Carotenoid biosynthesis and the evolution of carotenogenesis genes in rust fungi

Diseases caused by rust fungi pose a significant threat to global plant production. Although carotenoid pigments are produced in spores of nearly all rust species, the corresponding biosynthesis pathway(s) have not been investigated. Here, candidate genes for carotenoid biosynthesis in Puccinia graminis f. sp. tritici (Pgt) were identified, cloned and functionally complemented using specifically engineered strains of Escherichia coli. A part of the carotenoid biosynthesis pathway in rust fungi was elucidated, with only two genes, CrtYB and CrtI, catalysing the reactions from geranyl–geranyl diphosphate (GGPP) to γ-carotene. The CrtYB gene encodes a bi-functional lycopene cyclase/phytoene synthase, which catalyses the condensation of two GGPP into phytoene, as well as the cyclisation of the ψ-end of lycopene to form γ-carotene. The CrtI gene encodes a phytoene desaturase that carries out four successive desaturations of phytoene, through the intermediates phytofluene and neurosporene to lycopene. The evolution of carotenoid pigmentation in rust fungi, including Pgt, P. graminis avenae, P. graminis secalis (Pgs), P. graminis lolli, P. striiformis f. sp. tritici, P. striiformis f. sp. pseudohordei, P. striiformis f. sp. hordei, the “scabrum” rust (putative hybrids between Pgt and Pgs), P. triticina, and P. hordei, was investigated by phylogenetic analysis. Both CrtYB and CrtI were found to be closely related among rust fungi, other pathogenic fungi, and some aphids. Our results provide a springboard to increase the understanding of the physiological role(s) of carotenoid pigments in rust fungi, to better understand evolution within the Pucciniales, and to develop robust molecular diagnostics for rust fungi.     

Non-mevalonate isoprenoid biosynthesis: enzymes, genes and inhibitors

The essential steps of the novel non-mevalonate pathway of isopentenyl diphosphate and isoprenoid biosynthesis in plants are described. The first five enzymes and genes of this 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway are known. The herbicide fosmidomycin specifically blocks the second enzyme, the DOXP reductoisomerase. The DOXP/MEP pathway is also present in several pathogenic bacteria and the malaria parasite. Hence, all herbicides and inhibitors blocking this novel isoprenoid pathway in plants are also potential drugs against malaria and diseases caused by pathogenic bacteria.     

Microcystin biosynthesis in planktothrix: genes,evolution, and manipulation

Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases.     

Bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology

The discovery and chemical identification, in the 1920s, of the aliphatic polyester: poly(3-hydroxybutyrate), PHB, as a granular component in bacterial cells proceeded without any of the controversies which marked the recognition of macromolecules by Staudinger. Some thirty years after its discovery, PHB was recognized as the prototypical biodegradable thermoplastic to solve the waste disposal challenge. The development effort led by Imperial Chemical Industries Ltd., encouraged interdisciplinary research from genetic engineering and biotechnology to the study of enzymes involved in biosynthesis and biodegradation. From the simple PHB homopolyester discovered by Maurice Lemoigne in the mid-twenties, a family of over 100 different aliphatic polyesters of the same general structure has been discovered. Depending on bacterial species and substrates, these high molecular weight stereoregular polyesters have emerged as a new family of natural polymers ranking with nucleic acids, polyamides, polyisoprenoids, polyphenols, polyphosphates, and polysaccharides. In this historical review, the chemical, biochemical and microbial highlights are linked to personalities and locations involved with the events covering a discovery timespan of 75 years.     

Gibberellin biosynthesis and its regulation

The GAs (gibberellins) comprise a large group of diterpenoid carboxylic acids that are ubiquitous in higher plants, in which certain members function as endogenous growth regulators, promoting organ expansion and developmental changes. These compounds are also produced by some species of lower plants, fungi and bacteria, although, in contrast to higher plants, the function of GAs in these organisms has only recently been investigated and is still unclear. In higher plants, GAs are synthesized by the action of terpene cyclases, cytochrome P450 mono-oxygenases and 2-oxoglutarate-dependent dioxygenases localized, respectively, in plastids, the endomembrane system and the cytosol. The concentration of biologically active GAs at their sites of action is tightly regulated and is moderated by numerous developmental and environmental cues. Recent research has focused on regulatory mechanisms, acting primarily on expression of the genes that encode the dioxygenases involved in biosynthesis and deactivation. The present review discusses the current state of knowledge on GA metabolism with particular emphasis on regulation, including the complex mechanisms for the maintenance of GA homoeostasis.     

