, by both propionyl-CoA-dependent and propionyl-CoA-independent routes. Furthermore, we demonstrate that the methylmalonyl-CoA produced in the engineered yeast strains is used in vivo for the production of a polyketide product, a triketide lactone.

Acyl Coenzyme A↗

PubMed2005

New tool for metabolic pathway engineering in Escherichia coli: one-step method to modulate expression of chromosomal genes.

A simple and highly efficient method was developed to produce a library of Escherichia coli clones that express a particular chromosomal gene at a wide range of expression levels. The basic strategy was to replace all or part of the upstream region of a coding sequence containing the elements involved in its expression (promoter, operator, gene coding for a regulator, ribosome binding site, and start codon) with a PCR-generated library of expression cassettes.

Base Sequence↗

PubMed2004

Mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ionic regulation and developmental competence.

Mitochondria are the most abundant organelles in the mammalian oocyte and early embryo. While their role in ATP production has long been known, only recently has their contribution to oocyte and embryo competence been investigated in the human. This review considers whether such factors as mitochondrial complement size, mitochondrial DNA copy numbers and defects, levels of respiration, and stage-specific spatial distribution, influence the developmental normality and viability of human oocytes and preimplantation-stage embryos. The finding that mitochondrial polarity can differ within and between oocytes and embryos and that these organelles may participate in the regulation of intracellular Ca(2+)homeostasis are discussed in the context of how focal domains of differential respiration and intracellular-free Ca(2+)regulation may arise in early development and what functional implications this may have for preimplantation embryogenesis and developmental competence after implantation.

Blastocyst↗

PubMed2024

Strategies in engineering sustainable biochemical synthesis through microbial systems.

Growing environmental concerns and the urgency to address climate change have increased demand for the development of sustainable alternatives to fossil-derived fuels and chemicals. Microbial systems, possessing inherent biosynthetic capabilities, present a promising approach for achieving this goal. This review discusses the coupling of systems and synthetic biology to enable the elucidation and manipulation of microbial phenotypes for the production of chemicals that can substitute for petroleum-derived counterparts and contribute to advancing green biotechnology. The integration of artificial intelligence with metabolic engineering to facilitate precise and data-driven design of biosynthetic pathways is also discussed, along with the identification of current limitations and proposition of strategies for optimizing biosystems, thereby propelling the field of chemical biology towards sustainable chemical production.

Metabolic Engineering↗

PubMed2000

Engineering a living cell to desired metabolite concentrations and fluxes: pathways with multifunctional enzymes.

With molecular genetics enabling modulation of the concentrations of cellular enzymes, metabolic engineering becomes limited by the question of which modulations of the enzyme concentrations are required to bring about a desired pattern of cellular metabolism. In an earlier paper (Kholodenko et al. (1998). Biotechnol. Bioeng. 59, 239-247) we derived a method to determine the required modulations. This method, however, cannot be immediately applied to cellular pathways with enzymes catalyzing more than one step in metabolism (multifunctional enzymes). In the present paper we show to which extent the presence of multifunctional enzymes limits biotechological ambitions, which one might otherwise pursue in vain. In particular, it is impossible to change the concentration of a single intermediate and leave the rest of metabolism unperturbed if that intermediate interacts directly with a multifunctional enzyme. The analytical machinery of Metabolic Control Analysis is used to relate the desired and ensuing changes in the metabolic pattern. An explicit solution to this problem of engineering metabolism is then given in the form of a single matrix equation.

Animals↗

PubMed2003

[Co-expressions of phosphoenolpyruvate synthetase A (ppsA) and transketolase A (tktA) genes of Escherichia coli].

