Galactofuranose (Galf)-containing sugar chain contributes to the hyphal growth, conidiation and virulence of F. oxysporum f.sp. cucumerinum.

The ascomycete fungus Fusarium oxysporum f.sp. cucumerinum causes vascular wilt diseases in cucumber. However, few genes related to morphogenesis and pathogenicity of this fungal pathogen have been functionally characterized. BLASTp searches of the Aspergillus fumigatus UgmA and galatofuranosyltransferases (Galf-transferases) sequences in the F. oxysporum genome identified two genes encoding putative UDP-galactopyranose mutase (UGM), ugmA and ugmB, and six genes encoding putative Galf-transferase homologs. In this study, the single and double mutants of the ugmA, ugmB and gfsB were obtained. The roles of UGMs and GfsB were investigated by analyzing the phenotypes of the mutants. Our results showed that deletion of the ugmA gene led to a reduced production of galactofuranose-containing sugar chains, reduced growth and impaired conidiation of F. oxysporum f.sp. cucumerinum. Most importantly, the ugmA deletion mutant lost the pathogenicity in cucumber plantlets. Although deletion of the ugmB gene did not cause any visible phenotype, deletion of both ugmA and ugmB genes caused more severe phenotypes as compared with the ΔugmA, suggesting that UgmA and UgmB are redundant and they can both contribute to synthesis of UDP-Galf. Furthermore, the ΔgfsB exhibited an attenuated virulence although no other phenotype was observed. Our results demonstrate that the galactofuranose (Galf) synthesis contributes to the cell wall integrity, germination, hyphal growth, conidiation and virulence in Fusarium oxysporum f.sp. cucumerinum and an ideal target for the development of new anti-Fusarium agents.

Productive Amyrin Synthases for Efficient α-Amyrin Synthesis in Engineered Saccharomyces cerevisiae.

α-Amyrin is a plant-derived pentacyclic triterpenoid, with a lot of important physiological and pharmacological activities. The formation of α-amyrin from (3 S)-2,3-oxidosqualene is catalyzed by α-amyrin synthase (α-AS), a member of the oxidosqualene cyclase (OSC) protein family. However, α-amyrin is not yet commercially developed due to its extremely low productivity in plants. The engineered Saccharomyces cerevisiae with efficient α-amyrin production pathway could be used as an alternative and sustainable solution to produce α-amyrin from renewable raw materials. To efficiently improve α-amyrin production in S. cerevisiae, we identified two α-ASs, EjAS and MdOSC1 from Eriobotrya japonica and Malus × domestica, respectively, through strict bioinformatics screening criteria and phylogenetic analysis. The specific activities of purified EjAS and MdOSC1 were 0.0032 and 0.0293 µmol/min/mg, respectively. EjAS produced α-amyrin and ß-amyrin at a ratio of 17:3, MdOSC1 produced α-amyrin, ß-amyrin and lupeol at a ratio of 86:13:1, indicating MdOSC1 had significantly higher specific activity and higher ratio of α-amyrin than EjAS. Furthermore, MdOSC1 was introduced into S. cerevisiae combining with the increased supply of (3 S)-2,3-oxidosqualene to achieve the encouraging α-amyrin production, and the titer of α-amyrin achieved 11.97 ± 0.61 mg/L, 5.8 folds of the maximum production reported.

Synthetic and Therapeutic Applications of Ammonia-lyases and Aminomutases.

Ammonia-lyases and aminomutases are mechanistically and structurally diverse enzymes which catalyze the deamination and/or isomerization of amino acids in nature by cleaving or shifting a C-N bond. Of the many protein families in which these enzyme activities are found, only a subset have been employed in the synthesis of optically pure fine chemicals or in medical applications. This review covers the natural diversity of these enzymes, highlighting particular enzyme classes that are used within industrial and medical biotechnology. These highlights detail the discovery and mechanistic investigations of these commercially relevant enzymes, along with comparisons of their various applications as stand-alone catalysts, components of artificial biosynthetic pathways and biocatalytic or chemoenzymatic cascades, and therapeutic tools for the potential treatment of various pathologies.

Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene.

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to ß-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.

Role of cysteine residues in pseudouridine synthases of different families.

The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.

Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases.

Two new triterpene synthase cDNAs, named as OEW and TRW, were cloned from olive leaves (Olea europaea) and from dandelion roots (Taraxacum officinale), respectively, by the PCR method with primers designed from the conserved sequences found in the known oxidosqualene cyclases. Their ORFs consisted of 2274 bp nucleotides and coded for 758 amino acid long polypeptides. They shared high sequence identity (78%) to each other, while they showed only about 60% identities to the known triterpene synthases LUPI (lupeol synthase clone from Arabidopsis thaliana) and PNY (beta-amyrin synthase clone from Panax ginseng) at amino acid level. To determine the enzyme functions of the translates, they were expressed in an ERG7 deficient yeast mutant. Accumulation of lupeol in the cells of yeast transformants proved both of these clones code for lupeol synthase proteins. An EST (expression sequence tag) clone isolated from Medicago truncatula roots as a homologue of cycloartenol synthase gene, exhibits high sequence identity (75-77%) to these two lupeol synthase cDNAs, suggesting it to be another lupeol synthase clone. Comparatively low identity (approximately 57%) of LUP1 from Arabidopsis thaliana to either one of these clones leaves LUP1 as a distinct clone among lupeol synthases. From these sequence comparisons, now we propose that two branches of lupeol synthase gene have been generated in higher plants during the course of evolution.