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Results for “biosynthetic pathway engineering”

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PubMed2001

Genetic manipulation of carotenoid biosynthesis: strategies, problems and achievements.

Carotenoids, some of which are provitamin A, have a range of diverse biological functions and actions, especially in relation to human health. For example, carotenoids are known to be crucial for normal vision and have been associated with reducing the risk of several degenerative diseases including cancer. The putative advantage of modifying and engineering the carotenoid biosynthetic pathways is obvious: to provide sources for the isolation of desired carotenoids or to generate food plants with increased carotenoid content. This article reviews the studies of carotenoid production in heterologous microorganisms and the engineering of crop plants using manipulated carotenoid biosynthesis.

Candida↗

PubMed2003

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with high molar fractions of 3-hydroxyvalerate by a threonine-overproducing mutant of Alcaligenes sp. SH-69.

A threonine overproducing mutant of Alcaligenes sp. SH-69 was isolated and its ability to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), was investigated. The 3HV fraction in poly(3HB-co-3HV) produced from glucose as the sole carbon source exceeded 22 mol%, which is approximately six times higher than that achieved by the wild type under the same culture conditions. Furthermore, the addition of a relatively low concentration (10 mM) of propionic acid, valeric acid or levulinic acid to the glucose medium greatly increased the molar fraction of 3HV in the copolyester, to 38-77 mol%. The results suggest that metabolic engineering of the biosynthetic pathways supplying polyhydroxyalkanoate monomers, such as the threonine biosynthetic pathway, can lead to new poly(3HB-co-3HV)-producing strains.

Alcaligenes↗

PubMed2001

Production of 6-methylsalicylic acid by expression of a fungal polyketide synthase activates disease resistance in tobacco.

Salicylic acid (SA) has been shown to act as a signal molecule that is produced by many plants subsequent to the recognition of potentially pathogenic microbes. Increases in levels of SA often trigger the activation of plant defenses and can result in increased resistance to subsequent challenge by pathogens. We observed that the polyketide 6-methylsalicylic acid (6-MeSA), a compound that apparently is not endogenous to tobacco, can mimic SA. Tobacco leaves treated with 6-MeSA show enhanced accumulation of the pathogenesis-related (PR) proteins PR1, beta-1,3-glucanase, and chitinase and also develop increased resistance to tobacco mosaic virus. We transformed tobacco with 6msas, the 6-methylsalicylic acid synthase (6MSAS) gene from Penicillium patulum, to generate plants that constitutively accumulate 6-MeSA. Analysis of primary transformants and the first generation progeny of 6MSAS tobacco revealed that plants can be engineered to accumulate significant amounts of 6-MeSA as a conjugate. Levels of total 6-MeSA increased with plant age. Increased 6-MeSA accumulation correlated with increased levels of PR1 and chitinase proteins and resulted in enhanced resistance of NN genotype 6MSAS tobacco to tobacco mosaic virus. Our results demonstrate that a multistep biosynthetic pathway can be engineered into plants using a single fungal polyketide synthase gene. The functional expression of 6msas can be used to activate disease resistance pathways that normally are induced by SA.

Chitinases↗

PubMed2026

Identification and Catalytic Optimization of Pinene Oxidases in Paeoniflorin Biosynthetic Pathway.