Metabolic engineering is the analysis of metabolic pathway and designing rational genetic modification to optimize cellular properties by using principle of molecular biology. Aromatic metabolites such as tryptophan, phenylalanine, and tyrosine are essential amino acids for human and animals. In addition, phenylalanine is used in aspartame production. Escherichia coli and many other microoganism synthesize aromatic amino acids through the condensation reaction between phospho-enolpyruvate (PEP) and erythrose-4-phosphate(E4P) to form 3-deoxy-D-arabinoheptulosonate 7-phosphate(DAHP). But many enzymes compete for intracellular PEP, especially the phosphotransferase system which is responsible for glucose transport in E. coli. This system uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. To channel more carbon flux into the aromatic pathway, one has to overcome pathways competing for PEP. ppsA and tktA are the key genes in central metabolism of aromatic amino acids biosynthesis. ppsA encoding phosphoenolpyrucate synthetase A (PpsA) which catalyzes pyruvate into PEP; tktA encoding transketolase A which plays a major role in erythrose-4-phosphate (E4P) production of pentose pathway. We amplified ppsA and tktA from E. coli K-12 by PCR and constructed recombinant plasmids of them in pBV220 vector containing P(R)P(L) promoter. Because of each gene carrying P(L) promoter, four productions of ligation were obtained. The monoclonal host containing recombinant plasmids was routinely grown in Luria-Bertani (LB) medium added Ampicillin at 37 degrees C overnight, and then inoculated in LB (Apr) medium by 3%-5% in flasks on a rotary shaker at 30 degres C, induced at 42 degrees C for 4.5 hours when OD600 = 0.4, cells were obtained by centrifugation at 10,000 r/min at 4 degrees C. The results of SDS-PAGE demonstrated that the bands at 84kD and 73kD were more intensive than the same ones of the controls. The specific activity of PpsA in crude extracts was increased by 10.8-fold, and TktA, by 3.9-fold. When both genes were co-expressed in E. coli, the activity of PpsA varied from 2.1-9.1 fold comparing to control, but the activity of TktA was relatively stable(3.9-4.5 fold). Whatever the two genes were expressed respectively or cooperatively, both could promote the production of DAHP, the first intermediate of the common aromatic pathway, but co-expression was more effective on forming DAHP. The results demonstrate that co-expression of ppsA and tktA can improve the production of DAHP to near theoretical yield. This report details a different strategy based on co-expression of two genes in one vector in vivo to release the burden and paves the way for construction of genetic engineering bacteria for further research.

Electrophoresis, Polyacrylamide Gel↗

PubMed2000

Biocatalysis for pharmaceuticals--status and prospects for a key technology.

In reviewing how biocatalysis can be applied to improve chiral synthesis for pharmaceuticals it becomes clear that there will be many opportunities using a simple enzyme system but that many of the more useful applications will require the whole cell because of the requirement for cofactors. An assessment is made of the opportunities to apply metabolic engineering to construct de novo metabolic pathways for the biosynthesis of useful advanced intermediates and a conceptual example is provided for the biosynthesis of cis-aminoindanol. We predict that in the future novel pathways will be assembled for a one-step biosynthesis of many semisynthetic natural products.

Animals↗

PubMed2002

Metabolic flux analysis using mass spectrometry.

Detailed knowledge on carbon flux distributions is crucial for the understanding and targeted optimization of cellular systems. Analytical methods to identify the topology of metabolic networks and to quantify fluxes through its different pathways are therefore in the core of metabolic engineering. An elegant approach for metabolic flux analysis is provided by tracer experiments. In such studies tracer substrates with stable isotopes such as 13C are applied and the labeling pattern of metabolites is subsequently measured. Detailed flux distributions can be obtained by a combination of tracer experiments and stoichiometric balancing. In recent years, mass spectrometry (MS) has emerged as an interesting method for labeling measurements in metabolic flux analysis and provided valuable insights into the cellular metabolism. The present review provides an overview on current experimental and modeling tools for metabolic flux analysis by MS. The application of MS for flux analysis is illustrated by examples from the literature for various biological systems, including bacteria, fungi, tissue cultures and in vivo studies in humans.

Carbon Isotopes↗

PubMed2003

Advances in flux balance analysis.

Biology is going through a paradigm shift from reductionist to holistic, systems-based approaches. The complete genome sequence for a number of organisms is available and the analysis of genome sequence data is proving very useful. Thus, genome sequencing projects and bioinformatic analyses are leading to a complete 'parts catalog' of the molecular components in many organisms. The next challenge will be to reconstruct and simulate overall cellular functions based on the extensive reductionist information. Recent advances have been made in the area of flux balance analysis, a mathematical modeling approach often utilized by metabolic engineers to quantitatively simulate microbial metabolism.

Computational Biology↗

PubMed2005

Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA.

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo.

Aryl Hydrocarbon Hydroxylases↗

PubMed2003

Practical implications of lactate and pyruvate metabolism by lactic acid bacteria in food and beverage fermentations.

This article reviews the metabolism of pyruvate and lactate by lactic acid bacteria (LAB) involved in food and beverage fermentations with an emphasis on practical implications. First, the formation of pyruvate and lactate from a range of substrates, including carbohydrates, organic acids and amino acids, is briefly described. The catabolism of pyruvate and lactate by LAB is then reviewed. This is followed by a discussion of lactate degradation and racemisation by LAB from specific fermented foods and beverages. Finally, the impact of environmental factors and metabolic engineering on pyruvate and lactate metabolism by LAB is evaluated with regard to practical significance.

Beverages↗

PubMed2002

Brassica napus soluble epoxide hydrolase (BNSEH1).