Molecular and genetic studies of fusarium trichothecene biosynthesis: pathways, genes, and evolution

Trichothecenes are a large family of sesquiterpenoid secondary metabolites of Fusarium species (e.g., F. graminearum) and other molds. They are major mycotoxins that can cause serious problems when consumed via contaminated cereal grains. In the past 20 years, an outline of the trichothecene biosynthetic pathway has been established based on the results of precursor feeding experiments and blocked mutant analyses. Following the isolation of the pathway gene Tri5 encoding the first committed enzyme trichodiene synthase, 10 biosynthesis genes (Tri genes; two regulatory genes, seven pathway genes, and one transporter gene) were functionally identified in the Tri5 gene cluster. At least three pathway genes, Tri101 (separated alone), and Tri1 and Tri16 (located in the Tri1-Tri16 two-gene cluster), were found outside of the Tri5 gene cluster. In this review, we summarize the current understanding of the pathways of biosynthesis, the functions of cloned Tri genes, and the evolution of Tri genes, focusing on Fusarium species.     

Gibberellin biosynthesis inhibitors: Comparing growth-retarding effectiveness on apple

The relative growth inhibitory activities of paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2(1,2,4-triazol-1-yl)pentan-3-ol]; XE-1019 [(E)-(1-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol]; flurprimidol [-(1-methylethyl)--[4-(trifluoromethyloxy)-phenyl]-5-pyrimidine-methanol]; and triadimefon (a fungicide) [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone] were evaluated and compared by treating the root zone of young greenhouse-grown tissue-culture-propagated Gala (Malus domestica Borkh.) trees. At 0.25 mg/plant, only XE-1019 significantly reduced new stem length and number, area, and dry weight of leaves after 115 days. Paclobutrazol and flurprimidol both significantly reduced growth compared to controls when applied at 0.5 mg/plant, but XE-1019 was more effective. All three gibberellin (GA) biosynthesis inhibitors effectively retarded growth at a dosage of 1 mg/plant. Triadimefon applied at 10 mg/plant had essentially no effect on growth, but at 50 and 100 mg/plant it caused significant but less dramatic growth retardation when compared with the GA inhibitors. Major differences in effectiveness among the triazole GA biosynthesis inhibitors may be due to longevity of effect as well as to extent of inhibition.The use of a company or product name does not constitute an endorsement by USDA nor imply approval to the exclusion of other suitable products.     

Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: genes,enzymes and exopolysaccharide production engineering

The commercial gelling agent, gellan, is an extracellular polysaccharide (EPS) produced by Sphingomonas paucimobilis ATCC 31461. In recent years, significant progress in understanding the relationship between gellan structure and properties and elucidation of the biosynthesis and engineering of this recent product of biotechnology has been made. This review focuses on recent advances in this field. Emphasis is given to identification and characterization of genes and enzymes involved, or predicted to be involved, in the gellan biosynthetic pathway, at the level of synthesis of sugar-activated precursors, of the repeat unit assembly and of gellan polymerization and export. Identification of several genes, biochemical characterization of the encoded enzymes and elucidation of crucial steps of the gellan pathway indicate that possibilities now exist for exerting control over gellan production at any of the three levels of its biosynthesis. However, a better knowledge of the poorly understood steps and of the bottlenecks and regulation of the pathway, the characterization of the composition, structure and functional properties of gellan-like polymers produced either by the industrial strain under different culture conditions or by mutants are still required for eventual success of the metabolic engineering of gellan production. Journal of Industrial Microbiology & Biotechnology (2002) 29, 170–176 doi:10.1038/sj.jim.7000266 Received 11 February 2002/ Accepted in revised form 09 April 2002     