Paeoniflorin is a pharmacologically important cage-like monoterpene glycoside characteristic of Paeonia plants, yet its biosynthetic pathway has remained largely unresolved, hindering sustainable production. Here, we confirmed that paeoniflorin biosynthesis originates from α-pinene and identified three novel cytochrome P450 enzymes that catalyze pinene oxidation. CYP71AN126 catalyzes the hydroxylation of α-pinene at positions C4 and C10, followed by further oxidation of the alcohol to a ketone at C4, whereas CYP76A225/226 exclusively catalyze C10 hydroxylation. Virus-induced gene silencing (VIGS) assays demonstrated that silencing CYP71AN126, but not CYP76A225 and CYP76A226, significantly reduced the paeoniflorin content, indicating that C4 hydroxylation plays an important role in paeoniflorin biosynthesis, whereas C10 hydroxylation is not. Through the analysis of natural sequence and activity divergence among CYP71AN126 and CYP76A225/226, combined with protein structure prediction and site-directed mutagenesis, we identified L493 as a critical residue involved in regulating catalytic site specificity and substrate specificity of CYP71AN126. Mutation of L493 reduced or eliminated the formation of undesired C10 hydroxylation side-product and enhanced substrate specificity. These findings establish C4 oxidation of α-pinene as the critical committed step in paeoniflorin biosynthesis. Our study lays a foundation for elucidating the complete biosynthetic pathway of paeoniflorin in Paeonia and provides a target for enzyme engineering of CYP71AN126 aimed at the efficient production of paeoniflorin via synthetic biology approaches.

Paeonia genus↗

PubMed2000

Forest tree biotechnology.

The past year has seen the fruits of biotechnological manipulation of forest trees approach commercial application. Advances in somatic embryogenesis have brought mass clonal propagation of the top commercial trees closer to reality, and efficient gene transfer systems have been developed for a number of conifers and hardwoods. Radical alterations in the quantity and quality of lignin in wood have been shown to be possible in softwoods and hardwoods through identification of naturally occurring mutants, as well as by engineering the lignin biosynthetic pathway with transgenes. The potential environmental and social impacts of the release of transgenic trees have become an increasingly contentious issue that will require more attention if we are to use these technologies to their full advantage.

Biotechnology↗

PubMed2003

Total biosynthesis of hydrocortisone from a simple carbon source in yeast.

We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.

Animals↗

PubMed1998

The biosynthetic pathway of vitamin C in higher plants.

Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown-one of the few primary plant metabolic pathways for which this is the case. L-ascorbate is abundant in plants (found at concentrations of 1-5 mM in leaves and 25 mM in chloroplasts) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose an ascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.

Arabidopsis↗

PubMed2005

Bioactive C-glycosides from bacterial secondary metabolism.

C-Glycosides are commonly regarded as unusual structures, but they are far more prevalent among natural products than is imagined. This review discusses the C-glycosidic compounds produced by various bacteria, particularly the "biosynthetically talented" Streptomyces. The major structure types are presented, along with brief descriptions of the known biological and pharmacological properties of the compounds. Recent work has uncovered the genetic basis for the biosynthesis of several bacterial C-glycosides, and emphasis is placed on those cases where it has been possible to identify (at least provisionally) the C-glycosyltransferase in the pathway. Prospects for biosynthetic engineering, combinatorial biosynthesis, or glycorandomization in C-glycosidic natural products are briefly discussed.

Bacteria↗

PubMed2001

Biosynthetic pathway of cephabacins in Lysobacter lactamgenus: molecular and biochemical characterization of the upstream region of the gene clusters for engineering of novel antibiotics.