Epoxide hydrolase (EC 3.3.2.3) in plants is involved in the metabolism of epoxy fatty acids and in mediating defence responses. We report the cloning of a full-length epoxide hydrolase cDNA (BNSEH1) from oilseed rape (Brassica napus) obtained by screening of a cDNA library prepared from methyl jasmonate induced leaf tissue, and the 5'-RACE technique. The cDNA encodes a soluble protein containing 318 amino acid residues. The identity on the protein level is 85% to an Arabidopsis soluble epoxide hydrolase (sEH) and 50-60% to sEHs cloned from other plants. A 5 x His tag was added to the N-terminus of the BNSEH1 and the construct was over-expressed in the yeast Pichia pastoris. The recombinant protein was recovered at high levels after Ni-agarose chromatography of lysed cell extracts, had a molecular mass of 37 kDa on SDS/PAGE and cross-reacted on Western blots with antibodies raised to a sEH from Arabidopsis thaliana. BNSEH1 was shown to be a monomer by gel filtration analysis. The activity was low towards cis-stilbene oxide but much higher using trans-stilbene oxide as substrate with Vmax of 0.47 micro mol.min.mg-1, Km of 11 micro m and kcat of 0.3 s-1. The optimum temperature of the recombinant enzyme was 55 degrees C and the optimum pH 6-7 for trans-stilbene oxide hydrolysis. The isolation of BNSEH1 will facilitate metabolic engineering of epoxy fatty acid metabolism for functional studies of resistance and seed oil modification in this important oilcrop.

Amino Acid Sequence↗

PubMed2025

Engineering recombination machinery facilitates the construction of yeast cell factories.

Advances in genome editing have been promoted by programmable nucleases like CRISPR-Cas9, which triggers endogenous DNA repair mechanisms by inducing double-strand break (DSB). Cellular responses to DSBs are governed by competing repair pathways: error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination (HR). This review systematically compares the molecular mechanisms and key regulators of NHEJ and HR, with a focus on recent breakthroughs in recombination engineering in non-conventional yeasts. These advances address challenges in precise genome editing, enabling robust metabolic engineering of yeast cell factories for sustainable bioproduction.

Metabolic Engineering↗

PubMed2000

NMR adventures in the metabolic labyrinth within plants.

The era of metabolic engineering has begun, but there is only limited knowledge about metabolic fluxes and how they are regulated in plants. Particular challenges are the non-linearities between enzyme abundances, metabolite concentrations and metabolic fluxes, and the existence of metabolic networks that provide multiple routes between many important metabolites. NMR offers the means to distinguish and quantitate the fluxes along different routes to key metabolites. NMR can therefore help us understand and resolve the apparent paradox of, on the one hand, great metabolic flexibility evident in the natural responses of plants and, on the other hand, the unpredictable changes in metabolism reported in genetically engineered plants.

Carbohydrate Metabolism↗

PubMed2002

Engineering secondary metabolite production in plants.

Recent achievements have been made in the metabolic engineering of plant secondary metabolism. Various pathways have been altered using genes encoding biosynthetic enzymes or genes encoding regulatory proteins. In addition, antisense genes have been used to block competitive pathways, thereby increasing the flux towards the desired secondary metabolites.

Enzymes↗

PubMed2026

A Comprehensive Review on the Biosynthesis of Tropane Alkaloids.

Tropane alkaloids (TA) constitute a class of plant specialized metabolites with important pharmaceutical applications, including the anticholinergic agents hyoscyamine and scopolamine and the local anesthetic cocaine. Over the past decade, advances in genomics, structural biology, and synthetic biology have substantially revised our understanding of TA biosynthesis, leading to the identification of numerous key biosynthetic enzymes and evolutionary mechanisms. This review comprehensively summarizes current knowledge of TA biosynthesis from precursor formation to structurally diverse end products. We describe the pathway from putrescine to tropinone, the stereoselective metabolic branching mediated by Tropinone Reductases, and the downstream biosynthesis of medicinal tropane alkaloids, calystegines, and cocaine. Particular emphasis is placed on recent discoveries concerning catalytic mechanisms, structural determinants of substrate specificity, metabolic compartmentalization, and the convergent evolution of TA biosynthesis in Solanaceae and Erythroxylaceae. We further integrate advances in genomics, evolutionary biology, and metabolic engineering to highlight emerging strategies for microbial production and pathway redesign. By providing a comprehensive synthesis of recent progress and critical perspectives on unresolved questions, this review offers an updated framework for understanding TA biosynthesis and supports future research in plant specialized metabolism, synthetic biology, and natural product engineering.

Tropanes↗