LC-PUFA from photosynthetic microalgae: occurrence, biosynthesis, and prospects in biotechnology

Microalgae offer potential for numerous commercial applications, among them the production of long-chain polyunsaturated fatty acids (LC-PUFAs). These valuable fatty acids are important for a variety of nutraceutical and pharmaceutical purposes, and the market for these products is continually growing. An appropriate ratio of LC-PUFA of the ω-3 and ω-6 groups is vital for "healthy" nutrition, and adequate dietary intake has strong health benefits in humans. Microalgae of diverse classes are primary natural producers of LC-PUFA. This mini-review presents an introductory overview of LC-PUFA-related health benefits in humans, describes LC-PUFA occurrence in diverse microalgal classes, depicts the major pathways of their biosynthesis in microalgae, and discusses the prospects for microalgal LC-PUFA production.     

Gibberellin biosynthesis mutations and root development in pea

Dwarf mutants of pea (Pisum sativum), with impaired gibberellin (GA) biosynthesis in the shoot, were studied to determine whether the roots of these genotypes had altered elongation and GA levels. Mutations na, lh-2, and ls-1 reduced GA levels in root tips and taproot elongation, although in lh-2 and ls-1 roots the reduction in elongation was small (less than 15%). The na mutation reduced taproot length by about 50%. The roots of na plants elongated in response to applied GA(1) and recombining na with mutation sln (which blocks GA catabolism) increased GA(1) levels in root tips and completely restored normal root development. In shoots, Mendel's le-1 mutation impairs the 3beta-hydroxylation of GA(20) to the bioactive GA(1), resulting in dwarfism. However, GA(1) and GA(20) levels were normal in le-1 roots, as was root development. The null mutation le-2 also did not reduce root GA levels or elongation. The results support the theory that GAs are important for normal root elongation in pea, and indicate that a 3beta-hydroxylase gene other than LE operates in pea roots.     

Cellulases and related enzymes in biotechnology

Basic and applied research on microbial cellulases, hemicellulases and pectinases has not only generated significant scientific knowledge but has also revealed their enormous potential in biotechnology. At present, cellulases and related enzymes are used in food, brewery and wine, animal feed, textile and laundry, pulp and paper industries, as well as in agriculture and for research purposes. Indeed, the demand for these enzymes is growing more rapidly than ever before, and this demand has become the driving force for research on cellulases and related enzymes. The present article is an overview of the biotechnological state-of-the-art for cellulases and related enzymes.     

Green leaf volatiles: biosynthesis,biological functions and their applications in biotechnology

Plants have evolved numerous constitutive and inducible defence mechanisms to cope with biotic and abiotic stresses. These stresses induce the expression of various genes to activate defence‐related pathways that result in the release of defence chemicals. One of these defence mechanisms is the oxylipin pathway, which produces jasmonates, divinylethers and green leaf volatiles (GLVs) through the peroxidation of polyunsaturated fatty acids (PUFAs). GLVs have recently emerged as key players in plant defence, plant–plant interactions and plant–insect interactions. Some GLVs inhibit the growth and propagation of plant pathogens, including bacteria, viruses and fungi. In certain cases, GLVs released from plants under herbivore attack can serve as aerial messengers to neighbouring plants and to attract parasitic or parasitoid enemies of the herbivores. The plants that perceive these volatile signals are primed and can then adapt in preparation for the upcoming challenges. Due to their ‘green note’ odour, GLVs impart aromas and flavours to many natural foods, such as vegetables and fruits, and therefore, they can be exploited in industrial biotechnology. The aim of this study was to review the progress and recent developments in research on the oxylipin pathway, with a specific focus on the biosynthesis and biological functions of GLVs and their applications in industrial biotechnology.     

