The salicinoids are phenolic glycosides (PGs) characteristic of the Salicaceae family and are known defenses against insect herbivory. Common examples are salicin, salicortin, tremuloidin, and tremulacin, which accumulate to high concentrations in the leaves and bark of willows and poplars. Despite their important role in plant defense, their biosynthetic pathway is not known, although recent work has suggested that benzyl benzoate acts as a possible biosynthetic intermediate. We identified three candidate genes encoding BAHD-type acyltransferases that are predicted to produce benzylated secondary metabolites, named PtACT47, PtACT49, and PtACT54. Expression of PtACT47 and PtACT49 generally correlated with PG content in a variety of tissues and organs of wild type hybrid poplar plants. This correlation was also found in transgenic hybrid poplar where PG content varied with the level of expression of the condensed tannin regulator MYB134 transcript. In these plants, a suppression of PtACT47 and PtACT49 expression was correlated with lower PG content. In contrast, PtACT54 exhibited very low expression in all tissues tested, and this level of expression was not affected in MYB134 plants. In order to better understand their possible biochemical functions, cDNA cloning, heterologous expression, and in vitro functional characterization was performed on these three BAHD acyltransferases. Recombinant PtACT47 exhibited a low substrate selectivity and could utilize acetyl-CoA, benzoyl-CoA, and cinnamoyl-CoA as acyl donors with a variety of alcohols as acyl acceptors. This enzyme showed the greatest Km/Kcat ratio (45.8 nM-1 sec-1) and lowest Km values (45.1 µM) with benzoyl-CoA and salicyl alcohol, and was named benzoyl-CoA:salicyl alcohol O-benzoyltransferase (PtSABT). Recombinant PtACT49 utilized a narrower range of substrates, specifically benzoyl-CoA and acetyl-CoA and a limited number of alcohols. Its highest Km/Kcat (31.8 nM-1 sec-1) and lowest Km (55.3 µM) was observed for benzoyl-CoA and benzyl alcohol, and it was named benzoyl-CoA:benzyl alcohol O-benzoyltransferase (PtBEBT). Both enzymes were also capable of synthesizing plant volatile alcohol esters at trace levels, for example hexenyl benzoate. Recombinant PtACT54 shares low sequence identity with PtSABT (52.3%) and PtBEBT (52.5%) and exhibited only moderate BEBT-like properties. PtSABT and PtBEBT appear to be paralogs based on their high sequence identity (90.6%) and closely related yet distinct biochemical functions. They likely arose from gene duplication and subsequent functional diversification possibly by neofunctionalization. Wounding experiments showed that abiotic damage stimulated the synthesis of specific PGs, notably salicin and salicortin within 24-48hrs. This was accompanied by a proportional increase in the expression of PtSABT and PtBEBT. Furthermore, experiments using transgenic RNAi lines with knock-down suppression of PtBEBT, and PtSABT, and both genes simultaneously, provided the first direct evidence that BAHD acyltransferases are important in PG production. PtSABT suppression, both individually and in the double knock-down suppression, significantly lowered salicortin content, particularly in mature leaves. However, a reduced level of PtBEBT expression did not have a significant effect on the PGs measured. This could indicate that BEBT-like activity may be a shared function among closely related BAHDs. The suppression of multiple BEBT-like genes may be necessary to further delineate their functions. / Graduate / rjchedgy@uvic.ca

Du fait de l’apparition de microorganismes pathogènes ayant une résistance aux antibiotiques actuels, la recherche de nouvelles molécules bioactives possédant une application médicale est devenue une préoccupation mondiale. Saccharothrix algeriensis, une bactérie filamenteuse de l’ordre des actinomycètes a montré une étonnante capacité à produire des molécules bioactives qui appartiennent aux dithiolopyrrolones, ayant de remarquables propriétés à la fois antibiotiques et anticancéreuses. Lors de ce projet de thèse, l’identification du cluster de gènes de la voie de biosynthèse des dithiolopyrrolones chez Sa. algeriensis est envisagée. Suite au séquençage du génome de Sa. algeriensis, une approche génomique ou « genome mining » est suivie, cette approche a révélé un cluster thi potentiellement responsable de la voie de biosynthèse des dithiolopyrrolones chez Sa. algeriensis. Ce cluster contient 12 gènes, dont 8 gènes de biosynthèse, 3 gènes régulateurs et un gène transporteur. Les analyses in silico des gènes ont montré que la cystéine est le substrat d’une NRPS. Les analyses transcriptionelles ont montré que les trois gènes clés codent pour une NRPS, une thiorédoxine et une thioestérase qui pourraient être impliquées dans la biosynthèse des dithiolopyrrolones. Deux gènes actA et actB codant pour des acyltransférases putatives ont été identifiés. Les analyses transcriptionelles suggèrent qu’actA et actB pourraient être responsables de l’acylation de la pyrrothine. Finalement, la caractérisation de deux activités enzymatiques, acétyltransférase et benzoyltransférase, présentes dans l’extrait brut de Sa. algeriensis, ont permis de déterminer les paramètres optimaux (pH et T °C) de la réaction enzymatique. Enfin, les paramètres cinétiques de ces activités ont des valeurs complètements différentes, ce qui confirme la présence d’au moins deux activités différentes chez Sa. algeriensis. / Due to the emergence in the last decades of new and old infectious diseases to existing antibiotics, the research for new bioactive molecules which possess medical applications become a global occupation. Saccharothrix algeriensis, filamentous bacteria of actinomycetes order showed a surprising ability to produce bioactive molecules belongs to dithiolopyrrolones with remarkable properties of both antibiotics and anticancer. In this thesis, the identification of dithiolopyrrolones biosynthetic gene cluster in Sa. algeriensis was investigated. Through S. algeriensis genome sequencing, a genomics approach "genome mining" was followed, this approach has revealed a potentially thi cluster responsible for dithiolopyrrolones biosynthesis pathway in Sa algeriensis. This cluster contains 12 genes, including 8 biosynthesis genes, three regulatory genes and one transporter gene. The in silico analysis of this cluster showed that the cysteine is the substrate of the NRPS. The transcriptional analyzes showed that the three key genes which encode for NRPS, thioredoxin and thioesterase could be involved in dithiolopyrrolone biosyntheses. Two genes, actA and actB, encode for two putative acyltransferases were identified, the transcriptional analyzes suggests that these genes may be responsible for the acylation of pyrrothine core. The characterizations of two activities, acetyltransferase and benzoyltransferase, in the crude extract of Sa. algeriensis led the determination of the optimal parameters (pH and T °C) to detect these activities. Moreover, the effect of temperature and pH on these activities was determined. Finally, the kinetic parameters of these activities showed different values, which confirm the presence of, at least, two activities in Sa. algeriensis.