The cephabacins, one of the beta-lactam antibiotics, are produced by Lysobacter lactamgenus. The previous studies the cephabacin biosynthesis were limited to a gene cluster that encodes the gene products responsible for the biosynthesis of the cephem nucleus. The long-term goal of this research is to elucidate the metabolic diversity and biosynthetic pathway of cephabacins and to design and/or discover new pharmacologically active compounds by engineering the cephabacin biosynthetic pathway in L. lactamgenus. In this study, we have cloned and sequenced a 24-kb fragment of a DNA locus upstream of the previously reported but incomplete putative ORF9 of L. lactamgenus. This contains three putative ORFs (the complete ORF9, ORF10, and ORF11) transcribed in the same direction and one putative ORF (ORF12) in the opposite direction. The isolated DNA locus extends the previously cloned part of the DNA locus containing the genes responsible for biosynthesis of the cephem nucleus up to 45 kb. The 42-kb fragment of the 45-kb gene cluster is located between a potential TATA box just upstream of the ORF11 and a termination loop just downstream of the previously reported bla gene. The complete ORF9 contains three nonribosomal peptide synthetase (NRPS) modules and one polyketide synthase (PKS) module and the ORF11 contains one NRPS module. The complete ORF9 also contains a putative thioesterase domain at the C-terminal end. We predicted the amino acid specificity of the four NRPSs by generating specificity binding pockets and expressed one of the NRPSs to confirm the amino acid specificity. The adenylation domain of the NRPS1, which is the last module of the NRPSs, showed significant amino acid specificity for L-arginine. These findings are in perfect agreement with the composition that was expected for the structure of cephabacins which contain an acetate residue, an L-arginine, and one to three L-alanines at the C-3' position of the cephem nucleus of cephabacins. The ORF10, encoding a putative ABC transporter which might be involved in conferring resistance against cephabacins, was identified between the complete ORF9 and the ORF11. Therefore, the complete ORF9, ORF10, ORF11 reported here and the other genes previously reported constitute an operon for the biosynthesis of cephabacins in L. lactamgenus. Based on our results, the biosynthetic pathways of acetate and elongated peptide moieties and a mechanism by which cephabacins are assembled by connecting the peptide moiety synthesized by the gene products of the complete ORF9 and the ORF11 to the C-3' position of the cephem nucleus synthesized by the gene products of pcbAB, pcbC, cefE, cefF, and cefD have been elucidated.

Amino Acid Sequence↗

PubMed1994

Eubacteria show their true colors: genetics of carotenoid pigment biosynthesis from microbes to plants.

The opportunities to understand eubacterial carotenoid biosynthesis and apply the lessons learned in this field to eukaryotes have improved dramatically in the last several years. On the other hand, many questions remain. Although the pigments illustrated in Fig. 2 represent only a small fraction of the carotenoids found in nature, the characterization of eubacterial genes required for their biosynthesis has not yet been completed. Identifying those eukaryotic carotenoid biosynthetic mutants, genes, and enzymes that have no eubacterial counterparts will also prove essential for a full description of the biochemical pathways (81). Eubacterial crt gene regulation has not been studied in detail, with the notable exceptions of M. xanthus and R. capsulatus (5, 33, 39, 45, 46, 84). Determination of the rate-limiting reaction(s) in carotenoid biosynthesis has thus far yielded species-specific results (12, 27, 47, 69), and the mechanisms of many of the biochemical conversions remain obscure. Predicted characteristics of some carotenoid biosynthesis gene products await confirmation by studying the purified proteins. Despite these challenges, (over)expression of eubacterial or eukaryotic carotenoid genes in heterologous hosts has already created exciting possibilities for the directed manipulation of carotenoid levels and content. Such efforts could, for example, enhance the nutritional value of crop plants or yield microbial production of novel and desirable pigments. In the future, the functional compatibility of enzymes from different organisms will form a central theme in the genetic engineering of carotenoid pigment biosynthetic pathways.

Bacteria↗

PubMed2006

Role of energetic coenzyme pools in the production of L-carnitine by Escherichia coli.