Recent developments in the taxonomic affiliation and phylogenetic positioning of fungi: impact in applied microbiology and environmental biotechnology

The goal of modern taxonomy is to understand the relationships of living organisms in terms of evolutionary descent. Thereby, the relationships between living organisms are understood in terms of nested clades—every time a speciation event takes place, two new clades are produced. Life comprises three domains of living organisms, these are the Bacteria, the Archaea and the Eukaryota. Within the eukaryotic domain, the fungi form a monophyletic group of the eukaryotic crown group and are thus high up in the evolutionary hierarchy of life. Fungus-like organisms possess certain morphological features of fungi, such as the hyphal organization of the Oomycota or the spores and reproductive structures inside a fructification of plasmodiophorids (Plasmodiophoromycota) and slime moulds (Mycetozoa). The first group are algae which secondarily lost their plastids during evolution and contain cellulose in their cell walls. Both osmotrophic phyla, the Oomycota and the Plasmidiophoromycota belong to the Chromista and Rhizaria, respectively, whereas the last group, the cellular and plasmodial slime moulds (Mycetozoa) are phagotrophic amoeboid protists belonging to the Amoebozoa. These fungus-like organisms are not considered further in this review. The Fungi sensu stricto comprise a heterogenous, often inconspicuous group of microorganisms which (1) are primarily heterotrophic with an (2) osmotrophic style of nutrition containing (3) chitin and its derivatives in the cell wall. This review discusses species concepts and current strategies in fungal taxonomy, phylogenetic affiliations of miscellaneous fungus-like groups like the microsporidia, perspectives of fungal nomenclature, and their impact on natural product research.     

Virulence genes and the evolution of host specificity in plant-pathogenic fungi

In the fungal kingdom, the ability to cause disease in plants appears to have arisen multiple times during evolution. In many cases, the ability to infect particular plant species depends on specific genes that distinguish virulent fungi from their sometimes closely related nonvirulent relatives. These genes encode host-determining "virulence factors," including small, secreted proteins and enzymes involved in the synthesis of toxins. These virulence factors typically are involved in evolutionary arms races between plants and pathogens. We briefly summarize current knowledge of these virulence factors from several fungal species in terms of function, phylogenetic distribution, sequence variation, and genomic location. Second, we address some issues that are relevant to the evolution of virulence in fungi toward plants; in particular, horizontal gene transfer and the genomic organization of virulence genes.     

Metal accumulation by fungi: Applications in environmental biotechnology

Summary Fungi can accumulate metal and radionuclide species by physico-chemical and biological mechanisms including extracellular binding by metabolites and biopolymers, binding to specific polypeptides and metabolism-dependent accumulation. Biosorptive processes appear to have the most potential for environmental biotechnology. Biosorption consists of accumulation by predominatly metabolism-independent interactions, such as adsorptive or ion-exchange processes: the biosorptive capacity of the biomass can be manipulated by a range of physical and chemical treatments. Immobilized biomass retains biosorptive properties and possesses a number of advantages for process applications. Native or immobilized biomass can be used in fixed-bed, air-lift or fluidized bed bioreactors; biosorbed metal/radionuclide species can be removed for reclamation and the biomass regenerated by simple chemical treatments.     

Gibberellin biosynthesis: its regulation by endogenous and environmental signals

The hormone gibberellin (GA) plays an essential role in many aspects of plant growth and development, such as seed germination, stem elongation and flower development. In recent years, exciting progress has been made in understanding how the biosynthesis of this hormone is regulated by endogenous and environmental factors. This has resulted from isolation of genes encoding enzymes involved in GA biosynthesis and metabolism, which also enabled us to manipulate the pathway by modifying the expression of these genes in transgenic plants. In addition, new GA response mutants provided information about how signaling components are involved in feedback regulation of the GA biosynthetic pathway.     

Eight-carbon volatiles in mushrooms and fungi: properties, analysis, and biosynthesis

Eight-carbon volatiles are ubiquitous among fungi and characteristic of the fungal aroma. They are the product of the oxidation and cleavage of the fatty acid linoleic acid and are classified as oxylipins, molecules taking part in a wide range of biological processes. Their involvement in the fungal aroma, interactions with pests and pathogens, and reproductive events are reviewed here, as well as the enzymic systems involved in their biosynthesis.