The aim of this work was to understand the steps controlling the biotransformation of trimethylammonium compounds into L(-)-carnitine by Escherichia coli. The high-cell density reactor steady-state levels of carbon source (glycerol), biotransformation substrate (crotonobetaine), acetate (anaerobiosis product) and fumarate (as an electron acceptor) were pulsed by increasing them fivefold. Following the pulse, the evolution of the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration), in the synthesis of acetyl-CoA (ACS: acetyl-CoA synthetase and PTA: ATP: acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (ICDH: isocitrate dehydrogenase) and glyoxylate (ICL: isocitrate lyase) cycles was monitored. In addition, the levels of carnitine, the cell ATP content and the NADH/NAD(+) ratio were measured in order to assess the importance and participation of these energetic coenzymes in the catabolic system. The results provided an experimental demonstration of the important role of the glyoxylate shunt during biotransformation and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the results obtained for the NADH/NAD(+) pool indicated that it is correlated with the biotransformation process at the NAD(+) regeneration and ATP production level in anaerobiosis. More importantly, a linear correlation between the NADH/NAD(+) ratio and the levels of the ICDH and ICL (carbon and electron flows) and the PTA and ACS (acetate and ATP production and acetyl-CoA synthesis) activity levels was assessed. The main metabolic pathway operating during cell metabolic perturbation with a pulse of glycerol and acetate in the high-cell density membrane reactor was that related to ICDH and ICL, both regulating the carbon metabolism, together with PTA and ACS enzymes (regulating ATP production).

Acetates↗

PubMed2004

Para-position derivatives of fungal anthelmintic cyclodepsipeptides engineered with Streptomyces venezuelae antibiotic biosynthetic genes.

PF1022A, a cyclooctadepsipeptide possessing strong anthelmintic properties and produced by the filamentous fungus Rosellinia sp. PF1022, consists of four alternating residues of N-methyl-L-leucine and four residues of D-lactate or D-phenyllactate. PF1022A derivatives obtained through modification of their benzene ring at the para-position with nitro or amino groups act as valuable starting materials for the synthesis of compounds with improved anthelmintic activities. Here we describe the production of such derivatives by fermentation through metabolic engineering of the PF1022A biosynthetic pathway in Rosellinia sp. PF1022. Three genes cloned from Streptomyces venezuelae, and required for the biosynthesis of p-aminophenylpyruvate from chorismate in the chloramphenicol biosynthetic pathway, were expressed in a chorismate mutase-deficient strain derived from Rosellinia sp. PF1022. Liquid chromatography-mass spectrometry and NMR analyses confirmed that this approach facilitated the production of PF1022A derivatives specifically modified at the para-position. This fermentation method is environmentally safe and can be used for the industrial scale production of PF1022A derivatives.

Animals↗

PubMed2000

Rice transformation for crop improvement and functional genomics.

Although several japonica and some indica varieties of rice have already been transformed, there is significant scope for improvement in the technology for transformation of economically important indica varieties. Successful transformation of rice employing Agrobacterium and recent advances in direct gene transfer by biolistics, evidenced by transfer of multiple genes, have removed some of the serious impediments in the area of gene engineering. The transfer of genes for nutritionally important biosynthetic pathway has provided many opportunities for performing metabolic engineering. Other useful genes for resistance against pests, diseases and abiotic stresses have also been transferred to rice. But the limited knowledge about important target genes requires rapid progress in the field of functional genomics. Transgenic rice system can be applied to isolate new genes, promoters, and enhancers and their functions could be unravelled. The combination of novel regulatory systems for targeted expression and useful new genes should pave the way for improvement of rice and other cereals.

Journal Article↗

PubMed2002

The albonoursin gene Cluster of S noursei biosynthesis of diketopiperazine metabolites independent of nonribosomal peptide synthetases.

Albonoursin [cyclo(deltaPhe-DeltaLeu)], an antibacterial peptide produced by Streptomyces noursei, is one of the simplest representatives of the large diketopiperazine (DKP) family. Formation of alpha,beta unsaturations was previously shown to occur on cyclo(L-Phe-L-Leu), catalyzed by the cyclic dipeptide oxidase (CDO). We used CDO peptide sequence information to isolate a 3.8 kb S. noursei DNA fragment that directs albonoursin biosynthesis in Streptomyces lividans. This fragment encompasses four complete genes: albA and albB, necessary for CDO activity; albC, sufficient for cyclic dipeptide precursor formation, although displaying no similarity to non ribosomal peptide synthetase (NRPS) genes; and albD, encoding a putative membrane protein. This first isolated DKP biosynthetic gene cluster should help to elucidate the mechanism of DKP formation, totally independent of NRPS, and to characterize novel DKP biosynthetic pathways that could be engineered to increase the molecular diversity of DKP derivatives.

Amino Acid Sequence↗

PubMed2001

Biosynthesis of sialylated lipooligosaccharides in Haemophilus ducreyi is dependent on exogenous sialic acid and not mannosamine. Incorporation studies using N-acylmannosamine analogues, N-glycolylneuraminic acid, and 13C-labeled N-acetylneuraminic acid.

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted disease. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. The vast majority of H. ducreyi strains contain high levels of sialic acid (N-acetylneuraminic acid, NeuAc) in their LOS. Here we investigate the biosynthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of N-acylmannosamine and sialic acid analogues. Incorporation of sialosides into LOS was assessed by matrix-assisted laser desorption and electrospray ionization mass spectrometry. A Fourier transform ion cyclotron resonance mass spectrometer provided accurate mass measurements, and a quadrupole time-of-flight instrument was used to obtain characteristic fragment ions and partial carbohydrate sequences. Exogenously supplied N-acetylmannosamine analogues were not converted to LOS-associated sialosides at a detectable level. In contrast, exogenous (13)C-labeled N-acetylneuraminic acid ([(13)C]NeuAc) and N-glycolylneuraminic acid (NeuGc) were efficiently incorporated into LOS in a dose-dependent fashion. Moreover, approximately 1.3 microM total exogenous sialic acid was sufficient to obtain about 50% of the maximum production of sialic acid-containing glycoforms observed under in vitro growth conditions. Together, these data suggest that the expressed levels of sialylated LOS glycoforms observed in H. ducreyi are in large part controlled by the exogenous concentrations of sialic acid and at levels one might expect in vivo. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used.

Biological Transport↗

PubMed2005

Natural products to drugs: daptomycin and related lipopeptide antibiotics.

Daptomycin (Cubicin) is a lipopeptide antibiotic approved in the USA in 2003 for the treatment of skin and skin structure infections caused by Gram-positive pathogens. It is a member of the 10-membered cyclic lipopeptide family of antibiotics that includes A54145, calcium-dependent antibiotic (CDA), amphomycin, friulimicin, laspartomycin, and others. This review highlights research on this class of antibiotics from 1953 to 2005, focusing on more recent studies with particular emphasis on the interplay between structural features and antibacterial activities; chemical modifications to improve activity; the genetic organization and biosynthesis of lipopeptides; and the genetic engineering of the daptomycin biosynthetic pathway to produce novel derivatives for further chemical modification to develop candidates for clinical evaluation.

Anti-Bacterial Agents↗

PubMed2004

Accessing natural products by combinatorial biosynthesis.

Enhancement and selective production of the protein phosphatase IIa inhibitor phoslactomycin (PLM) B by rational engineering of the PLM biosynthetic pathway highlights the effectiveness of combinatorial biosynthesis as a promising way to prepare complex natural products and their analogs.

Combinatorial Chemistry Techniques↗

PubMed1998

Containment of herbicide resistance through genetic engineering of the chloroplast genome.

Glyphosate is a potent herbicide. It works by competitive inhibition of the enzyme 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS), which catalyzes an essential step in the aromatic amino acid biosynthetic pathway. We report the genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector. Southern blot analysis confirms stable integration of the EPSPS gene into all of the chloroplast genomes (5000-10,000 copies per cell) of transgenic plants. Seeds obtained after the first self-cross of transgenic plants germinated and grew normally in the presence of the selectable marker, whereas the control seedlings were bleached. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations of glyphosate. Chloroplast transformation provides containment of foreign genes because plastid transgenes are not transmitted by pollen. The escape of foreign genes via pollen is a serious environmental concern in nuclear transgenic plants because of the high rates of gene flow from crops to wild weedy relatives.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